Isothermal Amplification of Nucleic Acids | Chemical Reviews

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Isothermal Amplification of Nucleic Acids Yongxi Zhao,*,† Feng Chen,† Qian Li,‡ Lihua Wang,‡ and Chunhai Fan*,‡,§ †

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Key Laboratory of Biomedical Information Engineering of Education Ministry, School of Life Science and Technology, Xi’an Jiaotong University, Xianning West Road, Xi’an, Shaanxi 710049, China ‡ Division of Physical Biology, and Bioimaging Center, Shanghai Synchrotron Radiation Facility, CAS Key Laboraotory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China § School of Life Science & Technology, ShanghaiTech University, Shanghai 200031, China ABSTRACT: Isothermal amplification of nucleic acids is a simple process that rapidly and efficiently accumulates nucleic acid sequences at constant temperature. Since the early 1990s, various isothermal amplification techniques have been developed as alternatives to polymerase chain reaction (PCR). These isothermal amplification methods have been used for biosensing targets such as DNA, RNA, cells, proteins, small molecules, and ions. The applications of these techniques for in situ or intracellular bioimaging and sequencing have been amply demonstrated. Amplicons produced by isothermal amplification methods have also been utilized to construct versatile nucleic acid nanomaterials for promising applications in biomedicine, bioimaging, and biosensing. The integration of isothermal amplification into microsystems or portable devices improves nucleic acid-based on-site assays and confers high sensitivity. Single-cell and single-molecule analyses have also been implemented based on integrated microfluidic systems. In this review, we provide a comprehensive overview of the isothermal amplification of nucleic acids encompassing work published in the past two decades. First, different isothermal amplification techniques are classified into three types based on reaction kinetics. Then, we summarize the applications of isothermal amplification in bioanalysis, diagnostics, nanotechnology, materials science, and device integration. Finally, several challenges and perspectives in the field are discussed.

CONTENTS 1. Introduction 2. Isothermal Nucleic Acid Amplification 2.1. Exponential Amplification 2.1.1. Nucleic Acid Sequence-Based Amplification (NASBA) 2.1.2. Exponential Strand Displacement Amplification (E-SDA) 2.1.3. Exponential Rolling Circle Amplification (E-RCA) 2.1.4. Loop-Mediated Isothermal Amplification (LAMP) 2.1.5. Helicase-Dependent Amplification (HDA) 2.1.6. Recombinase Polymerase Amplification (RPA) 2.1.7. Exponential Amplification Reaction (EXPAR) 2.1.8. Whole Genome Amplification (WGA) 2.1.9. Emerging Exponential Isothermal Amplification 2.2. Linear Amplification 2.2.1. Linear SDA 2.2.2. Linear RCA 2.2.3. Transcription-Based Amplification 2.2.4. Signal Amplification Strategies

© 2015 American Chemical Society

2.3. Cascade Amplification 2.3.1. SDA-Combined Cascade Amplification 2.3.2. RCA-Combined Cascade Amplification 2.3.3. Additional Cascade Amplification 3. Bioanalytical Applications 3.1. Detection of Nucleic Acids 3.1.1. Detection of DNA 3.1.2. Detection of SNPs 3.1.3. Detection of DNA Methylation 3.1.4. Detection of Long RNAs 3.1.5. Detection of miRNAs 3.2. Detection of Proteins and Enzymes 3.2.1. Detection of Proteins 3.2.2. Detection of Enzymes 3.3. Detection of Cancer Cells and Pathogens 3.4. Detection of Small Molecules and Metal Ions 3.4.1. Detection of Small Molecules 3.4.2. Detection of Metal Ions 3.5. In Situ and Intracellular Analysis 3.6. Sequencing 4. Applications in Nanotechnology and Materials Science 4.1. Construction of Nucleic Acid Nanostructures 4.2. Construction of Nucleic Acid Hydrogels

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Received: July 25, 2015 Published: November 9, 2015 12491

DOI: 10.1021/acs.chemrev.5b00428 Chem. Rev. 2015, 115, 12491−12545

Chemical Reviews 4.3. Formation of Nucleic Acid-Templated Nanomaterials 5. Device Integration 5.1. Microfluidic Systems 5.2. Capillary Platforms 5.3. Paper-Based Platforms 5.4. Commercial Devices 6. Conclusion and Perspectives Author Information Corresponding Authors Notes Biographies Acknowledgments References

Review

Thousands of studies related to the isothermal amplification of nucleic acids have been published. However, the concept, applications, and perspectives of isothermal amplification of nucleic acids have not been comprehensively reviewed. The present comprehensive review of isothermal amplification of nucleic acids primarily covers published work in the past two decades. In this review, we first describe the development of isothermal amplification techniques and their classification into three types based on reaction kinetics. Furthermore, we summarize advances in isothermal amplification in biological research, diagnostic applications, nanobiotechnology, materials science, and device integration. Finally, challenges and perspectives in the field will be discussed.

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2. ISOTHERMAL NUCLEIC ACID AMPLIFICATION In contrast to PCR, which requires complex thermocycling to mediate denaturation, annealing, and subsequent extension, isothermal amplification can be performed at one reaction temperature under simple conditions (e.g., in a water bath). Many isothermal amplification techniques [e.g., exponential strand displacement amplification (E-SDA) and hyperbranched rolling circle amplification (HRCA)] are based on DNA replication. Others are based on enzyme-based digestion or enzyme-free nucleic acid assembly. In this section, we classify isothermal amplification techniques on the basis of their reaction kinetics: exponential amplification, linear amplification, and cascade amplification.

1. INTRODUCTION Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are essential for all forms of life. DNA stores genetic information and encodes the amino acid sequences of proteins responsible for cellular function. RNA plays various important roles in the coding, decoding, regulation, and expression of genes. Consequently, nucleic acids are used as important biomarkers for biological studies and medical diagnostics. In addition to their biological roles, nucleic acids can be used as high-affinity recognition molecules for ions, small molecules, proteins, and even whole cells.1−3 Nucleic acids can also be coupled with organic or inorganic materials for assembly and construction purposes.4,5 In view of these versatile functions and broad applications, methods have been developed to detect minute quantities of nucleic acids from complex media. Polymerase chain reaction (PCR) was the first and remains the most popular amplification technology for amplifying and detecting low-abundance nucleic acids. Although PCR has been widely employed in various fields, it requires large and expensive thermal cyclers, which largely limit the application of PCR in resource-limited settings and for point-of-care (POC) analysis. Isothermal amplification of nucleic acids has emerged as a promising alternative in which rapid and efficient amplification is achieved at a constant temperature without the thermocycling required in PCR.6−8 Additionally, isothermal amplification can be performed under simple conditions (e.g., water bath), on the cell surface, or even inside living cells, all of which are not feasible with PCR. Since the early 1990s, dozens of isothermal nucleic acid amplification techniques employing various amplification mechanisms have been developed. Most of these methods possess impressive sensitivity for the detection of nucleic acids, and some have achieved commercial success.9,10 More recently, isothermal amplification techniques have been expanded to detect a wide spectrum of targets including proteins, cells, small molecules, and ions. The combination of isothermal amplification and nanotechnology has also attracted intense attention. Isothermal amplification provides abundant nucleic acids as materials for constructing higher-ordered nucleic acid nanostructures, nucleic acidtemplated metal nanostructures, and nucleic acid hydrogels, which hold great promise in biosensing, bioimaging, and nanomedicine. Furthermore, whereas initial isothermal amplification methods were test tube based, more recent advances in microfabrication have led to the coupling of these methods with microfluidic chips, capillary platforms, and test paper. Integrated microfluidic systems have enabled single-cell or single-molecule analysis.

2.1. Exponential Amplification

More than ten types of isothermal exponential amplification methods have been developed by various laboratories. These mainly include nucleic acid sequence-based amplification (NASBA), E-SDA, HRCA, primer-generation rolling circle amplification (PG-RCA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), exponential amplification reaction (EXPAR), and whole genome amplification (WGA). Most of these amplification methods (e.g., NASBA, E-SDA, LAMP, HDA, and RPA) amplify nucleic acid templates using two or more primers, whereas others such as HRCA, PG-RCA, and EXPAR utilize one functional template to amplify the target nucleic acids as primers. In addition, although HRCA and LAMP can achieve exponential amplification using only DNA polymerase, others require additional enzymes or proteins. Importantly, these methods all exhibit high amplification efficiency comparable to that of PCR. A comparison of different isothermal amplification techniques is provided in Table 1. 2.1.1. Nucleic Acid Sequence-Based Amplification (NASBA). NASBA,11 also known as self-sustained sequence replication (3SR),12 is similar to transcription-mediated amplification (TMA). NASBA was designed in 1991 specifically to amplify single-stranded (ss) RNA sequences by mimicking retroviral RNA replication. NASBA requires three enzymes (reverse transcriptase, RNase H, and DNA-dependent RNA polymerase), whereas TMA utilizes a unique reverse transcriptase with intrinsic RNase H activity. The principle of NASBA is illustrated in Figure 1A. During the reaction, the target RNA first hybridizes to the forward primer and is converted to a complementary DNA (cDNA) intermediate by reverse transcriptase and RNase H. Then, the intermediate double-stranded (ds) cDNA containing a promoter region is formed using the second primer. Many antisense RNA strands 12492


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Table 1. Summary of Important Isothermal Amplification Methods method

required enzymes

primers

temperature (°C)

reaction time (h)

target

amplicon

efficiency

refs

106−109

11, 12

DNA

RNA, DNA dsDNA

107

1.5

DNA (RNA)

DNA

109

60

1−3

DNA (RNA)

DNA

4 2 2

60−65 37−65 37−42

8

DNA

106

39, 42, 45, 46 47, 666

pWGA

2: T7 gp4 primase and DNA polymerase

random primers 0

short DNA (RNA) DNA

∼60 copies of genomic DNA 109 107 10 copies of genomic DNA 106−108

19, 20, 256 24, 49, 227 25, 232, 371 26, 27 30, 349 35, 653

37

0.5−2

DNA

DNA

103−108

50

2

∼ 41

1.5−2

RNA (DNA)

E-SDA

2 or 3: reverse transcriptase and RNA polymerase (RNase H) 2: DNA polymerase and NEase

2 or 4

37

2

HRCA

2: ligase and DNA polymerase

2

60

PG-RCA

2: DNA polymerase and NEase

0

LAMP HDA RPA

1: DNA polymerase 2: DNA polymerase and helicase 2: DNA polymerase and recombinase

EXPAR

NASBA

followed by a new nicking reaction. The cycles of nicking and polymerization/displacement steps continuously produce a sscomplement of the target. The ss-complement from Pl-T1 acts as the template for primer P2, whereas the ss-complement from P2-T2 is the template for primer P1. These steps are repeated continuously throughout the amplification, resulting in exponential accumulation of the target sequences. In early versions of E-SDA, the incorporation of phosphorothioate into the recognition site of the amplicon during polymerization was required to ensure that the restriction endonuclease (REase) cut only one strand rather than two. This step has been eliminated by the use of NEase, which cuts only one strand of dsDNA. Although amplification is performed at a constant temperature, thermal denaturation (95 °C) for target generation is often required in E-SDA when sensing genomic DNA. Notably, the amplification kinetics are linear (linear SDA) rather than exponential when using only one primer (P1 or P2). 2.1.3. Exponential Rolling Circle Amplification (ERCA). In this review, E-RCA is defined as RCA-based exponential amplification methods including HRCA and PGRCA. In the mid-1990s, several studies21−23 reported that polymerase can utilize a circular template (called a “padlock probe”) to synthesize a long, repetitive ss-product, leading to the proposal of HRCA.24 In contrast to SDA, which requires NEase, only DNA polymerase is required during amplification in HRCA, but an additional ligase is required for the specific circularization of a padlock probe (with 3′-hydroxy and 5′phosphate) using the target sequence of the genomic DNA as the template in the ligation reaction. A primer (P1) complementary to the padlock probe subsequently initiates the extension reaction catalyzed by exo− DNA polymerase, generating a long ss-product with hundreds of closed-circle copies in a few minutes (Figure 1C). Sequential primer extension reactions are then induced by binding of the second primer P2 to the complementary sequence of the ss-product. As each primer extends to the downstream primer, strand displacement occurs and results in the production of tandem repetitive sequences of the original circularized probe. These displaced strands in turn serve as templates for the next cycle of extension from the first primer P1. As a result, continuously expanding DNA branches connected to the cyclized padlock probe are generated. The strand displacement process can also

to the target RNA are produced by transcription of this dscDNA by T7 DNA-dependent RNA polymerase and become templates for the original reverse transcription (RT) reaction. The newly synthesized antisense RNA and cDNA serve as templates for continuous cycling of the RT and transcription reactions, resulting in exponential accumulation of antisense RNA complementary to the target RNA. Generally, NASBA can achieve 109-fold amplification in 1.5− 2 h at 41 °C. This sensitivity is comparable to that of RTPCR.13 Another advantage of NASBA is that the amplified product is ssRNA, enabling simple and rapid hybridization detection without a denaturation step. Many assay approaches, such as gel electrophoresis, real-time fluorescence,14,15 colorimetric assays,16 and electrochemiluminescence (ECL),17 have been used to detect the NASBA amplicon. Multiplex detection by NASBA was also demonstrated as early as 1999.18 Occasionally, an additional step to remove the secondary structure of target RNA and for primer annealing at 65 °C is required before the amplification reaction. Both the amplification and detection processes require RNase-free conditions to prevent RNA degradation. 2.1.2. Exponential Strand Displacement Amplification (E-SDA). E-SDA was proposed19 in 1992 and subsequently improved20 in the same year by Walker et al. In contrast to NASBA, E-SDA is a DNA replication process that omits RT or transcription reactions and enables the exponential accumulation of dsDNA products from genomic DNA. Nearly 107-fold amplification is achieved in 2 h at 37 °C. The amplification mechanism of E-SDA is based on the continuous nicking and polymerization/displacement process catalyzed by endonuclease (Endo) and 5′ → 3′ exonuclease-deficient (exo−) DNA polymerase (Figure 1B). Prior to amplification, the target genomic DNA templates are prepared by displacement reactions with four primers20 or restriction enzyme-mediated cleavage.19 After heat denaturation in the presence of two primers (P1 and P2), two primer-template duplexes (Pl-T1 and P2-T2) with 5′ overhangs are formed. The 5′ overhangs of the two primers each contain one recognition sequence for nicking endonuclease (NEase). Then, DNA polymerase extends the 3′ ends of the duplexes to produce dsDNA containing complete nicking sites that will be nicked by NEase. After nicking, new 3′ ends are generated at the nick to initiate a new extension reaction with displacement of the downstream target strand 12493


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Figure 1. Principles of representative exponential amplification techniques. (A) Scheme of NASBA. Reproduced with permission from ref 12. Copyright 1990 National Academy of Sciences. (B) Scheme of E-SDA using two primers with a dsDNA target fragment. Reproduced from ref 19. Copyright 1992 National Academy of Sciences. (C) Scheme of HRCA using two primers. Reprinted with permission from ref 24. Copyright 1998 Nature Publishing Group. (D) Reaction mechanism of PG-RCA. Reprinted with permission from ref 25. Copyright 2009 Oxford University Press. (E) Principle of LAMP. (a) Primer design of the LAMP reaction. For ease of explanation, six distinct regions are designated on the target DNA, labeled F3, F2, F1, B1c, B2c, and B3 from the 5′ end. (b) Starting structure producing step. (c) Cycling amplification step. Reprinted with permission from ref 27. Copyright 2008 Nature Publishing Group. (F) Schematic diagram of HDA. Reprinted with permission from ref 30. Copyright 2004 European Molecular Biology Organization. (G) Schematic of the RPA process. Reprinted with permission from ref 35. Copyright 2006 Piepenburg et al. (H). Diagram of EXPAR of oligonucleotides. Reprinted with permission from ref 39. Copyright 2003 National Academy of Sciences.

2.1.4. Loop-Mediated Isothermal Amplification (LAMP). As shown above, NASBA is performed at a relatively low temperature of 40 °C, leading to a reduction of specificity. E-SDA overcomes this shortcoming using two or even four primers, but the buffer must be optimized for both DNA polymerase and NEase. HRCA uses only polymerase, but an additional ligation process before amplification is always required for the specific recognition of a target. To improve sequence specificity and simplify the amplification system, LAMP was first proposed by Notomi and co-workers in 2000.26 LAMP can achieve excellent specificity in a one-step reaction using a set of four target-specific primers, including a forward inner primer (FIP), backward inner primer (BIP), and two outer primers (F3 and B3), to recognize six distinct sites flanking the amplified DNA sequence (top left of Figure 1E).27 The FIP and BIP play major roles in LAMP and each contain two functional sequences (one for priming extension in the first stage and the other for self-priming in the second stage) corresponding to the sequences (sense and antisense) of the target dsDNA. The LAMP reaction is catalyzed by DNA polymerase with strand displacement activity and includes two

generate a discrete series of dsDNA fragments comprising one or multiple units of the circle. More than 109 copies of the circle can be produced within 90 min at approximately 60 °C. Unlike HRCA, PG-RCA25 does not require exogenous primers, which often cause nonspecific amplification by forming primer-dimers but uses the target nucleic acid as the primer to begin the rolling circle amplification (RCA) process on the circular template containing a nicking site (Figure D). Many “primers” accumulate during the nicking reaction by NEase, and then trigger additional RCA and nicking reactions. Therefore, the target products accumulate exponentially during the continuous RCA and nicking processes. Under optimized conditions, a remarkable sensitivity of 0.163 pg (∼60 molecules) of genomic DNA from Listeria monocytogenes has been demonstrated.25 Although PG-RCA does not use primers, NEase is required, and the generation of ss-targets with 3′hydroxy groups from genomic DNA samples is complex. In both HRCA and PG-RCA, the amplified products can be detected by gel electrophoresis and fluorescent dye-based fluorescent analysis. 12494


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lateral-flow strip sensing system are also employed in RPA to decrease background noise and enable instrumentation-free readout, respectively. Various RPA kits have been commercialized (TwistDx, Cambridge, U.K.) and applied to detect pathogens and viruses.36−38 Notably, ATP is required to provide energy for recombinase in RPA and helicase in HDA, respectively. 2.1.7. Exponential Amplification Reaction (EXPAR). EXPAR is a unique isothermal amplification method39 that exponentially synthesizes short oligonucleotides to act as primers. EXPAR employs a functional template rather than the primers used in other isothermal strategies. This template consists of two copies complementary to the target segment separated by the recognition sequence of NEase (Figure 1H). Upon hybridization to the template, the target oligonucleotide will form two types of transient duplexes instead of a stable duplex at a high reaction temperature (approximately 60 °C). One type of transient duplex (formed with the first copies of the template) can be extended by DNA polymerase to produce a stable template duplex. This primed template continues to generate oligonucleotide product (identical to the target oligonucleotide), which serves as a primer to create new stable template duplexes in turn via the cycle of three processes (nicking the primed template, dissociating the oligonucleotide product and re-elongating the primer). This amplification process, analogous to a molecular chain reaction, results in exponential accumulation of oligonucleotide products within a few minutes, with amplification levels as high as 108-fold. By contrast, the other type of transient duplex (formed with the second copy of the template) does not provide a primertemplate structure for extension by polymerase and rapidly dissociates. Both EXPAR and SDA use DNA polymerase and NEase to perform a similar three-step cyclic process. Product separation in EXPAR essentially depends on the transformation of thermostable duplex into unstable transient duplex at high reaction temperature, whereas product separation in SDA is triggered by polymerase-induced strand displacement. Although the phosphate group is labeled at the 3′-hydroxy end of the template as a blocker to prevent potential selfpriming by pairs of template molecules,39 nonspecific amplification still occurs in EXPAR due to primer-independent DNA synthesis and ab initio DNA synthesis.40,41 Despite this nonspecific amplification, EXPAR has been employed for the successful detection of various biomarkers.42−46 2.1.8. Whole Genome Amplification (WGA). The amplification methods described above are all limited to the amplification of specific sequences with defined primers or templates. In addition to these sequence-specific isothermal amplification methods, several WGA methods have been developed that use random primers or primase.47−50 The first example, reported in 2001,49 is multiply primed RCA, which was derived from HRCA for circular genomes and uses random hexamer primers to anneal with the denatured circular templates, producing multiple replication forks (Figure 2A). RCA reactions proceed by displacing the newly synthesized strands, and these displaced product strands subsequently lead to secondary priming events. A cascade of priming events consequently ensues, resulting in exponential accumulation of ds-products and 104-fold amplification of plasmid or phage DNA in a few hours. Due to its great processivity (up to 70 kb in length) and strong strand displacement activity, Phi 29 DNA polymerase is used in

stages: the starting structure-producing step (first stage, top right of Figure 1E) and the cycling amplification step (second stage, bottom of Figure 1E). In the first stage, four primers are used, whereas only the two inner primers are required during the second stage. In brief, a ssDNA is released by strand displacement DNA synthesis primed by an outer primer (F3) and then acts as the template for DNA synthesis primed by both BIP and B3, producing a stem-loop DNA structure. After initiation by one inner primer complementary to the loop on the product, the cycling amplification process is continued by each inner primer alternately, resulting in the geometric accumulation of 109 copies of the target sequence in less than an hour. The final products are stem-loop DNAs with different inverted target repeats and cauliflower-like structures with multiple loops; these products can be detected by realtime assays and even end-point methods.28 The high specificity and efficiency of LAMP has led to its application in the detection of pathogens, SNPs, and RNAs. 28,29 Many commercial test kits using this method are available from different suppliers (e.g., Eiken Chemical Corporation). 2.1.5. Helicase-Dependent Amplification (HDA). In contrast to PCR, the aforementioned isothermal amplification methods are incapable of amplifying longer DNA targets such as kilobase (kb) regions, which is required in many basic research and diagnostic applications. To overcome these shortcomings, HDA was devised in 2004 to mimic DNA replication.30 In vivo, DNA is replicated by DNA polymerases, and various accessory proteins including DNA helicase are used to separate the DNA duplex. In HDA, a DNA helicase is also employed to separate the target dsDNA and generate sstemplates for primer hybridization and subsequent extension by DNA polymerase (Figure 1F). The HDA reaction is a threestep cycle process (template separation, primer hybridization, and primer extension), similar to PCR. However, the dsDNA is unwound by DNA helicase rather than the thermocycling steps used in PCR. Over a million-fold amplification of the target sequence can be achieved by the simultaneous chain reaction at one temperature. In the first version of HDA, the reaction temperature was 37 °C due to the thermal instability of E. coli UvrD helicase. The development of thermostable proteins has enabled faster amplification at higher temperature (60−65 °C) and with improved sensitivity and specificity for pathogen and SNP detection.31−34 Commercial HDA kits are available from BioHelix Corporation. 2.1.6. Recombinase Polymerase Amplification (RPA). RPA is an isothermal amplification strategy35 that employs recombinase to separate a dsDNA target (Figure 1G). Recombinase catalyzes the hybridization of primer with the homologous template sequence. The recombinase-primer filaments scan the target dsDNA and promote strand exchange at cognate sites. The resulting structures are stabilized by ssDNA-binding proteins such as those used in HDA, to prevent primer displacement by branch migration. DNA polymerase recognizes the primer 3′-ends left by recombinase disassembly and initiates the primer extension reaction. The binding/ extension events of two opposing primers generate one complete copy of the amplicon together with the original template. The cyclic repetition of this process results in exponential amplification of the target sequence. In 40 min, millions of target copies accumulate at low temperature (37−42 °C) with a detection limit as low as a single target copy. In addition to the detection methods used in other isothermal techniques, a unique FRET-based fluorescent probe and a 12495


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ssDNA binding protein is used to bind the ssDNA template and interact with both helicase/primase and DNA polymerase. Typically, microgram range product yields from 1 to 10 ng of human genomic DNA are achieved after 1 h of incubation at 37 °C. Circular DNA can also be used as a template in pWGA with 108-fold amplification of as few as 100 copies of input. Despite its relatively low yield, the amplification time of pWGA (1 h) is much shorter than that of MDA (typically several hours). Additionally, pWGA avoids the initial heat denaturation of genomic DNA that is often performed in MDA. These two approaches provide significantly better genome coverage and lower amplification bias than PCR-based WGA. Commercial WGA kits based on MDA and pWGA are available from different suppliers. 2.1.9. Emerging Exponential Isothermal Amplification. Several new exponential amplification techniques have been developed relatively recently. In 2007, an asymmetric isothermal amplification, smart amplification process version 2 (SMAP 2), that incorporates mismatch-suppression technology was developed.52,53 The amplification scheme of SMAP 2 resembles that of LAMP. A mismatch-binding protein is employed to suppress mismatch-induced nonspecific amplification, enabling accurate SNP diagnostics. Beacon-assisted detection amplification was proposed in 2010.54,55 A molecular beacon (MB) containing the recognition sequence of NEase is designed as a template for hybridization with a specific DNA sequence (∼20 nt), resulting in the exponential accumulation of ssDNA containing target sequence and a highly amplified fluorescent signal. Isothermal and chimeric primer-initiated amplification of nucleic acids56,57 uses 5′-DNA-RNA-3′ primers and a scheme similar to E-SDA. Recently, cleavage-based RNA amplification, similar to EXPAR and SDA, was demonstrated with real-time fluorescent and colorimetric readout.58 As competitors, these new methods are still undergoing optimization and may serve as useful alternatives to wellestablished methods.

Figure 2. WGA strategies. (A) Scheme for multiply primed RCA. Reprinted with permission from ref 49. Copyright 2001 CSH Press. (B) MDA strategy and product characterization. (left) Scheme for MDA of genomic DNA. Secondary priming events are initiated from primary products. (right) Denaturing gel analysis of amplification product size. Reprinted with permission from ref 47. Copyright 2002 National Academy of Sciences. (C) Mechanism of pWGA reaction. Reprinted with permission from ref 50. Copyright 2008 Oxford University Press.

2.2. Linear Amplification

Though exponential amplification techniques provide high amplification efficiency and detection sensitivity, they suffer fast nonspecific amplification and complex design. Conversely, linear amplification strategies are more convenient and exclude the interference of nonspecific amplification. And they are suitable for various applications with promising potential despite the relatively low assay sensitivity. 2.2.1. Linear SDA. As described in section 2.1.2, linear SDA is initiated using only one primer.19 In contrast to the bidirectional nature of E-SDA, its amplification process (nicking and polymerization/displacement) is unidirectional, accumulating thousands of target copies. A similar method, circular strand displacement polymerization reaction,59 has been developed by designing target-specific hairpin fluorescent probes to achieve a detection limit of 6.4 × 10−15 M for ssDNA targets within 1000 s. These linear methods have been applied for the detection of multiple targets.59−61 2.2.2. Linear RCA. RCA generates a long ssDNA of repetitive target sequences. In this linear format, the second primer used in HRCA is unnecessary, and nucleic acid synthesis is catalyzed by Phi 29 DNA polymerase rather than Vent exo− DNA polymerase or Bst DNA polymerase in HRCA. The reaction temperature for RCA (∼37 °C) is lower than that for HRCA (∼60 °C). RCA achieves approximately 103-fold amplification in 1 h, and this efficiency can be further increased

multiply primed RCA. Pyrophosphatase is employed in the reaction mixtures to eliminate the accumulation of pyrophosphates, which inhibit enzyme activity by chelating Mg2+. In 2002, this system was improved for the amplification of linear whole genomes (Figure 2B) using random hexamer primers and Phi 29 DNA polymerase, and the resulting method was termed multiple displacement amplification (MDA).47 MDA can be directly applied to complex biological samples and even clinical samples.47,48,51 Approximately 20−30 μg of product with an average length of 10 kb is created from as few as 1−10 copies of human genomic DNA. These amplified products are amenable to a variety of downstream applications such as sequencing, SNP genotyping, comparative genomic hybridization, chromosome painting, and Southern blotting. Another WGA method is primase-based WGA (pWGA),50 which mimics the DNA replication of T7 bacteriophage in vivo. The T7 gp4 protein, a bifunctional protein with both helicase and primase activities, is used in pWGA to unwind the duplex genomic DNA (Figure 2C). The primase activity of T7 gp4 results in the generation of short RNA primers on a specific sequence site of the template, eliminating the need for random primers. Subsequent DNA synthesis is mediated by T7 DNA polymerase with high processivity. As in HDA and RPA, 12496


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reaction (HCR), was proposed by Pierce and Dirks in 2004.146 In HCR, two partially complementary monomer DNA building blocks with the same hairpin structure (long stem and short loop) are designed (Figure 3A). In the absence of a target, the

by the addition of ssDNA-binding proteins. In addition to bioanalytical applications,62−66 RCA has been utilized in applications in nanobiomedicine,67−69 nanotechnology,70,70 and materials science.72−77 Replacing Phi 29 DNA polymerase with RNA polymerase enabled rolling circle transcription (RCT) for the synthesis of a long RNA strand with a repetitive sequence for promising application in RNA interference (RNAi).21,78−81 2.2.3. Transcription-Based Amplification. Several other methods also utilize RNA polymerase to perform linear amplification and are classified as transcription-based amplification in this review. These methods include immuno-detection amplified by T7 RNA polymerase (IDAT),82 fluorescent amplification catalyzed by T7 polymerase technique (FACTT),83 signal-mediated amplification of RNA technology (SMART),84 and single primer isothermal amplification (SPIA).85 The T7 promoter sequence is incorporated into the DNA template and recognized by T7 RNA polymerase to catalyze the transcription reaction in vitro, accumulating many copies of RNA products. These methods permit highly sensitive detection of low-abundance targets (e.g., nucleic acids and proteins), with potential applications in RNA synthesis86 and RNAi,87 similar to RCT. 2.2.4. Signal Amplification Strategies. Isothermal amplification can also be achieved via signal amplification without relying on the generation of new nucleic acid products (DNA or RNA). In contrast to methods that employ polymerases, which are inhibited by the accumulation of pyrophosphate, these signal amplification strategies are not subject to product inhibition. Based on the signal-amplifying mechanism, these strategies can be classified into three types: nuclease-assisted, DNAzyme-assisted, and enzyme-free reactions. Nucleases can cleave the phosphodiester bonds of nucleic acids and include deoxyribonuclease (DNase), which cleaves DNA, and ribonuclease (RNase), which cleaves RNA, and can be further classified into Exos and Endos. Generally, nucleaseassisted strategies involve recycling cleavage of nucleic acids catalyzed by different nucleases, such as flap endonuclease (FEN),88 NEase,89−98 duplex-specific nuclease (DSN),99−104 REases,105,106 Exos,107−117 DNase I,118−121 and RNase H.122 NEase-assisted methods and Exo-assisted ones are widely used, and FEN-based methods (invader or invasive assay) have been commercialized for SNP genotyping and virus detection by Third Wave Technologies. In DNAzyme-assisted methods, metal ion-dependent DNAzymes are often used to perform nucleic acid cleavage. DNAzymes are DNA molecules that can catalyze a chemical reaction123−129 and activated by forming a compact structure in the presence of metal ions (e.g., Mg2+ and Pb2+). Abundant amplification is achieved in three steps (activation, cleavage, and turnover), offering excellent sensing performance for different analytes.130−133 A G-quadruplex DNAzyme with peroxidase mimicking catalytic activity has also been used for fast signal amplification by catalyzing the oxidation of nonnucleic acid substrates.134−136 On the basis of this special DNAzyme, colorimetric,137−141 fluorescent,142 chemiluminescent (CL),143,144 and electrochemical145 biosensing applications have been developed. In enzyme-free methods, neither a protein enzyme nor a DNAzyme is employed. Instead of a recycling cleavage reaction, only a hybridization process is involved in the amplification reaction. The first enzyme-free method, hybridization chain

Figure 3. Enzyme-free amplification strategies. (A) HCR system. Letters marked with * are complementary to the corresponding unmarked letter. Reprinted with permission from ref 146. Copyright 2004 National Academy of Sciences. (B) Scheme of CHA. The ssDNA C1 catalyzes the hybridization of hairpins H1 and H2 through a series of toehold-mediated strand displacement reactions (a, b, and c). Squares and arrows drawn on DNA strands represent 5′ termini and 3′ termini, respectively. Reprinted with permission from ref 161. Copyright 2011 Oxford University Press.

hybridization reaction is unable to proceed at room temperature due to the storage of potential energy in short loops protected by long stems. The target ssDNA triggers a chain reaction of alternating (two hairpin species) hybridization, producing a long nicked copolymer. This method introduces triggered DNA self-assembly as nanostructures147−151 and exploits DNA as an amplifying transducer for biosensing152−157 and bioimaging.148,158,159 Another enzyme-free signal amplification method is catalyzed hairpin assembly (CHA), which is achieved by modular circuits of DNA hybridization (self-assembling and disassembling) rather than chain reaction (Figure 3B)160,161 and results in a diversity of targets and analytical formats (fluorescent, colorimetric, and electrochemical signals).161,162 2.3. Cascade Amplification

Although linear amplification strategies are convenient and fast, they are limited by low signal gain and assay sensitivity. To overcome these limitations, techniques integrating two or more amplification methods have been developed. This so-called “cascade amplification” exhibits sensitivity comparable to those of some exponential amplification techniques. The key requirement for cascade amplification is that the product of the upstream method acts as the trigger for the downstream method and thus acts as a “bridge” to connect these modules. 2.3.1. SDA-Combined Cascade Amplification. Compared to dsDNA, ssDNA is more versatile for the design of cascade amplification. With the capacity to accumulate thousands of ssDNA products, SDA has been widely applied in different cascade amplification strategies. In 2006, Willner and co-workers first proposed SDA/DNAzyme combined 12497


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RCA-combined cascade amplification strategies with multiple reaction steps incorporating nuclease-assisted amplification methods have also been reported. The first method, based on FEN-catalyzed invasive cleavage followed by RCA, was devised by Smith and co-workers.186−188 Two-stage amplification of human genomic DNA on a surface provided sufficient sensitivity for SNP analysis. Two other nucleases (NEase189−191 and Exo192,193) have also been used to develop RCA-combined cascade amplification. An RCA-based biosensor coupling enzyme-free CHA with two separate amplification reactions was recently demonstrated.194 Despite requiring multiple reaction steps, these cascade methods afford high assay sensitivity. 2.3.3. Additional Cascade Amplification. The previously mentioned cascade amplification methods involve polymerasecatalyzed DNA synthesis. Polymerase-free cascade techniques are also well-established. One year after the development of the FEN-assisted invasive method, the serial invasive signal amplification reaction (SISAR) was proposed by coupling two invasive reactions in a homogeneous assay.195,196 This cascade version yields more than 107 reporter molecules for each target DNA within 4 h. The FEN-assisted invasive method was also combined with an NEase-assisted method in cascade enzymatic signal amplification (CESA).197 In addition to FEN, other nucleases can also be used for serial amplification and recycling amplification.198−200 Nuclease/DNAzyme-utilizing cascade approaches have also been demonstrated,201−204 and several enzyme-free cascades based on HCR and CHA were successively devised.147,205−207

cascade amplification by designing a unique template for the generation of DNAzyme.163 Thousands of DNAzyme molecules are produced, and each of these can oxidize lots of substrate molecules to generate an amplified optical signal. Thus, cascade signal amplification is achieved. Metal iondependent DNAzymes were subsequently combined with SDA for the cascade cleavage reaction.164,165 This type of cascade strategy has been applied to the construction of various biosensors.166−169 Notably, the former method requires additional steps for signal generation after nucleic acid amplification, whereas the latter method requires ribonucleotide modification in the DNA probe. In addition to DNAzyme, nucleases have also been integrated into SDA for cascade amplification. By incorporating lambda Exo (λ Exo), Xia et al. developed a one-step method called hairpin-mediated quadratic enzymatic amplification (HQEA).170 HQEA combines the target-recycled hybridization reaction and subsequent recycling cleavage reaction and exhibits high sensitivity for microRNA (miRNA) analysis at the single cell level. Instead of Exo, NEase was originally used in SDA. The integration of the NEase-assisted method into SDA should lead to a simple sensing system without requiring optimization of reaction buffer. This hypothesis has been confirmed by our group with the development of an enzyme synergetic isothermal quadratic DNA machine (ESQM).171,172 This DNA machine utilizes a specifically designed hairpin probe with two functional sections to bridge these methods and can perform cascade amplification under SDA reaction conditions. The signal enhancement of ESQM is dramatically higher than that of SDA. A related approach was devised by Ye et al.173 In addition, we proposed another SDA-incorporated cascade amplification without introducing extra enzymes to create a triplex SDA process.174 The product of the first SDA circularly induces the next SDA, and the product recycling process is equivalent to one SDA process. This method achieves a significantly higher amplified signal under SDA reaction conditions. Several other cascade amplification strategies incorporating SDA have been proposed by different groups.175−178 These methods split the cascade amplification process into two independent reaction steps, which extends the assay duration and complicates operation. 2.3.2. RCA-Combined Cascade Amplification. Similar to SDA, RCA generates a ssDNA product and has been widely combined with other amplification methods. In 2007, Willner and co-workers179 developed a RCA/DNAzyme cascade amplification in which a long DNAzyme chain is synthesized to perform a second amplification reaction. Several biosensors have been designed using this cascade amplification.180−183 Target sequence recycled RCA (TR-RCA)184 and dendritic RCA185 have also been devised as RCA cascades. In TR-RCA, a dumbbell padlock probe is designed to recognize the target sequence to activate RCA triggered by excess primer. This RCA process then displaces the target for activation of the next RCA process. Thus, cascade RCA is achieved by recycling the target sequence. In dendritic RCA, a hairpin structure is the key element. It can hybridize to the product of the target-primed RCA, leading to the release of a new target sequence. This released target sequence induces a new RCA process in the hairpin structure, resulting in cascade amplification. With a onestep amplification reaction, these three cascade strategies all provide a significantly amplified signal.

3. BIOANALYTICAL APPLICATIONS Thanks to convenient operation and high amplification efficiency, isothermal amplification techniques have become very promising alternatives to PCR for bioanalytical applications. Initially, nucleic acid (DNA and RNA) detection, SNP genotyping, and even DNA methylation analysis were successfully demonstrated by these techniques. With developments in molecular biology and the use of aptamers, antibodies, and DNAzymes, these methods have been widely employed for the biosensing of other analytes including proteins, cells, small biological molecules, and even ions. In situ and intracellular imaging by several isothermal methods have also been demonstrated. And these methods were recently applied for sequencing. In this section, we will summarize the broad bioanalytical applications of isothermal amplification. 3.1. Detection of Nucleic Acids

3.1.1. Detection of DNA. Most isothermal amplification techniques can amplify and detect DNA with excellent sensitivity. Even RT-utilizing NASBA can be used to sense ssDNA. Among these methods, LAMP, HDA, RPA, and E-SDA are widely used for genomic DNA detection due to their outstanding performance in sequence amplification. Complex samples from different species (human, bacteria, and even virus) have been successfully analyzed with these methods, demonstrating their promising biological, medical, and environmental applications. Signal amplification methods are also well-established for the detection of DNA (mainly oligonucleotides) as mentioned in section 2.2.4. For example, Plaxco and co-workers first reported Exo-assisted amplified DNA detection.108 In this system, Exo III catalyzes the stepwise removal of mononucleotides from the blunt 3′-hydroxyl termini of duplex DNAs rather than ssDNA, 12498


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induces invasive amplification, generating abundant amplified flaps. These flaps and P-oligo are ligated by T4 ligase using MBs (containing one nicking site) as templates, leading to the formation of a DNA duplex and fluorescence recovery. In the presence of NEase, recycling cleavage of the MBs occurs. Thus, a cascade-amplified fluorescent signal is obtained for a single target molecule. A detection limit of 1 fM was observed. 3.1.2. Detection of SNPs. Isothermal amplification is also suitable for SNP genotyping. In 1998, HRCA was developed for the detection of point mutations in small quantities of genomic DNA from human cell lines.24 A ligation reaction before the amplification process contributes to allele discrimination with high specificity. To overcome the problem of a nonspecific signal induced by primer artifacts in exponential HRCA, an improved method in which an energy transfer primer was designed.212,213 Other RCA-based strategies have also been demonstrated for the electrochemical,191,214 fluorescent,215,216 and even colorimetric182,217 detection of SNPs. In the colorimetric method developed by Li et al.,217 REase is added in RCA reaction mixture to digest the long RCA product into short DNA fragments that readily hybridize to AuNP-tagged DNA probes (Figure 5A). The aggregation of AuNP-tagged DNA probes produces a colorimetric signal. Without the additional ligation reaction, FEN-based invasive approaches can discriminate single-base differences during the amplification process.88,196,213 The high specificity is owing to FEN-catalyzed cleavage, which is dependent on an invaded structure and is sensitive to single-base mismatches. The mechanism of SISAR coupling two serial invasive reactions is presented in Figure 5B.196 This cascade invader assay permits the discrimination of single-base differences in the methylene tetrahydrofolate reductase gene using 20 ng of human genomic DNA. On the basis of FEN/RCA cascade methods, SNP analysis on surfaces and even in a DNA array format were demonstrated.188,218 In addition to FEN, other proteins or enzymes have also been utilized for accurate SNP detection based on isothermal amplification. The first protein is Thermus aquaticus MutS (Taq MutS), a mismatch binding protein used in SMAP 2.52,53 Taq MutS specifically binds the mismatched duplex of the extended discrimination primer and the target template and blocks the dissociation of this duplex (Figure 5C).52 Thus, mismatch amplification is prevented. With the use of this method, high-precision results were obtained from human blood samples without DNA purification. Another protein is the DNA repair protein Endo IV. Zhao et al. used Endo IV to propose a mismatch-directed signal amplification (MDSA) strategy by coupling λ Exo.219 In MDSA, Endo IV cleaves apurinic/apyrimidinic sites in DNA duplexes with different adjacent mismatched bases. By taking advantage of the unique discrimination properties of Endo IV, specific distinction between mutant type and wild type is achieved. λ Exo has been employed to perform the recycling cleavage reaction, resulting in an amplified fluorescent signal. A λ Exobased biosensor for SNP detection was recently proposed220 in which a fluorescent probe containing two introduced mismatches with the target ssDNA was designed (Figure 5D). Despite the two inserted mismatched bases, the probe/ target duplex with matched N:M was efficiently hydrolyzed by λ Exo, generating a fluorescent signal. In the presence of excess probes, the target recycling process leads to an amplified signal. By contrast, when M is mutated to a mismatched base, the λ Exo-catalyzed hydrolytic reaction is nearly completely inhibited. SNP analysis of PCR amplicons from human genomic DNA

and an MB containing Exo III-resistant 3′ protruding termini was designed as the signaling probe (Figure 4A). In the

Figure 4. DNA detection. (A) Exo III aided target recycling strategy. Reprinted from ref 108. Copyright 2010 American Chemical Society. (B) NEase assisted nanoparticle amplification for target DNA detection. Reprinted with permission from ref 91. Copyright 2009 John Wiley & Sons, Inc. (C) Working principle behind the detection of DNA on the basis of HCR amplification and the formation of pyrene excimers. Py = pyrene. Reprinted with permission from ref 152. Copyright 2011 John Wiley & Sons, Inc. (D) Principle of CESA for target DNA detection. There are three steps: invasive signal amplification by Afu endonuclease to generate amplified flaps, flap ligation by T4 ligase to form a nicking site, and nicking reaction by NEase to produce amplified signals. up = upstream probe, dp = downstream probe, T1 = target of interest. Reprinted with permission from ref 197. Copyright 2011 John Wiley & Sons, Inc.

presence of target ssDNAs, the MBs open to form duplex DNAs with blunt 3′-hydroxyl termini (in MBs). Then, Exo III cleaves the MBs in duplex DNAs from this terminus, liberating the fluorophore before releasing the target. The released target then hybridizes to another MB probe for the next digestion cycle. In this way, a single target copy generates an amplified fluorescent signal. Detection limits of 10 pM and 20 aM were obtained within 30 min at 37 °C and 24 h at 4 °C, respectively. Endo-assisted and DNAzyme-based amplified detection of DNA have also been demonstrated.91,105,208−211 Notably, Liu et al. reported NEase-assisted nanoparticle amplification for ultrasensitive and selective DNA detection.91 In this method, when the target DNA is complementary to the DNA linker, recycling cleavage of these linkers is performed by NEase, preventing the formation of cross-linked structures of DNAAuNP probes (Figure 4B). Moreover, Huang et al. designed pyrene-labeled hairpin probes for HCR amplification (Figure 4C), demonstrating the fluorescent detection of DNA at concentrations as low as 0.256 pM.152 Owing to the longer lifetime of pyrene (up to 100 ns) compared to chromophores (less than 10 ns) in biological fluids, this method also allowed the efficient analysis of cell media samples with significantly minimized environmental effects. Zou et al. reported a novel cascade amplification, CESA, for ultrasensitive DNA detection.197 As depicted in Figure 4D, the target DNA (T1) first 12499


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assay, Zhu et al. demonstrated the sensitive detection of methylated DNA.225 After sodium bisulfite treatment, the cytosine (C) residues in DNA fragments were transformed to uracil, whereas 5-methylcytosine (5mC) remained unchanged (Figure 6A). Thus, the treated unmethylated DNA was not

Figure 5. SNP detection. (A) Schematic illustration of the RCA reaction and AuNP assembly based assay. Reprinted from ref 217. Copyright 2010 American Chemical Society. (B) Schematic representations of SISAR. (top) Proposed secondary structure of the overlapping substrate of the primary reaction. (middle) Proposed secondary structure of the overlapping substrate of the secondary reaction. (bottom) The X-structure formed by the uncut primary probe and the secondary probe, which contributes to the background of the reaction. Reprinted with permission from ref 196. Copyright 2000 National Academy of Sciences. (C) The mechanism of allele discrimination as mediated by Taq MutS. SNP typing with a wild-type allele-specific primer, using the wild-type allele (left) and the mutant allele (right) as templates. Reprinted with permission from ref 52. Copyright 2007 Nature Publishing Group. (D) Schematic depiction of the principle of the enzyme-mediated DNA detection at single-base difference level by using a double-mismatch/fluorophore-modified DNA probe and λ Exo. M represents the polymorphic base in the target strand. N is the base opposite M in the probe. Reprinted with permission from ref 220. Copyright 2015 Royal Society of Chemistry. (E) Reaction scheme of the KRAS genotyping assay after HDA. Reprinted from ref 221. Copyright 2013 American Chemical Society.

Figure 6. Detection of DNA methylation. (A) Schematic illustration of DNA methylation assay using the short linear probe and two-stage isothermal amplification. Reprinted from ref 225. Copyright 2013 Elsevier. (B) Principle of the COEXPAR method. Reprinted from ref 42. Copyright 2015 American Chemical Society. (C) Scheme of DNA methylation assay based on ligation-mediated HRCA. Reprinted from ref 227. Copyright 2012 American Chemical Society.

was realized by this approach with a high discrimination factor (320). Exploiting cooperative hybridization for increased binding affinity, Pompa et al. demonstrated the specific detection of cancer-related point mutations based on HDA.221 Human genomic DNA was amplified by HDA using one biotinylated primer, and the products were captured by streptavidin-labeled paramagnetic beads. After alkaline denaturation and magnetic separation, beads with biotinylated ssproducts (target in Figure 5E) were obtained. The discriminating probe bound both the biotinylated target and the AuNP probe, forming a cooperative hybridization structure to stabilize the hybrid (red circle). However, proper hybridization of the probe with the target was impaired by the point mutation (light blue). This colorimetric assay is instrument-free and suitable for POC applications. In addition, DNA probes with unique structures have been designed for specific SNP detection using SDA-based methods.167,222−224 3.1.3. Detection of DNA Methylation. Several isothermal amplification strategies have recently been employed for the detection of DNA methylation which is closely associated with various diseases. Using a two-stage isothermal amplification

complementary to the SDA template and only methylated DNA fragments initiated SDA. The accumulation of numerous ssDNA triggers induced the recycling cleavage of fluorescent probes by an additional NEase in the second reaction. This method can distinguish methylation levels as low as 0.1% from a DNA mixture with a detection limit of 0.78 pM. Xu et al. reported a chemical oxidation cleavage-triggered EXPAR method (COEXPAR) for gene-specific methylation detection.42 EXPAR requires triggers with 3′-hydroxyl groups to initiate the amplification reaction. In COEXPAR, a p53 gene fragment (54-mer) with only one methylation locus was used as a target model (Figure 6B). This gene fragment was first treated with NaIO4/LiBr solution followed by hot piperidine. Due to the lower redox potential of 5mC compared to C, the C5−C6 double bond of 5mC was selectively epoxidized in the presence of NaIO4 and then brominated at C6 by LiBr.226 The 12500


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Figure 7. Detection of long RNAs. (A) Schematic of a RNA invasive cleavage assay using FEN. Reprinted with permission from ref 195. Copyright 2001 Nature Publishing Group. (B) The principle of cleavage-based RNA amplification and verification experiments. (a) Scheme of cleavage-based RNA amplification. (b) PAGE analysis results. (c) The photograph of colorimetric result; “+” including 10−8 mol RNA target; “−” negative control (a sample of H2O). Reprinted with permission from ref 58. Copyright 2013 Nature Publishing Group. (C) RNA detection mechanism by 3-WJ probe and PG-RCA. (left) 3-WJ probes (primer and template) are designed to specifically form a 3-WJ structure on target RNA; however, they do not interact with each other without target RNA because their complementary sequence is only 6−8 bases. Addition of DNA polymerase and NEase initiates SDA reaction to generate signal primers. (right) The generated signal primers can be detected by PG-RCA. Reprinted with permission from ref 232. Copyright 2011 Oxford University Press.

enabled direct RNA quantitation with a low detection limit of 90 copies of HIV viral RNA (Figure 7A).195 Tang et al. recently reported a new isothermal signal amplification strategy named cleavage-based RNA amplification for mRNA detection.58 This strategy includes two processes, the cleavage of target mRNA and serial SDA reaction, as illustrated in Figure 7B. A specific DNAzyme (fCat) cleaves the target mRNA and yields a forward fragment with a 2′,3′ cyclic phosphate group, which is then removed by T4 polynucleotide kinase (PNK) to generate a 3′ hydroxyl fragment. DNA polymerase recognizes this 3′ hydroxyl group to perform the first SDA reaction (cycle 1) in the presence of NEase. The products of the first SDA (Ts) hybridize to Ta probes, inducing a second SDA reaction (cycle 2). The products of the second SDA (Rs) contain a peroxidasemimicking DNAzyme sequence (blue) and the antisense sequence of the partial fCat catalytic core (red). After Rs annealing with fCat, the third SDA process is initiated (cycle 3), producing Ts sequences that act as a primer in the second SDA. These cycles result in exponential amplification with the accumulation of the G-quadruplex product Rs, which can be tested by both colorimetric and fluorescent reporters. A detection limit of 69.2 aM was obtained, and the assay of total RNA (0.3 ng) from HepG2 cells was demonstrated without contamination by genomic DNA. In addition to direct methods, isothermal amplification techniques have been used to indirectly detect long RNAs using assistant probe or RT processes. With three-way junction (3WJ) formation, SMART was proposed for specific RNA detection.84 Utilizing this 3-WJ structure to recognize RNA,

methylated target DNA was cleaved at 5mC by piperidine treatment, generating a ssDNA break with a 27-mer fragment and a 26-mer fragment. One of these two fragments acted as a trigger to induce EXPAR. The amplification reaction was monitored using the fluorescent dye SYBR Green I (SG I), and the guanine monomer-induced voltammetric response was also recorded. Low detection limits of 411 aM by fluorescence and 576 aM by electrochemistry were obtained. Despite the good performance of these methods, DNA methylation analysis of human genome samples by COEXPAR or the above method has not been reported. By employing HRCA, Zhang et al. successfully demonstrated the detection of methylated genomic DNA from cancer cells.227 As illustrated in Figure 6C, genomic DNA treated with bisulfite was denatured and annealed with a linear padlock probe to induce the ligation of the linear padlock probe. Two primers were then added to initiate the HRCA process monitored by SG I. As little as 2 ng of genomic DNA from human lung cancer cell lines was detected by this method. 3.1.4. Detection of Long RNAs. In this review, long RNAs are RNA molecules with hundreds or even thousands of nucleotides, primarily messenger RNA (mRNA), ribosomal RNA (rRNA), and viral RNA. NASBA and SPIA were developed for the amplification of RNA and have also been used to assay long RNAs. NASBA has been widely used due to the high sensitivity conferred by exponential signal gain.17,228−230 Some NASBA-based commercial kits have been used in the clinical diagnosis of pathogens.231 Without requiring transcription, FEN-assisted invasive amplification 12501


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Figure 8. MiRNA detection. (A) Reaction scheme of the hairpin ribozyme iHP-let7 responsive to miRNA let-7. Reprinted from ref 240. Copyright 2004 American Chemical Society. (B) Illustration of the target-primed branched RCA reaction and fluorescence detection of miRNA. Reprinted with permission from ref 243. Copyright 2009 John Wiley & Sons, Inc. (C) Schematic representation of the EXPAR with let-7a miRNA as the trigger. Reprinted with permission from ref 46. Copyright 2010 John Wiley & Sons, Inc. (D) Illustration of ERA strategy for miRNA detection. Reprinted from ref 45. Copyright 2014 American Chemical Society. (E) Schematic illustration of E-SDA initiated by miRNA let-7a. Reprinted from ref 256. Copyright 2014 American Chemical Society. (F) The scheme of the HQEA strategy based on Bst polymerase-induced strand-displacement reaction and λ Exoaided recycling reaction. Reprinted from ref 170. Copyright 2013 American Chemical Society. (G) Schematic illustration of enzymeassisted amplified detection of specific miRNA based on DNA polymerase and NEase. Reprinted from ref 173. Copyright 2013 American Chemical Society. (H) Schematic illustration of miRNA detection based on target-triggered 3-WJ structure and ESQM. Reprinted from ref 171. Copyright 2014 American Chemical Society. (I) Schematic representation of miRNA direct detection based on DSNSA. Reprinted from ref 99. Copyright 2012 American Chemical Society.

8A). After activation by the miRNA, the ribozyme catalyzes the recycling cleavage reactions of fluorescent probes, similar to DNAzyme, achieving a high signal:noise ratio (20-fold). Because of the displacement hybridization, this method exhibits high sequence specificity. A modified invader assay was also developed for the quantitation of miRNAs.241 Two stem-loop probes were required to recognize miRNA for the formation of a dumbbell-like structure by base-stacking interactions. Then, multiple probe cleavages by FEN were induced with an amplified fluorescent signal in response to a single target RNA. This assay achieved the detection of 103 lysed cells or 50 ng of total cellular RNA. However, these two methods suffer from relatively low sensitivity and the complex design of probes. RCA was introduced for miRNA analysis in 2006.242 The target miRNA was first used as a template for the ligation of the padlock probe by DNA ligase and then as a primer to initiate the RCA reaction. The amplified product was separated by gel electrophoresis and detected by a radioactive assay. A few nanograms of total RNA was detectable. An improved strategy, branched RCA, was subsequently developed by Cheng et al. (Figure 8B).243 Instead of T4 DNA ligase, they utilized T4 RNA ligase 2 to greatly improve the specificity of the miRNAtemplated ligation of the padlock probes. Even a single-base difference was accurately distinguished. A second primer was added to trigger the branched reaction, increasing the yield of amplified product. By using SG I, homogeneous analysis was

CD4 mRNA in human mRNA samples was detected by a cascade method combining SDA and PG-RCA (Figure 7C).232 Polymerization-based amplification techniques, including HDA, LAMP, RPA, and SDA, have been used for long RNA detection with an extra RT reaction before amplification.233−239 Theoretically, all polymerization-based methods can be used for long RNA detection in combination with an RT reaction. 3.1.5. Detection of miRNAs. miRNAs are a class of endogenous small noncoding RNA molecules (approximately 22 nucleotides) with 3′-hydroxy groups that are found in many species. miRNAs function as negative post-transcriptional regulators of gene expression by forming an active RNAinduced silencing complex with several relevant proteins. Accumulating evidence indicates that the aberrant expression of miRNAs is closely related to various human diseases, including cancer. Thus, miRNAs are considered as clinically important biomarkers. Unfortunately, the unique characteristics of miRNAs, such as high sequence homology (even single nucleotide variation) among family members and low abundance in real samples, have complicated the development of sensitive and specific assays. In contrast to long RNAs, miRNAs are poor templates for RT, making them inaccessible by NASBA or RT-combined methods. In 2003, Famulok et al. demonstrated the detection of miRNAs by isothermal amplification.240 A signal-amplifying ribozyme was designed to recognize the target miRNA (Figure 12502


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the absence of target miRNA was observed in EXPAR, LAMP, and E-SDA. SDA-combined cascade strategies have also been devised for miRNA detection to avoid nonspecific amplification. One strategy is HQEA, which is presented in Figure 8F.170 A twostep enzymatic cascade results in quadratic fluorescent signal amplification. The first step is initiated by the target miRNA, which anneals with an MB probe on the loop region, opening the stem and freeing the fluorescent signal. Then, an engaging primer hybridizes to the opened stem, triggering polymerization and displacement of the target miRNA. The target miRNA can bind to another MB to initiate the next cycle. Thus, linear amplification is achieved with the accumulation of thousands of duplex MBs. The duplex MB is nicked by NEase with the generation of 5′-phosphate termini, inducing the second amplification. Following nicking, λ Exo recognizes the 5′-phosphate termini and catalyzes the stepwise degradation of the MB strand in the duplex. The other strand binds excess MB to form duplex MB, initiating the next cleavage cycle. Thus, quadratic amplification is achieved, enhancing the fluorescent signal. This method exhibits ultrahigh sensitivity with a detection limit of 1 aM, comparable to the sensitivity of EXPAR-based methods. Application of this method in crude extract of MCF-7 and PC3 cells and even patient tissues was demonstrated. Another strategy was developed by Yin et al.173 As illustrated in Figure 8G, the reaction system consists of three DNA units (hairpin template, short primer, and MB). One stem strand (denoted in blue) in the hairpin template is complementary to the 5′ region of the MB. Once the target miRNA anneals with the hairpin template to initiate the SDA process (stopped by the 18-carbon spacer block as a loop of hairpin template), this stem strand is displaced and flanks as an arm. The arm bound to the MB triggers a target recycling reaction in a similar manner as in HQEA. Therefore, this method produces a cascade enzymatic amplification, providing strong fluorescence proportional to the miRNA concentration. With a low detection limit of 1 fM, this method was used to quantify miR-141 in cancerous cell lysates. Although it can discriminate miR-141 from family members, this method is not capable of the single-base discrimination. We then designed a cascade biosensor for highly selective miRNA detection using a target-triggered 3-WJ structure and ESQM.171 As shown in Figure 8H, the stable 3-WJ structure formed by the 3-WJ primer and 3-WJ template with the target miRNA was designed to improve the sequence specificity. Even a single-base mismatch in both ends of the miRNA could disrupt this structure. The 3-WJ primer could not initiate an amplification reaction in the absence of target, but when the target was present, the 3-WJ primer induced the SDA process along the 3-WJ template, accumulating abundant ssDNA products (Recycling I). A hairpin probe was designed to anneal with the ssDNA product and be complementary to MB, acting as a “bridge” connecting SDA and the subsequent NEase-assisted recycling cleavage process (Recycling II). ESQM offers a polynomial amplification format with high assay signal. An attomolar detection limit was achieved within 30 min, and the analysis of small RNA extracted from cancer cells was achieved. The homologous sequences of single-base mismatches, even at both ends as well as the middle, were welldiscriminated from the target miRNA by the 3-WJ probes. Nuclease-assisted methods are also suitable for the rapid and simple analysis of miRNA. Yin et al. created duplex-specific nuclease signal amplification (DSNSA) for the rapid and direct

also achieved. A detection limit of 10 fM was estimated, and this method was successfully applied to total RNA samples extracted from healthy human lung tissue. For the analysis of clinical samples, we proposed a dumbbell probe-mediated RCA (D-RCA) strategy for highly sensitive detection of miRNAs in colon tumor tissue.244 The dumbbell probe significantly improved both the sensing specificity and sensitivity. The stem-loop structure in the dumbbell probe enhanced the hybridization specificity via competition between stem region and loop/target binding. A detection limit as low as 1 fM and direct quantification of miRNA in 100 pg of total RNA from colon tumor tissue was demonstrated using D-RCA. Several other RCA-based miRNA biosensors were subsequently devised.181,245−252 Most of these sensors are compatible with complex RNA samples. Other polymerization-based isothermal amplification strategies have been employed for the detection of miRNA. In contrast to RCA-based methods, which require an additional ligation process and extended assay time (several hours), most of these methods enable rapid miRNA assays within 1 h. Jia et al. first reported that EXPAR is suitable for the efficient amplification of miRNAs.46 The amplification template was designed to contain two repeat sequences (complementary to target miRNA) separated by a nicking site (Figure 8C). The amplification reaction was triggered after hybridization between the miRNA and template. SG I was utilized for real-time monitoring of the EXPAR product. As little as 10 aM synthetic miRNA and even 10 pg of total RNA from human brain were detected within 30 min. However, only homologous sequences with mismatched bases near the 3′ end were efficiently distinguished from the target sequence. The low specificity of the assay was attributed to the ability of DNA polymerase to recognize the 3′-hydroxy of not only stable duplexes (high Tm) but also transient duplexes (low Tm) to initiate the extension reaction. To further improve the sensing performance (sensitivity and multiplex detection), two EXPAR-based methods have been developed for end-point analysis by combining silver nanoclusters or quantum dots (QDs).43,177 By including two repair enzymes (uracil-DNA glycosylase and Endo IV) in EXPAR (Figure 8D), Zhou et al. demonstrated selective miRNA analysis with efficient inhibition of nonspecific amplification from template- or primer-independent DNA synthesis.45 However, this method requires an extended assay time (>3 h) and is subject to other types of nonspecific amplification. Colorimetric and electrochemical biosensors based on EXPAR have also been proposed.253,254 LAMP has also been employed for miRNA detection using miRNA as one outer primer to induce the LAMP reaction.255 E-SDA was also recently introduced for the analysis of miRNA.256 As illustrated in Figure 8E, after hybridization of the target miRNA and Primer 1, both Primer 1 and miRNA are extended by exo-DNA polymerase to produce a chimeric duplex. Next, NEase induces a strand break in Primer 1, generating abundant ssDNA T* by subsequent nicking and a polymerization/displacement process. T* can hybridize with Primer 2 to trigger another linear SDA, resulting in the accumulation of T, which has the same sequence as the target miRNA, except for the replacement of uracil by thymine. Thus, exponential amplification is achieved by the continuous production of T and T*. The formation of dsDNA can be monitored in real time using SG I. Commercial human serum samples diluted only 2-fold were successfully analyzed using this method. Notably, high-level nonspecific amplification in 12503


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quantification of miRNAs in biological samples.99 Instead of ssDNA or ssRNA, DSN preferably cleaves dsDNA or DNA in DNA-RNA hybrid duplexes.257 Moreover, the cleavage rate of perfectly matched duplexes is significantly higher than that of mismatched duplexes. As shown in Figure 8I, a Taqman probe complementary to the target miRNA is designed to form DNARNA hybrid duplexes. Then, DSN binds to these duplexes and digests the Taqman probe, leading to the restoration of fluorescence, and free miRNA anneals with the next copy of the Taqman probe. In this way, an amplified fluorescent signal from one target molecule was obtained. Within 30 min, the direct detection of femtomolar miRNA was achieved by DSNSA. A multiplex assay of different cancer cells was also demonstrated. Several improved strategies utilizing DSN have been serially developed for the fluorescent, colorimetric, and electrochemical detection of miRNA.103,258−261 Exo- and NEase-assisted cyclic signal amplification have also been proposed for ultrasensitive miRNA assays.262−264 Additionally, enzyme-free HCR and CHA have been employed to construct fluorescent, electrochemical, and ECL biosensors for miRNA detection.265−272 Ascribed to the unique properties, nanomaterials have also been used in amplification-based miRNA detection. For example, silver nanoclusters43 and QDs177 have been employed as fluorescent reporters. Fiammengo et al. designed a DNAAuNP probe by immobilizing fluorescently labeled DNA on PEGylated AuNP as shown in Figure 9A.100 The fluorescence was efficiently quenched by AuNP. In the presence of the target miRNA, DSN enabled recycling cleavage of DNA in DNAAuNP probes, yielding a highly amplified signal due to the separation of fluorophores from the gold surface. The PEGbased passivation layer prevented DSN inhibition, and they achieved directly quantified cancer-related miRNAs in total RNA samples extracted from cell cultures. Due to the preferential affinity for long ssDNAs over short fragments, WS2 nanosheets have also been used to mediate fluorescence quenching in DSN-based methods as shown in Figure 9B.101 In addition to AuNPs and WS2 nanosheets, graphene oxide (GO) has been widely adopted in nuclease-assisted,120,273 enzymefree,154,274 and even polymerization-based275−277 signal amplification strategies for miRNA assay. Cui et al. constructed GOprotected DNA probes for multiplex miRNA analysis on the basis of DNase I-assisted signal amplification.120 Liu et al. designed a GO fluorescent switch to protect target miRNA from degradation and demonstrated accurate miRNA detection with EXPAR amplification (Figure 9C).276 Numerous recent studies have established that miRNAs also exist in clinical samples of plasma and serum in a remarkably stable form as circulating miRNAs, which are considered potential blood-based cancer biomarkers.278−280 Several research groups have developed amplification-based sensing systems for circulating miRNA detection. Li et al. proposed a hairpin probe-based RCA for isothermally sensitive detection of serum miRNAs.281 Direct and accurate distinction of nonsmallcell lung carcinoma patients from healthy subjects was achieved via the quantification of serum miR-486-5p expression using this method. Gao et al. demonstrated direct detection of miRNAs in serum by a DSN-assisted electrochemical biosensor.102 Yang et al. also designed two biosensors for serum miRNA analysis based on HCR and RCA.254,282 The 2′ hydroxyl group of the 3′ end ribose is methylated in plant miRNAs and other important small RNAs (siRNA and piRNA).283−285 This methylation remarkably inhibits enzymatic reactions including extension, ligation, and RT.171,286−289

Figure 9. MiRNA detection combined with nanomaterials. (A) Direct miRNA quantification using DNA-AuNP probes and DSN. Reprinted from ref 100. Copyright 2014 American Chemical Society. (B) Schematic illustration of the miRNA assay based on WS2 nanosheet mediated fluorescence quenching and DSNSA. Reprinted from ref 101. Copyright 2014 American Chemical Society. (C) Illustration of the graphene fluorescence switch-based cooperative EXPAR for target miRNAs. Reprinted from ref 276. Copyright 2014 American Chemical Society.

The inhibition of DNA polymerase-catalyzed extension biases the results of the analysis of these methylated small RNAs by the above methods when using the target as a primer. To overcome this bias, we designed a target-triggered 3-WJ hybrid structure for unbiased recognition of small RNAs.171 In the 3WJ hybrid structure, the 3-WJ primer rather than the target miRNA is used as a primer to initiate the extension reaction, followed by ESQM-mediated amplification as shown in Figure 8H. The successful detection of human miRNA and plant miRNA confirmed the universality of our strategy for different small RNAs. 3.2. Detection of Proteins and Enzymes

Proteins and enzymes perform a vast array of functions in living organisms as basic functional materials, and specific proteins have been implicated in many diseases. The detection of target proteins is significant in fundamental research and biomedical applications. However, these proteins are often present at low 12504


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Figure 10. Protein detection based on immuno-isothermal amplification. (A) Schematic of immuno-RCA assay. (top left) A reporter Ab conjugated to an oligonucleotide binds to a test analyte that is captured on a solid surface by covalent attachment or by a capture Ab. (top right) A DNA circle hybridizes to a complementary sequence in the oligonucleotide. (bottom left) The resulting complex is washed to remove excess reagents, and the DNA tag is amplified by RCA. (bottom right) The amplified product is labeled in situ by hybridization with fluor-labeled oligonucleotides. Reprinted with permission from ref 290. Copyright 2000 National Academy of Sciences. (B) Illustration of FACTT. Reprinted with permission from ref 83. Copyright 2006 Nature Publishing Group. (C) Schematic depiction of the sandwiched immunoassays with the DNA-based HCR. Reprinted from ref 155. Copyright 2012 American Chemical Society. (D) Principle (top), evaluation (middle), and performance (bottom) of the binding-induced CHA. Reprinted from ref 306. Copyright 2013 American Chemical Society. (E) Principle and performance of the target-induced proximity amplification based on NEase-assisted recycling reaction. Reprinted from ref 307. Copyright 2014 American Chemical Society. (F) Schematic illustration of triplebinder assembly of Mg2+-dependent DNAzyme along with autocatalytic cleavage of MB-HP on the electrochemical biosensor. Reprinted from ref 308. Copyright 2015 American Chemical Society.

concentrations in real samples, demanding detection strategies with high sensitivity. The high signal amplification of isothermal amplification methods fulfills this requirement. In contrast to nucleic acids, proteins cannot be directly amplified. Extensive efforts have been devoted to the expansion of nucleic acid amplification techniques for protein detection, including antibody-DNA affinity probes, aptamers, and other nucleic acid probes. 3.2.1. Detection of Proteins. 3.2.1.1. Antibody-Based Protein Detection. Antibodies (Abs) are widely used as ligands to recognize target proteins via specific binding to antigenic epitopes. Antibody-based protein detection systems are versatile and powerful tools for fundamental research and clinical diagnostics. Among these detection methods, enzymelinked immunosorbent assays are a routine method that suffers from the limitations of low sensitivity and complex operations. After devising HRCA for SNP analysis, Ward et al. adapted RCA to “immuno-RCA” in 2000 by incorporating an immunoassay for protein detection.290 In immuno-RCA, the immobilized antibodies are used to capture target proteins from sample solutions, and the 5′ end of the primer is attached to the Ab for the target protein as shown in Figure 10A. Circular DNA hybridizes to the primer as the template for the RCA reaction in the presence of DNA polymerase, producing a long ssDNA

product consisting of multiple copies of sequences complementary to the circular DNA. The amplified product can be detected in different ways, including the direct incorporation of labeled nucleotides and the hybridization of labeled complementary probes. Thus, an amplified signal for one protein molecule was achieved with a detection limit of 0.1 pg/mL prostate-specific antigen (PSA) in a microspot assay. This assay sensitivity is 3 orders of magnitude higher than that of standard immunoassays for PSA. An improved multiplexed microarray format was developed for protein profiling.291 A total of 75 cytokines were tested simultaneously on glass arrays by RCA. Immuno-RCA has been subsequently improved and widely applied for the analysis of various proteins.292−299 Cheng et al. introduced the biotin−streptavidin system to bind three primers to one target molecule and used oligonucleotide functionalized QDs (QD-probe) for an electrochemical readout.292 Both the biotin−streptavidin system and the QD-probe remarkably enhanced the amplification efficiency, and a detection limit of 0.27 aM with a broad linear calibration range (1 aM to 1 pM) was demonstrated using human vascular endothelial growth factor (VEGF) as a model protein. We designed two nanoRCA-based immunoassay platforms by employing primer-labeled AuNPs and multiwalled carbon nanotubes.293,294 Using primer-encapsulating liposomes, Ou 12505


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Therefore, the strand exchange reaction between OT DNA and C DNA is inhibited in the absence of the target protein. When the target molecule is present, C DNA is brought sufficiently close to OT DNA by the binding of the two ligands to the target. This binding greatly accelerates the strand displacement reaction by increasing the local concentration of C DNA, releasing O DNA as the output. This free output DNA initiates the CHA amplification by cyclical opening of H1 and H2, forming several H1−H2 complexes. These complexes then displace the fluorescent probe to allow detection. This method achieved homogeneous detection of streptavidin without separation and washing. Zong et al. also demonstrated a onestep, wash-free protein assay by proximity hybridizationtriggered signal amplification (Figure 10E).307 Target-induced proximity hybridization opened the MB, which is the substrate of recycling cleavage by NEase. As little as 24 pg/mL CEA was detected. Five clinical serum samples were also analyzed, and the results were consistent with the reference values. Another proximity method was developed for electrochemical immunosensing based on DNAzyme-assisted signal amplification.308 As depicted in Figure 10F, Liu et al. designed a PLA and 3-WJ structure-induced RCA for the ultrasensitive electronic monitoring of concanavalin A.309 These homogeneous PLAbased amplification strategies do not require washing or phase separations, are convenient to standardize, and are suitable for automation. 3.2.1.2. Aptamer-Based Protein Detection. Aptamers are another type of affinity ligand commonly used to connect target proteins and isothermal amplification strategies. They are synthetic short ss-DNA or ss-RNA molecules that generally have high affinity and specificity for specific targets (proteins, peptides, small organics, inorganic ions, metabolites, and even whole cells or viruses) and are derived from the systematic evolution of ligands by exponential enrichment techniques.310 Compared to Abs, aptamers offer several advantages, including improved thermal stability, lower cost, longer shelf life, easier modification, and simpler conjugation with nucleic acid amplification. Generally, the binding of aptamers to their specific targets changes the aptamers’ secondary and tertiary structure, thus facilitating the transduction of target recognition events to amplification reactions. In 2005, King et al. first reported aptamer-mediated isothermal amplification for protein detection;311 RCA was utilized with human thrombin as a model target. However, two types of aptamers were required in this design, limiting its application to other proteins with only one aptamer. To facilitate the development of aptamer-RCA for protein targets, Ellington and co-workers designed a novel structure-switching aptamer that can be circularized by DNA ligase upon binding to its protein target (Figure 11A).312 This aptamer acts as a template for the RCA process. PDGF was specifically quantified at low-nanomolar levels even in a background of cellular lysate. This method can be readily adapted to other proteins and does not require two or more affinity ligands. Some modified structure-switching aptamer probes or other aptameric recognition systems have also been designed for RCA-based amplification analysis of various proteins. RCA products have been detected by several techniques, including gel electrophoresis,313 fluorescence,314,315 electrochemistry,316,317 ECL,318 diffractometry,319 colorimetry,320,321 and surface plasmon resonance.322,323 Polymerization-based amplification strategies such as SDA and EXPAR have also been employed for protein detection. Yu

et al. devised a cascade immuno-RCA (liposome-RCA immunoassay) for the ultrasensitive detection of protein (down to 0.08 fg/mL PSA).295 Both the chemical conjugation of antibodies with DNA and the biotinylation of antibodies significantly affect their antigen-binding affinity. To overcome these shortcomings, a fusion protein was constructed with two functional domains, one for the covalent linkage of DNA and the other for the binding of specific antibodies.297 This fusion protein used to develop a novel immuno-RCA for the sensitive detection of human interferon-γ (IFN-γ) with a detection limit of 62.5 pg/mL. In addition to immuno-RCA, two T7 RNA polymerase-based immuno-isothermal amplification strategies, IDAT and FACTT, have been proposed by Greene and co-workers.82,83 Compared to IDAT, which uses radioactive isotopes, FACTT uses the fluorescent RNA-intercalating dye RiboGreen, which increases fluorescence intensity more than 1000-fold upon binding to RNA fragments. As shown in Figure 10B, streptavidin bridges the biotinylated ds-template and the detection antibody. Then, T7 RNA polymerase recognizes the T7 promoter sequence in the ds-template to catalyze the transcription reaction in vitro, producing many copies of ssRNA products. The amplified RNA is monitored by RiboGreen, eliminating the need for radioactivity and tedious electrophoresis. Highly sensitive detection of 0.05 pg/mL (∼0.5 fM) Her2 in human serum was achieved by FACTT. High-throughput assays are also available in the 384-well plate format. Immuno-HCR has also been reported by Tang et al. as an electrochemical immunoassay.155,300 As shown in Figure 10C, large amounts of ssDNA initiators are loaded on an individual AuNP, and antibody-labeled magnetic beads are used for the separation.155 DNA hairpins (H1* and H2*) are labeled with ferrocene moieties at both terminals. Each molecule of target protein can induce multiple HCR processes initiated by the initiators, and a single HCR process accumulates numerous ferrocene molecules to produce an electrochemical signal. Immunoglobulin G was employed as the model target with a detection limit as low as 0.1 fg/mL. The results of the analysis of 15 clinical serum specimens using this method were in good agreement with those of a commercialized ECL immunoassay method. Compared to immuno-RCA and FACTT, immunoHCR achieves ultrasensitive detection via an enzyme-free amplification approach. LAMP, Exo-assisted methods, and DNAzyme-assisted methods have also been introduced for immunoassays.301−303 Although the aforementioned methods have been widely applied, they require extensive and carefully controlled separation. To avoid this problem, Landegren and co-workers devised the proximity ligation assay (PLA) for homogeneous protein detection.304,305 In PLA, two DNA-labeled Abs bind to the same target protein, stabilizing the hybridization of these two DNA strands or between these two DNA strands and other probes by increasing their local concentrations. Inspired by PLA, Li et al. demonstrated that protein binding induces DNA strand displacement and described a binding-induced DNA circuit using CHA amplification (Figure 10D).306 Two specific affinity ligands were used to recognize the target molecule. One was conjugated to the output DNA motif (OT DNA) formed by the prehybridization of the output DNA (O DNA) and the support DNA (T DNA), and the other was labeled with the competing DNA motif (C DNA). The sequence of C DNA is complementary to T DNA and has the same length as O. 12506


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prepared by prehybridization with blocker DNA to prevent nonspecific folding in the absence of targets. The binding of target protein to the aptamer dissociated the blocker DNA from the probe, making the probe fold into its binding secondary structure. The probe with 3′ termini functioned as a primer to initiate the SDA reaction. The SDA products served as EXPAR triggers for hybridization with the EXPAR template, inducing exponential amplification. A detection limit of 0.9 pM PDGFBB was reported for this target-triggered two-stage amplification. NEase-assisted recycling cleavage signal amplification has also been utilized to develop amplified aptasensors for protein assays. The structure-switch aptamer is commonly designed to contain a hairpin that cages the trigger sequence used in the recycling cleavage reaction. When the target protein binds the aptamer, the hairpin structure is disrupted, making the trigger sequence available. Xing et al. inserted the thrombin aptamer into a hairpin probe (aptamer hairpin switch) that changes to an open form upon binding to thrombin (Figure 11D).96 The binding interaction releases the ssDNA (trigger), which then hybridizes with the signal probe. In the presence of NEase, recycling cleavage of the signal probe occurs, generating an amplified fluorescent signal with a detection limit of 100 pM in a homogeneous format. Similar assays have been developed for various proteins using alternative signal reporters including stem-loop probes,333−335 functionalized AuNPs,95,336 and DNAzymes.202 Among these assays, two novel colorimetric methods have been established using an HRP-mimicking DNAzyme202 or DNA-AuNPs.95 Target binding-induced conformational changes can also trigger an Exo-mediated recycling cleavage reaction. Duplexswitching aptamers with 3′-end overhangs are often used for Exo-assisted amplified analysis of proteins.337−340 In these assays, the binding of target results in the dissociation of aptamer sequences from the duplex switching, and the aptamers then act as targets for digestion. Another kind of switching aptamer is the split aptamer designed by Willner and coworkers.341 As shown in Figure 11E, a thrombin aptamer is split into two subunits. One serves as a FRET probe labeled with fluorophores at each end. Both subunits, ssDNAs, are resistant to digestion by Exo III in the absence of thrombin. When thrombin is present, the two subunits and thrombin form a Gquadruplex that is cleaved by Exo III. The digestion of the FRET probe releases thrombin to bind to another subunit pair. Thus, the recycling cleavage reaction is realized together with the amplified signal. As little as 89 pM thrombin can be detected within 12 min. A similar aptasensor was also proposed by this group for the detection of VEGF.342 HCR and CHA have also been modified for enzyme-free amplified detection of proteins. Yu et al. used an aptamer probe to form a long duplex on the surface of an electrode by HCR and demonstrated electrochemical sensing of IFN-γ.343 A HCR-mediated bioluminescence and two CHA-based fluorescent aptasensors for thrombin analysis have also been developed.344−346 Overall, aptamers have been widely utilized as improved alternatives to Abs owing to their excellent qualities, and amplified protein detection by isothermal methods has been well-established using aptamer probes. Three types of aptamer probes are frequently adopted: hairpins caging an aptamer sequence, duplexes prehybridizing with a blocker, and split aptamers with two subunits. Upon binding to their target molecules, the signal amplification process is induced.

Figure 11. Protein detection based on aptamer-combined isothermal amplification. (A) Design of the conformation-switching aptamer and RCA-based detection for PDGF. Reprinted from ref 312. Copyright 2007 American Chemical Society. (B) Schematic illustration of homogeneous fluorescent detection of PDGF-BB based on the structure-switching-triggered isothermal circular target-displacement polymerization signal amplification. Reprinted from ref 324. Copyright 2011 American Chemical Society. (C) Scheme for sensitive detection of PDGF-BB with aptamer-based target-triggering two-stage amplification (SDA and EXPAR). Reprinted from ref 44. Copyright 2012 American Chemical Society. (D) Schematic representation of homogeneous aptamer and NEase-assisted fluorescence signal amplification assay for protein. Reprinted from ref 96. Copyright 2012 American Chemical Society. (E) Amplified fluorescent sensing of thrombin using two aptamer subunits and Exo III-catalyzed regeneration of thrombin. Reprinted with permission from ref 341. Copyright 2012 John Wiley & Sons, Inc.

and co-workers designed a self-blocked bifunctional fluorescent probe for the homogeneous detection of PDGF.324 This probe connects the aptamer with a hairpin DNA as the signal reporter, acting as the template (Figure 11B). The interaction between PDGF-BB and the aptamer domain leads to the displacement of hairpin DNA and a fluorescent signal. Then, the primer hybridizes to the opened hairpin and initiates the extension reaction, releasing the aptamer-binding PDGF. The released PDGF can bind another aptamer, resulting in the recycling amplification reaction driven by the polymerase extension-mediated displacement of the target. As little as 0.18 nM PDGF was detected even in the presence of a significantly higher concentration (7−200 times) of interference proteins. A similar target recycling design has also been used for the detection of other targets (VEGF and IFN-γ) via improved SDA approaches.325−328 By contrast, we observed that the formation of thrombin-aptamer complexes was a barrier to the polymerase and inhibited primer extension in both fluorescent and gel-electrophoresis experiments.329 Zhang et al. have constructed several novel aptasensors for lysozyme analysis by developing various SDA cascade amplification strategies.176,330−332 To improve the sensitivity, EXPAR was employed to follow SDA as a two-stage amplification.44 As shown in Figure 11C, a structure-switching aptamer probe was 12507


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3.2.1.3. DNA-Binding Protein Detection. DNA-binding proteins are unique proteins that contain DNA-binding domains for interacting with DNA sequences. Transcription factors (TFs), which bind to specific dsDNA sequences, have been studied most intensively. TFs modulate the transcription of genes by binding to sequences close to related promoters and are thus important targets in biological and biomedical research. In contrast to the detection of other proteins, TF assays using isothermal amplification methods do not require Abs or aptamers. Lu et al. first demonstrated the amplified detection of TFs by RCA.347 However, this method required an Endo recognition site in the TF binding site, limiting its universality. Yin et al. achieved universal detection of TFs by near-infrared fluorescent solid-phase rolling circle amplification (NIRF-sRCA).348 As shown in Figure 12A, the TF-bound DNA is separated by gel

based colorimetric assay with AuNP-DNA probes has also been proposed (Figure 12C).350 This method exhibited high sensitivity with a detection limit of 3.8 pM when used to measure TNF-α-induced endogenous NF-κB p50 in the nuclear extracts of HeLa cells. To improve sensitivity, transcriptionassisted amplification has been combined with EXPAR to construct a cascade amplified biosensor.351 The G-quadruplex DNAzyme produced by EXPAR was used to further increase the CL signal. A detection limit as low as 6.03 fM was obtained with a broad dynamic range (5 orders of magnitude), and the analysis of NF-κB p50 in crude cell nuclear extracts was demonstrated. 3.2.2. Detection of Enzymes. Enzymes are unique proteins that act as biological catalysts of chemical reactions with high specificity. They are required in nearly all metabolic processes to sustain life, and some are important disease biomarkers. In this section, we focus on the analysis of enzyme activities by isothermal amplification methods. 3.2.2.1. Detection of Telomerase. Telomerase is a ribonucleoprotein that elongates telomeres with a telomeric repeat sequence (e.g., “TTAGGG” in vertebrates) at the 3′ ends of chromosomes using its endogenous RNA template.352,353 The elongation of telomeres by this RT process protects chromosomal ends from natural shortening during cell differentiation, leading to extension of cellular lifespan and cancerous transformation. Elevated telomerase activity is observed in approximately 90% of analyzed tumors. Telomerase is considered a biomarker for early stage cancer diagnosis and a drug target for therapy. In 2004, Willner and co-workers introduced DNAzymeassisted signal amplification to analyze telomerase activity.134,354,355 They first designed a catalytic beacon that caged the DNAzyme sequence (A and B) and included a primer (C) for telomerase (Figure 13A).354 In the presence of telomerase, the telomere repeat units are synthesized and hybridize with the loop sequence of the catalytic beacon, resulting in beacon opening and generation of the DNAzyme. A colorimetric amplified signal is obtained. Telomerase activity was detected in as few as 500 HeLa cells. Two DNAzyme-based CL methods were subsequently developed for surface detection.134,355 One used DNAzyme-AuNPs as catalytic labels that were captured on the Au surface by telomere repeat sequences (Figure 13B).134 An amplified fluorescent assay with higher sensitivity (200 HeLa cells) has been achieved by DNAzyme-mediated recycling cleavage.356,357 Exo-mediated recycling cleavage reaction has also been introduced. Wang et al. used T7 Exoassisted signal amplification for the fluorescent analysis of telomerase activity after the extension reaction.358 Similarly, Li et al. developed a homogeneous electrochemical assay of telomerase activity (Figure 13C).359 Another electrochemical strategy has been developed based on AuNP-supporting HCR (Figure 13D).360 To further improve the performance, polymerization-based amplification was employed. Ding et al. designed a SDA-based fluorescent biosensor using an MB as both the signal reporter and the template for the SDA reaction, permitting the measurement of telomerase activity in as few as 4 HeLa cells.361 We proposed a novel cascade strategy for a telomerase activity assay with high sensitivity (3 HeLa cells).174 This method integrated two consecutive isothermal amplification processes, a 3-WJ-triggered DNA-machine (3-WJ-DNAM) and a base-stacking hybridization-assisted “biological circuit” DNAmachine (BSHBC-DNAM), into a one-step reaction. As shown

Figure 12. Detection of DNA-binding proteins. (A) Schematic illustration of the procedures for detecting the activity of TFs with NIRF-sRCA. Reprinted from ref 348. Copyright 2014 American Chemical Society. (B) Principle of target-converted HDA assay for TFs detection. Reprinted from ref 349. Copyright 2013 American Chemical Society. (C) Schematic illustration of EXPAR-based colorimetric assay for the detection of TFs. Reprinted from ref 350. Copyright 2012 American Chemical Society.

electrophoresis and isolated for array hybridization with the circle template for RCA. During the amplification reaction, the biotin-labeled dUTPs are incorporated in the sRCA products, which capture the NIRF-labeled streptavidin. Thus, TFs can be quantified by NIRF imaging. As little as 6.94 ng (∼140 fmol) of NF-κB p50 was detected by this method with successful application in as little as 0.625 μg of cell nuclear extracts. Cao et al. designed a hairpin probe containing a TF binding site in the stem region to convert the detection of TFs into HDA.349 As illustrated in Figure 12B, the hairpin probes were completely digested by Exo III and Exo I in the absence of target TFs. However, the specific binding of TFs to hairpin probes blocked the digestion reaction. The intact hairpin probes served as templates for the HDA reaction upon the addition of enzymes and primers. SG I was employed for realtime monitoring of the amplification process, and a zerobackground signal was observed due to the complete digestion of templates and the decrease in primer dimers. An EXPAR12508


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Figure 13. Detection of telomerase activity. (A) Analyzing telomerase activity by a functional DNA beacon that self-generates a DNAzyme. Reprinted from ref 354. Copyright 2004 American Chemical Society. (B) Amplified CL detection of telomerase activity using DNAzymefunctionalized AuNPs. Reprinted from ref 134. Copyright 2004 American Chemical Society. (C) Principle of the homogeneous electrochemical strategy for the detection of telomerase activity based on T7 Exo-aided target recycling amplification. Reprinted from ref 359. Copyright 2015 American Chemical Society. (D) Schematic illustration of AuNPs triggered mimic-HCR dual signal amplification electrochemical assay for telomerase activity detection. Reprinted from ref 360. Copyright 2015 American Chemical Society. (E) Schematic illustration of telomerase activity assay by cascade isothermal signal amplification: (top left) telomerase extension reaction step, (bottom left) 3WJ-DNAM, and (right) BSHBCDNAM. Reprinted with permission from ref 174. Copyright 2013 Elsevier. (F) Schematic diagram of the EXPIATR assay using (top) the NFRP primer as a fluorescence probe and (bottom) SYBR Green I dye probe. Reprinted from ref 362. Copyright 2013 American Chemical Society.

quencher. SG I was also used to monitor the reaction without labeling of the NFRP primer. The reaction buffer was systematically optimized to integrate telomerase extension and exponential amplification in a closed-tube reaction. Compared to the telomere repeat amplification protocol (TRAP), which is based on PCR and considered the gold standard for detecting telomerase activity, EXPIATR exhibits higher sensitivity (single cancer cell or less) and reduces the assay time to ∼25 min (∼1.5−2 h for TRAP). Weizmann and co-workers observed that abundant cellular proteins in complex samples impair assay specificity due to nucleic acids interference, challenging the application of nucleic acid amplification-based assays to real clinical specimens.363 To address this problem, they introduced AuNPs into EXPIATR and demonstrated improved sensitivity (5-fold) of telomerase activity detection in protein-rich complex samples.363 AuNPs also improved the exponential amplification specificity. The potential mechanism of these improvements has been investigated in detail in their work. Inspired by EXPIATR, Zhang et al. also developed two similar methods for single-cell telomerase analysis based on cascade amplification integrating SDA, EXPAR, and DNAzyme.364,365 3.2.2.2. Detection of DNA Methyltransferase. DNA methyltransferases (MTases) catalyze DNA methylation by transferring the methyl group from S-adenosyl methionine (SAM) to a target adenine or cytosine in a specific DNA site. Aberrant expression of DNA MTase is associated with multiple diseases.366−368 High DNA MTase activity leads to hypermethylation, which can silence tumor suppressor genes and promote malignant transformations. DNA MTase is considered a potential target in anticancer therapy. Clearly, effective and accessible methods to detect MTase activity are important for

in Figure 13E, the telomerase product was added to the 3′ terminus of the TS-primer, which then formed the 3-WJ structure via hybridization with the 3-WJ probes (3-WJ primer and 3-WJ template). The 3-WJ structure was capable of initiating the 3-WJ-DNAM (as a SDA process), continuously generating “DNA triggers” (Product 1). In the BSHBCDNAM, these “DNA triggers” annealed with Primer 1 to stabilize the hybridization (5-bp) between Primer 1 and Primer 2 via base-stacking interaction. These stable duplexes induced two SDA reactions along each 3′ end. One SDA reaction was a target (“DNA trigger”) recycling process, whereas the other accumulated Product 2 for binding to the MB. Each telomerase-catalyzed elongation event was thus efficiently converted into a highly amplified fluorescent signal. Inhibition of telomerase activity by 3′-azido-3′-deoxythymidine was also evaluated by this method to demonstrate its potential application in drug screening. Telomerase activity analysis at the single-cell level was recently demonstrated. Inspired by E-SDA, Weizmann and Tian described a novel exponential isothermal amplification of telomere repeat (EXPIATR) assay that permits rapid (25 min), real-time detection of telomerase activity in a single HeLa cell.362 The scheme of EXPIATR is similar to that of E-SDA. As illustrated in Figure 13F, both a nicking telomerase substrate (NTS) primer and a nicking fluorescent reporter probe (NFRP) primer are involved in the sensing system for exponential amplification, as in E-SDA. As the NTS primer is extended by telomerase, the extension product serves as the template. With the help of Bst 2.0 polymerase and Nt.BspQ I NEase, exponential amplification was achieved using the NFRP primer and the free NTS primer. A real-time fluorescent signal was obtained via opening of the stem in the NFRP primer and consequent separation of fluorescein from the Iowa Black 12509


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to cleave a Dam-methylated probe and to catalyze the nicking process in SDA, achieving colorimetric analysis using an SDADNAzyme signal cascade without DpnI.169 As shown in Figure 14B, the recognition site (5′-G-A-T-C-C-3′) of Nt.AlwI contains the recognition site of Dam MTase (5′-G-A-T-C-3′). The methylation of 5′-G-A-T-C-3′ by Dam MTase blocks nicking by Nt.AlwI and subsequent signal amplification. Conversely, the nicking reaction proceeds when Dam MTase is absent, inducing the SDA process as well as DNAzymemediated signal generation. The absence of additional Endo and buffer optimization in our method increases its convenience despite acting as a signal-off system. The EXPAR-based exponential amplification of DNAzyme has also been demonstrated for the fluorescent assay of MTase activity.98 RCA-based methods have also been employed for the assay of MTase activity. Zeng et al. utilized PG-RCA to achieve ultrasensitive detection of Dam MTase activity with a low detection limit of 1.29 × 10−4 U/mL.371 The hairpin probe was methylated and cleaved by Dam and Dpn I coupled reaction, releasing a ssDNA as the primer to initiate the PG-RCA reaction (Figure 14C). The corresponding products contained the sequence of HRP-mimicking DNAzyme, which catalyzes the oxidation of luminol by H2O2 to yield a CL signal. The inhibition of Dam MTase activity was investigated to assess its applicability in screening antimicrobial drugs. Other RCA-based CL assays incorporating HCR have also been developed.372 An electrochemical biosensor for the detection of M.SssI MTase activity has been proposed as an immuno-RCA assay.373 In addition to polymerization-based amplification methods, NEase-assisted,97,98 Exo-mediated,374−377 and DNAzymebased378 recycling cleavage have been introduced for amplified analysis of DNA MTase activity. Xing et al. first evaluated Dam activity in E. coli in different growth stages using an Exo IIImediated target recycling reaction (Figure 14D).377 Enzymefree signal amplification was recently used to assay the effect of environmental phenolic hormones on MTase activity.379 3.2.2.3. Detection of Alkaline Phosphatase and Polynucleotide Kinase. Alkaline phosphatase (ALP) catalyzes the dephosphorylation of substrates such as nucleotides, whereas polynucleotide kinase (PNK) catalyzes the phosphorylation of nucleic acids at their 5′ hydroxyl ends. Both enzymes play critical roles in a majority of cellular events, and aberrant activities of these enzymes have been associated with serious human disorders and diseases.380−383 Therefore, the measurement of their activities and screening for potential inhibitors are of great importance in many diagnostic and clinical assays.393 Miao et al. first employed λ Exo-assisted signal amplification for ALP activity detection.113 Two complementary phosphorylated probes were used. One was immobilized on the Au electrode (Figure 15A), and the other was treated by ALP to remove its phosphate group and immersed in a mixed solution on a Au electrode. After hybridization between the two DNA probes, λ Exo performed the recycling cleavage of immobilized probes, resulting in a decrease in the electrochemical signal. In addition to λ Exo, Exo III,384,385 and DNAzyme386,387 have been introduced to perform recycling amplification for a fluorescent assay of T4 PNK activity. Coupled with a λ Exocatalyzed cleavage reaction, enzyme-free CHA (Figure 15B)388 and HCR389 have also been applied to the amplified detection of T4 PNK activity. However, these methods all require multistep processing and extended assay time.

early disease diagnosis and the development of antimethylation therapies.369 Li et al. first demonstrated the analysis of MTase activity by a two-stage signal amplification cascade.168 As illustrated in Figure 14A, this cascade combining SDA with DNAzyme is

Figure 14. Detection of DNA MTase activity. (A) Schematic diagram of the Dam MTase activity assay using SDA and DNAzyme cascade amplification. Reprinted from ref 168. Copyright 2010 American Chemical Society. (B) Schematic illustration of methylation-blocked cascade amplification based label-free colorimetric biosensor for Dam MTase activity assay. Reprinted with permission from ref 169. Copyright 2014 Elsevier. (C) Schematic illustration of Dam MTase assay using hairpin probe-based PG-RCA-induced CL. Reprinted from ref 371. Copyright 2013 American Chemical Society. (D) (top) Schematic diagram of the assay for Dam activity based on Exo IIImediated target recycling. (middle) Measurement of the activity of Dam in E. coli cells. (bottom) The activity of Dam at different growth stages of E. coli cells and in Dam negative E. coli cells, and the inhibition of Dam activity by gentamycin (30 μM) and benzylpenicillin (30 μM). Reprinted from ref 377. Copyright 2014 American Chemical Society.

triggered by DNA adenine methylation (Dam) MTase as a model target. Dam MTase catalyzes the methylation of N6adenine in the palindromic site 5′-G-A-T-C-3′ (red in DNAMac1), generating the methylated duplex DNA 5′-G-Am-T-C3′, which serves as the substrate for DpnI Endo. After cleavage by DpnI, DNA-Mac1 is split into three parts. The part containing segment B is fully complementary to region I of DNA-Mac2, inducing the SDA reaction. The SDA product is designed to be an HRP-mimicking DNAzyme that generates the colorimetric readout. However, DNA-Mac2 is blocked by DNA-Mac1 in the absence of Dam MTase, preventing it from forming an active SDA duplex. This method exhibits higher sensitivity (detection limit of 0.25 U/mL) than previously reported assays without signal amplification,368,369 and it can be used to screen MTase inhibitors. A methylation-stimulated DNA machine was proposed for the detection of M.HpaII MTase.370 We utilized the methylation-sensitive NEase Nt.AlwI 12510


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The nicked products were also ligated by T4 DNA ligase. When the ligation and nicking process reached a dynamic equilibrium, multiple MBs were cut. Eventually, an amplified fluorescent signal was obtained. The phosphorylation, ligation, and nicking processes were all achieved in a single sensing system. With high sensitivity (0.00001 U/ml T4 PNK), the proposed strategy can be used in a complex biological matrix and to screen T4 PNK inhibitors. Although not homogeneous, two similar methods have been developed based on NEase-assisted signal amplification following λ Exo-catalyzed cleavage reaction.391,392 SDA and RCA have also been employed for multistep analysis of T4 PNK activity.180,393,394 3.2.2.4. Detection of Other Enzymes. Various isothermal amplification techniques have been employed to assay the activity of other enzymes including ligase, DNA glycosylase, recombinase, and topoisomerase (Top). He et al. utilized ligation-triggered SDA to accumulate DNAzymes, demonstrating label-free colorimetric biosensing of DNA ligase.395 Human 8-oxoG DNA glycosylase 1 (hOGG1), an important repair enzyme, has also been analyzed by a DNAzyme-based colorimetric assay.396 Fluorescent detection of hOGG1 activity was then achieved by Exo III-assisted signal amplification to increase the sensitivity.397 Activity analysis of human TopI and recombinase at the single-molecule level has been performed using RCA.398−400

Figure 15. Detection of ALP and PNK activities. (A) Schematic illustration of the electrochemical detection of ALP by two DNA probes coupled with λ Exo. Reprinted with permission from ref 113. Copyright 2011 Elsevier. (B) Schematic illustration for the detection of PNK activity based on the coupled λ Exo cleavage reaction and CHA of bimolecular beacons. Reprinted from ref 388. Copyright 2014 American Chemical Society. (C) Schematic illustration of the ligationnicking coupled reaction-mediated signal amplification strategy for one-step highly sensitive assay of T4 PNK activity. Reprinted with permission from ref 390. Copyright 2013 Elsevier.

The homogeneous analysis of these enzyme activities based on signal amplification was first demonstrated by us.390 As shown in Figure 15C, one (Oligo B) of two short oligonucleotides complementary to the loop sequence of the MB was phosphorylated by T4 PNK and ligated to the other (Oligo A) by DNA ligase using the MB as the template. The ligated DNA triggered recycling cleavage of the MB by NEase.

3.3. Detection of Cancer Cells and Pathogens

The identification of cancer cells and pathogens relies primarily on the detection of specific nucleic acids or proteins as molecular biomarkers, but cell lysis and the subsequent extraction of targets are typically required. The direct detection

Figure 16. Detection of cancer cells and pathogens. (A) Schematic representation of improved CL strategy for cancer cell detection based on the NEase assisted strand circular amplification process. Reprinted with permission from ref 405. Copyright 2010 Royal Society of Chemistry. (B) Schematic illustration of a highly sensitive colorimetric method for the detection of rare cancer cells based on cell-triggered NEase-assisted signal amplification. Reprinted from ref 406. Copyright 2014 American Chemical Society. (C) Isolation and detection of cancer cells in whole blood using RCA-generated long multivalent DNA aptamer-based microfluidic device. The zoomed-in box illustrates a captured cell is bound by several long DNA molecules via multiple aptamer domains (red color). Reprinted with permission from ref 416. Copyright 2012 National Academy of Sciences. (D) MHCR for cancer cells detection. Reprinted from ref 157. Copyright 2014 American Chemical Society. (E) Schematic illustration of PLAcombined RCA cascade amplification for pathogens assay. Reprinted with permission from ref 418. Copyright 2012 John Wiley & Sons, Inc. 12511


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Figure 17. Aptamer-based detection of small molecules. (A) (left) DNAzyme ligase and substrate. The ligase is shown in black, the allosteric, ATPbinding domain is in red, and the padlock probe is in blue. An arrow indicates the ligation junction. (right) Schematic of DNAzyme-mediated RCA. The aptazyme is activated by ATP and ligates a padlock probe. RCA is initiated from the 3′ end of the aptazyme, and the elongated aptazyme is visualized using fluorescent oligonucleotide probes labeled with Cy3. Reprinted from ref 422. Copyright 2005 American Chemical Society. (B) Schematic representation of the colorimetric detection of a specific target by using (top) an RNA-cleaving allosteric DNAzyme, (middle) RCA, and (bottom) PNA and DiSC2 (5). Reprinted with permission from ref 424. Copyright 2009 John Wiley & Sons, Inc. (C) The amplified analysis of cocaine by an autonomous aptamer-based machine using SDA. Reprinted from ref 60. Copyright 2007 American Chemical Society. (D) Amplification strategy based on a DNA-protective nanomaterial through DNase-catalyzed recycling of target. Reprinted with permission from ref 118. Copyright 2010 John Wiley & Sons, Inc. (E) Schematic representation of fluorescence anisotropy (FA) assay of ATP via HCR. Double-headed curved arrows represent the relative rotational variation of the fluorophore. Reprinted from ref 453. Copyright 2013 American Chemical Society.

has also been achieved.406 As illustrated in Figure 16B, the specific binding of the hairpin aptamer probes (HAPs) to CCRF-CEM cells changes the conformation of the HAPs, resulting in recycling cleavage of the linker DNAs. The cleaved linker DNAs cannot assemble free DNA-AuNPs, and no color change is observed. In the absence of target cells, linker DNAs remain intact for the assembly of DNA-AuNPs, producing a color change from red to purple. The linear response of this method ranges from 102 to 104 CCRF-CEM cells with a low detection limit of 40 cells. SDA and RCA have been employed for the electrochemical,407−409 CL,410,411 and ECL,412,413 detection of cancer cells. SERS and colorimetric assays using RCA-nicking cascade amplification have also been reported.414,415 Importantly, inspired by marine creatures using long multiple adhesive domains to effectively capture flowing food particulates, Zhao et al. designed an RCA-based multivalent aptamer network for the capture of cells with higher efficiency.416 As illustrated in Figure 16C, the biotinylated primer-circular template duplex was immobilized on the microfluidic device to induce the RCA reaction, generating a long multiple aptamer network on the surface. This aptamer network increased the accessibility and

of intact cells and pathogens by omitting these tedious steps is more convenient and efficient.401−404 In this section, we focus on the development of methods for the direct detection of intact cancer cells and pathogens based on isothermal amplification techniques. Using a cell-binding aptamer as the recognition element, amplified detection of cancer cells was achieved based on NEase-assisted signal amplification. For example, Bi et al. utilized a duplex probe caging the aptamer to recognize Ramos cells and trigger an amplification reaction (Figure 16A).405 The binding of a Ramos cell to the aptamer released the other DNA strand in the duplex probe to serve as the trigger for the nicking of hairpin substrate containing two HRP-mimicking DNAzyme units. Thus, the DNAzyme was activated, resulting in a CL signal. The Fe3O4−Au core−shell nanoparticle was used to separate the DNAzyme from the unbound hemins to circumvent the background catalytic reaction. An estimated detection limit of 67 cells/mL was achieved using this method. The same group then constructed a similar NEase-based biosensor for Ramos cells using dendrimer/QD nanoclusters as ECL reporters.93 The detection of CCRF-CEM cells based on NEase-assisted signal amplification by a colorimetric aptasensor 12512


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reaction proceeded, the immobilized aptazyme was elongated to produce long repetitive ssDNAs, which captured the fluorescent probes. A detection limit of 1 μM ATP was obtained. Li et al. expanded the RCA-based method for colorimetric ATP sensing using an RNA-cleaving allosteric DNAzyme and a colorimetric reporting mechanism based on a peptide nucleic acid (PNA) and an organic dye.424 As illustrated in Figure 17B, the binding of ATP to the allosteric DNAzyme activated the cleavage reaction, releasing a DNA molecule as the primer to initiate a RCA reaction. The RCA products were detected by the color change from blue to purple upon hybridization with a complementary PNA in the presence of DiSC2 (5) (3,3′-diethylthiadicarbocyanine). Several other RCA-based aptasensors have also been proposed for the assay of cocaine,425,426 OTA,427 and GTP.428 In addition, SDA-based methods were employed for the detection of cocaine60,61,429−432 and ATP.433 Notably, Willner and co-workers were the first to achieve SDA-based fluorescent detection of cocaine (Figure 17C).60 A block DNA (1a) annealed with SDA template (3) was used to inhibit background amplification due to uncontrolled folding. When cocaine bound to the aptamer sequence in the SDA template, the block DNA was displaced by the self-assembly of the SDA template upon activation of the SDA reaction. MB was used as the fluorescent reporter. This method is much simpler and does not require the DNAzymes used in the two RCA-based methods described above. Another polymerization-based method, LAMP, has been used for the electrochemical and ECL detection of OTA.434,435 Nuclease-based amplification strategies were subsequently employed for the simple and convenient detection of small molecules. Lu et al. first reported an amplified aptamer-based assay based on DNase I-assisted signal amplification.118 Graphene sheets were used to adsorb the fluorophore-labeled aptamer (ssDNA) for the protection of aptamer from DNase Icatalyzed cleavage and quenching of the fluorescent signal (Figure 17D). Upon binding to the target molecule, the aptamer formed a rigid structure and dissociated from the graphene substrate. Then, DNase I cleaved the dissociated aptamer, releasing the target and liberating the fluorophore. Ultimately, the target recycling cleavage of fluorophore-labeled aptamers leads to significant amplification of the fluorescent signal. Good assay sensitivity was achieved with detection limits of 40 nM for ATP and 100 nM for cocaine. Nuclease-based biosensors using DNase I, 1 1 9 , 4 3 6 Exos, 4 3 7 − 4 4 3 and NEases444−447 have also been developed for the analysis of different small molecules. In addition to nucleases, DNAzymeassisted signal amplification strategies have been expanded to colorimetric,448−450fluorescent,133,451 and CL145 detection of small molecules. Even enzyme-free HCR and CHA have been implemented in small-molecule assays.161,452−454 Yang et al. reported a novel aptamer-based fluorescence anisotropy assay for ATP based on HCR signal amplification (Figure 17E).453 In addition to aptamers, enzymes can also recognize small biomolecules. T4 DNA ligase and E. coli DNA ligase specifically employ ATP and NAD+, respectively, as cofactors. Utilizing these characteristics, Zhang et al. demonstrated highly specific and sensitive detection of ATP and NAD+ based on DNAzymeassisted signal amplification.455 The enzyme strand of DNAzyme was split into two fragments (DNA 1 and DNA 2) that were inactive to the MB substrate (Figure 18A). Despite their displacement by invasive DNA, a zero-background signal was observed in the absence of target biomolecules. However, DNA 1 and DNA 2 could be ligated to produce an intact

frequency of interactions with target cancer cells (containing many aptamer-binding sites on the cell surface), significantly improving the cell capture efficiency compared to monovalent aptamers or antibodies. The captured cells maintained high purity and were detected by the fluorescent label. Furthermore, the captured cells could be easily released for other studies by cleaving the DNA strands containing recognition sites for restriction enzymes. Utilizing multibranched HCR (mHCR), Zhou et al. also demonstrated the multivalent capture and detection of cancer cells with nanostructured electrochemical biosensors.157 As shown in Figure 16D, the aptamer, initiator, and two hairpins (H1 and H2) form the mHCR product containing long multiple branched arms and multiple biotins. An individual cancer cell can bind to many mHCR products with the aptamers and be captured on the gold electrode by multiple hybridizations between the branched arms and DNA tetrahedral probes. The biotins in the mHCR products are used to attach avidin-HRPs for signal amplification. The DNA tetrahedral probe-modified gold electrode provides superior hybridization conditions for multivalent binding, and as few as 4 cancer cells can be detected by this method. Another HCRbased electrochemical biosensor for cancer cell detection was proposed by Liu et al.417 The detection of intact pathogens based on isothermal amplification strategies has been reported. As illustrated in Figure 16E, Nilsson and co-workers demonstrated the sensitive detection of Bacillus globigii spores by PLA-combined RCA cascade amplification.418 Oligonucleotide-tagged magnetic nanobeads were used as signal reporters. This method was then expanded to on-chip detection of B. globigii spores and Vibrio cholerae.419 Detection limits as low as 500 B. globigii spores and 2 pM V. cholerae were achieved. In addition, SDAbased lateral flow biosensors were proposed for pathogen detection by Zeng et al.420,421 After binding to Salmonella enteritidis, two aptamers (A-aptamer and biotin-labeled Captamer) were separated by streptavidin-coated magnetic beads.420 The A-aptamer served as the template to trigger the SDA reaction, and the products of this reaction were detected by the lateral flow test. As few as 10 colony-forming units of S. enteritidis were detected in this study, comparable to the detection limit of PCR. 3.4. Detection of Small Molecules and Metal Ions

Like proteins and cells, small molecules and metal ions cannot be directly amplified by isothermal amplification techniques. Various functional nucleic acids such as aptamers and DNAzymes are also required to expand these techniques to the sensitive detection of small molecules and metal ions. Here, we summarize advances in small molecule and ion assays based on isothermal amplification techniques. 3.4.1. Detection of Small Molecules. Various significant small molecules, including cocaine, ATP, Ochratoxin A (OTA), nicotinamide adenine dinucleotide (NAD+), and L-histidine, have been detected by isothermal amplification techniques. Most of these molecules trigger amplification reactions via aptamers. As early as 2005, Ellington and co-workers introduced RCA for the detection of ATP.422 A DNA aptazyme previously reported by their group was used to bind ATP to trigger the ligation of a 63-mer padlock probe (Figure 17A). The autoligation of a 3′-phosphorothioate end on a 5′-iodine residue was activated by up to 460-fold by ATP.423 As the RCA 12513


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based method458 have also been developed for label-free fluorescent and colorimetric detection, respectively. Like ligases, some DNAzymes are activated by small molecule cofactors. A representative example is L-histidinedependent nucleic acid cleaving DNAzyme. Using this DNAzyme, Willner and co-workers reported the colorimetric and CL detection of L-histidine.459 On the basis of NEaseassisted signal amplification, the amplified detection of Lhistidine was demonstrated as shown in Figure 18C.94 A SERS assay of L-histidine via the SDA cascade has also been developed.460 3.4.2. Detection of Metal Ions. Contamination by metal ions such as Pb2+ and Hg2+ is a serious concern to human health. The level of metal ions in the ambient environment has increased in the last few decades. Inductively coupled plasma mass spectrometry (ICP-MS) is a powerful technique for the detection of metal ions. However, it is rather expensive and complex, requiring large equipment and limiting on-site analysis. Some biosensors have been proposed for convenient and rapid metal ion assays.461−469 Most utilize nucleic acids for target recognition. Recently, isothermal amplification techniques have been employed to further improve the detection sensitivity of nucleic acid-based biosensors. Using Pb2+-dependent DNAzyme, Willner and co-workers first demonstrated the amplified detection of Pb2+ based on catalytic signal amplification by HRP-mimicking DNAzyme.459 A detection limit of 10 nM Pb2+ was obtained, significantly lower than the maximum contamination level (72 nM) in drinking water defined by the U.S. Environmental Protection Agency. UO22+ was detected in a similar manner.470 By integrating a Cu2+-dependent DNA-cleaving DNAzyme and an HRP-mimicking DNAzyme, Yin et al. designed an allosteric dual-DNAzyme unimolecular probe for colorimetric Cu2+ detection.471 Using GO as a superquencher, Zhao et al. demonstrated Pb2+-recycling cleavage by Pb2+-dependent DNAzyme with a low detection limit of 300 pM Pb2+.472 Polymerization-based,172,473,474 nuclease-assisted,475,476 and enzyme-free477,478 amplification techniques have been introduced for the highly sensitive detection of Pb2+. Notably, our group has proposed a novel biosensing platform based on ESQM.172 As shown in Figure 19A, this platform integrates an SDA module and an NEase-assisted signal amplification module into a one-step system to achieve ultrasensitive Pb2+ analysis (detection limit of 30 fM) within a short assay time (40 min). The use of this system to detect Pb2+ in complex environmental water samples yielded results consistent with those obtained by ICP-MS. Functional nucleic acids other than DNAzymes have been used to recognize Hg 2+ and Ag+ for inducing signal amplification. Hg2+ specifically binds to thymine-thymine (TT) mismatches in DNA duplexes to form a stable T-Hg2+-T complex.479−481 Similarly, a C-Ag+-C complex is formed by Ag+.482,483 Based on the T-Hg2+-T complex, Willner and coworkers achieved colorimetric analysis of Hg2+ at the nanomolar level by SDA-DNAzyme cascade amplification (Figure 19B).166 Other polymerization-based amplification methods have been employed to develop fluorescent biosensors for Hg2+ or Ag+.175,484−489 In particular, Bi et al. investigated Hg2+- and Ag+-triggered ligase activity and then constructed an RCA-based molecular logic gate for the detection of these two metal ions (Figure 19C).487 Nuclease-assisted490−492 and DNAzyme-mediated132,493 amplification techniques have also been employed. An Exo III-based electrochemical method

Figure 18. Detection of small molecules without aptamers. (A) Design strategy of the DNAzyme cascade for amplified fluorescence detection of biological small molecules. Reprinted from ref 455. Copyright 2011 American Chemical Society. (B) Schematic representation of targettriggered ligation-RCA assay for NAD+. Reprinted with permission from ref 456. Copyright 2012 Royal Society of Chemistry. (C) Schematics of DNAzyme-based sensing system for the detection of LHistidine by using enzymatic signal amplification strategy. Reprinted from ref 94. Copyright 2011 American Chemical Society.

DNAzyme strand by the corresponding DNA ligase in the presence of a target. With the introduction of invasive DNA, the intact DNAzyme was separated from C-DNA by competitive hybridization. Therefore, an amplified signal was produced by DNAzyme-catalyzed recycling cleavage of the MBs. Within 25 min (cleavage reaction), as little as 100 pM ATP and 50 pM NAD+ were detected. In contrast to aptamers, this method can easily distinguish ATP from its analogues (adenosine, AMP, and ADP). Inspired by the general requirement for a ligation process in RCA, we reported the ultrasensitive detection of NAD+ by target-triggered ligationRCA (Figure 18B).456 A detection limit of 1 pM was obtained with a broad linear range of 6 orders of magnitude. A homogeneous biosensor based on a ligation-nicking coupled reaction was also proposed by our group for a highly sensitive ATP assay with a detection limit of 5 pM.390 Although requiring multiple steps, an RCA-based assay457 and an SDA12514


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demonstrated using a PNA-directed padlock probe assembly.498 However, extraneous primers were still added in the above methods. To overcome this shortcoming, Nilsson and coworkers developed target-primed RCA for in situ genotyping of individual DNA molecules.63 As illustrated in Figure 20B, the target dsDNA was first restriction digested. Then, the ss-target sequence with a free 3′ end was produced by λ Exo-catalyzed cleavage to act as a template for the cyclization of the padlock probe. This 3′ end sequence protruding beyond the proberecognition site was removed by the 3′-5′ exonucleolytic activity of Phi29 polymerase and then served as the primer to initiate the extension reaction by Phi29 polymerase. Using the target as a primer, this method avoids the topological inhibition of replication and also ensures efficient retention of RCA products at the original location, significantly improving the amplified signal and assay specificity. The distribution of individual normal and mutant human mitochondrial genomes with single-base differences has been determined using this method. Nilsson and co-workers extended target-primed RCA to the detection and genotyping of individual mRNA molecules in human and mouse cells and tissues.64 Deng et al. recently devised toehold-initiated RCA for the in situ visualization of individual miRNAs in single cells with excellent specificity (Figure 20C).65 A modified method was then proposed by Ge et al.499 Prehybridization of the RCA product with multiple fluorescent probes was performed to detect single influenza viral RNA copies in cells.500 Combined with the PLA technique, RCA has been employed for in situ observation of protein−protein interactions,501,502 protein−DNA interactions,503 and protein modifications.504−506 Owens et al. demonstrated the visualization of histone modifications at specific gene loci with single-cell resolution in paraffinembedded tissue sections (Figure 20D).505 In situ analysis by HCR has also been reported.148,158,159,507 Significantly, Pierce and co-workers achieved the imaging of five target mRNAs simultaneously in fixed whole-mount and sectioned zebrafish embryos with robust performance (Figure 20E).158 Tan and co-workers demonstrated the in situ selfassembly of aptamer-induced fluorescent nanodevices for targeting live cell membranes in complex extracellular environments (Figure 20F).148 Other isothermal amplification techniques including LAMP,508−510 SDA,511 and transcription-based amplification512 have also been extended to in situ analysis. Very recently, HCR and the other enzyme-free amplification strategy CHA were performed in live cells for amplified imaging of miRNA or mRNA. Using hsa-miR-21 as a model target, Weizmann et al. reported real-time miRNA imaging inside live cells based on HCR or a cascade hybridization reaction.513 Two programmable DNA hairpin probes, HP1 and HP2, were designed to be labeled with the fluorescent dyes Cy3 and Cy5 (Figure 21A). As hsa-miR-21 initiated the HCR reaction, the self-assembly of HP1 and HP2 via repeating multiple hybridizations brought the two dyes into close proximity to generate a FRET signal. The FRET-based method has been demonstrated to effectively prevent false positive signals that can be observed in quencher/dye systems due to probe accumulation and/or degradation.514 Additionally, CHA-based mRNA imaging has been implemented by Tan and co-workers (Figure 21B).515 The target-triggered recycling displacement assembly of H1 and H2 yields multiple signal outputs for the intracellular mRNA. Notably, DNA probes in these two studies were transfected into live cells using the commercial reagent

Figure 19. Detection of metal ions. (A) Schematic illustration of ESQM and its application for Pb2+ and Dam MTase detection. Reprinted with permission from ref 172. Copyright 2014 Nature Publishing Group. (B) The analysis of Hg2+ ions by a DNA-based machine utilizing 3 as a track, and the autonomous synthesis of the HRP-mimicking DNAzyme units by repeated replication/nicking cycles. Reprinted with permission from ref 166. Copyright 2008 John Wiley & Sons, Inc. (C) (top) Diagrammatic representation of DNA ligase activity triggered by Hg2+, which further initiates RCA reaction. (bottom) Fluorescence spectra of FAM corresponding to difference concentrations of Hg2+ in “YES” gate and “NOT” gate, respectively. The arrows indicate the signal changes with increases in the Hg2+ concentration (0, 10−10, 10−9, 10−8, 10−7, and 10−6 M). Insets: corresponding calibration curves for various concentrations of Hg2+. Reprinted from ref 487. Copyright 2013 Royal Society of Chemistry. (D) Illustration of the Hg2+-triggered digestion of the designed e-T-rich probe by Exo III monitored by the immobilizationfree electrochemical monitoring protocol. Reprinted from ref 491. Copyright 2013 American Chemical Society.

achieved ultrasensitive detection of Hg2+ as low as 0.2 nM (Figure 19D). By binding to its aptamer, K+ has also been detected via nuclease-assisted signal amplification.494,495 3.5. In Situ and Intracellular Analysis

Isothermal amplification techniques also play important roles in in situ and intracellular analysis. In early 2001, Ward et al. first used RCA for in situ visualization of target DNA sequences and point mutations in interphase nuclei and DNA fibers.496 As shown in Figure 20A, long probes were employed to associate the denatured target sequences (via hybridization and/or ligation) and functioned as primers to initiate RCA. The RCA products could be visualized with fluorophore-labeled decorator probes. These long probes were synthesized with two 3′ ends to stabilize the association of the RCA products on the target DNA molecules and increase the detection efficiency. Then, an improved method was proposed for the in situ detection of gene copy number and single-base mutations in individual cells.497 REases and Exo III were used to digest genomic dsDNA to produce ssDNA for hybridization and ligation of the RCA probe. The primer was then added to initiate the RCA reaction. The complete removal of the nontarget DNA strand by Exo III significantly increased the efficiency of heat denaturation-mediated ssDNA generation. Under DNA nondenaturing conditions, RCA-based imaging of single-copy sequences within human genomic DNA was 12515


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Figure 20. In situ analysis. (A) Schematic for detecting small target sequences (left) or point mutations (right) using RCA. Reprinted with permission from ref 496. Copyright 2001 National Academy of Sciences. (B) Schematic representation of in situ target-primed RCA. Arrowheads indicate 3′ ends. Reprinted with permission from ref 63. Copyright 2004 Nature Publishing Group. (C) Schematic representation of toeholdinitiated RCA for visualizing individual miRNAs in situ inside cells. RCA products are shown as green dots in an A549 cell with its nucleus stained by 4′,6-diamidino-2-phenylindole (DAPI; blue), and the outline of A549 cell is marked with a dotted line. Reprinted with permission from ref 65. Copyright 2014 John Wiley & Sons, Inc. (D) Schematic of the ISH-PLA method for detecting H3K4me2 of the MYH11 promoter. Reprinted with permission from ref 505. Copyright 2013 Nature Publishing Group. (E) Multiplexed in situ hybridization using fluorescent HCR in situ amplification. Reprinted with permission from ref 158. Copyright 2010 Nature Publishing Group. (F) Construction of fluorescent DNA nanodevices on target living cell surfaces based on an aptND platform, where (top) three types of fluorescent DNA nanodevices, preformed using HCR-based self-assembly upon initiation by aptamer-tethered trigger probes, are anchored on target cell surfaces, or (bottom) aptamer seed probes initiate in situ self-assembly of fluorescent DNA nanodevices on target cell surfaces by either (I) cascading alternative hybridization of two partially complementary monomers or (II) HCR. Reprinted with permission from ref 148. Copyright 2013 John Wiley & Sons, Inc.

similar to that of nonamplified libraries. An improved method named linear amplification of DNA (LinDA) was then developed for the generation of sequencing libraries using less DNA material (< 30 picograms).521,522 Instead of lowefficiency blunt-end ligation, T-tailing was used to attach T7 promoter-containing primers to the genomic DNA with high efficiency (Figure 22B).521 Klenow polymerase then repaired gaps and displaced strands to generate dsDNA with one T7 promoter at each end. Subsequent in vitro transcription amplification by T7 RNA polymerase produced RNA transcripts, followed by RT and second-strand synthesis. After removing the primers, the amplified DNA was used for highthroughput sequencing or the next round of LinDA. The transcriptome has also been amplified by transcriptionbased amplification for single-cell RNA sequencing. Yanai et al. demonstrated that multiplexed linear transcription amplification yields more reproducible and sensitive sequencing results compared with a PCR-based amplification method.523 Phi29transcriptome amplification (PTA), which is similar to multiply primed RCA, has also been developed for full-length RNA sequencing of single cells.524 As shown in Figure 22C, a phosphorylated oligo-dT primer was designed for dscDNA generation of the full-length sequence and its intramolecular circularization (possibly including partial concatemers). Phi29 DNA polymerase executed the multiply primed RCA reaction for whole DNA amplification. After random fragmentation, the amplicons were ligated to Illumina adapters for NGS sequencing library preparation. On the basis of target-primed

Lipofectamine. Nearly simultaneously, Wu et al. constructed a novel AuNP/cationic peptide carrier for the highly efficient delivery of HCR probes (H1 and H2) into live cells (Figure 21C) and demonstrated ultrasensitive mRNA imaging with FRET.516 3.6. Sequencing

As described in section 2.1.8, isothermal WGA techniques can efficiently amplify whole genomic DNA using random primers. This amplified DNA is sufficient input material for sequencing library preparation.517−519 Transcription-based linear amplification has also been recently applied to the preparation of a sequencing library without random primers. Using a T7 RNA polymerase-assisted linear amplification system, Stunnenberg et al. demonstrated linear amplification for deep sequencing (LADS) to produce representative libraries for Illumina nextgeneration sequencing (NGS).520 As shown in Figure 22A, LADS relies on the ligation of two different sequencing adapters to blunt end-repaired and A-tailed genomic dsDNA fragments. The T7 promoter sequence is present in one of the adapters to permit the T7 RNA polymerase-mediated transcription of size-selected dsDNA fragments in vitro. Therefore, many RNA transcripts accumulate in a linear manner and are subsequently converted to cDNA followed by second-strand cDNA synthesis using adapter-specific primers, thus ensuring that the library only contains full-length fragments with two distinct adapters. Compared to PCR-amplified libraries, which suffer from severely biased representation of AT- or GC-rich fragments, the sequence coverage in LADS is much higher and 12516


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4. APPLICATIONS IN NANOTECHNOLOGY AND MATERIALS SCIENCE Because of their unique properties, such as convenient modification and engineering versatility, nucleic acids have been increasingly utilized as programmable building blocks for nanomaterials and nanoarchitecture.530−536 Isothermal amplification techniques enable the rapid and cost-effective preparation of various building blocks to construct nucleic acid nanostructures and hydrogels. Amplified products also serve as scaffolds for the self-assembly of nucleic acid-protein or nucleic acid-nanoparticle materials with nanoscale or microscale precision. These nucleic acid-based structures and materials hold great promise in a broad range of applications (e.g., drug delivery). Recent advances in these applications are summarized below. 4.1. Construction of Nucleic Acid Nanostructures

Among isothermal amplification techniques, RCA and RCT are particularly attractive in nanotechnological applications due to the ability to generate micrometer-long strands of repetitive units. These reduplicated units can self-assemble into nanoscale structures and incorporate functional labels (e.g., fluorophore) or serve as templates for hybridization with nucleic acid probes to form multifunctional nanostructures. In 2006, Yan and co-workers first demonstrated the efficient amplification of complex DNA structures containing stable Holliday junctions or multiple crossovers, providing a foundation for RCA-based nucleic acid nanoconstruction.537,538 Hammond et al. recently reported the synthesis of selfassembled RNA microsponges as carriers for RNAi based on RCT.80 As illustrated in Figure 23A, padlock probes encoding complementary sequences of the antisense and sense siRNA sequences were first ligated with T7 promoter primers. Then, T7 RNA polymerase was used to perform RCT by recognizing the T7 promoter, yielding hairpin RNA transcripts. After a sufficiently long reaction time of 20 h, these hairpin RNA products self-assembled into sponge-like microspheres, which were efficiently ingested by cells and protected from RNA degradation during transport to the cytoplasm. After digestion to produce siRNAs by Dicer in vivo, the hairpin RNAs silenced the expression of the target genes. The ingestion of a single RNA microsponge by a cell can generate more than half a million copies of siRNA, resulting in higher transfection efficiency compared to commercial nanoparticle-based delivery systems. Inspired by this work, Tan and co-workers then used RCA to construct multifunctional DNA nanoflowers for targeted therapy and imaging.68 They designed a special RCA template to produce a long DNA strand that was decorated with serial aptamers and drug-loading sites (CG or GC sequences) for the anticancer drug doxorubicin (Dox) (Figure 23B). A fluorophore was also incorporated into the RCA product during the amplification process using fluorescently labeled dUTP. The liquid crystallization and dense packaging of building blocks drove the assembly of long-strand RCA products into nanoflowers rather than relying on hybridization by Watson−Crick base pairing. By adjusting parameters such as assembly time and template sequences, the size of the nanoflower can be varied over a wide range. The concatemer aptamers and tremendous drug loading sites enhanced the binding affinity of the nanoflower to target cells and conferred a high drug payload capacity, respectively. Eventually, selective cancer cell bioimaging and targeted drug delivery were achieved by RCA-based DNA nanoflowers. Subsequent incorporation of

Figure 21. Intracellular analysis. (A) Illustration of the cascade hybridization reaction for the imaging of hsa-miR-21 in live cell. Reprinted from ref 513. Copyright 2015 American Chemical Society. (B) Illustration of CHA-based signal enhancement for specific mRNA imaging in living cells. After transfection into live cells, the reaction is initiated by specific target mRNA to repeatedly yield many H1−H2 duplexes, which further destabilize Reporter moiety and fluoresce inside the cells. Reprinted from ref 515. Copyright 2015 American Chemical Society. (C) Illustration of intracellular HCR for mRNA detection. Reprinted from ref 516. Copyright 2015 American Chemical Society.

RCA, in situ sequencing has been demonstrated for RNA analysis in preserved tissue and cells (Figure 22D).525 By exploiting DNA template walking, Lao et al. proposed a solidphase in situ isothermal amplification approach to generate billions of monoclonal colonies in less than 30 min, demonstrating NGS applications with a cost-effective and simple workflow (Figure 22E).526 In addition to NGS, the application of isothermal amplified strategies (LAMP, SDA, and NASBA) in pyrosequencing has also been reported by Zhou et al.527−529 12517


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Figure 22. Sequencing applications. (A) Workflow of LADS. Reprinted with permission from ref 520. Copyright 2011 Nature Publishing Group. (B) Schematic illustration of the LinDA protocol and its key features. Reprinted with permission from ref 521. Copyright 2012 Nature Publishing Group. (C) Flowchart for PMA with oligo-dT priming. Reprinted with permission from ref 524. Copyright 2013 National Academy of Sciences. (D) Procedure for targeted in situ sequencing based on RCA. Reprinted with permission from ref 525. Copyright 2013 Nature Publishing Group. (E) Isothermal template walking. Nicked templates were captured by surface-immobilized primers. Reprinted with permission from ref 526. Copyright 2013 National Academy of Sciences.

reported.87,549−553 Shapiro et al. reported one-pot production of chemically modified RNA nanoparticles functionalized with siRNAs by T7 RNA polymerase-mediated amplification (Figure 23D).87 A mixture of DNA templates was employed to encode RNA strands that formed RNA nanoparticles directly in the transcription reaction without purification and post-transcriptional assembly. The 2′-modified NTPs were incorporated in the transcripts, resulting in increased resistance of the RNA nanoparticles to RNases in human blood serum. After functionalization with siRNAs at either the 5′ or 3′ end, these RNA nanoparticles induced significant gene silencing in transfected cells, whereas no silencing effect was observed for the transcription mixture lacking siRNAs. HCR-based targeted drug carriers termed aptamer-tethered DNA nanotrains (aptNTrs) were recently constructed by Tan and co-workers.554 As shown in Figure 23E, a chimeric aptamer-tethered trigger probe was designed to initiate HCR amplification, generating self-assembled long aptNTrs with tandem “boxcars”. Anthracycline anticancer drugs such as Dox preferentially bind the loading sites in the boxcars, thus quenching the drug fluorescence. Upon selective binding to target cells, these aptNTrs are transported into cells via endocytosis, followed by drug unloading and recovery of the fluorescent signal. Using a mouse xenograft tumor model, Tan and co-workers demonstrated that HCR-based aptNTrs are a promising targeted drug transport platform for cancer theranostics with the merits of easy design and high payload capacity. Hsing and Xuan reported self-sustained assembly of quenched dsDNA substrates into fluorescent dendritic nanostructures by a nonlinear HCR system (Figure 23F).147 HCR and CHA have also been

three dye molecules in the RCA amplicon produced aptamerconjugated FRET nanoflowers for traceable targeted drug delivery and multiplexed cellular imaging by single-wavelength excitation.69 A polyvalent aptamer system for targeted drug delivery based on RCA has also been proposed.539 Via self-assembly of nucleic acid hexagon or tetragon subunits, the presynthesized RCA long strand was employed to form single-DNA nanotubes by Willner and co-workers.540 Sleiman and co-workers subsequently constructed RCAtemplated DNA nanotubes using DNA triangular rungs as building blocks in a series of studies.541−545 These DNA nanotubes were more resistant to nuclease degradation with increased cell uptake and were functionalized with other block particles, suggesting their potential for applications in bioimaging and drug delivery. We have created RCA-based DNA origamis including DNA nanowires, nanoplates, and nanoribbons using a simpler assembly process and fewer staple strands. And we have also decorated the nanoribbon with CpG oligonucleotides for a high cellular immunological response.70,546,547 Weizmann et al. recently reported the onepot enzymatic synthesis of DNA nanoribbons by RCA reactions using two circular templates (scaffold template and staple template) and demonstrated the intracellular application of the nanoribbons as a pH sensor and an efficient siRNA nanocarrier (Figure 23C).548 Most of these studies have been summarized previously in another review.66 Transcription-based amplification methods and enzyme-free methods have also been used to construct nucleic acid nanostructures. The assembly of RNA nanostructures using amplified RNA products by transcription has been widely 12518


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Figure 23. Construction of nucleic acid nanostructures. (A) Schematic showing the process of RCT for the self-assembled RNAi-microsponge. Reprinted with permission from ref 80. Copyright 2012 Nature Publishing Group. (B) Schematic illustration of noncanonical self-assembly of multifunctional DNA NFs based on RCA. Reprinted from ref 68. Copyright 2013 American Chemical Society. (C) Schematic representation of the enzymatic synthesis of DNR structures. (a) DNR-T structures assembled from RCA scaffold strand and three 32-nt staple strands. (b) DNR-S structures assembled from RCA scaffold strand and one synthetic 96-nt staple strand. (c) Generation of DNR nanostructure by enzymatic reactions in one pot. Reprinted from ref 548. Copyright 2015 American Chemical Society. (D) Schematic representation of cotranscriptional assembly leading to the formation of RNA NP (nanoring) functionalized with six siRNAs and their further purification. Functional siRNAs can be released by Dicer nuclease. Reprinted from ref 87. Copyright 2012 American Chemical Society. (E) Schematics of the self-assembly of aptamer-tethered DNA nanotrains (aptNTrs) for transport of molecular drugs. AFM images (1−3) showed the morphologies of the corresponding nanostructures [1, M1 + M2; 2, sgc8-NTrs; 3, sgc8-NTrs loaded with molecular drugs (Dox)]. Reprinted with permission from ref 554. Copyright 2013 National Academy of Sciences. (F) Components and reaction pathway for the triggered chain-branching growth of fluorescent DNA dendrimers. The lengths of different domains are indicated. DNA sequences drawn in the same color are either identical or complementary. Reprinted with permission from ref 147. Copyright 2014 American Chemical Society.

reported.565 Two complementary circular DNA templates are prepared to produce amplified transcripts that then hybridize to each other to form the initial membrane with a thin, ringlike structure (Figure 24C). After the complementary RCT process, the water in the solution evaporates, resulting in self-assembly of the RNA membrane on the tube wall (Figure 24C). By adjusting the RNA base pairing, the structural and functional properties of this RNA membrane can be controlled without using any polymer support or complexation. This tunable RNA membrane enables the controlled release of drugs and siRNA by enzymatic activity, suggesting potential therapeutic applications. Inspired by the recent work of PCR-based DNA hydrogels,556 we speculate that HDA and RPA can also be employed for hydrogel construction.

used for the self-assembly of other DNA nanostructures such as binary molecular trees.160,555 Despite requiring the denaturation of dsDNA at high temperature, PCR-based construction of DNA nanostructures has been reported.556−558 Encouraged by these efforts, we believe that HDA and RPA can also be extended to the field of nanotechnology because their amplification scheme is similar to that of PCR. 4.2. Construction of Nucleic Acid Hydrogels

Several isothermal amplification methods have been used to build nucleic acid hydrogels, which have attracted great attention in the development of biosensors.559−564 Luo and co-workers first reported the formation of fascinating DNA hydrogels based on RCA and multiprimed chain amplification (MCA, similar to HRCA) (Figure 24A).72 These two processes together result in a physically linked DNA hydrogel with mechanical meta-properties by creating extremely long DNA molecules, whereas the RCA process produces only a viscous solution. The resulting hydrogel presents liquidlike properties out of water and solidlike properties in water and conforms to the shape of containers in water. This work provided a new strategy for DNA hydrogel formation using polymerasemediated amplification. Yin et al. subsequently constructed shape-controlled hydrogels via directed self-assembly of RCA products.73 Primer-modified PEG hydrogels were prepared to generate and attach “giant” DNA glues to the surface of hydrogel cubes, inducing the self-assembly of cube dimers (Figure 24B). This method permits the construction of highly programmable and controllable three-dimensional structures for diverse applications in materials science. The self-assembly of RNA membranes based on complementary RCT has been

4.3. Formation of Nucleic Acid-Templated Nanomaterials

The amplified products of isothermal techniques are also ideal scaffolds or templates for the assembly of proteins or inorganic nanospecies. For instance, the assembly of AuNPs on RCA long strand products into one-dimensional (1D)74,566 and threedimensional (3D) arrays75 has been reported. Willner and coworkers constructed periodic DNA−protein nanostructures by assembling proteins on RCA products.71,76 These studies have been reviewed elsewhere.66 Cleaved RCA products with only one copy sequence can mediate the aggregation of DNA-AuNP probes into a crosslinked network, providing a colorimetric change in the presence of a mutation (Figure 5A).217 Similarly, the assembly of DNAfunctionalized nanoparticles by amplified products of EXPAR and HDA has also been reported (Figure 5E).221,350,567,568 Nuclease-assisted signal amplification methods have been 12519


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Figure 24. Construction of hydrogels. (A) Schematic diagram of the stepwise approach for DNA hydrogel synthesis based on RCA and MCA processes. Reprinted with permission from ref 72. Copyright 2012 Nature Publishing Group. (B) Fabrication of hydrogel cubes with uniform giant DNA glue modification. (a) Schematic of the fabrication process of hydrogel cubes uniformly modified with giant DNA. Phase contrast (b, e), fluorescent (c, f), and SEM (d, g) images of hydrogels carrying short 56-nt single-stranded DNA primers (b−d) or amplified single-stranded giant DNA (e−g). The gels in c and f were stained with SYBR Gold before imaging. Scale bar, 200 μm in b, c, e, and f; scale bar, 10 μm in d and g. Reprinted with permission from ref 73. Copyright 2013 Nature Publishing Group. (C) Fabrication of a self-assembled free-standing RNA membrane. (Top) Hybridization of RNA transcripts by the cRCT process. (Middle) Schematic drawing of the fabrication processes of the self-assembled RNA membrane. (Bottom) Detailed description of the EISA by hybridization of the RNA strands of initial membrane and free RNA transcripts, resulting in self-assembly of the RNA membrane on the tube wall during the evaporation. Reprinted with permission from ref 565. Copyright 2014 Nature Publishing Group.

co-workers demonstrated the generation of electrically conductive metal nanowires by metalizing stretched RCA products between two electrodes.77 Aligned AuNPs along the stretched RCA products were employed to facilitate the metallization. The metal nanowires formed by the silver and gold solutions resulted in a remarkable drop in resistance (from TΩ to kΩ for silver and from TΩ to Ω for gold), allowing the electrical detection of 10 ng of genomic DNA from E. coli.

employed to circularly digest excess DNA linkers designed to bridge DNA-AuNP probes, preventing linker-mediated nanoparticle assembly.91,95,114 Overall, the assembly or aggregation of nanoparticles with amplified products provides interesting biophysical and optical phenomena for the development of biosensors. Isothermal amplified products have also been applied as templates for the synthesis or growth of metal nanoparticles. For example, we used RCA products to capture Cy3functionalized AuNPs to promote the reduction of Ag+ as a SERS “hot spot”, demonstrating ultrasensitive protein microarray analysis by silver enhancement.569−571 HCR-mediated silver nanoparticle growth via chemical reduction was subsequently reported for the label-free SERS detection of DNA.572 Utilizing EXPAR or RCA, fluorescent DNA-scaffolded silver nanoclusters have also been synthesized, permitting nucleic acid analysis with high sensitivity.43,573 The formation of another type of fluorescent metal nanoparticle, DNAtemplated copper nanoparticles (CuNPs), based on RCA was reported by Wang et al.574 DNA hybridization enabled the conversion of the periodic RCA products to concatemeric dsDNA scaffolds, which then templated the synthesis of CuNPs using ascorbate as the reducing agent. Compared to monomeric dsDNA-templated CuNPs, the concatemeric dsDNA-templated CuNPs exhibited enhanced fluorescence stability (∼2 times) and improved sensitivity (∼104-fold). Moreover, Nilsson and

5. DEVICE INTEGRATION Isothermal nucleic acid amplification techniques are often integrated with miniaturized and/or portable devices including microfluidic chips, capillary platforms, and test strips. Commercial devices and diagnostic kits are available for POC analysis. In this section, we will discuss successes in integrating various components with isothermal amplification. 5.1. Microfluidic Systems

Since its inception, microfluidic technology has drawn great attention because of its advantages of reduced sample/reagent consumption, integrated functions, high portability, and short assay time.402,575−578 Compared to traditional detection assays, miniaturization minimizes the risk of sample contamination and enhances sensing performance.579−582 Without requiring thermal cycling, isothermal microsystems can be designed to be simple and portable with low energy consumption for 12520


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Figure 25. Integrated microfluidic systems. (A) Overview of the MEQ-LAMP. Reprinted with permission from ref 598. Copyright 2012 John Wiley & Sons, Inc. (B) Working mechanism for RCA-based digital quantification system. Reprinted with permission from ref 611. Copyright 2006 Nature Publishing Group. (C) Digital isothermal quantification of nucleic acids via simultaneous chemical initiation of RPA reactions on SlipChip. Reprinted from ref 36. Copyright 2011 American Chemical Society. (D) (Top) Schematic representation of the aptamer and NEase-assisted signal amplification assay for membrane protein on single living cells and (bottom) basic principle of microfluidic droplet system. Reprinted from ref 634. Copyright 2014 American Chemical Society.

shown in Figure 25A, this chip contains a single microfluidic chamber for performing the LAMP reaction and electrochemical measurement. The platinum counter and reference electrodes and a gold working electrode are contained in the MEQ-LAMP system, and the dsDNA-binding dye methylene blue is used to monitor the LAMP reaction in real time. The binding of methylene blue by the LAMP amplicons decreases the number of the free methylene blue molecules, resulting in decreased redox current. Using this MEQ-LAMP platform, Soh et al. demonstrated rapid and quantitative detection of as few as 16 copies of genomic DNA from Salmonella enterica enterica Typhimurium. Recently, pH-sensing microfabricated systems have been created by integrating ion-sensitive field effect transistor sensors.601,602 Instead of indirect measurements such as fluorescent and electrochemical signals, these pH-sensing systems directly measure hydrogen ions released during nucleotide incorporation for real-time monitoring of the LAMP reaction, allowing simple and label-free analysis of SNPs and bacteria. Furthermore, digital microfluidic devices have been introduced to perform digital LAMP (dLAMP) for absolute quantification without requiring a standard curve or external calibrator, which are required in real-time quantitative LAMP (qLAMP).603−608 Significantly, Nixon et al. observed that dLAMP was more resistant to inhibitors than qLAMP.604 Sun et al. also demonstrated significantly increased robustness in digital measurements versus the real-time kinetic format606 and reported the mechanistic evaluation of the pros and cons of digital RT-LAMP for quantification of HIV-1 viral load.605

process automation and integration in a single device, superior to PCR. SDA-employing microfluidic amplification systems were reported in the late 1990s. Burns et al. first developed an integrated analysis device with fluorescence detectors for the amplified detection of nanoliter DNA samples using SDA.583 In another design, SDA primers were anchored on a microelectronic chip array for the amplification and detection of multiple targets in an open format.584 Other isothermal amplification strategies have been successively integrated into microfluidic chips. In particular, LAMP has been widely applied. For instance, the integration of LAMP and electrophoresis separation in microfluidic chips for nucleic acid assays has been reported.585−588 Optical end-point analysis was subsequently employed for simple detection with improved sensitivity.589−591 The introduction of paper into the microfluidic device for LAMP reactions permitted stable test results for longer than 2 months.589 By integrating miniaturized optical detectors into the chips592−595 or using potable optical readers,596,597 real-time monitoring of LAMP reactions has been demonstrated for the POC detection of pathogens. In particular, the simultaneous analysis of multiple viruses in one system593 and the integration of a flow-through membrane for nucleic acid preparation (isolation, concentration, and purification)597 were achieved. Electrochemical microfluidic platforms have also been fabricated for real-time detection based on LAMP.598−600 Importantly, Soh et al. developed the microfluidic electrochemical quantitative LAMP (MEQ-LAMP) system for single-step, rapid testing of pathogenic DNAs.598 As 12521


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microbes in the human subgingival crevice using MDA in microfluidic devices.636 The same research group has also reported digital MDA for the enumeration of total nucleic acid contamination.637 Using the marine cyanobacterium Prochlorococcus as a model system, Chisholm et al. demonstrated increased single-cell sequencing efficiency and genome assembly with optimal genome recovery based on microfluidic MDA.638 Zhang et al. recently developed a microwell displacement amplification system (MIDAS) for massively parallel polymerase cloning and genome sequencing of single cells.639 In MIDAS, each reactor spatially confines an MDA reaction within a 12 nL volume, thus reducing amplification bias and facilitating de novo assembly of near-complete microbial genomes. Zhang et al. also demonstrated the detection of single-copy number changes in primary human adult neurons at 1- to 2-Mb resolution.

RCA-based digital microfluidic chips have also been developed.609,610 Nilsson and co-workers first reported the digital quantification of RCA amplicons for the detection of nucleic acids or proteins at the single-molecule level.611 In this system (Figure 25B), target-induced RCA amplicons are hybridized with fluorescent molecule-tagged probes, which are then pumped into the thermoplastic microchannel for analysis by a standard confocal fluorescence microscope. Multiplex detection is achieved via the hybridization of probes of distinct colors, and highly precise quantification with a dynamic range of 7 orders of magnitude can be achieved. RCAassisted measurements of endogenous enzyme activity can enable the quantification of even a single malaria-causing Plasmodium parasite in unprocessed blood and saliva on a droplet microfluidic platform.612 Other RCA-employed microfluidic systems have been established, primarily for single-cell or single-molecule analysis.416,613−621 Other isothermal amplification strategies, including HDA,622 RPA,36,623−626 and NABSA,627−633 also play important roles in integrated microfluidic platforms for the detection of pathogenic DNA or RNA. Notably, Ismagilov and co-workers reported digital RPA reactions on a SlipChip by adding a chemical initiator to each reaction compartment (Figure 25C).36 With a simple slipping step after pipet loading, more than 1000 nanoliter-scale RPA reactions were simultaneously initiated. This device was used to amplify and count a single target molecule in the genomic DNA of methicillin-resistant Staphylococcus aureus. Reinholt et al. developed a surfacemodified microchannel with thymidine oligonucleotide probes for the efficient isolation of mRNA, which was then amplified by NASBA, demonstrating the successful detection of mRNA from as few as 30 C. parvum oocysts.631 By integrating a membrane-based sampling module, a sample preparation cassette, and a 24-channel qNASBA chip, a multifunctional microfluidic platform was also proposed for the extraction and amplification of DNA/RNA molecules from aquatic microorganisms, providing compatible results with microplate readers common in laboratories.632 Recently, based on NEase-assisted signal amplification, Tang et al. reported the highly sensitive and homogeneous detection of membrane proteins on single live cells in microfluidic droplets.634 As shown in Figure 25D, samples containing HeLa cells and DNA probes were introduced into the microfluidic device along with enzymes and reaction buffer. Upon mixing with the oil stream, monodisperse droplets of approximately 300 pL were formed that were suitable for the encapsulation of single cells. Then, NEase-assisted signal amplification occurred in each droplet via membrane protein-triggered conformation alteration (Figure 25D). Using tyrosine kinase-7 as a model analyte, they demonstrated single-cell analysis based on the high-throughput microfluidic platform with the elimination of several washing and separation steps. MDA-integrating microfluidic devices have also been developed for single-cell analysis. Marcy et al. first reported nanoliter reactors to improve the MDA of genomic DNA from single cells.635 They used a microfluidic device to isolate individual Escherichia coli cells and perform MDA-mediated genome amplification in 60 nL reactions. The smaller reaction volume (nanoliter scale) decreased nonspecific synthesis from contaminant DNA templates and primer dimers and greatly reduced amplification bias, enabling high-throughput pyrosequencing of individual E. coli cells. Simultaneously, Marcy et al. reported another study on single-cell genetic analysis of

5.2. Capillary Platforms

In contrast to microfluidic chips, which require relatively complex and costly microfabrication, capillary tubes can be prepared in a convenient and inexpensive manner and have been employed to perform isothermal amplification reactions in small volumes. Liu et al. first proposed an integrated capillaryarray microsystem for the extraction, amplification, and detection of DNA from Mycobacterium tuberculosis.640 As illustrated in Figure 26A, different samples or reagents were sequentially injected into parallel polytetrafluoroethylene capillaries as droplets by a multichannel syringe pump. The whole analytical process was implemented by passing the sample solution through capillaries. Using this automatic microsystem, they achieved the parallel detection of 10 samples with a low detection limit of 10 bacteria, and they successfully

Figure 26. Integrated capillary platforms. (A) Schematic of the processes of the μLAMP assay for M. tuberculosis detection. Reprinted with permission from ref 640. Copyright 2013 American Chemical Society. (B) Schematics of cLAMP device. Two types of sample introduction (left and middle): (left) capillary force and positive pressure-driven introduction and (middle) introduction by displacement. (right) Schematic representation of a typical result of cLAMP. Reprinted from ref 641. Copyright 2014 American Chemical Society. (C) (left) Integrated capillary LAMP (icLAMP) with preloaded reagents. A membrane used to extract DNA from blood samples was inserted into the microcapillary before fluidic operations. (middle) 200 nL of Blood sample was introduced into the microcapillary and contacted with the FTA card with the aid of a pipet tip and a piston. DNA from blood sample was extracted and trapped onto the FTA card. (right) A visual readout of the fluorescent signal after the LAMP reaction with a hand-held flashlight. Reprinted from ref 642. Copyright 2014 American Chemical Society. 12522


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Figure 27. Paper-based platforms. (A) Schematic of DNA detection on a paper strip via DNA ligation, RCA, and DNA hybridization. DNA3 is the intended DNA target. MG: microgel. Reprinted with permission from ref 651. Copyright 2009 Royal Society of Chemistry. (B) Lateral flow detection of RPA product. Reprinted from ref 655. Copyright 2014 American Chemical Society. (C) Schematic illustration of paper-based platforms for Pb2+ detection using CHA. SA, streptavidin; TZ, the test zone; CZ, the control zone. Reprinted with permission from ref 478. Copyright 2013 Royal Society of Chemistry. (D) Schematic illustration of paper-based platforms for Hg2+ detection using Exo III-assisted signal amplification. SA, streptavidin; TZ, the test zone; CZ, the control zone. Reprinted from ref 490. Copyright 2014 American Chemical Society.

system enable the collection, amplification, and detection of nucleic acids from bacteria, with important implications for POC nucleic acid detection. In addition, a simple DNA extraction device has been designed for on-field detection of genetically modified organisms by employing a visual LAMP assay primarily consisting of a silica gel membrane filtration column and a modified syringe.644 Importantly, other isothermal amplification methods can also be introduced in capillary-based detection platforms for nucleic acids and other analytes.

analyzed clinical samples with 96.8% clinical sensitivity (positive detection rate) and 100% specificity (negative detection rate). Other capillary-based LAMP microsystems have been reported for the simple detection of nucleic acids.641−643 Significantly, Zhang et al. demonstrated the straightforward, robust, and multiplexed detection of nucleic acids by a microcapillary LAMP (cLAMP) system.641 Samples or reagents and segments of water droplets were introduced and housed in glass or plastic capillaries, which isolated each amplification reaction to prevent contamination (Figure 26B). The ends of the capillaries were sealed by a set of segments of pure water (liquid), air (gas), and epoxy glue (solid). The LAMP reaction was powered by a pocket warmer, and the fluorescent signal was visualized by a hand-held flashlight. This cLAMP system was used to simultaneously detect two HIV RNA targets in plasma samples. Without relying on an external power supply or bulky equipment, this cLAMP system holds great promise for on-site applications, particularly in resource-poor settings. An integrated cLAMP (icLAMP) system was subsequently developed that contains a DNA extraction card and preloaded reagents for sample-to-answer screening of SNP typing.642 A Flinders Technology Associates (FTA) membrane was inserted into the microcapillary to extract DNA from blood samples before the introduction of preloaded reagents (Figure 26C). A positive-displacement pipet tip containing a piston was connected to this integrated microcapillary to facilitate the handling of blood samples and preloaded reagents. After DNA extraction and purification, the FTA card was immersed in a LAMP mixture, and the microcapillary ends were sealed. The capillary was placed in an oven to initiate the LAMP reaction, and the visible amplified signal was detected by a hand-held UV-flashlight. Utilizing this icLAMP system, the detection of the CYP2C19 gene from an untreated, freshly drawn blood sample was achieved. Minor modifications of the icLAMP

5.3. Paper-Based Platforms

Due to its attractive features of portability, abundance, simple fabrication, and low cost, test paper has drawn increasing interest as an ideal supporting material for developing sensing devices, particularly in the field of POC diagnostic applications.645−649 Ali et al. reported RCA reactions on paper strips for sensitive DNA detection based on their previous work.650,651 As illustrated in Figure 27A,651 poly(Nisopropylacrylamide) microgels (MGs) coupled with DNA oligonucleotides (DNA1) were spotted onto a paper strip to allow sequential target hybridization, DNA ligation, RCA process, and probe capture. The target-triggered amplified products at the spotted bioactive material zone were visualized via fluorescence imaging. Ali et al. also proposed an effective patterned paper aptasensor by printing functional RCA amplicons on filter paper using a simple inkjet printing technique.652 Other isothermal nucleic acid amplification techniques including SDA,653 RPA,654,655 and NASBA656 were subsequently introduced to paper-based assay platforms. Notably, based on SDA or cascade SDA with AuNPs as color indicators, Zeng et al. proposed a series of lateral-flow biosensors for the amplified detection of nucleic acids, bacteria, and human pluripotent stem cells.420,421,657−659 Crannell et al. proposed a 12523


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simple lateral flow strip to detect RPA amplicons from Cryptosporidium DNA extracted from the stool samples of animals and patients.655 As shown in Figure 27B, dual-labeled (biotin and FITC at each end) RPA amplicons were first captured at a detection region by streptavidin, followed by binding of anti-FITC conjugated gold nanoparticles. A positive control line was also designed to capture any anti-FITC gold nanoparticles that were not captured at the detection line. This assay achieved visual detection of as few as 100 Cryptosporidium oocysts/mL in stool samples with excellent specificity and exhibited sensing performance comparable to the gold standard method, PCR. In another study, Rohrman et al. investigated the inhibition of RPA by background DNA and developed a novel lateral flow-based method to enrich the target DNA for HIV detection.654 Signal amplification strategies have also been employed for the construction of paper strip biosensors. Zeng et al. utilized a paper strip for CHA-based detection of Pb2+, with a detection limit of 10 pM in the absence of instrumentation (Figure 27C).478 A disposable strip biosensor for the ultrasensitive detection of Hg2+ has also been constructed based on Exo IIIassisted signal amplification.480 In the presence of Hg2+, a THg2+-T base-pairing interaction (Hairpin DNA and Assistant DNA) initiated a toehold-mediated strand displacement reaction, forming a duplex with blunt 3′ termini (Figure 27D). As Exo III catalyzed the cleavage reaction, the Assistant DNA was released to initiate the next cycle, generating large amounts of ssDNA products. Via sandwich hybridization, these ssDNA products and AuNPs-DNA probe 1 (as tracers) were captured at the test zone (CZ), and visual detection of as little as 1 pM Hg2+ was achieved with remarkable specificity. This paper-based biosensor is also capable of monitoring spiked Hg2+ in environmental water samples with good recovery and accuracy.

progress. In general, these devices were developed based on a specific amplification method, but most (except turbidimeters) are suitable for other amplification strategies.

6. CONCLUSION AND PERSPECTIVES Over the past 20 years, various isothermal nucleic acid amplification techniques and signal amplification strategies have been successively developed for the simple and rapid detection of nucleic acids with remarkably high sensitivity. Benefiting from rapid advances in biotechnology, chemistry, and nanotechnology, these isothermal methods have been expanded to detect targets ranging from DNA and RNA to cells, proteins, small molecules, and even ions. Applications of these methods in sequencing and in situ or intracellular bioimaging have also been amply demonstrated. The introduction of nanomaterials with unique electronic and optical properties has significantly improved assay performance, including robustness and specificity. Amplicons produced using isothermal amplification methods have recently been utilized for the construction of versatile nucleic acid nanomaterials, demonstrating their promising applications in biomedicine, bioimaging, and biosensing. Moreover, the integration of isothermal amplification into microsystems or portable devices has facilitated the development of nucleic acid-based POC diagnosis with high assay sensitivity. With reduced system complexity, diagnostic devices and kits have become commercially available. Single-cell and single-molecule analyses have also been implemented based on integrated microfluidic systems. Table 2 summarizes the main applications of isothermal nucleic acid amplification. Table 2. Main Applications of Isothermal Nucleic Acid Amplification application

5.4. Commercial Devices

biosensing

Several types of devices are commercially available. Since 1999, the BD ProbeTec ET System has been supplied by BD Diagnostic Systems (Sparks, USA), offering high-throughput real-time SDA assays. Although not portable, this semiautomated platform is an easy-to-use tool in pathogen diagnosis.660,661 On the basis of NASBA or TMA, three nucleic acid testing systems, SAMBA System (Diagnostics for the Real World), NucliSENS EasyQ System (BioMerieux), and Tigris DTS System (HOLOGIC Gen-Probe), are commercially available. Notably, the Tigris DTS system is the first to automate all testing phases from sample preparation, amplification, and detection to the reporting of results. Coris BioConcept (Gemblux, Belgium) have marketed several lateral flow devices for the diagnosis of pathogens.662 To monitor the LAMP reaction in real time, two turbidimeters, LA-320C and LA-500, are provided by EIKEN CHEMICAL (Tokyo, Japan). However, these devices all require external power, limiting their on-site applications. One portable turbidimeter, the illumipro10 Incubator Reader (Meridian Bioscience, Nice, France), has been commercialized recently. Other available optical portable instruments can be divided into two types: real-time fluorometers and bioluminescence-based instruments. Realtime fluorometers include the Genie series (OptiGene, Horsham, U.K.) and Twista (TwistDX, Cambridge, U.K.), and bioluminescence-based instruments are developed by Lumora (Cambridgeshire, U.K.). The development of fully integrated (termed Next Generation) and digital devices is in

bioimaging diagnostics sequencing nanotechnology miniaturized and POC systems single-cell and single-molecule analysis

description amplified detection of nucleic acids, proteins, cells, small molecules, ions, etc. in situ or intracellular imaging of nuclear DNA, mitochondrial DNA, mRNA, miRNA, protein, etc. clinical testing of pathogenic DNA, SNPs, DNA methylation, antigens, etc. preparing sequencing libraries for DNA and RNA sequencing constructing nucleic acid nanomaterials for drug delivery, etc. integration with microfluidic chips, capillary platforms, or test strips as microsystems amplifying whole genome or transcriptome from single cells; detecting protein, protein modification, DNA, mRNA, or miRNA from single cells or at the singlemolecule level

Despite this progress, several challenges remain to be addressed. The first challenge is the amplification bias and nonspecific amplification inherent in exponential strategies. Some proteins, nanomaterials, or reagents can improve amplification accuracy and/or reduce sequence-dependent bias.363,663−667 Reducing reaction volume to the nanoliter scale using microsystems inhibits nonspecific DNA synthesis and improves amplification uniformity in MDA by increasing the effective concentration of the template genome.635,639 Recently, a new amplification method, multiple annealing and looping-based amplification cycles (MALBAC), that amplifies genomic DNA from single human cells in microliter reactions with less bias than previous methods was reported.668 The 12524


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reduced bias in MALBAC results from a five-cycle quasilinear preamplification with more evenly distributed initial priming, which accumulates hairpin amplicons for subsequent PCR amplification.669 Despite these efforts, amplification bias remains a major challenge in single-cell nucleic acid analysis.638,668 Encouraged by these improvements, we expect that the integration of various techniques including nanomaterials, droplet microfluidic chips, and nonexponential preamplification into one multifunctional system will minimize amplification bias. In addition, the development of novel DNA polymerases by enzyme engineering may also contribute to the inhibition and even elimination of amplification bias.670,671 Another challenge is the application of these methods to intracellular and in vivo analysis. The enzyme-free amplification techniques HCR and CHA have very recently been successfully introduced to provide high signal gain for the imaging of intracellular mRNA or miRNA.513,515,516 However, intracellular amplified analysis of non-nucleic acid biomolecules (e.g., proteins), protein−protein or nucleic acid−protein interactions and in vivo analysis have not been reported. To convert the detection of non-nucleic acid targets into nucleic acid amplification events, additional recognition components (e.g., Abs) and amplified components must be delivered simultaneously into live cells or whole organisms. Moreover, the implementation of enzyme-mediated amplification strategies in live cells or organisms remains challenging despite their high amplification efficiency and excellent versatility. A promising route is the utilization of intracellular enzymes such as polymerases to perform amplification reactions.672−676 Transporting the required enzymes into cells may be another possibility.677−679 Furthermore, there are several challenges to the integration of portable devices for POC diagnosis despite the emergence of commercial products. For instance, neither paper-based platforms nor microfluidic chips are reagent-free assays. The addition of reagents is required before paper testing, and reagents are usually preloaded in microfluidic chips. The development of sample-to-answer devices relies on further progress in diverse fields in engineering and the physical and biological sciences. Another problem is the nonspecific adsorption and limited reaction efficiency on the surface of chips and other solid supports. We recently developed a generic sensing platform based on 3D-nanostructured DNA probes.680−687 Because of their mechanical rigidity and structural stability, these 3D-nanostructured probes can be readily assembled on gold surfaces in an upright and ordered orientation with well-controlled probe spacing, thus ensuring high hybridization efficiency and resistance to nonspecific adsorption. The introduction of ordered DNA nanostructured probes into integrated microsystems may be a promising solution for these problems. Finally, we hope this review will offer readers an overview of isothermal amplification and stimulate interest, new ideas, and discoveries in this fascinating field.

Biographies

Yongxi Zhao earned his M.S. and his Ph.D. from Xi’an Jiaotong University in 2005 and 2009, respectively. During his doctoral period, he moved to University of Washington, Seattle, under the joint educational project. Since 2009, he joined the faculty at Xi’an Jiaotong University and completed his postdoctoral research at Shanghai Institute of Applied Physics, Chinese Academy of Science from 2012 to 2014. He is now a professor of School of Life Science and Technology, Xi’an Jiaotong University. His current research are focused on biosensors, bioimaging, and nanobiotechnology,

Feng Chen received his B.S. in Biomedical Engineering from Xi’an Jiaotong University in 2011 and is currently pursuing his Ph.D. at Xi’an Jiaotong University. His research interests include developing novel optical biosensors for disease biomarkers and constructing nanocarriers for biomedical applications.

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected].

Qian Li received her B.E. in Fine Chemical Technology at Dalian University of Technology in 2003. She obtained her M.S. at Utrecht University in 2006 and Ph.D. at University of Groningen in 2011. She

Notes

The authors declare no competing financial interest. 12525


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Foundation of Shaanxi Province (Grant 2013JQ2017), the Chinese Academy of Sciences and the Fundamental Research Funds for the Central Universities.

carried out her postdoctoral research at Shanghai Institute of Applied Physics (SINAP), Chinese Academy of Sciences, and joined the faculty of Division of Physical Biology at SINAP and the Center of Bioimaging at the Shanghai Synchrotron Radiation Facility (SSRF) in 2014 as an assistant professor. Her current research interests are focused on functional DNA nanostructures.

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Lihua Wang received her Ph.D. in 2003 from Shanghai Institute of Applied Physics (SINAP), Chinese Academy of Sciences (CAS). She is now a full professor of the Division of Physical Biology at SINAP and the Center of Bioimaging at the Shanghai Synchrotron Radiation Facility (SSRF). Her research interests are focused on developing novel biosensors based on nanomaterials, as well as bioimaging based on super-resolution microscopy and synchrotron radiation.

Chunhai Fan obtained his B.S. and Ph.D. from the Department of Biochemistry at Nanjing University in 1996 and 2000, respectively. After his postdoctoral research at University of California, Santa Barbara (UCSB), he joined the faculty at Shanghai Institute of Applied Physics (SINAP), Chinese Academy of Sciences (CAS) in 2004. He is now Professor and Chief of the Division of Physical Biology at SINAP and the Center of Bioimaging at the Shanghai Synchrotron Radiation Facility (SSRF). He is also an Adjunct Professor at ShanghaiTech University. He is an elected fellow of the Royal Society of Chemistry (FRS) and the International Society of Electrochemistry (ISE). His research interests are biosensors, bioimaging, and DNA nanotechnology. He was recently recognized as one of the High Cited Researchers in 2014 and 2015 by Thomson Reuters.

ACKNOWLEDGMENTS Part of the research work described in this review was supported by the National Natural Science Foundation of China (Grants 21475102 and 21390414), the Ministry of Science and Technology (Grants 2012CB932600, 2013CB933802, and 2013CB932803), the Natural Science 12526


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Chemical Reviews

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