Chemical and Biological Sensing using Hybridization Chain Reaction

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Review

Chemical and Biological Sensing using Hybridization Chain Reaction Erik E. Augspurger, Muhit Rana, and Mehmet Veysel Yigit ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00208 • Publication Date (Web): 07 May 2018 Downloaded from http://pubs.acs.org on May 7, 2018

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Chemical and Biological Sensing using Hybridization Chain Reaction Erik E. Augspurger†, Muhit Rana†, and Mehmet V. Yigit †, ‡, *

† Department of Chemistry, University at Albany, State University of New York, 1400 Washington Avenue, Albany, New York 12222, United States.

‡ The RNA Institute, University at Albany, State University of New York, 1400 Washington Avenue, Albany, New York 12222, United States.

*Correspondence: Tel: (1) 518-442-3002 [email protected]

Keywords: programmable, hybridization chain reaction, sensor, detection, nanoparticle, DNA, RNA, protein, metal ions, biomarker, metabolite

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Page

Table of Contents

Abstract

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Introduction

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Chemical and Biological Sensing Using HCR

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Nucleic Acid Detection

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Fluorescence-based Detection

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Labeled Approaches

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Label-free Approaches

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Electrochemical-based Detection

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Nanoparticle-based Detection

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Colorimetric Nanoparticle Approach

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Fluorometric Nanoparticle Approach

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Electrochemical Nanoparticle Approach

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SERS Nanoparticle Approach

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Metabolite Detection

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ATP Detection

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Glutathione Detection

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Ochratoxin A Detection

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Protein Detection

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Metal Ion Detection

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Conclusion and Future Perspectives

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Vocabulary

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References

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Figures

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Tables

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Table of Contents Figure

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Abstract. Since the advent of its theoretical discovery more than 30 years ago, DNA nanotechnology has been used in a plethora of diverse applications in both the fundamental and applied sciences. DNA-based technologies’ recent prominence in the scientific community is largely due to the programmable features stored in its nucleobase composition and sequence, which allow it to assemble into highly advanced structures. DNA nanoassemblies are also highly controllable due to the precision of natural and artificial base-pairing, which can be manipulated by pH, temperature, metal ions and solvent types. This programmability and molecular-level control have allowed scientists to create and utilize DNA nanostructures in one-, two- and threedimensions (1D, 2D and 3D). Initially, these 2D and 3D DNA lattices and shapes attracted a broad scientific audience because they are fundamentally captivating and structurally elegant, however, transforming these conceptual architectural blueprints into functional materials is essential for further advancements in the DNA nanotechnology field. Herein, the chemical and biological sensing applications of a 1D DNA self-assembly process known as known as hybridization chain reaction (HCR) are reviewed. HCR is a one-dimensional (1D) double stranded (ds) DNA assembly process initiated only in the presence of a specific short ssDNA (initiator) and two kinetically trapped DNA hairpin structures. HCR is considered an enzymefree isothermal amplification process, which shows substantial promise and offers a wide range of applications for in situ chemical and biological sensing. Due to its modular nature, HCR can be programmed to activate only in the presence of highly specific biological and/or chemical stimuli. HCR can also be combined with different types of molecular reporters and detection approaches for various analytical readouts. While the long dsDNA HCR product may not be as structurally attractive as the 2D & 3D DNA networks, HCR is highly instrumental for applied biological, chemical and environmental sciences, and has therefore been studied to foster a variety of objectives. In this review, we have focused on nucleic acid, protein, metabolite and heavy metal ion detection using this 1D DNA nanotechnology via fluorescence, electrochemical and nanoparticle-based methodologies.

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No longer is deoxyribonucleic acid (DNA) known solely as the carrier of genetic information.1-3 The

nanotechnological

applications

of

DNA

have

grown

exponentially

since

its

conceptualization more than thirty years ago.4-9 Specifically, the folding and assembly of DNA into well-defined two- and three-dimensional (2D and 3D) structures, known as DNA origami, have gained recent prominence in the scientific community. These captivating architectures are assembled using various forms of DNA, including a long single-stranded (ss) viral DNA that folds into precise shapes by virtue of specific base-pairing with short ssDNA staple strands. This “scaffolding DNA origami” technique has enabled researchers to fold DNA into a map of the western hemisphere and a smiley face, among other iconic 2D structures.10 In addition, reactivity of the modified nucleobases or backbones has been used to anchor proteins and nanoparticles onto complex DNA networks for template-assisted assembly.11-13 Other researchers have developed self-assembly pathways14,15 to program metastable hairpins for rational 3D DNA designs, such as a tetrahedron.16,17 Furthermore, precise assembly of short DNA building blocks enabled the construction of nanoscale 2D-networks and discrete 3D DNA nanostructures.18,19 Because of the highly predictable and controllable interactions within DNA or with external factors, the scientific community is witnessing extraordinary and diverse DNA constructs through a variety of approaches. In parallel with these advancements in molecular architecture using DNA as the template or building block, notable progress has also been made in the development of functional DNA systems through aptamer and DNAzyme technologies.20-24 One prominent example in applied DNA/RNA science is a methodology known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX), whereby a short DNA/RNA strand with a specific nucleic acid sequence is selected against a specific target molecule.25-27 The resulting functional nucleic acid molecules have been utilized in a variety of diverse goals, with broad application in inhibition, biosensing and biomedicine.28-31 In particular, the number of biosensors developed in recent years using functional nucleic acid technology has grown exponentially.32 A recent study by Plaxco and coworkers demonstrated the application of aptamer-based sensors in a living animal for the detection of the antibiotic kanamycin.33 Another study by Lu, Chan and coworkers demonstrated the photoacoustic imaging of thrombin in living mice using a DNA aptamer design.34 Since

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aptamers can be extremely specific with low immunogenicity, they are being tested in clinical trials with greater frequency and attracting significant attention for drug development.35,36 The field of functional DNA will continue to be a major focus in the new scientific era due to its costefficient and ease of synthesis, its modification with a variety of chemical moieties and its thermal and chemical stability.37 Although the developments in DNA molecular architecture and functional DNA selection are significant, it is equally important for scientists to merge these fields and build complex systems with functional properties.38 To meet this demand, 2D and 3D DNA shapes have been evaluated for a number of biomedical applications.39 For example, a recent study reported a DNA nanosuitcase design that was used to encapsulate and release a siRNA payload intracellularly in response to an endogenous biomolecule.40 Other contemporary developments include a tetrahedral nanostructure of DNA cages designed to shuttle metal complexes to tumor tissues using in vivo models.41 In addition to biomedical applications, other interesting studies report the construction of soft materials with tunable electronic properties; for example, the plasmonic interactions of nanoparticles have been tuned by controlling their spacing using DNA origami.4245

While these preliminary demonstrations of utilitarian 2D and 3D DNA nanostructures show

great promise, the development of functional soft materials using 1D DNA nanotechnology specifically for their sensing applications, has been particularly noteworthy. The chemical and biological sensing approaches reviewed herein are based upon Hybridization Chain Reaction (HCR), considered to be a significant discovery in DNA nanotechnology for the development of functional DNA nanosystems. Introduced by Dirks and Pierce in 2004, HCR is a 1D DNA polymerization process initiated with a small ssDNA molecule (initiator).46 The system includes two metastable DNA hairpins (H1 & H2) that are activated and assembled to form a long double strand (ds) DNA product only in the presence of the initiator. Early studies used a ssDNA as the initiator, but subsequent studies tested other biological/chemical molecules as the initiator to program this system for a multitude of applications. From the sensor development standpoint, HCR is considered an enzyme-free, isothermal amplification process that can be triggered by highly specific biological and/or chemical stimuli. Briefly, HCR progresses as follows: two metastable kinetically trapped DNA hairpins (H1 & H2) do not hybridize to each other when they coexist in an aqueous solution; rather, they start

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binding to each other and assemble to form a large DNA concatemer upon the introduction of the initiator. This process occurs when the initiator opens and activates the first hairpin, H1, (Figure 1, Step 1) which immediately hybridizes with and opens the second hairpin, H2, (Figure 1, Step 2) forming a nicked dsDNA with a ssDNA ‘sticky end’, which induces the opening of another H1 and triggers the perpetuation of the HCR process (Figure 1, Step 3). As a result, long dsDNA polymers assemble using hairpins as monomers (Figure 1, Step 4). This process continues until the supply of hairpins is exhausted or the size of the dsDNA product reaches a threshold. Depending upon the application, each initiator strand can be programmed to trigger its own HCR with separate hairpin pairs, thereby creating a plethora of different DNA polymers or, alternatively, several initiators can be designed so that a fragment of their sequence is identical, and they activate the same hairpin pair. This programmable DNA polymerization process has received considerable attention due to its modular nature. Because a single initiator can activate numerous hairpins, the HCR technique was originally regarded as an alternative amplification procedure to current methods, such as polymerase chain reaction (PCR). While PCR requires enzymes and heating/cooling cycles for amplification, HCR is an enzyme-free technique that progresses isothermally.47,48 In addition, HCR can be used to detect endogenous biomarkers without isolating them from their original environment.49 Moreover, although HCR’s most common applications are for nucleic acid detection, HCR was originally tested for the detection and discrimination of small metabolites in combination with aptamer technology. For instance, the discrimination of ATP (adenosine-5’-triphosphate) from GTP (guanosine-5’-triphosphate) was possible by combining the HCR technique with a DNA aptamer specific to ATP, Figure 2.46 HCR is also regarded as alternative to other isothermal amplification methods such as rolling circle amplification (RCA) and catalytic hairpin assembly (CHA).50,51 RCA is an isothermal amplification process which generates long ssDNA and RNA from single nucleotides. In contrast to HCR, RCA requires enzymes and ligation process for its amplification. Furthermore, HCR utilizes short ssDNA for assembly of a dsDNA polymer whereas RCA requires nucleotide triphosphates (NTPs) for the assembly of ssDNA or RNA polymer.50 Because there are differences in the final product and precursors, each oligonucleotide-based amplification method has been practical for different goals.50 CHA, on the other hand, is a spontaneous hybridization process between two hairpins that are kinetically-trapped due to the complementary regions

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being embedded within the hairpin stems.51 With the introduction of an input strand, the CHA is initiated through a toehold-mediated strand displacement. Ultimately, the input strand is displaced in the final dsDNA assembly. In HCR, however, the reaction starts only in the presence of the input strand, which is retained in the dsDNA body. Although CHA and HCR, developed by the same research group, are similar processes and HCR is considered a precursor to CHA, we have focused solely on the chemical and biological sensing applications of HCR in this review.16,46 In addition to its sensing potential, HCR has been employed to create a molecular autonomous motor capable of in situ locomotion and has been influential in the development of novel theranostics approaches.

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Despite these diverse applications, the current use of HCR

remains predominantly sensor development-oriented;56-58 therefore, in this review, the chemical and biological sensing aspect of HCR is presented. Chemical and Biological Sensing Using HCR. The examples of HCR-based sensor design for bioprocess monitoring and biomolecule, small molecule and metal ion sensing are innumerable. Specifically, HCR has been used as a programmable in situ amplification technique for the detection of mRNAs within living cells and higher organisms.49,59,60 Kubota et al. demonstrated the simultaneous detection of whole-cell microorganisms (Bacteria, Archaea and Methanosaetaceae) for phylogenetic identification via in situ-HCR.61 Other studies have applied the HCR technique in combination with various nanoparticle formulations for intracellular mRNA imaging.62,63 Our lab has exploited the programmability and signal amplification features of HCR for the detection of multiple DNA/RNA -based biomarkers, and toxic heavy metal ions.64-66 Others have incorporated HCR as the signal amplification step in their protocol for the detection of metabolites,67 toxins61 and proteins.68-71 In this review, we have compiled some of the notable works where HCR was successfully implemented into the sensing schemes for nucleic acids, metabolites, proteins and metal ions using a variety of different protocols Nucleic Acid Detection. In addition to their biological functions, certain RNA and DNA molecules have been regarded as therapeutic targets72 and molecular markers for disease diagnostics, prognostics, infection, forensics and contamination.73-79 Furthermore, quantification of RNA expression in living systems as well as monitoring their cellular and exosomal localization are imperative in

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order to fundamentally understand cellular trafficking in vivo.80-82 Therefore, there remains significant demand for highly sensitive systems that not only function within the cellular environment but also are capable of identifying in situ RNA processes in real-time. Since HCR has the capability to operate in isothermal conditions without requisite enzymes, it exhibits enormous potential for intracellular and in vivo RNA detection and trafficking. In addition to its applications in mRNA imaging in living systems,49,59,60,62,63 HCR has been evaluated for the detection of various nucleic acid-based disease biomarkers using both optical spectroscopy and electrochemistry with and without nanoparticles. In this section, we summarize key analytical approaches and methodologies that have been developed using HCR technology for nucleic acid detection. Fluorescence-based Detection. HCR-based detection using both labeled and label-free fluorescence spectroscopy approaches were reviewed and are summarized below. Labeled Approaches: When first discovered in 2004, HCR was thoroughly characterized against a ssDNA molecule referred to as the initiator (I). The HCR mechanism was studied using gel electrophoresis and the fluorescence quenching properties of 2-aminopurine (2AP), which was attached to the first DNA hairpin (H12AP) in its ssDNA sticky end, Figure 3.46 2AP is a fluorescence probe used commonly for DNA conformational changes because it fluoresces in ssDNA form, but is quenched when in dsDNA form.83 The DNA sequences in their protocol were systematically optimized until the HCR design resulted in a system containing a 24-mer initiator DNA and two 48-mer hairpins. The two hairpins remained stable and unreacted in the absence of the target initiator sequence and the fluorescence was therefore observed in H12AP. When the initiator was introduced, however, the HCR process was initiated and as a result, the fluorescence of 2AP was quenched due to the participation of H12AP in dsDNA polymerization, Figure 3d. The HCR protocol was illustrated using I, H1 and H2, and the control experiments demonstrated that the process is highly specific to the particular target initiator ssDNA sequence. Fluorescence in situ hybridization (FISH) strategies have been applied in a variety of platforms to allow for tissue and intracellular mRNA expression imaging.60,84 In 2010, Pierce et al. utilized an HCR design to create a fluorescent in situ amplification protocol for the simultaneous mapping of five different mRNAs in zebrafish embryos.60 This multiplexed approach was based on RNA polymerization of fluorescently labeled metastable RNA hairpins

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(monomers) through orthogonal HCR amplification. In a zebrafish total RNA background, each 81-nt long probe contained a 50-nt recognition sequence that partially hybridized to its specific mRNA target at its complementary location. This enabled the generation of a 26-nt sticky end initiator sequence that triggered HCR amplification in the presence of the HCR-requisite hairpins, thereby creating a distinct fluorophore labeled HCR product. In this design, the nucleic acid sequences were rationally designed with the hypothesis that they would penetrate the samples prior to polymerization. The hairpins used in this protocol were altered from the basic HCR design, from DNA to RNA, to allow for polymerization in the in vivo restrictive hybridization conditions where RNA hybridization is more stable than DNA; each hairpin was a 52-mer RNA sequence with 10-nt sticky end, 16-bp stem and 10-nt loop, and the 5’-end of H2 was labeled with a fluorophore. Since this technique allowed for simultaneous mapping in the same sample, the time required to map all five mRNA was the same as it would be for one mRNA. Although this in situ HCR study resulted in the successful imaging of target mRNAs using RNA hairpins, the protocol was advanced even further using DNA hairpins, which are considered to be more stable than RNA because they are not as prone to enzymatic degradation.84 In addition, RNA synthesis is more expensive than DNA synthesis, so another goal of this revised study from a practical standpoint was to reduce the cost. In the milder experimental conditions, the scientists utilized 72-mer DNA hairpins designed with 12-nt sticky end, 24-bp stem and 12-nt loop. This new approach lowered the overall cost of the system and increased the fluorescent signal per target. With this modified protocol, four mRNA targets were mapped simultaneously in whole-mount zebrafish embryos. Furthermore, Pierce et al. applied this optimized and advanced in situ HCR system for the mRNA mapping of eight sample types: bacteria, whole-mount nematode larvae, whole-mount fruit fly embryos, whole-mount sea urchin embryos, whole-mount zebrafish larvae, whole-mount chicken embryos, whole-mount mouse embryos and formalin-fixed paraffin-embedded human tissue sections. Their study incorporated probe sets containing between two and ten probes, each possessing two initiators for the same amplifier to map 24 target mRNAs.59 Parallel in situ HCR experiments have also been conducted in the imaging of three different mRNA targets (egfp, Twist1 & Pax2) in intact mouse embryos.49

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Additionally, there are innumerous other fluorescence-based nucleic acid detection methodologies reported using HCR. Recently, Pierce et al. incorporated HCR into northern blot for the multiplexed and simultaneous detection of as little as 25 amol of three endogenous human miRNAs (miR-16, miR-18a and miR-30a).85 The authors designed three nucleic acid probes complementary to the target miRNA molecules, which were used along with fluorophore labeled DNA hairpins to trigger HCR and assemble fluorescent dsDNA polymers. The authors concluded that the HCR northern blots, in contrast to the current northern blot approaches, offer straightforward multiplexing while achieving multiple target detections and signal amplifications in parallel. Pyrene excimer probes have also been tested with HCR for target DNA detection in complex biological matrices. In this study, 48-mer hairpin probes with 18-bp stem and 6-nt loop regions were labeled with pyrene molecules at their ssDNA terminals. After the target DNA-triggered HCR process, the pyrene monomers came into close proximity, thereby creating excimers that emit at wavelength 485 nm, resulting in target DNA sensing in the fM detection range.86 The reaction time in this study was reported to be approximately 1 hour and pyrene-excimer formation can be measured ratiometrically for more precise detection and minimal interference from the environment. In a similar scenario, Huang et al. used pyrene and the fluorescence of β-cyclodextrintethered cationic polymers (cationic polyβ-CD) to detect a target DNA with a 0.1 nM LOD.87 In this study, H2 was labeled with a pyrene on its ssDNA terminal, Figure 4. Due to the strong attraction between the inherently negatively charged (-) ssDNA and the positively charged (+) cationic polyβ-CD, the pyrene was able to enter into the polyβ-CD cavity, thereby increasing the fluorescence due to the pyrene emission enhancement. However, in the presence of the target DNA and HCR-requisite hairpins, HCR was triggered, and DNA polymerization was observed. Since the steric hindrance of the dsDNA product prevented access of the pyrene into the polyβCD cavity, a significantly lower fluorescence signal was therefore observed. In contrast to the aforementioned pyrene-excimer approach,86 this method relies on electrostatic interactions and can therefore be more vulnerable to environmental factors in complex matrices. Separately, a dual amplification strategy was tested by Xiang et al. using HCR and the targetrecycling features of catalytic hairpin assembly (CHA) for the detection of oncogenic miR-141 with a 0.3 fM LOD.88 The authors designed the hairpins with FAM (fluorophore) and Dabcyl

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(quencher) labels located on opposite ends for a fluorescence quenching and recovery (F-Q) design for miRNA analysis. The close proximity to Dabcyl in the stem-loop conformation quenched the FAM fluorescence. The hybridization of miR-141, however, unfolded the hairpins and triggered HCR in the system, thereby forming the dsDNA HCR product and allowing for fluorescence recovery due to the separation of the FAM and Dabcyl probes. This classical F-Q approach provides a notable sensitivity because, in contrast to amplification-free (1:1, target:probe) approaches, one miR-141 results in the opening of multiple quenched hairpins. Additionally, a separate fluorescence quenching and recovery (F-Q) design has been implemented with HCR to create a biomolecule-to-fluorescent color encoder. Using a 24-mer target ssDNA, the proximity of FAM and TAMRA fluorophore labels with BHQ-1 and BHQ-2 quencher labels was altered upon the initiation of the HCR process. The overall change in the fluorescence wavelength and intensity were used for the detection of the target DNA in the environment.89 This approach is similar with the aforementioned approach,88 except two fluorescent reports and quenchers were used in the detection scheme. Recently, HCR was coupled with an aptamer (LZH8) for a surface biomarker expressed on cancerous exosomes.90 In this study, the hairpins were fluorescently labeled and the HCR process on the exosomal surface was validated by fluorescence measurements. The authors demonstrated that the HCR process enabled a 10-times greater fluorescent-labeling on target HepG2 exosomes than the control non-target Hu1545 exosomes. The signal amplification for exosome detection is interesting from a bio-diagnostic perspective, however, it could be challenging to control the HCR-product size and density, therefore limiting the quantification of exosomes. Label-Free Approaches: In addition to the aforementioned labeled approaches, the HCR technique has also been exploited for label-free detection using intercalating fluorescent reporters. HCR was used as the signal amplifier in an assay where magnetic beads (MBs) were utilized to isolate a target RNA (miR-141) from bovine serum.47 This HCR on MBs approach was capable of specifically discriminating base-pair mismatches between members of same miRNA family, miR-141 and other miR-200 members. The MBs were functionalized with a capture probe capable of binding to the target DNA sequence. Subsequently, HCR was initiated upon introduction of the HCR-requisite hairpins, forming dsDNA polymers on the MB surface. The duplexes were denatured with heat and magnetic separation was performed to isolate the

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HCR dsDNA products. These products were then quantified by the fluorescence emitted from SYBR Green fluorescent dyes intercalated in the duplex. As little as 0.55 fmol target miRNA was detected with this approach, which was ~230-fold more sensitive than previous MB protocols. In another study, a fluorescent amine molecule, 5,6,7-trimethyl-1,8-naphthyridin-2-ylamine (ATMND), was bound to the stem of the HCR-requisite hairpins through H-bonding, thereby quenching its fluorescence.91 In the presence of a target DNA, HCR occurred and released ATMND from the hairpins, recovering the fluorescence and allowing for the detection of the target DNA with a 0.2 nM LOD. This HCR method was also implemented to detect a 107-mer PCR DNA product of the KRAS gene. The detection of KRAS is highly interesting from a biodiagnostic standpoint, however, two amplification steps (PCR & HCR) were used in this assay in contrast to only HCR in other studies. In a separate study, a visual method was demonstrated by combining HCR with Gquadruplex structures in the presence of hemin, H2O2, and ABTS (2,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonicacid)) to sense a target DNA with a 7.5 nM LOD.92 The authors later adapted this approach for fluorescence detection using a dsDNA intercalating fluorescent molecule providing a LOD of 4 nM. The authors developed a visual-chip platform for instrument-free detection of the target sequence. As also indicated by the authors, a new set of probes must be assembled for a new target molecule. Electrochemical-based Detection. The remarkable sensitivity achieved with electrochemistry makes electrochemical approaches attractive for detecting minute copies of molecular targets.93 Electrochemical-based nucleic acid sensors using HCR as a signal amplifier have garnered significant attention in the scientific community as their detection sensitivity has the potential to be as low as the aM level. Recently, Zuo et al. designed two parallel electrochemical biosensors by coupling tetrahedral DNA nanostructure probes with HCR technology.94 One of the sensors was designed for a target DNA molecule and the other for miRNA-122b, with LODs of 100 aM and 10 aM, respectively. The sensitivity in this approach was three orders of magnitude greater than previous supersandwich amplification techniques. In this study, target recognition probes were immobilized to self-assembled 3D tetrahedral DNA nanostructures on a gold electrode (AuE) surface, Figure 5a. When present, the target partially hybridizes to the recognition probes, creating a DNA

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overhang that acts as the HCR initiator strand. The main difference between the DNA and RNA biosensors was the introduction of a nucleic acid hairpin on a nanostructure scaffold, which opens only in the presence of the target miRNA-122b. Thereafter, the addition of biotin-labeled hairpins and the subsequent HCR process resulted in dsDNA polymers, which captured avidinmodified horseradish peroxidase enzymes that generated an electro-catalytic signal in the presence of H2O2. Another study reported the detection of a DNA sequence from the protozoan C. parvum with a label-free electrochemical biosensor by measuring the capacitance signal at an electrodesolution interface, Figure 5b. In this study, the electrical capacitance signal increased after HCR occurred on the electrode surface, allowing for the electrochemical detection of the DNA sequence with a 0.1-0.5 µM LOD.95 Because this approach is label-free, the LOD was determined to be within the µM range. Additionally, a label-free homogenous electrochemical biosensor has been developed using HCR and a negatively charged (-) indium tin oxide (ITO) electrode that detected the let-7a miRNA with a 1pM LOD.96 When present, the electrochemical indicator methylene blue (MetB) diffused over the ITO surface, resulting in a large electrochemical signal, Figure 6a. However, through the rational design of the HCR-requisite hairpins containing split G-quadruplex sequences, the target let-7a miRNA acted as the initiator and HCR dsDNA-products were formed with numerous G-quadruplex structures. Subsequently, the majority of the MetB intercalated into the polymers and G-quadruplex structures, resulting in a lower electrochemical signal. Detection of a single nucleotide difference was demonstrated using let-7f and let-7g, indicating that this technique was highly sequence-specific. A separate G-quadruplex-based electrochemical DNA assay was fabricated by combining isothermal exponential amplification (EXPAR) with HCR to detect the avian influenza A (H7N9) virus DNA with a 9.4 fM LOD.97 The HCR products in this design generated polymer nanowires consisting of stable G-quadruplex units tethered to a AuE surface, Figure 6b. The system resulted in the formation of HRP-like DNAzymes in the presence of hemin. When H2O2 was added to the system, the electrochemical signals were obtained by monitoring the reduction current generated by the catalytic oxidation of 3.3′, 5.5′-tetramethylbenzidinesulfate (TMB) from the DNAzymes. In another miRNA detection study, Yuan et al. utilized the amplification properties of target

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assisted polymerization nicking reaction (TAPNR) and HCR, along with the electronic and chemical properties of silver nanoclusters AgNCs98,99 for the detection of miRNA-199a with a 0.64 fM LOD.100 In this study, cytosine (C)-rich hairpins were used as the HCR monomers. The miRNA-199a target recognition initiated the HCR process, producing long dsDNA polymers. Thereafter, incubation of the dsDNA HCR products with AgNO3 and NaBH4 resulted in the in situ synthesis of AgNCs on the AuE surface in the C-rich loop DNA positions. Cyclic voltammetry and differential pulse voltammetry were used for the measurement of the electrochemical signal upon target binding. In addition to TAPNR, HCR has also been utilized in conjunction with isothermal strand-displacement polymerase reaction (ISDPR) to electrochemically detect a nucleic target molecule with a 0.02 fM LOD.101 It should also be noted that the combination of HCR’s signal enhancement properties with other amplification methods is not limited to the aforementioned studies. HCR has also been applied in conjunction with electrochemiluminescence (ECL), a process where a measurable light is emitted from an exergonic reaction of electrochemically generated intermediates.102 Chai et al. incorporated the HCR process into an ECL electrochemical approach to detect the Escherichia coli uropathogen (6S rRNA gene) with a 15 fM LOD. To accomplish this, thiolated DNA capture probes were attached to a AuE surface and used as the platform for in-situ HCR. Partial hybridization of the target nucleic acid to the capture probe occurred, creating a 15-nt initiator strand that commenced the HCR process. Numerous ECL probe Ru(phen)32+ molecules were thereafter intercalated into the grooves of the dsDNA HCR product, generating an amplified ECL signal with respect to the target concentration.103 Similar lightgenerating chemiluminescence (CL) nucleic acid biosensors have also been fabricated through the instantaneous derivatization reaction between the CL reagent, 3,4,5-trimethoxylphenylglyoxal (TMPG), and the guanine nucleotides within HCR products.104,105 It is also noteworthy to stress that even though the electrochemical approaches are exploited due to their greater sensitivity over other analytical approaches, the LODs in each electrochemical approach are reported to be highly different. This variability is attributed to the other techniques used in each study, i.e., labeled vs. label-free approach, or with/out an additional amplification step. Nanoparticle-based Detection.

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In addition to their applications as drug delivery vectors,106-110 nanoparticles are regarded as highly powerful probes for molecular detection.111 In particular, gold nanoparticles (AuNPs) have been thoroughly studied due to their intriguing properties, which are ideal for the development of optical sensors.112-116 For example, their unique surface plasmon properties and facile surface functionalization make them attractive probes for colorimetric sensor development.117,118 Moreover, AuNPs are efficient fluorescence-quenchers and have therefore been extensively studied for fluorescence-based detection as well.119,120 To detect nucleic acids, the nucleic acid-based recognition elements can be covalently and noncovalently attached to the surface of AuNPs.121-124 In addition to AuNPs, metal oxide nanoparticles also interact with nucleic acids in varying and quantifiable degrees, resulting in either an alteration in their physicochemical properties or a folding of the target oligonucleotides in a manner whereby both can be monitored spectroscopically. Furthermore, 2D nanoparticles such as nano-graphene oxide (GO), MoS2 or WS2, can adsorb and home fluorophore-labeled ssDNA strands on their planar surface, while efficiently quenching the strands’ fluorescence.125,126 The HCR protocol has been coupled with various types of nanoparticles for the programmable and sensitive detection of a multitude of target molecules. In this section, we will review a plethora of scientific studies reporting nucleic acid sensors that have been developed in combination with HCR and nanoparticles using colorimetric, fluorescence, electrochemical and Raman spectroscopy -based detection methods. Colorimetric Nanoparticle Approach. Due to their surface plasmon properties, AuNPs are ideal templates for designing colorimetric nanoprobes. For instance, negatively charged ~13-nm sized AuNPs repel each other and their colloidal Au solution is a wine-red color. However, gradual aggregation of AuNPs with an external stimulus can change the color of the solution slowly from red to purple to gray to colorless. This visually monitored transition can be induced with various additives such as cations, positively charged materials, nucleic acid hybridization or antigen-antibody binding. Due to their cost-effective and instrument-free characteristics, colorimetric approaches using AuNPs have been implemented in a variety of different protocols. It is essential, however, to increase the sensitivity of this visual detection methodology in order to meet the detection limit regulations for each target of choice. The HCR process has the potential to address this challenge in light of its intrinsic signal amplification features.127 Furthermore, the programmability

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properties of HCR enables one to use a single AuNP construct for the detection of multiple targets simultaneously or individually, in multiple combinations. In this section, colorimetric sensors developed using HCR in combination with various nanoparticle formulations will be reviewed. He et al. designed a AuNP and HCR methodology for the discrimination of ssDNAs from dsDNAs by exploiting the difference in their binding affinities to the AuNP surface.128 When unreacted, the HCR-requisite hairpins contain a ssDNA sticky end, which adsorbs to the AuNP surface and protects the AuNPs from salt-induced aggregation and color transition, Figure 7a. Addition of the target DNA (initiator) activates and opens H1, triggering the HCR process. As a result, the hairpins separate from the AuNPs and form dsDNA HCR products that do not adsorb to the AuNPs, causing the AuNPs to aggregate under the experimental salt concentration, which can be monitored visually. The incorporation of the HCR process in this nanoparticle-based DNA detection approach increased the sensitivity to 100 pM from 10 nM, which was attained using an HCR-free sandwich assay. The experiments were also successfully tested for the recognition of a single nucleotide difference in the target sequence, indicating the sensitivity of this HCR approach. In a separate study, instead of salt-induced aggregation, the AuNP color transition was achieved via the crosslinking of AuNPs. This catalytic AuNP aggregation study was performed using AuNPs functionalized with HCR-requisite hairpins that triggered HCR with the addition of the target DNA in the system, resulting in a 157 pM LOD.129 In contrast to the aforementioned approach128 where the AuNPs were used solely as reporters, in this approach, the AuNPs were used both as signal reporters and the substrate for immobilizing the hairpins. Another report implemented the HCR-induced dsDNA polymerization on DNAfunctionalized AuNPs for the detection of a target DNA with a 0.5 nM LOD.130 In this study, the capture DNA probe, immobilized on the AuNPs through thiol-Au chemistry, was capable of capturing the target DNA through hybridization, thereby creating a ssDNA overhang, Figure 7b. Upon addition of the hairpins, this overhang acts as the initiator and opens the first hairpin, triggering the HCR process. The resulting dsDNA product on AuNPs enhances the repulsion between polymer-tethered AuNPs and protects them against salt-induced aggregation, maintaining the original red color of the suspension. In the absence of the target, however, HCR does not occur on the AuNP surface and thus, the AuNPs are subject to salt-induced aggregation

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and the associated red-to-purple-gray visual color transition. Though the signal readout is colorimetric, this approach is vastly different than the previous two approaches. The first approach was performed through salt-induced aggregation without immobilizing the HCR components on AuNP surface.128 In the second approach, the hairpin-functionalized AuNPs were cross-linked in the presence of the target, inducing color transition.129 However, in the last approach, the AuNPs were used as the substrate for immobilizing the HCR-product, which protected the AuNPs from salt-induced aggregation.130 Recently, our lab combined the programmability and signal amplification features of HCR for the colorimetric detection of three circulating breast cancer associated miRNAs (oncomiRs), miR-10b, miR-21 and miR-141.65 In this system, a 36-mer DNA capture strand was immobilized on the surface of ~15-nm sized AuNPs, Figure 8a. The capture strand has a separate binding affinity to the each of the three oncomiRs in distinct 12-nt long regions throughout its 36-nt long sequence, c10b for miR-10b, c21 for miR-21 and c141 for miR-141. Each oncomiR therefore partially binds to the specific position on the capture probe, creating a ssDNA overhang. Three rationally designed bridging ssDNAs, referred to as ‘programming units’, subsequently bind to their specific target’s DNA overhang to prepare the system for the HCR process. Upon binding to their target, each programming unit generates the exact same ssDNA overhang, which serves as the initiator and triggers HCR by activating the hairpins in the system, Table 1. As a result, long dsDNA polymers form on the AuNP surface. These DNA polymers protect the AuNPs from Mg2+-induced aggregation and inhibit the color transition in the colloidal suspension. However, in the absence of the target sequence, HCR is not initiated and DNA polymerization on the AuNPs does not occur. Therefore, the AuNPs cannot resist Mg2+-induced aggregation and the resultant visible color transition. This method enables one to use only a single AuNP design for the detection of three oncomiR targets simultaneously or individually, in seven distinct combinations by selectively adding the appropriate programming units, Figure 8b. As little as 20 fmol of the target oncomiR was detected with this approach. Furthermore, the detection of endogenous miR-10b in a total RNA pool isolated from a breast cancer cell line was also demonstrated, Figure 8c. This approach is similar with the aforementioned approach130 in terms of salt-induced aggregation, however, using a bridging ssDNA between the target and the hairpins enabled us to use only one set of hairpins for multi-analyte detection. Later, this HCR coupled with AuNP approach was advanced for the detection of four DNA

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targets in sixteen different combinations using a single AuNP construct.64 The methodology was designed for the detection and phenotyping of four DNA Ebolavirus subtype biomarkers, Bundibugyo, Taï Forest, Sudan and Zaire. Four ssDNA targets (TB, TT, TS and TZ) were identified from the full genome sequences of the ebolavirus subtypes known to cause disease in humans (TB for Bundibugyo, etc.). The programming units (IB, IT, IS and IZ) were specially designed for each specific target (IB for TB, etc.) and used for the reprogrammable detection studies. In this study, the AuNPs were functionalized with capture strands capable of specifically binding to each target in separate complementary regions, Figure 9a. The binding of the target DNA strand creates a sticky ssDNA end, which is thereafter able to hybridize to its corresponding programming unit and prepare each system for the HCR process with identical ssDNA overhangs. In the presence of the HCR-requisite hairpins, HCR occurs and a long dsDNA polymer assembles on the AuNP surface, protecting the AuNP from Mg2+-induced aggregation and inhibiting the color transition. However, in the absence of the target(s) HCR does not occur, and an obvious visible color transition is observed. This approach enabled the detection of as little as 400 amols (24 × 106 molecules) of target DNA with the naked eye. Furthermore, this same methodology was adapted for the detection and classification of each target subtype in human urine Figure 9b. Highlighting the programmability nature of HCR, scientists are able to rapidly reprogram a single nanoprobe for the colorimetric detection of each target or groups of targets in various combinations by solely changing the initiator DNA sequence, or programming unit, and keeping the remainder of the system’s characteristics constant. These programmable features of HCR polymerization allow for a cost-effective and easy alternative to many other colorimetric sensing protocols. Examples of nanoparticle-based colorimetric detection of nucleic acids using HCR are multifaceted,131,132 and we anticipate development of a multitude of additional nanoparticle formulations in the near future. Fluorometric Nanoparticle Approach. In addition to the colorimetric approach, HCR technology has been used in conjunction with various types of nanoparticles in the development of fluorescent nanosensors for nucleic acid detection. Recently, Zhao et al. proposed a fluorescence polarization (FP) assay using streptavidin-coated SiO2 nanoparticles (SA-SiNPs) and HCR for the detection of a DNA target molecule with a 35 pM LOD.133 In this study, one hairpin was labeled with biotin and the other

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with a fluorescent dye. After recognition of the target DNA, the HCR process was initiated with the hairpins, assembling a fluorescently labeled dsDNA product that was immobilized on SASiNPs through the biotin-streptavidin interactions, Figure 10a. This system was also successfully tested for target DNA detection in human serum. This biotin-streptavidin interaction was also implemented for the detection of an Escherichia coli uropathogen target DNA with a 0.4 nM LOD using a novel copper nanoparticle (CuNP) and HCR approach.134 In this study, the biotinylated capture DNA strands were immobilized on streptavidin-coated beads, Figure 10b. The hybridization of the target strand to the capture strand freed a 15-nt overhang initiator sequence, which triggered HCR and formed a dsDNA polymer on the beads. After centrifugation, Cu2+ was used to form CuNPs on the HCR products. These dsDNA-templated copper nanoparticles were thereafter used as fluorophores for the detection of the target DNA. In a separate study, noncovalent π-π stacking interactions of core– shell Fe3O4 polydopamine nanoparticles (Fe3O4@PDA NPs) were used to separate targetinitiated fluorescently labeled HCR products, enabling the fluorescent detection of the target DNA with a 0.05 nM LOD.135 Due to their extraordinary ssDNA adsorbing and fluorescence quenching abilities, 2D nanomaterials such as graphene oxide (GO), MoS2 and WS2, to name a few, have been widely used for the development of nucleic acid based fluorescent nanosensors.136-142 GO-based silver nanocluster (AgNC) sensing platforms have previously been reported,143 and to increase their sensitivity, Han et al. incorporated HCR in this GO-based AgNC protocol to detect an HIVtarget DNA with a 1.18 nM LOD.144 In this study, AgNCs were utilized as the fluorophores and were formed at the ends of the hairpins using the reduction of AgNO3 with NaBH4, Figure 11a. Addition of GO to the system resulted in the adsorption of the unreacted hairpins onto its surface via non-covalent π-π stacking. In the absence of the target DNA, the AgNC-tagged hairpins were also adsorbed onto the GO surface, quenching the AgNC fluorescence. However, in the presence of the target DNA, HCR was initiated, and a dsDNA polymer assembled, preventing GOadsorption and thus inhibiting the fluorescence quenching of the AgNC fluorophores. A similar methodology was also reported using FAM fluorophore labeled hairpin probes for the detection of the let-7a miRNA. In this study, however, the dsDNA polymers remained tethered to the GO surface due to the ssDNA sticky end of the HCR product, but were still able to fluoresce due to the outward length of the polymer. In the absence of the target, the

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fluorescently labeled hairpins were fully adsorbed on GO and the fluorescence was quenched.145 The authors observed signal recovery with as low as 1 pM of the target miRNA. This sensitivity was reported to be more than two orders of magnitude greater than other GO-based detection approaches with 1:1 binding design. 2D nanomaterials have also been used in conjunction with HCR nanotechnology for the in situ two-color detection of miRNAs.146 In 2016, Tang et al. demonstrated the detection of miR21 and let-7a in the same cell using four hairpins (H1-H4). FAM-labeled (green) H1 and H2 hairpins were designed for miR-21 detection and ROX-labeled (red) H3 and H4 hairpins were designed for let-7a detection. First, the hairpins were adsorbed on the GO surface forming a hairpin probe/GO nanoformulation, Figure 11b. In this system, GO acted as both the fluorescence quencher and probe carrier for the intracellular probe delivery via non-destructive clathrin-mediated endocytosis. In the presence of the target miRNAs, HCR thereafter occurred intracellularly, recovering a green/red fluorescence signal that correlated to the expression of each specific miRNA in MCF-7 living cells. Likewise, another study reported the detection of human telomerase RNA (hTR) in a living cell utilizing a similar probe/GO complex to deliver and initiate intracellular HCR.147 Analogous to GO, biodegradable MnO2 nanosheets have also been used as fluorescence quenchers and intracellular delivery agents of HCR hairpins for miRNA imaging in HeLa cells.148 In this study, hairpins were labeled with a Fluorescence Resonance Energy Transfer (FRET) pair, FAM and TAMRA. After the intracellular uptake, the MnO2 nanosheets degraded, exposing the hairpins for miR-21 triggered HCR. The HCR dsDNA product then brought the FAM and TAMRA probes in close proximity so that FRET could occur, correlating to the successful detection of the target miR-21. Electrochemical Nanoparticle Approach. Electrochemical approaches have also exploited the advantages of HCR to improve the sensitivity of nanoparticle-based nucleic acid detection. Ling et al. used positively charged (+) AuNPs for the detection of a target dsDNA without a denaturation step. In this study, ferrocene (Fc) modified hairpins assembled to a AuE surface upon recognition of the target dsDNA sequence, Figure 12a. The subsequent addition of (+) AuNPs amplified the electrochemical signal after absorbing to the negatively charged (-) dsDNA HCR product, resulting in a 2.6 pM LOD.149

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In a similar study, Tang et al. developed an electrochemical methodology using HCR in conjunction with silver nanoparticles (AgNPs) for the detection of HIV-related gene fragments.150 The HCR products were used as the recognition sites for AgNPs to amplify the observed electrochemical signal in the system. Briefly, a capture strand anchored on a AuE surface selectively bound to the target DNA strand, which thereafter hybridized with a rationally designed detection probe, creating an HCR-initiator ssDNA sticky end overhang. After the HCR dsDNA product formed in the presence of the HCR-requisite hairpins, the electrode was submerged in CTAB-capped silver colloids, allowing (+) metal AgNPs to adsorb on the (-) dsDNA product. An electrochemical signal was thereafter observed with respect to the target concentration with a 0.5 fM LOD. This method, in contrast to aforementioned approach,149 is label-free and carried out using unmodified hairpins instead of Fc-modified hairpins. Another AgNP-based electrochemical biosensor was developed by recording the silver stripping current from 4-nm sized AgNPs attached to HCR-requisite hairpins.151 In this study, a hairpin strand complementary to the target DNA was anchored to a tetrahedral nanostructure assembled on a AuE surface to increase the reactivity and accessibility of the reaction, Figure 12b. When present, the target hybridizes with and opens the hairpin, which then captures DNAmodified 13 nm-sized AuNPs and initiates the HCR process. The activation and immobilization of AgNP-tagged hairpins on this AuNP seed region thereafter generates a silver stripping current peak in relation to the target concentration. This designed protocol was able to detect the target miRNA with a 2 aM LOD. A human miRNA (hsa-miR-17-5p) was also detected by applying this methodology in HUVEC, HeLa, HK-2 and MCF-7 cell lysates. This approach has higher sensitivity than the previous two methods149,150 because it has dual amplification. An intermediate AuNP serves a platform for multiple HCR polymerization sites, thus one target molecule triggers multiple HCR-products, offering much higher sensitivity. This elegant approach can also be extended to the detection of other biological molecules. In a separate scenario, an ECL biosensor was recently fabricated by Li et al. via catalysis of luminol using HCR in conjunction with AuNPs for the detection of HIV-type 1 DNA with a 5fM LOD.152 Similarly, AuNPs electrodeposited onto a bare glassy carbon electrode (GCE) were used as the platform for HCR to occur in the presence of a target miRNA. Hemin was thereafter intercalated into the dsDNA HCR products to catalyze the oxidation of hydrogen peroxide (H2O2) and allow for the ECL detection of microRNA-155 with a 1.67 fM LOD.153

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SERS Nanoparticle Approach. Surface-enhanced Raman spectroscopy (SERS) is a highly sensitive technique used for molecular detection through surface adsorbed Raman reporters. Recently, HCR has been coupled with SERS for a variety of detection schemes. Yang et al. reported the detection of a circulating miRNA in human serum using miRNA-triggered HCR and ion-mediated cascade amplification.154 In this study, AgNPs were immobilized on the HCR product and dissolved upon oxidation. The dissolved silver ions were used to control the gapping between 4-aminobenzenethiol (4-ABT) encoded gold nanoparticles (AuNP@4-ABT) in the environment and form ‘hot spots’ that generated the SERS signals. This SERS approach offered the detection of a miRNA with 0.3 fM LOD. Although this method was exploited for miRNA detection, it can be adapted for the detection of other biomolecules through hybridization or aptasensor designs. Separately, Luo et al. reported the detection of miR-141 with a wide linear range of 10-15 to 10-7 M using an HCR-SERS bio-barcode. In this study, AuNPs were used as both the signal carrier and enhancer, whereas the magnetic beads (MBs) were used as the signal carrier and substrate for immobilizing the HCR-AuNPs with SERS reporters. The target miR-141 initiated the HCR reaction and formation of HCR-AuNPs on the MBs. The experiments were performed in the presence of other miRNAs and results demonstrated that the approach is highly sensitive to the target miR-141. Therefore, this SERS approach is ideal for the detection of miR-141 in a wide concentration spectrum. However, measuring the subtle differences in the miR-141 concentration could present a challenge with the current state of this approach.155 In another SERS study, Han et al. demonstrated the detection of a DNA sequence specific to Bacillus thuringiensis (Bt) transgene with a LOD of 50 pM using SERS nanoparticles and HCR.156 The Bt gene has been used for insect control in agricultural products and its detection is important for bio-security and food safety. In this design, biotinylated hairpins were activated in the presence of the target DNA sequence and immobilized on a solid substrate. Later, the HCR product on the substrate was treated with both streptavidin and biotin-labeled AuNPs. The final HCR-product with AuNPs deposition was subject to laser exposure to record the SERS readouts. The results demonstrated the detection of the target DNA and its discrimination from DNA with single nucleotide differences. This report, along with others, demonstrates that HCR is

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compatible with SERS for the detection of biological samples with both high sensitivity and specificity. Metabolite Detection. Human metabolism is composed of complex and multi-step reactions. Aberrant levels of metabolites, the final or intermediate metabolism products, can be indicative of various biological abnormalities. Many metabolites are also harmful to humans and some have even been linked to rheumatoid arthritis, non-alcoholic fatty liver disease, cardiovascular disease and numerous other diseases.157-160 Since metabolites are key indicators of overall human health, early metabolite detection may help to prevent the progression and severity of a myriad of human diseases. In particular, significant effort has been made in sensing various metabolites such as adenosine triphosphate (ATP),161 thiamine 5′-pyrophosphate (TPP)162 and fructose 1, 6bisphosphate (FBP)163 using aptamer-based designs. HCR technology, however, can help provide an increased sensitivity to these aptamer platforms and utilizing the programmability features of this process allows for multi-analyte metabolite detection. ATP Detection. As previously mentioned, Pierce et al. exploited the HCR process in the detection of ATP and illustrated this process’ specificity features by successfully discriminating ATP from GTP, Figure 2.46 In a later study, this HCR methodology was incorporated in conjunction with AuNPs to develop an ATP colorimetric aptasensor with a 1.0 nM LOD.164 In this study, the ssDNA ends of the hairpins noncovalently bound to the AuNPs and therefore protected them from salt-induced aggregation. However, when present, ATP reacted with a 25mer long recognition (aptamer) site on H1 and subsequently triggered HCR. The dsDNA HCR products were unable to adsorb to the AuNP surface, thereby allowing the AuNPs to participate in salt-induced aggregation and a visual color transition was observed. This sensor design was able to discriminate the ATP target from CTP, GTP and UTP.164 In another study, ATP was electrochemically detected by combining HCR with the assembly of electroactive silver nanotags on a AuE surface. When present, ATP bound to the molecular recognition site of a dsDNA aptamer-initiator complex, enabling HCR on the AuE surface in the presence of the HCR-requisite hairpins. The electrochemical signal was then generated via the silver nanotags on the HCR dsDNA product, allowing for a 30 fM LOD.165 Surface plasma resonance (SPR) strategies have also been employed with HCR for metabolite detection. One SPR protocol reported the detection of DNA and ATP with LODs of

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0.30 fM and 0.48 nM, respectively.166 To detect ATP, a thiol-modified ssDNA capture probe capable of binding the initiator strand was anchored on a Au chip and an ATP aptamer partially hybridized to the initiator strand was attached to a MB, Figure 13a. In the presence of ATP, the ATP aptamer bound to the ATP, which released the initiator, allowing it to hybridize to the capture strand on the Au chip. The initiator strand subsequently triggered HCR on the Au chip in the presence of the Fc-modified HCR-requisite hairpins. Therefore, the resulting Fc-modified dsDNA product assembled only in the presence of ATP. This method enabled the discrimination of ATP from CTP, GTP and UTP analogs. Another SPR biosensor was also developed using streptavidin-coated AuNPs along with cascade multiple cycle amplification techniques including HCR, aptamer-based target-triggering nicking enzyme signaling amplification (T-NESA) and nicking enzyme signaling amplification (NESA) to detect adenosine with a 4 fM LOD.167 Glutathione Detection. In addition to these nucleotides/sides, HCR has been used to detect other metabolites such as biothiols. The cellular metabolite, glutathione (GSH), for example, is an essential biothiol and antioxidant involved in redox reactions. GSH’s health implications have been extensively studied and abnormal concentrations of GSH are linked to many human diseases, including Alzheimer’s and Parkinson’s disease.168 Separately, many biothiols, such as GSH and cysteine (Cys) have been known to bind to Hg2+ and form stable [biothiol:Hg2+] complexes via Hg–S bonds.169 One of the first reports to implement HCR in the detection of biothiols was performed by Chu et al., where a fluorescent nanosensor was developed using HCR along with the ssDNA quenching abilities of GO.67 The authors also exploited the highly specific Thymine-Hg2+Thymine (T-Hg2+-T) interactions into their detection scheme, Figure 13b. In their study, an initiator strand with T-T bp mismatches was rationally designed to form a stem loop structure only in the presence of Hg2+. Addition of Cys or GSH intercalated the Hg2+, forming the stable [biothiol:Hg2+] complex. As a result, the stem loop structure was denatured, creating a ssDNA initiator strand, which was then able to trigger HCR in the presence of fluorescently labeled HCR-requisite hairpins. Thereafter, the dye-labeled HCR dsDNA product continued to fluoresce even after the addition of GO into the system. However, without the target Cys or GSH, HCR could not occur and the ssDNA species were adsorbed to the GO surface, quenching the fluorescence of the system. This protocol was able to detect Cys and GSH with LODs of 0.08 nM and 0.1 nM, respectively.

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A similar methodology employed the Hg2+ binding capabilities of biothiols to displace Hg2+ from a stem-loop T-Hg2+-T complex of the initiator strand and trigger HCR on a AuE surface. The electroactive species [Ru(NH3)6]3+ then intercalated into the grooves of the HCR dsDNA product and were used as probes for the electrochemical detection of GSH with a 0.6 nM LOD.170 Ochratoxin A Detection. The HCR technique has also been implemented to detect many challenging small toxin molecules prevalent in the food industry. Ochratoxin A (OTA), for example, is a mycotoxin that contaminates cereal food worldwide and has nephrotoxic, hepatotoxic, neurotoxic, teratogenic and immunotoxic effects.171 Li et al. utilized HCR along with OTA aptamer-modified magnetic nanoparticles (AMNPs) and fluorescent perylene probes to optically detect OTA with a 0.10 pM LOD, which is more sensitive than most of the previous OTA-detection methods.172 In their study, the OTA-aptamer combined with the linear HCR dsDNA polymer products to create branched/dendritic DNA structures on AMNPs. The resulting structure aggregated the perylene probes and quenched their fluorescence via electrostatic interactions. However, in the presence of OTA binding, the HCR dsDNA product and aggregated-probes were released from the AMNP surface. After magnetic separation, methanol was used to disaggregate the perylene probes, allowing them to fluoresce. Additionally, another group used DNAzyme peroxidase-mimicking activity along with linear HCR polymer growth to develop an OTA colorimetric aptasensor with a 0.01 nM LOD.173 Protein Detection The expression of certain protein can be symptomatic of a variety of biological disorders. Identification of these protein-based biomarkers in the circulation or cellular surface is also vital in disease diagnostics and prognostics. Serum prostate specific antigen (PSA), for example, is a clinical disease marker for prostate cancer (PCa) and is found in the circulation, whereas another PCa-associated protein, prostate specific membrane antigen (PSMA), is overexpressed on PCa cell membranes.174 Separately, HER2 and CDK8 are protein-based therapeutic and diagnostic markers in breast and colorectal cancer, respectively.

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Due to their far-reaching

implications, the sensitive detection of these serum or cell surface proteins is not only important in order to fundamentally understand many biological processes, but it is also vital for clinical decision-making. Therefore, HCR technology has recently been evaluated in several protein detection schemes.

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Multiple HCR aptasensors have been reported for the fluorometric detection of proteins. One study detailed the detection of human IgG with a 14 aM LOD using biotinylated initiators and anti-IgG assembled to streptavidin-magnetic nanobeads on a glass substrate.177 In the presence of the target IgG and HCR-requisite hairpins, HCR occurred on the nanobeads and the nanobeadHCR assembly was magnetically separated. Subsequent intercalation of the HCR product with SYBR green fluorescent dyes allowed for the fluorescence detection of IgG. Likewise, Li et al. developed a fluorescent methodology for platelet-derived growth factor BB (PDGF-BB) detection with a limit of 1.25 pM by using an aptamer-target binding site placed on H1 for molecular recognition of PDGF-BB. In this system, SYBR green dyes and GO were used as fluorescent probes and fluorescent quenchers, respectively.178 In a similar fluorescence quenching scenario, Wang et al. utilized HCR coupled with AuNPs to detect the anterior gradient homolog 2 (AGR2) cancer protein with a 2.65 pM LOD.179 In the absence of AGR2 in their system, its unbound aptamer acted as an initiator and triggered HCR by activating fluorescently labeled hairpins. The resulting fluorescent HCR dsDNA product was unable to adsorb on AuNPs and therefore, fluorescence quenching was not observed. In the presence of AGR2, however, the aptamer bound to its target and was unable to serve as an initiator to the HCR process. Thus, the fluorescently labeled hairpins were adsorbed on AuNPs, quenching the overall fluorescence of the system. Similar to the aforementioned approach,178 AuNPs instead of GO, serve as fluorescent quenchers for the reporters used in the assay. Separately, the HCR technique has also been implemented in the development of electrochemical sensors for protein detection. Zhang et al. designed an electrochemical immunoassay using primary antibodies immobilized on a AuNP-modified glass carbon electrode (GCE). In their study, the target protein was sandwiched between the GCE and a secondary antibody conjugated to HCR dsDNA MetB and ferrocene (Fc) signal-labeled products. MetB and Fc were used for the detection of alpha-fetoprotein (AFP) and PSA with LODs of 0.25 pg mL−1 and 0.17 pg mL−1, respectively.71 In a similar scenario, HCR was coupled with doxorubicin hydrochloride and MetB in the fabrication of a multiple immunosensor used to detect AFP and CEA with LODs of 0.02 pg mL−1 and 0.03 pg mL−1, respectively.180 Both of these studies were performed by the same group, however, in the second approach, a different set of molecules were used as signal reporters, which resulted in higher sensitivity.

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The HCR technique has also been utilized in conjunction with DNAzymes for electrochemical protein detection. Tang et al. developed an impedimetric immunosensor for the detection of CEA with a 0.42 pg mL-1 LOD.181 In this study, hemin-binding aptamers and AuNPs were used for the assembly and tethering of the HCR products for signal enhancement. This protocol was also successfully used in six clinical human serum specimens.181 Similarly, [heminG-quadruplex/DNAzymes] systems have been used in combination with HCR using MB-based cascade amplification182 and aptamer proximity probes183 for the colorimetric detection of thrombin with LODs of 15 pM and 1.9 pM, respectively. In all of these three studies, H2O2mediated oxidation of reporter molecules are used either for colorimetric or electrochemical detection. Likewise, a simultaneous detection methodology of four protein-based cancer biomarkers was demonstrated using an immunosensor developed by monitoring the HCR process on a graphene/gold (GR–Au) hybrid film.184 In this study, AFP, CEA, carbohydrate antigen and PSA were concurrently detected with LODs of 62, 48, 77 and 60 fg mL−1, respectively. Each biomarker was immobilized on the GR–Au surface via its corresponding capture antibody and was sandwiched to a biotin labeled-detection antibody via gold magnetic nanomaterials (Au/SiO2–Fe3O4). The initiator was then introduced through biotin-streptavidin interactions, triggering HCR and tethering the HCR dsDNA products onto the GR–Au surface. Each product had a different redox probe attached to it, allowing for the individual or simultaneous detection of each protein using a single pass differential pulse voltammetry. Additionally, Bai et al. designed an ECL immunosensor by using enzymatic catalysis and an anti-IgG immobilized L-cysteine functionalized reduced graphene oxide (L-cys-rGO) GCE surface that could detect IgG with a 33 fg mL−1 LOD.185 When present, IgG bound to the biotin labeled anti-IgG, which subsequently bound to streptavidin and a biotin-labeled initiator strand. The resulting assembly initiated the HCR process in the presence of ssDNA building blocks labeled with glucose oxidase (GOD). The GOD-labeled HCR products were therefore able to catalyze glucose and produce H2O2, behaving as luminol’s co-reactant to amplify the resulting ECL signal. Similar chemiluminescence (CL) protocols have been used in the detection of thrombin using magnetic particles with a 9.7 fM LOD,186 and the prohormone brain natriuretic peptide and cardiac troponin I using HCR in combination with rolling chain amplification.187 Scientific reports detailing the electrochemical detection of proteins using HCR are

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multifaceted. Other electrochemical thrombin detection techniques have been reported using HCR in combination with DNAzymes,188 HRP enzyme amplification189 and exonuclease target recycling.190 In addition, the HCR technique has been applied to detect folate receptor using copper nanoparticle (CuNPs) with a 3 pg mL−1 LOD,191 IgG and PSA via AuNPs with LODs of 0.1 and 50 fg mL−1, respectively192 and CEA via DNA nanopolylinker193 and DNA-AuNPs194 with LODs of 1.2 and 3.2 fg mL−1, respectively. Metal ion Detection. Determining the environmental, agricultural and biological concentrations of different metal ions is essential to humanity. The selective and sensitive detection of each metal ion, however, is relatively challenging from a practical standpoint. Currently, there are a variety of approaches to the development of metal ion sensors, but perhaps most intriguing with respect thereto is the utilization of the distinctive interactions between metal ions and nucleic acids. For instance, certain cations can interact and form distinct complexes with nucleic acids.195 Metal ions can induce conformational changes in specific DNA structures and even catalyze permanent cleavages in DNA/RNA sequences. One example of this is the formation of G-quadruplex structures with guanine-rich DNA sequences using potassium ions (K+).196 Numerous studies have also reported the selection of DNAzymes against lead, copper, uranium and lanthanide ions.197-200 Equally noteworthy, inorganic mercury (Hg2+) and silver (Ag+) bind to specific pyrimidine base pairs, forming interactions stronger than typical Watson-Crick base pairs; Hg2+ and Ag+ form T-Hg2+-T201,202 and C-Ag+-C linkages, respectively.203,204 The aforementioned interactions between certain metal ions and DNA have been utilized to develop a multitude of different sensors. To further increase their sensitivity and programmability, the HCR technique has recently been incorporated in a plethora of metal ion detection schemes. Li et al. combined HCR with the ssDNA-adsorption and fluorescencequenching properties of GO to detect Hg2+ with a 0.3 nM LOD.205 In this approach, a T-rich ssDNA initiator and fluorescently labeled hairpins were rationally designed, Figure 14. In the absence of Hg2+, the hairpins were adsorbed to the GO surface, thereby quenching their fluorescence. However, addition of Hg2+ bridged the initiator and the first hairpin to each other through T-Hg2+-T linkages, activating the HCR process. The resulting HCR dsDNA product was unable to adsorb to the GO surface and the fluorescence of the system was therefore recovered.

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In this study, the difference in the adsorption affinity of ssDNA and dsDNA was exploited for the fluorometric detection of Hg2+. Recently, our lab combined AuNPs and HCR technology for the programmable and colorimetric detection of Hg2+ and Ag+.66 In this system, AuNPs were functionalized with capture DNA strands which were able to bind to Hg2+ and Ag+ through T-Hg2+-T and T-Ag+-T bridges, respectively, Figure 15a. Two initiator strands were rationally designed, one initiator to program the nanosensor for Hg2+ and the other for Ag+, therefore triggering HCR on the AuNPs only in the presence of the corresponding target metal ion and the HCR-requisite hairpins. The individual and simultaneous detection of each metal ion was demonstrated in four combinations with detections as low as 10 pM, Figure 15b. The system was tested for selectivity using different metal ions and was found to be extremely specific to the programmed settings, Hg2+ or Ag+, Figure 15c. The nanosensor was also tested for the detection of the targets in biological and environmental samples. In a similar scenario, we recently modified this nanotechnology for the development of colorimetric AND and OR logic gates using Hg2+ and Ag+ as the input signals.206 HCR has also been used in combination with silver nanowires to detect Hg2+ and hemin/Gquadruplex nanowires to detect Ag+.207,208 Furthermore, the detection of copper,209 lead,210 and uranyl ions211 has also been demonstrated using HCR. Conclusion and Future Perspectives. Contemporary advancements in DNA nanotechnology have resulted in the construction of DNA origami nanostructures,10 molecular motors,52 drug-delivery systems,212 DNA-based biocomputing circuits213 and molecular logic gate nanodevices.214 Equally noteworthy are the functional bio-systems built using a 1D DNA nanotechnology process known as Hybridization Chain Reaction (HCR). The applications of HCR are multifaceted, however its impact on the development of chemical and biological sensors is remarkable because HCR offers an enzymefree amplification feature alternative to polymerase chain reaction (PCR). Furthermore, unlike PCR, HCR is not restricted by enzyme inhibiting factors or coexisting materials commonly found in serum and since HCR is a signal transduction amplifier, it intensifies the amplitude of the signal instead of replicating the target molecule. Therefore, HCR offers a simple and costeffective method for detecting low copies of a myriad of target molecule. In this review, we have provided a conspectus of scientific reports regarding nucleic acid, metabolite, protein and metal ion detection using HCR nanotechnology, Table 2. Although we

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have reviewed a significant number of original studies concerning HCR-based sensing, this review is not an exhaustive compendium of the entire spectrum of molecular target types evaluated. For example, HCR has also been tested for the detection of lipopolysaccharides,215 cytokines,216 base excision repair enzyme activity,217 post-translational modifications,218 telomerase activity,219 DNA methylation220 and gene expression during symbiosis.221 Furthermore, it is worth noting that HCR can be used in the development of a highly specific sensor provided that the initiator strand can be merged with a molecular recognition site, thereby triggering the self-assembly process. It is our contention that the applications of HCR are in their infancy and we anticipate far more novel and complex applications thereof for chemical and biological sensing in the near future. HCR is a 1D DNA self-assembly process that provides signal-amplification and programmability features to many molecular detection approaches. However, there are still several directions that need attention in order to achieve precise and reproducible signal readout. One challenge is to control the size of the HCR-assembly, the dsDNA polymer formed in response to the initiator, in the sensing schemes. In general, the HCR-product doesn’t have a uniform size, but rather a size ranging from hundreds of base-pairs to thousands. This gives a wide range of variability among different tests for the same target molecule. Thus, it is often challenging to predict or control the signal amplification process using HCR. With that in mind, the control over the size of the HCR-process will offer more accurate and precise quantification of the targets by taking the amplification degree in to consideration. Additionally, HCR provides a significant signal enhancement in the absence of any enzymeamplification steps. Yet, the detection of low copies of RNAs for disease diagnostic can still be a challenge. The dsDNA assembly with HCR has a threshold; beyond this threshold, larger sizes of dsDNAs are not observed, and even if observed, they cannot be easily incorporated into the detection scheme. Thus, another amplification step is often necessary with these types of samples, which makes the sensing approach challenging for real-world sample analyses. Another major challenge is using HCR for multi-analyte detection. In a typical approach, one set of hairpins (H1 & H2) is designed for each target. For example, for 5 targets, 10 hairpins are required for the HCR process. This not only limits its use for multi-analyte detection but also brings another issue about potential ‘cross-talk’ between the hairpins, which could activate HCR even in the absence of a target. Therefore, the interference and ‘cross-talking’ of each hairpin

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should be minimized in order to avoid a false-positive result. In order to avoid designing multiple hairpins and minimize potential ‘cross-talking’, our lab has used only one set of universal hairpins for multi-analyte detection by incorporating a bridging ssDNA (bDNA) between the hairpin 1 (H1) and the target. This approach enables using one set of hairpins for many different targets; however, for every single target, a different bDNA needs to be designed. Although it is easier to design a short bDNA than a new set of hairpins, it is still another experimental step in the detection scheme. Therefore, a computational toolkit that can offer rational hairpin designs and evaluate their interference with each other could potentially provide remarkable simplicity and a broader usage. Another potential challenge in the HCR process is that it is run under restricted experimental conditions (temperature, pH and ionic strength) in order to avoid denaturation of the hairpins. The unintended opening of a hairpin and assembly of a set of hairpins can, in principle, initiate the reaction and result in a false-positive result. Thus, it is important to control the experimental parameters, which are often difficult for certain detection scenarios. In point-of-care detection, for example, it is important to obtain rapid readout for on-site decision-making. A rapid HCR process with uniform size could facilitate more frequent use by the sensor community for the sensitive detection of biological and chemical molecules. Many of HCR’s sensing applications are related to various bio-diagnostic applications. For example, the detection of disease-relevant RNAs and proteins have been reported and correlated to particular human diseases, such as cancer.64,65,71,85,88,181 The majority of studies are focused on detection of disease biomarkers in lysed cells, biological fluids or buffered environment, however, there are a number of reports studying miRNA detection in live and fixed cells.60,84,146 The transportation of HCR components, particularly hairpins, is unfortunately challenging due to its size and charge; therefore, a practical delivery approach will likely increase its use in in vitro settings for the detection of overexpressed RNAs or protein markers. Acknowledgements. This work was supported by the USDA National Institute of Food and Agriculture (NIFA), AFRI project (2018-67021-27973, 2017-07822). Vocabulary: Hybridization chain reaction: One-dimensional dsDNA assembly process initiated in the presence of a specific short ssDNA. Isothermal amplification: Amplification of DNA strands without heating and cooling cycles.

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DNA origami: Folding and assembly of DNA into well-defined two- and three-dimensional structures. Aptamer: ssDNA or RNA molecule that binds to a specific target molecule. DNAzyme: DNA molecule with catalytic activity. Systematic Evolution of Ligands by Exponential Enrichment (SELEX): Combinatorial method for producing specific oligonucleotides against a target molecule. Immunogenicity: Ability of a molecule to provoke an immune response. Endogenous: Originating or produced within a living system. Initiator: Molecule that triggers a polymerization reaction. Hairpin: ssDNA or RNA with an intramolecular stem-loop base-pairing. Concatemer: Long dsDNA that contains multiple copies of the same DNA sequence. References.

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Figures:

Figure 1. Hybridization Chain Reaction initiation by activation of metastable H1 and H2 hairpins with the initiator strand (I). The initiator binds and activates H1, exposing a sticky end capable of activating and binding to H2. The opening of H2 activates and binds to another H1, thereby triggering the perpetuation of the HCR process. As a result, a long dsDNA product assembles from two hairpin monomers only in the presence of the initiator.

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Figure 2. a) ATP detection using the HCR-hairpin and DNA aptamer assembly, releasing a sticky end that triggers HCR after ATP binds with the DNA aptamer. b) ATP (lane 6) and GTP (lane 7, control) induced HCR. Lane 8: DNA ladder. Reprinted with permission from ref. [46]. Copyright (2004) National Academy of Sciences.

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Figure 3. Schematics of the HCR process triggered with a sequence-specific initiator strand. a) H1 and H2 hairpins, b) opening of H1 with initiator, and c) opening of H2 with I:H1 complex. d) Fluorescence quenching confirming the HCR process. Reprinted with permission from ref. [46]. Copyright (2004) National Academy of Sciences.

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Figure 4. Schematic representation of the amplified DNA detection based on HCR in combination with cationic polyβ-CD enhanced pyrene fluorescence. Reprinted with permission from ref. [87]. Copyright (2016) Royal Society of Chemistry.

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Figure 5. a) Schematic illustration of the electrochemical miRNA-122b detection; probes are immobilized on a 3D tetrahedral scaffold and the signal is amplified by HCR. Reprinted with permission from ref. [94]. Copyright (2014) American Chemical Society. b) Schematic illustration of the HCR process on a metal surface and target detection using an electrochemical SPR approach. Reprinted with permission from ref [95]. Copyright (2014) Elsevier.

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Figure 6. a) Schematic illustration of the electrochemical miRNA detection using HCR and methylene blue on an ITO surface. Reprinted with permission from ref. [96]. Copyright (2015) American Chemical Society. b) HCR and EXPAR amplification process on a AuE surface for electrochemical target DNA detection. Reprinted with permission from ref. [97]. Copyright (2015) Elsevier.

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Figure 7. Schematic illustration of the colorimetric detection of a target oligonucleotide using HCR and AuNPs with a) noncovalent, and b) covalent surface attachments. Reprinted with permission from ref [128] and [130], respectively. Copyright (2013) American Chemical Society and (2014) Elsevier.

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Figure 8. a) Schematic representation of the colorimetric and reprogrammable detection of three different oncomiRs using AuNPs and HCR. b) Programming the AuNPs for the simultaneous detection of the three oncomiRs in combination of two and three in groups. c) Colorimetric detection of endogenous miR-10b. Reprinted with permission from ref. [65]. Copyright (2016) Royal Society of Chemistry.

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Figure 9. a) Schematic illustration of the colorimetric and reprogrammable detection of four different ebola virus-associated DNA targets using AuNPs and HCR. b) Detection and classification of the DNA target subtypes in human urine samples using the programmability properties of HCR. Reprinted with permission from ref. [64]. Copyright (2017) John Wiley and Sons.

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Figure 10. Schematic representation of the a) fluorescence polarization assay for nucleic acid detection using SA-SiNPs and HCR, and b) label-free DNA detection using Cu2+ reduction on the HCR product. Reprinted with permission from refs. [133] and [134], respectively. Copyright (2015) and (2014) Elsevier.

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Figure 11. Schematic illustration of the a) target DNA detection using AgNCs labeled hairpins, graphene oxide and HCR, and b) intracellular multicolor miRNA detection using graphene oxide and HCR. Reprinted with permission from refs. [144] and [146], respectively. Copyright (2017) Elsevier and (2016) Royal Society of Chemistry.

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Figure 12. Schematic illustration of the electrochemical nucleic acid detection using HCR and AuNPs a) without a dsDNA denaturation step, and b) with AgNP-decorated hairpins. Reprinted with permission from refs. [149] and [151], respectively. Copyright (2015) Elsevier and (2015) Royal Society of Chemistry.

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Figure 13. Schematic illustration of the a) SPR assay for ATP detection, and b) fluorescence approach for biothiol analysis using HCR. Reprinted with permission from refs. [166] and [67], respectively. Copyright (2014) Royal Society of Chemistry.

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Figure 14. Schematic illustration of the graphene oxide and HCR-based fluorescent sensor for Hg2+ detection. Reprinted with permission from ref. [205]. Copyright (2014) American Chemical Society.

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Figure 15. a) Schematic illustration of the colorimetric and reprogrammable detection of Hg2+ and Ag+ using HCR and AuNPs. b) Detection of different concentrations of Hg2+ and Ag+. c) Specificity of the colorimetric assay with various metal ions. Reprinted with permission from ref. [66]. Copyright (2017) Royal Society of Chemistry.

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Table 1: H1&H2 hairpin pair design and an initiator sequence for triggering HCR.

DNA strand

HCR Oligonucleotide Designs (Reference: 64-66, 206)

H1

5'-TTAACCCACGCCGAATCCTAGACTCAAAGTAGTCTAGGATTCGGCGTG-3'

H2

5'-AGTCTAGGATTCGGCGTGGGTTAACACGCCGAATCCTAGACTACTTTG-3'

Initiator sequence (I) 5'-AGTCTAGGATTCGGCGTGGGTTAA-3'

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Table 2: Analytical approaches using HCR for chemical and biological sensing. Analytical Method

Target (Type)

Signal Reporter

Platform

Nanoparticle

Limit of Detection or Performance

Reference

Nucleic Acid Detection Fluorescence

mRNA

Five Fluorophores

In Situ

N/A

N/A

Fluorescence

miRNA

Alexa Dyes

RNA Extract

N/A

25 amol

85

Fluorescence

DNA

Pyrene Excimer

In Solution/Human Serum

N/A

fM range

86

Fluorescence

DNA

Pyrene

In Solution

N/A

0.1 nM

87

Fluorescence

miRNA (miR141)

FAM/Dabcyl

In Solution

N/A

0.3 fM

88

Fluorescence

DNA

FAM/BHQ-1 In Solution TAMRA/BHQ-2

N/A

250 nM (tested)

89

Fluorescence

miRNA (miR141)

SYBR Green

Bovine Serum

N/A

0.55 fmol

47

Fluorescence

DNA (KRAS gene)

ATMND

In Solution

N/A

0.2 nM

91

ABTS

In Solution

N/A

7.5 nM/4 nM

92

Visual/Fluorescence DNA

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Electrochemistry

DNA/miRNA

HRP

Gold Electrode

N/A

100 aM/10 aM

94

Electrochemistry

DNA (C. parvum)

Label-free

Metal Surface

N/A

0.1-0.5 µM range

95

Electrochemistry

miRNA (let7a)

Label-free MetB

Indium Tin Oxide Electrode

N/A

1 pM

96

Electrochemistry

DNA (H7N9)

TMB

Gold Electrode

N/A

9.4 fM

97

Electrochemistry

miRNA (miRNA199a)

Label-free AgNC

Gold Electrode

N/A

0.64 fM

100

ECL

DNA (6S rRNA gene)

Ru(phen)32+

Gold Electrode

N/A

15 fM

103

128

Nanoparticle-based approaches In Solution

AuNP

50 pM (spectroscopic); 100 pM (visual)

Colorimetric

DNA

Label-free (AuNP)

Colorimetric

DNA

AuNP (DNAfunctionalized)

In Solution

AuNP

157 pM

129

Colorimetric

DNA


In Solution

AuNP

0.5 nM (visual)

130

Colorimetric

Three miRNAs


In Solution

AuNP

20 fmol

65

Colorimetric

Four DNA strands (Ebola)


In Solution

AuNP

400 amol

64

67


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Page 68 of 71

Fluorometric

DNA

FAM

In Solution

SiO2 NP

35 pM

133

Fluorometric

DNA (E. Coli)

CuNPs as Fluorophores

Streptavidin Beads

CuNP

0.4 nM

134

Fluorometric

DNA (HIV)

AgNCs as Fluorophores

In Solution

GO-based AgNC

1.18 nM

144

Fluorometric

miRNA (let7a)

FAM

In Solution

GO

1 pM

145

Fluorometric

Two miRNAs

FAM & ROX

In Situ

GO

N/A

146

Fluorometric (FRET)

miRNA (miR21)

FAM and ROX

In Situ

MnO2 nanosheet

N/A

148

Electrochemistry

dsDNA

Ferrocene

Gold Electrode

AuNP

2.6 pM

149

Electrochemistry

DNA (HIV)

Label-free (AgNP)

Gold Electrode

AgNP

0.5 fM

150

Electrochemistry

miRNA

Label-free (AgNP)

Gold Electrode (Endogenous miRNA)

AgNP and AuNP

2 aM

151

ECL

DNA (HIV)

Luminol

Gold Electrode

AuNP

5 fM

152

ECL

miRNA (miR155)

Hemin

Carbon Electrode

AuNP

1.67 fM

153

SERS

miRNA

AuNP@4-ABT

Human Serum

AgNP and AuNP

0.3 fM

154

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ACS Sensors

SERS

miRNA (miR141)

AuNP@ROXDNA

Magnetic Beads (RNA Extract)

AuNP

0.17 fM

155

SERS

DNA (Bt gene)

AuNP@ROX

Gold Electrode

AuNP

50 pM

156

Fluorescence

ATP

2-aminopurine

In Solution

N/A

N/A

46

Colorimetric

ATP

AuNP (labelfree)

In Solution

AuNP

1 nM

164

Electrochemistry

ATP

AgNC (labelfree)

Gold Electrode

AgNC

30 fM

165

SPR

ATP

Ferrocene

Gold Chip

N/A

0.48 nM

166

Fluorescence

GSH and cysteine (Cys)

FAM

In Solution

GO

0.1 nM (GSH) 0.08 nM (Cys)

67

Electrochemistry

GSH

[Ru(NH3)6]3+

Gold Electrode

N/A

0.6 nM

170

Fluorescence

OTA

Perylene

In Solution

Magnetic NP

0.10 pM

172

Human IgG

SYBR Green

Magnetic Beads

N/A

14 aM

177

Metabolite detection

Protein detection Fluorescence

69


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Page 70 of 71

Fluorescence

PDGF-BB

SYBR Green

In Solution

GO

1.25 pM

178

Fluorescence

AGR2

Flurophore

In Solution

AuNP

2.65 pM

179

0.25 pg mL−1 (AFP); 0.17 pg mL−1 (PSA) 0.02 pg mL−1 (AFP); 0.03 pg mL−1 (CEA)

Electrochemistry

AFP and PSA

MetB and Ferrocene

Glassy Carbon Electrode

N/A

Electrochemistry

AFP and CEA

MetB and DOX


N/A

Electrochemistry

CEA

4-chloro-1naphthol

Glassy Carbon Electrode (Human serum)

AuNP

0.42 pg mL-1

175

184

71

180

Electrochemistry

AFP, CEA, CA and PSA

Four Redox Probes

Au-Graphene Film

AuNPMagnetic NP

62 (AFP); 48 (CEA); 77 (CA); and 60 (PSA) fg mL−1

Electrochemistry

IgG

Luminol


GO

33 fg mL−1

185

Fluorescence

Inorganic Mercury

FAM

In Solution

GO

0.3 nM

205

Colorimetric

Inorganic Mercury and Silver


In Solution

AuNP

10 pM visual

66

Metal ion detection

70


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ACS Sensors

Table of Contents Figure:

71


Chemical and Biological Sensing using Hybridization Chain Reaction

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