Aptamers Facilitating Amplified Detection of Biomolecules - Analytical

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Aptamers Facilitate Amplified Detection of Biomolecules Feng Li, Hongquan Zhang, Zhixin Wang, Ashley M Newbigging, Michael S Reid, Xing-Fang Li, and X. Chris Le Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac5037236 • Publication Date (Web): 14 Oct 2014 Downloaded from http://pubs.acs.org on October 19, 2014

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Analytical Chemistry

Aptamers Facilitate Amplified Detection of Biomolecules Feng Li†§, Hongquan Zhang†§, Zhixin Wang†, Ashley M. Newbigging†, Michael S. Reid‡, Xing-Fang Li†, and X. Chris Le†‡*

†

Department of Laboratory Medicine and Pathology, ‡Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G3

* Corresponding author. Phone (780) 492-6416; Email: [email protected]

§ These authors contributed equally.

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CONTENTS 1. Introduction 2. Selecting aptamers amenable to amplified detection of biomolecules 3. Aptamer-facilitated assays using nucleic acid amplification 3.1 PCR-assisted assays 3.1.1 Aptamer-based immuno-PCR assays 3.1.2 Homogeneous assays incorporating aptamer binding and PCR amplification 3.2 Assays incorporating isothermal amplification 3.2.1 Assays incorporating rolling-circle amplification 3.2.2 Assays incorporating strand displacement amplification 3.2.3 Nicking Endonuclease-assisted amplification assays 3.2.4 Exonuclease-assisted amplification assays 3.3 Amplification assays based on toehold-mediated DNA strand displacement 4. Aptamer-facilitated assays using signal amplification 4.1 Signal amplification through enzymes 4.2 Signal amplification through DNAzymes 4.3 Signal amplification through nanomaterials 4.3.1 Aptamer-functionalized metallic nanoparticles 4.3.2 Nanofabrication techniques for amplified aptamer assays 5. Conclusions and Perspectives

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1. INTRODUCTION Since they were first reported by two independent research groups1,2 in 1990, aptamers have been widely used in analytical detection, separation, diagnostics, imaging and therapeutics. Aptamers are synthetic single-stranded DNA or RNA molecules. They are shorter than 100 nucleotides (n.t.) in length, and they generally have high binding affinity and selectivity for specific targets, ranging from macromolecules (e.g., proteins) to small molecules (e.g., metal ions). A number of recent reviews have summarized applications of aptamers to diagnostics,3,4 molecular imaging,5 therapeutics,6 and sensing.7,8 Our present review is unique in that our focus is on recent bioanalytical techniques that harness special properties of aptamers for signal amplification. Rather than providing a comprehensive review on all analytical applications of aptamers, we emphasize the special features of aptamers and discuss how they enable the amplified detection of biomolecules. Most of these approaches would not be achievable with the use of any other affinity ligand. Signal amplification is a key component in ultra-sensitive assays to detect trace amounts of analytical targets, such as various macro- and small molecules of biological significance, disease biomarkers, natural toxins, bacterial pathogens or viruses, and environmental contaminants.9-11 Two general strategies have been developed to achieve amplified detection: amplification of the target molecule or its surrogate molecule and amplification of the detection signal. In the first strategy, a binding event triggers an amplification process that generates more of the target or its surrogate. The second strategy relies on a catalytic reporter (e.g., enzymes in enzyme-linked immunosorbent assays) or a physical enhancement process (e.g., surface enhanced Raman scattering) to increase the signal resulting from a binding event.

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Aptamers have several intrinsic properties that allow for amplified detection of various biomolecules. (1) The binding of aptamers to their specific targets depends on their secondary and tertiary structures.10,12 Therefore, a variety of structure-switching mechanisms can be developed to facilitate the transduction of target recognition events to either target- or signalbased amplifications. (2) Aptamers are single-stranded nucleic acids and thus are ideal amplifiable surrogates for any non-nucleic acid targets.11,13,14 This extends the benefits of many amplification techniques originally designed only for nucleic acids to other molecules. For example, aptamers can be manipulated and amplified by a broad range of enzymes, such as polymerases, ligases, endonucleases and exonucleases, enabling the use of diverse enzymeassisted amplification mechanisms. Enzyme-free amplification techniques can also be designed using structure-switching aptamers, allowing for signal amplification of biomolecules and implementation of assays under resource limited conditions. (3) Aptamers are highly negatively charged biopolymers.15 Therefore, when binding to protein targets that are usually larger and much less charged, the resulting aptamer-protein complexes introduce substantial changes to electrophoretic mobility. Large differences in electrophoretic mobility between the aptamerprotein complexes and the unbound aptamers and proteins enable their electrophoretic separation and subsequent signal amplification. (4) Aptamers have relatively low molecular weights and simpler structures as compared to protein-based affinity ligands. When conjugated to nanomaterials or immobilized on a solid support, aptamers can form compact clusters. This allows multivalent affinity interactions, which enhances target binding.16,17 (5) Aptamers have unique adsorptive features on many metallic- and carbon-based nanomaterials, which can be tailored into switches to trigger signal amplifications.18 (6) Aptamers can be engineered to enhance their target selectivity and avoid cross-reactivity.

Non-specific binding to sample

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matrix components is commonly encountered in antibody-based amplification assays. Selectivity of aptamers can be enhanced by adding negative (counter) selections during the aptamer selection process or by using post-selection chemical modifications to alter aptamer sequences and structures. 19,20 These unique properties of aptamers enable the development of various strategies for amplified detection of biomolecules. These strategies benefit from aptamers that possess properties unavailable to other affinity ligands. Here, we illustrate how these properties of aptamers are explored to design signal amplification strategies and how these strategies are used for amplified detection of biomolecules.

2.

SELECTING

APTAMERS

AMENABLE

TO

AMPLIFIED

DETECTION

OF

BIOMOLECULES Most aptamers are generated through an in vitro evolution process, known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX, Figure 1), from a large number of random DNA or RNA sequences (up to 1016 sequences).19-21 This artificial evolutionary process does not require the use of any animals or cell lines that are required for generation of antibodies; thus, the production of aptamers is much easier than the production of antibodies. Once the sequence is identified, aptamers can be easily produced in large quantities through chemical synthesis. Being oligonucleotides, aptamers share the following common properties of DNA and/or RNA, including programmability through Watson-Crick base pairing, predictable secondary or tertiary structures, high chemical stability and site-specific modification of various functional groups. Moreover, chemically modified nucleotides that expand the natural genetic alphabet can be incorporated into aptamers, which greatly enhances binding affinity, selectivity, stability and functionality.22,23 5



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Although many aptamers have been identified over the past two decades, most current aptamer-based assays are concentrated on a few well known model targets,24 including thrombin, adenosine, platelet derived growth factor BB (PDGF-BB), and vascular endothelial growth factor. The lack of well-characterized aptamers with high binding affinity and selectivity has long been a bottleneck. Chemically modified or artificial nucleotides have been incorporated into the SELEX process to expand the target diversity of aptamers,25-27 leading to the creation of thousands of high quality aptamers. Gold and coworkers25 incorporated chemically modified uridine analogs (e.g., benzyl, naphthyl, tryptamino and isobutyl) into the DNA library for SELEX and generated thousands of slow off-rate modified aptamers (SOMAmers). Dissociation constants (Kd) were at the sub-nanomolar level for complexes between these aptamers and human proteins that covered the full isoelectric spectrum of the predicted human proteome. Importantly, an application of these SOMAmers enabled simultaneous detection of 813 protein biomarkers with picomolar (pM) level detection limits and an overall dynamic range spanning seven orders of magnitude.25 This excellent performance is mainly because modifications with hydrophobic functional groups allow aptamers to possess many “protein-like” properties, which dramatically improves their binding affinity and selectivity compared to aptamers with only natural bases.25,26 Recent advances in high-throughput sequencing (also known as Next Generation Sequencing) techniques and bioinformatics have also accelerated the identification of aptamers in the SELEX process.28,29 Traditionally,, bacterial cloning and Sanger sequencing techniques were used but identified only a few aptamer sequences from the enriched DNA pool obtained after selection. However, high-throughput sequencing provides quantitative data to identify millions of binding aptamer sequences in each round of selection.30-33 High quality aptamers can

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then be effectively selected in a significantly reduced number of rounds.30,34 Because of the great expansion in aptamer diversity through modification and an accelerated discovery processes, it is now possible to expand aptamer facilitated amplification strategies to many more targets beyond the classic models. SELEX can also be designed to directly generate aptamers with special features, which are critical in designing many amplification strategies. Two distinct aptamer sequences that bind to different epitopes of targets are required for many signal amplification mechanisms, such as aptamer-based immuno-PCR, enzyme-linked aptamer assays, proximity ligation assays35 and binding-induced DNA assembly strategies36. To meet such needs, Gong et al.37 generated aptamer pairs for integrin αVβ3 by using two different subunits of integrin as targets in an additional round of SELEX. Ochsner et al.38 used a “sandwich” SELEX strategy by using previously formed complexes of a protein and a primary SOMAmer as the new targets to select the second SOMAmer. Furthermore, structure-switching properties can also be directly implanted into aptamers through the SELEX process. Nutiu and Li first reported direct selection of structure-switching aptamers for ATP and GTP from a DNA library.39 The random sequence region of DNA library sequences were split by a region with a fixed sequence that can hybridize to a complementary oligonucleotide labeled with biotin. The duplex of library and complementary strands was captured on avidin-coated beads in the absence of target molecules. Upon exposure to the target molecule, the aptamer portion of the duplex bound to the target and caused the structure of the aptamers to change. This resulted in the dehybridization and release of the aptamer from the complementary strand. The released aptamers were collected and amplified for the next round of selection. Finally, structure-switching aptamers can be identified for subsequent applications. Other signal amplification components can also be incorporated into

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aptamers through SELEX. For example, the Soh group added DNA enzyme (DNAzyme) sequences to the center of DNA library sequences during SELEX to generate structure-switching aptamers that can amplify detection signal automatically upon target binding through the activation of DNAzymes.40 These in vitro strategies for aptamer discovery and the resulting special properties unique to aptamers enable the design of various signal amplification strategies not viable with other affinity ligands. In the following sections, we will describe these aptamerenabled signal amplification strategies in detail.

Figure 1. Schematic illustration of a typical SELEX process for aptamer discovery. Aptamers for a target of interest are identified through an in vitro SELEX process, which contains iterative rounds of incubation, partition and amplification. A typical SELEX process includes four components: (1) a library of random single-stranded sequences of DNA or RNA (approximately 1015 sequences) is incubated with a target to form oligonucleotide-target complexes; (2) unbound oligonucleotides are removed; (3) bound oligonucleotides are amplified to generate a new enriched single-stranded DNA/RNA pool as the input for the next round of selection; (4) aptamer sequences are identified and characterized from the enriched pool of the last round.

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3. APTAMER-FACILITATED ASSAYS USING NUCLEIC ACID AMPLIFICATION TECHNIQUES Highly sensitive and specific detection of nucleic acids can be readily achieved through a variety of nucleic acid amplification techniques including PCR, rolling circle amplification (RCA), strand displacement amplification (SDA), exonuclease-assisted signal amplification and nicking endonuclease-assisted signal amplification. Unlike nucleic acids, proteins and other biomolecules cannot be directly amplified, making it challenging to detect trace amounts proteins and other biomolecules. To expand nucleic acid amplification techniques to non-nucleic acid targets, extensive efforts have been devoted to couple immunoassays with nucleic acid amplification techniques by using nucleic acid-containing affinity probes. Due to their dual identities as both nucleic acids and affinity ligands, aptamers are ideal for converting the detection of proteins and other molecules into nucleic acid amplification.

3.1 PCR-assisted assays PCR is the most widely used method to amplify and quantify nucleic acids. Because PCR amplification is exponential, it enables the detection of as few as 10 nucleic acid molecules. As nucleic acid affinity ligands, aptamers possess a number of unique features that enable the use of PCR for the detection of non-nucleic acid biomolecules. For instance, aptamers can serve as both target-binding molecules and PCR templates in immuno-PCR assays, and can be easily extended or modified when required. Some of the unique features of aptamers explored to develop various aptamer-based amplification assays include the ability to switch structures, a strong negative charge and their stability from enzymatic degradation when bound to target.

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3.1.1 Aptamer-based immuno-PCR assays Immuno-PCR combines the exponential amplification power of PCR with the versatile and specific immunorecognition of ELISA to achieve highly sensitive detection of antigens.41 Conventional immuno-PCR requires antibodies conjugated with specific DNA sequences to be amplified by PCR to generate a detection signal. The conjugation is usually cumbersome and laborious which negatively impacts the applicability of immuno-PCR. This can be largely circumvented by using aptamers as the affinity ligands because aptamers can be easily extended and modified for direct amplification by PCR. Tok and coworkers demonstrated the use of DNA aptamers as both target-recognizing and signal-generating molecules by developing an immunoPCR assay for thrombin detection (Figure 2A).42 Antibody-coated magnetic beads were first used to capture thrombin from sample solutions. An aptamer probe consisting of a 15-nt antithrombin aptamer, a poly-A15 linker and a short 20-nt primer was then introduced to bind to the captured thrombin. After unbound aptamers were washed away, the bound aptamers were dissociated by heating and then subjected to real-time PCR detection. A LOD in the low pM range was obtained for the detection of thrombin in 10% serum. A similar format was applied to the detection of other proteins, such as human IgG and platelet derived growth factor BB (PDGF-BB). 43-45 To facilitate the removal of unbound components and aptamers, Soh et al. used a microfluidic device that trapped the antibody-coated magnetic beads by applying a highgradient magnetic field.45 A LOD of 66 fM was achieved for PDGF-BB in 20% serum. Aptamerbased immuno-PCR assays were also developed for the detection of E. coli and cancer cells.46,47 SOMAmers were also used to develop an immuno-PCR method for protein detection.48 In this method, an anti-epidermal growth factor receptor (EGFR) SOMAmer was labeled with a biotin moiety through a photo-cleavable linker.48 The biotinylated SOMAmer was then introduced to

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bind to EGFR in diluted serum samples, followed by the capture of all bound and unbound SOMAmers onto a streptavidin-coated plate. Unbound sample components were removed by washing and the captured proteins were labeled with another biotin moiety. All SOMAmers were then released from the plate by photo-induced cleavage of the linker between the SOMAmer and biotin. The released SOMAmer-protein complexes were captured on a second streptavidincoated plate. After the removal of unbound SOMAmers, the bound SOMAmers were dissociated from the complexes by using an alkaline buffer and quantitated by real-time PCR. This assay was able to distinguish EGFR from its drug-bound complex because of the SOMAmer’s high specificity. A dynamic range from 2.5 to 600 ng/mL EGFR was obtained. Structure-switching aptamers have been used to develop immuno-PCR methods for target molecules that can only be bound by a single aptamer molecule.49,50 When hybridized to complementary sequences, these aptamers exist as a duplex. Upon binding to their target molecules, the aptamers switch structures into specific secondary structures and dissociate from their complementary sequences.39 Xu and coworkers used structure-switching aptamers to demonstrate the sensitive detection of Ochrotoxin A (Figure 2B).49 A biotinylated antiOchrotoxin A aptamer was immobilized on the surface of a streptavidin-coated PCR tube. The complementary sequence was then introduced to hybridize with the aptamer. Upon target addition, the aptamers bound to the target and switched structures. This disrupted the duplex structure which released the complementary sequences from the tube surface. After the released complementary sequences were washed away, the remaining bound complementary sequences were amplified by real-time PCR. Ochrotoxin A was then quantified by measuring the increase of cycle numbers of PCR reaction due to the decreased amounts of DNA templates. This method

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was able to detect as low as 1 fg/mL of Ochrotoxin A. An assay using a similar structureswitching aptamer was developed to detect Aflatoxin B1 with a LOD of 25 fg/mL.50 Aptamers are highly negatively charged. Proteins are usually larger and much less charged than aptamers. As a result, protein-aptamer complexes can have a large differences in electrophoretic mobility compared to unbound aptamers and proteins. Based on this fact, we developed an affinity aptamer amplification assay for protein detection by using free zone capillary electrophoresis to separate the protein-aptamer complexes from unbound aptamers.15 Reverse transcriptase of human immunodeficiency virus (HIV-RT) was chosen as a model target to demonstrate the proof of concept. Anti-HIV-RT aptamers were first introduced to bind to HIV-RT, forming a protein-aptamer complex. The solution was then injected into the capillary for separation. Because complexes migrate faster than free aptamers, this allowed us to collect the fraction containing only the complexed molecules. The complexed aptamers were then amplified by PCR and analyzed by gel electrophoresis. Using capillary electrophoresis for separation obviates solid phase immobilization and the corresponding background from unspecific adsorption, resulting in high sensitivity. We were able to detect as few as 180 molecules of HIV-RT in a sample volume of 10 nL. Multiplexed protein detection was also demonstrated using a similar concept.51 Aptamers bound to protein molecules can be protected from nuclease degradation. Wang and coworkers utilized this property to develop an aptamer-based exonuclease protection assay for ultrasensitive protein detection.52 The binding of an aptamer to a thrombin molecule protected the aptamer from exonuclease I degradation. Exonuclease I was added to degrade unbound aptamer probes. The solution was then heated to inactivate the nuclease and release bound probes. For detection, the released probes served as connectors for enzymatic ligation of

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two subsequently added DNA sequences to form a new DNA sequence. Detection of the new DNA sequence by real-time PCR represented the detection of the target protein. The detection of as few as several hundred thrombin molecules was achieved. DNase I was also used to develop an analogous assay for the detection of ovine follicle-stimulating hormone α subunit with a LOD of 10 fM.53

3.1.2 Homogeneous assays incorporating aptamer binding and PCR amplification Homogeneous assays are carried out in solution without the need for separation or washing, which simplifies sample analysis.11 Because no washing steps are involved to remove unbound aptamer probes, it is not possible to directly amplify aptamer probes in the same way as aptamerbased immuno-PCR assays. Therefore, aptamer-based homogeneous assays with PCR amplification usually involve the conversion of aptamer-target binding events into the generation of new amplifiable DNA sequences; this can be achieved by incorporating DNA ligation or extension events into the assay design. Proximity ligation assays (PLA) generates a new amplifiable DNA sequence from the binding of a target molecule by two aptamer probes without separation or washing.35 PLA was originally developed by Landegren and coworkers to detect PDGF-BB by using two aptamer probes (Figure 2C). The two probes were prepared by extending anti-PDGF-BB aptamers with additional sequences at either the 5' or 3' end. When the two probes bound the same PDGF-BB molecule, the free ends of the probes were brought into close proximity. This allows an added connector DNA strand to juxtapose the two ends, which does not occur in the absence of the target molecule. The two ends were enzymatically ligated together, forming a new DNA sequence. This new sequence was then measured by real-time PCR for quantitative detection of PDGF-BB. The detection of as few as 2400 PDGF-BB molecules was achieved. The use of PLA 13



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to detect thrombin was also demonstrated.35,54 Using proximity probes prepared by a single aptamer, PLA also detected adjacent cell surface antigens.55 Motivated by PLA, we introduced the concept of binding-induced DNA assembly (BINDA).36,56 Distinct from DNA self-assembly that is dependent on spontaneous hybridization between complementary sequences, BINDA refers to DNA assembly that only occurs when a specific target triggers a binding event. BINDA has been demonstrated as an excellent strategy to develop versatile homogeneous assays.56-59 Using aptamers as affinity ligands, we used BINDA to develop a homogeneous assay for ultrasensitive detection of PDGF-BB (Figure 2D).36 Two probes were made by tagging two biotinylated aptamers with DNA motif-F or motif-R using streptavidin as a connecting molecule. The two motifs each contained a short complementary sequence so hybridization is unstable in the absence of target molecules. To further minimize self-hybridization between the two complementary sequences, we designed a pair of blocking oligonucleotides to compete with the two complementary sequences for hybridization. Upon the addition of PDGF-BB molecules, the binding of the two probes to a single target molecule placed the two DNA motifs in close proximity. This dramatically increased their local concentrations and lead to the hybridization between the complementary regions of the DNA motifs. Because motif-F consisted of a DNA hairpin structure at the 3' end, the assembly of the DNA motifs placed the 3' end of the hairpin sequence adjacent to 5' end of the complementary sequence of motif-R, allowing the two motifs to be enzymatically ligated together. This formed a new DNA sequence which was then detected by real-time PCR, enabling the ultrasensitive detection of PDGF-BB. A detection limit of 1 fM was achieved for PDGF-BB detection. Target-induced aptamer structure switching was also used to develop PCR-based homogeneous assays to detect protein targets. Yang and Ellington designed an aptamer that

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switched structures upon target binding and enabled the ligation of an additional sequence to the 3' end of the aptamer, forming a new DNA sequence.60 This new sequence was detected by realtime PCR. Low-pM detection limits were achieved for detection of thrombin and PDGF-BB. The binding of a protein molecule by a single aptamer is sufficient to trigger amplified detection of the target protein.

Figure 2. Amplification assays using PCR. (A) Immuno-PCR assay using aptamers as both target-recognizing and signal-generating molecules.42 (B) Structure switching aptamers achieve detection of non-nucleic acid targets by PCR.49 (C) Proximity ligation assay enables homogeneous detection of proteins and other biomolecules by using PCR.35 (D) Application of binding-induced DNA assembly for the detection of proteins by PCR.36

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3.2 Assays incorporating isothermal amplification PCR requires thermal cycling to achieve exponential amplification, which may not be favorable for resource-limited scenarios and on-site analysis. Complementing PCR, a variety of isothermal amplification techniques, including rolling circle amplification (RCA), strand displacement amplification (SDA), exonuclease-assisted signal amplification and nicking endonucleaseassisted signal amplification, have been developed to detect nucleic acids. These techniques have been extended to the detection of non-nucleic acid targets by taking advantage of special properties of aptamers.

3.2.1 Assays incorporating rolling-circle amplification RCA is an isothermal nucleic acid amplification process that synthesizes a long single-stranded nucleic acid product from continual and unidirectional replication of a circular, single-stranded nucleic acid template.61,62 Similar to immuno-PCR, the combination of RCA with ELISA is a common approach to develop RCA-based assays for the amplified detection of proteins and other targets.63-69 These assays usually use immobilized antibodies or aptamers to capture target molecules from sample solutions. Aptamer probes containing a primer region for annealing to the circular template of RCA are then introduced to bind to the captured target molecules. Unbound aptamer probes are washed away and RCA is performed. The RCA products are detected using various signal generation techniques. Yu and coworkers first described this approach using electrochemical detection to quantify RCA products.63 The assay detected as low as 10 fM PDGF-BB with a dynamic range of 4 orders of magnitude. Other techniques for detecting RCA products, including fluorescence detection, diffractometric detection,64 Gquadruplex-mediated

colorimetric

detection,65,69

chemiluminescence,66

surface

plasmon

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resonance and quartz crystal microbalance sensors,67,68 have also been incorporated into this assay format for amplified detection of various targets. Structure-switching aptamers are also used to develop aptamer-based RCA assays. For example, by switching their structures upon target binding, hybridized aptamers dissociate from their complementary sequences, resulting in the release of primers or circular templates for RCA. Assays based on this mechanism can be performed either on solid supports or in homogeneous solutions. For assays involving the use of solid supports, the duplexes between aptamers and complementary sequences are immobilized on solid supports by conjugation of either aptamers or complementary sequences.70-74 Upon binding to their targets, aptamers switch structure and dissociate from their complementary sequences. The dissociated complementary sequences that are either released in solution or immobilized on the solid supports are then detected by RCA using padlock probes or pre-prepared circular probes. Alternatively, Yin and coworkers prepared complementary sequences as circular structures that hybridized with aptamers conjugated to the solid phase.75 The binding of aptamers to target molecules dissociated the circular DNA from the solid phase. The remaining circular DNA sequences then served as templates for the extension of unbound aptamers using RCA. They were able to detect as low as 10 fM human vascular endothelial growth factor (VEGF) by using AuNP-enhanced silver staining to detect RCA products. Zhang and coworkers integrated aptamer complementary sequences into a polycatenated nanostructure that contained multiple interlocked circular structures, enabling the generation of multiple RCA products from a single nanostructure.76 The binding of aptamers to target molecules can also separate aptamers from their complementary sequences in homogeneous solutions.77-79 Since duplexes between aptamers and complementary sequences are not immobilized, both free and hybridized complementary sequences are present in the solution.

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Padlock probes are then added to detect these free complementary sequences by RCA. The hybridized sequences are not able to undergo RCA because padlock probes cannot hybridize to duplexed sequences. Yu et al. applied this approach to the homogeneous detection of PDGF-BB with a detection limit of 6.8 pM.77 Specially designed aptamers can also directly trigger RCA. Ellington et al. designed a structure-switching aptamer that can circularize upon target binding (Figure 3A).80 A 13-nt sequence was added to the 3'-end of an anti-PGDF-BB aptamer via a linker. This 13-nt sequence was complementary to the aptamer and disrupted its binding secondary structure by forming a hairpin. The aptamer was further modified by adding a 6-nt sequence to its 5'-end and altering the linker sequence so that target binding resulted in a closed ligation junction. Therefore, enzymatic ligation generated a circular DNA that served as a RCA template. This structureswitching aptamer was used for the homogeneous detection of PGDF-BB and a detection limit of 0.4 nM was achieved. The same research group also designed an allosteric DNAzyme that transduced target binding into the generation of a circular template for RCA (Figure 3B).81 The allosteric DNAzyme consisted of an anti-ATP DNA aptamer linked to a DNAzyme in such a way that the DNAzyme can only be activated when the aptamer bindsto ATP. The selected DNAzyme catalyzes DNA ligation and thus upon ATP binding, the allosteric DNAzyme enabled ligation and circularization of a DNA substrate which formed a padlock probe. The allosteric DNAzyme was used for amplified detection of ATP by RCA. Up to a 1500-fold increase in signal was obtained after 10 min of RCA. Aptamers are single-stranded nucleic acids and thus can be constructed into circular structures to directly serve as RCA templates. Di Giusto et al. described a proximity extension assay enabling homogeneous detection of various proteins (Figure 3C).82 They selected thrombin

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to demonstrate the proof of concept because there are two known aptamers that bind to different epitopes of thrombin. They constructed the first aptamer into a circular structure and added second aptamer to the 3'-end of a short RCA primer via a linker. In the absence of the target molecule, the annealing of the primer to the circular aptamer is unstable. When the two aptamers bound to the same thrombin molecule, it brought the primer in close proximity to the circular aptamer, which allowed the primer to anneal to the circular aptamer and initiate RCA. A detection limit of 30 pM was achieved for thrombin. Li and coworkers also used a circular aptamer for amplified protein detection by RCA.83 They found that the protein target binding to the aptamer inhibited phi 29 DNA polymerase from reading the bound domain. As a consequence, the presence of the target can suppress RCA proportional to the target concentration. They developed a simple colorimetric assay for thrombin using this principle. Aptamers can adsorb on graphene oxide (GO) and desorb upon target binding. Li and coworkers developed a RCA-mediated homogeneous assay for the amplified detection of proteins and small molecules using GO (Figure 3D).84 Aptamers containing RCA primers adsorbed to a GO surface disabled the occurrence of RCA. The binding of target molecules to aptamers released the aptamers from GO, thereby exposing the primers for RCA template annealing and RCA initiation. The detection of RCA products represented amplified detection of target molecules. The assay was used to detect thrombin and ATP.

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Figure 3. Assays incorporating rolling circle amplification (RCA). (A) RCA assay using a structure-switching aptamer that circularizes upon target binding.80 (B) Allosteric DNAzyme transducing target binding into generation of a circular RCA template.81 (C) Proximity extension assay using a circularized aptamer probe.82 (D) RCA-mediated assay based on target bindinginduced desorption of aptamers from graphene oxide (GO).84

3.2.2 Assays incorporating strand displacement amplification Strand displacement amplification (SDA) relies on the combined use of a strand-displacing DNA polymerase and a nicking endonuclease for isothermal amplification of nucleic acids.85 SDA starts with primer polymerase extension reaction that creates a new complementary strand with a nicking site that is cleaved by a nicking endonuclease. The 3'-end of the nick can be further extended by the DNA polymerase, which displaces the downstream strand and regenerates the nicking site. This process cycles continuously, resulting in isothermal amplification of nucleic 20


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acids. SDA can enable exponential amplification when the newly synthesized DNA serves as a SDA primer.86 Because of its enormous amplification power, SDA is widely used to detect nucleic acids. Attempts to use SDA for detection of other targets mainly focus on the conversion of target binding into DNA detection, in which aptamers have been extensively used. Structure-switching aptamers are predominantly used in SDA for amplified detection of proteins and small molecules. One strategy is to design the aptamers into hairpins.87-90 Yu and coworkers described a target-induced SDA assay for the amplified detection of cocaine (Figure 4A).87 An anti-cocaine aptamer split into halves was designed as a structure-switching aptamer. The first half of the aptamer was extended to form a hairpin to cage the primer-annealing region. Binding of the split aptamer to cocaine disrupted the hairpin and freed the primer-annealing region. This triggered primer polymerase extension which displaced the second half of the aptamer from the binding complex, and released cocaine. The released cocaine could bind to another split aptamer and initiate new amplification cycles. Besides split aptamers, the same research group further demonstrated the detection of PDGF-BB by a regular aptamer designed to form a hairpin in a similar manner.88 The concept also allowed the detection of other targets by using other signal generation approaches, such as FRET.89,90 One important feature of these methods is that amplification cycles are driven by polymerase extension-mediated displacement of target molecules from complexes. In contrast, the formation of protein-aptamer complexes was found to inhibit primed polymerase extension, and this allowed the development of another method for amplified detection of thrombin using SDA.91 Structure-switching aptamers prepared with complementary sequences can also aid in developing SDA-assisted methods. Zhang et al. added a complementary sequence to the 5’-end of the anti-PDGF-BB aptamer which switched the aptamer into a non-binding secondary

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structure (Figure 4B).92 The binding of the target protein to the aptamer dissociated the complementary sequence and allowed the aptamer to fold into its binding secondary structure. The 3'-end of the aptamer then served as a primer and initiated SDA of the sequence that was added to the 5'-end of the aptamer. The newly synthesized SDA products acted as primers and annealed to EXponential Amplification Reaction (EXPAR) templates (which contained two copies of the same sequence), resulting in exponential amplification. This two-stage amplification detected as low as 0.9 pM PDGF-BB. Similarly, Wang et al. designed a structureswitching aptamer for the amplified detection of cocaine.93 Instead of using EXPAR for the second stage of amplification, SDA products were designed to be DNAzymes that initiated second stage amplification by catalyzing the cleavage of its substrates. This type of structure switching aptamer was also immobilized on magnetic beads, enabling the incorporation of washes and additional amplification stages.94-97 Sandwiching of a single target molecule by two aptamers can also achieve amplified detection of proteins by SDA. Tan et al. developed a proximity-dependent homogeneous method for protein detection.98 They designed a pair of proximity probes by extending two anti-thrombin aptamers with a specific oligonucleotide. Each oligonucleotide contained a short complementary sequence and a sequence for subsequent SDA amplification. The complementary sequences were short so hybridization was unstable in the absence of the target molecule. When two probes bound to a single thrombin molecule, it brought the two complementary sequences into close proximity, resulting in hybridization between the two sequences and initiation of SDA amplification. This assay obtained a detection limit of 1 pM for thrombin. The binding of two types of aptamers, unlabeled and biotinylated, to a single cell also enabled the development of a lateral flow biosensor for pathogens.99

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3.2.3 Nicking Endonuclease-assisted amplification assays Nicking endonucleases (NEases) are a class of enzymes that catalyze the hydrolysis of only one strand of a duplex at specific recognition sites, i.e.; nicking one strand and leaving the other strand intact. NEases are used to develop Nicking Enzyme Signal Amplification (NESA) assays for nucleic acids. In NESA, the target DNA hybridizes with a signalling DNA, forming the recognition site of a NEase and initiating NEase-catalyzed hydrolysis of the signalling DNA. Consequently, the target DNA is released for the next cycle of reaction, resulting in recycled signal amplification. Aptamers can be used in NEase-assisted amplification assays to detect targets other than nucleic acids. The main strategy for the development of these NEase-assisted amplification assays is using structure-switching aptamers to generate sequences for NESA. One common aptamer design is to use a hairpin to cage the target sequence of NESA. Upon target binding, the formation of aptamer-protein complexes disrupts the hairpin, making the target sequence available to NESA. Xue et al. extended a thrombin aptamer to form a hairpin probe (Figure 4C).100 The binding of the aptamer to thrombin opened the hairpin, which enabled hybridization with a signalling sequence that is dual labeled with a fluorophore and a quencher. This initiated repeated (cyclic) cleavage of the signalling sequence which generated an amplified fluorescence signal. A detection limit of 100 pM was obtained. Similar assays were developed for other proteins and small molecules using alternative signal reporters such as molecular beacons, DNAfunctionalized AuNPs and DNAzymes.101-103 Hairpin aptamer probes have also been used to develop assays for the detection of membrane proteins on cancer cells.104,105 Complementary sequences can hybridize with aptamers to construct structure-switching aptamers for NEase-assisted amplification assays. When the aptamer binds the target, the

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complementary sequence dissociates and serves as the target sequence for NESA.106,107 Based on this principle, Hun et al. developed an amplification assay for lysozyme using chemiluminescence detection.106 The complementary sequence dissociated upon target binding and hybridized with DNA-functionalized nanoparticle chemiluminescent probes, which initiated cyclic cleavage of the probes from magnetic beads and resulted in an amplified chemiluminescent signal. The detection of complementary sequences by NESA can be further improved by coupling it to secondary amplifications, such as strand displacement amplification and another NESA.108,109 Graphene oxide (GO) is known to preferentially adsorb single-stranded DNA; thus GO can act as a scaffold for aptamer probes and fluorescently-labeled signalling DNA to develop assays for small molecules and proteins. Huang et al. extended an adenosine aptamer with a 10nt additional sequence and designed a fluorescently-labeled signal DNA consisting of a complementary sequence to the 10-nt addition.110 In the absence of target molecules, GO strongly adsorbed both aptamer and signal DNA, which disabled hybridization between them. Fluorescence polarization of the adsorbed signal DNA probe was high due to the large size of GO. Upon target binding, the aptamer dissociated from GO, which enabled hybridization between the aptamer and signal DNA. This duplex contained the recognition site of a nicking enzyme that triggered the cyclic cleavage of the fluorescently-labeled end of the signal DNA, resulting in a substantial decrease in fluorescence polarization. The assay detected as low as 2 pM adenosine. Thrombin detection was also demonstrated using the assay.

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Figure 4. Amplification assays incorporating strand displacement amplification (SDA) and/or nicking endonuclease-assisted signal amplification. (A) Amplified detection of cocaine using 25



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polymerase extension-mediated displacement of target molecules87. (B) Amplified detection of PDGF-BB using SDA.92 (C) Amplified detection of thrombin using nicking endonucleaseassisted signal amplification.100 Reprinted from references 87, 92, and 100.

3.2.4 Exonuclease-assisted amplification assays Exonucleases (Exos) are a family of enzymes that catalyze the stepwise removal of mononucleotides at either the 3'-end or 5'-end from single-stranded or double-stranded DNA. Three Exos, named Exo-III, RecJf, and Exo-I, have mainly been used to develop aptamer-based amplification assays for proteins and small molecules. These assays commonly rely on structureswitching aptamers that respond differently to Exo-catalyzed hydrolysis upon target binding. In these assays, signaling sequences can be the aptamers themselves or complement sequences to aptamers. Target binding enables repeated (cyclic) hydrolysis of the signaling sequences, resulting in an amplified signal for target detection. Exo-III removes mononucleotides from the 3'-end of duplexed DNA. Exo-III is much less active in the hydrolysis of unpaired 3'-ends. Therefore, duplexed aptamers with 3' overhangs are resistant to Exo-III digestion. If target binding switches the aptamer into a folded secondary structure where the 3'-end is paired, Exo-III hydrolyzes the aptamer. Willner and coworkers demonstrated the combination of split aptamers with Exo-III’s selectivity for the amplified detection of proteins (Figure 5A). 111 Thrombin was chosen to demonstrate the proof of concept. An aptamer for thrombin was split into two subunits. One subunit was labeled with a quencher and fluorophore at its 3'- and 5'-ends, respectively. In the absence of thrombin, both subunits were single stranded and therefore resistant to Exo-III digestion. The binding of the split aptamer to a thrombin molecule allowed the two subunits to assemble into a G-quadruplex, pairing the

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quencher-labeled 3'-end with the 5'-end of the other subunit. Exo-III was then able to cleave the 3'-end, restoring fluorescence. The cleavage also disrupted the protein-aptamer complex, releasing thrombin to bind to another split aptamer. This process cycled approximately 1000 times in 12 min. As low as 89 pM thrombin was detected. A similar assay was also used to detect VEGF.112 Exo-III assisted assays were also developed using structure-switching aptamers. Liu et al. designed a hairpin anti-ATP aptamer with a 3' overhang to protect unbound aptamers from ExoIII digestion (Figure 5B).113 Upon target binding, the aptamer folded into its secondary structure, pairing the 3'-end. Exo-III then hydrolyzed the 3'-end of the aptamer, destroying the complex and freeing ATP for further binding. The cyclic cleavage of the aptamer resulted in amplified electrochemical detection of ATP. Xu et al. designed a similar assay for ATP detection by using fluorescence detection.114 An alternative design also used aptamers with a hairpin; however, target binding disrupted the hairpin structure of the aptamer but did not enable pairing of its 3'end.115,116 Instead of enzymatic cleavage of the 3'-end of the aptamer, the structure change exposed a complementary sequence to a signaling sequence. This initiated cyclic cleavage of the signaling sequence by Exo-III. This strategy amplified the detection of lysozyme and thrombin. Gao et al. also designed aptamers with a hairpin, but added an additional sequence to the 3'end.117 Before target binding, the aptamer was single stranded. The added sequence of the aptamer hybridized to a signalling sequence, causing the enzymatic hydrolysis of the signalling sequence. Upon target binding, the formation of protein-aptamer complex prevented Exo-III from accessing the 3'-end of the hybridized signalling sequence, inhibiting hydrolysis. The free signalling sequence was then measured using DNA-functionalized AuNPs, enabling amplified protein detection.

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Assays were also developed using another type of structure-switching aptamer that relies on complementary sequences.118-122 The 3'-ends of aptamers were usually modified or designed to have an overhang. This modification and hybridization to the complementary sequence inhibited digestion by Exo-III. Upon target binding, the conformational changes of the aptamer dissociated aptamer sequences from their complementary sequences, initiating Exo-III-assisted digestion and signal amplification. Hu et al. designed a structure-switching aptamer for adenosine detection.118 When the aptamer bound to adenosine the complementary sequence dissociated, which then served as a DNA target for digestion of a signalling DNA. Target binding can also fold the aptamer into a structure cleavable by Exo-III; this allowed the development of a cascade recycling amplification assay for PDGF-BB.119 Yang et al. designed an aptamer where the 3'-end of the complementary sequence was extended to hybridize with a cleavage-triggering sequence.120 Target binding exposed the 3'-end of this sequence for Exo-III digestion. RecJf removes mononucleotides from single-stranded DNA in a 5' to 3' direction. Structure-switching aptamers can be designed for RecJf-aided cyclic cleavage; these aptamers are commonly generated with complementary sequences. Tong et al. hybridized a complementary sequence to an aptamer for Ochratoxin A, inhibiting RecJf digestion of the aptamer.123 The formation of target-aptamer complexes disrupts the duplex, allowing the aptamer to fold into its binding secondary structure which is susceptible to RecJf digestion. RecJf digestion then initiated target recycling amplification. A similar aptamer design was used to develop RecJf-assisted assays for Ochratoxin A and thrombin, but using different means for signal generation.124-127

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Exo-I is also a single-stranded DNA specific exonuclease that catalyzes the removal of mononucleotides in a 3' to 5' direction. Wu et al. used a similar aptamer design to that of RecJfaided methods to develop an assay for the amplified detection of staphylococcal enterotoxin B.128 Target binding placed the aptamer into an Exo-I cleavable structure, which then enabled target recycling. Aptamers can also be protected from Exo-I digestion when the 3' end of aptamers are caged as a duplex or G-quadruplex upon target binding.129 This can separate unbound aptamers from bound aptamers which can then be detected by Exo-III-assisted recycling amplification.

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Figure 5. Exonuclease-assisted amplification assays. (A) Amplified detection of thrombin using split aptamers and Exonuclease-assisted signal amplification. Reprinted with permission from reference 111. (B) Using hairpin aptamer probes to enable exonuclease-assisted signal amplification.113 30


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3.3 Amplification assays based on toehold-mediated DNA strand displacement. Toehold-mediated DNA strand displacement is a process of displacing one or more prehybridized strands from a DNA duplex containing a toehold overhang.130 The toehold refers to a single-stranded domain (typically 5-8 nt) where strand displacement is initiated through branched chain migration. The rate of strand displacement can be modulated by a factor of 106 by altering the length and sequence of the toehold. A variety of dynamic DNA nano-devices were recently designed using toehold-mediated DNA strand displacement, including DNA circuits, autonomous DNA motors, and DNA walkers.130 Two main strand displacement reactions, DNA circuits and Hybridization Chain Reaction (HCR), have also been used for enzyme-free, amplified detection of nucleic acids. The inclusion of aptamers for target recognition in these reactions extends toehold mediated DNA strand displacement strategies to detect targets other than nucleic acids. DNA circuits can be applied to the detection of proteins and small molecules by using structure-switching aptamers. Chen and coworkers used an anti-ATP aptamer with a toehold sequence capable of initiating a catalytic DNA hairpin circuit (Figure 6A).131 This DNA circuit consisted of two hairpins designed to hybridize together only after the first hairpin had been opened by the aptamer via toehold mediated strand displacement. However, the toehold region of the aptamer was initially inhibited by a complementary sequence. Only when the aptamer bound to the target, would the complementary sequence dissociate and liberate the toehold region of the aptamer which could then open the first hairpin and allow subsequent hybridization between the two hairpins. The hybridization of two hairpins then released the aptamer for the next cycle of amplification. Structure-switching aptamers were also coupled to an entropy-driven DNA circuit

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for amplified detection of ATP.132 Target binding released the complementary sequences hybridized to the aptamers that operated as input DNA to trigger the circuit amplification. The interaction of the input DNA with substrate exposed the toehold region for a so-called “fuel” DNA, thereby allowing the “fuel” DNA to displace the signal DNA and the input DNA for the next cycle of displacement. A detection limit of 20 nM was obtained for ATP detection. The entropy-driven DNA circuit was also used for amplified detection of thrombin by using a sandwich binding format.133 HCR was introduced by Dirks and Pierce and is a hybridization cascade which forms double-stranded DNA duplexes by using hairpins that are stable in solution but can undergo continuous assembly when triggered by an input DNA (Figure 6B).134 Measuring the duplex products allows amplified detection of the input DNA. HCR was coupled with aptamers to extend HCR to the detection of molecules other than DNA. For example, Dirks and Pierce designed an anti-ATP aptamer to contain a hairpin by extending the aptamer with an additional DNA sequence that served as the input DNA for subsequent HCR. In the absence of ATP, the input DNA sequence was caged in the hairpin, and thus could not trigger HCR. However, in the presence of the target, target binding altered the aptamer structure by opening the hairpins which released the toehold region of the input DNA. This initiated a cascade HCR reaction which allowed the formation of DNA duplexes up to thousands of base pairs long. In a similar design, Xu et al. demonstrated the application of HCR to the amplified detection of thrombin using bioluminescence to measure products generated from HCR.135 Besides using aptamers with hairpins, HCR can also use structure-switching aptamers hybridized with their complementary sequences. Yang el al. hybridized a complementary sequence to an anti-ATP aptamer, which blocked the complementary sequence’s toehold region.136 Target binding caused the dissociation

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of the complementary sequence which freed the toehold region to serve as the input DNA to trigger HCR. This assay detected 100 nM ATP by using fluorescence anisotropy to measure the HCR products. Song et al. also demonstrated HCR coupled with aptamer recognition in a sandwich binding format.137 Toehold-mediated strand displacement relies on DNA toeholds to enhance the strand displacement rate. We demonstrated a new strand displacement strategy termed binding-induced DNA strand displacement in which target binding, instead of toeholds, was used to accelerate strand displacement.138,139 By coupling it to DNA circuits, we demonstrated amplified detection of proteins.139 In binding-induced DNA strand displacement, affinity ligands were conjugated to a competing DNA oligonucleotide and a support DNA oligonucleotide pre-hybridized with an output DNA (Figure 6C). Because the competing DNA contained fewer complementary bases than the output DNA, the rate of output DNA displaced by the competing DNA was extremely slow in the absence of a target. The binding of two affinity ligands to a single target molecule dramatically increases the local concentration of the two DNA strands conjugated to the affinity ligands, thereby significantly increasing the displacement rate. The displaced output DNA then triggered the subsequent catalytic hairpin DNA circuit, enabling amplified detection of proteins.

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Figure 6. Amplification assays based on toehold-mediated DNA strand displacement. (A) Amplified detection of biomolecules using a structure-switching aptamer and catalytic DNA circuit.131 (B) Amplified detection of biomolecules using a hairpin aptamer probe and hybridization chain reaction.134 (C) Amplified detection of biomolecules using binding-induced DNA strand displacement.139

4. APTAMER-FACILITATED ASSAYS USING SIGNAL AMPLIFICATION Amplified detection can also be achieved by using catalytic components and nanomaterials to increase the signals that result from a single binding event.140 Classically, protein-based enzymes have been used as the catalytic labels in assays such as ELISA. In recent years, DNA/RNA enzymes, diverse nanomaterials and advanced nanofabrication techniques have also been used as

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highly efficient amplifiable labels/techniques for diverse assays.141-146 Aptamers are advantageous for use in these amplified assays, not only because aptamers offer an alternative to antibodies as affinity ligands, but more importantly, because a large number of powerful amplification mechanisms, e.g. those based on structure-switching properties of aptamers, can only be achieved with aptamers. In this section, we will focus on discussing emerging roles of aptamers in designing signal amplification strategies.

4.1 Signal amplification through enzymes In a typical ELISA design, a target protein is captured by an antibody immobilized on a solid support and then sandwiched with a second antibody conjugated to a signal-amplifying enzyme. Replacing antibodies with aptamers in ELISA assays is beneficial because aptamers have longer shelf-life and are easier to conjugate with other molecules (e.g., probes for detection). Moreover, aptamers can be engineered to have better binding affinity and selectivity than antibodies by taking potential interferences from the sample matrix into consideration during the SELEX process (known as negative selection) or by chemically modifying either the aptamer sequences or structures.140 In 1996, Drolet et al. described the first example of Enzyme Linked Aptamer Assays (ELAA) to detect human VEGF on microtiter plates.141 Since then, both sandwich (Figure 7A) and competitive (Figure 7B) ELAA assays have been developed for protein, bacteria, virus and small molecules.142-146 In all sandwiched ELAA assays, horseradish peroxidase (HRP) has been used as the enzyme to generate a signal. In some cases, target molecules themselves are enzymes and possess strong catalytic activities and can directly be quantified by triggering catalytic reactions to amplify detection signals.147 However, the activities of these enzymes are prone to interferences when complex matrix such as human serum is present. To improve assay 35



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specificity and sensitivity for such enzyme targets, Zhao et al. developed an aptamer capturing approach (Figure 7C).147 Target-specific aptamers were conjugated on magnetic beads and served as affinity probes to capture and separate minute amounts of the target enzyme. The captured enzymes catalyzed the subsequent conversion of fluorogenic substrates to fluorescent products, enabling a sensitive measurement of the active enzyme. Two important enzymes, human thrombin and human neutrophil elastase, have been detected with high sensitivity and specificity. As low as 2 fM thrombin and 100 fM human neutrophil esterase was detected from human serum samples.

Figure 7 Enzyme-linked aptamer assays (ELAAs). (A) Sandwiched ELAA.141,142,145 One aptamer is immobilized on a solid support as a capture probe and the other aptamer is conjugated with a reporter enzyme. The sandwiched binding between target molecule and the two aptamer probes captures the reporter enzymes on the solid surface. After washing off unbound enzymes, substrate is added and converted into signal generating products, which amplifies the detection signals. (B) Competitive ELAA.143,144 The aptamer is immobilized on a solid support that can capture both target molecule and the signal generating probe which is an enzyme modified form of the target molecule. As the binding of unmodified targets displaces the enzyme modified probes, signal attenuation is monitored which is proportional to target concentrations. (C)

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Aptamer capturing assay for enzyme analysis.147 Target enzymes are captured by an aptamercoated magnetic bead and separated from the sample matrix. Enzyme-specific substrates are then added and converted into signal generating products to amplify the detection of the target enzyme.

Enzymes have also been combined with structure-switching aptamers to develop novel signal amplification strategies. For example, by integrating structure-switching aptamers with invertase, an enzyme that converts sucrose into glucose, Xiang and Lu developed a general strategy to use commercially available, pocket-sized personal glucose meters to detect a broad range of non-glucose targets with high sensitivity and specificity (Figure 8).148 A biotinylated DNA aptamer is conjugated to magnetic beads through streptavidin-biotin binding. Another DNA probe conjugated to invertase is assembled onto the DNA functionalized magnetic beads through the hybridization to the aptamer. The aptamer switches structure in the presence of its target, resulting in the release of the DNA-invertase probe from the magnetic bead. The released invertase catalyzes the conversion of sucrose to glucose, the latter being detected by a personal glucose meter. This system enabled assays for a panel of non-glucose targets, including cocaine (3.4 µM detection limit), adenosine (18 µM), interferon-gamma (2.6 nM) and uranium (9.1 nM).148 Such a sensitive yet simple, low-cost and portable detection system is potentially useful for point-of-care disease diagnostics and on-site environmental monitoring. Many other redox enzymes, e.g. HRP, alkaline phosphatase, and glucose dehydrogenase, have also been coupled with aptamers and used as catalytic labels to amplify electronic or electrochemical signals for a diverse set of targets.149 This aspect has been recently reviewed.149.150

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Figure 8. Schematic illustrating the principle of using personal glucose meters and structureswitching aptamers to detect non-glucose targets. Reprinted with permission from reference 148.

4.2 Signal amplification through DNAzymes DNAzymes can also amplify detection signals in aptamer-based assays. DNAzymes are attractive because they are capable of amplifying fluorescent, chemiluminescent, colorimetric and electrochemical signals for metal ions and nucleic acids.10,151,152 Applications of DNAzymes to assays for other targets require affinity ligands to transduce target binding to the activation of DNAzymes. In such cases, aptamers are advantageous compared to other affinity ligands because aptamers are also single-stranded nucleic acids and therefore can be easily integrated into DNAzymes by simply extending DNAzyme sequences with aptameric sequences. 38


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Furthermore, structure-switching strategies (e.g., allosteric mechanisms) can also be incorporated into aptamer-DNAzyme (also known as aptazyme) designs to amplify optical or electrical signals for target detection. The

hemin/G-quadruplex

horseradish

peroxidase-mimicking

DNAzyme

(HRP-

DNAzyme) is one of the most frequently used biocatalytic labels for amplified biosensing.152 The HRP-DNAzyme can generate a colorimetric signal or a chemiluminescence signal; the former is by catalyzing the oxidation of 2,2’-azino-bis(3-ethylbenzthiazolin-6-sulfonic acid) (ABTS2-) with H2O2 to the colored ABTS·- and the latter is by catalyzing the oxidation of luminol with H2O2.152 HRP-DNAzyme also electrocatalyzes the reduction of H2O2.152,153 These optical and electrochemical properties make HRP-DNAzyme an ideal signal generator in aptamer assays. A common strategy for aptamer-HRP-DNAzyme assays is to deactivate HRPDNAzyme by caging it into a hairpin or by using a complementary DNA sequence to block its catalytic core.153-155 In these designs, aptamer sequences are either incorporated into the hairpin structure or used to serve as part of the blocking sequence.153,154 Upon binding to its target, the aptamer switches its structure, which either opens up the hairpin or releases the blocking sequence; this restores the enzymatic activity of the HRP-DNAzyme (Figure 9A). For example, Teller et al. developed a series of aptamer-DNAzyme hairpins for amplified sensing of adenosine monophosphate (AMP) and lysozyme.154 In their designs, the stem of a hairpin contained the inactivated HRP-DNAzyme and the loop region included an aptamer sequence. The aptamer bound to either AMP or lysozyme targets, which opened the hairpin and activated the HRPDNAzyme, thus generating an amplified colorimetric signal by catalyzing the H2O2-mediated oxidation of ABTS2-. Compared to commonly used fluorophore-quencher labels in aptamer

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beacons, HRP-DNAzymes significantly improve the analytical performance of aptamer-based assays.152 An alternative strategy for aptamer-HRP-DNAzyme assays is to split the active HRPDNAzyme into two inactive parts.156 Each part of the DNAzyme sequence is extended with a partial aptamer sequence that has also been separated into two parts. In the presence of the target molecule, the binding of the two partial aptamers to the same target reassembles the two partial HRP-DNAzymes (Figure 9B). This restores the catalytic activity of HRP-DNAzyme and enables optical or electrochemical signal amplification. Willner and coworkers developed such an assay for the ultrasensitive detection of ATP and mercury.156 They incorporated their aptamerDNAzyme system with CdSe/ZnS Quantum Dots (QDs) to generate Chemiluminescence Resonance Energy Transfer (CRET) signals to further enhance assay sensitivity and multiplexity. Another strategy uses RNA-cleaving DNAzymes as catalytic labels for amplified aptamer assays. Since they were isolated in Joyce’s laboratory157 in 1994, RNA-cleaving DNAzymes have been selected and engineered for sensitive detection of metal ions and a few amino acids that serve as cofactors at the catalytic core of DNAzymes.151,158 Aptamers have been engineered into the core sequences of RNA-cleaving DNAzymes to control their enzymatic activity through target binding-induced structure-switching of aptamers (generally known as allosteric aptazymes).158-161 Lu and coworkers took advantage of allosteric aptazymes and tailored them to the sensitive detection of adenosine through the design of hairpin-structured fluorogenic substrates (Figure 9C).159 An adenosine-specific aptamer is inserted into the core sequence of a 10-23 nt DNAzyme so that in the absence of target adenosine, the DNAzyme domain cannot form a stable and active structure to catalyze the cleavage of the hairpin substrate. However, in the presence of its target the aptamer region bound to adenosine and formed a compact structure,

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which restored the stem-loop and activity of the DNAzyme. The activated DNAzyme catalyzed the cleavage of the hairpin-structured DNA-RNA chimeric substrate and thereby released the fluorophore-labeled DNA strand for an amplified detection signal. Through this design, as low as 500 nM adenosine could be detected, which is lower than those of other reported fluorescent adenosine sensors.159

Figure 9. DNAzymes for amplified aptamer assays. (A) Principle of a hairpin-based aptamerDNAzyme sensor.154 A DNA hairpin structure is designed by incorporating a structure-switching aptamer and HRP-mimicking DNAzyme. In the presence of the target molecule, the binding between the target and aptamer switches the secondary structure of aptamer and thus releases the DNAzyme. Once released from hairpin structure, DNAzyme activity is restored and amplifies the detection signal by catalyzing the H2O2-mediated oxidation. (B) Principle of split aptamer-

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DNAzyme sensors.156 HRP-mimicking DNAzyme is split into two inactive parts and each is conjugated with a partial aptamer sequence. In the presence of the target molecule, the binding between target and two partial aptamers assembles the DNAzyme and restores its catalytic activity to amplify the detection signal. (C) Principle of a RNA-cleaving DNAzyme assay.159 An aptamer is inserted into the core sequence of a RNA-cleaving DNAzyme so that in the absence of its target, adenosine, the DNAzyme domain cannot form a stable and active structure to catalyze the cleavage of the hairpin substrate. In the presence of the target molecule, the aptamer region binds to the target and forms a compact structure, which restores the stem-loop and activates the DNAzyme. The activated DNAzyme catalyzes the cleavage of the hairpin DNARNA chimeric substrate and thereby releases the fluorophore-labeled DNA strand to amplify the detection signal.

RNA enzymes can also be incorporated into aptamers to amplify detection signals for various targets.162,163 For example, Lam and Joyce converted the RNA ligase, R3C, to an aptazyme by replacing the central stem-loop of the enzyme with an aptamer specific for theophylline.163 In the presence of theophylline, the aptamer domain switched into a welldefined structure that supported the catalytic activity of the RNA enzyme. The enzyme catalyzed the formation of a phosphodiester bond between two RNA substrates, one bearing a 3’-hydroxyl and the other bearing a 5’-triphosphate. The ligated product contained the catalytic core sequence of another RNA ligase and thus initiated cross-replication. Cross replication employs two RNA enzymes that catalyze each other’s synthesis from a total of four substrates. Consequently, an exponential amplification profile can be achieved from this autocatalysis process. For quantification of the products, all DNA probes were labeled with [5’-32P] and analyzed by PAGE.

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This strategy enabled the quantitative detection of theophylline with a range of 2-500 µM and a detection limit of 80-fold lower than the Kd of the aptamer-ligand interaction.

4.3 Signal amplification through nanomaterials One of the largest categories of signal amplification strategies for bioassays relies on using nanoscale materials, such as metallic nanoparticles, silica-based nanoparticles and carbon-based nanomaterials. All have controllable size-dependent properties, tunable chemical compositions and the ability to enhance optical and electrochemical signals.16,17,164-166 Integration of aptamers on various nanomaterials provides advantages over other affinity ligands. For example, aptamer functionalized nanomaterials generally have higher valences due to the relatively small molecular size and simple structure of aptamers, thus enhancing the affinity bindings.16,17 Many aptamers are able to switch structures upon binding to their targets, providing the potential to develop novel signal transduction mechanisms on nanomaterials.166 The binding of an aptamer to its target can trigger a change in the aptamer-nanomaterial interaction, which can be taken advantage of when designing signal amplification strategies.18 The recent advances of nanofabrication techniques have also led to novel amplification strategies for aptamer assays. In this section, we focus on discussing the use of aptamer functionalized metallic nanoparticles and nanofabrication techniques for amplified aptamer assays.

4.3.1 Aptamer functionalized metallic nanoparticles Aptamer functionalized metallic nanoparticles can serve as carriers to enable mass-based amplification for biosensing.167,168 For example, gold nanoparticles (AuNPs) are clusters of a large number of elemental atoms, where one 10 nm AuNP contains ~30 000 Au atoms.167 Zhao et al. developed an aptamer-linked thrombin assay by using AuNP amplification and Inductively 43



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Coupled Plasma-Mass Spectrometry (ICP-MS) detection (Figure 10A).167 Two DNA aptamers for thrombin are conjugated onto either a magnetic bead or an AuNP. In the presence of thrombin, sandwiched binding between thrombin and the two aptamers assembles the AuNPs onto magnetic beads. After washing away unbound AuNPs, the bound aptamer functionalized AuNPs are released by heating and detected using ICP-MS. Since each AuNP contains a large number of Au atoms, as low as 0.5 fmol thrombin can be detected. Mass-based amplification can also be achieved through the incorporation of aptamers, because hundreds to thousands of aptamer oligonucleotides can be attached to metallic nanoparticles with sizes from 10 nm to 100 nm. He et al. developed an ultrasensitive electrochemical amplification strategy for thrombin based on this principle (Figure 10B).168 A AuNP bearing hundreds of aptamer molecules is captured in a microtiter plate well by sandwiched binding between antibody, thrombin and aptamer. Once unbound aptamer-AuNPs are washed away, the adenine bases from the aptamers are released from the AuNPs, by acid or enzyme digestion, and are subjected to differential pulse voltammetry analysis on a pyrolytic graphite electrode. Thousands of adenosine bases are released from a single binding event, resulting in a very low detection limit of 0.1 ng/mL for thrombin. Aptamer functionalized metallic nanoparticles also display unique resonance scattering properties and can be used to amplify colorimetric signals in aptamer-based assays.169-173 Willner and coworkers developed a simple sandwich binding strategy to capture AuNPs on a silica monolayer through aptamer-thrombin bindings.169 Strong absorbance intensities corresponded quantitatively to the amount of thrombin present. To further amplify the scattering signal, AuNPs were enlarged by depositing gold on the AuNP surface through the catalytic reduction of HAuCl4 (Figure 10C). Similarly, Li et al. developed an ultrasensitive densitometric method for the

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detection of PDGF by catalytically depositing silver on aptamer functionalized AuNPs.170 In this work, two aptamers that bind to different epitopes of the same target were required to generate an amplified signal. To enable the detection with only one aptamer, Yu and coworkers developed an aptamer beacon strategy by conjugating biotin to one end of the hairpin aptamer.171 The biotinylated aptamer was then immobilized on a disposable plastic (polycarbonate) substrate (Figure 10D). Upon target binding, the hairpin of the aptamer was opened and the biotin end exposed. By incubating the opened beacon with nanogold-streptavidin conjugates that bind the terminal biotin groups, the authors directly visualized and quantified the target with a standard flatbed scanner. To further amplify the detection signal, silver enhancement can be used to enlarge nanogold probes and thus enhance the scattering signals. As low as 10 nM thrombin and 5 nM mercury ions were successfully detected in buffer, human serum and urine samples. When using aptamer-functionalized metallic nanoparticles to analyze target proteins in a complicated sample matrix, such as human serum, an important issue is nonspecific adsorption of interfering proteins that significantly contribute to the background signal.172 To overcome this problem, Li et al. developed a competitive protection strategy to eliminate nonspecific protein adsorption by assembling short DNA oligonucleotides (protecting DNA oligonucleotides) that strongly bind to exposed surfaces of AuNPs by hybridizing with DNA aptamers.170 Only targets that can bind specifically with the aptamer and switch the aptamer’s structure can displace the protecting DNA oligonucleotides. This novel aptamer-AuNP probe detected human thrombin that was separated from other serum proteins by gel electrophoresis and western blotting. Furthermore, the protecting DNA oligonucleotides can also serve as a linker DNA to assemble multiple layers of aptamer-AuNPs on the surface, thus amplifying the detection signal (Figure 10E). As low as 6 ng of thrombin was detected in human serum using this strategy.

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The unique plasmonic optical properties of aptamer-functionalized metallic nanoparticles enable their use in amplifying signals in Surface Enhanced Raman Scattering (SERS) assays for small molecules, proteins, viruses and cells.174-180 SERS is an attractive technique to design amplified aptamer assays with due to its ultrahigh sensitivity (up to single molecule detection) and high multiplexity.181 To generate amplified Raman signals, Raman hotpots must be created on a metallic surface, such as a gold or silver film. This can be achieved either by bringing plasmonic nanoparticles, such as AuNPs or silver nanoparticles (AgNPs), carrying Raman reporters into close proximity with the metallic surface or by directly creating nanostructures, e.g. nanopillars, on the metallic surface.174-180 For example, Kim et al. developed an aptamermediated SERS amplification strategy by using a bi-functional adenosine-specific doublestranded DNA aptamer to control a SERS hot spot between a gold surface and a AuNP attached to the aptamer via a biotin-avidin linkage (Figure 10F).174 Specifically, a partially hybridized double-stranded DNA aptamer was anchored to the gold film via a thiolate group. The DNA strand not anchored to the gold film was biotinylated and linked, through an avidin moiety, to an AuNP labelled with a Raman reporter molecule, 4-aminobenzenethiol (4-ABT). In the presence of adenosine, the binding between adenosine and its aptamer dehybridized the double strand DNA, resulting in the folding of 4-ABT bearing AuNP onto the gold film. The resulting close proximity between the AuNP and gold film created a hotspot for SERS and thus amplifies the detection signal 4-fold. Aptamer functionalized nanoparticles can also be used to amplify detection signals in Surface Plasmon Resonance (SPR) platforms. SPR provides fast detection of a wide range of biological targets and molecular interactions.182 The generation of optical signals in SPR relies on the detection of small changes in refractive index due to the specific adsorption of analytes

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through affinity ligands immobilized on gold films. Aptamers are widely used as affinity ligands in SPR due to their relatively small weight and ease of modifications.182-187 Although target biomolecules can be directly detected without fluorescence or catalytic labels in aptamer-SPR, detection limits of only low nanomolar range are possible.182 Nanoparticle-based labels are frequently used in sandwich binding formats to amplify the detection signal.183-187 Due to their larger size (> 10 nm) than many molecules, nanoparticles introduce larger changes in refractive index upon molecular binding. Recent studies have further revealed that the shape of nanoparticles can also have a large effect on SPR signals. For example, by comparing different nanoparticle shapes and sizes on an aptamer-SPR system, Kwon et al. found that quasi-spherical AuNPs with a size of ~40 nm could push the detection limit to 1 aM for thrombin, whereas gold nanocages and nanorods of the same size could only achieve detection limits of 1 fM and 10 aM, respectively.183 Shortly after this discovery, the same group found that gold nanotubes showed the same signal enhancement as quasi-spherical AuNP in the same aptamer-SPR system. Thus, they further developed an ultrasensitive detection method for B-type natriuretic peptide (BNP), a cardiac biomarker, by integrating a BNP-specific aptamer with antibody-coated gold nanotubes in the aptamer-SPR platform.184 Nanoparticles can also enhance the detection signals in SPRphase imaging (SPR-PI). Measuring the phase change of reflected light rather than the intensities can improve the sensitivity of SPR.185 Corn and coworkers demonstrated that the use of DNA/aptamer functionalized metallic or silica nanoparticles in SPR-PI can greatly enhance sensitivity for the detection of nucleic acids and proteins.185 They also incorporated a DNAzyme with nanoparticle-enhanced aptamer-SPR-PI to achieve ultrasensitive detection of thrombin.161 Aptamer functionalized metallic nanoparticles are also widely used as electrochemical labels to amplify detection signals in aptamer assays. Due to the large effective surface area,

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metallic nanoparticles are considered to be more efficient electrochemical labels than enzymes or DNAzymes.149 For example, Willner and co-workers described the use of aptamer functionalized Pt nanoparticles (PtNPs) as catalytic labels to amplify the detection signal for proteins.188 PtNPs are able to catalyze the electrochemical reduction of H2O2 with high efficiency, and the cathodic currents enabled the amplified detection of thrombin with a detection limit of 1 nM. Other nanomaterials, including AuNPs, AgNPs, quantum dots (QDs) and carbon nanotubes, have been adopted to amplify the detection signals in voltammetric, potentiometric and impedimetric aptasensors for proteins and small molecules.168,189-193 To further amplify the detection signals, polystyrene microbeads assembled with multiple layers of CdTe QDs have been used to enhance the sensitivity of electrochemiluminescent detection of thrombin.194 Catalytic deposition of silver ions onto aptamer-AuNP conjugates has been used to amplify voltammetric signals for quantifying the expression levels of human epidermal growth factor receptor 2 (HER2) from SKBR-3 breast cancer cells.195

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Figure 10. Use of aptamer functionalized metallic nanoparticles for amplified aptamer assays. (A) Mass amplification achieved using aptamer functionalized AuNP as an amplifiable elemental tag with ICP-MS detection.167 (B) Mass amplification achieved by digesting target binding aptamer-AuNPs, followed by electrochemical detection of released adenosine bases.168 (C) Amplified colorimetric aptamer assay using sandwiched aptamer binding and reduced metal ion deposition onto aptamer-AuNPs.169 Once enlarged, the resonance scattering signals from aptamer-AuNPs is greatly enhanced. (D) Amplified colorimetric aptamer assay achieved by coupling structure-switching aptamers with silver enhancement.171 (E) Amplified colorimetric aptamer assay achieved by assembly of multiple layers of aptamer/DNA functionalized AuNPs

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upon target bindings.170 (F) Surface enhanced Raman scattering for amplified aptamer assays using a structure-switching aptamer to control the distance between the gold film and DNAAuNP labeled with a Raman reporter.174

4.3.2 Nanofabrication techniques for amplified aptamer assays Nanofabrication is the design and manufacturing of devices with dimensions in the nanometer range. Recent advances in both top-down and bottom-up nanofabrication techniques, including atomic layer deposition, sol-gel nanofabrication, molecular self-assembly and vapor-phase deposition, have allowed researchers to engineer aptamer modified nanodevices for amplified assays.196-206 One such example is the development of nanostructured microelectrodes (NMEs) that exhibit very high levels of analytical sensitivity.196 By depositing lead on silicon dioxide and controlling the plating conditions during electrodeposition, Kelley and coworkers developed a variety of 3-D NMEs. The nanostructured microelectrode sensors demonstrated a limit of detection as low as 10 aM for nucleic acids, corresponding to a 106 times improvement over traditional electrodes.197 To expand this powerful electrochemical detection platform to nonnucleic acid targets, they developed a neutralizer displacement assay through incorporation of aptamers into NMEs.198 In this assay, an aptamer probe conjugated to the surface of a NME is bound by a neutralizer. The neutralizer is a conjugate of PNA and a cationic amino acid that neutralizes the charge of the probe. In the presence of a target molecule, the neutralizer is displaced by target-aptamer binding. By monitoring the large change in surface charge on NMEs, the authors detected a diverse set of targets with excellent detection limits (e.g. 10 fM for thrombin) without further amplification. Many nanofabricated devices, including nanopores and nano-field-effect transistors (nano-FETs), have demonstrated ultrahigh sensitivity by counting aptamer-protein binding 50


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events at single molecule levels.199-204 For example, by covalently attaching a DNA aptamer through a disulfide bond to a single cysteine near the mouth of a protein nanopore, Rotem et al. developed a sensitive approach for quantifying thrombin.199 Because a single aptamer-thrombin binding event can be monitored by detecting the change in ionic current through the aptamer modified nanopores, the detection signal is greatly amplified. Moreover, this strategy enables the possibility of designing arrays of aptamer modified nanopores, which could allow high throughput target analysis.201 Similarly, aptamer modified silicon nanowire FETs and carbon nanotube FETs have been applied to monitor single molecule binding events for ultrasensitive protein and cell analysis.202-204

5. CONCLUSIONS AND PERSPECTIVES In the recent 20 years, aptamers have been well-recognized as improved alternatives to antibodies due to their comparable binding affinity and selectivity, aptamer’s longer shelf-life, and ease of production and site-specific chemical modification of aptamers. However, less credit has been given towards aptamers for their unique properties amenable to amplified detection of biomolecules. As nucleic acids, aptamers possess several advantages that other affinity ligands cannot match. In this review, we described a number of intrinsic properties of aptamers that enable various signal amplification mechanisms that are of great value to the detection of trace amounts of biomolecules. Aptamers are affinity ligands that can switch into well-defined secondary and tertiary structures upon binding to their target. This is one of the most extensively used properties of aptamers in designing amplified assays for biomolecules. Generally, three main strategies are frequently adopted, including hybridizing an aptamer sequence with a complementary sequence, caging an aptamer sequence into a stem-loop hairpin, and splitting an aptamer sequence into two 51



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partial aptamers. Upon binding to their target molecules, aptamers switch from hybridized, caged or split forms into well-defined aptamer conformations. This results in the release of the complementary sequence, the opening of the hairpin structure, or the assembly of the two DNA strands, all of which can serve as surrogates to further amplify detection signals for the target molecules. Aptamers are nucleic acids that can be manipulated with various enzymes, such as polymerases, ligases, exonucleases, and endonucleases, to allow enzyme-assisted amplification. Therefore, aptamers can act as surrogates for their specific targets in various enzyme-driven DNA/RNA amplification techniques, including polymerase chain reaction, rolling circle amplification, strand displacement amplification, exonuclease-assisted signal amplification, and nicking endonuclease-assisted signal amplification. Furthermore, aptamers can be protected from enzymatic degradation upon binding to their targets. This property has also been used to create templates for subsequent enzyme amplifications. Aptamers can be programmed through Watson-Crick base pairings to allow the detection of target molecules through enzyme-free DNA amplifications. Enzyme-free DNA amplification techniques utilizing toehold-mediated DNA strand displacement, such as hybridization chain reactions and catalytic DNA circuits, are attractive methods for the detection of nucleic acids. Aptamers are used to expand such techniques to non-nucleic-acid targets mainly through structure-switching mechanisms to release or expose DNA toeholds that trigger subsequent enzyme-free DNA amplifications. Aptamers are single-stranded biopolymers bearing a highly negatively charged sugarphosphate backbone on one side and tandem heterocyclic nucleobases on the other. These chemical properties can be tailored into amplified bioassays. First, as a result of the dense

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negative charges, aptamers can be used to bind with protein targets and modulate electrophoretic mobilities of proteins, enabling separation and detection of proteins. Second, the dense negative charges and tandem nucleobases also lead to unique interactions between aptamers and various metallic and carbon-based nanomaterials. Particularly, unbound aptamers can strongly adsorb onto the surfaces of AuNPs, AgNPs, graphene oxides and carbon nanotubes, whereas aptamers bound to their targets can be effectively desorbed from surfaces of nanomaterials. This unique “on/off” property can be used to trigger subsequent signal amplifications. Third, because of their relatively small molecular size and simple chemical structure, dense layers of aptamers can be formed when conjugated to the surfaces of nanomaterials, leading to high valences and enhanced binding affinities. This property of aptamers is particularly useful when applying aptamer functionalized nanomaterials as catalytic labels for amplified detection of biomolecules. Many additional features can be engineered into aptamers through their in vitro selection process to expand their applicability to the amplified detection of biomolecules. For example, the use of modified nucleosides and next generation DNA sequencing have greatly accelerated the SELEX process and expanded aptamer target sets. By incorporating structure switching and signal generation components, such as DNAzymes, into the SELEX process, researchers can now directly evolve aptamers that are able to amplify detection signals upon target binding. SELEX processes can also include negative selection controls that would minimize nonspecific binding and take into account of potential sample matrix interference. We envision that in the near future, more properties of aptamers will be explored through SELEX as well as other molecular engineering techniques. These aptamers will be continuously tailored into signal amplification strategies for the detection of diverse range of biomolecules. The aptamer-facilitated signal amplification techniques will play important roles in various

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applications, ranging from fundamental chemical and biomedical research, to biomarker discovery and validation, and to point-of-care analysis.

■ ASSOCIATED CONTENT ■ AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest. Biographies Feng Li received his B.Sc. degree from Tianjin University (China) in 2006 and his Ph.D. degree from the University of Alberta (Canada) in 2013 under the direction of Professor X. Chris Le. He has recently been appointed Assistant Professor in Department of Chemistry at Brock University, St. Catharines (Canada). His current research focuses on developing novel diagnostic tools for disease biomarkers. He is also interested in constructing DNA-assembled nanomaterials for diagnostic and medical applications. Hongquan Zhang received his B.Sc. degree in 1997 and M.Sc. degree in 1999 from Northwest University (China) and his Ph.D. degree in 2009 from University of Alberta (Canada). After three years of research experience as a research associate in Institute for Biological Sciences, National Research Council of Canada, and at University of Alberta, he was appointed Assistant Professor in 2012 in Department of Laboratory Medicine and Pathology, University of Alberta. His research interests include (a) binding-induced DNA assembly and its applications to detection of biological targets, (b) construction of binding-induced DNA nanomachines and nanodevices, and (c) generation, modification, and manipulation of functional nucleic acids. Zhixin Wang received his B.Sc. (2004) and M.Eng. (2006) from Wuhan University (China) and his Ph.D. (2010) from the Research Centre for Eco-Environmental Sciences, Chinese Academy of Sciences (Beijing, China). He is currently a postdoctoral fellow in the Department of Laboratory Medicine and Pathology at the University of Alberta. His research focuses on the selection of DNA aptamers for cancer biomarkers and the development of bioanalytical assays for studying DNA and protein interactions. Ashley M. Newbigging received her B.Sc. in Medical Laboratory Science in 2014 from the University of Alberta (Canada). She began research shortly after in the Department of 54


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Laboratory Medicine and Pathology and is continuing with her M.Sc. in Analytical and Environmental Toxicology. Her current research is focused on the development of a bindinginduced DNA assay for the detection of HER2 protein associated with breast cancer. Michael S. Reid received his B.Sc. in Chemistry from Thompson Rivers University (Canada) in 2012. He is currently working towards a PhD in Analytical Chemistry at the University of Alberta. His current research is focused on developing ultrasensitive homogeneous protein assays. Xing-Fang Li is Professor in the Faculty of Medicine and Dentistry, University of Alberta. She received B.Sc. (1983) from Hangzhou University (China), M.Sc. (1986) from Chinese Academy of Sciences (Beijing), M.Sc. (1990) from Brock University (Canada), and Ph.D. (1995) from the University of British Columbia (Canada). She received the W.A.E. McBryde Medal from the Canadian Society for Chemistry in 2010 and Killam Annual Professorship (2012) and Mentoring Award (2014) from the University of Alberta. She is elected Fellow of Chemical Institute of Canada. Her research interests include studies of protein interaction with small molecules, selection of DNA aptamers, detection of microbial pathogens, characterization of new drinking water disinfection by-products, and studies of water contaminants and human health effects. X. Chris Le is Distinguished University Professor with cross appointments in departments of Laboratory Medicine and Pathology, Chemistry, and Public Health Sciences. He is Canada Research Chair in Bioanalytical Technology and Environmental Health. He is an elected Fellow of the Royal Society of Canada (Academy of Science). He leads an interdisciplinary team developing ultrasensitive analytical techniques and studying human health effects in relation to the environment. His research in bioanalytical chemistry focuses on the development of DNA and protein binding assays, fluorescence biosensing techniques, signal amplification approaches, and separation and targeted metallomics, for measuring proteins, DNA damage, chemical speciation, and molecular interactions. He is an Associate Editor for Environmental Health Perspectives and an Editorial Advisory Board member for Analytical Chemistry.

■ ACKNOWLEDGMENTS We thank the Natural Sciences and Engineering Research Council of Canada, the Canadian Institutes of Health Research, the Canada Research Chairs Program, Alberta Health, and Alberta Innovates for financial support.

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