Hybridization chain reaction: a versatile molecular tool for biosensing, bioimaging, and biomedicine

Abstract

Developing powerful, simple and low-cost DNA amplification techniques is of great significance to bioanalysis and biomedical research. Thus far, many signal amplification strategies have been developed, such as polymerase chain reaction (PCR), rolling circle amplification (RCA), and DNA strand displacement amplification (SDA). In particular, hybridization chain reaction (HCR), a type of toehold-mediated strand displacement (TMSD) reaction, has attracted great interest because of its enzyme-free nature, isothermal conditions, simple protocols, and excellent amplification efficiency. In a typical HCR, an analyte initiates the cross-opening of two DNA hairpins, yielding nicked double helices that are analogous to alternating copolymers. As an efficient amplification platform, HCR has been utilized for the sensitive detection of a wide variety of analytes, including nucleic acids, proteins, small molecules, and cells. In recent years, more complicated sets of monomers have been designed to develop nonlinear HCR, such as branched HCR and even dendritic systems, achieving quadratic and exponential growth mechanisms. In addition, HCR has attracted enormous attention in the fields of bioimaging and biomedicine, including applications in fluorescence in situ hybridization (FISH) imaging, live cell imaging, and targeted drug delivery. In this review, we introduce the fundamentals of HCR and examine the visualization and analysis techniques for HCR products in detail. The most recent HCR developments in biosensing, bioimaging, and biomedicine are subsequently discussed with selected examples. Finally, the review provides insight into the challenges and future perspectives of HCR.

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Sai Bi

Sai Bi received her PhD degree from Qingdao University of Science and Technology in 2010. She worked as a postdoctoral fellow at Nanjing University from 2012 to 2014. She is currently a professor in College of Chemistry and Chemical Engineering at Qingdao University. Her research interests focus on biochemical analysis, DNA nanotechnology, molecular imaging and targeting therapy.

Shuzhen Yue

Shuzhen Yue is now an MS candidate majoring in Analytical Chemistry in the College of Chemistry and Chemical Engineering at Qingdao University. She received her BS in Chemistry from Liaocheng University in 2015. Her current research interests focus on the assembly of DNA nanostructures and their applications in biosensing, bioimaging and targeted cancer therapy.

Shusheng Zhang

Shusheng Zhang is currently a professor at Linyi University. He received his PhD degree in 1999 from Nanjing University. From 1990 to 2012, he was a professor in Qingdao University of Science and Technology. He joined Linyi University in 2012. His scientific interests involve biosensors, bioimaging and cancer therapy, and single molecule analysis.

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1 Introduction

DNAs, as genetic information carriers and protein encoders, play central roles in life processes that translate the genetic code into proteins and further regulate diverse cellular functions.^1,2 In addition to their biological functions, DNAs have been considered functional materials for the construction of DNA nanostructures^3–6 and circuits.⁷ More recently, functional nucleic acid structure-based amplification techniques have emerged as attractive tools in the fields of biotechnology. These nucleic acid amplification techniques can be divided into two categories: thermocycling amplification techniques, which include polymerase chain reaction (PCR)⁸ and ligase chain reaction (LCR),⁹ and isothermal amplification techniques, including helicase-dependent amplification (HDA),¹⁰ rolling circle amplification (RCA),¹¹ and strand displacement amplification (SDA), such as enzyme-mediated SDA (e.g., polymerase-mediated SDA)¹² and enzyme-free SDA (e.g., toehold-mediated strand displacement [TMSD] amplification).¹³ Compared to thermocycling amplification methods, which often involve the use of thermal cyclers and suffer from externally driven melting and rehybridization, isothermal amplification protocols can be implemented under simple conditions (e.g., at a constant temperature).¹⁴ In particular, TMSD amplification is one of the most popular isothermal amplification strategies because of its advantages, which include being enzyme-free and having high amplification efficiency and controllable kinetics. In a typical TMSD, the reaction is initiated at the complementary single-stranded overhang region (termed as a toehold), and a long single-stranded DNA (ssDNA) displaces a short one and hybridizes with a third strand to form a double-helix structure with more complementary base pairs.^15–17 The kinetics of the strand displacement process has been determined to be regulated by the number and species of bases in the toehold region.¹³ Theoretically, the rate of strand displacement can be accelerated 10⁶-fold using TMSD.¹⁷

Among TMSD systems, hybridization chain reaction (HCR) is a simple and efficient isothermal amplification process proposed by Dirks and Pierce in 2004.¹⁹ In a typical HCR, an initiator triggers a cascade of hybridization events between two species of DNA hairpins, leading to the formation of a nicked double helix with tens to hundreds of repeated units until the hairpins are exhausted. Given the advantages of its enzyme-free nature, efficient isothermal amplification, ultrahigh sensitivity and structural flexibility, HCR has emerged as a powerful molecular tool with versatile applications in biosensing, bioimaging, and biomedicine. Biosensing, signal transduction, or transforming input molecules into readable signal outputs, can readily be achieved via incorporation of HCR products with functional moieties such as fluorophores,^25–28 nanoparticles (NPs),²⁹ and electrochemical reagents.^30,31 To date, HCR has been used for the sensitive detection of various targets, including nucleic acids (DNAs and RNAs),³² proteins,^33,34 enzyme activities,^35,36 small biomolecules,^37–39 metal ions,^40,41 and even tumour cells.^42,43 In addition to target analyte detection, recognition molecules (e.g., antibodies [Abs] and aptamers) have been combined with HCR to develop novel methods for in situ or intracellular imaging to better understand the biological processes, and these methods also can be applied to disease diagnosis and therapy.^20,21,45,128 More recently, HCR applications have expanded to include biomedicine; for example, HCR products have been used as drug delivery carriers with high payload capacities.^23,24,46 Although the applications of HCR in biosensing have undergone tremendous development during the last few decades, the most significant HCR developments in bioimaging and biomedicine have occurred in recent years. Given its advantages (efficient isothermal amplification, ultrahigh sensitivity, structural versatility, and enzyme-free nature), HCR holds great promise as a powerful molecular biotechnology tool for versatile applications in nanomedicine and nanotechnology.

In this review, we first describe the fundamental principles of HCR and demonstrate visualization and analysis techniques of HCR products. Then, we discuss recent representative works using HCR, which are classified into three sections based on their application fields: biosensing, bioimaging, and biomedicine. Finally, the challenges and outlook of HCR development are considered.

2 Fundamentals of HCR

2.1 Basic features of HCR

As a target-triggered amplification system, HCR was first demonstrated and named by Dirks and Pierce in 2004.¹⁹ They introduced a novel strategy in which DNA can accomplish the roles of recognition and signal amplification simultaneously by binding to a substrate. In a typical HCR process, the recognition of a target initiates the cross-opening of two DNA hairpins to form DNA polymeric nanowires (Fig. 1). Briefly, two species of hairpins (H1 and H2) caught in a kinetic trap can coexist metastably in the absence of an initiator (I) (Fig. 1a). The introduction of I (a*–b*) opens H1 via a TMSD reaction in which I binds to the sticky end of H1 (domain a) and undergoes a branch migration (Fig. 1b). The newly exposed domain (c–b*) of H1 acts as another initiator to interact with the sticky end of H2 (domain c*), resulting in the release of sequence a*–b* of H2 that is identical to I and, thus, subsequently opens H1 (Fig. 1c). Finally, DNA polymeric nanowires can be produced by the cross-opening of two DNA hairpins until the supply of H1 or H2 is exhausted. Dirks and Pierce also showed that the molecular weight of the HCR product is inversely proportional to the amount of initiator. In addition, based on aptamer recognition, they demonstrated a versatile application of HCR in the detection of adenosine triphosphate (ATP). In 2007, the same group proposed an autonomous polymerization motor in which the target triggered a four-way branch migration to perform HCR, resulting in the formation of a long nicked double helix.⁴⁷ Unlike the traditional analytical technique of PCR, HCR is an isothermal and non-enzymatic amplification strategy. Another significant difference between HCR and PCR is that HCR is a probe-amplification technique, whereas PCR is a target-amplification technique.³² Thus, HCR can effectively reduce false-positive results and cross-contamination from amplicons, which often occur in PCR.

Fig. 1 Fundamentals of HCR. The initiator (I) triggers a chain hybridization reaction between alternating H1 and H2 hairpins to form a nicked double helix. Reprinted with permission from ref. 19. Copyright (2004) National Academy of Sciences.

The crucial elements of HCR are two auxiliary hairpins that contain three domains: toehold, stem, and loop. In the hairpin structure, the short loop is protected by the long stem to store potential energy.¹⁹ The kinetics of HCR can be precisely controlled by changing the length and binding strength of the toehold sequence. Thus, to programmably control the HCR process, the sequence and length of the toehold are crucial factors in the rational design of an HCR. Generally, HCR cannot be effectively initiated if the toehold domain is too short. It has been found that the reaction rate remains stable and almost unchanged when the toehold length is approximately 6–10 nucleotides.¹⁸ Recently, Yung and co-workers established a four-point set of guidelines for designing the lengths and GC contents of the hairpin toehold and stem domains to improve the kinetics of HCR.⁴⁸ Up to now, a variety of novel strategies for activation and control of the HCR process have been reported such as photoactivation,^49,50 pH control,⁵¹ and proximity-dependent initiation.⁵²

2.2 Nonlinear HCR

In most cases, HCR is fundamentally a linear growth mechanism in response to an initiator. In recent years, more complicated sets of monomers have been designed to develop nonlinear HCR, such as branched HCR and even dendritic systems. Compared with linear HCR, nonlinear HCR can readily achieve quadratic or exponential growth kinetics, thereby facilitating construction of amplified biosensing platforms with ultrahigh sensitivity and excellent selectivity. For example, based on triggered HCR, LaBean's group proposed a dendritic DNA nanostructure for the first time.⁵³ Although this system could be used to detect an arbitrary target, many problems remained such as the thermal process (from 25 °C to 30 °C), leakage of hairpins, and need for a relatively long stem sequence (15 base pairs), which could lead to linear growth rather than dendritic growth.

Alternatively, Hsing's group developed a target DNA-triggered nonlinear HCR protocol using two double-stranded DNAs (dsDNAs) as substrates (substrate-A and substrate-B) and two ssDNAs as assistants (assistant-A and assistant-B) (Fig. 2A).²⁷ The A and B substrates were designed with toehold and bulge loop domains. A trigger DNA initiated successive TMSD reactions, resulting in the self-assembly of double-stranded substrates into dendritic DNA structures via exponential growth. Moreover, the same group successfully applied this strategy to immobilization-free and enzyme-free electrochemical detection of DNA, which offered a limit of detection as low as 100 fM and excellent specificity for detecting single-nucleotide polymorphisms.⁵⁴ Although the above target-triggered nonlinear HCR strategies could achieve exponential amplification for nucleic acid detection and efficiently controlled system leakage, the introduction of double-stranded substrates required purification steps to remove the unreacted ssDNAs. A hairpin-based hyperbranched HCR (HB-HCR) strategy was also proposed (Fig. 2B).²⁸ This system consisted of two super-hairpins (SH1 and SH2), two hairpins (H1 and H2), and two assistant ssDNAs (AS1 and AS2). In the super-hairpin structures, two base pairs were pre-programmed in the bulged loop, which divided the stem into two domains and effectively avoided the spontaneous initiation of HB-HCR in the absence of an initiator. The introduction of a target DNA triggered a cascade of TMSD events, resulting in the self-assembly of hyperbranched and nicked double-stranded DNA structures. This HB-HCR demonstrated exponential growth kinetics and allowed reliable detection of target DNA at concentrations as low as 10⁻¹³ M. More recently, through the integration of catalytic hairpin assembly (CHA) with HCR, a triggered and catalytic system for the self-assembly of hyperbranched DNA structures was reported by the same group.⁵⁵ In addition, HCR has been applied to build a variety of complex DNA nanostructures, such as ladder-like and ring-like DNA nanostructures⁵⁶ and DNA hydrogels.⁵⁷

Fig. 2 Nonlinear HCR systems. (A) Self-assembly of DNA dendrimers via triggered chain-branching growth. Reprinted with permission from ref. 27. Copyright (2014) American Chemical Society. (B) HB-HCR. Inset: Atomic force microscopy (AFM) images of assembled products in (a) the absence and (b) presence of initiator DNA (I). Adapted with permission from ref. 28. Copyright (2015) John Wiley and Sons.

2.3 Visualization and analysis techniques of HCR products

Since numerous signal readout techniques are available, the HCR process and its products can be flexibly monitored and detected. Gel electrophoresis is one of the most common ways to verify the formation of HCR products, but it is rarely used for quantitative determination.^19,21 AFM has been applied to observe the morphology of HCR products, such as nanowires (Fig. 5A and 13A)^23,58,59 and dendritic or hyperbranched structures (Fig. 2B).^27,28,55 The length and height of assembled HCR products can also be obtained directly from AFM.

Optical detection methods provide effective tools for the quantitative determination of analytes. Optical readout techniques mainly include fluorescence, chemiluminescence, bioluminescence, colorimetry, surface plasmon resonance (SPR), and surface-enhanced Raman scattering (SERS). HCR-based fluorescence methods can be classified into four types: (1) fluorophores that are chemically conjugated to the hairpins;^25–28,60 (2) a label-free fluorescent strategy in which the fluorescent dyes (e.g., SYBR Green I) intercalate into the grooves of dsDNA;⁶¹ (3) a fluorescence resonance energy transfer (FRET) strategy in which a fluorescence donor and an acceptor are labelled at the appropriate positions of hairpins;⁶² and (4) NPs used as fluorescent signal reporters, such as quantum dots (QDs),^63,84 silver nanoclusters (AgNCs),⁶⁴ and copper NPs (CuNPs).⁴³ For chemiluminescence, a signal is usually generated by immobilizing horseradish peroxidase (HRP) molecules on the HCR products; HRP catalyses the oxidation of luminol by H₂O₂.³⁵ DNAzyme-based chemiluminescence strategies, such as the hemin/G-quadruplex HRP-mimicking DNAzyme⁵⁹ and manganese porphyrin (MnTMPyP)-dsDNA mimicking peroxidase,³⁴ have also attracted increasing attention in recent years. For bioluminescence, HCR can be used for the homogeneous detection of various targets.⁶⁵ Additionally, colorimetric assays can be mainly divided into two major classes. The first is gold nanoparticle (AuNP)-based assays. In this case, colorimetric and spectroscopic analysis of HCR products can be realized based on the different aggregative behaviours of AuNPs induced by the analytes.^29,66 The other class of HCR-amplified colorimetric methods is based on HRP⁴² or hemin/G-quadruplex HRP-mimicking DNAzyme⁵⁹ catalysed colorimetric systems, such as the oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS²⁻) or 3,3′,5,5′-tetramethylbenzidine (TMB) by H₂O₂. In addition, SPR⁶⁷ and SERS⁶⁸ have also emerged as powerful optical techniques for quantitative analysis of HCR products.

In addition to optical techniques, electrochemical methods have been used extensively to study HCR. HCR-based electrochemical strategies can be broadly classified into four varieties: (1) hybridization with hairpins that are chemically modified with electroactive indicators;³¹ (2) employment of HRP molecules⁶⁹ or DNAzymes⁷⁰ (e.g., hemin/G-quadruplex HRP-mimicking DNAzyme) immobilized on HCR products to catalyse the H₂O₂-mediated oxidization of electrochemical substrate; (3) label-free electrochemical strategies in which electrochemical indicators (e.g., methylene blue [MB],^30,33 doxorubicin [DOX],⁷¹ or hexaammineruthenium(III) chloride ([Ru(NH₃)₆]³⁺, RuHex))⁷² are intercalated into the double-stranded HCR products; and (4) incorporation of NPs, especially silver NPs (AgNPs), into the HCR products via labelling at DNA hairpins⁷³ or electrostatic adsorption.⁷⁴ Moreover, HCR-based photoelectrochemical (PEC) detection methods have been identified as powerful tools for biological analysis.^75,76 Additionally, the HCR process can be monitored by quartz crystal microbalances (QCMs), which have been demonstrated to be a simple and label-free method for sensitive and real-time detection of nucleic acids.⁷⁷

3 Applications of HCR in biosensing assays

HCR exploits DNAs as amplification transducers that can amplify an output signal over hundreds of times. Thus, combined with its advantages of isothermal and enzyme-free conditions, HCR has become an ideal analytical method for ultrasensitive and selective detection of various targets, offering new perspectives for point-of-care (POC) diagnostics. Herein, we classify the HCR amplification biosensing methods into five categories based on the type of analyte: nucleic acids, protein molecules, enzyme activities, tumour cells, and small molecules.

3.1 Detection of nucleic acids

Nucleic acids, including DNAs and RNAs, are fascinating biopolymers that have been considered as specific markers for the early diagnosis of diseases, especially cancers. Therefore, sensitive and specific detection of nucleic acids is of great significance for biological and biomedical studies and clinical diagnosis. To achieve this goal, a variety of HCR-based strategies have been developed for nucleic acid detection, and they can be artificially divided into two classes based on whether they occur in solution or on a solid support.

3.1.1 Solution-based detection. In solution-based (homogeneous) HCR for nucleic acid detection, a DNA or RNA target is used as an initiator to trigger the cross-opening of two kinetically trapped hairpins, resulting in the formation of DNA polymeric nanowires. HCR products, which contain hundreds of repeat units, provide an excellent isothermal amplification platform for nucleic acid detection via the incorporation of various labels to generate signals. These strategies mainly include fluorescent and colorimetric assays. For a practical application of HCR-based fluorescence biosensors, the binding of labels for signal transduction is a crucial factor to be considered. Tan's group proposed a DNA detection system based on pyrene-labelled hairpins for HCR (Fig. 3A).²⁵ The two hairpin probes, H1 and H2, were modified with pyrene moieties at the 3′- and 5′-ends. The two pyrene residues were spatially separated from each other in the absence of the target, resulting in strong monomer emission peaks (at 375 and 398 nm). When the target was present, it activated the HCR process, forming a nicked double helix, and bringing the pyrene unit modified on one hairpin close to the pyrene on the adjacent hairpin. Because of the transition from the pyrene monomer to the pyrene excimer, a shift in the emission from 375 and 398 nm to a longer wavelength (485 nm) can be observed. This method achieved the sensitive detection of DNA at concentrations as low as 256 fM and was successfully applied to quantitation of nucleic acids in complex biological fluids. Another prominently homogeneous example is based on the different fluorescence quenching effects of graphene oxide (GO) between fluorophore-labelled hairpins and HCR products (Fig. 3B).²⁶ Compared with other GO-based fluorescent assays for nucleic acid detection, which use a 1 : 1 (target : probe) binding model,^78,79 the sensitivity of this method was improved by more than 2 orders of magnitude. Luminescent NPs have also attracted considerable interest as fluorescent reporters for label-free biosensors. By combining highly fluorescent AgNCs as reporters with HCR amplification, a fluorescent biosensing platform was established for the detection of let-7a microRNA with a detection limit of 0.78 nM and the identification of single-nucleotide polymorphisms in the let-7 microRNA family.⁶⁴

Fig. 3 HCR-based homogeneous fluorescent assays for nucleic acid detection. (A) Pyrene-excimer probes based on HCR for DNA detection. Reprinted with permission from ref. 25. Copyright (2011) John Wiley and Sons. (B) GO-assisted HCR for microRNA detection. Reprinted with permission from ref. 26. Copyright (2012) American Chemical Society.

Colorimetric assays have attracted substantial interest for nucleic acid detection because of their advantages of low cost, easy operation, and practicality.⁸⁰ AuNPs can be considered one of the most competitive materials in colorimetric biosensing.^81–83 There are two major mechanisms of HCR-based colorimetric assays using AuNPs. The first is the use of nucleotide-modified AuNPs as signal reporters. Based on this mechanism, an HCR-amplified colorimetric approach has been proposed (Fig. 4A).⁶⁶ In this method, AuNPs were modified with capture DNA probes. The target oncomiR specifically hybridized with the capture DNA, forming a RNA/DNA heteroduplex that exposed a sticky RNA end to bind with a specific initiator to initiate HCR on the AuNPs. The resulting long double helix on the surface of the AuNPs protected the AuNPs from aggregation induced by Mg²⁺ ions, resulting in a red colour. Conversely, since no HCR product was assembled on the AuNPs in the absence of a target, an immediate colour transition (within seconds) or black precipitate (after an hour) can be observed upon Mg²⁺ treatment, which results from aggregation of the AuNPs. This visual strategy permitted the sensitive and selective analysis of three circulating oncomiRs separately and simultaneously with a detection limit of 20 fM. The other, more notable HCR-based colorimetric method mechanism is based on the different binding capacities of ssDNA and dsDNA toward unmodified AuNPs, which facilitate label-free analysis. Since no covalent functionalization of AuNPs is required, this type of strategy shows notable benefits for a visual analysis of nucleic acids (Fig. 4B).²⁹ Target DNA concentrations as low as 100 pM can be identified by the naked eye, and 50 pM concentrations can be detected by ultraviolet (UV)-vis measurement.

Fig. 4 HCR-based colorimetric assays for nucleic acid detection. (A) HCR-amplified colorimetric approach using nucleotide-modified AuNPs as reporters. Reprinted with permission from ref. 66. Copyright (2016) Royal Society of Chemistry. (B) HCR-amplified colorimetric method based on different binding capacities of ssDNA and dsDNA towards unmodified AuNPs. Reprinted with permission from ref. 29. Copyright (2013) American Chemical Society. (C) HCR-triggered enzyme cascade amplification for colorimetric detection of target DNA (T). Reprinted with permission from ref. 85. Copyright (2016) American Chemical Society.

More recently, Yang's group developed an ultrasensitive colorimetric platform based on HCR-triggered enzyme cascade amplification (Fig. 4C).⁸⁵ The two hairpins used in this strategy were modified with glucose oxidase (GOx) and HRP. Then addition of the nucleic acid target (T) initiated HCR, resulting in the formation of GOx/HRP enzyme pairs. By combining HCR amplification with enhanced catalytic performance of the GOx/HRP enzyme pairs for the oxidation of ABTS²⁻ by H₂O₂, a low detection limit of 5.2 fM was achieved for the nucleic acid target.

Catalytic units are often used as amplifying labels in biosensing strategies. Catalytic nucleic acids (DNAzymes or ribozymes), which are isolated through systematic evolution of ligands by exponential enrichment (SELEX), can be considered to be ideal candidates for developing bioanalytical platforms because of their good structural flexibility and high catalytic activity.⁹⁰ Specifically, DNAzymes have been coupled with HCR to act as effective catalysts for amplified fluorescence, chemiluminescence, colorimetric, and electrochemical detection of nucleic acids. Willner's group combined a DNAzyme with HCR for DNA detection for the first time (Fig. 5A).⁵⁸ In this system, the two hairpins included Mg²⁺-dependent DNAzyme subunits that were partially “caged” by the stem regions. In the presence of target DNA, HCR was activated to generate polymeric DNA nanowires consisting of catalytically active Mg²⁺-dependent DNAzyme units. The activated DNAzyme nanostructures catalytically cleaved the substrates that were labelled with fluorophore/quencher (F/Q) pairs at their 3′- and 5′-ends, respectively, resulting in the generation of fluorescence and allowing sensitive detection of DNA with a detection limit of 10 fM. In a related system, an HCR-based colorimetric and chemiluminescent biosensing platform was demonstrated by the same group using hemin/G-quadruplex HRP-mimicking DNAzymes as amplifying labels (Fig. 5B).⁵⁹ The hairpin structures contained three-fourths and one-fourth of the HRP-mimicking DNAzyme sequence, which were both in inactive configurations in the absence of a target. The target DNA triggered an autonomous cross-opening of the hairpins, resulting in formation of DNA nanowires consisting of numerous hemin/G-quadruplex HRP-mimicking DNAzymes that could catalyse the H₂O₂-mediated oxidation of ABTS²⁻ or luminol to generate a colorimetric or chemiluminescent signal for target DNA detection. G-Quadruplexes that kinetically conjugated on DNA nanowires with precise positioning were studied by Wang et al., who determined that they provided a solid support for label-free detection of various targets via signal amplification from HCR and hemin/G-quadruplex HRP-mimicking DNAzyme.⁹¹

Fig. 5 (A) Mg²⁺-dependent DNAzyme-based and (B) hemin/G-quadruplex DNAzyme-based HCR for nucleic acid detection. Inset of (A): AFM image and cross-sectional analysis of the resulting DNAzyme nanowires via HCR. Adapted with permission from ref. 58 and 59. Copyright (2011) and (2012) American Chemical Society.

3.1.2 Solid-phase detection. In HCR-based solid-phase strategies for nucleic acid analysis, HCR is implemented on a surface, such as an electrode, microparticles or NPs, microwell plate, glass chip, or microfluidic device. The main advantage of solid-phase assays is that they can easily separate an analyte from the complex sample matrix. Moreover, these assays can be flexibly applied for optical or electrochemical detection, and they hold great potential for high-throughput analysis and development in POC settings.

As a convenient, sensitive, and cost-effective detection technique, HCR-based electrochemical sensors have attracted substantial attention. Yang and co-workers introduced an enzyme-free and conjugation-free electrochemical genosensor (Fig. 6A).⁸⁶ In this method, the capture probes were immobilized on the surface of an electrode via Au–S bonds. Upon the introduction of target DNA, sandwiched hybridization between the target and initiator strands initiated HCR, resulting in the formation of linear, electronegative DNA concatemers. Then, the electrochemical reporter [Ru(NH₃)₆]³⁺ interacted with the DNAs through electrostatic adsorption. This HCR-based electrochemical biosensor could accurately detect the breast cancer type 1 (BRCA1) gene with ultrahigh sensitivity (as low as 1 aM) and specifically discriminate single-base mismatched sequences.

Fig. 6 HCR-based electrochemical strategies for nucleic acid detection. (A) Enzyme-free and conjugation-free electrochemical genosensor for detection of the BRCA1 gene. Reprinted with permission from ref. 86. Copyright (2016) Elsevier. (B) Incorporation of tetrahedral DNA nanostructured probes with HCR amplification for microRNA detection. Reprinted with permission from ref. 69. Copyright (2014) American Chemical Society.

Controlling the density and orientation of molecular probes on an electrode surface is the main challenge associated with electrochemical biosensors. To address this issue, a probe-conjugated three-dimensional (3D) tetrahedral DNA nanostructure-immobilized electrode was developed to increase the accessibility and reactivity of probes and avoid the introduction of spacer molecules (Fig. 6B).⁶⁹ Target DNA was hybridized with the tetrahedral probe on the electrode surface, which then triggered the HCR to assemble HRP molecules and produce an electrocatalytic signal for quantitative analysis of the target DNA. Based on the synergetic effect of the tetrahedral probe and HCR, the detection limits of DNA and microRNA were as low as 100 aM and 10 aM, respectively. Similarly, another tetrahedral DNA nanostructure-based HCR-amplified electrochemical biosensor was constructed using AuNPs as initiator carriers, and its detection limit was as low as 2 aM microRNA.⁷³

For optical solid-phase assays, microparticles or NPs, glass chips or microwell plates are usually employed as carriers to immobilize HCR products. For instance, Zhang and co-workers reported an amplification strategy for ultrasensitive and selective detection of DNA on magnetic particles that combined HCR with enzyme enhancement.⁸⁷ In this method, one biotin-modified hairpin (Bio-H1) was first immobilized on streptavidin (SA)-coated magnetic beads. Upon addition of target DNA, the Bio-H1 was opened, and a sticky end was exposed to specifically hybridize to the toehold of the other kind of biotin-modified hairpin (Bio-H2), resulting in the initiation of HCR. Then, avidin-conjugated HRP molecules were attached to the resulting HCR products, and the fluorescence signal of bi-p,p′-4-hydroxyphenylacetic acid that was generated via HRP catalysis of hydroxyphenylacetic acid (HPA) was measured. Ultrasensitive detection of the target DNA was achieved, with a detection limit as low as 8.1 × 10⁻¹⁶ M.

Although this method achieved ultrahigh sensitivity, it suffered from the disadvantages of expensive labelling costs (e.g., the modification of biotin and avidin) and sophisticated processes for signal molecule immobilization (e.g., the biotin–avidin interaction). As an alternative, a label-free electrochemiluminescence (ECL) protocol was developed for DNA detection by intercalating Ru(phen)₃²⁺ into the dsDNA grooves of the HCR products and achieved an ultrasensitive analysis of femtomolar DNA.⁸⁸ In addition, a label-free fluorescence strategy was proposed for a high-throughput detection of RNA based on branched HCR polymerization with binding to SYBR Green I dye.⁶¹ In recent years, SPR has emerged as a powerful optical tool for detecting a wide range of biomolecules.⁸⁹ Accordingly, HCR-based SPR amplification strategy has been developed for label-free detection of DNAs and has achieved high sensitivity and selectivity.⁶⁷ A comparison of different HCR strategies for detecting nucleic acids is listed in Table 1. Although all the strategies are based on HCR, they demonstrate different detection limits. Generally, sensitivity of HCR-based sensors is related to not only HCR amplification and signal readout methods but also the protocols that integrate with other isothermal amplification techniques and detection models (e.g., “signal-on” or “signal-off”). In addition, reaction conditions, especially stability of the designed hairpins, are of great significance to determine a detection limit. For instance, unstable hairpins partially open in the absence of a target would potentially reduce signal gain.

Table 1 HCR strategies for detecting nucleic acids

Target	Method	Signal transduction	Detection limit	Ref.
Solution-based detection
DNA	Pyrene molecules modified hairpins for HCR	Fluorescence	256 fM	25
microRNA	GO-Assisted HCR	Fluorescence	1 pM	26
microRNA	HCR modulated DNA-hosted AgNCs as signal reporters	Fluorescence	0.78 nM	64
DNA	Mg^2+-Dependent DNAzyme-based HCR assay	Fluorescence	10 fM	58
DNA	Nonlinear HCR using dsDNAs as substrates	Fluorescence	0.15 nM	27
DNA	Hairpin-based HB-HCR	Fluorescence	100 fM	28
microRNA	Self-assembly of hyperbranched DNA structures through integrating CHA with HCR	Chemiluminescence	0.72 pM	55
oncomiRs	HCR-amplified assay using nucleotide-modified AuNPs as signal reporters	Colorimetry	20 fmol	66
DNA	Unmodified AuNPs as signal reporters that have different binding capacities toward hairpin monomers and double-stranded HCR products	Colorimetry	50 pM (UV-vis detection)	29
			100 pM (naked eye detection)
DNA	HCR-triggered enzyme cascade amplification	Colorimetry	5.2 fM	85
DNA	Assembly of HCR products containing hemin/G-quadruplex HRP-mimicking DNAzymes as amplifying labels	Colorimetry and chemiluminescence	100 fM	59
DNA	Target-triggered nonlinear HCR	Electrochemistry	100 fM	54
DNA	Self-assembly of dendritic DNA nanostructure	Polyacrylamide gel	10 nM	53
Solid-phase detection
DNA	HCR-based enzyme-free and conjugation-free genosensor using Ru(NH3)6 as electrochemical reporter	Electrochemistry	1 aM	86
DNA	Probe-conjugated 3D tetrahedral DNA nanostructure modified electrode for HCR	Electrochemistry	100 aM (DNA)	69
microRNA			10 aM (microRNA)
microRNA	Tetrahedral DNA nanostructure modified electrode using AuNPs as initiator carriers for HCR	Electrochemistry	2 aM	73
microRNA	Tetrahedral DNA nanostructure modified electrode using metal-catalyst-free click chemical signal amplification via HCR	PEC	27 aM	75
DNA	HCR-based label-free strategy using cyclometalated iridium(III) complex as signal indicator	PEC	9.0 fM	76
DNA	HCR with enzyme-amplification using magnetic particles as carriers	Fluorescence	0.81 fM	87
RNA	Branched HCR-based label-free detection platform on 96-well plate	Fluorescence	1 pM	61
DNA	Label-free and real-time QCM with dissipation monitoring (QCM-D) biosensing platform	QCM	0.1 nM	77
DNA	HCR-based label-free biosensor using Ru(phen)3^2+ as ECL reporter	ECL	15 fM	88
DNA	HCR-based label-free SPR assay	SPR	0.3 fM (DNA)	67

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3.2 Detection of proteins

Proteins are essential for the maintenance of life, and protein dysfunction has been demonstrated to cause many diseases.⁹² In addition, many proteins exist in quite low abundance in real samples. Therefore, development of signal amplification methods for highly sensitive and specific detection of proteins is very significant for bioanalysis and biomedicine. HCR, as a fascinating enzyme-free isothermal amplification strategy, has been widely exploited for the ultrasensitive detection of proteins. Using recognition mechanisms, these methods can be divided into two types: Ab-based and aptamer-based strategies.

3.2.1 Ab-Based strategies. Abs, which specifically bind to antigenic epitopes, have been widely used as ligands for quantitative determination of proteins in fundamental research and clinical diagnostics. The most common Ab-based method used to detect proteins is the enzyme-linked immunosorbent assay (ELISA). Improvement of detection sensitivity is still in high demand. Alternatively, immuno-HCR strategies have been developed through the conjugation of specific DNAs to Abs for HCR amplification. The principles underlying these assays are mainly as follows: (1) formation of the capture Ab (Ab1)/target protein/secondary Ab (Ab2) sandwich immunocomplex on the surface of a solid substrate (e.g., magnetic beads, glass slides, or electrodes); (2) initiation of HCR by an oligonucleotide trigger that is linked to Ab2; and (3) quantitative measurement of the immuno-HCR products via optical or electrochemical techniques. Based on these principles, Choi and co-workers developed a fluorescence-based strategy for detecting secreted cytokines and chemokines from a single human mononuclear cell (Fig. 7).⁶⁰ Compared with other fluorescence methods without HCR amplification, the detection limit and sensitivity were improved by 200-fold on average. Furthermore, an electrochemical immuno-HCR biosensor was constructed using AuNPs as DNA initiator carriers for signal amplification.³¹ Employing AuNPs greatly increased the amount of DNA initiators, resulting in more HCR products and a good signal amplification capacity for determining the target protein at concentrations as low as 0.1 fg mL⁻¹.

Fig. 7 Ab-Based immuno-HCR for protein detection. Reprinted with permission from ref. 60. Copyright (2011) American Chemical Society.

3.2.2 Aptamer-based strategies. Aptamers are short, ssDNA or RNA molecules derived from a random population of DNA/RNA sequences by SELEX.^93,94 As another type of affinity ligand, aptamers have been frequently used to recognize a wide range of targets, including proteins, small molecules, and even whole cells, with high affinity and specificity.^95,96 Compared to Abs, aptamers have many advantages such as easy synthesis and modification, low cost, long storage time, and nontoxicity. In recent years, various HCR-based aptasensors have been developed for protein detection. A typical aptasensor for the detection of protein (platelet-derived growth factor BB [PDGF-BB]) that coupled HCR with aptamer recognition is demonstrated in Fig. 8A.⁶³ Target aptamers first initiated hybridization events between biotinylated DNA hairpins to form long nicked double helices, which then reacted with SA–QDs to form QD–DNA conjugates. Based on target-aptamer recognition, the QD–DNA conjugates specifically bound to PDGF-BB that was previously captured by Abs on the surface of a well, facilitating fluorescence detection of PDGF-BB with a detection limit of 50 pM. Moreover, a universal aptamer-based SERS biosensing platform was proposed using HCR for signal amplification (Fig. 8B).⁶⁸ This strategy resulted in the design of a DNA triple-helix probe based on Watson Crick and Hoogsteen base pairings.⁹⁷ Binding of the target to the aptamer perturbed the triple-helix structure, dissociating the universal trigger, which was captured on the surface of silicon microbeads (SiMBs) and initiated the HCR. AuNPs were then conjugated to the resulting HCR products to generate SERS-active hot-spots. This SERS biosensor has been applied to the determination of multiple analytes, including thrombin, adenosine, and cancer cells.

Fig. 8 HCR-based aptasensors for protein detection. (A) Fluorescence detection of PDGF-BB through the coupling of aptamer recognition with HCR. Reprinted with permission from ref. 63. Copyright (2012) Royal Society of Chemistry. (B) Construction of HCR-based universal SERS aptasensors for the detection of multiple analytes. Reprinted with permission from ref. 68. Copyright (2014) American Chemical Society.

In addition to heterogeneous assays, a homogeneous aptasensing strategy was developed for protein analysis by combining target-triggered HCR with GO-based selective fluorescence quenching.⁹⁸ Based on the different adsorption capacity of GO for ssDNAs and double-stranded HCR polymer products, a strong fluorescence signal could be detected when the target protein was present in a sample, resulting in the highly sensitive and selective detection of PDGF-BB with a detection limit of 1.25 pM. A comparison of different HCR strategies for detecting proteins is provided in Table 2.

Table 2 HCR strategies for detecting proteins and enzyme activities

Target	Method	Signal transduction	Detection limit	Ref.
Cytokines	Immuno-HCR through linking an oligonucleotide initiator to Ab2	Fluorescence	0.27–2.2 pM	60
Human IgG	Immuno-HCR using AuNPs as DNA initiator carriers for signal amplification	Electrochemistry	0.1 fg mL^−1	31
PDGF-BB	HCR-based aptasensor	Fluorescence	50 pM	63
Thrombin	An universal aptasensor using DNA triple-helix probe	SERS	18 pM (thrombin)	68
Adenosine			1.5 nM (adenosine)
CEM cells			10 cells per mL (CEM cell)
PDGF-BB	Homogeneous aptasensing strategy combining HCR with GO-based selective fluorescence quenching	Fluorescence	1.25 pM	98
T4 PNK	HCR-based FCBA	Fluorescence	4 × 10^−6 U mL^−1	106
DNA MTase	HCR-based branched RCA	Chemiluminescence	0.52 U mL^−1	35
Uracil-DNA glycosylase (UDG)	Nuclease-responsive HCR strategy using TWJ with specific nuclease sites	Fluorescence	4.3 × 10^−5 U mL^−1 (UDG)	36
CpG methyltransferase M.SssI			0.019 U mL^−1 (M.SssI)

---

3.3 Detection of enzyme activities

Enzymes are unique proteins that can catalyse biochemical reactions with high rates and selectivity and play a vital role in almost all life processes.^99,100 Additionally, some enzymes (e.g., telomerase,^101,102 DNA methyltransferase [MTase],¹⁰³ and polynucleotide kinase [PNK]^104,105) have been found to be biomarkers of diseases. Therefore, developing assays for detecting enzyme activity is important for biomedical and clinical research. To date, various HCR-based biosensing platforms have been reported for highly sensitive and selective analysis of enzyme activity. Zhang and co-workers proposed an HCR-based flow cytometric bead assay (FCBA) for ultrasensitive detection of T4 PNK activity.¹⁰⁶ In the absence of T4 PNK, the 5′-OH terminus of the blocking DNA cannot be phosphorylated. Thus, the blocking DNA will not be digested by λ exonuclease, resulting in the prevention of HCR. In contrast, when T4 PNK was present in a sample, HCR was triggered on the surface of the magnetic beads. The fluorophores that accumulated on the magnetic beads could be directly analysed using a flow cytometer, and excellent analytical performance for determination of T4 PNK activity was achieved, with a detection limit of 4 × 10⁻⁶ U mL⁻¹. Detecting DNA MTase activity and its inhibitors has also been achieved by HCR.³⁵ Moreover, a universal nuclease-responsive HCR strategy has been developed.³⁶ Through the rational design of a three-way junction (TWJ) with specific nuclease sites, this method can be readily used to detect a wide range of DNA-modified enzymes, which can be further expanded to the construction of advanced target-responsive DNA devices for diverse applications. A comparison of different HCR strategies for determining enzyme activities is shown in Table 2.

3.4 Detection of tumour cells

Expression levels of some biomarkers, such as receptors, proteins or specific enzymes, on tumour cell surfaces or within tumour cells differ significantly from their normal values, offering opportunity for an early clinical diagnosis and treatment of cancers.⁹² To date, many studies have reported the detection of cancer cells through recognition of biomarkers on the cytomembrane.^107,108 Unlike detection of other tumour markers (e.g., nucleic acids and proteins) within the cells, which is often associated with tedious treatment steps, such as cell lysis and target extraction, the direct detection of whole cancer cells is relatively convenient and efficient. Since the cell surface is complex and contains many different molecules, especially proteins,¹⁰⁹ developing enzyme-free amplification strategies for cancer cell detection is highly desirable. For this purpose, a multibranched HCR (mHCR) was developed for detecting cancer cells through aptamer recognition (Fig. 9A).⁴² This mHCR was obtained by joining typical HCR to produce long products with multiple reporters for signal amplification and multiple branched arms for multivalent binding on a DNA tetrahedron nanostructured gold electrode. Because of the synergetic effect of mHCR amplification and multivalent binding, this biosensing platform achieved ultrahigh sensitivity with a detection limit as low as four MCF-7 cells.

Fig. 9 HCR-based strategies for tumour cell detection. (A) mHCR for electrochemical detection of tumour cells. Reprinted with permission from ref. 42. Copyright (2014) American Chemical Society. (B) Label-free detection of tumour cells through metallization of CuNPs on HCR products. Reprinted with permission from ref. 43. Copyright (2015) Royal Society of Chemistry.

A label-free strategy was also developed through the metallization of NPs on HCR products (Fig. 9B).⁴³ In this method, a partially complementary duplex was first formed through hybridization of aptamer sequence (DNAa) with a messenger DNA (DNAb). In the presence of target cells, the aptamers specifically bound to the targets, releasing DNAb to trigger HCR. The resultant long duplex DNA oligomers were employed as templates for the in situ synthesis of CuNPs with strong fluorescence, which were further used as a readout signal to achieve label-free quantitative determination of tumour cells with a detection limit of 50 cells.

3.5 Detection of small molecules

Currently, a number of functional nucleic acids, such as aptamers and DNAzymes, have been employed to expand HCR-based isothermal amplified techniques for detecting small molecules, including small biomolecules and metal ions. For small biomolecule detection, most strategies have relied on aptamer recognition, in which target molecules trigger the HCR upon binding to aptamers. Based on this mechanism, a variety of biomolecules, such as ATP, adenosine and glutathione (GSH), have been sensitively and selectively detected by combining HCR with SA assembly as a molar mass amplifier,¹¹⁰ exonuclease III (Exo III)-assisted DNA recycling,³⁹ and specific thymine–Hg²⁺–thymine (T–Hg²⁺–T) coordination,³⁸ respectively.

As another kind of small molecules, metal ions have also been determined to exert serious effects on human health and environmental quality.¹¹¹ Thus, construction of facile, sensitive, and reliable methods for detecting metal ions has attracted substantial interest. Traditional techniques for metal ion analysis include inductively coupled plasma mass spectrometry (ICP-MS)¹¹² and atomic absorption spectroscopy.¹¹³ However, these methods usually require sophisticated instruments, complicated procedures, and relatively large amounts of sample. Recently, nucleic acid-based biosensors have been developed for metal ion detection with improved sensitivity. Generally, the interactions between metal ions and nucleic acids can be divided into two types: the specific response of DNAzymes or ribozymes to metal ions (e.g., Pb²⁺, Mg²⁺, and Cu²⁺)⁹⁰ and the specific bridges between metal ions and nucleosides or ligandosides that form stable metal ion-coordination base pairs (e.g., T–Hg²⁺–T¹¹⁴ and C–Ag⁺–C¹¹⁵). Based on these mechanisms, HCR amplification strategies have been developed in which initiator DNA is generated in response to target metal ions, which, in turn, initiate HCR, and pM-level sensitivity has been achieved.^41,116

4 Applications of HCR in bioimaging and biomedicine

Comprehensively understanding biological pathways is critical for the development of biological systems and their applications in clinical research. In recent years, imaging analysis has emerged as one of the most promising techniques for a complete understanding of biological processes and effective diagnosis and treatment of diseases. Some isothermal amplification techniques, such as RCA,^117,118 SDA,¹¹⁹ and loop-mediated isothermal amplification (LAMP),^120,121 have been used for bioimaging analysis. However, these methods need enzymes and often require complicated operations. More recently, HCR-based isothermal amplification techniques have been extended to image various targets, such as extracellular matrix,⁴⁴ tumour cells,^21,22 and RNAs,^{20,45,62,122,123} demonstrating these techniques' advantages of signal amplification and simplicity and their enzyme-free nature. In this section, we focus on the development of HCR-based bioimaging methods, including fluorescence in situ hybridization (FISH) imaging and live cell imaging (cell surface and intracellular imaging). In addition, the applications of HCR in biomedicine are discussed.

4.1 FISH imaging based on HCR

In early studies, FISH offered an effective tool for mapping RNAs within cells. In a typical FISH method, fluorophore-labelled oligodeoxynucleotides are employed as probes, which enter the fixed cells or tissues by means of permeation and hybridize with target RNAs to generate a response signal. Through FISH, images of intracellular RNA expression and distribution can be obtained to elucidate relevant pathological processes and verify their functions in clinical diagnosis. However, since only one signal probe hybridizes to the target RNA, traditional FISH methods suffer from low signal intensity. Therefore, to improve the signal level, it is highly desirable to develop a method for targeting the same analyte with multiple probes.¹³⁰ In recent years, HCR-based FISH (HCR-FISH) techniques have been developed to generate sufficient signal-to-background ratios, where a polymerization reaction between two fluorophore-labeled hairpins is initiated upon recognizing a single given target, thus achieving amplified fluorescence activation imaging. It has been reported that the mean signal obtained by HCR-FISH is ∼200-fold higher than using direct-labeled probes without HCR amplification.¹²²

Using HCR-FISH, Pierce's group conducted a series of innovative studies.^{20,122,124–128} They first proposed an HCR-FISH method to map five target mRNAs simultaneously in fixed whole-mount and sectioned zebrafish embryos (Fig. 10A).²⁰ This strategy mainly involved two stages: the detection stage and the amplification stage. In the detection stage, target mRNAs hybridized in situ to the complementary RNA probes, and then, the samples were washed to remove the unreacted probes. In the amplification stage, orthogonal initiators triggered the in situ HCR to form distinct fluorophore-tethered DNA polymers, and the optical signals generated by all the target mRNAs were imaged simultaneously. As shown by the results in Fig. 10A, this HCR-FISH method exhibited deep sample penetration, high signal-to-background ratios, and sharp signal localization. The same group subsequently introduced the next generation of in situ HCR for imaging RNA expression.¹²² Unlike first-generation HCR-FISH, in this system, DNA probes and amplifiers were employed instead of RNA probes and amplifiers to improve reagent durability and decrease reagent costs. In addition, permissive in situ amplification conditions were established, and the DNA hairpins were engineered to increase the signal gain per amplifier. Recently, HCR-FISH has also been applied for detecting environmental microorganisms.¹²⁹

Fig. 10 (A) HCR-FISH used for simultaneous mapping of multiple target mRNAs in fixed whole-mount and cross-sectioned zebrafish embryos. Adapted with permission from ref. 20. Copyright (2010) Nature Publishing Group. (B) Wash-free HCR-FISH used for in situ detection of target mRNA in cancer cells. Reprinted with permission from ref. 62. Copyright (2016) Royal Society of Chemistry.

The above mentioned HCR-FISH methods required repeated washing steps to remove unbound probes and hairpins and were, thus, time consuming and required complicated procedures. Very recently, Wang introduced a wash-free HCR-FISH technique using FRET, which successfully eliminated the need for multiple washing steps and prevented false-positive signals caused by probe accumulation or degradation in quencher/dye systems (Fig. 10B).⁶² This system contained three DNA hairpins: H1, H2, and H3. H1 was partially complementary to the target mRNA, whereas H2 and H3 were modified with a fluorescence acceptor (carboxytetramethylrhodamine [TAMRA]) and donor (fluorescein amidite [FAM]), respectively, at the appropriate positions. As a result, H1 could be opened by the target mRNA, which further initiated HCR via the cross-opening of H2 and H3, facilitating the generation of amplified FRET signals. Therefore, washing steps were not needed in this FRET HCR-FISH strategy, which significantly simplified the operations and reduced experimental time.

In particular, a proximity-dependent initiation of HCR (proxHCR) was proposed by Söderberg et al.⁵² When hairpin-conjugated Abs bound in close proximity, an initiator strand was revealed to activate the HCR, which resulted in generation of fluorescence signals. This proxHCR protocol was successfully applied to the sensitive detection of protein interactions and post-translational modifications of native proteins in fixed cells and tissues and is promising as a robust alternative to proximity ligation assays.

4.2 Live cell imaging based on HCR

The above mentioned HCR-based imaging methods mainly focused on fixed cells or tissue sections. To obtain detailed spatial-temporal information on dynamics related to cellular functions and diseases, live cell imaging methods are required.

4.2.1 Cell surface imaging. The self-assembly of signal probes on cell surfaces with high affinity is important for cell surface imaging. Cell aptamers are short single-stranded RNA or DNA sequences that can specifically bind to a certain cell line with high affinity.¹⁰⁹ Tan's group reported HCR-based aptamer-tethered DNA nanodevices (aptNDs) that specially targeted live cells in a complicated environment (Fig. 11A).²¹ Fluorescent aptNDs were constructed on target live cell surfaces by anchoring and in situ self-assembly models, highlighting the potential to build nanodevices for bioimaging on cell surfaces through specific aptamer–target interactions. Very recently, the HCR-programmed construction of linear QDs-aptamer polymers (QAPs) was developed for signal-amplified molecular imaging of a specific type of cancer cell (Fig. 11B).²² In comparison with QD-aptamer monomer (QAM)-treated target cancer cells, the photoluminescence signals generated by QAPs were significantly improved, which could be attributed to multivalent binding and multi-QD-based signal amplification.

Fig. 11 HCR-based cell surface imaging. (A) Construction of fluorescent DNA aptNDs on target cell surfaces using HCR via an anchoring model (a) and an in situ self-assembly model (b). Reprinted with permission from ref. 21. Copyright (2013) John Wiley and Sons. (B) Fabrication of linear QAPs for molecular imaging of cancer cells. Reprinted with permission from ref. 22. Copyright (2016) American Chemical Society.

4.2.2 Intracellular imaging. Recently, imaging techniques have been developed for intracellular imaging in live cells. Compared to extracellular analysis, these methods require transportation of DNA probes into cells. Thus, efficient delivery of probes into the cytoplasm is a key step for intracellular imaging. Several inherent factors, such as charges and sizes of the DNA probes, are highly important for cellular uptake. For example, the negatively charged phosphate backbones of nucleic acids repel the negatively charged cell surface, resulting in inadequate and inefficient cellular association. In addition, oligonucleotides that are longer than 25 bases tend to self-hybridize, which affects their uptake by cells.¹⁴⁰ Thus, developing efficient approaches for probe internalization is urgently needed to enable intracellular nucleic acid delivery for in vivo applications.

Recently, HCR-based imaging strategies have been proposed to investigate the dynamic expression and distribution of intracellular nucleic acids, enzyme activities, and small molecules in live cells with the assistance of nanomaterials (e.g., AuNPs,⁴⁵ GO,^123,131 mesoporous silica nanospheres [MSNs],¹³² and ZnO NPs¹³³). In general, hairpins were assembled on the nanomaterials through electrostatic interaction, π-stacking, or hydrogen bonding. After transporting the conjugates into cells, HCR was initiated upon the recognition of targets, resulting in generation of long double helices with strong fluorescence. Jiang et al. developed an electrostatic DNA nanoassembly for ultrasensitive imaging of mRNA in live cells based on HCR amplification (Fig. 12A).⁴⁵ In this strategy, the fluorescence donor and acceptor-labelled DNA hairpins were electrostatically assembled on cationic peptide-coated AuNPs, which were efficiently delivered into live cells through an endocytosis-independent mechanism. Because of efficient quenching of the fluorescence signals of the fluorophores by AuNPs, the DNA hairpins that assembled on the AuNPs produced very weak fluorescence signals in the absence of target mRNA. In contrast, presence of target mRNA triggered the HCR, resulting in dissociation of the hairpins from the particles and the recovery of FRET signals, indicating target mRNA expression. Similarly, via electrostatic interaction between DNA probes and positively charged group-modified MSNs, a GSH-initiated HCR was proposed for sensitive monitoring of GSH expression in single cancer cells.¹³² In addition, using GO as a probe carrier and fluorescence quencher, Tang's group proposed an HCR-amplified fluorescence imaging of microRNA in live cells (Fig. 12B).¹²³ This method also achieved simultaneous imaging of two types of microRNAs in live cells, which would efficiently avoid false positive results. Moreover, a similar work has been reported by the same group using GO for fluorescence imaging of intracellular telomerase activity based on HCR amplification.¹³¹

Fig. 12 (A) HCR-based imaging of mRNA in live cells. Reprinted with permission from ref. 45. Copyright (2015) American Chemical Society. (B) Two-color imaging of microRNA in live cells based on HCR and GO. Reprinted with permission from ref. 123. Copyright (2016) Royal Society of Chemistry.

4.3 Applications of HCR in biomedicine

In recent decades, cancer has emerged as one of the most devastating diseases and results in high death tolls worldwide. Currently, surgical intervention, radiotherapy, and chemotherapy are the main cancer treatment methods. However, these methods often kill normal cells or tissues, resulting in a requirement for insufficient doses of anticancer reagents and eventual treatment failure or even patient death.¹³⁴ Therefore, developing efficient approaches for targeted therapy is essential. Ligand-targeted therapeutics (LTTs), such as radioimmunotherapy, has been considered to be a successful means to improve selectivity of anticancer drugs with cancer cells.¹³⁴ In addition to Ab recognition, aptamer-based targeted drug delivery techniques have been developed.^135–137 Compared to immunotherapeutics, aptamers have significant advantages in terms of their size and preparation. In addition, aptamers can transport themselves or anticancer reagents across a cell membrane via the endocytosis pathway.¹³⁸ Thus, aptamers are ideal recognition molecules for targeted drug delivery.

In recent years, aptamer-tethered DNA assemblies based on HCR have been devised that allow construction of enzyme-free platforms for targeted drug delivery with easy procedures and high drug payload capacities. In aptamer-HCR strategies, aptamers have been used as recognition elements to specifically bind to target cells, whereas HCR products with abundant spatially addressable sites have been employed as drug carriers that could be readily internalized by target cells for drug delivery and disease treatment. Chronic accumulation of nanomaterials is avoided because of the intrinsic biodegradability of nucleic acids. Moreover, assembly of aptamer-HCR-based drug carriers is implemented under enzyme-free and isothermal conditions, making this technique suitable for clinical applications. Based on this mechanism, Tan's group reported the first aptamer-HCR example of targeted anticancer drug delivery through construction of aptamer-tethered DNA nanotrains (aptNTrs) and demonstrated that these structures constitute a promising platform for a precise treatment of cancer (Fig. 13A).²³ Recently, an activatable theranostic approach has been developed through a structure-switching aptamer-triggered HCR on cell surfaces (Fig. 13B).⁴⁶ DNA polymers assembled on the target cells accumulated high-load prodrugs, which were then taken up and transported into cells for selective cancer therapy.

Fig. 13 Applications of HCR in biomedicine. (A) Construction of aptNTrs for targeted anticancer drug delivery. Reprinted with permission from ref. 23. Copyright (2013) National Academy of Sciences. (B) Activatable theranostic approach based on a structure-switching aptamer-triggered HCR. Reprinted with permission from ref. 46. Copyright (2015) American Chemical Society.

Structures with multiple binding sites have higher affinity and specificity compared to their monovalent counterparts, which can improve the cellular uptake ability of nanocarriers.¹³⁹ Thus, self-assembly of multivalent DNA nanostructures as drug carriers is desirable for efficient drug delivery. Because of multivalent effects, the multiple binding approach has achieved enhanced binding affinity via HCR, facilitating cellular internalization with no loss of selectivity.²⁴

5 Conclusions and perspectives

In this review, we have summarized development of HCR-based isothermal amplification strategies by highlighting several examples. Based on the unique properties of its enzyme-free and isothermal nature, controllable synthetic routes, and good biocompatibility, HCR has been demonstrated to be a powerful amplification tool in the fields of biosensing, bioimaging, and biomedicine.

Although HCR-based methods have undergone substantial development, biosensing applications still face many challenges. (1) In an HCR, design of the hairpins is a crucial factor. Indeed, a small variation in the hairpins may lead to failure of the HCR process. Substantial effort has been expended to construct creative molecular design systems. For example, based on simulation and experimental validation, a set of four-point design guidelines have been proposed which can be used to design the lengths and GC contents of toehold and stem domains of a hairpin.⁴⁸ Although substantial progress has been achieved, systematic guidelines for designing hairpin sequences remain lacking. (2) Generally, HCR can be combined with various signal readout techniques, such as fluorescence, chemiluminescence, colorimetry, and electrochemistry, to achieve sensitive detection of analytes. However, strategies based on HCR amplification alone often cannot provide sufficient sensitivity for biosensing. To further improve sensitivity, various enzyme-assisted HCR strategies have been proposed with detection limits at the sub-attomole level, which is even lower than that achieved by PCR. Although these enzyme-assisted HCR methods have ultrahigh sensitivities, given the harsh procedures and reaction conditions of the enzymes required, employing enzymes in HCR is incompatible with the goal to improve the detection sensitivity and limits its applications in complex sample analyses. Therefore, an ideal HCR-based biosensing strategy should be completely enzyme-free. Alternatively, developing functional nucleic acids, especially DNAzymes, promotes the establishment of enzyme-free HCR paradigms. HCR-mediated synthesis of DNAzyme products, such as hemin/G-quadruplex wires, not only improves detection sensitivity but also overcomes the drawbacks of enzyme-assisted HCR strategies. (3) For heterogeneous HCR, controlling the density and orientation of DNA probes on the surface of solid substrates is an important issue that must be overcome because it could cause nonspecific adsorption and crowding effects, and limit reaction efficiencies. Although employment of a DNA tetrahedron as a scaffold on solid surfaces has solved this problem to a certain degree because of its structural stability and mechanical rigidity, programmable and controllable immobilization of molecular probes on solid supports remains challenging. (4) In recent years, branched or even dendritic HCR systems have been developed through triggered self-assembly of complicated DNA monomers, achieving quadratic or exponential growth mechanisms. However, these nonlinear HCR strategies are still limited. Therefore, integration of HCR with other isothermal mechanisms to construct more complicated DNA structures is highly desirable. (5) Development of aptamers has benefitted HCR amplification methods, which have been extended to detect a wide range of analytes ranging from nucleic acids to proteins, small molecules, and even whole cells. However, a limited number of aptamers have been selected, which is an obstacle to developing aptamer-HCR strategies. (6) More importantly, currently, most HCR-based bioanalytical methods remain in the laboratory stage, and their practical applications are still challenging. In-depth studies on HCR should be implemented to establish a foundation for future clinical applications. Other fields, such as physical and engineering sciences, should focus on designing simple and sensitive “sample to answer” settings based on HCR to facilitate POC applications.

For bioimaging and biomedicine, HCR-based isothermal protocols provide exciting opportunities for bioimaging in fixed cells and tissues, or even live cells. Furthermore, nanometre-sized HCR products are being used as carriers for targeted drug delivery, highlighting their future utility in biomedicine. However, applications of HCR in bioimaging and biomedicine remain in their infancy, and several issues still need to be addressed. (1) Transportation of HCR products into cells is a crucial step for intracellular imaging or drug delivery. However, cellular uptake of HCR products carrying imaging reagents or drugs is not easy because of the charge and size of nucleic acids. Additionally, few target recognition probes (e.g., aptamers) can be internalized into live cells. Nevertheless, encouraged by ongoing advancements, we expect that a greater variety of transport modes for nucleic acid probes will be developed and that more aptamers will be selected in future HCR studies. (2) Because of the high molecular weight of HCR products, it is difficult to avoid its nonspecific binding to normal cells during cell targeting and drug delivery, which may lead to false signals in bioimaging and kill normal cells or tissues in biomedical applications. Another challenge for the use of HCR in biomedicine is lack of efficient and accurate drug release mechanisms, which would certainly reduce therapeutic effects. (3) The in situ imaging of intracellular non-nucleic acid bioanalytes (e.g., proteins and small molecules) is rarely reported. Moreover, developing enzyme-mediated HCR strategies to study nucleic acid–protein and protein–protein interactions in vivo should be considered. Employing intracellular enzymes or the transportation of enzymes into cells may resolve this issue.

Although many challenges lie ahead of us, we believe that HCR-based isothermal non-enzymatic platforms can be readily integrated with a wide variety of nanomaterials, synthetic and analytical approaches, and precision manufacturing technologies to stimulate development of DNA nanotechnology and further enhance the progress of these strategies in practical applications, such as clinical diagnostics and therapeutics, energy storage and transfer, and molecular computation.

Acknowledgements

The authors appreciate the financial support from the National Natural Science Foundation of China (21535002 and 21375056), the Special Funds of Taishan Scholar Program of Shandong Province (tsqn20161028), and the Shandong Provincial Natural Science Foundation (ZR2016QZ001).

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