Hybridization Chain Reaction Products Research Articles

Branched hybridization chain reaction\u2014using highly dimensional DNA nanostructures for label-free, reagent-less, multiplexed molecular diagnostics

The specific and multiplexed detection of DNA underpins many analytical methods, including the detection of microorganisms that are important in the medical, veterinary, and environmental sciences. To achieve such measurements generally requires enzyme-mediated amplification of the low concentrations of the target nucleic acid sequences present, together with the precise control of temperature, as well as the use of enzyme-compatible reagents. This inevitably leads to compromises between analytical performance and the complexity of the assay. The hybridization chain reaction (HCR) provides an attractive alternative, as a route to enzyme-free DNA amplification. To date, the linear nucleic acid products, produced during amplification, have not enabled the development of efficient multiplexing strategies, nor the use of label-free analysis. Here, we show that by designing new DNA nanoconstructs, we are able, for the first time, to increase the molecular dimensionality of HCR products, creating highly branched amplification products, which can be readily detected on label-free sensors. To show that this new, branching HCR system offers a route for enzyme-free, label-free DNA detection, we demonstrate the multiplexed detection of a target sequence (as the initiator) in whole blood. In the future, this technology will enable rapid point-of-care multiplexed clinical analysis or in-the-field environmental monitoring.

Mesoporous Silica Containers and Programmed Catalytic Hairpin Assembly/Hybridization Chain Reaction Based Electrochemical Sensing Platform for MicroRNA Ultrasensitive Detection with Low Background.

In this work, based on mesoporous silica containers (MSNs) with the programmed enzyme-free DNA assembly amplification of catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR), an ultrasensitive electrochemical sensing platform with low background is developed for the detection of microRNA (miRNA). Herein, the electrochemical reporter methylene blue (MB) was sealed in the pores of MSNs by the double-stranded DNA (dsDNA) gate of hairpin DNA H1 and anchor DNA. In the absence of target, neither the CHA nor the HCR process happened, which enabled a low background. After target was added, DNA H1 was displaced from the MSNs surface and participated in the CHA process with the assistance of hairpin DNA H2, which accelerated the release of MB from the MSNs pore. Meanwhile, the CHA products H1-H2 were hybridized with the capture probes (SH-CP) on the electrode surface, which further initiated the HCR process. The released MB from the MSNs will effectively intercalate into long dsDNA polymers of HCR products, resulting in a significant electrochemical response. Taking miRNA-21 as the model target, the proposed sensing platform achieves a satisfactory detection limit down to 0.037 fM, which is lower than that of electrochemical assay with amplification methods. In addition, the strategy shows good selectivity against other miRNAs and is capable in practical analytes. Benefitting from the features of being label-free and enzyme-free and having low background, high sensitivity, and selectivity, this strategy shows great potential in bioanalysis and clinical diagnostics.

MoS2-loaded G-quadruplex molecular beacon probes for versatile detection of MicroRNA through hybridization chain reaction signal amplification

A molecular beacons (MBs) loaded on molybdenum disulfide (MoS2) nanosheets as fluorescence probes for sensitive and versatile detection of microRNAs (miRNAs) through hybridization chain reaction (HCR) has been designed. MoS2 was used as a adsorbent to capture the MBs and a selective fluorescence quencher to reduce the background signal. In the absence of miRNAs, HCR could not be triggered due to the stability of MB probes. The probes attached to the MoS2 surface, efficiently quenching fluorescence of the G-quadruplex/Thioflavin T. However, the presence of target miRNAs triggers the HCR process to generate large amount of HCR products. Meanwhile, the HCR products of long nanowires chain with abundant G-quadruplexes could not be adsorbed on the surface of MoS2, and therefore detach from the MoS2. Consequently, Thioflavin T could be embedded in G-quadruplexes and produced strong fluorescence signal. This fluorescence emission signal could achieve detection of miRNA as low as 4.2 pM and a wide linear ranges from 0.1 to 100 nM. In addition, a versatile fluorescence probe has been developed for detection of miRNA-21 by changing the miRNA-recognition domain of MB. Thus, the fluorescent probe would be a potential alternative tool for biomedical research and clinical molecular diagnostics.

The ultra-sensitive visual biosensor based on thermostatic triple step functional nucleic acid cascade amplification for detecting Zn2+

Here, we present an ultra-sensitive visual biosensor based on thermostatic triple step functional nucleic acid cascade amplification for detecting Zn2+. A Zn2+-assisted cDNAzyme assay is conducted and the Zn2+ is successfully converted into nucleic acids to achieve the first circular amplification within 1 h. The cleavage products prompted Hybridization Chain Reaction (HCR), forming multiple DNA nanowire structures with branched chains. And the Strand Displacement Amplification (SDA) were further empowered by the HCR products with the addition of the amplification enzyme and the endonuclease. In just 80 min, the second and third-order signal amplifications are reached. With the addition of Hemin and chromogenic substrates, the visual signal output is achieved in 15 min based on the large amount of G-quadruplex (G4) DNAzymes. This biosensor can detect levels as low as 1.075 pM Zn2+, showing a good linear range within 10 pM–100 nM. It shows considerable potential for the Zn2+ quantitative detection.

Label-Free in Situ Monitoring of the DNA Hybridization Chain Reaction by Using Sequence-Selective Minor-Groove-Binding Fluorophores.

A new label-free in situ monitoring system for the hybridization chain reaction (HCR) based on DNA minor-groove-binding fluorophores [Hoechst 33258 (Hoe) or quinone cyanine-dithiazole (QCy-DT)] has been developed. Use of two unmodified hairpin oligodeoxyribonucleotides containing incomplete double-stranded AATT sequences enabled target-dependent formation of probe binding sites-that is, AATT double strand-in the HCR product, together with fluorescence enhancement of minor-groove-binding fluorophores in situ. This system allows target DNA to be detected through the fluorescence enhancement of Hoe and QCy-DT in real time and in situ. Further development of a label-free, isothermal detection system might provide a cost-effective and user-friendly method for nucleic acid detection.

A novel label-free fluorescent detection of histidine based upon Cu2+-specific DNAzyme and hybridization chain reaction.

A novel label-free fluorescent sensor for histidine was developed based upon Cu2+-specific DNAzyme, hybridization chain reaction(HCR) and triplex DNA. Cu2+ can bind to the histidine, in the presence of histidine, leading to the inhibition of the cleavage of substrate strand of Cu2+-dependent DNAzyme, then the intact substrate strand trigger the HCR between H1 and H2. The HCR product can be recognized by triplex-forming oligonucleotide (TFO) through triplex formation and reported by the fluorescence of berberine, the fluorescence intensity of the sensing system was proportional to the concentration of histidine during the range of 5.7-455 nmol L-1, with a detection limit of 2.0 nmol L-1.

Electrochemical detection of arsenic contamination based on hybridization chain reaction and RecJf exonuclease-mediated amplification

Arsenic (As) is a type of highly toxic substance, which is widely distributed in environment due to both natural and anthropogenic sources. For health concerns, the allowable arsenic level must be strictly regulated. In this study, an ultrasensitive electrochemical biosensor for arsenic contamination is developed with signal amplification mediated by hybridization chain reaction (HCR) and RecJf exonuclease catalyzed reaction. DNA assembly is firstly achieved on the surface of gold electrode prior to the analysis, which generates tremendous charge-transfer resistance (Rct). In the presence of As3+, aptamer sequence specifically binds As3+ and DNA dissociation occurs. The release of HCR product significantly decreases Rct, which could be further enhanced by RecJf exonuclease catalyzed digestion. Superior analytical performance for As3+ detection is obtained including the limit of detection (LOD) as low as 0.02 ppb and a wide linear range from 0.1 to 200 ppb, which complies with WHO regulation. It can be envisioned that in combination with a portable electrochemical instrumentation, the sensing strategy is suitable for field application of arsenic contamination monitoring.

An unmodified gold nanorods-based DNA colorimetric biosensor with enzyme-free hybridization chain reaction amplification

A novel and simple colorimetric biosensor was developed by combining the unique optical properties of unmodified gold nanorods (AuNRs) with the amplification strategy of hybridization chain reaction (HCR), which was capable of detecting DNA sensitively and selectively. The detection mechanism is based on the dispersion/aggregation of AuNRs under the high concentration of salt. When target DNA introduces, the hybridization of hairpin DNA is triggered and a nicked double-helix DNA as HCR product forms which sticks tightly with AuNRs through a strong electrostatic adsorption and protects AuNRs from aggregation in high salt concentration condition. When no target DNA appears, AuNRs undergo aggregation due to the weak protection of hairpin DNA. The approach is able to detect target DNA in a range of 0–60 nM with a detection limit of 1.47 nM and exhibits high selectivity to distinguish fully matched and single-base mismatched DNA. Results demonstrate this method is label-free, modification-free, and enzyme-free, which holds great promise for routine sensing applications.

A label-free light-up fluorescent sensing platform based upon hybridization chain reaction amplification and DNA triplex assembly

A label-free light-up fluorescent sensing strategy using hybridization chain reaction (HCR) amplification and DNA triplex assembly has been developed. Remarkably, the proposed fluorescence assay is successfully applied to the determination of avian influenza A (H7N9) virus DNA and thrombin. Herein, in the presence of targets, the target DNA/initiator triggers a cascade of hybridization events between H1 and H2 that yields nicked double helices analogous to alternating copolymers. With the additions of triplex-forming oligonucleotide (TFO) and berberine, the triplex structures form between HCR products and TFO. Then, a large amount of berberine can bind to the triplex structures and the sensing system exhibits a dramatic increase in the fluorescence intensity at 530 nm. Under optimal conditions, the label-free fluorescent sensing platform shows sensitive responses to H7N9 virus DNA and thrombin in the range of 0.2–100 nM and 0.5–200 nM, respectively. The detection limits of H7N9 virus DNA and thrombin are as low as 0.14 nM and 0.32 nM, respectively. Owing to the simplicity and low cost, the proposed strategy can be used in other biomarkers assays, providing a promising tool for clinical diagnosis and biomedical detection.

Chemical and Biological Sensing Using Hybridization Chain Reaction.

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

Target-triggering multiple-cycle signal amplification strategy for ultrasensitive detection of DNA based on QCM and SPR

Detection of ultralow concentrations of nucleic acid sequences is a central challenge in the early diagnosis of genetic diseases. Herein, we developed a target-triggering cascade multiple cycle amplification for ultrasensitive DNA detection using quartz crystal microbalance (QCM) and surface plasmon resonance (SPR). It was based on the exonuclease Ⅲ (Exo Ⅲ)-assisted signal amplification and the hybridization chain reaction (HCR). The streptavidin-coated Au-NPs (Au-NPs-SA) were assembled on the HCR products as recognition element. Upon sensing of target DNA, the duplex DNA probe triggered the Exo Ⅲ cleavage process, accompanied by generating a new secondary target DNA and releasing target DNA. The released target DNA and the secondary target DNA were recycled. Simultaneously, numerous single strands were liberated and acted as the trigger of HCR to generate further signal amplification, resulting in the immobilization of abundant Au-NPs-SA on the gold substrate. The QCM sensor results were found to be comparable to that achieved using a SPR sensor platform. This method exhibited a high sensitivity toward target DNA with a detection limit of 0.70 fM. The high sensitivity and specificity make this method a great potential for detecting DNA with trace amounts in bioanalysis and clinical biomedicine.

Modified beacon probe assisted dual signal amplification for visual detection of microRNA

In a recent study, we reported a novel assay for the detection of microRNA-21 based on duplex-specific nuclease (DSN)-assisted isothermal cleavage and hybridization chain reaction (HCR) dual signal amplification. The Fam modified double-stranded DNA products were generated after the HCR, another biotin modified probe was digested by DSN and released from the magnetic beads after the addition of the target miRNA. The released sequence was then combined with HCR products to form a double-tagging dsDNA, which can be recognized by the lateral flow strips. In this study, we introduced a 2-OMethyl-RNA modified beacon probe (2-OMe-MB) to make some improvements based on the previous study. Firstly, the substitution of modified probe combined on magnetic beads avoids the fussy washing steps for the separation of un-reacted probes. Furthermore, the modification of 2-OMe on the stem of the probe avoided the unnecessary cleavage by DSN, which greatly reduce the background signal. Compared to the previous work, these improvements save us a lot of steps but possess the comparable sensitivity and selectivity.

Hybridization chain reaction and DNAzyme-based dual signal amplification strategy for sensitive colorimetric sensing of acetylcholinesterase activity and inhibitor screening in rat blood

We have constructed a novel colorimetric sensing platform for quantitative detection of acetylcholinesterase (AChE) activity and its inhibitor (donepezil) in rat blood, which integrates the signal amplification of DNAzyme and hybridization chain reaction (HCR) with the assembly of gold nanoparticles (AuNPs). Herein, in the presence of AChE and its substrate acetylthiocholine (ATCh), the sensing system exhibits a dramatic decrease in absorbance at 522 nm, where AChE hydrolyzes ATCh into thiocholine, the resulting thiols capture Cu2+ from DNAzyme, and then the substrate strand of DNAzyme acts as an initiator to trigger HCR process. The HCR products can induce the assembly of AuNPs via DNA hybridization, accompanying by a sharp color-change from red to blue. When donepezil is added, the enzymatic activity of AChE is suppressed, resulting in an increase in the absorbance at 522 nm. Under optimal conditions, the colorimetric sensing platform shows sensitive responses to AChE and donepezil in the range of 0.02–1.5 mU mL−1 and 0.5–100 nM, respectively. The detection limits of AChE and donepezil are as low as 5 μU mL−1 and 0.5 nM, respectively. Owing to high sensitivity of the proposed method, various nonspecific interactions can be eliminated with a sufficient dilution, indicating that our strategy has great potential for practical application in neurobiology and pharmacology fields.

Ultrasensitive Faraday cage-type electrochemiluminescence assay for femtomolar miRNA-141 via graphene oxide and hybridization chain reaction-assisted cascade amplification

In this study, a novel electrochemiluminescence (ECL) biosensor for sensitive detection of femtomolar miRNA-141 was constructed on the basis of Faraday cage-type strategy via graphene oxide (GO) and hybridization chain reaction (HCR)-assisted cascade amplification. A capture probe (CP) was immobilized on Fe3O4@SiO2@Au nanoparticles as capture unit, which could catch the miRNA-141, and the immobilization of the signal unit (Ru(phen)32+-HCR/GO) was allowed via nucleic acid hybridization. The prepared biosensor exhibited two advantages for signal amplification: firstly, GO could lap on the electrode surface directly, extending Outer Helmholtz Plane (OHP) of the sensor due to the large surface area and good electronic transport property; secondly, HCR-assisted cascade amplification was designed by anchoring all HCR products on the GO surface, then embedding Ru(phen)32+ as a signal readout pathway. All these signal molecules could take part in electrochemical reactions, thus further enhancing the ECL signal drastically. Therefore, the proposed sensor constructed by integrating HCR with Faraday cage-type strategy displayed an ultrasensitive detection platform for the miRNA-141 with a low detection limit of 0.03 fM. In addition, this proposed biosensor provides a universal platform for analysis of other microRNAs.

Quantum dots-Ru complex assembling dyads for cancer cell detection and cellular imaging based on hybridization chain reaction

An easy and fluorescent strategy is developed for the detection of cancer cells and cellular imaging. This assay relies on the quenching of Ru complex toward quantum dots (QDs) and the amplification of hybridization chain reaction (HCR). In the presence of target cancer cells, folate-modified single-stranded DNA (F-DNA) links on the cell membranes through high affinity of folate and receptors. Hybridization reaction between pre-prepared HCR products and F-DNA immobilizes HCR products on the surfaces of target cells, resulting in large amount of Ru complex into HCR products and strong red fluorescence on the surfaces of target cells. Meanwhile, QDs-Ru complex assembling dyads in supernate are reduced, leading to the fluorescence restoration of QDs. Taking human cells lines HeLa as a model analyst, the linear response for HeLa cells in a concentration range from 100 to 105 cells mL−1 is obtained with a detection limit of 100 cells mL−1. The specificity of this assay for the detection of cancer cells is demonstrated against several cell competitors. Additionally, this assay is successfully applied to inhibitor screening for cancer cells in combination with cellular imaging, demonstrating the university of this design.

Hybridization chain reaction-based flow cytometric bead sensor for the detection of emetic Bacillus cereus in milk

Emetic Bacillus cereus is a common foodborne pathogen that can cause vomiting after ingesting the contaminated food. Detection of emetic B. cereus plays an important role in food industry. In this study, a flow cytometric bead assay based on hybridization chain reaction (HCR) were developed to detect emetic B. cereus. Forward primer labeled magnetic beads (MBs) could capture single-strand DNA (ssDNA), which were generated through the asymmetric polymerase chain reaction from the partial cereulide-encoded gene of emetic B. cereus. Then the ssDNA initiated HCR to self-assemble with two fluorescent hairpin probes. Flow cytometry was then performed to monitor the HCR products of MBs. Under optimal experimental conditions, the limit of detection (LOD) for emetic B. cereus was 7.6×100CFU/mL with a liner range from 7.6×100CFU/mL to 7.6×106CFU/mL in pure culture. The proposed strategy can be used in spiked milk with LOD of 9.2×102CFU/mL without enrichment. The high specificity of the assay was verified using closely related strains, such as B. subtilis, non-emetic B. cereus. The proposed strategy offers a great promise for simple, low-cost, sensitive, and specific pathogen diagnosis in a real food matrix.

Lateral flow nucleic acid biosensor for sensitive detection of microRNAs based on the dual amplification strategy of duplex-specific nuclease and hybridization chain reaction.

MicroRNAs (miRNAs) constitute novel biomarkers for various diseases. Accurate and quantitative analysis of miRNA expression is critical for biomedical research and clinical theranostics. In this study, a method was developed for sensitive and specific detection of miRNAs via dual signal amplification based on duplex specific nuclease (DSN) and hybridization chain reaction (HCR). A reporter probe (RP), comprising recognition sequence (3’ end modified with biotin) for a target miRNA of miR-21 and capture sequence (5’ end modified with Fam) for HCR product, was designed and synthesized. HCR was initiated by partial sequence of initiator probe (IP), the other part of which can hybridize with capture sequence of RP, and was assembled by hairpin probes modified with biotin (H1-bio and H2-bio). A miR-21 triggered cyclical DSN cleavage of RP, which was immobilized to a streptavidin (SA) coated magnetic bead (MB). The released Fam labeled capture sequence then hybridized with the HCR product to generate a detectable dsDNA. This polymer was then dropped on lateral flow strip and positive result was observed. The proposed method allowed quantitative sequence-specific detection of miR-21 (with a detection limit of 2.1 fM, S/N = 3) in a dynamic range from 100 fM to 100 pM, with an excellent ability to discriminate differences in miRNAs. The method showed acceptable testing recoveries for the determination of miRNAs in serum.

A dual amplification strategy for ultrasensitive detection of microRNA

Here, we present a dual amplification strategy based on a duplex-specific nuclease and a hybridization chain reaction, which has been designed for the highly sensitive detection of miRNAs. In the first step, a loop amplification strategy based on a duplex-specific nuclease was employed to obtain a large number of ssDNA reporters that then act as initiators to trigger the next amplification step. The second step involves a hybridization chain reaction based on two hairpin DNAs denoted as H1 and H2. Doxorubicin-CdTe quantum dots, which function as detecting indicators that intercalated into the hybridization chain reaction products, lead to significantly amplified electrochemiluminescence signal readout. This dual amplification strategy enabled the sensitive detection of let-7d from 10 aM to 10nM with a detection limit of 10aM, and offered an excellent capacity to discriminate between microRNA family members. This assay therefore offers great potential applications in biomedical research and early clinical diagnosis.

Ultra-sensitive fluorescent sensor for intracellular miRNA based on enzyme-free signal amplification with carbon nitride nanosheet as a carrier.

A novel ultra-sensitive fluorescent sensor for monitoring microRNA (miRNA) in living cells was constructed by utilizing a hybridization chain reaction (HCR) as the signal amplification with a carbon nitride nanosheet (CNNS) as a carrier. The Cy5-labeled hairpin DNA could be adsorbed onto the surface of CNNS, resulting in fluorescence quenching of Cy5. When treated with complementary miRNA, the fluorescence was recovered because miRNA could efficiently trigger an HCR, which led to the release of the HCR products from the CNNS. This intracellular HCR strategy can be used for ultra-sensitive monitoring of intracellular miRNA. The main advantages of the proposed method are its simplicity, high sensitivity, high specificity and low toxicity for monitoring low-level biomarkers.

Ultrasensitive electrochemical biosensor for silver ion based on magnetic nanoparticles labeling with hybridization chain reaction amplification strategy

Silver ion (Ag+) is a highly toxic heavy metal ion to aquatic organisms and accumulates in the human body via the food chain. Therefore, fast and accurate detection of Ag+ in water and food resources has become a critical issue within the scope of human health. Herein, we developed an ultrasensitive electrochemical biosensor for detection of Ag+ based on magnetic Fe3O4@gold core-shell nanoparticles (Fe3O4@Au NPs) labeling with hybridization chain reaction (HCR) amplification strategy. In this sensing strategy, the magnetic Fe3O4@Au NPs were selected for labeling with HCR product and enrichment on the surface of magnetic gold electrode. Thiolated-oligonucleotide (S1) was firstly immobilized on the surface of Fe3O4@Au NPs through Au–S chemical bond. In the presence of Ag+, cytosine-rich DNA oligonucleotide S2 hybridized with S1 to form an intramolecular duplex, in which Ag+ can selectively bind to cytosine–cytosine mismatches forming C–Ag+–C complex. The exposed stem of the C–Ag+–C complex opened two alternating ferrocene-labeled DNA hairpins (H1 and H2) in turn and triggered HCR to form a supersandwich DNA structure on the surface of Fe3O4@Au NPs. The HCR products modified Fe3O4@Au NPs were brought to the surface of magnetic gold electrode for direct electrochemical measurements. The proposed strategy led to a low detection limit of 0.5fM and a wide dynamic range of 1fM–100pM for target Ag+. The developed biosensor was highly selective and its practical applicability in tap water and lake water samples was also investigated with a satisfactory result.