Hybridization Chain Reaction Products Research Articles

Ultrasensitive colorimetric detection of circulating tumor DNA using hybridization chain reaction and the pivot of triplex DNA

This work presents an amplified colorimetric biosensor for circulating tumor DNA (ctDNA), which associates the hybridization chain reaction (HCR) amplification with G-Quadruplex DNAzymes activity through triplex DNA formation. In the presence of ctDNA, HCR occurs. The resulting HCR products are specially recognized by one sequence to include one GGG repeat and the other containing three GGG repeats, through the synergetic effect of triplex DNA and asymmetrically split G-Quadruplex forming. Such design takes advantage of the amplification property of HCR and the high peroxidase-like catalytic activity of asymmetrically split G-Quadruplex DNAzymes by means of triplex DNA formation, which produces color signals in the presence of ctDNA. Nevertheless, in the absence of ctDNA, no HCR happens. Thus, no triplex DNA and G-Quadruplex structure is formed, producing a negligible background. The colorimetric sensing platform is successfully applied in complex biological environments such as human blood plasma for ctDNA detection, with a detection limit corresponding to 0.1 pM. This study unambiguously uses triplex DNA forming as the pivot to integrate nucleic acid amplification and DNAzymes for producing a highly sensitive signal with low background.

A colorimetric sensing platform based upon recognizing hybridization chain reaction products with oligonucleotide modified gold nanoparticles through triplex formation.

A novel colorimetric sensing strategy for biomolecule assay has been developed, which integrates the signal amplification of the hybridization chain reaction (HCR) with the assembly of gold nanoparticles (AuNPs) through triplex formation. In the presence of targets, the HCR process can be triggered, the HCR products are specifically recognized by triplex-forming oligonucleotide (TFO) functionalized AuNPs, accompanying the aggregation of AuNPs and a dramatic absorbance change at 522 nm. In addition, the DNA hairpin probes can form rigid triplex structures with TFO-functionalized AuNPs in the absence of targets, resulting in a negligible background signal. By taking advantage of this new biosensor platform, a broad range of targets, involving nucleic acids, small molecules and proteins, have been successfully determined with high sensitivity and selectivity, which may hold great potential for practical application.

A DNA-stabilized silver nanoclusters/graphene oxide-based platform for the sensitive detection of DNA through hybridization chain reaction

A silver nanoclusters (AgNCs)/graphene oxide (GO)-based fluorescence sensor was developed for label-free DNA detection through hybridization chain reaction (HCR). A DNA sequence associated with the human immunodeficiency virus (HIV) was selected as a model target. Two DNA probes, hairpin probe 1 (H1) and hairpin probe 2 (H2), were partially complementary. GO was used as an adsorption material to capture the hairpin probes and a selective fluorescence quencher was used to reduce the background signal. Upon addition of AgNO3 and NaBH4, the AgNCs were synthesized at the terminals of the H1 and H2 probes. In the absence of target DNA (THIV), hybridization chain reaction (HCR) could not be triggered due to the stability of H1 and H2 probes. The hairpin probe-protected AgNCs attached to the GO surface, efficiently quenching fluorescence of the AgNCs. Therefore, the system showed very low background. In presence of THIV, the target triggered the chain-like assembly of H1 and H2 through HCR, generating a long chain of H1 and H2 complexes. The HCR product (AgNCs nanowires) could not be adsorbed on the surface of GO; hence, it generated a strong fluorescent signal based on the concentration of the target. Under the optimized conditions, the detection limit of the fluorescence sensor was 1.18nM, and hence it can be applied to clinical samples.

Highly sensitive chemiluminescence biosensor for protein detection based on the functionalized magnetic microparticles and the hybridization chain reaction

An ultrasensitive chemiluminescence (CL) biosensor for the detection of protein is developed in this study based on the functionalized magnetic microparticles (MMPs) and the hybridization chain reaction (HCR). First, the primer hybridized with the thrombin aptamer conjugated on the surface of MMPs. Then the HCR was triggered by part of the primer and its products were assembled on the surface of the MMPs. Through the interaction between streptavidin and biotin, the streptavidin-horseradish peroxidase (SA-HRP) was coupled with the HCR products. In the presence of thrombin, the HCR products conjugating with SA-HRP were released from the surface of MMPs after the aptamer recognized and bound to its target molecule. So the released SA-HRP in the supernatant produced a significant chemiluminescence imaging signal after the addition of H2O2-luminol. The detection limit of thrombin with this method could be as low as 9.7fM. Besides, the sensing strategy was modified by changing the adding order of reagents that was then successfully applied in the detection of thrombin in complex sample. What's more, the DNA detection also could be carried out with this method, which demonstrated the universality of the proposed sensing strategy.

Enzyme-free fluorescent biosensor for the detection of DNA based on core–shell Fe 3 O 4 polydopamine nanoparticles and hybridization chain reaction amplification

A novel, highly sensitive assay for quantitative determination of DNA is developed based on hybridization chain reaction (HCR) amplification and the separation via core-shell Fe3O4 polydopamine nanoparticles (Fe3O4@PDA NPs). In this assay, two hairpin probes are designed, one of which is labeled with a 6-carboxyfluorescein (FAM). Without target DNA, auxiliary hairpin probes are stable in solution. However, when target DNA is present, the HCR between the two hairpins is triggered. The HCR products have sticky ends of 24 nt, which are much longer than the length of sticky ends of auxiliary hairpins (6 nt) and make the adsorption much easier by Fe3O4@PDA NPs. With the addition of Fe3O4@PDA NPs, HCR products could be adsorbed because of the strong interaction between their sticky ends and Fe3O4@PDA NPs. As a result, supernatant of the solution with target DNA emits weak fluorescence after separation by magnet, which is much lower than that of the blank solution. The detection limit of the proposed method is as low as 0.05 nM. And the sensing method exhibits high selectivity for the determination between perfectly complementary sequence and target with single base-pair mismatch. Importantly, the application of the sensor for DNA detection in human serum shows that the proposed method works well for biological samples.

Enzyme-free and isothermal detection of microRNA based on click-chemical ligation-assisted hybridization coupled with hybridization chain reaction signal amplification.

An enzyme-free and isothermal microRNA (miRNA) detection method has been developed based on click-chemical ligation-assisted hybridization coupled with hybridization chain reaction (HCR) on magnetic beads (MBs). The click-chemical ligation between an azide-modified probe DNA and a dibenzocyclooctyne-modified probe DNA occurred through the hybridization of target miRNA (miR-141). HCR on MBs was performed by the addition of DNA hairpin monomers (H1 and H2). After magnetic separation and denaturation/rehybridization of HCR products ([H1/H2] n ), the resulting HCR products were analyzed by the fluorescence emitted from an intercalative dye, allowing amplification of the fluorescent signal. The proposed assay had a limit of detection of 0.55 fmol, which was 230-fold more sensitive than that of the HCR on the MBs coupled with a conventional sandwich hybridization assay (without click-chemical ligation) (limit of detection 127 fmol). Additionally, the proposed assay could discriminate between miR-141 and other miR-200 family members. In contrast to quantitative reverse transcription polymerase chain reaction techniques using enzymes and thermal cycling, this is an enzyme-free assay that can be conducted under isothermal conditions and can specifically detect miR-141 in fetal bovine serum.

Enzyme-free and label-free fluorescence aptasensing strategy for highly sensitive detection of protein based on target-triggered hybridization chain reaction amplification.

Proteins are of great importance in medical and biological fields. In this paper, a novel fluorescent aptasensing strategy for protein assay has been developed based on target-triggered hybridization chain reaction (HCR) and graphene oxide (GO)-based selective fluorescence quenching. Three DNA probes, a helper DNA probe (HP), hairpin probe 1 (H1) and hairpin probe 2 (H2) are ingeniously designed. In the presence of the target, the aptamer sequences in HP recognize the target to form a target-aptamer complex, which causes the HP conformation change, and then triggers the chain-like assembly of H1 and H2 through the hybridization chain reaction, generating a long chain of HP leading complex of H1 and H2. At last the fluorescence indicator SYBR Green I (SG) binds with the long double strands of the HCR product through both intercalation and minor groove binding. When GO was added into the solutions after HCR, the free H1, H2 and SG would be closely adsorbed onto GO surface via π-π stacking. However, the HCR product cannot be adsorbed on GO surface, thereby the SG bound to HCR product gives a strong fluorescence signal dependent on the concentration of the target. With the use of platelet-derived growth factor BB (PDGF-BB) as the model analyte, this newly designed protocol provides a highly sensitive fluorescence detection of PDGF-BB with a limit of detection down to 1.25 pM, and also exhibit good selectivity and applicability in complex matrixes. Therefore, the proposed aptasensing strategy based on target-triggered hybridization chain reaction amplification should have wide applications in the diagnosis of genetic diseases due to its simplicity, low cost, and high sensitivity at extremely low target concentrations.

A fluorescence polarization assay for nucleic acid based on the amplification of hybridization chain reaction and nanoparticles

An amplified fluorescence polarization (FP) assay based on hybridization chain reaction (HCR) and nanoparticles was developed for the detection of nucleic acid. In this work, the target nucleic acid could trigger the autonomous cross-opening the biotin-modified hairpin probe H1 and fluorescein-labeled hairpin probe H2 to form long fluorescent nicked double-strand polymers (dsDNA). Thus the FP value was increased because the rotation rate of fluorescein slowed down by the formation long HCR products with a larger molecular mass. After then introduction of streptavidin-coated the SiO2 nanoparticles (SA-SiNPs), the HCR-produced dsDNA products and SA-SiNPs were combined via the interactions of biotin and streptavidin, generating much larger fluorescent complexes. Thus, a further amplified FP signal was obtained. Under optimized conditions, the developed method exhibits a linear response to nucleic acid in the concentration range of 0–2.5nM, and the limit of detection is 35pM. Moreover, the FP assay also showed high precision, pronounced specificity and the applicability in complicated human serum matrix.

A simple colorimetric DNA detection by target-induced hybridization chain reaction for isothermal signal amplification

A novel DNA detection method is presented based on a gold nanoparticle (AuNP) colorimetric assay and hybridization chain reaction (HCR). In this method, target DNA hybridized with probe DNA modified on AuNP, and triggered HCR. The resulting HCR products with a large number of negative charges significantly enhanced the stability of AuNPs, inhibiting aggregation of AuNPs at an elevated salt concentration. The approach was highly sensitive and selective. Using this enzyme-free and isothermal signal amplification method, we were able to detect target DNA at concentrations as low as 0.5nM with the naked eye. Our method also has great potential for detecting other analytes, such as metal ions, proteins, and small molecules, if the target analytes could make HCR products attach to AuNPs.

DNA-based hybridization chain reaction for an ultrasensitive cancer marker EBNA-1 electrochemical immunosensor

An ultrasensitive and selective electrochemical immunosensor was developed for the detection of Epstein Barr virus nuclear antigen 1 (EBNA-1). Firstly, a suspension of graphene sheets (GS) and multi-walled carbon nanotubes (MWCNTs) was prepared with the aid of chitosan (CS) solution and then modified on a glassy carbon electrode (GCE). Gold nanoparticles (AuNPs) were then electrodeposited onto the surface of the GS–MWCNTs film by cyclic voltammetry (CV) to immobilize the captured antibodies. After that, specific sandwich immunoreactions were formed among the captured antibody, EBNA-1, and secondary antibody, DNA-coated carboxyl multi-wall carbon nanotubes (DNA–MWCNTs–Ab2). DNA initiator strands (S0) and secondary antibodies linked to the MWCNTs and double-helix DNA polymers were obtained by hybridization chain reaction (HCR), and here S0 on the MWCNTs propagates a chain reaction of hybridization events between two alternating hairpins to form a nicked double-helix. Finally, electroactive indicator doxorubicin hydrochloride was intercalated into the CG–GC steps between the HCR products and could produce an electrochemical signal, which was monitored by differential pulse voltammetry (DPV). Under optimum conditions, the amperometric signal increased linearly with the target concentrations (0.05–6.4ngmL−1), and the immunosensor exhibited a detection limit as low as 0.7pgmL−1 (S/N=3). The proposed method showed acceptable stability and reproducibility, as well as favorable recovery for EBNA-1 in human serum. The proposed immunosensor provides a novel avenue for signal amplification and potential applications in bioanalysis and clinical diagnostics.

A cytometric bead assay for sensitive DNA detection based on enzyme-free signal amplification of hybridization chain reaction

A versatile flow cytometric bead assay (CBA) is developed for sensitive DNA detection by integrating the advantages of hybridization chain reaction (HCR) for enzyme-free signal amplification, flow cytometry for robust and rapid signal readout as well as magnetic beads (MBs) for facile separation. In this HCR–CBA, a biotinylated hairpin DNA (Bio-H1) is firstly immobilized on streptavidin-functionalized MBs. Upon the addition of target DNA, each target would hybridize with one Bio-H1 to open its hairpin structure and subsequently initiate a cascade of hybridization events between two species of fluorescent DNA hairpin probes (H1*/H2*) to form a nicked double helical DNA structure, resulting in amplified accumulation of numerous fluorophores on the MBs. Finally, the fluorescent MBs are directly analyzed by flow cytometry. This technique enables quantitative analysis of the HCR products anchored on the MBs as a function of target DNA concentration, and analysis of each sample can be completed within few minutes. Therefore, the HCR–CBA approach provides a practical DNA assay with greatly improved sensitivity. The detection limit of a model DNA target is 0.5pM (3σ), which is about 3 orders of magnitude lower compared with traditional hybridization methods without HCR. Furthermore, the signal of complementary target can be clearly distinguished from that of single-base mismatched sequences, indicating the high specificity of the HCR–CBA. Moreover, this strategy is also successfully applied to the DNA analysis in complex biological samples, showing great potential in gene analysis and disease diagnosis in clinical samples.

Graphene Surface-Anchored Fluorescence Sensor for Sensitive Detection of MicroRNA Coupled with Enzyme-Free Signal Amplification of Hybridization Chain Reaction

A new enzyme-free signal amplification-based assay for microRNA (miRNA) detection is developed by using hybridization chain reaction (HCR) coupled with a graphene oxide (GO) surface-anchored fluorescence signal readout pathway. MiRNAs can efficiently initiate HCR between two species of fluorescent hairpin probes. After HCR, both of the excess hairpin probes and the HCR products will be anchored on the GO surface. The fluorescence of the hairpin probes can be completely quenched by GO, whereas the HCR products maintain strong fluorescence. Therefore, integrating HCR strategy for signal amplification with selective fluorescence quenching effects of GO provides a versatile miRNA assay.

Triggered amplification by hybridization chain reaction

We introduce the concept of hybridization chain reaction (HCR), in which stable DNA monomers assemble only upon exposure to a target DNA fragment. In the simplest version of this process, two stable species of DNA hairpins coexist in solution until the introduction of initiator strands triggers a cascade of hybridization events that yields nicked double helices analogous to alternating copolymers. The average molecular weight of the HCR products varies inversely with initiator concentration. Amplification of more diverse recognition events can be achieved by coupling HCR to aptamer triggers. This functionality allows DNA to act as an amplifying transducer for biosensing applications.