DNA-based Hybridization Chain Reaction Research Articles

Planar intercalated copper (II) complex molecule as small molecule enzyme mimic combined with Fe3O4 nanozyme for bienzyme synergistic catalysis applied to the microRNA biosensor

Enzyme mimics have been developed for bioassay of nucleic acids, with some of them involving complicated labeling. Herein, we report a label-free bioassay for ultrasensitive electronic determination of microRNA at an ultralow concentration based on target-triggered long-range self-assembly DNA-based hybridization chain reaction (HCR) protocol coupled with bienzyme mimics synergistic catalysis strategy. In this work, a planar intercalation molecule, copper (II) complex, is applied for the first time as a small molecule enzyme mimic as well as intercalation molecule in microRNA biosensor for signal amplification. Fe3O4 nanozyme were used as a separate and enriched target under magnetic field, and also in combination with HCR protocol detected in 3,3’,5,5’-tetramethylbenzidine+hydrogen peroxide (TMB+H2O2) system to improve the sensitivity of the biosensor. Under optimal conditions, these strategies present good electrochemical behaviors for the detection of microRNA with a wide range from 100 aM to 100 nM and at relatively low detection limit of 33 aM This remarkable sensitivity can make this proposed approach a promising scheme for development of next-generation microRNA sensors without the need of enzyme labeling or fluorophore labeling.

DNA-based hybridization chain reaction and biotin–streptavidin signal amplification for sensitive detection of Escherichia coli O157:H7 through ELISA

This study reported on a novel sandwich enzyme linked immunosorbent assay (ELISA) for the sensitive determination of Escherichia coli O157:H7 (E. coli O157:H7) by using DNA-based hybridization chain reaction (HCR) and biotin–streptavidin signal amplification. The anti-E. coli O157:H7 polyclonal antibody (pAb) was immobilized in the ELISA wells. The anti-E. coli O157:H7 monoclonal antibody (mAb) and initiator strand (DNA1) were labeled on gold nanoparticle (AuNP) to form a mAb–AuNP–DNA1 complex. In the presence of the target E. coli O157:H7, the sandwiched immunocomplex, which is pAb–E. coli O157:H7–mAb–AuNP–DNA1, could be formed. Two types of biotinylated hairpin were subsequently added in the ELISA well. A nicked double-stranded DNA (dsDNA) that contained abundant biotins was formed after HCR. Detection was performed after adding horseradish peroxidase–streptavidin and substrate/chromogen solution. Under optimal conditions, E. coli O157:H7 could be detected in the range of 5×102 CFU/mL to 1×107 CFU/mL; the limit of detection was 1.08×102 CFU/mL in pure culture. The LOD of the novel ELISA was 185 times lower than that of traditional ELISA. The proposed method is considerably specific and can be applied in the detection of whole milk samples inoculated with E. coli O157:H7. The coefficient of variation of in pure culture and in whole milk was 0.99–5.88% and 0.76–5.38%, respectively. This method offers a promising application in the detection of low concentrations of food-borne pathogens.

Ultrasensitive electrochemical sensor for Hg2+ by using hybridization chain reaction coupled with Ag@Au core–shell nanoparticles

A novel electrochemical biosensor for Hg2+ detection was reported by using DNA-based hybridization chain reaction (HCR) coupled with positively charged Ag@Au core-shell nanoparticles ((+)Ag@Au CSNPs) amplification. To construct the sensor, capture probe (CP ) was firstly immobilized onto the surface of glass carbon electrode (GCE). In the presence of Hg2+, the sandwiched complex can be formed between the immobilized CP on the electrode surface and the detection probe (DP) modified on the gold nanoparticles (AuNPs) based on T-Hg2+-T coordination chemistry. The carried DP then opened two ferrocene (Fc) modified hairpin DNA (H1 and H2) in sequence and propagated the happen of HCR to form a nicked double-helix. Numerous Fc molecules were formed on the neighboring probe and produced an obvious electrochemical signal. Moreover, (+)Ag@Au CSNPs were assembly onto such dsDNA polymers as electrochemical signal enhancer. Under optimal conditions, such sensor presents good electrochemical responses for Hg2+ detection with a detection limit of 3.6pM. Importantly, the methodology has high selectivity for Hg2+ detection.

A simple and ultrasensitive electrochemical biosensor for detection of microRNA based on hybridization chain reaction amplification

Abstract MicroRNAs (miRNAs) play vital roles in regulating gene expression and cellular processes. Dysregulation of miRNAs has been associated with a variety of diseases, which make them promising biomarkers. In this work, a simple and ultrasensitive electrochemical biosensor was developed for detection of target miRNA using DNA-based hybridization chain reaction (HCR) and biotin-strepavidin based amplification. In the presence of target miRNA, the hairpin DNA capture probes immobilized on gold electrode surface were opened. Then DNA initiator strands hybridized with the opened capture probes and triggered HCR, finally, a long nicked double-helix DNA structure was developed. Streptavidin is employed for signal amplification via binding with biotin labeled on the two hairpin DNA probes which take part in the HCR. The proposed biosensor presented a high sensitivity for determination of target miRNA down to 0.56 fM with a linear range from 1 fM to 100 pM and high specificity that discriminates target miRNA from mismatched miRNAs. The method was successfully applied to the determination of miRNA spiked into human total RNA samples. Therefore, the biosensor might have potential clinical application for miRNA detection in biomedical research and clinical diagnosis.

DNA-based hybridization chain reaction for signal amplification and ultrasensitive chemiluminescence detection of gibberellic acid

A novel chemiluminescent (CL) immunoassay protocol for the sensitive determination of Gibberellic acid (GA) using DNA-based hybridization chain reaction (HCR) is described. The carboxyl terminated magnetic beads (MBs) were modified with GA antibody and the gold nanoparticles (AuNPs) signal probes were labeled with the GA antibody and DNA initiator strands. In the presence of target GA, the sandwiched immunocomplex was formed between the immobilized antibody on the MBs and the signal antibody on the AuNPs. When the two complementary stable species of DNA hairpins, which are G-rich DNA, were added the hybridization events were happened, thereby resulting forming the long nicked double-helix. Numerous double G-rich DNA were formed on the AuNPs, each of which produced a CL signal within the applied CL reaction. The CL signal was obtained via the instantaneous derivatization reaction between a specific CL reagent, 3,4,5-trimethoxyl-phenylglyoxal (TMPG), and the G-rich DNA on immunocomplex. Under optimal conditions, GA can be detected in the concentration ranged from 0.01ng/mL to 70ng/mL, and the limit of detection was 0.003ng/mL. The reproducibility and selectivity of the developed method were also investigated. In addition, the real samples were assayed by using the developed immunoassay, and the recovery was 96.0–102.0%.

DNA-based hybridization chain reaction amplification for assaying the effect of environmental phenolic hormone on DNA methyltransferase activity

In this work, a novel electrochemical protocol with signal amplification for determination of DNA methylation and methyltransferase activity using DNA-based hybridization chain reaction (HCR) was proposed. After the gold electrode was modified with dsDNA, it was treated with M.SssI MTase, HpaII endonuclease, respectively. And then the HCR was initiated by the target DNA and two hairpin helper DNAs, which lead to the formation of extended dsDNA polymers on the electrode surface. The signal was amplified by the labeled biotin on the hairpin probes. As a result, the streptavidin-alkaline phosphatase (S-ALP) conjugated on the electrode surface through the specific interaction between biotin and S-ALP. ALP could convert 1-naphthyl phosphate into 1-naphthol and the latter could be electrochemically oxidized, which was used to monitor the methylation event and MTase activity. The HCR assay presents good electrochemical responses for the determination of M.SssI MTase at a concentration as low as 0.0067unitmL−1. Moreover, the effects of anti-cancer drug and environmental phenolic hormone on M.SssI MTase activity were also investigated. The results indicated that 5-fluorouracil and daunorubicin hydrochloride could inhibit the activity, and the opposite results were obtained with bisphenol A and nonylphenol. Therefore, this method can not only provide a platform to screen the inhibitors of DNA MTase and develop new anticancer drugs, but also offer a novel technique to investigate the possible carcinogenesis mechanism.

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.

Signal Amplification Strategy with Synergistic Catalysis of Hollow Pt Nanochains and Hemoglobin for Electrochemical Immunosensor

A novel signal amplification strategy for ultrasensitive detection of α-fetoprotein (AFP) based on the synergistic catalysis of hollow Pt nanochains (HPtNCs) and hemoglobin (Hb) with DNA-based hybridization chain reaction (HCR) has been reported in this work. Firstly, numerous biocatalysts of Hb and redox probes of thionine (Thi) were carried onto the double-stranded DNA polymers based on HCR with high affinity. It is worth pointing out that HCR could effective immobilize Hb and Thi under mild conditions maintaining the Hb's three-dimensional structure and avoiding the leakage of Thi. Significantly, the electrochemical signal was greatly enhanced by the synergistic catalysis of HPtNCs and Hb. With a sandwich-type immunoreaction, the linear dynamic range of AFP by differential pulse voltammetry was determined to be from 0.1 pg mL−1 to 50 ng mL−1 with the detection limit of 0.03 pg mL−1 (S/N = 3).

Enhanced DNA sensing via catalytic aggregation of gold nanoparticles

A catalytic colorimetric detection scheme that incorporates a DNA-based hybridization chain reaction into gold nanoparticles was designed and tested. While direct aggregation forms an inter-particle linkage from only one target DNA strand, catalytic aggregation forms multiple linkages from a single target DNA strand. Gold nanoparticles were functionalized with thiol-modified DNA strands capable of undergoing hybridization chain reactions. The changes in their absorption spectra were measured at different times and target concentrations and compared against direct aggregation. Catalytic aggregation showed a multifold increase in sensitivity at low target concentrations when compared to direct aggregation. Gel electrophoresis was performed to compare DNA hybridization reactions in catalytic and direct aggregation schemes, and the product formation was confirmed in the catalytic aggregation scheme at low levels of target concentrations. The catalytic aggregation scheme also showed high target specificity. This application of a DNA reaction network to gold nanoparticle-based colorimetric detection enables highly-sensitive, field-deployable, colorimetric readout systems capable of detecting a variety of biomolecules.

Sensitive electrochemical monitoring of nucleic acids coupling DNA nanostructures with hybridization chain reaction

Methods based on metal nanotags have been developed for metallobioassay of nucleic acids, but most involve complicated labeling or stripping procedures and are unsuitable for routine use. Herein, we report the proof-of-concept of a novel and label-free metallobioassay for ultrasensitive electronic determination of human immunodeficiency virus (HIV)-related gene fragments at an ultralow concentration based on target-triggered long-range self-assembled DNA nanostructures and DNA-based hybridization chain reaction (HCR). The signal is amplified by silver nanotags on the DNA duplex. The assay mainly consists of capture probe, detection probe, and two different DNA hairpins. In the presence of target DNA, the capture probe immobilized on the sensor sandwiches target DNA with the 3′ end of detection probe. Another exposed part of detection probe at the 5′ end opens two alternating DNA hairpins in turn, and propagates a chain reaction of hybridization events to form a nicked double-helix. Finally, numerous silver nanotags are immobilized onto the long-range DNA nanostructures, each of which produces a strong electronic signal within the applied potentials. Under optimal conditions, the target-triggered long-range DNA nanostructures present good electrochemical behaviors for the detection of HIV DNA at a concentration as low as 0.5fM. Importantly, the outstanding sensitivity can make this approach a promising scheme for development of next-generation DNA sensors without the need of enzyme labeling or fluorophore labeling.

DNAzyme-based magneto-controlled electronic switch for picomolar detection of lead (II) coupling with DNA-based hybridization chain reaction

A novel magneto-controlled electrochemical DNA biosensor is designed for the ultrasensitive detection of lead coupling a lead-specific DNAzyme with DNA-based hybridization chain reaction (HCR). To construct such a magnetic lead sensor, DNAzyme-based molecular beacons, selective to cleavage in the presence of Pb2+, are initially immobilized onto magnetic beads, which were used as the recognition elements. Upon addition of target lead, catalytic cleavage of substrate DNA segments in the double-stranded DNAzymes results in the capture of the initiator strands via the conjugated catalytic strands on magnetic beads. The captured DNA initiator strands trigger the hybridization chain reaction between two alternating hairpin DNA structures labeled with ferrocene to form a nicked double-helix on the magnetic beads. Numerous ferrocene molecules are formed on the neighboring probes, each of which produces an electrochemical signal within the applied potential. Under optimal conditions, the electrochemical signal of the magnetic lead sensor increases with the increasing lead level in the sample, and exhibits a linear response over a Pb2+ concentration range of 0.1–75nM with a detection limit of 37pM. Quantitative measurement of Pb2+ in the complex sample demonstrates the selectivity of the sensor scheme and points favorably to the application of such technologies to the analysis of environmental samples. The unique combination of a DNAzyme with hybridization chain reaction makes it possible to change the DNAzyme to select for other compounds of interest. This work represents the initial steps toward the creation of a robust field sensor for lead in groundwater or drinking water.

DNA-Based Hybridization Chain Reaction for Amplified Bioelectronic Signal and Ultrasensitive Detection of Proteins

This work reports a novel electrochemical immunoassay protocol with signal amplification for determination of proteins (human IgG here used as a model target analyte) at an ultralow concentration using DNA-based hybridization chain reaction (HCR). The immuno-HCR assay consists of magnetic immunosensing probes, nanogold-labeled signal probes conjugated with the DNA initiator strands, and two different hairpin DNA molecules. The signal is amplified by the labeled ferrocene on the hairpin probes. In the presence of target IgG, the sandwiched immunocomplex can be formed between the immobilized antibodies on the magnetic beads and the signal antibodies on the gold nanoparticles. The carried DNA initiator strands open the hairpin DNA structures in sequence and propagate a chain reaction of hybridization events between two alternating hairpins to form a nicked double-helix. Numerous ferrocene molecules are formed on the neighboring probe, each of which produces an electrochemical signal within the applied potentials. Under optimal conditions, the immuno-HCR assay presents good electrochemical responses for determination of target IgG at a concentration as low as 0.1 fg mL(-1). Importantly, the methodology can be further extended to the detection of other proteins or biomarkers.

Nanogold-based bio-bar codes for label-free immunosensing of proteins coupling with an in situ DNA-based hybridization chain reaction

A label-free, non-enzyme immunosensing strategy is designed for ultrasensitive electronic detection of disease-related proteins (carcinoembryonic antigen as a model) by using gold nanoparticle-based bio-bar codes and an in situ amplified DNA-based hybridization chain reaction.