Junyang Zhuang

A rolling circle amplification-based DNA machine for miRNA screening coupling catalytic hairpin assembly with DNAzyme formation

A novel DNA nanomachine based on the linear rolling circle amplification strategy was designed for sensitive screening of microRNA (miRNA) at an ultralow concentration coupling catalytic hairpin assembly (CHA) with DNAzyme formation.

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 Pb(2+), 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 Pb(2+) concentration range of 0.1-75 nM with a detection limit of 37 pM. Quantitative measurement of Pb(2+) 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.

Magnetic Bead-Based Enzyme-Chromogenic Substrate System for Ultrasensitive Colorimetric Immunoassay Accompanying Cascade Reaction for Enzymatic Formation of Squaric Acid-Iron(III) Chelate

This work reports on a simple and feasible colorimetric immunoassay with signal amplification for sensitive determination of prostate-specific antigen (PSA, used as a model) at an ultralow concentration by using a new enzyme-chromogenic substrate system. We discovered that glucose oxidase (GOx), the enzyme broadly used in enzyme-linked immunosorbent assay (ELISA), has the ability to stimulate in situ formation of squaric acid (SQA)-iron(III) chelate. GOx-catalyzed oxidization of glucose leads to the formation of gluconic acid and hydrogen peroxide (H2O2). The latter can catalytically oxidize iron(II) to iron(III), which can rapidly (<1 min) coordinate with the SQA. Formation of the iron-squarate complex causes the color of the solution to change from bluish purple to bluish red accompanying the increasing absorbance with the increment of iron(III) concentration. On the basis of the SQA-iron(III) system, a new immunoassay protocol with GOx-labeled anti-PSA detection antibody can be designed for the detection of target PSA on capture antibody-functionalized magnetic immunosensing probe, monitored by recording the color or absorbance (λ = 468 nm) of the generated SQA-iron(III) chelate. The absorbance intensity shows to be dependent on the concentration of target PSA. A linear dependence between the absorbance and target PSA concentration is obtained under optimal conditions in the range from 1.0 pg mL(-1) to 30 ng mL(-1) with a detection limit (LOD) of 0.5 pg mL(-1) (0.5 ppt) estimated at the 3Sblank level. The sensitivity displays to be 3-5 orders of magnitude better than those of most commercialized human PSA ELISA kits. In addition, the developed colorimetric immunoassay was validated by assaying 12 human serum samples, receiving in good accordance with those obtained by the commercialized PSA ELISA kit. Importantly, the SQA-based immunosensing system can be further extended for the detection of other low-abundance proteins or biomarkers by controlling the target antibody.

Nanogold-Functionalized DNAzyme Concatamers with Redox-Active Intercalators for Quadruple Signal Amplification of Electrochemical Immunoassay

A novel and in situ amplified immunoassay strategy with quadruple signal amplification was designed for highly efficient electrochemical detection of low-abundance proteins (carcinoembryonic antigen, CEA, as a model) by using nanogold-functionalized DNAzyme concatamers with redox-active intercalators. To construct such an in situ amplification system, streptavidin-labeled gold nanoparticles (AuNP-SA) were initially used for the labelling of initiator strands (S0) and detection antibody (mAb2) with a large ratio (mAb2-AuNP-S0), and then two auxiliary DNA strands S1 and S2 were designed for in situ propagation of DNAzyme concatamers with the hemin/G-quadruplex format. The quadruple signal amplification was implemented by using the avidin-biotin chemistry, nanogold labels, DNA concatamers, and DNAzymes. In the presence of target CEA, the sandwiched immunocomplex was formed between the immobilized primary antibodies on the electrode and the conjugated detection antibodies on the mAb2-AuNP-S0. The carried S0 initiator strands could progress a chain reaction of hybridization events between alternating S1/S2 DNA strands to form a nicked double-helix. Upon addition of hemin, the hemin-binding aptamers could be bound to form the hemin/G-quadruplex-based DNAzymes. The formed double-helix DNA polymers could cause the intercalation of numerous electroactive methylene blue molecules. During the electrochemical measurement, the formed DNAzymes could catalyze the reduction of H2O2 in the solution to amplify the electrochemical signal of the intercalated methylene blue. Under optimal conditions, the electrochemical immunoassay exhibited a wide dynamic range of 1.0 fg mL(-1) to 20 ng mL(-1) toward CEA standards with a low detection limit of 0.5 fg mL(-1). Intra-assay and inter-assay coefficients of variation (CV) were less than 8.5% and 11.5%, respectively. No significant differences at the 0.05 significance level were encountered in the analysis of 14 clinical serum specimens between the developed immunoassay and commercialized electrochemiluminescent (ECL) method for detection of CEA.

Target-induced nano-enzyme reactor mediated hole-trapping for high-throughput immunoassay based on a split-type photoelectrochemical detection strategy.

Photoelectrochemical (PEC) detection is an emerging and promising analytical tool. However, its actual application still faces some challenges like potential damage of biomolecules (caused by itself system) and intrinsic low-throughput detection. To solve the problems, herein we design a novel split-type photoelectrochemical immunoassay (STPIA) for ultrasensitive detection of prostate specific antigen (PSA). Initially, the immunoreaction was performed on a microplate using a secondary antibody/primer-circular DNA-labeled gold nanoparticle as the detection tag. Then, numerously repeated oligonucleotide sequences with many biotin moieties were in situ synthesized on the nanogold tag via RCA reaction. The formed biotin concatamers acted as a powerful scaffold to bind with avidin-alkaline phosphatase (ALP) conjugates and construct a nanoenzyme reactor. By this means, enzymatic hydrolysate (ascorbic acid) was generated to capture the photogenerated holes in the CdS quantum dot-sensitized TiO2 nanotube arrays, resulting in amplification of the photocurrent signal. To elaborate, the microplate-based immunoassay and the high-throughput detection system, a semiautomatic detection cell (installed with a three-electrode system), was employed. Under optimal conditions, the photocurrent increased with the increasing PSA concentration in a dynamic working range from 0.001 to 3 ng mL(-1), with a low detection limit (LOD) of 0.32 pg mL(-1). Meanwhile, the developed split-type photoelectrochemical immunoassay exhibited high specificity and acceptable accuracy for analysis of human serum specimens in comparison with referenced electrochemiluminescence immunoassay method. Importantly, the system was not only suitable for the sandwich-type immunoassay mode, but also utilized for the detection of small molecules (e.g., aflatoxin B1) with a competitive-type assay format.

Enzyme-catalyzed silver deposition on irregular-shaped gold nanoparticles for electrochemical immunoassay of alpha-fetoprotein.

A new and disposable electrochemical immunosensor was designed for detection of alpha-fetoprotein (AFP), as a model analyte, with sensitivity enhancement based on enzyme-catalyzed silver deposition onto irregular-shaped gold nanoparticles (ISGNPs). The assay was carried out with a sandwich-type immunoassay protocol by using ISGNP-labeled anti-AFP antibodies conjugated with alkaline phosphatase (ALP-Ab(2)) as detection antibodies. The enzymatically catalytic deposition of silver on the electrode could be measured by stripping analysis in KCl solution due to the Ag/AgCl solid-state voltammetric process. Several labeling protocols including spherical gold nanoparticle-labeled ALP-Ab(2) and ISGNP-labeled ALP-Ab(2) were investigated for determination of AFP, and improved analytical properties were achieved with the ISGNP labeling. With the ISGNP labeling method, the effects of incubation time and incubation temperature for antigen-antibody reaction, and deposition time of silver on the current responses of the electrochemical immunosensors were also monitored. Under optimal conditions, the electrochemical immunosensor exhibited a wide dynamic range from 0.01 ng mL(-1) to 200 ng mL(-1) with a detection limit of 5.0 pg mL(-1) AFP. The immunosensor displayed a good stability and acceptable reproducibility and accuracy. No significant differences at the 95% confidence level were encountered in the analysis of 10 clinical serum samples between the developed immunoassay and the commercially available electrochemiluminescent method for determination of AFP.

Immobilization-Free Programmable Hairpin Probe for Ultrasensitive Electronic Monitoring of Nucleic Acid Based on a Biphasic Reaction Mode

This work designs a novel programmable hairpin probe (PHP) for the immobilization-free electrochemical detection of nucleic acid by coupling polymerase/nicking-induced isothermal signal amplification strategy with a biphasic reaction mode for the first time. The designed PHP (including a target-recognition region, a template sequence for enzymatic reaction and an inactivated anti-streptavidin aptamer) could program multiple isothermal reactions in the solution phase accompanying in situ amplified detectable signal at the electrode surface by the labeled ferrocene tag on the PHP. Upon addition of target analyte into the detection solution, target DNA initially hybridized with the recognition region on the PHP. Replication-induced strand-displacement generated an activated anti-streptavidin aptamer with the assistance of polymerase. Then, the polymerase/nicking enzymes could cleave and polymerize repeatedly the replication product, thus resulting in the formation of numerous template-complementary DNA initiator strands. The released initiator strands could retrigger the programmable hairpin probe to produce a large number of activated anti-streptavidin aptamers, which could be captured by the immobilized streptavidin on the electrode, thus activating the electrical contact between the labeled ferrocene and the electrode. Going after the aptamers, the carried ferrocene could produce the in situ amplified electronic signal within the applied potentials. Under optimal conditions, the electrochemical signal increased with the increasing target DNA concentration in the dynamic range from 5 fM to 10 pM with a detection limit (LOD) of 2.56 fM at the 3sblank criterion. Importantly, the methodology with high specificity was also validated and evaluated by assaying 6 target DNA-spiked human serum and calf thymus DNA samples, and the recoveries were 95-110%. Further work for the programmable hairpin probe could be even utilized in a real world sample to detect miRNA-21 at femtomol level.

Carbon nanospheres-promoted electrochemical immunoassay coupled with hollow platinum nanolabels for sensitivity enhancement

Two nanostructures including carbon nanospheres-graphene hybrid nanosheets (CNS-GNS) and hollow platinum nanospheres (HPtNS) were first synthesized by using direct electrolytic reduction and wet chemistry methods, respectively. Thereafter, a specific sandwich-type electrochemical immunoassay was designed for determination of carcinoembryonic antigen (CEA) by using HPtNS-labeled horseradish peroxidase-anti-CEA conjugates (HRP-anti-CEA) as molecular tags and anti-CEA-assembled CNS-GPS as sensing probes. Compared with pure graphene nanosheets, the presence of carbon nanospheres on the graphene increased the surface coverage of the substrate, and enhanced the immobilized amount of primary antibodies. Several labeling protocols, such as HRP-anti-CEA, solid platinum nanoparticle-labeled HRP-anti-CEA, and hollow platinum nanospheres-labeled HRP-anti-CEA, were investigated for determination of CEA and improved analytical features were obtained with hollow platinum nanosphere labeling. With the HPtNS labeling method, the effects of incubation time and pH on the current responses of the immunosensors were also studied. The strong attachment of biomolecules to the CNS-GPS and HPtNS resulted in a good repeatability and intermediate precision down to 10.2%. The dynamic concentration range spanned from 0.001 ng mL(-1) to 100 ng mL(-1) CEA with a detection limit of 1.0 pg mL(-1) at the 3S(blank) level. No significant differences at the 0.05 significance level were encountered in the analysis of 10 clinical serum samples between the developed immunoassay and the commercially available electrochemiluminescent method for determination of CEA.

In Situ Generation of Electron Donor to Assist Signal Amplification on Porphyrin-Sensitized Titanium Dioxide Nanostructures for Ultrasensitive Photoelectrochemical Immunoassay

An ultrasensitive photoelectrochemical (PEC) immunoassay protocol for quantitative detection of low-abundant proteins at a low potential was designed by utilizing porphyrin-sensitized titanium dioxide (TiO2) nanostructures. Experimental results demonstrated that the water-soluble 5,10,15,20-tetra(4-sulfophenyl)-21H,23H-porphyrin (TSPP) could be bound onto titanium dioxide via the sulfonic group. TSPP-sensitized TiO2 nanostructures exhibited better photoelectrochemical responses and stability in comparison with TiO2 nanoparticles alone under continuous illumination. Using carcinoembryonic antigen (CEA) as a model analyte, a typical PEC immunosensor by using TSPP-TiO2 as the affinity support of anti-CEA capture antibody (Ab1) to facilitate the improvement of photocurrent response was developed. Bioconjugates of secondary antibody and glucose oxidase with gold nanoparticles (Ab2/GOx-AuNPs) was introduced by an antigen-antibody immunoreaction. AuNP acted as a powerful scaffold to bind with bioactive molecules, while GOx catalyzed glucose to in situ generate hydrogen peroxide (H2O2). The generated H2O2 as a sacrificial electron donor could be oxidized by the photogenerated holes to assist the signal amplification at a low potential under light excitation, thus eliminating interference from other species coexisting in the samples. Under optimal conditions, the PEC immunosensor showed a good linear relationship ranging from 0.02 to 40 ng mL(-1) with a low detection limit of 6 pg mL(-1) CEA. The precision, reproducibility, and specificity were acceptable. In addition, the method accuracy was also evaluated for quantitatively monitoring human serum samples, giving results matching with the referenced CEA ELISA kit.

Portable and quantitative monitoring of heavy metal ions using DNAzyme-capped mesoporous silica nanoparticles with a glucometer readout

A portable and quantitative monitoring protocol for sensitive detection of lead ions is designed, based on target-responsive cargo release from Pb2+-specific DNAzyme-capped mesoporous silica nanoparticles (MSNs), by coupling with a widely accessible personal glucose meter (PGM). Initially, glucose molecules are loaded into the pores of the MSNs, the pores are then capped with Pb2+-specific DNAzymes. Upon target introduction, the molecular gates open, resulting in release of the cargo from the pores. The released glucose can be quantitatively monitored using a portable PGM. Under optimal conditions, the as-prepared sensing platform presents good analytical properties for the determination of the target Pb2+ ions, and allows detection of Pb2+ at concentrations as low as 1.0 pM. Importantly, the portable sensing platform has the advantages of simple, on-site, user-friendly and low-cost assessment and has tremendous potential for quantitative detection of non-glucose targets by the public.