Wenqiang Lai

Sandwich-type immunosensors and immunoassays exploiting nanostructure labels: A review

Methods based on sandwich-type immunosensors and immunoassays have been developed for detection of multivalent antigens/analytes with more than one eptiope due to the use of two matched antibodies. High-affinity antibodies and appropriate labels are usually employed for the amplification of detectable signal. Recent research has looked to develop innovative and powerful novel nanoparticle labels, controlling and tailoring their properties in a very predictable manner to meet the requirements of specific applications. This articles reviews recent advances, exploiting nanoparticle labels, in the sandwich-type immunosensors and immunoassays. Routine approaches involve noble metal nanoparticles, carbon nanomaterials, semiconductor nanoparticles, metal oxide nanostructures, and hybrid nanostructures. The enormous signal enhancement associated with the use of nanoparticle labels and with the formation of nanoparticle-antibody-antigen assemblies provides the basis for sensitive detection of disease-related proteins or biomolecules. Techniques commonly rely on the use of biofunctionalized nanoparticles, inorganic-biological hybrid nanoparticles, and signal tag-doped nanoparticles. Rather than being exhaustive, this review focuses on selected examples to illustrate novel concepts and promising applications. Approaches described include the biofunctionalized nanoparticles, inorganic-biological hybrid nanoparticles, and signal tage-doped nanoparticles. Further, promising application in electrochemical, mass-sensitive, optical and multianalyte detection are discussed in detail.

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.

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.

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.

Hybridization-induced isothermal cycling signal amplification for sensitive electronic detection of nucleic acid.

This works reports a new signal-on amplification strategy for sensitive electronic detection of nucleic acid based on the isothermal circular strand-displacement polymerization (ICSDP) reaction. The assay mainly involves a hybridization of ferrocene-labeled hairpin DNA with blocker DNA, a strand-displacement process with target DNA, and an ICSDP-based polymerization reaction. The signal is amplified by the labeled ferrocene on the hairpin probe with target recycling. Upon addition of target analyte, the blocker DNA is initially displaced by target DNA from the hairpin/blocker DNA duplex owing to the difference of the folding free energy, then the newly formed target/blocker DNA duplex causes the ICSDP reaction with the aid of the primer and polymerase, and then the released target DNA retriggers the strand-displacement for target recycling. Numerous ferrocene molecules are close to the electrode surface due to the reformation of hairpin DNA, each of which produces an electronic signal within the applied potentials, thereby resulting in the amplification of electrochemical signal. Under the optimal conditions, the ICSDP-based amplification method displays good electrochemical responses for detection of target DNA at a concentration as low as 0.03pM.

Mesoporous nanogold–MnO2–poly(o-phenylenediamine) hollow microspheres as nanotags and peroxidase mimics for sensing biomolecules

A new electrochemical immunosensor was designed for the determination of carcinoembryonic antigen (CEA) with sensitivity enhanced by using nanogold-poly(o-phenylenediamine)-manganese dioxide organic-inorganic hybrid nanostructures (GNPM) as nanotags and peroxidase mimics. Initially, mesoporous poly(o-phenylenediamine)-manganese dioxide (PPD-MnO2) hollow microspheres were synthesized by an inorganic/organic interfacial polymerization technique. Then gold nanoparticles were assembled onto the surface of PPD-MnO2, which were used for the labelling of the anti-CEA detection antibody (pAb2). The prepared GNPM nanotags were characterized using transmission electron microscopy (TEM), field emission scanning electron microscopy (FESEM), UV-vis absorption spectroscopy, N2 adsorption-desorption isotherm measurements and Fourier transform infrared spectroscopy (FTIR). The assay was carried out with a sandwich-type immunoassay format in pH 5.5 acetic acid-buffered saline solution containing 2.5 mmol L-1 H2O2. Experimental results indicated that the electrochemical immunosensor exhibited a wide dynamic range from 0.01 to 80 ng mL-1 towards the target CEA with a detection limit (LOD) of 6.0 pg mL-1. The immunosensor also displayed a good stability and acceptable reproducibility and selectivity. In addition, the methodology was evaluated by assaying 10 clinical serum samples, providing a good relationship between the electrochemical immunosensor and the commercialized electrochemiluminescent (ECL) method for determination of CEA.