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

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 <i>anti</i>-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 <i>anti</i>-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 <i>anti</i>-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 3<i>s</i>_blank 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

Plasmonic AuNP/g‑C₃N₄ Nanohybrid-based Photoelectrochemical Sensing Platform for Ultrasensitive Monitoring of Polynucleotide Kinase Activity Accompanying DNAzyme-Catalyzed Precipitation Amplification

A convenient and feasible photoelectrochemical (PEC) sensing platform based on gold nanoparticles-decorated g-C₃N₄ nanosheets (AuNP/g-C₃N₄) was designed for highly sensitive monitoring of T4 polynucleotide kinase (PNK) activity, using DNAzyme-mediated catalytic precipitation amplification. To realize our design, the AuNP/g-C₃N₄ nanohybrid was initially synthesized through in situ reduction of Au­(III) on the g-C₃N₄ nanosheets, which was utilized for the immobilization of hairpin DNA₁ (HP₁) on the sensing interface. Thereafter, a target-induced isothermal amplification was automatically carried out on hairpin DNA₂ (HP₂) in the solution phase through PNK-catalyzed 5′-phosphorylation accompanying formation of numerous trigger DNA fragments, which could induce generation of hemin/G-quadruplex-based DNAzyme on hairpin DNA₁. Subsequently, the DNAzyme could catalyze the 4-chloro-1-naphthol (4-CN) oxidation to produce an insoluble precipitation on the AuNP/g-C₃N₄ surface, thereby resulting in the local alternation of the photocurrent. Experimental results revealed that introduction of AuNP on the g-C₃N₄ could cause a ∼100% increase in the photocurrent because of surface plasmon resonance-enhanced light harvesting and separation of photogenerated e^–/h⁺ pairs. Under the optimal conditions, the percentage of photocurrent decrement (Δ<i>I</i>/<i>I</i>₀, relative to background signal) increased with the increasing PNK activity in a dynamic working range from 2 to 100 mU mL^–1 with a low detection limit (LOD) of 1.0 mU mL^–1. The inhibition effect of adenosine diphosphate also received a good performance in PNK inhibitor screening research, thereby providing a useful scheme for practical use in quantitative PNK activity assay for life science and biological research