Signal-on photoelectrochemical immunoassay mediated by the etching reaction of oxygen/phosphorus co-doped g-C3N4/AgBr/MnO2 nanohybrids.

We designed a signal-on photoelectrochemical (PEC) immunoassay for the sensitive monitoring of prostate-specific antigen (PSA) based on the etching reaction of hydrogen peroxide (H2O2) toward oxygen/phosphorus co-doped graphitic C3N4/AgBr/MnO2 nanosheets (OP-g-C3N4/AgBr/MnO2). Initially, glucose oxidase (GOX)-labeled detection antibodies were introduced into the capture antibody-coated microplate with a sandwich-type immunoreaction in the presence of PSA. Then, the as-generated H2O2 from the decomposition of glucose by GOX etched the manganese dioxide (MnO2) nanosheets into manganese ions (Mn2+), thereby causing the exposure of the underlying OP-g-C3N4/AgBr. Meanwhile, H2O2 could be also used as an electron scavenger, and restrain the recombination of the electron-hole pairs of OP-g-C3N4/AgBr. Two advantages of H2O2 enhanced the photocurrent synergistically. Under optimum conditions, the PEC immunoassay showed high sensitivity toward target PSA within a dynamic working range of 0.05-50 ng mL-1 with a limit of detection of 17 pg mL-1. In addition, our system possessed high specificity, favorable selectivity, and good stability. Relative to commercialized PSA ELISA kits, the accuracy of our strategy was acceptable. More importantly, our strategy can be easily extended to screen other biomarkers by controlling the corresponding antibodies.

Horseradish peroxidase-encapsulated DNA nanoflowers: An innovative signal-generation tag for colorimetric biosensor.

Herein a versatile colorimetric biosensing platform was designed for sensitive, specific, and rapid screening of cancer-derived exosomes (HepG2 cell-derived) by introducing horseradish peroxidase -encapsulated DNA nanoflowers (HRP-DF) as the biorecognition elements and signal-generation tags. HRP was concurrently associated with the growing ultralong-chain DNA and the magnesium pyrophosphate crystals (Mg2PPi) during the rolling circle amplification (RCA), thereby ultimately leading to the direct fixation of HRP in DFs. For demonstration, a linear DNA circular molecule encoding the complementary sequence of CD63 aptamer (a nucleic acid sequence that can highly bind to exosomes) was used as a starting amplification template to obtain HRP-DF with the high biorecognition ability of exosomes. Upon addition of target exosomes, a sandwiched reaction was carried out between the cholesterol-modified DNA probes-conjugated magnetic bead (MB) and the HRP-DFs, accompanying formation of ternary complexes (MB-exosomes-HRP-DF). After simple magnetic separation, the HRP carried on the ternary complexes (MB-exosomes-HRP-DF) initiated oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS, nearly colorless) into green-colored oxidized ABTS in the presence of hydrogen peroxide (H2O2). The obvious color change (from nearly colorless to dark green) of ABTS-H2O2 system can be easily observed with the naked eye and accurately monitored by UV-visible spectrometry, which was also proportional to the concentration of exosomes. Impressively, HRP-DF-based biosensing platform exhibited satisfactory colorimetric responses toward target exosomes within the working range from 5.0 × 103 to 5.0 × 106 particles/µL at a low detection limit of 3.32 × 103 particles/µL. Combined with a one-step sandwich reaction, magnetic separation and HRP-DF-based color-changing, this system had the advantages of acceptable accuracy, strong anti-interference ability and good reproducibility.

ZIF-8-Assisted NaYF4:Yb,Tm@ZnO Converter with Exonuclease III-Powered DNA Walker for Near-Infrared Light Responsive Biosensor.

This work reports a ZIF-8 (ZIF: Zeolitic Imidazolate Framework)-assisted NaYF4:Yb,Tm@ZnO upconverter for the photoelectrochemical (PEC) biosensing of carcinoembryonic antigen (CEA) under near-infrared (NIR) irradiation on a homemade 3D-printed device with DNA walker-based amplification strategy. The composite photosensitive material NaYF4:Yb,Tm@ZnO, as converter to transfer NIR import to photocurrent output, was driven from annealed NaYF4:Yb,Tm@ZIF-8. Yb3+ and Tm3+-codoped NaYF4 (NaYF4:Yb,Tm) converted NIR excitation into UV emission, matching with the absorption of ZnO for in situ excitation to generate the photocurrent. Upon target CEA introduction, the swing arm of DNA walker including the sequence of CEA aptamer carried out the sandwiched bioassembly with CEA capture aptamer on the G-rich anchorage DNA tracks-functionalized magnetic beads. Thereafter, DNA walker was triggered, and the swing arm DNA was captured by the G-rich anchorage DNA according to partly complementary pairing and Exonuclease III (Exo III) consumed anchorage DNA by a burnt-bridge mechanism to go into the next cycle. The released guanine (G) bases from DNA walker enhanced the photocurrent response on a miniature homemade 3D-printed device consisting of the detection cell, dark box, and light platform. Under optimal conditions, NaYF4:Yb,Tm@ZnO-based NIR light-driven PEC biosensor presented high sensitivity and selectivity for CEA sensing with a detection limit of 0.032 ng mL-1. Importantly, our strategy provides a new horizon for the development of NIR-based PEC biosensors in the aspect of developing MOF-derived photoelectric materials, flexible design of a 3D-printed device, and effective signal amplification mode.

Double Photosystems-Based 'Z-Scheme' Photoelectrochemical Sensing Mode for Ultrasensitive Detection of Disease Biomarker Accompanying Three-Dimensional DNA Walker.

A new double photosystems-based 'Z-scheme' photoelectrochemical (PEC) sensing platform is designed for ultrasensitive detection of prostate-specific antigen (PSA) by coupling with a three-dimensional (3D) DNA walker. Two photosystems consist of CdS quantum dots (photosystem I; PS I) and BiVO4 photoactive materials (photosystem II; PS II), whereas gold nanoparticles (AuNPs) photodeposited on high-active {010} facets of BiVO4 are used as the electron mediators to promote electron transfer from conduction band of PS II to valence band of PS I. 3D DNA walker-based amplification strategy is carried out between hairpin DNA1 conjugated onto the AuNP, hairpin DNA2 labeled with CdS quantum dot (QD-H2), and DNA walker complementary with the PSA aptamer modified to a magnetic bead (Apt-MB). Upon addition of target, DNA walker strand is displaced from DNA walker/Apt-MB to open hairpin DNA1 on AuNP@BiVO4. In the presence of QD-H2, DNA walker induces the hybridization of DNA1 with DNA2 on the gold nanoparticles step by step, thereby resulting in the assembly of CdS QDs on the AuNP@BiVO4 to form Z-scheme double photosystems with strong photocurrent. Under optimum conditions, the Z-scheme PEC sensing system exhibits good photocurrent responses toward target PSA within the working range of 0.01-50 ng mL-1 at a low detection limit of 1.5 pg mL-1. Good reproducibility and accuracy are acquired for analysis of target PSA and human serum specimens relative to the commercial PSA ELISA kit. Importantly, our strategy provides a new horizon for photoelectrochemical in vitro diagnostics.

A conventional chemical reaction for use in an unconventional assay: A colorimetric immunoassay for aflatoxin B1 by using enzyme-responsive just-in-time generation of a MnO2 based nanocatalyst.

The authors describe a colorimetric immunoassay for the model nalyte aflatoxin B1 (AFB1). It is based on the just-in-time generation of an MnO2 nanocatalyst. Unlike previously developed immunoassay, the chromogenic reaction relies on the just-in-time formation of an oxidase mimic without the aid of the substrate. Potassium permanganate (KMnO4) is converted into manganese dioxide (MnO2) which acts as an oxidase mimic that catalyzes the oxidation 3,3',5,5'-tetramethylbenzidine (TMB) by oxygen to give a blue colored product. In the presence of ascorbic acid (AA), KMnO4 is reduced to Mn(II) ions. This results in a decrease in the amount of MnO2 nanocatalyst. Hence, the oxidation of TMB does not take place. By adding ascorbate oxidase, AA is converted into dehydroascorbic acid which cannot reduce KMnO4. Based on these observations, a colorimetric competitive enzyme immunoassay was developed where ascorbate oxidase and gold nanoparticle-labeled antibody against AFB1 and magnetic beads carrying bovine serum albumin conjugated to AFB1 are used for the determination of AFB1. In presence of AFB1, it will compete with the BSA-conjugated AFB1 (on the magnetic beads) for the labeled antibody against AFB1 on the gold nanoparticles. This makes the amount of ascorbate oxidase/anti-AFB1 antibody-labeled gold nanoparticles, which conjugated on magnetic beads, reduce, and resulted in an increase of ascorbic acid. Under optimal conditions, the absorbance (measured at 652 nm) decreases with increasing AFB1 concentrations in the range from 0.1 to 100 ng mL-1, with a 0.1 ng mL-1 detection limit (at the 3Sblank level). The accuracy of the assay was validated by analyzing spiked peanut samples. The results matched well with those obtained with a commercial ELISA kit. Conceivably, the method is not limited to aflatoxins but has a wide scope in that it may be applied to many other analytes for which respective antibodies are available. Graphical abstract Schematic illustration of ascorbate oxidase (AOx)-mediated potassium permanganate (KMnO4)-responsive ascorbic acid (AA) for visual colorimetric immunoassay of aflatoxin B1 (AFB1) by coupling with hydrolytic reaction of AOx toward AA and the KMnO4-Mn(II)-TMB system [note: 3,3',5,5'-tetramethylbenzidine: TMB].

Bioresponsive Release System for Visual Fluorescence Detection of Carcinoembryonic Antigen from Mesoporous Silica Nanocontainers Mediated Optical Color on Quantum Dot-Enzyme-Impregnated Paper.

An all-in-one paper-based analytical device (PAD) was successfully developed for visual fluorescence detection of carcinoembryonic antigen (CEA) on CdTe/CdSe quantum dot (QD)-enzyme-impregnated paper by coupling with a bioresponsive controlled-release system from DNA-gated mesoporous silica nanocontainers (MSNs). The assay was carried out in a centrifuge tube by using glucose-loaded MSNs with a CEA aptamer and a QD-enzyme-paper attached on the lid. Initially, single-strand complementary DNA to a CEA aptamer was covalently conjugated to the aminated MSN, and then glucose (enzyme substrate) molecules were gated into the pore with the help of the aptamer. Glucose oxidase (GOD) and CdTe/CdSe QDs were coimmobilized on paper for the visual fluorescence signal output. Upon target CEA introduction in the detection cell, the analyte specifically reacted with the immobilized aptamer on the MSN to open the pore, thereby resulting in the glucose release. The released glucose was oxidized by the immobilized GOD on paper to produce gluconic acid and hydrogen peroxide, and the latter quenched the fluorescence of CdTe/CdSe QDs, which could be determined by the naked eye on a portable smartphone and a commercial fluorospectrometer. Under optimal conditions, the PAD-based sensing system enabled sensitive discrimination of target CEA against other biomarkers or proteins in a linear range of 0.05-20 ng mL-1 with a limit of detection of 6.7 pg mL-1 (ppt). In addition, our strategy displayed high specificity, good reproducibility, and acceptable accuracy for analyzing human serum specimens with a commercial human CEA ELISA kit. Importantly, this methodology offers promise for simple analysis of biological samples and is suitable for use in the mass production of miniaturized devices, thus opening new opportunities for protein diagnostics and biosecurity.

Hybridization chain reaction-based colorimetric aptasensor of adenosine 5'-triphosphate on unmodified gold nanoparticles and two label-free hairpin probes.

This work designs a new label-free aptasensor for the colorimetric determination of small molecules (adenosine 5'-triphosphate, ATP) by using visible gold nanoparticles as the signal-generation tags, based on target-triggered hybridization chain reaction (HCR) between two hairpin DNA probes. The assay is carried out referring to the change in the color/absorbance by salt-induced aggregation of gold nanoparticles after the interaction with hairpins, gold nanoparticles and ATP. To construct such an assay system, two hairpin DNA probes with a short single-stranded DNA at the sticky end are utilized for interaction with gold nanoparticles. In the absence of target ATP, the hairpin DNA probes can prevent gold nanoparticles from the salt-induced aggregation through the interaction of the single-stranded DNA at the sticky end with gold nanoparticles. Upon target ATP introduction, the aptamer-based hairpin probe is opened to expose a new sticky end for the strand-displacement reaction with another complementary hairpin, thus resulting in the decreasing single-stranded DNA because of the consumption of hairpins. In this case, gold nanoparticles are uncovered owing to the formation of double-stranded DNA, which causes their aggregation upon addition of the salt, thereby leading to the change in the red-to-blue color. Under the optimal conditions, the HCR-based colorimetric assay presents good visible color or absorbance responses for the determination of target ATP at a concentration as low as 1.0nM. Importantly, the methodology can be further extended to quantitatively or qualitatively monitor other small molecules or biotoxins by changing the sequence of the corresponding aptamer.