CRISPR/Cas12a-based MUSCA-PEC strategy for HSV-1 assay.

In the photoelectrochemical sensing, constant potential excitation to get the photoelectrochemical signal is the main excitation signal mode. Novel method for photoelectrochemical signal obtaining is needed. Inspired by this ideal, a photoelectrochemical strategy for Herpes simplex virus (HSV-1) detection with multiple potential step chronoamperometry (MUSCA) pattern was fabricated using CRISPR/Cas12a cleavage coupled with entropy-driven target recycling. In the presence of target, HSV-1, the Cas12a was activated by the H1-H2 complex obtained by entropy-driven, then digesting the circular fragment of csRNA to expose single-stranded crRNA2 and alkaline phosphatase (ALP). The inactive Cas12a was self-assembled with crRNA2 and activated again with the help of assistant dsDNA. After multiple rounds of CRISPR/Cas12a cleavage and magnetic separation, MUSCA, as a signal amplifier, collected the enhanced photocurrent responses generated by catalyzed p-Aminophenol (p-AP). Different from the reported signal enhancement strategies based on photoactive nanomaterials and sensing mechanisms, MUSCA technique endowed the strategy with unique advantages of direct, fast and ultrasensitive. A superior detection limit of 3 aM toward HSV-1 was achieved. This strategy was successfully applied for HSV-1 detection in Human serum samples. The combination of MUSCA technique and CRISPR/Cas12a assay brings broader potential prospect for the detection of nucleic acids.

Ti3C2@WSe2 as photoelectractive materials coupling with recombinase polymerase amplification for nucleic acid detection.

The photoelectrochemical biosensor based on double redox cycle amplification technology coupled with Tungsten diselenide and MXene-modified electrode was developed. Signal amplification technology is a commonly used method to improve the sensitivity. In this article, in the double redox cycle amplification method, p-aminophenol is used as the signal molecule, and squaric acid acts as a redox indicator. Tris(2-carboxyethyl) phosphine is an excellent reducing agent, which greatly improves the photoelectric response. Tungsten diselenide and MXene are used as photosensitive materials. In the presence of model target, respiratory syncytial virus RNA, the recombinase polymerase amplification process occurred because there is amplification template, so that the signal molecule p-aminophenol will be produced, leading to redox cycle was carried out, and the photocurrent signal is improved. In the absence of syncytial virus RNA, the photocurrent signal is low. The detection range of the biosensor is from 0.2 fM to 80 fM, and the detection limit reaches 30 aM for respiratory syncytial virus RNA. The method of introducing redox cycle amplification and Tungsten diselenide, MXene into photoelectrochemical biosensing provides a new idea for future biological analysis and has application potential.

Self-enhancement photoelectrochemical strategy for kanamycin determination with amino functionalized MOFs.

Based on the self-enhanced photoelectrochemical quality of Cu-MOF-NH2, a photoelectrochemical biosensor for kanamycin detection with a Cu-MOF-NH2 modified electrode was constructed. This biosensor took full advantage of the low electron hole recombination rate due to the ligand-to-metal charge-transfer mechanism of excited electron transfer in Cu-MOF-NH2. The kanamycin aptamer was modified onto Cu-MOF-NH2 by Schiff base reaction. In the presence of kanamycin, the holes on the amino group in Cu-MOF-NH2 oxidize kanamycin, making kanamycin itself as a signal enhancement substance, and achieving the effect of self-enhancement. The dynamic monitoring range for kanamycin is 0.5 to 650 nM, and the detection limit is 0.1 nM. The sensor has been successfully applied to the determination of kanamycin in fish with recoveries of 95.7-105.0% and RSD of 1.5-4.0%. This work provides a broad path for the development of self-enhanced photoelectrochemical sensors.

Chemiluminescence assay for kanamycin based on target recycling strategy.

A chemiluminescence (CL) sensing strategy for kanamycin residue detection in fish samples was established based on luminol-functionalized gold nanoparticles as CL nanoprobe materials combined with DNA hairpin structure and carboxyl-modified magnetic beads. Relying on nucleic acid amplification technology, the system can successfully realize the recycling of kanamycin, so that the biosensor can release a large number of luminol-functionalized gold nanoparticles with excellent CL performance even at a low residual levels of kanamycin. The biosensor strategy showed a good linear relationship with kanamycin in the range 0.09-130 nM, the detection limit was as low as 0.04 nM. This method proves the excellent performance of the sensing strategy and provides a low-cost and high-sensitivity CL analysis strategy for the detection of kanamycin and even other antibiotics.

ZnSe nanodisks:Ti3C2 MXenes-modified electrode for nucleic acid liquid biopsy with photoelectrochemical strategy.

ZnSe nanodisks:Ti3C2 MXene complex was prepared for the first time. Based on its remarkable photoelectrochemical performance, combined with the enzyme-free toehold-mediated strand displacement reaction, a photoelectrochemical biosensor for the detection of the non-small-cell cancer biomarker ctDNA KRAS G12D was developed. ZnSe nanodisks were in situ grown on Ti3C2 MXene surface by two-step hydrothermal method. The high conductivity and adjustable band gap of MXene significantly enhanced the photoelectric response of ZnSe. Subsequently, the photoelectrochemical biosensor was prepared by combining with the signal amplification function of p-aminophenol and the enzyme-free toehold-mediated strand displacement reaction on the modified ITO electrode surface. Under the optimized conditions, the linear detection range is 0.5 ~ 100.0 fM, and the detection limit is 0.2 fM, which realizes the sensitive detection of KRAS G12D. The photoelectrochemical biosensor constructed opens up a new pathway for the preparation of new Mxene-based composite materials and the research of photoelectrochemical biosensor. Nucleic acid liquid biopsy with ZnSe nanodisks:Ti3C2 MXene photoelectroactive modified electrode.

Photoelectrochemical Sensing of α-Synuclein Based on a AuNPs/Graphdiyne-Modified Electrode Coupled with a Nanoprobe.

We developed a method for photoelectrochemical (PEC) sensing based on a AuNPs/graphdiyne, as a low background signal composite material, modified electrode coupled with a nanoprobe (probe DNA/DA/MBA/WSe2) for sensitive α-synuclein (α-Syn) detection. A tungsten selenide (WSe2) nanoflower was first produced with a one-pot solvothermal method and employed as a signal amplification element and the modified substrate of the nanoprobe. The synergy effect between the WSe2 nanoflower and graphdiyne (GDY) can reduce the photoinduced electron-hole recombination and expedite the spatial charge separation. Due to the synergistic effect of AuNPs/GDY and WSe2, this detection strategy provides a high signal-to-noise ratio and good performance. The signal indicator, dopamine/4-mercaptophenyl boronic acid/WSe2 (DA/MBA/WSe2), was generated with the recognition of boron-diol. In the presence of the α-Syn oligomer, the target triggered cycle I strand displacement amplification and achieved the conversion of the α-Syn oligomer to a massive output of false-target DNA (FT). The output FT was used for the cycle II catalytic hairpin assembly onto the electrode which was modified with AuNPs/GDY and triple-stranded DNA (TsDNA); thereby, plenty of PEC nanoprobes which are composed of probe DNA and the signal indicator are captured, and the photocurrent response is produced correspondingly. This PEC biosensor generated a strong photocurrent with low blank (27.6 nA) and was sensitive to α-Syn oligomer. The limit of detection was 3.3 aM, and the relative standard deviation (RSD) was 3.7% at 100 aM. Moreover, it also has good selectivity, indicating promising potential in clinical diagnostics.

An enzyme linked aptamer photoelectrochemical biosensor for Tau-381 protein using AuNPs/MoSe2 as sensing material.

Alzheimer's disease is a worldwide health problem and it has attracted extensive attention. Tau protein is an important biomarker in the pathogenesis of Alzheimer's disease. Herein, we devise a in situ enzyme catalysis generating electron donor photoelectrochemical (PEC) biosensor for Tau-381 protein. Tau-381 protein aptamer is immobilized onto the surface of AuNPs/MoSe2 nanosheets modified electrode. In the presence of Tau-381 protein, an aptamer-protein duplex is formed. Meanwhile, the Tau-381 antibody and the protein G/AP (protein G labeled with alkaline phosphatase) are captured with the affinity interaction between Tau-381 protein and Tau-381 antibody, Tau-381 antibody and protein G/AP. The electron donor, ascorbic acid, is in situ produced by the catalyzing of ascorbic acid 2-phosphate in the PEC detection solution. As a result, low blank noise and strong photocurrent response are engendered. The photocurrent response is related to the concentration of Tau-381 protein. The detection range of Tau-381 protein is from 0.5 fM to 1.0 nM with detection limit of 0.3 fM. This in situ generating electron donor PEC biosensor can detect various targets by simply alternating antibody, antigen, or aptamer commercially. Thus, this work represents a simple and general sensing protocol.

Chemiluminescence assay for Listeria monocytogenes based on Cu/Co/Ni ternary nanocatalyst coupled with penicillin as generic capturing agent.

Bacterial pathogen control is important in seafood production. In this study, a Cu/Co/Ni ternary nanoalloy (Cu/Co/Ni TNA) was synthesized using the oleylamine reducing method. It was found that Cu/Co/Ni TNA greatly enhanced the chemiluminescence (CL) signal of the hydroxylamine-O-sulfonic acid (HOSA)-luminol system. The CL properties of Cu/Co/Ni TNA were investigated systemically. The possible CL mechanism also was intensively investigated. Based on the enhanced CL phenomenon of Cu/Co/Ni TNA, a Cu/Co/Ni TNA, penicillin, and anti-L. monocytogenes (Listeria monocytogenes) antibody-based sandwich complex assay for detection of L. monocytogenes was established. In this sandwich CL assay, penicillin was employed to capture and enrich pathogenic bacteria with penicillin-binding proteins (PBPs) while anti-L. monocytogenes antibody was adopted as the specific recognition molecule to recognize L. monocytogenes. L. monocytogenes was detected sensitively based on this new Cu/Co/Ni TNA-HOSA-luminol CL system. The CL intensity was proportional to the L. monocytogenes concentration ranging from 2.0 × 102 CFU ml-1 to 3.0 × 107 CFU ml-1 and the limit of detection wa 70 CFU ml-1 . The reliability and potential applications of our method was verified by comparison with official methods and recovery tests in environment and food samples.

Photoelectrochemical aptasensor with low background noise.

In photoelectrochemical (PEC) detection, enhancing the PEC signal and depressing the blank signal are conducive to improve the sensitivity. Because the carbon nanotube (CNT) effectively transfers photogenerated electrons from SnSe to the electrode, the composite nanomaterial CNTs/SnSe generates a strong PEC signal. Methionine (Met), AuNPs, and probe DNA are woven together forming a nanoprobe which is used as a quencher to quench the PEC signal of CNTs/SnSe. When the nanoprobe and CNTs/SnSe are modified onto the electrode, there is a low blank signal. In the presence of metastatic breast cancer cells, the cells interact with the aptamer of dsDNA; concomitantly, cDNA is released to trigger catalytic hairpin assembly (CHA). As a result, a new dsDNA which has an overhang is formed. The nanoprobe on the surface of the electrode hybridizes with the newly formed dsDNA. Subsequently, the nanoprobe is released from the surface of the electrode and the quenching effect between the nanoprobe and the CNTs/SnSe disappears. The PEC aptasensor is linear in the concentration range of 300-5,000 cells/mL, and the detection limit is 180 cells/mL under optimized conditions. The relative standard deviation (RSD) is 3.6% at 10,000 cells/mL. This work demonstrates a promising strategy using CNTs/SnSe as the photoactive material and Met-AuNPs as the quencher to establish a PEC aptasensor with a high PEC response and low blank signal. It can be used to detect bioactive substances at ultralow levels prospectively. Graphical abstract.

Electron Acceptors Co-Regulated Self-Powered Photoelectrochemical Strategy and Its Application for Circulating Tumor Nucleic Acid Detection Coupled with Recombinase Polymerase Amplification.

Biosensor working in a self-powered mode has been widely concerned because it produces a signal when the bias potential is 0 V. However, the self-powered mode is used only when the materials have self-powered properties. Conversion of non-self-powered to self-powered through molecular regulation can solve this problem effectively. Here, we fabricated a self-powered photoelectrochemical mode based on co-regulation of electron acceptors methylene blue (MB) and p-nitrophenol (p-NP). AuNPs@ZnSe nanosheet-modified gold electrode (AuNPs@ZnSeNSs/GE) gave a small photocurrent at 0 V. In the presence of MB and p-NP, AuNPs@ZnSeNSs/GE gave the strongest photocurrent at 0 V. Accordingly, an electron acceptor co-regulated self-powered photoelectrochemical assay was fabricated. As proof-of-concept demonstrations, this assay was applied for prostate cancer circulating tumor nucleic acid biomarker, KLK2 and PCA3, detection combined with in situ recombinase polymerase amplification strategy. This assay generated a strong photocurrent and was sensitive to the variation of KLK2 and PCA3 concentration. The limits of detection were 30 and 32 aM, respectively. We anticipate this electron acceptor co-regulated self-powered photoelectrochemical mode to pave a new way for the development of self-powered sensing.

One-step fabrication of trimetallic alloy nanozyme catalyzer for luminol-H2O2 chemiluminescence and its application for miRNA-21 detection coupled with miRNA walking machine.

PtCuCo trimetallic alloys (PtCuCo-TAs) are synthesized by one-step reduction. The chemiluminescence (CL) properties of PtCuCo-TAs are studied systemically. PtCuCo-TAs show good catalyzing for luminol-H2O2 system. A CL platform is developed for the detection of miRNA-21 using PtCuCo-TAs as nanozyme catalyzer. In the CL detection platform, H1 (Hairpin DNA1) is immobilized onto magnetic beads (MBs) firstly. In the presence of miRNA-21, H1 is opened. H2 (Hairpin DNA2) then hybridizes with H1. Meanwhile, a "cleat" in the end of miRNA-21 with a fewer bases complementary is formed to prevent miRNA-21 dissociating from H1. This miRNA-21 hybridizes to another H1. When cpDNA-PtCuCo-TAs which consisted with cDNA (Complementary strand of probe DNA) and pDNA-PtCuCo-TAs (PtCuCo-TAs labeled with probe DNA) are added, the ssDNA region of H1 reacts with the toehold domain of probe DNA and cDNA is released resulting pDNA-PtCuCo-TAs being captured. With this process repeatedly, a lot of pDNA-PtCuCo-TAs are captured onto MBs. After separation and washing, the precipitate and H2O2 are put into the 96-well and luminol solution is injected. The CL signal is produced by PtCuCo-TAs catalyzing luminol-H2O2 system. The amount of miRNA-21 is detected with CL signal. This CL platform performs with limit of detection 0.167 fM and has good selectivity over other RNA.

Photoelectric current as a highly sensitive readout for potentiometric sensors.

We report here a general strategy to read out potentiometric signals via a photoelectrochemical method. The photocurrent at a working electrode coated with a ZnSe/r-GO composite can be modulated by a polymeric membrane ion-selective electrode that works as a reference electrode.

An assay for Staphylococcus aureus based on a self-catalytic ampicillin-metal (Fe3+)-organic gels-H2O2 chemiluminescence system with near-zero background noise.

A self-catalytic ampicillin-metal (Fe3+)-organic gels (AMP-MOGs (Fe))-H2O2 CL system, which is not influenced by transition metal ions, was studied. A method for CL detection of Staphylococcus aureus (S. aureus) based on the AMP-MOGs (Fe)-H2O2 CL system was achieved. A superior detection limit of 31 CFU mL-1 toward S. aureus was obtained with near-zero background noise.

A design for the photoelectrochemical detection of miRNA-221 based on a tungsten diselenide-cysteine-dopamine nanoprobe coupled with mismatched catalytic hairpin assembly target recycling with ultra-low background noise.

A strategy for the photoelectrochemical detection of miRNA with ultra-low background noise was developed using tungsten diselenide-cysteine-dopamine (WSe2/Cys/DA) as a nanoprobe coupled with mismatched catalytic hairpin assembly target recycling. A superior detection limit of 3.3 aM toward miRNA-221 was achieved.

Photoelectrochemical determination of ractopamine based on inner filter effect between gold nanoparticles and graphitic carbon nitride-copper(II) polyphthalocyanine coupled with 3D DNA stabilizer.

Copper(II) polyphthalocyanine (CuPPc) was combined with graphitic carbon nitride (g-C3N4) to form a heterojunction with enhanced photoelectrochemical (PEC) signal. A sensitive PEC method was developed for determination of ractopamine based on a PEC inner filter effect between gold nanoparticles (AuNPs) and the g-C3N4/CuPPc. A gold electrode was modified with g-C3N4/CuPPc and the DNA was linked to the AuNPs. Initially, the PEC signal is weak due to the inner filter effect between the AuNPs and g-C3N4/CuPPc. In the presence of ractopamine, it interacts with the aptamer and the complementary chain (C chain) is released. This triggers the entropy-driven cyclic amplification and results in the release of the substrate B chain (SB chain) from three-dimensional DNA stabilizer. The probe is released from the electrode due to the interaction of probe DNA and the SB chain. As a result, the PEC signal increases linearly in the 0.1 pmol·L-1 to 1000 pmol·L-1 ractopamine concentration range. The detection limit is 0.03 pM, and the relative standard deviation is 3.4% (at a 10 pmol·L-1 level; for n = 11). The method has been successfully applied to the determination of ractopamine in pork samples. Graphical abstract Schematic presentation of detection method based on PEC inner filter effect between AuNPs and the g-C3N4/CuPPc being fabricated for ractopamine. 3D DNA was used as stabilizer to decrease the PEC blank signal.

Photoelectrochemical platform for MicroRNA let-7a detection based on graphdiyne loaded with AuNPs modified electrode coupled with alkaline phosphatase.

In this work, a photoactive material, Graphdiyne (GDY) loaded with AuNPs (AuNPs-GDY), was successfully synthesized. The fabricated material made use of the natural band-gap structure of GDY, which could produce hole-electron pairs and the plasmon resonance effect of AuNPs to obtain a high photoelectrochemical (PEC) response. AuNPs-GDY PEC response changed with the mass ratio of GDY to tetrachloroauric acid. When the mass ratio of GDY to tetrachloroauric acid was 1:2.5, AuNPs-GDY exhibited the best PEC performance. Thus, the best one was selected as the photoactive material to establish a PEC biosensor for microRNA detection. The PEC biosensor used the alkaline phosphatases as catalyzer to generate ascorbic acid in situ, which provided a low background signal and a high PEC response. The cancer marker, MicroRNA let-7a, was chosen as a target model. Under optimal condition, potential 300 mV and pH 8.0, the PEC biosensor had a detection limit of 3.3 × 10-19 M and a good linearity with microRNA let-7a concentration ranged from 1.0 × 10-18 M to 1.0 × 10-10 M. This PEC biosensor opened a promising platform using GDY to fabricate analytical method and detect microRNA at ultralow levels for diagnoses.

A photoelectrochemical sensing strategy based on single-layer MoS2 modified electrode for methionine detection.

MoS2, a typical transition metal disulfide, is widely used in the photoelectrochemical (PEC) sensor construction. In general, MoS2 based PEC sensor are "signal-on" strategies. Surprisingly, we discovered that the PEC response of MoS2 was quenched by methionine greatly. Based on this discovery, a reduction PEC sensing strategy utilized MoS2 modified electrode for methionine detection was fabricated for the first time. Experimental factors, such as, bias potential, volume of MoS2 and pH were studied. Under optimized conditions, the decreased intensity of the photocurrent signal was proportional to the logarithmic value of methionine concentrations from 0.1 nM to 1 µM with the detection limit of 0.03 nM. Moreover, this method exhibited good performance of excellent selectivity. And it showed potential applications in the practical determination of methionine in real-life sample. This strategy not only expands the PEC detection method but also provides a simple, rapid response, good selectivity and high sensitivity way to detect methionine.

Nanogapped Au(core) @ Au-Ag(shell) structures coupled with Fe3O4 magnetic nanoparticles for the detection of Ochratoxin A.

Contamination of mycotoxins has been a general problem of food safety and afflicts people all around the world. Ochratoxin A (OTA), an abundant and representative food-contaminating mycotoxin, has a detrimental effect on our health. Herein, an ultrasensitive surface-enhanced Raman scattering (SERS) aptasensor was fabricated for OTA detection. Au(core)@Au-Ag(shell) nanogapped nanostructures (Au@Au-Ag NNSs) were coupled with Fe3O4 magnetic nanoparticles (Fe3O4 MNPs) by OTA aptamer and its complementary DNA sequence. The distance between two nanoparticles become far away in presence of the OTA due to the affinity of aptamer. This effect can obviously bring about the change of Raman intensity which can be used for the detection of OTA. Compared with pure metal nanoparticles, Au@Au-Ag NNSs can effectively enhance the Raman intensity of Raman signal molecule (4-MBA) via the ultrasmall nanogap, which contribute to the sensitivity of the sensor system. Additionally, the utilization of Fe3O4 MNPs offers a green, economical and facile technology to capture targets from solution. The specially modified aptamer and its complementary strand establish a bridge connecting between Au@Au-Ag NNSs and Fe3O4 MNPs. In this study, OTA concentration with a linear range of 0.01-50 ng mL-1 and the limit of detection (LOD) was as low as 0.004 ng mL-1. The proposed SERS aptasensor can combine both of these above type of materials and provide a rapid, sensitive, and low-cost methodology for numerous future applications of point-of-care diagnostics.

Magnetic Separation-Based Multiple SELEX for Effectively Selecting Aptamers against Saxitoxin, Domoic Acid, and Tetrodotoxin.

In this study, a novel magnetic separation-based multiple systematic evolution of ligands by exponential enrichment (SELEX) was applied to select aptamers simultaneously against three kinds of marine biotoxins, including domoic acid (DA), saxitoxin (STX), and tetrodotoxin (TTX). Magnetic reduced graphene oxide (MRGO) was prepared to adsorb unbound ssDNAs and simplify the separation step. In the multiple SELEX, after the initial twelve rounds of selection against mixed targets and the subsequent four respective rounds of selection against each single target, the three resulting ssDNA pools were cloned, sequenced, and analyzed. Several aptamer candidates were selected and subjected to the binding affinity and specificity test. Finally, DA-06 ( Kd = 62.07 ± 19.97 nM), TTX-07 ( Kd = 44.12 ± 15.38 nM), and STX-41 ( Kd = 61.44 ± 23.18 nM) showed high affinity and good specificity for DA, TTX, and STX, respectively. They were also applied to detect and quantify DA, TTX, and STX successfully. The other two multitarget aptamers, DA-01 and TTX-27, were also obtained, which can bind with either DA or TTX. These aptamers provide alternative recognition molecules to antibodies for biosensor applications.

Photoelectrochemical platform for cancer cell glutathione detection based on polyaniline and nanoMoS2 composites modified gold electrode.

Herein, a visible light photoelectrochemical (PEC) platform based on polyaniline (PANI) and nanoMoS2 composites as optoelectronic material for glutathione detection without any auxiliary of biomolecules or labeled materials was developed. Firstly, the nanoMoS2 was prepared via a simple ultrasound exfoliation method. The PANI was synthesized by chemical oxidative polymerization method. Then composite of PANI and nanoMoS2 was used to modify gold electrode. It was found that the composite membrane showed excellent PEC properties. And glutathione enhanced the PEC signal greatly. Based on this finding a method for glutathione detection was fabricated. Under the optimum conditions, the linear response of glutathione concentrations ranged from 1.0 × 10-10 to 1.0 × 10-4 mol L-1 was obtained with a detection limit of 3.1 × 10-11 mol L-1. The relative standard deviation was 2.9% at 2.0 × 10-9 M (n = 10). This method showed high sensitivity and simpleness which opened up a new promising signal-on PEC platform for future bioassay.