Construction And Application Of Photoelectrochemical Biosensors Based On Bismuth Functional Nanocomposites And Novel Signal Amplification Strategies

Photoelectrochemical（PEC）bioassay,as a newly analytical method,is constructed based on the PEC process and biological recognition process,with features such as high sensitivity,low cost,simple equipment and easy miniaturization,which has attracted great interest among the community.With the increasing demand for sensitive bioassay,it is vital to explore the effective methods for significantly improving the analytical performance of sensors.The integration of photosensitive material with excellent performance and new signal amplification mechanism would be an effective strategy to meet the above requirements of bioassay.In this paper,on the basis of the ternary Bi₂S₃/Ag₂S/TiO₂ NTs,double Bi-based heterojunction and other nanomaterials with excellent performance,the sensitive PEC biosensing methods was proposed by innovatively introducing a series of signal amplification techniques,such as enzyme assisted electron donor consumption,enzymatic generation,photogenerated hole-induced chemical redox cycling amplification（RCA）,PEC-CC RCA,energy transfer and steric hindrance.The proposed biosensors have achieved the sensitive detection of biomarkers carcinoembryonic antigen（CEA）.,myoglobin（Myo）and so on,and applied in biochemical analysis,providing the new methods for sensitive detection of disease markers.The main innovations and research contents are as follows.1.Constructionofelectrondonorconsumptionstrategy-based photoelectrochemicalbiosensorandhighlysensitivedetectionof carcinoembryonic antigenIn this work,we prepared ternary Bi₂S₃/Ag₂S/TiO₂ NTs as the photoelectrode,and the ascorbic acid oxidase（AAO）-ascorbic acid（AA）bioevent-based electron donor consumption mode in PEC bioassay for the first time.Combining of the enzymatic consumption of AAO with the steric effect of SiO₂ for dual signal quenching,the proposed biosensor was used to detect CEA.Compared to the Ag₂S and Bi₂S₃,ternary Bi₂S₃/Ag₂S/TiO₂ NTs displayed a higher PEC signal.The photocurrent response could be greatly reduced by the decreased amount of AA resulting from the catalytic oxidation of AA by the AAO.The target CEA present could competitively conjugate with the probe of the functional SiO₂ labeled with NH₂-aptamer and AAO,which is employed as the crucial factor of signal change,thereby causing the enhancement of photocurrent.This method achieves the highly sensitive CEA detection with the linear range from 0.1 pg/mL to 10 ng/mL.The LOD is experimentally measured to be 0.03pg/mL.Moreover,the proposed biosensor has been applied to determine the content of CEA in human serum samples.The results were consistent with the results provided by Xinyang Central Hospital.The recoveries ranged from 90.0%to 110.0%,and the relative standard deviations（RSDs）are not more than 4.7%.2.Photogenerated hole-induced chemical redox cycling on Bi₂S₃/Bi₂Sn₂O₇heterojunction:Toward general amplified split-type photoelectrochemical immunoassayThis work reports the elegant bridging of enzymatic generation of electron donor with photogenerated hole-induced chemical RCA for innovative PEC immunoassay,by the aid of a heterojunction photoelectrode with split-type strategy.Specifically,the system was exemplified by the alkaline phosphatase（ALP）catalytic generation of AA,the redox cycling of AA by tris（2-carboxyethyl）phosphine（TCEP）as reductant,and the use of a novel Bi₂S₃/Bi₂Sn₂O₇ heterojunction and myocardial infarction marker Myo as the photoelectrode and the target,respectively.After the immunoreaction and ALP-induced production of AA,the subsequent oxidation of AA by the photogenerated holes of the Bi₂S₃/Bi₂Sn₂O₇ heterojunction could be cycled via the regeneration of AA by TCEP from the oxidized product of dehydroascorbic acid,leading to easy signal amplification for the sensitive immunoassay of Myo.The linear relationship ranges from 4.0×10^-13 to 1.0×10^-7 g/mL with the LOD of 1.0×10^-13 g/mL.The designed biosensor is successfully used to assay the real sample.The obtained results are in good agreement with the reference values from ROCHE ECL analyzer of Xinyang Central Hospital.The recovery ranges from 88.0%to 110.0%,and RSDs were no more than5.5%.It is believed that this work provided a basis for further design and development of general RCA-based PEC immunoassays.3.Photoelectrochemical-chemical-chemical redox cycling for advanced signal amplification:Proof-of-concept toward new photoelectrochemical bioanalysis and ultrasensitive detection of myoglobinSignal amplification is essential for ultrasensitive PEC bioanalysis.Exploration of the facile and efficient route for multiple signal amplification is highly appealing.Herein,we present the concept of PECCC redox cycling as an advanced signal amplification route and a proof-of-concept toward ultrasensitive PEC bioanalysis.The system operated upon the bridging between the enzymatic generation of signaling species AA from a sandwich immunoassay and the PECCC redox cycling among the ferrocenecarboxylic acid as redox mediator,the AA,and the TCEP as reducing agent at the Bi₂S₃/Bi₂WO₆ photoelectrode.Exemplified by Myo as target,the proposed system achieved efficient regeneration of AA and thus signal amplification toward the ultrasensitive split-type PEC immunoassay.The linear range is of 1.0×10^-13-1.0×10^-7 g/mL with an experimental detection limit of 3.0×10^-14 g/mL.The assay results in real samples are consistent with the standard values from the hospital,and the recoveries are in the range of 80.0 to 103.1%with RSDs of less than 6.0%,indicating acceptable accuracy for the detection in real samples.This work first exploits the PECCC redox cycling,and provides a new route for the research of PEC bioassay on the basis of advanced redox cycling.4.Ru（NH₃）₆³⁺/Ru（NH₃）₆²⁺-mediated redox cycling:Toward enhanced triple signal amplification for photoelectrochemical immunoassayHerein we report an effective Ru（NH₃）₆³⁺/Ru（NH₃）₆²⁺-mediated PECCC RCA strategy toward enhanced triple signal amplification for advanced split-type PEC immunoassay application.Specifically,ALP label was confined via a sandwich immunorecognition to convert 4-aminophenyl phosphate to the signal reporter4-aminophenol（AP）,which was then directed to interact with Ru（NH₃）₆²⁺as redox mediator and TCEP as reducing agent in the detection buffer.Upon illumination,the system was then operated upon the oxidation of Ru（NH₃）₆²⁺by the photogenerated holes on the Bi₂S₃/BiVO₄ photoelectrode,starting the chain reaction in which the Ru（NH₃）₆²⁺was regenerated by Ru（NH₃）₆³⁺-enabled oxidization of AP to p-quinoneimine,which was simultaneously recovered by TCEP.Exemplified by interleukin-6（IL-6）as the analyte,the Ru（NH₃）₆³⁺/Ru（NH₃）₆²⁺-mediated,AP-involved PECCC RCA coupled with ALP enzymatic amplification could achieve triple signal amplification toward the ultrasensitive PEC IL-6 immunoassay.The linear ranges from5.0×10^-15 to 1.0×10^-9 g/mL.The LOD is found to be 2.0×10^-15 g/mL experimentally.The method has been successfully applied to the determination of IL-6 in human serum samples.the recoveries range from 84.9 to 110.3%with RSDs of less than 7.6%.This protocol could offer a new perspective for the further exploration of advanced RCA-based PEC bioassay.5.Quenching effect of exciton energy transfer from CdS:Mn to Au nanoparticles:A highly efficient photoelectrochemical strategy for microRNA-21 detectionA novel and simple PEC biosensing method for microRNA-21（miRNA-21）detection is reported based on energy transfer（ET）between CdS:Mn doped structure（CdS:Mn）and AuNPs.In this protocol,TiO₂-CdS:Mn hybrid structure was equipped by the successive ionic layer adsorption and reaction technique using Cd²⁺/Mn²⁺and S^2-,which was then used as a sensing platform for hairpin DNA immobilization.Integrating the significant ET effect between CdS:Mn and AuNPs with the configuration changes caused by hybridization between hairpin DNA and miRNA-21,this system was used to detect the target.In the absence of miRNA-21,the immobilized DNA was in the hairpin form.In this state,the photocurrent of the electrode was greatly depressed,due to the effective ET effect produced by short interparticle distance between CdS:Mn and AuNPs.In the presence of miRNA-21,the hairpin DNA hybridized with miRNA-21 and changed into a more rigid,rodlike double helix,which forced the AuNPs away from the electrode surface,leading to obvious recover of photocurrent because of the vanished damping effect.This method realized the sensitive detection of miRNA-21 in a linear range of 1.0 fM to 10.0 pM with a low detection limit of 0.5 fM.Moreover,this method has been successfully used to detection the miRNA-21 in human serum samples,the recovery ranges from 90%to103.3,and the %RSDs are no more than 6.3%.