Construction And Application Of Photoelectrochemical Immunosensor Based On Modified ZnO Nanomaterials

Photoelectrochemistry（PEC）biosensing is a new type of biological analysis technology using PEC active materials as converters and biomolecules as recognition elements.As an important branch of PEC biosensing,PEC immunosensor is a detection method established by the combination of PEC analysis and immunoassay.Because the PEC analysis technology use the specific waveband or wavelength light as excitation source,and current or voltage as the detection signal,it can not only reduce the background signal and improve the sensitivity,but also realize the analysis of the target at a lower applied potential.Therefore,PEC immunosensors have unique advantages such as high sensitivity,strong specificity,good selectivity,fast response speed,and easy miniaturization.In PEC immunosensors,the photoactive materials with high photoelectric conversion efficiency,good biocompatibility,and strong stability it is crucial to the performance of the sensor.As one of many photoactive materials,zinc oxide（ZnO）nanomaterials not only have the above advantages,but also equipped with excellent properties such as high electron mobility,easy adjustment,and easy synthesis.However,as a wide-bandgap semiconductor nanomaterial,ZnO has a short photoelectron lifetime,low utilization of visible light,and the electron-hole pairs of it is easy recombination,which limit its application in the field of PEC bioanalysis and photocatalysis.This paper focuses on the modification of ZnO by different methods to synthesize ZnO-based nanocomposites.Combined with different signal amplification strategies and analysis modes,the constructed PEC immunosensor could realize the sensitive and accurate detection of disease markers such as Prostate-specific antigen（PSA）and Cardiac troponin I,（c Tn I）.The main innovations and research contents are as follows:1.An ingenious photoelectrochemical（PEC）immunoassay platform was constructed based on the in-situ destruction of plasma metal in metal/semiconductor hybrids for the sensitive detection of prostate specific antigen（PSA）.In the ZnO-Ag-Bi₂S₃nanocomposites,Ag NPs could act as electronic relays to accelerate the electron transfer between ZnO and Bi₂S₃.Furthermore,due to the localized surface plasmon resonance（LSPR）effect of Ag NPs,the ZnO-Ag-Bi₂S₃ nanocomposite exhibits strong absorbance and excellent photocurrent response in the visible region.However,H₂O₂,as an etchant for plasma metal Ag,could weaken the effect of Ag NPs,leading to the decrease of photocurrent intensity.Glucose oxidase（GOx）-loaded ZIF-67@Ca O₂ was used to label PSA-Ab₂ to form the signal amplification probe.In the existence of O₂,GOx could catalyze glucose to produce H₂O₂ and gluconic acid.The ZIF-67 could be degraded by the generated gluconic acid,resulting in the unprotected Ca O₂ hydrolyzation to generate H₂O₂.The H₂O₂ generated by the enzymatic reaction and the hydrolysis reaction synergistically etched the Ag NPs in the ZnO-Ag-Bi₂S₃ nanocomposite,which weakened the LSPR effect and affected the photocurrent intensity.In addition,the formation of the electromagnetic field induced by LSPR is hindered,which is not conducive to electron transfer.Under the optimized conditions,the immunosensor exhibited good linearity in the concentration range of PSA from 1.0×10^-14-1.0×10^-8 g m L^-1,and the detection limit was as low as 5.0×10^-15 g m L^-1.The method was applied to the determination of PSA in human serum samples,and the results were consistent with those obtained by Xinyang Central Hospital.The recovery was ranged from 83.0%to 110%with the RSDs no more than 8.4%.2.An innovative PEC immunosensor was developed to sensitively detect c Tn I by integrating Ag₂S/ZnO nanocomposites as photoactive materials,chemical redox cycle as signal amplification strategy with the split-type assay format.In order to achieve the split analysis mode,the immunoreaction was carried out in 96-well plate.In the procedure of immunoreaction,the signal label of alkaline phosphatase（ALP）and anti-c Tn I polyclonal antibody（anti-c Tn I）functionalized gold nanoparticles（ALP-Au NPs-anti c Tn I）could hydrolyze ascorbic acid 2-phosphate（AAP）to generate the signal species ascorbic acid（AA）.After that,the generated AA was added into the detection solution,which could serve as electron donor to capture the holes from the excited state of Ag₂S/ZnO nanocomposities.The holes could oxidate AA and trigger chemical redox cycling reaction.In such a cycling system,the tris（2-carboxyethyl）phosphine（TCEP）served as reduction reagent and interacted with the oxidation product of AA to promote the regeneration of AA,and thus resulting in the PEC response enhanced.In addition,the split analysis mode could avoid such adverse factors such as time-consuming and laborious,large diffusion barrier and irreversible damage to biomolecules,and could eliminate the interference between immune recognition and PEC detection,which is conducive to improving detection sensitivity.The detection limit is 3.0×10^-15 g m L^-1and the linear range of the split type immunosensor is 1.0×10^-14-1.0×10^-9 g m L^-1.The detection results of c Tn I in human serum samples were consistent with the report provided by Xinyang Central Hospital.The recoveries range from 80%to 110%and RSDs was lower than 6.4%.Besides,given the diversity of the redox cycling reaction and photoactive materials that could be integrated into such a split-type assay mode,this work could be easily extended to develop the general ultrasensitive PEC bioanalysis.3.Based on luminol chemiluminescence system,digital multimeter and capacitor were integrated into the circuit,and a portable PEC immune sensing platform for highly sensitive detection of PSA was constructed.In the platform,the ZnO-Au-Cu₂O nanomaterial was used to construct the photo-anode and the Pt as cathode.In order to realize the excitation of photoactive materials,the luminol molecules were encapsulated inside of MSNs using mesoporous silica nanospheres（MSNs）as the carrier and GOx as the sealer.The controlled release of luminol achieved under appropriate condition and interacted with H₂O₂ produces chemiluminescence as the excitation source.The Ab₂-GOx-MSNs-luminol probe was synthesized and introduced to the photoelectrode surface through immune recognition reaction,which shortened the distance between the excitation light source and the light receptor.When glucose presented in the detection solution,GOx could oxidize glucose to produce the co-reaction agent（H₂O₂）of luminol chemiluminescence system.At the same time,GOx separated from the surface of MSNs,causing luminol to be released.In addition,horseradish peroxidase（HRP）was added into the solution to enhance the energy of the excitation light source.When the photonanomaterial was excited,electron-hole pairs were formed and photocurrent could be generated in the external circuit.Electrons flowing through the external circuit were temporarily stored in the capacitor and charged to the capacitor.When the charging was completed,the capacitor discharged and amplified the electrical signal.Finally,the amplified signal was displayed directly on a digital multimeter and transmitted to computer.Under optimized conditions,the linear detection range of PSA by the portable sensor was 1.0×10^-14-1.0×10^-7 g m L^-1,and the detection limit（LOD）was 4.5×10^-15g m L^-1.The sensor could be applied to the detection of actual samples,the recovery within the range of 88%-117%,RSDs were no more than 9.3%.This work provides a simple way for the highly sensitive analysis of biomarkers,and provides ideas for the miniaturization of PEC detection devices.