Functional Nanomaterials-based “Signal-on” Photoelectrochemical Biosensor Construction And Applications

As the growing number of cancer cases in the world,biosensor plays a vital role in precision medicine,clinical diagnostis and so on.The basic biosensor is made up of two modules,including the exquisitely tailored sensitive element and transducer module,in which those two sensing modules must be shrewdly balanced.The sensitive element recognizes and binds the analytes,then sets the thresholds of signal detection,and the transducer module transmits and filters the signals,then reports a cellular response.Among various biosensor,the biosensor based on nucleic acid and enzyme as the sensitive element have become hot topics in the cross field of biochemical analysis,because of their high sensitivity,low energy consumption and easy miniaturization.With the help of sensitive elements to identify the target,the biosensor,combined with the functional nanomaterials and signal amplication strategy,provides a promising way to realize the trace or ultra trace target detection.This paper aims to construct the photoelectrochemical?PEC?biosensor and investigate the related application.The thesis is mainly summaried as the following section:1.Ligating Dopamine as Signal Trigger onto the Substrate via Metal-Catalyst-Free Click Chemistry for“Signal-on”Photoelectrochemical Sensing of Ultralow MicroRNA LevelsThe efficiency of photon-to-electron conversion is extremely restricted by the electron-hole recombinant.Here,a new photoelectrochemical?PEC?sensing platform has been established based on the signal amplification of click chemistry?CC?via hybridization chain reaction?HCR?for highly sensitive microRNA?miRNA?assay.In this proposal,a preferred electron donor dopamine?DA?was first assembled with designed ligation probe?probe-N₃?via amidation reaction to achieve DA-coordinated signal probe(P_DA-N₃).The P_DA-N₃ served as a flexible trigger to signal amplification through efficiently suppressing the electron-hole recombinant.Specifically,the P_DA-N₃ can be successfully ligated into the trapped hairpins?H1 and H2?via the superior ligation method of metal-catalyst-free CC,in which the electron donor DA was introduced into the assay system.Moreover,the enzyme-free HCR,employed as a versatile amplification way,ensures that lots of P_DA-N₃ can be attached to the substrate.This PEC sensing for miRNA-141 detection illustrated the outstanding linear response to a concentration variation from 0.1 fmol L^-1 to 0.5 nmol L^-1 and a detection limit down to 27 amol L^-1,without additional electron donors.The sensor is further employed to monitor miRNA-141 from prostate carcinoma cell?22Rv1?,showing good quantitative detection capability.This strategy exquisitely influences the analytical performance and offers a new PEC route to highly selective and sensitive detection of biological molecules.2.Fabrication of Pt/Cu₃?PO₄?₂ Ultrathin Nanosheet Heterostructure for Photoelectrochemical MicroRNA Sensing Using Novel G-Wire-Enhanced StrategyHerein,we focus on preparing a highly efficient photocatalytic material to construct a signal-on photoelectrochemical?PEC?sensing platform in view of the rigorous demand of accurate miRNA quantification.The well-dispersed Pt nanoclusters-coated copper phosphate ultrathin nanosheets?PtNCs/Cu₃?PO₄?₂NSs?were first successfully synthesized as a photoelectrode material.Because of the ultrathin two-dimensional lamellar structure of Cu₃?PO₄?₂NSs with a 1.3 nm thickness,as well as the homogeneous size and abundant PtNCs loaded on Cu₃?PO₄?₂NSs,the resultant PtNCs/Cu₃?PO₄?₂NSs were employed as a photoelectrode material for the first time and revealed outstanding photocatalytic activity in PEC sensing as a substrate.As a well-designed protocol,we realized accurate miRNA quantification via a novel signal amplification strategy based on G-wire superstructure exponentially ligating a signal probe,which possesses efficient and simple operation compared to the traditional amplification method.Moreover,the electron donor is generated in situ by lactate oxidase?LOx?labels catalyzing lactate for H₂O₂ production,boosting the efficient separation of electron–hole pairs for further signal amplification.Impressively,this PEC sensing platform is commendably utilized to determine miRNA-141 from prostate carcinoma cell line 22Rv1.This study,considering the excellent PtNCs/Cu₃?PO₄?₂NSs combined with G-wire superstructure for exponential signal amplification strategy,paves a new path in biosensing and clinical diagnosis.3.Label-Free Photoelectrochemical“Off-On”Platform Coupled with G-Wire-Enhanced Strategy for Highly Sensitive MicroRNA Sensing in Cancer CellsMicroRNA?miRNAs?quantification,especially at low abundance,is vital for disease diagnosis,prognosis,and therapy.Herein we develop a distinctive label-free“off-on”configuration for photoelectrochemical?PEC?sensing platform fabrication,coupled with DNA four-way junction?4J?architecture as well as G-wire superstructure for signal amplification.In addition,ultrathin copper phosphate nanosheets?CuPi NSs?coating Au nanoparticles?Au-CuPi NSs?serve as a highly efficient photocathode substrate.To improve the sensitivity,and avoid the false positive signals,the quencher,gold nanoparticles?GNPs?,is utilized to switch off the PEC signal because of the commendable surface plasmon resonance?SPR?absorption.Subsequently,ingenious DNA 4J architecture is applied to export proportional c-myc regions for target quantification.Assisted with the G-wire superstructure formation,the enhancer 5,10,15,20-tetra?4-sulfophenyl?-21H,23H-porphyrin?TSPP?is coupled on the substrate to switch on the PEC signal,thus realizing the miRNA assay with persuasive accuracy,high sensitivity,and low detection limit.In addition,we execute the miRNA detection in prostate carcinoma cell line 22Rv1,and acquire desirable quantitative capability.Remarkably,the prepared PEC sensing platform not only realizes the highly efficient miRNAs quantification,but also uncovers a marvelous horizon for sensing platform fabrication.4.Highly Sensitive Electrochemiluminescenc Assay of Acetylcholinesterase Activity Based on Dual Biomarkers Using Pd–Au Nanowires as Immobilization PlatformOne-dimensional Pd–Au nanowires?Pd–Au NWs?were prepared and applied to fabricate an electrochemiluminescence?ECL?biosensor for the detection of acetylcholinesterase?AChE?activity.Compared with single-component of Pd or Au,the bimetallic nanocomposite of Pd–Au NWs offers a larger surface area for the immobilization of enzyme,and displays superior electrocatalytic activity and efficient electron transport capacity.In the presence of AChE and choline oxidase?ChOx?,acetylcholine?ATCl?is hydrolyzed by AChE to generate thiocholine,then thiocholine is catalyzed by ChOx to produce H₂O₂ in situ,which serves as the coreactant to effectively enhance the ECL intensity in luminol-ECL system.The detection principle is based on the inhibited AChE and reactivated AChE as dual biomarkers,in which AChE was inhibited by organophosphorus?OP?agents,and then reactivated by obidoxime.Such dual biomarkers method can achieve credible evaluation for AChE activity via providing AChE activity before and after reactivation.The liner range for AChE activity detection was from 0.025 U·L^-1 to 25 kU·L^-1 with a low detection limit down to 0.0083 U L¹.5.Cyclovoltammetric Acetylcholinesterase Activity Assay after Inhibition and Subsequent Reactivation by Using a Glassy Carbon Electrode Modified with Palladium Nanorods Composited with Functionalized C₆₀ FullereneA glassy carbon electrode?GCE?was modified with a nanocomposite consisting of tetraoctylammonium bromide?TOAB?,C₆₀ fullerene,and palladium nanorods?PdNRs?.The PdNRs were hydrothermally prepared and had a typical width of 20±2nm.The nanocomposite forms stable films on the GCE and exhibits a reversible redox pair for the C₆₀/C₆₀-system while rendering the surface to be positively charged.The modified GCE was applied to fabricate an electrochemical biosensor for detecting acetylcholinesterase?AChE?by measurement of the amount of thiocholine formed from acetylthiocholine,best at a working voltage of-0.19 V?vs.SCE?.The detection scheme is based on?a?measurement of the activity of ethyl paraoxon-inhibited AChE,and?b?measurement of AChE activity after reactivation with pralidoxime?2-PAM?.Compared to the conventional methods using acetylthiocholine as a substrate,the dual method presented here provides data on the AChE activity after inhibition and subsequent reactivation,thereby yielding credible data on reactivated enzyme activity.The linear analytical range for AChE activity extends from 2.5 U L^-1 to 250 kU·L^-1,and the detection limit is 0.83 U L^-1.