Research On Photoelectrochemical Biosensors Based On Noval Photoactive Materials And Enzyme-assisted Signal Amplification For MicroRNA-141 Detection

Photoelectrochemical（PEC）biosensor as a potential assay method was developed based on the photoelectric conversion effect of photoactive materials.The performance of the PEC biosensor was significantly influenced by the photoelectric characteristics of the photoactive material.Meanwhile,the appropriate signal amplification strategy laid a foundation for further improving the PEC biosensor detection performance.For constructing highly effective PEC bioassay,it is highly essential and significant to exploit functional photoactive materials,develop effective signal amplification strategies,and design novel PEC detection method.Nucleic acid probe technology as a powerful tool for qualitative and quantitative analysis of specific RNA and DNA fragments,was widely used in biological analysis.In this paper,a series of PEC biosensors were constructed based on the innovative photoactive materials and the enzyme-assisted nucleic acid amplification strategy designed to match with them to realize sensitive detection of micro RNA-141 and improve the analytical performance of sensors.1.[Ru（dcbpy）₂dppz]²⁺/Fullerene Cosensitized PTB7-Th for Ultrasensitive Photoelectrochemical microRNA AssayIn this work,a new cosensitization photoelectrochemical（PEC）strategy was established by using a donor-acceptor-type photoactive material,poly{4,8-bis[5-（2-ethylhexyl）thiophen-2-yl]benzo[1,2-b:4,5-b’]dithiophene-2,6-diyl-alt-3-fluoro-2-[（2-ethylhexyl）carbonyl]thieno[3,4-b]thiophene-4,6-diyl}（PTB7-Th）as signal indicator,which was cosensitized with bis（4,4’dicarboxyl-2,2’-bipyridyl）（4,5,9,14-tetraazabenzo[b]triphenylene）ruthenium（II）([Ru（dcbpy）₂dppz]²⁺)embedded in the grooves of the DNA duplex and fullerene(nano-C₆₀)immobilized on the surface of DNA nanoflowers for micro RNA assay.[Ru（dcbpy）₂dppz]²⁺and nano-C₆₀ could effectively enhance the photoelectric conversion efficiency of PTB7-Th as a result of well-matched energy levels among nano-C₆₀,[Ru（dcbpy）₂dppz]²⁺and PTB7-Th,leading to a clearly enhanced photocurrent signal.Meanwhile,a target recycling magnification technique based on duplex-specific nuclease（DSN）was applied in this work to obtain higher detection sensitivity.The proposed biosensor demonstrated excellent analytical properties within a linear detection range of 2.5 fmol/L to 2.5 nmol/L and a limit of detection down to 0.83 fmol/L.Impressively,this cosensitization PEC strategy offers an effective and convenient avenue to effectively increase the photoelectric conversion efficiency of a photoactive material,resulting in a remarkably improved photocurrent signal for ultrasensitive and highly accurate detection of various targets.2.A Novel“Signal On”Photoelectrochemical Strategy Based on Dual Functional Hemin for microRNA AssayIn the previous work,we used the cosensitized structure significantly improves the photocurrent signal of photoelectrochemical（PEC）biosensor.Usually,the substrate material brings unneglected background signal,which is not conducive to improving the accuracy and sensitivity of the PEC biosensor.Commonly,signal quencher utilized in PEC biosensing system played a single role of unidirectional signal quenching effect,which would lead to narrow detection range and limited sensitivity for PEC assay.In this work,a novel“signal on”PEC biosensor was constructed by dual functional hemin as signal quencher and electronic mediator for ultrasensitive target micro RNA-141 assay with the assistance of T7 Exonuclease（Exo）-initiated target amplification technology.Firstly,meso-porous TiO₂-hemin（MTiO₂-hemin）was employed as a substrate,in which hemin used as the signal quencher and MTiO₂ used as the photoactive material,to obtain an extremely weak initial signal with low background.Then,with the assistance of T7Exo-initiated target recycle amplification technology,a little of target could be transduced to numerous H1 probe,which could further hybridize with H2 labeled CdS quantum dots（QDs）to form the Z-scheme structure of MTiO₂-hemin-CdS QDs.In view of the facile conversion between Fe³⁺and Fe²⁺in Z-scheme structure,the hemin could be also used as a redox shuttle mediator for significantly enhancing the PEC signal.The presented biosensor exhibited good sensitivity for the quantitative detection of micro RNA in range from 0.25 fmol/L to 2.5 nmol/L with a limit of detection of 139 amol/L.Impressively,the designed strategy paved a new prospect for effective PEC bioassay and early clinical diagnostics of cancers.3.A Novel Photoelectrochemical Biosensor Based on Single-Enzyme Assisted Dual Recycle Amplification Strategy and Bi₂WO₆ as Photoactive MaterialIn the previous work,we construct photoelectrochemical（PEC）biosensor with the aid of enzyme-assisted recycle amplificatiob strategy.Generally,the reported enzyme-assisted recycle amplification strategies can only achieve the single recycle of target,while the remaining substrates are not recycled,which results in the waste of raw materials.Herein,a novel single-enzyme assisted dual recycle amplification strategy based on T7 exonuclease（Exo）and strand displacement reaction（SDR）was designed to fabricate PEC biosensor for the ultrasensitive detection of micro RNA-141.Compared with traditional enzyme assisted dual recycle amplification strategy,the presented method could effectively refrain enzyme interference reaction,reduce the environment sensitivity and save cost.For this system,hairpin DNA1（H1）decorated on magnetic beads（MB）as the substrate hybridized with target micro RNA-141 to form the H1/micro RNA-141heteroduplex.With the introduction of H2 labeled SiO₂（H2-SiO₂）,SDR was triggered between H2-SiO₂ and H1,thus micro RNA-141 was displaced from the H1/micro RNA-141 heteroduplex and the H1/H2-SiO₂ duplex was formed,realizing the reuse of target.In the presence of T7 Exo,the H1/H2-SiO₂ duplex was digested with the release of output DNA labeled SiO₂（output DNA-SiO₂）as mimic target.To enhance the target conversion rate,H1-MB was intactly released and hybridized with H2-SiO₂ again,which was inclined to initiate more T7 Exo digestion and freed abundant output DNA-SiO₂.Through such a single-enzyme assisted dual recycle amplification process,a tiny micro RNA-141could induced substantial output DNA-SiO₂,effectively improving the target amplification efficiency and detection sensitivity of PEC biosensor.The hairpin structure of H3 immobilized on electrode was opened in the presence of output DNA-SiO₂,making SiO₂ close to the electrode surface,which significantly decrease the PEC signal.Based on the proposed strategy and Bi₂WO₆ as photoactive material,the constructed PEC biosensor exhibited a wide detection linear range from 0.25 fM to 2.5 nM with a low detection limit of 83 aM.Moreover,this work firstly features a single-enzyme assisted dual recycle amplification process for constructing ultrasensitive PEC biosensor,which opens a new route for the detection of biomarkers.