Photoelectrochemical Biosensors Constructed Based On Novel Signal Amplification Strategy For Detection Of MiRNA-21

miRNA can be used as an effective biomarker for early diagnosis and treatment of diseases,and the development of sensitive and rapid miRNA detection technologies is of great importance in biology and medicine.Photoelectrochemical（PEC）biosensor is an emerging sensing technology developed by combining the PEC process with a biomolecular recognition process.This method uses the photocurrent changes generated by the target directly or indirectly changing the photoactive material or electrolyte environment to achieve quantitative detection of the target.It is considered as an important analytical tool for miRNAs due to its advantages of low background interference,high sensitivity,simple and rapid instrumentation as well as good selectivity.Under the increasing requirements of detection sensitivity,it is particularly important to construct efficient PEC biosensors.Therefore,the development of high-performance photoactive materials,the design of simple yet efficient signal amplification methods and the exploration of novel signal response sensing systems are current research hotspots in the field of PEC bioanalysis.Based on the above exploration,this study constructes three signal amplification sensing platforms for the determination of tumor biomarkers miRNA-21.The major contents are concluded as follows:1.This work presented a photoelectrochemical sensing platform based on the biocatalytically induced formation of Cu S/Cu O-Au NPs heterojunctions with the aid of 3D DNA walker signal amplification technology.Compared with conventional amplification methods,3D DNA Walker has higher walk efficiency and signal amplification capability.With target introduction,the WS strand hybridized with TS strand to form a Mn²⁺-specific DNAzyme,resulting in TS strand digestion and HRP release.In the presence of H₂O₂,HRP catalyzed Na₂S₂O₃ to produce H₂S.The generated H₂S reacted with Cu O-Au NPs complexes modified on indium tin oxide（ITO）electrodes,leading to the in situ formation of Cu S/Cu O-Au NPs heterojunctions and a substantial increase in photocurrent.Under optimal conditions,the sensor exhibited good analytical performance in the concentration range from 0.004 p M to400 p M with a low detection limit of 2.1 f M.2.This work developed a highly sensitive novel PEC sensing platform for the detection of miRNA-21 by combining the BSA promoted the pohotocurrent of（2Z,2′Z）-2,2′-（1,4-phenylene）bis（3-（4-（bis（4-methoxyphenyl）amino）phenyl）acrylonitr ile）（TPDCN）and the Td T enzyme-mediated chain extension reaction as a signal amplification strategy.H1 was opened by the miRNA-21,and successively reacted with H2 and H3 to form Y-type DNA nanostructures.Meanwhile,target molecule was released and triggered CHA cycle,thus multiple Y-type DNA nanostructures was yield.Then,multiple biotin-linked d ATPs was incorporated into the Y-type DNA double-arm extension strand after a template-free DNA extension reaction catalyzed by Td Tase.Afterwards,a large amount of Ag@BSA-SA were assembled on the Y-type DNA dendrimer by strong binding interaction between biotin and avidin.After reacted with HNO₃,Ag NPs in Ag@BSA-SA was etched and then multiple BSA was released from the magnetic probes.When BSA was transfered to the TPDCN-modified electrode,the photocurrent response is enhanced.The biosensor has a wide linear range from 0.01 to 10,000 p M with a detection limit as low as 4.7 f M.3.Based on the Fermi energy level matching principle,a novel self-powered PEC biosensor was successfully established with Bi₂S₃ as the photoanode material and hole transporting material（N,N′-bis（1-naphthyl）-N,N′-diphenyl-1,1′-biphenyl-4,4′-diamine）（NPB）as the photocathode material,using for the sensitive detection of miRNA-21.In the process of detection,miRNA-21 was hybridized with the hairpin probe H1,exposing the G-base-rich fragment of H1.Upon addition of hemin,this fragment was coupled with hemin to form the hemin/G-tetraspanin complex.The power of the sensing platform is increased due to the redox reaction occurring on hemin.The biological recognition process of the target miRNA-21 occured on the photocathode,which can avoid the false positives caused by the hole redox small molecules generated on the surface of the photoanode.Thus,it can enhance the accuracy of the sensing detection.The constructed dual-photoelectrode self-powered PEC biosensor showed good linearity between the maximum output power density(P_max)and the logarithm of the miRNA-21 concentration in the range of 0.10 to10,000 p M,with the detection limit as low as 69 f M.The method achieved sensitive quantitative detection of miRNA-21 in human serum samples.