Study On Signal-on Electrochemical Biosensor Based On Multiple Amplification Techniques

Since the beginning of the new century,social development has been extremely rapid,and the people's material living standards have rapidly been improved.Health issues have been valued increasingly by the people.However,the increasing incidence of cancer and other diseases threatens people's health.The early diagnosis and treatment of diseases can greatly improve the survival rate of patients,so it is urgent to develop disease detection techniques.On the other hand,the concentration of disease-associated markers such as nucleic acids,enzymes in the body are extremely low,so that the relevant markers can not be detected and quantified by ordinary methods.Electrochemical biosensors are a combination of biomolecular recognition technology and electrochemical response that convert physical and chemical signals into electrical signals and record them.Due to simple structure,good stability,low sensitivity,low cost,and ease portability detection,electrochemical biosensors are very popular in the field of disease detection.In this paper,nucleic acid amplification,functional nano-materials and biological enzymes were applied to construct electrochemical biosensors to achieve high-specificity,ultra-sensitive detection of the target.The details are as follows:1.A novel electrochemical biosensor for detection of DNA species related to oral cancer based on a particular host-guest recognition-assisted strategy for signal tag in situIn order to achieve the increase of sensor sensitivity.Herein,a novel host-guest recognition among tryptophan?Trp?-linked DNA,methyl viologen(MV²⁺)and cucurbit[8]uril?CB[8]?was introduced to construct a biosensor for the sensitive assay of oral cancer gene based on nicking enzyme signaling amplification?NESA?and Pt&Pd-MoS₂ nanomaterial for the first time.A bioconjugates containing alkaline phosphatase?ALP?and complementary strand?S1?was designed as signal probe,The S3 fragment labeled with Trp was obtained by transformation and amplification of the target under the action of NESA.The S3 fragment could be immobilized on the surface of the electrode modified by CB[8]and MV²⁺complexes in advance through host-guest recognition.The complementary on the signal probe hybridized with S3 to achieve signal probe immobilization and catalyzed the 1-naphthyl phosphate monosodium salt monohydrate?NPP?to in situ generate massive electroactive substance 1-naphthol,resulting in the generation of significant peak current with Pt&Pd-MoS₂ nanomaterial as a catalyst.With target recycling amplification and highly efficient catalysis of ALP,the proposed biosensor for target DNA exhibited a satisfactory linear range from 10fmol·L^-1 to 10 nmol·L^-1.Importantly,the admirable performance of the proposed method shows a potential in biomedical research and clinical diagnosis.2.A sensitive electrochemical biosensor for PDGF-BB baesd on rolling circle amplification and streptavidin-biotin binding systemIn this work,we successfully builted an aptasensor for the detection of platelet-derived growth factor-BB?PDGF-BB?based on rolling circle amplification?RCA?and streptavidin-biotin binding system.In the detection process,the PDGF-BB was firstly recognized by the specially designed double hairpin aptamer strand?dHP?releasing the closed part.Then the primer strand which was labeled with biotin could hybridize with the releasing part of dHP.Under the action of T4 DNA ligase and Phi29dna polymerase,the RCA was started.Meanwhile,in the process of RCA,the target was squeezed out to achieve the cycling of PDGF-BB.The signal of PDGF-BB was amplified and converted to the long-chain nucleic acid labeled with biotin.The exporting long-chain nucleic acid were immobilized on electrodes which was pre-modified with streptavidin.In addition,the by adding primer complementary strand which was part complementary with long-chain nucleic.The immobilized single-stranded DNA could be polymerized into double-stranded,and further increased the amount of adsorbed methylene blue?MB?,resulting in the electrochemical signal amplification.And the detection range was from 1 pmol·L^-1 to 10 nmol·L^-1,the limit of detection was 0.39 pmol·L^-1.Furthermore,the sensor exhibited good selectivity and applicability in complex matrixes,indicating the potential in practical application.3.One step signal-on electrochemical biosensor for sensitive target detection:signal amplification by target recycling and entropy-driven DNA walkerTimely and accurate detection of DNA has great signification in genetic diseases diagnosis.In this work,we designed a sensitive electrochemical biosensor for the one-step signal-on detection of target DNA.We constructed the whole biosensor on the glass carbon electrode?GCE?and took the HIV as a model to investigate its performance.In the presence of target HIV DNA,Fixing Hairpin 1?FH₁?was opened by HIV DNA.And one leg of the swing arm was immobilized by recognizing the exposed toehold region in FH₁.With the participation of Hairpin 1?H₁?,the target was released via a strand displacement reaction,achieving the target recycling.At this point,the concentration of the swing arm near the surface of the biosensor significantly increased,and the other leg of the swing arm opened Fixing Hairpin 2?FH₂?to expose a toehold region for the ferrocene-modified hairpin(H_fc).Then,H_fc replaced the leg through a strand displacement reaction to achieved migration of walker.After many cycles,a large amount of H_fc were immobilized on the surface of electrode,resulting in a strong electrochemical signal.The employed biosensor achieved signal amplification by target recycling and entropy-driven DNA walker,It's detection range was 0.1pmol?L^-1-10 nmol?L^-1 and presented a satisfactory sensitivity(32 fmol·L^-1)and a high selectivity.