| At present,with the increasing concern in the medical and health fields such as disease diagnosis,precision medicine,and health monitoring,biosensing technology is developing flourishingly in the field of clinical and laboratory diagnosis.Biosensors have great potential because of their selectivity,sensitivity,and rapid response.However,in the application of biosensing technology,there is still a problem of dependence on expensive instruments and high cost,which cannot meet the needs of efficient and rapid detection of disease biomarkers in practical applications.Therefore,the development of efficient,sensitive,specific,portable,and rapid biosensors is a hot topic of current research.This thesis has established four biosensor systems,combining biosensing technology with nanomaterials,smart devices,and electrochemical technologies to explore the principle of sensor fabrication and improve the sensitivity,stability and portability of the sensor.The application in clinical testing is discussed,including the following parts:1.A novel nano-electrochemical immunosensor was designed for the heart failure marker,N-terminal pro brain natriuretic peptide(NT-proBNP),and it was used for the detection of NT-proBNP in the serum.The antibody(primary antibody)was immobilized on the surface of the electrode by sandwiching the composite nanomaterial AuNPs/PDDA/RGO on the glassy carbon electrode,and the antigen marker(NT-proBNP)and an enzyme-labeled antibody(secondary antibody)was bind to the electrode surface by the "sandwich method".Under the high efficiency of the enzyme-catalyzed reaction,the catalytic TMB is oxidized by H2O2,and the oxidation product generates a reduction current signal on the surface of the electrode.The experimental results show that the quantitative detection of antigenic markers can be achieved by using the nano-composite electrochemical immunosensor.The limit of detection(S/N=3)is 8 pg/ml.The method utilizes the nano composite material to enhance the coverage of the antibody at the sensing interface,and at the same time promote the adsorption of the interface antibody,and has stronger sensitivity and stability than the traditional single nano material immunosensor,which solves the problems of the low concentration of NT-proBNP in blood sample from patients.2.A smartphone assisted pressure-measuring-based diagnosis system(SPDS)based on smartphone-assisted and 8-channel air pressure sensing devices was developed and used for rapid diagnosis of myocardial infarction.With SPDS,each batch of samples can be efficiently and quickly tested(detection time is less than 20 minutes),combined with smartphone to complete each analysis,storage and output The experimental results showed that the fabricated SPDS achieved a lower limit of detection of 0.014 ng/mL cTnI,0.16 ng/mL CK-MB and 0.85 ng/mL Myo.The system was applied to the comparative evaluation of 50 clinical blood samples and their chemiluminescence detection results,showing good sensitivity and stability.In addition,SPDS has the advantages of portability,high accuracy and less time consumption,compared to traditional methods for acute myocardial infarction,such as ECG,imaging and chemiluminescence,making it have a good promotion value in the hospital of low-developing areas,community and bedside measurements.3.An electrochemical DNA sensor based on homogeneous hybridization and competitive assembly was developed and used for the detection of Cryptococcus neoformans.Through the efficient and rapid binding of the capture probe to the signal probe,and the target double-stranded DNA(dsDNA)in a homogeneous system,a sandwich structure is formed on the surface of the sensor,and the target sequence is subjected to current detection by enzyme-catalyzed catalysis,which achieves the diagnosis of Cryptococcus neoformans.The results showed that the method was applied to the detection of double-stranded DNA in clinical samples of Cryptococcus neoformans.The current value and the concentration of double-stranded DNA showed a good linear relationship in the range of 5 pmol/L-1 nmol/L.The limit of detection(S/N=3)is 800 fmol/L.During the construction of probe recognition interface,the mechanism of recognition of DNA probe and dsDNA was revealed by simple difference between homogeneous and heterogeneous systems.It was realized without complex sample pretreatment such as enzymatic digestion.The selective quantitative detection of ssDNA and dsDNA provides a research basis and new ideas for breaking the detection of disease dsDNA by DNA electrochemical sensors.4.A self-signal amplified DNA electrochemical sensor based on repetitive sequence was designed and constructed and used for rapid detection of Tubercle Bacillus(TB).By using the feature of multiple repeats in the TB gene sequence,a specific probe is designed,and a highly specific repeat sequence is used as a target,for identifying the target sequence with high-density recognition,thereby enhancing amplification of the signal of the sensor.The results showed that the quantitative detection of the artificially synthesized TB gene containing two repeats using this DNA electrochemical sensor showed a good linearity,the current value versus the DNA concentration,in the target sequence concentration range of 0.7 pmol/L to 10 pmol/L.The limit of detection(S/N=3)is 15 finol/L,which has high sensitivity.At the same time,the high specificity of the TB repeat sequence(1034 bp)is utilized in the design of the sensor,which ensures the high specificity of the target,and thus the preliminary detection of the DNA fragment of the plasmid fragment and the tuberculosis sputum specimen is realized.The method utilizes the unique repeat structure of microorganisms and fabricates a novel "sandwich" amplification system according to the simple structure design of natural structure,which realizes highly sensitive detection of a small number of samples,and reduces the pre-processing process of complex samples and time.It provides a research basis for the POCT application of DNA electrochemical sensors. |
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