Study Of Novel Biosensors Of Carcinoembryonic Antigen And Cadmium Ion Aptamer Based On Nanomaterials

Electrochemical biosensors can realize the effective detection of biomolecules through specific recognition between biomolecules and conversion of other signals into electrical signals.Therefore,it is significant to develop an instant,sensitive,and efficient electrochemical biosensor for detecting samples.Many nanomaterials show high electron transfer performance and good biocompatibility,which have been applied to biosensing.In addition,aptamers have the characteristics of simple synthesis,non-immunogenicity,and effective identification of ionic targets.They have also been widely used in the detection and analysis by means of sensors.In this dissertation,we intended to use nanomaterials to fabricate a protein biosensor and a nucleic acid biosensor to meet the needs of different types of biological detections.The main contents of this thesis are as follows:1.An ultrasensitive electrochemical immunosensor of carcinoem-bryonic antigen based on gold-label silver-stain signal amplificationA novel gold-label silver-stain electrochemical immunosensor was developed based on a polythionine-gold nanoparticle?PTh-Au?modified glassy carbon electrode as a platform and secondary antibody labeled Au NPs?Ab₂-Au?as an immumoprobe for carcinoembryonic antigen?CEA?detection.By means of the sandwich-type biosensor,anodic stripping voltammetry was adopted to detect the silver stripping signal when the Ab₂-Au nanoparticles of the formed immunocomplexes were stained with silver.This immunosensor showed a wide linear calibration of 0.1-120 ng m L^-1?R²=0.99856?and a low detection limit of 0.055 ng m L^-1.Moreover,this immunoassay also presented the advantages of good reproducibility,stability and selectivity.2.Fabrication and application of cadmium ion aptsensor based on functionalized black phosphorus nanosheetsFirstly,a novel strategy for the rapid and efficient preparation of black phosphorus nanosheets was developed.The Tween 20-assisted liquid was used to exfoliate the bulk black phosphorus and then the supernatant was collected after centrifugation.The characterizations showed that the Tween20-assisted liquid displayed the best exfoliating effect compared with the reported literature.The UV-Vis further indicated that black phosphorus nanosheets showed good dispersibility in Tween 20-assisted liquid.TEM,SAED,and Raman characterizations proved its good structural characteristics.In addition,the A_g¹/A_g²=0.44>0.2 showed the dispersion liquid was a fresh black phosphorus nanosheet solution,further demonstrating that the dispersion liquid possessed good stability.Subsequently,a random DNA library was fixed with streptavidin magnetic beads and then incubated with cadmium ion target.Later,elution,magnetic separation,emulsion PCR amplification,and preparation of the single-stranded library were performed to screen the aptamers specifically binding cadmium ion.After successive selection of 9 rounds,the product of the final round was subjected to high-throughput sequencing,secondary structure analysis,and affinity determination.The results showed that after high-throughput sequencing,6 aptamers with the highest enrichment were selected for analysis and determination.The lowest Kd value of the aptamer Cd-3 was 81.39±8.60?M.Based on the above research,a novel electrochemical biosensor based on functionalized black phosphorus nanosheets was fabricated for the detection of cadmium ions.In the fabricated aptsensor,poly-lysine-functionalized black phosphorus nanosheets?PLL-BP?and gold nanoparticles?Au NPs?as the sensing interface were applied to effectively amplify the detection signal.Using sulfhydryl primers as fixed primers and cadmium ion aptamer as recognition probes,the aptamers were effectively immobilized on the electrode surface by hybridization.Later,the aptamers were released from the electrode surface through competitive binding of cadmium ions,which lead to the sensing interface displaying the change.Under the best conditions,the differential pulse voltammetry?DPV?was used for detection.The linear response range of this aptsensor was 0.01n M-1?M.The linear equation was:?I??A?=2.227lg C+7.696,linear correlation coefficient R²=0.991,and the detection limit is 0.0037 n M.The biosensor was used to check water samples,and it was showed that the sensor displayed a good stability,sensitivity and specificity.