Construction And Applications Of Ultrasensitive Biosensor System For Protein Detection

In recent years,with the development of science and technology,protein detection is becoming more and more important in medical diagnosis,drug development,ecological security and so on.The traditional immunoassay method has low sensitivity and poor specificity.In this case,protein biosensors have attracted more and more attention due to their high sensitivity,low cost and high stability.Based on these,we developed two biosensor system for protein detection.Firstly,we report the use of the distinctive metal-to-ligand charge-transfer(MLCT)absorption properties for the visual detection of alkaline phosphatase(ALP)activity.In the presence of ALP,the substrate ascorbic acid 2-phosphate(AAP)can be enzymatically hydrolyzed to release ascorbic acid(AA),which in turn reduces Cu2+ to Cu+.Subsequently,the complexation of Cu+ with the BCA ligand generates the chromogenic Cu(BCA)+ reporter,accompanied by the color change of colorless-to-purple of the solution with a sharp absorption band at 562 nm.Needless of sequential multistep operations and elaborately designed colorimetric probe,the proposed MLCT-based method allows a fast and sensitive visual detection of ALP activity within a broad linear range of 0-200 mU mL-1,with a detection limit of 1.25 mU mL-1.Results also indicate that it is highly selective and has great potential for the screening of ALP inhibitors in drug discovery.More importantly,it shows a good analytical performance for the direct detection of the endogenous ALP levels of undiluted human serum samples.Secondly,we report a terminal deoxynucleotidyl transferase(TdT)initiated on-chip polymerization biosensor for ultrasensitive protein detection.In this approach,two different thrombin-binding aptamers were chosen to capture the thrombin,and form a sandwich structure for improved specificity.One amino-modified aptamer was immobilized on pretreated silicon chip and the 3’-terminal of another aptamer,which was exposed on the chip surface,can be incorporated with fluorescein labeled dATPs under the catalysis of TdT and result in signal amplification.Under optimal conditions,the sandwich biosensor presented a great linear relationship between the fluorescence intensity and the logarithm of thrombin concentration in the range from 100 fM to 0.1 μM,with a low detection limit of 8.35 fM.In addition,the biosensor rendered satisfactory analytical performance for the thrombin in the presence of human serum.These results demonstrate that both two biosensors show excellent sensitivity and selectivity,and hold great potential for diagnosis and prognosis in disease diagnosis,clinical prognosis and environmental monitoring.