Surface Enhanced Raman Spectroscopy Technology In The Application Of Protease Detection

Surface enhanced Raman spectroscopy?SERS?has become a highly effective analysis method,which has several advantages,such as narrow frequency band,weak aqueous background,perfect stability and high selectivity.SERS is a fingerprint identification technique based on Raman scattering.It has high sensitivity,can be in situ,real-time monitoring.At the same time SERS can be used in aqueous system,particularly suitable for the study of the biological sample.In recent years,SERS has been widely applied in the interface and the analysis of surface science,materials,biology,medicine,food safety,environmental monitoring,and other fields,Including drugs identification,food safety inspection,chemical interaction research,and frescoes,diamond jewelry appraisal,air and water pollution detection and so on.With the continuous development of optoelectronics,nanotechnology and the analysis of the biology,the study on bio-sensing was more and more attention based on the SERS.In this paper,we will propose two SERS-based bio-sensing methods with high sensitivity and selectivity for detection of protease.The main research contents as follows:1.We report an aptamer-based SERS sensing method for tracing thrombin via constructing a bio-recognition,which is forming Ag nanoprism array/thrombin /Ag particles sandwich configuration with abundant “hot spots”.The specific scheme as follows: firstly,we prepared Ag nanoprism array on a smooth glass slide with vacuum deposition of metal on a close-packed polystyrene nanosphere pre-patterned substrates.Secondly,the top surface of the Ag nanoprism array was immobilized with athiolated aptamer?5'-SH-C₆-GGTTGGTGTGGTTGG-3'?,which can specially recognize thrombin.Then the SERS substrate connected with the aptamer was treated with SH-?CH₂?₂-OH in the third step for the purpose of blocking the unoccupied Agnanoprism surface.Fourthly,thrombin was added and captured by the aptamer due to specific recognition.At last,4-MBA marked silver nanoparticles?4-MBA@Ag NPs?built up a connection with the substrate via the carboxyl group of 4-MBA that can react with the amino of thrombin,to form a sandwich structure.Thrombin analysis was performed by monitoring the intensity variation of a SERS signal of 4-MBA with the change of thrombin concentration.2.We develop a simple and effective SERS strategy based on the enzymatic hydrolysis reaction and the electrostatic assembling of Ag NPs for the detection of protease.To determination of trypsin,a hexa-peptide?CHRDDG?with two opposite electronic charged sequences is designed at two ends.Specially,a short peptide working as a protease-catalyzed substrate was first immobilized onto the surface of an Ag film via an Ag–S bond,which forms a double charged monolayer.Next,we assembled a Raman probe?4-mercaptobenzoic acid,4-MBA?on the peptide-assembled Ag film surface to work as a SERS signal reporter.To respond to protease,the peptide would be cleaved by protease exactly at the middle location of arginine by the trypsin-catalyzed hydrolysis reaction and the peptide fragment containing the negatively charged residue would depart from the chip.Accordingly,the chip surface would be positively charged.Then,a signal amplification step using Ag NPs was carried out in which the positive charged chip can capture the citrate-cladded Ag NPs?negative-charged?through the electrostatic adsorption.As a result,by forming a particle-on-a-film configuration,the 4-MBA signal would be greatly improved and we can quantitatively determine trypsin by the SERS signal gain.