Aptamer Functionalized Polydimethylsiloxane(PDMS) Film-SERS Detection Methods Of Two Foodborne Pathogenic Bacteria

Foodborne disease is an important factor threatening global public health,which has caused serious impacts and hazards on human health.Foodborne pathogens are the main causes of foodborne diseases.Traditional detection methods of food borne pathogens are usually time-consuming in operation steps and processes,and have some defects in sensitivity and specificity.Therefore,it is of great significance to develop a more rapid,convenient,sensitive and specific detection methods for ensuring food safety.Surface-enhanced Raman spectroscopy（SERS）is a non-destructive and rapid detection technology with high sensitivity.Based on the"hot spot"effect,preparing a substrate with a good SERS effect is a key factor in the development of SERS technology.It is very important to select a stable and economical solid support material of SERS substrate for rapid detection of foodborne pathogens.In this study,gold nanoparticles were used as SERS substrates,and aptamers were used as recognition molecules.A method based on aptamer functionalized Polydimethylsiloxane（PDMS）-SERS for the detection of foodborne pathogens was developed and applied to the detection of real samples.The main work includes:Firstly,a method based on aptamer functionalized PDMS film for sandwich-type was developed for the detection of V.parahaemolyticus.At first,the surface of PDMS film was chemically modified by piranha and APTES,and Au-PDMS film was prepared by connecting gold nanoparticles.Then,the gold-sulfur bond was used to immobilate aptamers on the Au-PDMS film to form the capture substrate.At the same time,we had prepared Raman signal molecule 4-MBA connecting gold nanoparticles modified by aptamers of V.parahaemolyticus as signal molecular probes.When the target existed,the sandwich structure of"capture substrate-target-signal molecular probe"was formed based on the specific binding of the aptamers and targets,and the detection of V.parahaemolyticus was achieved by measuring the Raman intensity.The results showed the signal of 4-MBA at 1592 cm^-1 was linearly related to the V.parahaemolyticus concentration in the range between 1.2×10²cfu/m L¹.2×10⁶ cfu/m L（y=544.68x-944.13,R²=0.9948）,with a detection limit of 12cfu/m L.The method had been successfully applied in spiked prawn sample testing.Secondly,a method based on aptamer non immobilized Au-PDMS film for detection of V.parahaemolyticus was developed.In order to further improve the convenience of detection,the Au-PDMS film was firstly prepared through the reduction of chloro auric acid by PDMS precursor.Then cysteamine was connected to the Au-PDMS film by gold-sulfur bond to make the surface positively charged.Then,the signal molecule probe（4-MBA was modified by gold-sulfur bond and gold nanoparticles modified by aptamers）was adsorbed on the surface of the film by electrostatic interaction.When the target existed,based on the specificity of the aptamer and target,the signal molecular probe combined with the target specificity and fell off the surface of the film.The detection of V.parahaemolyticus was achieved by measuring the change of Raman intensity.The results showed the signal of 4-MBA at 1592 cm^-1 was linearly related to the V.parahaemolyticus concentration in the range between 3.3×10²cfu/m L³.3×10⁶ cfu/m L（y=650.78x-1063.2,R²=0.9913）with a detection limit of 33 cfu/m L.The method had been successfully applied in spiked prawn sample testing.Finally,a method based on aptamer functionalized PDMS film was developed for the simultaneous detection of two pathogenic bacteria.The Au-PDMS film modified by two kinds of aptamers were used as the solid-state capturing substrate,and two types of signal molecular probes modified by aptamers,4-MBA and NBA were prepared,and a dual sandwich structure based on captured solid substrates and dual targets was established for SERS detection of Vibrio parahaemolyticus and Salmonella typhimurium.The results showed the signal of4-MBA at 1592 cm^-1 was linearly related to the V.parahaemolyticus concentration in the range between 2.5×10² cfu/mL².5×10⁶ cfu/m L（y=567.24x-485.86,R²=0.9883）.The detection limit is 25 cfu/mL.In the same way,The results showed the signal of NBA at 592cm^-1 was linearly related to the S.typhimurium concentration in the range between 4.5×10²cfu/m L⁴.5×10⁶ cfu/m L（y=324.57x-514.03,R²=0.9892）.The detection limit is 45 cfu/m L.The method had been successfully applied in spiked prawn sample testing.