Construction And Performance Of Ultrasensitive Bacterial SERS Detection Based On Aptamer-recognition And Signal Amplification Strategy

Bacterial infection and side effects caused by antibiotics have posed a serious threat to human health and public health.It is of great significance to develop rapid and sensitive clinical diagnosis of bacteria for the timely prevention and accurate treatment of bacteria.In order to meet the urgent needs of bacteria detection,this thesis focus on the design and development of surface-enhanced Raman scattering(SERS)biosensors which intergarated high affinity reconition of nucleic acid aptamers and signal amplification of catalytic hairpin assembly(CHA).The main research contents and innovations are as follows:(1)SERS biosensor for S.aureus detectionTo meet the needs of facile and accurate detection of bacteria,a potable and high-sensitive SERS biosensor based on aptamer recognition and catalytic hairpin assembly(CHA)signal amplification was proposed for point-of-care testing of S.aureus detection.The portable SERS biosensor contains three parts:recognition probes,SERS capture substrate,and SERS tags.The recognition probe is an Apt-P ds DNA assembled by an aptamer(Apt)of S.aureus and a primer ss DNA(P);the SERS capture substrate is prepared by modifying hairpin ss DNAs H1 on a silver nanorod(Ag NRs)SERS substrate;the SERS tag is obtained by modifying hairpin ss DNAs H2 and Raman molecules DTNB on the surface of gold nanoparticles(Au NPs).When the target S.aureus is present,the Apt in the Apt-P ds DNAs specifically bind S.aureus and releases P to hybridize with H1 on the capture substrate.Since the formation of H1-P ds DNAs,the hairpin of H1 which opened can further hybridize with H2 which modified on the SERS tags to form the H2-H1 ds DNAs and displaces P from the H1-P ds DNAs.The released ss DNAs turn on the next P-triggered CHA and realize the signal amplification.The feasibility of the strategy was verified by gel electrophoresis,and the one-step test rout and incubation time of 135 min were optimized.The bacterial SERS biosensor shows high sensitivity,specificity,uniformity,repeatability on S.aureus detection,which has a good linear calibration curve ranging from 10 to 10~7 CFU/m L with a detection limit of 4.46CFU/m L.The SERS biosensor shows a good recovery rate(98.15-104.67%)for testing S.aureus spiked in milk,which indicates that the SERS biosensor is expected for the reliable detection of trace bacteria in real samples.(2)Hydrogel-based flexible SERS biosensor for S.aureus detectionTo meet the needs of sensitive and accurate in situ detection and analysis of bacteria on skin and other surfaces,the flexible SERS biosensor which base on hydrogel and Ag nanoparticles(Ag NPs)is proposed for S.aureus detection.The Ag NPs are embedded in the hydrogel to prepare the SERS-active Ag-AG substrate,and the Apt-P recognition probe and hairpin DNA H1 are co-modified on the substrate to prepare the flexible SERS capture substrate.The SERS tags co-modified Au NPs with hairpin ss DNAs H2 and and Raman molecules DTNB are also prepared and loaded into the hydrogel.When S.aureus incubates with the SERS capture substrate,Apt in the Apt-P modified on Ag NPs combined with the target bacteria and detached from the surface of Ag NPs.Then,the P on the Ag NPs hybridize with H1 and open the H1 hairpin structure to trigger the CHA with the H2 on SERS tags to achieve CHA signal maplification and capture the SERS tags.Finally,the S.aureus is detected qualitatively and quantitatively by measuring the SERS signal of DTNB.The optimal sensing strategy,including the preparation of SERS-active Ag-AG substrate,sensing routs,concentration ratio of Apt-P and H1 and the incubation time were studied and obtained.The SERS sensor shows good performance on S.aureus detection,i.e.,possessing a linear calibration curve ranging from 10 to 10~7 CFU/m L,a detection limit as low as 3.98 CFU/m L,high specificity,and good recovery rate within 91.51%to 105.84%.The flexible biosensor detection results of wound exudate detection model based on pig skin show that the flexible SERS biosensor realized the detection of S.aureus in wound exudate by attaching the flexible biosensor to a wound on the surface of the pig skin,which provides the possibility and technical support for the detection of actual wound exudate.