Detection Of Staphylococcus Aureus Based On Aptamer/Fluorescent Silica Nanoparticles

Staphylococcus aureus (S. aureus), a spherical gram-positive bacterium, is the most common pathogen in human purulent infection. Recently, the U.S. Centers for Disease Control has reported that infections caused by S. aureus accounted second only to Escherichia coli. Currently, many biosensing methods have been developed for detection of S. aureus, especially by taking advantage of the antibody-antigen recognition, including atomic force microscopy measurement, surface plasmon resonance, fluorescencebased immunoassay, electrochemical biosensing etc. However, shortcomings with the production, stability, and modification of antibodies and low abundance of antigens on the surface of S. aureus still limit their extensive application. Aptamers can represent an alternative to antibodies as recognition agents and provide several advantages over antibodies such as good stability, simple synthesis, easy labeling, wide applicability, high resistance against denaturation, and high sensitivity, which provides a new thought for the development of rapid and sensitive detection method for S. aureus. However, the reported S. aureus detection method based on aptamer mainly focused on electrochemical sensing method and fluorescence spectrometric method. These methods are complex, and cannot achieve high throughput detection. Dual-colour FCM can qualitatively and quantitatively analyse the cells or particles. By compared with the traditional detection methods, it displays the advantages of wash-free and high speed and is becoming an advanced method for quantitative analysis cells and paticles. Meanwhile, the dielectrophoresis online enrichment technology can effectively enrich and separate cells, bacteria and other complex biochemical samples, which provide a new platform for the rapid and sensitive detection of bacteria. Aiming at the rapid and sensitive detection of S. aureus, this thesis mainly focuses on development of aptamer-based detection methods for S. aureus by coupling fluorescent nanoparticle-based labeling technology with our previous reported dual-color flow cytometric analysis and dielectrophoresis online enrichment technology.1. Aptamer/fluorescent silica nanoparticles (Aptamer/FSiNPs) based dual-color flow cytometry for detection of S. aureusBy selecting aptamer as the recognition probe and combining with our developed fluorescent nanoparticles label based dual-color flow cytometric analysis, we here developed a sensitive and specific determination strategy for S. aureus detection. In this protocol, an aptamer, having high affinity to S. aureus, was first covalently immobilized onto chloropropyl functionalized FSiNPs through a click chemistry approach to generate Aptamer/FSiNPs. The specific recognition capability of the Aptamer/FSiNPs to S. aureus was first assessed through confocal laser scanning microscopy imaging. Next, S. aureus was incubated with Aptamer/FSiNPs, and then stained with SYBR Green I. Upon target binding and nucleic acid staining with SYBR Green I, the S. aureus was determined using dual-color flow cytometry. The method decreased false positives of dual-color flow cytometry assay. It was demonstrated that these Aptamer/FSiNPs could efficiently recognize and fluorescently label target S. aureus. Through multiparameter determination with flow cytometry, this assay allowed for detection of as low as1.5×102and7.6×102cells mL-1S. aureus in buffer and spiked milk, respectively. By compared with the Aptamer/FITC based flow cytometry, it showed higher sensitivity.2. Aptamer/FSiNPs based dielectrophoretic enrichment technology for detection of S. aureus.By selecting aptamer as the recognition probe and combining with our developed fluorescent nanoparticles label based dielectrophoresis online enrichment technology, we presented a fast, convenient, continuous and sensitive detection method for S. aureus. In this method, S. aureus aptamer was labbeled with FSiNPs, and then the S. aureus was introduced into the pDEP-based microfluidic chip after incubation with Aptamer/FSiNPs. The Aptamer/FSiNPs probe labelled S. aureus accumulated in the electrode gap by applying an AC voltage in a positive dielectrophoresis (pDEP) frequency region, and the signal was collected by inverted fluorescence microscope CCD imaging system. The detection limit of S. aureus was determined to be93cells mL-1. The aptamer/FSiNPs based dielectrophoretic enrichment technology could achieve the online enrichment and detection of S. aureus, and this method showed higher sensitivity compared with the traditional detection methods.