Drug Susceotibility Test And Antibiotics Detection On Surface Plasmon Resonance Imaging Platform

In the treatment of infectious diseases, the rational use of antibiotics is very important. It usually need antimicrobial susceptibility test (AST) before clinical treatment of infectious diseases, otherwise it will lead to the abuse of antibiotics and the bacterial drug resistance enhancement. However, current conventional AST method is cumbersome, time-consuming. Therefore, it is essential to get fast drug susceptibility test results before the treatment of microbial infections. In addition, the environmental pollution problem caused by the antibiotics abuse has aroused people’s attention increasingly. The antibiotics residue in the environment will cause serious negative impact on human health and ecological environment, thus it is very critical to establish the rapid real-time monitoring technique for trace antibiotics detection in the environment. Surface plasmon resonance (SPR) technology can real-time monitor the biomolecular interaction, and response sensitively to refractive index change on the surface of sensor chip, it is widely used due to the advantages of free-label and high sensitivity, etc. As the extension of the SPR technology, surface plasmon resonance imaging (SPRi) technology can achieve high-throughput analysis, SPRi has broad prospect for application in fields such as drug susceptibility test, antibiotics detection and recognition. This paper attempts to establish drug susceptibility test and antibiotics detection method on a platform based on SPRi technology, it mainly includes the following two parts:(1) The in situ microbial growth monitoring method on SPRi gold sensing chip surface with the PDMS array microcavity construction was established. We found that the growth condition of bacteria can be observed real-time through SPRi signal change, we did the experiment regarding the growth curves of different concentrations E. coli suspension, and further compared the different kinds of antibiotics sensitivity on E. coli, tested the minimal inhibition concentration (MIC) of oxytetracycline. This in situ AST-SPRi method for microbial cultivation in a small volume of PDMS microcavity can accelerat the exchange between bacteria and nutrient substance, so it not only reduced the reagent consumption, but also accelerated the growth rate of bacteria. Secondly, SPRi sensor is sensitive to refractive index change on the chip surface, E. coli adsorption and deposition on the surface of SPRi gold chip would produce significant signal during the breeding process, it can further shorten the testing time within 5-6 h to get AST results. What’s more, the oxytetracycline MIC value obtained by SPRi monitoring method was consistent with the turbidimetry test results with high accuracy. The AST-SPRi technology integrated cultivation and detection can realize microbe droplets automatic monitoring, meanwhile it can avoid the interference of sample color or turbidity itself. So it has good application prospect on practical drug susceptibility test.(2) We developed a method that preparing molecularly imprinted polymer (MIP) thin film on the surface of SPR sensor chip through in situ photo-polymerization, the method has the advantages of short time, good uniformity, nanoscale controllable thickness, etc. With the antibiotic ciprofloxacin (CIP) as a small molecule model, the properties of CIP molecularly imprinted SPR sensor chips were studied, such as sensitivity and selectivity. CIP molecularly imprinted film had good selectivity to ciprofloxacin and its structurally analogue, imprinting factors of ciprofloxacin and ofloxacin were 2.63 and 3.80 respectively, much higher than the imprinting factors of azithromycin, dopamine and penicillin. The SPR response had good linear relation with CIP concentration over the range 10-11 mol/L-10-7 mol/L. From the results of repetitive experiment, it also showed good repeatability and stability. On the basis of this rapid photo-polymerization method, the SPRi sensor chip with molecularly imprinted polymers (MIPs) array of three different polymer sites was fabricated and the performance of the MIPs-SPRi sensor to antibiotics was tested, the MIPs-SPRi sensor had different response patterns to ciprofloxacin and azithromycin. The MIPs-SPRi sensing technology revealed the ability to recognize different antibiotic molecules, and it is a potential method for realizing multiple antibiotics qualitative and quantitative analysis simultaneously.