Construction Of A High-Throughput Microfluidic System For Long-Term Bacterial Colony Monitoring And Antibiotics Testing

Standard bacterial tests such as growth inhibition tests are the only convenient investigation method to detect the impacts of water soluble drugs, e.g. antibiotics, on bacteria. State of the art methods for determining the inhibiting effect of toxic components are cumbersome, because they are not feasible for parallelization and high-throughput. Therefore, biosensor with miniaturization, integration, intelligent and on-line monitoring have been become more important. In recent years, microfluidic technology caused widespread attention in biosensors'field because of its automation, low consumption of costly reagents and power, minimized handling of hazardous materials, short reaction times, portability and versatility in design, and capability for parallel operation.In this study, a high-throughput microfluidic system is presented. The microfluidic chip is comprised of seven parallel main channels. Each channel contains thirty two square-shaped microchambers connected to the main channels through 30μm wide capillaries and uniformly distributed micropillars. After simulation studies on samples loaded into the microchambers, and the solute exchange between the microchambers and main channels, the long-term culture of Escherichia coli (E. coli) HB101 in the microchambers is realized. Using the principle that L-arabinose (L-Ara) can induce recombinant E. coli HB101 pGLO to synthesize green fluorescent protein (GFP), we can online monitor growth status of bacterial colony in the microchambers, test deviation of the fluorescence intensity in different initial cells densities, quantitative analysize the inhibition of different concentration of tetracycline and erythromycin to the expression level of bacteria, and real-time observe morphological change of bacteria. The main results and conclusion are as follow:(1) Recombination E.coli HB101, pGLO was prepared and was used to express GFP steadily. The growth curve of E.coli HB101, pGLO was measured by optical density, which can supply basis for incubating bacteria on chip. The growth curve is important for understanding the growth laws and selecting bacterial in the optimal growth period for the subsequent experiment.(2) A high-throughput PDMS microfluidic chip containing seven parallel channels and 32 microchambers in single channel was prepared. Micropillar structure designed between the microchambers and main channel is used for reducing the influence of flow to bacteria in the microchamber. According to the theory of small molecule diffusion and the molecule simulated experimental results, we can find that the exchange of solute between microchambers and main channel can support the growth of bacteria in microchambers. On the basis of the permeation and sorption of PDMS, injection of bacteria is achieved by the continuous flow by the syringe pump. Once loaded, the bacteria can hardly escape from microchambers. These results are based for bacterial growth in chips and antibiotic testing.(3) E.coli HB101, pGLO can grow in the microfludic chip by supplement of medium by syringe pump. And the fluorescence changes were observed online. Compared with the traditional methods, the linear growth time of bacteria on the chip is longer, which indicated that the environment of growth on the chip under continuous supplement of medium and remove of metabolite is superior to the traditional experiment for E.coli HB101, pGLO. High or low initial loading densities of the bacterial cell suspension induce the same maximum growth rates during the log-phase, which can provide the guarantee for a high-throughput microfluidic system for long-term bacterial colony monitoring and antibiotic testing. The results demonstrate that higher initial loading densities of the bacterial colony cause bacterial cell to enter log-phase proliferation sooner.(4) The quantitative analysis of inhibition of different concentration of tetracycline and erythromycin to the expression level of GFP of bacteria was studied. And, the results of observation to the morphological change of bacteria indicated that E.coli can grow to long filamentous bacteria only when the concentration of tetracycline is at 3μg/mL The average length of bacteria is more than 50μm. But bcterial morphology under other antibiotic's concentration is similar to blank test.