Integrated Microreactors Of Low Density Bacterial Culture And Detection For Antibiotic Susceptibility Analysis

The over-time abuse of antibiotics in medical treatment and stock breeding has led to its high-level residue in sewage plant,natural water and soil.The resultant pollution has been a tremendous threat to human health and ecosystem.To reduce the overuse of antibiotics at the source,it is an effective way to take the advantage of the bacterial antibiotic sensitivity analysis results to supervise the rational usage of antibiotics,and the supervision could guarantee the environmental health and safety.At present,the conventional antibiotic sensitivity analysis method is time-consuming,and generally could only detect the free floating bacteria.As for the free-floating bacteria in low concentration,and the bacteria within biofilm,there's no effective detection tool in conventional method for antibiotics sensitivity analysis.The research utilized the in-situ test ability of microfluidic technology for continuous fluid in micro scale,to carry out label-free bacteria counting for ultra low concentration bacteria,and established an microfluidic system integrated continuous flow cultivation and antibiotics sensibility analysis,so that its advantage of precise control on conditions could be taken to culture and in-situ test the biofilm formed by low preliminary bacteria in sub-microliter scale without label.This study provides an effective platform for the rapid detection of antibiotic sensitivity,in addition,it lays the foundation for the study of biofilm contamination in micro scale.The capacitively coupled contactless conductivity detection device with highly integrated electrodes and detection circuit was developed as the solution for the difficult detection on sample in low concentration and minute sample.The space between excitation electrode,shielding electrode and detection electrode has been optimized.The signal-to-noise ratio(SNR)under different excitation frequencies was discussed.The linear relationship and detection limit of the device in different buffer solutions have also been tested.The device performs extraordinarily in the single bacteria count analysis of E.coli at the concentration of 10⁴?10⁶cells/m L.To ensure the universal application of the device,for the rapid detection of high concentration bacteria,the concentration of 10⁶?10⁸cells/m L of E.coli was detected after optimizing the background solution of bacteria by using 19 mg/L PBS as background solution.The results showed that the viability of E.coli was not affected by electrical detection under excitation frequency of 60?120 k Hz,and the survival rate was higher than 96%.To count the bacteria in 10⁴?10⁸cells/m L by above method,the sample amount is only 50?L.In view of the long period of antibiotic sensitivity detection caused by low preliminary concentration and slow bacterial culture,a microfluidic device integrating rapid culture and dynamic monitoring was developed.A 3-D chip consisting of upper channel layer,middle culture chamber layer and lower gas path layer,is designed and fabricated.Free floating bacteria is rapidly cultured on the chip with continuous flow in micro scale,and the minimum inhibitory concentration(MIC)of Amoxicillin on E.coli is determined by the six different antibiotic concentrations formed at the same time.The channel layer is not only to provide continuous flow medium,but could also form antibiotic concentration gradient with its specified structure.The 70?m×100?m rhombic microarray structure in the channel is designed to promote mixture,so that the length of the mixing channel could be shortened to 9 mm.The mixture is stimulated by COMSOL and verified by fluorescein experiment.For a single culture chamber,the diameter is 1 mm and the depth is 1.2 mm,with a flange structure in the upper channel,the influence of shear stress on bacteria caused by the continuous flow could be avoided.The gas path layer consists of 36 arrays,which are located at the bottom of the culture chamber.Each array consists of 25 channels with a width of 15?m and a depth of 5?m.On the optimized 3-D chip,E.coli was cultured in micro scale by continuous flow method,and the results of growth curve showed that the delay time was shortened to0.06 h,indicating that bacteria adapt to the micro scale environment sooner than ever.On this basis,the inhibitory effect of Amoxicillin on E.coli was quantitatively analyzed.The MIC of Amoxicillin on E.coli was determined within 6 h under the condition of low sample concentration.To deal with the observation difficulty in its early age of formation,a microfluidic device integrating rapid cultivation of biofilm and in-situ monitoring was developed,so by it the growth characteristics under the effect of shear force in its early formation age and the stress of antibiotics could been studies.A 12 mm²culture chamber was designed and fabricated for continuous flow cultivation of biofilm.A 54?m×40?m micro column array was set up in the culture chamber to reduce the average flow velocity and avoid the direct impact of the flowing medium on bacteria.This method can promote the formation speed of biofilm in micro scale,and realize the in-situ observation and characterization of biofilm.The results showed that when the shear rate of culture medium was higher than 83.3 s^-1,with the increase of continuous flow velocity,the speed of biofilm formation would be gradually accelerated.In the process of biofilm formation,E.coli was exposed to Amoxicillin of the same concentration at different stages of culture(1 h,3 h,5 h,7h),which could promote the formation of biofilm in different degrees.In addition,the characteristics of biofilm formation by mixed bacteria in the active sludge were studied.The results showed that the mixed bacteria were easier to form biofilm,and was not sensitive to Amoxicillin.