Research Of Pathogens Enrichment,Detection And Culture Based On Microfluidic Chips

It is known that pathogens can lead to serious infectious diseases worldwide and cause enormous economic losses,they can trigger explosive epidemics,such as influenza A virus,Zika,Ebola,and the recent COVID-19 outbreaks.Therefore,a better monitoring of pathogens is critical for prevention and control strategies.However,the pathogens concentration is frequently low in most samples,which result in failure of nucleic acid detection.Solutions for the above urgent problems could be solved with microfluidic platform.The microfluidic platform integrates the basic operating units of sample preparation,reaction,separation and detection into a several square centimeters chip.Moreover,the conventional methods such as shake flasks,petri dishes,bioreactors and multi-well plates are still applied to culture and investigate pathogens.However,the operating procedures of conventional methods are complicated and time-consuming and can only provide limited control on pathogens culture.The development of microfluidic systems can perfectly solve complicated and time-consuming problems in conventional methods.We designed and fabricated different functional microfluidic chips to study pathogens enrichment,pathogens detection and three-dimensional cultivation of pathogens.Our platform can provide new approaches for pathogens research,the specific research content and results are as follows:（1）Herein,we develop an 3D electrostatic microfluidic platform and an electrophoretic microfluidic platform to rapidly and label-free enrich pathogens.The microfluidic device consists of five functional layers:two electrode substrates,two poly（dimethylsiloxane）（PDMS）channel layers and one filter membrane.We inject virus samples into the microfluidic chip when the voltage is applied and the enriching flow is closed,negative charged pathogens are transferred from the sample channel to the enriching channel under the electrostatic field or electrophoretic field.We collect the enriched virus from enriching flow when all samples passed and the voltage is turned off.In order to investigate the unique feature of the 3D device,we performed numerical modeling of the electric field and the trajectories of simulation particle.The trajectories of particle under different electrostatic field forces and different sample flow rates are studied.The results of electrostatic field show that the particle remains in sample channel when voltage is 1V or 5 V.However,when the voltage increased to 10 V and sample flow rate increased to 600μL h^-1,the particle can move from sample channel to enrichment channel.In addition,the particle can pass from sample channel into enrichment channel when voltage is 20 V or 30 V.The result of electrophoretic field show that the particle can move from sample channel to enrichment channel under different electrostatic field forces and different sample flow rates.As the voltage increase,the maximum sample flow rate increase.Particles can still be enriched when the voltage value is increased to 30 V,and the sample flow rate is increased to an ultra-high 35000μL h^-1.Compared with the electrostatic field,the electrophoretic field reflects a stronger ability of enrichment.These results demonstrate the feasibility of our platform,and the results of particle trajectories can provide a theoretical basis for subsequent experiments.We enriched the H1N1 virus sample with different concentrations by using electrostatic microfluidic chip.According to simulation results,we set the voltage as 30 V and the flow rate400μL h^-1.We characterized the virus by using scanning electron microscope（SEM）.We found that the bulky sample has large amount of impurity leading to virus hard be identified.After enrichment,the virus can be observed clearly.However,the enrich efficiency of microfluidic chip can’t be verified by SEM results.Therefore,in order to demonstrate the enrich efficiency of the device,we compared the threshold cycle（C_T）values of real-time PCR between primary samples and enriched samples.The result reveals that C_T values of primary samples and enriched samples are slightly differences when there are high virus concentrations.However,when samples are diluted to 10⁴ X,the C_T values without enrichment are 4–5 cycles delayed rather than the enriched sample.It demonstrates that the effect of enrichment come out when virus samples are diluted to 10⁴ X.In addition,the virus can’t be detected when samples are diluted to 10⁵ X and 10⁶ X,while the virus can be detected after enrichment.It demonstrates that our method can increase the limit of detection with 100-fold higher based on real-time PCR quantified analysis.（2）The process of traditional amplification is complex and troubled with amplification bias,and electrophoresis or sequencing is required for further analysis.To simplify the amplification process and reduce amplification bias,we built a droplet microfluidic amplification platform combined with fluorescence reading.To demonstrate the feasibility of our platform,we performed Multiple Displacement Amplification（MDA）,Multiple Annealing and Looping Based Amplification Cycles（MALBAC）in droplets and tubes within limited samples synchronously.We found that MDA and MALBAC in droplets was highly effective even though the initial templet was 125 pg（～20 cells）based on the fluorescent readouts.Furthermore,droplet method could dramatically reduce the amplification bias and retain the high accuracy of replication than traditional tube method.To further demonstrate the effectiveness of the platform in pathogen detection,we detected the H1N1 virus sample with different concentrations.The fluorescent results showed that viral nucleic acids can be amplified within droplets and can still be detected when the viral samples are diluted to 10⁵ X.We successfully built a droplet microfluidic amplification platform combined with fluorescence reading,the platform can simplify amplification process and reduce amplification bias.Our results provided insights that will allow future decision making regarding WGA protocols.（3）To further study the growth characteristics of pathogens,we present a flexible and multifunctional microfluidic device,which allow to generate and separate individual hydrogel microspheres and facilitate real-time monitoring of pathogens.To optimize the size of alginate droplets produced in our platform,we investigated different flow rates between continuous phase and disperse phase in a given microchannel geometry.Monodisperse alginate droplets with different sizes are produced by adjusting the flow rate of oil at a fixed alginate flow rate(F_alg)of 30μL h^-1.The results demonstrate that the diameter of alginate droplets is decreased as F_oil/F_alg increased,low CV values demonstrate that the droplets are sufficiently monodisperse and the device is stable and high-efficiency.To evaluate the efficiency of demulsification in our device and optimize appropriate alginate microspheres,we analyzed the size and CV of alginate microspheres after demulsification.The increase of microspheres diameters compared with droplets are observed due to the affinity of the carboxyl group of alginate for water molecules.After droplets are broken,the uniformity of alginate microspheres become worsen in the process of swelling.The results of microspheres characterization demonstrate that alginate microspheres are appropriate in subsequent culture experiment.Thus,our platform achieves rapid transformation from oil phase to aqueous phase and produces massive monodispersed alginate microspheres.According to the results of microsphere’s characterization,alginate microspheres with diameter size of 135μm are used during bacterial cells immobilization and growth.We collected fluorescence images of individual alginate microsphere cultivated on the microfluidic device every 60 min.We clearly observed an increasing trend of bacterial cells appear after 120min cultivation.However,when the culture time is increased to 480 min,bacterial cells leaked out of the alginate microsphere.The cell concentration was so high that alginate microparticles disintegrated.The results indicate that the alginate microsphere is stable for up to 480 min in our device,this immobilized alginate microsphere has potential in biomimetic cell culture engineering.The technique will become a powerful tool for rapid screening and parallel observation of microbial cultures for drug discovery or testing.Furthermore,the platform has potential applications to couple with biomimetic material to provide feasibility for cell culture engineering system.