Rapid Identification Of Bacterium And Its Associated Antibiotic Specificity Assay With An Integrated Microfluidic Chip

Objectives: Infectious diseases are a leading cause of death in clinical. Over95%of these deaths are caused by the lack of proper diagnosis and treatment. However,rapid pathogen identification and its associated antibiotic specificity assay remains apressing issue today since related conventional methods are tedious, cost intensive andtime consuming, typically requiring from48to72h. In turn, microfluidic chip(lab-on-chip), with its integration, high-throughput, short reaction time, less reagentconsumption, miniaturization and other advantages, offers real alternatives capable offilling this technological gap. In particular microfluidic devices capable of integratedmultitarget analysis, normally performed in a centralized laboratory, are able to providehealthcare workers and outpatients with important infecting pathogen-relatedinformation. The purpose of this work lies in fabricating a integrated microbeads-basedmicrofluidic platform used for more rapid identification of bacteria that benefits earlydiagnostics of infectious diseases, while simultaneously providing reliable and timelyinformation of antibiograms that contributes to effective treatment of infectiousdiseases.Methods: In this study, the microfluidic chip was consisted of four units. One unit was utilized for the identification of bacteria in sample (unit a), and the other threeunits (unit b, c, and d) were used to test the susceptibility of the identified bacteria tothree antibiotics in parallel. In the unit a, a high-specificity and high-capture-rateimmune-microbead chamber has been integrated into chip’s microchannel, on-linedetection and quantitative analysis for bacteria enriched has been carried out byimmunofluorescence. And the main components of the unit b/c/d were mixturechambers, concentration gradient generator (CGG) and monitoring chamber array. Thebacterial sample was injected into the mixture chambers via the inlet with theconnection of a pump. Subsequently various antibiotics were pumped into the mixturechambers at different concentrations by CGG. Viable cells staining and online monitorstatus of bacterial survival in the monitoring chamber array were followed by afluorescence microscope after the treatments with different antibiotics. Herein, taking E.coliO157as the detection target, firstly, E. coliO157suspention with different initialcells densities was assayed on the unit a，and E. coliO157was captured, enriched andquantified with microbeads-based immunocapture and immunofluorescence assay. Thetraditional method PCM was also needed in parallel. Next， the sensitivity, accuracyand the specificity of chip-unit a for the identification of E. coli O157were evaluated.Then the suspension and three antibiotics (ceftriaxone, norfloxacin and ampicillin) wereinjected into unit b, c, and d, respectively, and co-cultured in the mixture chambers ofthe three parallel units. Finally, we tested deviation of the fluorescence intensity indifferent monitoring chambers, analyzed the inhibition of different concentration ofantibiotics to the survival of E. coliO157. The antibiotic sensitivity tests were alsodetermined by the absorbance based assay performed in the96-well.Result: In this study, we have designed and built a microfluidic chip on theidentification and antibiotic sensitivity test of E.coliO157successfully. In the unit a, E.coli O157at a cell density range of101-105CFU/μL could be detected within30min,and the method of quantitating bacteria by the fluorescence intensity of reaction was setup successfully through the drawing of standard curve of bacteriaconcentration-fluorescence intensity. And the high specificity of this microbiological identification unit was proved though the performance for different kinds of bacteria. Inthe unit b, c, and d, the concentration gradient generators could produce a0:1:2:3:4drugconcentration, and the effects of three antibiotics on E. coli O157were evaluated within4-8hours simultaneously. The results of quantitative analysis for bacteria and antibioticsensitivity test on the microdevice were consistent with the conventional bench-topsystems assay.Conclusion: From the above-mentioned, the integrated microbeads-basedmicrofluidic chip is sensitive, rapid, reliable and high effective for the identification ofbacteria and the assay of antibiograms of antibiotics. This work highlights theadvantages of the microdevice in terms of assay time and the integration of multipleassays, as well as the great potential in constructing a portable device for rapid detectionof pathogens and multiple drugs sensitivity test in clinic.