Rapid detection of airborne microorganisms is necessary for prevention and disease control. Conventional methods of microorganism detection in aerosols are laborious and time-consuming. Multiplex gene assay has become a valuable tool in rapid detection and diagnostic applications. In this study, we propose a microfluidic droplet-based detection platform combined with the loop-mediated isothermal amplification (LAMP) method and calcein dye for quick fluorescent-based detection of bacteria for point-of-care applications. For proof of concept, E. coli K12 strain was selected and an assay for the uidA gene detection was developed. The assay can detect E. coli concentrations of as low as 3.2x10⁶ cells/ml. The temperature-controlling module can maintain a constant temperature at 65 C to perform a LAMP assay. Hence, we can integrate the developed assay with microfluidic droplets to enable sensitive and rapid bacterial pathogen detection.

<b>(a) Specificity </b> Lane 1: No template; Lane 2: B. subtilis heat lysate (negative control); Lane 3: E. coli heat lysate; Lane 4: E Coli uidA gene PCR product (positive control); <b>(b) Detection Limit</b> Lane 1: E. coli heat lysate OD = 40; Lane 2: E. coli heat lysate OD = 4; Lane 3: E. coli heat lysate OD = 0.4; Lane 4: E. coli heat lysate OD = 0.04; Lane 5: E Coli heat lysate OD = 0.004; Lane 6: E. coli heat lysate OD = 0.0004 Lane 7: No template (negative control);<b> (c) Fluorescent Intensity.</b> Sample with dye - 154.81 ± 12.58 RFU, Negative control with dye - 83.72 ± 7.189 RFU, and Sample without dye - 2.97 ± 0.37. The box images show the fluorescence intensity

(a) Specificity Lane 1: No template; Lane 2: B. subtilis heat lysate (negative control); Lane 3: E. coli heat lysate; Lane 4: E Coli uidA gene PCR product (positive control); (b) Detection Limit Lane 1: E. coli heat lysate OD = 40; Lane 2: E. coli heat lysate OD = 4; Lane 3: E. coli heat lysate OD = 0.4; Lane 4: E. coli heat lysate OD = 0.04; Lane 5: E Coli heat lysate OD = 0.004; Lane 6: E. coli heat lysate OD = 0.0004 Lane 7: No template (negative control); (c) Fluorescent Intensity. Sample with dye - 154.81 ± 12.58 RFU, Negative control with dye - 83.72 ± 7.189 RFU, and Sample without dye - 2.97 ± 0.37. The box images show the fluorescence intensity

a) Experimental setup, the PDMS chamber was filled with Novac 7500™, the outlet was blocked with Eppendorf<b>™</b> tube, and the inlet was pressurized using a syringe pump at 5μL/h. b) Experiment results over 30 minutes of operation

a) Experimental setup, the PDMS chamber was filled with Novac 7500™, the outlet was blocked with Eppendorf™ tube, and the inlet was pressurized using a syringe pump at 5μL/h. b) Experiment results over 30 minutes of operation

Jing Dai

Appearances