Label-free counting of Escherichia coli cells in nanoliter droplets using 3D printed microfluidic devices with...

This study describes for the first time the development of 3D printed microfluidic devices with integrated electrodes for label-free counting of E. coli cells incorporated inside droplets based on capacitively coupled contactless conductivity detection (C⁴D). Microfluidic devices were fully fabricated by 3D printing in the T-junction shape containing two channels for disperse and continuous phases and two sensing electrodes for C⁴D measurements. The disperse phase containing E. coli K12 cells and the continuous phase containing oil and 1% Span^® 80 were pumped through flow rates fixed at 5 and 60 μL min^-1, respectively. The droplets with incorporated cells were monitored in the C⁴D system applying a 500-kHz sinusoidal wave with 1 V_pp amplitude. The generated droplets exhibited a spherical shape with average diameter of 321 ± 9 μm and presented volume of 17.3 ± 0.5 nL. The proposed approach demonstrated ability to detect E. coli cells in the concentration range between 86.5 and 8650 CFU droplet^-1. The number of cells per droplet was quantified through the plate counting method and revealed a good agreement with the Poisson distribution. The limit of detection achieved for counting E. coli cells was 63.66 CFU droplet^-1. The label-free counting method has offered instrumental simplicity, low cost, high sensitivity and compatibility to be integrated on single microfluidic platforms entirely fabricated by 3D printing, thus opening new possibilities of applications in microbiology.