Live bacterial specific fluorescence quantification via switchable exogenous electric field induced change in membrane potential.

We have demonstrated the feasibility of using exogenous electric-field enabled fluorescence measurement to detect and quantify live Escherichia coli K12 bacterial sample in a cuvette on a bespoke sensor platform. The exogenous electric field (100 V/cm) across the cuvette switched off and on to induce changes in the bacterial membrane potential in live bacterial cells. This allowed the voltage-sensitive dye (VSD) dipropylthiodicarbocyanine iodide (diS-C3-(5)) to enter and then be expelled from the live bacterial membrane. The movement of the diS-C3-(5) in and out of live cells were simultaneously captured by fluorescence measurement via a custom sensor platform. The detection and quantification of live bacteria was demonstrated over a range of concentrations (OD600nm = 0 to 1.0) with a measurement duration of 180 s (R2 = 0.85, linear fit). Between the concentrations of OD600nm = 0.25 to 1.0, live bacteria yielded VSD signal that ranged from 3.56 ± 0.62 to 16.54 ± 0.28%. In comparison, dead bacteria (sonicated for 30 s) yield VSD signal that ranged -0.09 ± 0.74 to 0.93 ± 0.71%. In the event where commercial adenosine triphosphate (ATP) kit was not able to differentiate between live and dead bacteria, resulting in a false positive. However, the proposed method is able to differentiate and quantify live and dead bacteria, hence avoids both false negative and positive results. More importantly, this work has re-purposed and expanded the role of voltage-sensitive dyes from niche microscopy visualization to the broader family of fluorescence-based bacterial biosensors and quantification assays.