Statistical Analysis of DNA/RNA Macroscopic Fluorescence in 0.15 mL Fluid Drops for Rapid Bacteria Detection via a Handheld, Low-Cost, Small Volume Diagnostic Device: BacteroBug™

Rising numbers of pathogens and pandemics in the 21st (SARS-I, Ebola, SARS-II, mPox) create the need for rapidly triaging of infectious patients via small, handheld, low-cost devices to detect pathogen load. This work uses 0.15 mL drops of bacterial solutions to test macroscopic fluorescence for bacterial load detection, based on the hypothesis that the macroscopic intensity of green fluorescence from SYBER-safe stains scales lineraly with bacterial concentration.

Calibrated solutions of 100,000 E.Coli CFUs were incubated and serially diluted in PBS to create four solutions: 1.0, 10-1, 10-2 , and 10-3. Metered drops of 0.15 mL from each solution are applied to LabVida slides, with 0.15mL of SYBR green stain, and photographed while illuminated with 470 nm of UV blue light. The green fluorescence to blue light excitation ratio is measured via RGB analysis for each drop. The measured ratio of Green to Blue intensity(Ig/Ib) reaches an average of 8.5 ± 2.1 for 5k E.Coli CFU/mL, and 3.6 ± 0.34 for 500 E.Coli CFU/mL, a significant 250% increase in fluorescence with bacterial load. The background RGB analysis yields an Ig/Ib = 1.8 ± 0.25 Ig/Ib, thus a factor 2 to 4 lower than in drops. Thus, a reproducible fluorescence factor, Ig/Ib, can be correlated with bacterial loads of 100 - 100,000 CFU/mL, in the BacteroBug device.