Analysis of Pathogen Detection using Macroscopic Fluorescence from DNA Stains on Bacterial Solution Drops*

In medical settings, approximately 40% of bacterial cultures sampled from bodily fluids yield false positives due to pathogen contamination from the skin or the environment during collection (NIH). This results in a 20% increase in laboratory charges and a 40% rise in intravenous antibiotic costs. Such overdiagnosis also leads to excessive antibiotic usage, incurring approximately $20 billion annually for U.S. hospitals.

The present work aims to design, test, and optimize a handheld, low-cost, and rapid diagnostic device for detecting pathogens in small fluid volumes. The device, referred to as InnovaBug™, operates using a principle called macroscopic fluorescence. Fluorescence emission is captured via digital images from sets of four 0.1 mL pathogen-solution drops deposited on superhydrophilic strips. These drops are impregnated with DNA- or RNA-specific fluorescent dyes. SYBR Safe Green dyes are utilized for detecting DNA from bacteria. The net green fluorescence produced is expected to scale proportionally with the quantity of pathogens.

To validate this principle, various sets of identically metered solution drops are digitally photographed to quantify a ratio defined as green fluorescence (540 nm) emission to blue illumination (470 nm) emission, denoted as Rg/b. Rg/b values are subsequently correlated to the calibrated count of bacteria in drops sampled from different bacterial dilutions. This is achieved by extracting a color intensity spectrum in the form of an RGB (Red-Green-Blue) spectrum from the images.

The blue and green emissions are integrated into the RGB spectrum and scaled by the intensity recorded for the corresponding color channels.

Bacterial detection has been successfully replicated in four independent laboratories. The primary focus of the present work lies in measuring Rg/b under various conditions when drops are applied and assessing the impact of microfluidics on both bacterial detection and bacterial load as measured via the Rg/b ratio.