Gelatin-Based Microfluidic Channel for Quantitative E. Coli Detection Using Blue Fluorescence of 4-Methyl-Umbelliferone Product and a Smartphone Camera

Abstract

Escherichia coli <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">(E. coli)</i> is a foodborne pathogen that can produce potent toxins, causing severe illnesses due to contaminated food and water consumption. This research has utilized a fluorescence measurement to quantify <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">E. coli</i> colonies from blue fluorescence emitted by 4-methyl-umbelliferone (4MU). The 4MU is the product of the catalytic reaction between beta-D-glucuronidase (GUD) secreted by multiple strains of <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">Escherichia coli</i> and its substrate 4-methylumbelliferyl-beta-D-glucuronide (MUG). Here, we apply the 4MU enzymatic reaction and propose simple instrumentation for label-free, real-time, <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">in-situ, and</i> quantitative <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">E. coli</i> measurement. The detection platform consists of a smartphone camera, an ultraviolet light source for fluorescence excitation, and MUG suspended microfluidic channels. The underlining mechanism for the proposed <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">E. coli</i> measurement is the passive diffusion process of the MUG secreted by <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">E. coli</i> and the GUD suspended in the gelatin, forming the blue fluorescence 4MU product in the channels. We have also proposed a cost-effective and eco-friendly fabrication method for preparing the MUG suspended gelatin microfluidic channels using a laser printer. Gelatin is an ultraviolet light-absorbing material in nature, providing an embedded optical filter. Here, we demonstrate that a smartphone camera can be utilized to image the fluorescence emission of the 4MU excited by the ultraviolet light in the gelatin film. The proposed <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">E. coli</i> detection technique allows the amount of <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">E. coli</i> colonies to be quantified without liquid sampling, cell-culturing, inoculation, and sophisticated equipment. Furthermore, the proposed method has a trade-off between response time and detection limit.