Biosensors for Detection of Pathogenic Microorganisms

Maria T. Dulay

The ubiquity of pathogenic microorganisms, such as bacteria, viruses and parasites, in food, water, and blood makes it necessary to have effective testing methods for such microorganisms. Conventional microbiological testing methods are time consuming in that they involve a morphological evaluation of the microorganism as well as tests for their viability and often an amplification step, and these methods lack the high sensitivity needed to detect low concentrations where even a single pathogenic organism in complex biological environments that may include other nonpathogenic organisms can be an infectious dose. To address the need for rapid and sensitive detection of microorganisms, our lab has been working on the development and application of cell-imprinted polymers for microorganism capture and detection. This research area began with imprinting of PDMS (polydimethylsiloxane), a viscoelastic polymer, with a variety of microorganisms, including bacteria and viruses.

The focus of this project is to develop cell imprints on polymers, including silane-based ones, whose properties can be modified by changing a variety of reaction parameters, which is an advantage over the use of PDMS. Briefly, a cellimprinted polymer is prepared by first creating a target bacteria template by adsorption of the bacteria onto an inert surface. Polymer reaction solution is poured over the template and the resulting polymer forms around the fixed bacteria, creating an imprint of the bacteria on the polymer, which is peeled from the template and used to capture the target bacteria from a suspension. This approach is just one of the ways that we imprint bacteria into these OSX polymers. The unique shape and chemical fingerprint of the targeted bacteria are captured in the polymer during imprinting. Capture of the targeted bacteria is achieved without laminar flow at room temperature or 30°C. Different detection schemes are coupled to the imprinted polymer biosensor, including optical methods.

Figure 1. Fluorescent images of captured (A) inactivated E. coli-GFP (OD600 0.38 solution) and (B) inactivated S. typhimuriumGFP (OD600 0.38 solution) on an inactivated E. coli-GFP-imprinted OSX polymer.

Preliminary results using E. coli-GFP-imprinted sol-gel polymers as shown in Figure 1 demonstrate the potential of these imprinted OSX polymers for the capture of target bacteria within 30 minutes at 30° C by gravitational settling (no laminar flow) in the imprinted area of the polymer. Furthermore, differentiation between the targeted chemically inactivated bacterium E. coli, and non-targeted S. typhimurium and native E. coli is demonstrated with high selectivity compared to imprinted PDMS. By designing cell-imprinted OSX polymers as biosensors for microorganisms such as bacteria, we hope to have a rapid, sensitive, and inexpensive device for the detection of pathogenic microorganisms in different environments. The use of these devices as point-of-care biosensors is one application that we are pursuing. Figure 2 show SEM images of E. coli-GFP-imprinted OSX polymers and a template.

Figure 2. SEM images of (A) imprinted OSX-1 polymer with heat-fixed E. coli-GFP (OD600 1.0 suspension) on polystyrene substrate, (B) glutaraldehyde-inactivated E. coli-GFP on OSX-2 polymer prepared with E. coli-GFP (OD600 0.44 suspension) on Thermanox plastic, and (C) bright-field image of glutaraldehyde-inactivated E. coli-GFP template at 40X magnification.