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<p>The key objective of this proposed work is to establish the proof of principle of a kitchen hygiene wipe capable of detecting foodborne Escherichia coli (E. coli) infection, signaling the bacterial infection via a colorimetric change in the wipe; and evaluate its effectiveness in the kitchen hygiene practice. The wipe is made from a nonwoven, highly porous polymer nanofiber matrix. Biosensing occurs via polydiacetylene (PDA)-based synthetic vesicles decorating the nanofiber surfaces, which include a receptor molecule selective for E. coli. These synthetic vesicles experience a blue-to-red color change in the response to binding of the target compound, as shown in Figure 1.</p><p> This key objective will be accomplished via the following specific aims:</p><p> 1. Develop and characterize an electrospun nonwoven, highly porous polymer nanofiber matrix containing a surface decoration of PDA synthetic vesicles that contain a glycolipid-based biological receptor molecule capable of absorbing E. coli.</p><p> 2. Integrate the electrospun nanofiber-vesicle matrix into a hygiene wipe capable of detecting E. coli.</p><p> 3. Assess usability and efficacy of the microbial-sensing wipe in real-time monitoring of foodborne bacterial infection.</p>
<p>Methodology: The following methodology will be taken to accomplish the specific aims:</p><p>Specific Aim 1: Develop and characterize electrospun nanofiber-based biosensors containing a surface decoration of PDA synthetic vesicles capable of adsorbing E. coli.</p><p>Task 1.1 Vesicle Fabrication and Evaluation: Initial efforts will focus on the identification and incorporation of the appropriate glycolipid receptor molecule that will bind to E. coli, according to methodologies well described in the literature (Pires et al., 2011). (de Oliviera et al., 2013). (Su, Li, Jiang, & Cao, 2005). The bioactivity and sensitivity of these vesicles will then be evaluated via UV-vis spectroscopy. The selectivity will be evaluated by combining the vesicles with other bacterial species common to the skin flora. The effect of solution pH and temperature on the sensitivity and selectivity of the sensor will also be evaluated.</p><p>Task 1.2 Fabrication and Characterization of Electrospun Nanofiber-Vesicle Matrix: Electrospinning will be used to both fabricate the fibers and direct the positioning of the vesicles to the surface of the fibers. A variety of electrospinning approaches will be investigated, including coaxial electrospinning and emulsion electrospinning. In addition, various biocompatible polymers will be explored to serve as the supporting nanofiber matrix, including, but not limited to, polycaprolactone, cellulose acetate, and polylactic acid. These nanofiber/PDA vesicle constructs will be evaluated by SEM to characterize the vesicle/matrix morphology, and by AFM, to characterize the binding interaction between the vesicles and the fiber surface.</p><p>Task 1.3: Evaluation of Selectivity and Sensitivity of Electrspun Nanofiber-Vesicle Matrix for Detecting E. coli: Sensitivity will be evaluated by combining, stepwise, aqueous solutions of E. coli, and measuring the resulting colorimetric response. The effect of pH and temperature will also be evaluated. Sensitivity of the sensor constructs will be compared to that of the unincorporated vesicles, to explore any dampening effects incurred by incorporation into the matrix. The selectivity of the sensors will be evaluated by exposing the sensors to bacteria that are not indicative of foodborne bacterial infection. The effect of PDA vesicle colorimetric response to binding, if any, will be measured. Selectivity of the sensor constructs will also be compared to that of the unincorporated vesicles.</p><p>Specific Aim 2: Integrate electrospun nanofiber-vesicle matrix into smart hygiene wipes capable of detecting E. coli.Task 2.1 Fabrication of Smart Wipes: Effective nanofiber/PDA matrices capable of detecting E. coli will be electrospun onto a standard hygiene wipe substrate. It will create hygiene wipes layered with PDA-added nanofibers. The electrospinning will be varied by spinning time to incorporate the PDA-nanofibers on the wipe substrate at different weight ratios.</p><p>Task 2.2 Evaluation of the Sensitivity and Selectivity of Smart Wipes: Sensitivity of the smart wipes will be evaluated in the lab setting by exposing the wipes to aqueous solutions of E. coli, and testing the resulting colorimetric change. The selectivity of the smart wipes will be also evaluated by exposing the wipes to bacteria that are not indicative of foodborne bacterial infection.</p><p>Specific Aim 3: Assess usability and efficacy of the microbial-sensing nanofiber technology in real-time monitoring of foodborne bacterial infection.</p><p>Task 3.1: Scale-Up of Smart Wipe: Upon the successful verification of nanofiber biosensing technology from the lab work described in specific aim 1 and 2, a field testing will be performed at the Aspen Grille to evaluate sensitivity and selectivity of the smart wipe in the food processing practice. Aspen Grille is a student-operated restaurant conveniently located on the CSU campus. One of the PIs is the faculty of CSU's Hospitality Management Program and will have access to the restaurant's kitchen and personnel. For the field testing, ten 3"x3" nonwoven PDA-added nanofiber swatches will be produced in the PI's lab and patched on the surface of 100% cotton kitchen wipes. This practice is designed to mitigate the low quantity of electrospun nanofibers that can be produced in the lab.</p><p>Task 3.2: Field Evaluation on Usability and Performance: Aspen Grille personnel will be asked to test for visual monitoring of bacterial existence on kitchen countertops. Taking a parsimonious approach, the kitchen surface will first be cleaned by trained personnel to the restaurant's hygiene standard. The kitchen personnel will then wipe the cleaned countertops using the smart biosensing wipe and visually examine colorimetric changes on the wipe. This experiment will be conducted at the end of a daily kitchen operation, once a day, in a 2-week span. The tested specimen will be collected in a designated separate zip lock bag each day. The frequency, size, and location of bacterial contamination on the wipes will be measured, so as to determine contamination patterns. Followed by the 2-week long experiment, focus groups will be convened with the kitchen personnel who has participated in the experiment during any day of the experiment period, to understand their evaluations on the performance and effectiveness of the smart wipe for the kitchen safety and hygiene.</p>