The first is the chitosan hydrogel which houses our engineered yeast cells and glucose for binding to GOx. Chitosan is biodegradable, biocompatible and regarded safe for human dietary use (Mohammed 2017). Chitosan is a biopolymer processed from chitin, which is found in crustacean shells such as shrimp. To prepare chitosan, insoluble chitin is processed commonly in acetic acid over heat, which causes deacytelation and renders it soluble. As it polymerizes, the gel is rendered a highly interconnected matrix of pores. Chitosan medium molecular weight (448877, Sigma) was selected for initial experiments. Viscosity, or resistance to flow, is directly related to molecular weight. To ensure cellular material was retained without leakage as well as to provide viscosity high enough to retain cellular material in the chitosan gel, medium molecular weight was selected for initial experiments. The gel was prepared at 0.5 wt%/v 0.1M acetic acid. The low weight percent ensures large pore size for ample diffusion of molecules. Bovine serum albumin is cast with the cellular material into the gel to aid in undisrupted flow of proteins.
The agarose hydrogel lies against the skin, uptakes sweat and ISF and acts as a buffer against pH buildup and charge due to polarized molecules gathering at the skin. Agar is a biopolymer generally obtained from red algae consisting of agaropectin and agarose. Agaropectin has more charged side groups and therefore can interact in unwanted ways with DNA or proteins. For this reason, agar is purified to agarose which is much more neutral and has a lower chance of interacting with biomolecules. An agarose with a high electroendosmosis (EEO) value, or movement of liquid through a gel, is desired to optimize the speed of detection. We selected a medium EEO for molecular biology with an EEO >= 0.15 (A6877, Sigma) due to budget constraints for initial testing. The paper after which we modelled the system used Agarose type IV for molecular biology with an EEO >0.30 for which we plan to use in the future to optimize the system. The agarose powder is prepared in a potassium phosphate buffer and heated until dissolution.
The cryogel was not made for this phase of the project. The pilocarpine drug delivery would be designed as per Kim & al. This hydrogel is a cryogel which is optimized for controlled pilocarpine drug delivery chosen for osmotic properties among others. When induced by a small current sent from the PCB via conductive inks, pilocarpine is repelled and delivered into the skin, inducing sweat and an electroosmotic flow of molecules which allows sampling of the ISF in addition to sweat.
Silver/ Silver Chloride Ink (Ag/AgCl) and Carbon/ Graphite Paste (CP) were selected to carry current from the biosensor to the printed circuit board. Ag/AgCl ink is durable and a well-characterized reference electrode. CP is necessary to carry current from the Prussian Blue (PB) oxidation reaction to the Ag/AgCl ink. We chose to incorporate Prussian Blue into the carbon/ graphite paste by mortar and pestle with paraffin as a binder as per Moscone.
The electrodes were screen-printed by hand using a DIY screen-printing frame with mesh. The electrode pattern was printed on plastic and cut with an exacto knife into a stencil. Conductive inks used were Ag/AgCl Medical Grade Electrically Conductive Ink (117-23, Creative Materials, MA) with Thinner (102-03, Creative Materials, MA) until desired viscosity was attained. Carbon/ Graphite Paste (901969, Sigma) activated with Prussian Blue (PB), (234125, Sigma) acts as the working and counter electrode. The Prussian Blue electrodes are to be constructed and tested via cyclic voltammeter in iGEM Concordia futures.
Ag/AgCl ink was thinned to desired viscosity and screen-printed onto Silhouette temporary tattoo paper. It was then cured at 80*C for 4min. Carbon/Graphite paste activated with Prussian Blue is screen-printed over the working and counter electrode. An insulator ink is silk-screened over all electrode areas to prevent heat or charge transfer. Chitosan solution(o.5 wt%) in acetic acid was prepared. Yeast cells engineered for fentanyl detection are mixed with Bovine Serum Albumin (BSA), D-Glucose and chitosan hydrogel solution. The resulting mixture is cast onto the working Prussian Blue electrode. The same solution without cells is cast on the counter electrode. The agarose hydrogel containing buffer is prepared and silk-screened over the whole area.
May 23, 2019 - May 24, 2019
May 24, 2019 - May 27, 2019
May 27, 2019 - June 8, 2019
June 8, 2019 - June 20, 2019
June 20, 2019 - July 1, 2019
July 1, 2019 - July 5, 2019
July 5, 2019 - July 11, 2019
July 11, 2019 - July 13, 2019
July 13, 2019 - July 18, 2019
August 18, 2019 - August 21, 2019
September 19, 2019 - September 20, 2019
September 20, 2019 - September 30, 2019
September 30, 2019 - October 20, 2019
July 1, 2019 - July 11, 2019
July 11, 2019 - July 23, 2019
July 23, 2019 - August 11, 2019
August 11, 2019 - August 17, 2019
August 17, 2019 - August 19, 2019
August 19, 2019 - August 30, 2019
August 30, 2019 - September 8, 2019
September 8, 2019 - October 13, 2019
