Stabilization of Horseradish Peroxidase Using Epoxy Novolac Resins for Applications with Microfluidic Paper-Based Analytical Devices

Microfluidic paper-based analytical devices (microPADs) are an emerging platform for point-of-care diagnostic tests for use by untrained users with potential applications in healthcare, environmental monitoring, and food safety. These devices can be developed for a multitude of different tests, many of which employ enzymes as catalysts. Without specialized treatment, some enzymes tend to lose their activity when stored on microPADs within 48 hours, which is a major hurdle for taking these types of devices out of the laboratory and into the real world. This work focused on the development of simple methods for stabilizing enzymes by applying polymers to chromatography paper. The longterm stabilization was exlored and SU-8 of various concentrations was found to stabilize horseradish peroxidase for times in excess of two weeks. A variety of microPAD fabrications, enzyme dispensing methods, and substrate delivery techniques were explored

Paper-based standard addition assays

Standard addition assays conducted on paper-based microfluidic devices are introduced as an alternative to external standards for calibrating quantitative tests. To demonstrate this technique, a colorimetric, paper-based, standard addition assay was optimized for the determination of glucose concentrations in the range of 0 to 5 mM. Comparable results were obtained from the assay via digital image colorimetry under three different lighting conditions

Paper Microzone Plates as Analytical Tools for Studying Enzyme Stability: A Case Study on the Stabilization of Horseradish Peroxidase Using Trehalose and SU-8 Epoxy Novolac Resin

Paper microzone plates in combination with a noncontact liquid handling robot were demonstrated as tools for studying the stability of enzymes stored on paper. The effect of trehalose and SU-8 epoxy novolac resin (SU-8) on the stability of horseradish peroxidase (HRP) was studied in both a short-term experiment, where the activity of various concentrations of HRP dried on paper were measured after 1 h, and a long-term experiment, where the activity of a single concentration of HRP dried and stored on paper was monitored for 61 days. SU-8 was found to stabilize HRP up to 35 times more than trehalose in the short-term experiment for comparable concentrations of the two reagents, and a 1% SU-8 solution was found to stabilize HRP approximately 2 times more than a 34% trehalose solution in both short- and long-term experiments. The results suggest that SU-8 is a promising candidate for use as an enzyme-stabilizing reagent for paper-based diagnostic devices and that the short-term experiment could be used to quickly evaluate the capacity of various reagents for stabilizing enzymes to identify and characterize new enzyme-stabilizing reagents

Reagent pencils: A new technique for solvent-free deposition of reagents onto paper-based microfluidic devices

Custom-made pencils containing reagents dispersed in a solid matrix were developed to enable rapid and solvent-free deposition of reagents onto membrane-based fluidic devices. The technique is as simple as drawing with the reagent pencils on a device. When aqueous samples are added to the device, the reagents dissolve from the pencil matrix and become available to react with analytes in the sample. Colorimetric glucose assays conducted on devices prepared using reagent pencils had comparable accuracy and precision to assays conducted on conventional devices prepared with reagents deposited from solution. Most importantly, sensitive reagents, such as enzymes, are stable in the pencils under ambient conditions, and no significant decrease in the activity of the enzyme horseradish peroxidase stored in a pencil was observed after 63 days. Reagent pencils offer a new option for preparing and customizing diagnostic tests at the point of care without the need for specialized equipment

A Kinetic Isotope Effect and Isotope Exchange Study of the Nonenzymatic and the Equine Serum Butyrylcholinesterase-Catalyzed Thioester Hydrolysis

Formylthiocholine (FTC) was synthesized and found to be a substrate for nonenzymatic and butyrylcholinesterase (BChE)-catalyzed hydrolysis. Solvent (D₂O) and secondary formyl-H kinetic isotope effects (KIEs) were measured by an NMR spectroscopic method. The solvent (D₂O) KIEs are ^D₂O<i>k</i> = 0.20 in 200 mM HCl, ^D₂O<i>k</i> = 0.81 in 50 mM HCl, and ^D₂O<i>k</i> = 4.2 in pure water. The formyl-H KIEs are ^D<i>k</i> = 0.80 in 200 mM HCl, ^D<i>k</i> = 0.77 in 50 mM HCl, ^D<i>k</i> = 0.75 in pure water, ^D<i>k</i> = 0.88 in 50 mM NaOH, and ^D(<i>V</i>/<i>K</i>) = 0.89 in the BChE-catalyzed hydrolysis in MES buffer at pH 6.8. Positional isotope exchange experiments showed no detectable exchange of ¹⁸O into the carbonyl oxygen of FTC or the product, formate, under any of the above conditions. Solvent nucleophile-O KIEs were determined to be ¹⁸<i>k</i> = 0.9917 under neutral conditions, ¹⁸<i>k</i> = 1.0290 (water nucleophile) or ¹⁸<i>k</i> = 0.989 (hydroxide nucleophile) under alkaline conditions, and ¹⁸(<i>V</i>/<i>K</i>) = 0.9925 for BChE catalysis. The acidic, neutral, and BChE-catalyzed reactions are explained in terms of a stepwise mechanism with tetrahedral intermediates. Evidence for a change to a direct displacement mechanism under alkaline conditions is presented