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

Abstract

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.

Wax-Printed Fluidic Time Delays for Automating Multi-Step Assays in Paper-Based Microfluidic Devices (MicroPADs)

“…Examples of multi-step assays performed on microPADs range from simple enzymatic assays [12][13][14], to more complex paper-based enzyme-linked immunosorbent assays (ELISAs) for diagnostic applications [15][16][17][18][19]. Two examples of multi-step enzymatic assays that we explored in this work include a horseradish peroxidase (HRP) assay [13,[20][21][22], and an enzyme inhibition assay for toxic heavy metal ions [23,24]. The HRP assay requires a multi-step process in which a chromogenic reagent is first incubated with HRP, followed by a sulfuric acid quench.…”

“…The HRP assay requires a multi-step process in which a chromogenic reagent is first incubated with HRP, followed by a sulfuric acid quench. While the HRP assay alone can be used to study enzyme stability [21], it is more commonly used in coupled reactions [14], or as a reporter enzyme in microPADs [13,22]. The enzyme inhibition assay can be used to determine the concentration of toxic heavy metal ions, such as silver (I) and mercury (II), in water samples with limits of detection as low as 0.001 ppm [23].…”

Molecular Methods for Pathogenic Bacteria Detection and Recent Advances in Wastewater Analysis

“…However, the shelf life of paperbased device limits its further applications. Some paper-based devices contain reagents with a short shelf life, such as enzymes, and thus need to be stored in a refrigerator or freezer to maintain the activity of the reagents [158,159]. Studies about the shelf life of enzymes on paper-based devices have yielded some promising results and proven techniques, although further research is still needed.…”

Long-term stabilization of hydrogen peroxide by poly(vinyl alcohol) on paper-based analytical devices

“…Despite the recognition of its importance by researchers, only a few studies have focused on the stability of enzymes 35–37 and antibodies 38 on paper substrates. The stabilization of horseradish peroxidase (HRP) was achieved by trehalose and SU-8 epoxy novolac resin 35 whereas poly(vinyl alcohol) (PVA) has also been used as a reagent for stabilization of enzymes in the PADs 39–41 . However, to the best of our knowledge, no other successful stabilization of chemical reagents in PADs has yet been achieved, which is unfortunate.…”