A rapid enzyme assay for � -galactosidase using optically gated sample introduction on a microfabricated chip

Abstract

The ability to perform enzyme assays on microchips is demonstrated using optically gated sample introduction. The hydrolysis of fluorescein mono- beta- d-galactopyranoside (FMG) by beta- d-galactosidase ( beta-Gal) is continuously monitored using a microchip for 5 to 10 min. The outcome of the reaction was analyzed by performing serial on-chip separations of fluorescent substrate, FMG, and product, fluorescein. Kinetic information about beta-Gal has been successfully obtained by varying the concentration of FMG. beta-Gal enzymes from two different sources including bovine liver and E. coli., have been examined and compared to each other and to results obtained using traditional assay methods. In addition, the competitive inhibition of beta-Gal by phenylethyl beta- d-thiogalactoside (PETG) and beta-lactose has been studied using this technique. PETG is found to have higher inhibition than lactose in the hydrolysis. This separation-based enzyme assay technique avoids the possible fluorescence interference between FMG and fluorescein, which is a problem with the traditional plate assay method. Additionally, the amount of the enzyme and substrate required with this technique is at least four orders of magnitude lower than the traditional plate assay method. By using optically gated sample introduction, microchips allow continuous serial injections and separations without any potential switch, thus making this technique ideal as a sensor for enzyme assays. This technique should therefore be valuable for high-throughput screening in the drug discovery industry.

Near-infrared laser illumination transforms the fluorescence absorbing X-Gal reaction product BCI into a transparent, yet brightly fluorescent substance

“…Indeed, such quenching blocks photoactivation in tissue areas of very high BCI precipitation, and one should not attempt to overcome this using continuous or intense exposure. Recent work indicates the use of a fluorescent product formation through β-galactosidase enzymatic activity [5,26] in continuous monitoring for plate assay methods or flow cytometry [2,6,20]. However, the reaction products do not allow imaging with transmitted or reflected light, and the photostability of one of these products (fluorescein) is possibly inferior to the photoactivated BCI product presented here.…”

“…Our data clearly imply that diverse applications could benefit from studying such properties more closely. Such applications are relevant for diagnostics of human diseases and drug discovery exploiting minichip-based essays, as recently proposed for the fluorescent β-galactosidase reaction [26].…”

“…Several other substrates resulting in red, green or purple modifications of XGal have been developed, and fluorescent substrates have been recently introduced [5,6,24]. Even substrates useful for nuclear magnetic resonance (NMR) are now available, increasing the versatility of β-galactosidase [26,29]. However, none of these modifications allows simple timing through direct observation of reaction progression.…”

High-throughput enzyme assay on a multichannel microchip using optically gated sample introduction

“…This provides a very rapid injection time which is repeatable at high repetition rate. Recently, this sample introduction method also has been used in a single channel on a CE chip to perform rapid monitoring for the hydrolysis of fluorescein mono-β-D-galactopyranoside (FMG) by β-galactosidase (β-Gal) and to evaluate the effect of specific inhibitors on enzyme activity [24]. With the advantage of serial injections and separations, optically gated sample introduction has been used to continuously monitor enzyme reaction rates with little intervention from an operator and also avoiding signal interference between FMG and the reaction product, fluorescein.…”

“…As both the substrate, FMG, and the product, fluorescein, are fluorescent, interference when detecting either has been a problem for plate assay methods used to study this hydrolysis. This problem can be overcome by determining substrate or product concentrations after on-chip electrophoretic separation of FMG and fluorescein with optically gated sample introduction and LIF detection for FMG and fluorescein, as shown previously [24].…”