A 96-Well Electrochemical Method for the Screening of Enzymatic Activities

Abstract

The rapid electrochemical screening of enzyme activities in bioelectronics is still a challenging issue. In order to solve this problem, we propose to use a 96-well electrochemical assay. This system is composed of 96 screen-printed electrodes on a printed circuit board adapted from a commercial system (carbon is used as the working electrode and silver chloride as the counter/reference electrode). The associated device allows for the measurements on the 96 electrodes to be performed within a few seconds. In this work, we demonstrate the validity of the screening method with the commercial laccase from the fungus Trametes versicolor. The signal-to-noise ratio (S/N) is found to be the best way to analyze the electrochemical signals. The S/N follows a saturation-like mechanism with a dynamic linear range of two decades ranging from 0.5 to 75 ng of laccase (corresponding to enzymatic activities from 62 × 10(-6) to 9.37 × 10(-3) μmol min(-1)) and a sensitivity of 3027 μg(-1) at +100 mV versus Ag/AgCl. Laccase inhibitors (azide and fluoride anions), pH optima, and interfering molecules could also be identified within a few minutes.

High‐Throughput Electrochemical Screening Assay for Free and Immobilized Oxidases: Electrochemiluminescence and Intermittent Pulse Amperometry

“…Electrochemical assays were performed with an AndCare 9600 sensor (Alderon Biosciences Inc., Beaufort, USA), which allowed a potential to be applied on the 96 electrodes at the same time. Measurements were performed by intermittent pulse amperometry (IPA): series of millisecond pulses of potential were applied to the working electrode, separated by periods during which the circuit was open . Electrodes were used once before recycling of the printed circuit board by washing with acetone as described previously …”

“…intermittent pulse amperometry (IPA): series of millisecond pulses of potential were applied to the working electrode, separated by periods during which the circuit was open. [26] Electrodes were used once before recycling of the printed circuit board by washing with acetone as described previously. [26] Preparation of Enzyme-Modified Electrodes…”

“…[26] Electrodes were used once before recycling of the printed circuit board by washing with acetone as described previously. [26] Preparation of Enzyme-Modified Electrodes…”

“…This leads to lower sensitivities or to a limited dynamic range. To overcome these limitations, we previously proposed an approach to detect 96 enzymatic activities within a short timeframe (1 to 3 min) by using intermittent pulse amperometry (IPA) …”

“…To overcome these limitations, we previously proposed an approach to detect96enzymatic activities within as hort timeframe (1 to 3min) by using intermittent pulse amperometry (IPA). [26,27] This method is appliedt os creen the activities of dehydrogenasea nd laccase frome nzymel ibraries by measuring electron transfer between the enzymesa nd the electrodes. The system consists of 96 working carbon electrodes and 96 Ag/ AgCl counter-referencee lectrodes screen printed onto ap rinted circuitb oard that operates as ac onnector with the dedicated electrochemical reader.…”