Development and implementation of an extended-gate ion-sensitive field-effect-transistor (EGFET) biosensor for the detection of β-lactamase activity

Antimicrobial resistance (AMR) is a major threat to global public health. A leading cause behind the acceleration of AMR is the inappropriate and unnecessary use of antibiotics. To enable informed prescribing behaviours and to preserve the efficacy of existing antibiotics, better diagnostic technology is required that enables analysis of bacterial susceptibility, at the time of prescription. One of the most common forms of resistance is to β-lactam antibiotics such as penicillins. This resistance is primarily caused by β-lactamase enzymes, which catalytically hydrolyse β-lactam drugs, rendering them ineffective. Current methods for point-of-care detection of these enzymes often use pH-sensitive dyes to detect solution acidification caused by β-lactam hydrolysis. However, these assays are typically qualitative and have been shown to be ambiguous for cases of low-level enzyme expression. This work describes the development and testing of an extended-gate ion-sensitive field-effect transistor (EGFET) as the transduction element of an acidimetric β-lactamase assay. The EGFET uses a discrete, commercial metal-oxide-semiconductor field-effect transistor (MOSFET) coupled to an IrO electrode which is capable of producing high pH sensitivities above 60 mV/pH with a less than two second time-response. This device was used to perform quantitative measurements of β-lactamase activity. Measurements were demonstrated for a β-lactamase blend containing as low as 0.4-0.7 IU β-lactamase I and 0.06-0.1 IU β-lactamase II. Additionally, this work demonstrates the use of a surface-bindable β-lactam antibiotic which reacts in the presence of β-lactamase enzymes in both buffer and physiological media (undiluted urine). By localising the hydrolysis reaction to the sensor surface, it is anticipated that the combination of the surface-tethered antibiotic and EGFET will increase the sensitivity of the assay. Such high-sensitivity, quantitative measurements of β-lactamase activity in a miniaturised device, could ultimately provide an unambiguous assay for point-of-care detection of β-lactam resistance.