Toxin detection by a miniaturized in vitro protein expression array.

Abstract

A novel concept for toxin detection is presented that is based on the inhibitory effects of certain toxins on protein synthesis. Biological synthesis of proteins is called protein expression, which consists of the steps of DNA transcription and protein translation. In the toxin detection concept, a group of proteins are expressed simultaneously in a device consisting of an array of miniaturized wells. The expression yields of these proteins are inhibited by presence of a toxin. Differential inhibitory effects of each toxin on the expression of proteins in wells result in a response pattern (or signature) of the array device, which can be used as a tool for detection and identification of known and unknown agents. The concept has been demonstrated by in vitro protein expression of three proteins, including green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), and luciferase. The production yields of these proteins in a cell-free medium were inhibited differentially by two toxin simulants, tetracycline (TC) and cycloheximide (CH). We confirmed that TC has an inhibitory effect on the production of GFP and CAT in the E. coli expression system whereas CH has a negligible effect. Moreover, the degree of TC's inhibitory effect on GFP is different from that on CAT. We also observed an opposite disparity; TC has a negligible inhibitory effect on the luciferase production in the rabbit reticulocyte expression system, whereas CH has a significant inhibitory effect. In addition, we obtained a response pattern in a 3 x 4 array device; the results suggest the feasibility of toxin detection based on the mechanisms of toxin actions. The device and method are also likely applicable to high-throughput drug screening, evident from the fact that TC is an antibiotic medicine.