A Simple, Fast and Portable Method for Electrochemical Detection of Adenine Released by Ricin Enzymatic Activity

Abstract

International authorities classify ricin toxin present in castor seed as a potential agent for use in bioterrorism. Therefore, the detection, identification, and characterization of ricin in various sample matrices are considered necessary actions for risk assessment during a suspected exposure. This study reports a portable electrochemical assay for detecting active ricin based on the adenine electro-oxidation released from herring sperm DNA substrate by its catalytic action. Also, kinetic parameters were calculated, and the values were K_m of 3.14 µM and K_cat 2107 min^-1. A linear response was found in optimized experimental conditions for ricin concentrations ranging from 8 to 120 ng/mL, and with a detection limit of 5.14 ng/mL. This proposed detection strategy emphasizes the possibility of field detection of active ricin in food matrices and can be applied to other endonucleolytic activities.

Keywords: depurination reaction; herring sperm DNA; kinetic analysis; ricin; square wave voltammetry.

Figure 1

Protocol for detecting active ricin using the herring sperm DNA (hsDNA) substrate.

Figure 2

(a) Square wave voltammetry (SWV) for oxidation of 25 μM of adenine at different pH values (4.2–6.6) at the screen printed electrodes (SPEs) in acetate buffer; (b) anodic peak current obtained changing pH values.

Figure 3

(a) SWV with varying concentrations of adenine up to 80 µM (pH 4.6) in the hsDNA sample. The arrow represents increasing adenine concentrations; (b) analytical curve of adenine; (c) progressive curve of adenine released after depurination reaction from hsDNA (8–60 µM) catalyzed by ricin at 100 ng/mL; (d) kinetic curve fitted for ricin catalysis on the hsDNA substrate.

Figure 3

(a) SWV with varying concentrations of adenine up to 80 µM (pH 4.6) in the hsDNA sample. The arrow represents increasing adenine concentrations; (b) analytical curve of adenine; (c) progressive curve of adenine released after depurination reaction from hsDNA (8–60 µM) catalyzed by ricin at 100 ng/mL; (d) kinetic curve fitted for ricin catalysis on the hsDNA substrate.

Figure 4

(a) SWV voltamograms of hsDNA treated with increasing concentrations of active ricin from 0 to 120 ng/mL; (b) released adenine after depurination from hsDNA catalyzed by different ricin levels.