A choline oxidase amperometric bioassay for the detection of mustard agents based on screen-printed electrodes modified with Prussian Blue nanoparticles

Abstract

In this work a novel bioassay for mustard agent detection was proposed. The bioassay is based on the capability of these compounds to inhibit the enzyme choline oxidase. The enzymatic activity, which is correlated to the mustard agents, was electrochemically monitored measuring the enzymatic product, hydrogen peroxide, by means of a screen-printed electrode modified with Prussian Blue nanoparticles. Prussian Blue nanoparticles are able to electrocatalyse the hydrogen peroxide concentration reduction at low applied potential (-50 mV vs. Ag/AgCl), thus allowing the detection of the mustard agents with no electrochemical interferences. The suitability of this novel bioassay was tested with the nitrogen mustard simulant bis(2-chloroethyl)amine and the sulfur mustard simulants 2-chloroethyl ethyl sulfide and 2-chloroethyl phenyl sulfide. The bioassay proposed in this work allowed the detection of mustard agent simulants with good sensitivity and fast response, which are excellent premises for the development of a miniaturised sensor well suited for an alarm system in case of terrorist attacks.

Figure 1.

Photo of an SPE modified with PBNPs. Inset: SEM imagine of the working electrode modified with PBNPs (PBNP diameter 95 ± 15 nm). The 5 cent euro coin reference has a diameter of 22.25 mm.

Figure 2.

Bioassay response by varying the amount of choline oxidase. Choline at final concentration of 0.5 mM, applied potential: −50 mV vs. Ag/AgCl, 0.05 M phosphate buffer + 0.1 M KCl, pH 7.4. Measurements were carried out in triplicate.

Figure 3.

Choline calibration plot. Choline oxidase concentration: 180 mU/mL; applied potential: −50 mV vs. Ag/AgCl; 0.05 M phosphate buffer + 0.1 M KCl, pH 7.4. Measurements were carried out in triplicate.

Figure 4.

Original recording obtained using the bioassay. Applied potential −50 mV vs. Ag/AgCl; Choline oxidase concentration: 180 mU/mL. Signal recorded in 0.05 M phosphate buffer solution + 0.1 M KCl, pH 7.4 (grey line) and in a solution of choline (0.5 mM) in 0.05 M phosphate buffer solution + 0.1 M KCl, pH 7.4 in absence (green line) and in presence of bis(2-chloroethyl)amine 0.25 mM (blue line) and 25 mM (pink line).

Figure 5.

(A) Study of percentage inhibition vs. incubation time. Choline oxidase concentration: 180 mU/mL; choline concentration: 0.5 mM; bis(2-chloroethyl)amine concentration: 2.5 mM; phosphate buffer 0.05 M + 0.1 M KCl, pH 7.4; potential: −50 mV vs. Ag/AgCl; (B) Study of percentage inhibition vs. enzymatic units. Choline concentration: 0.5 mM; bis(2-chloroethyl)amine concentration: 2.5 mM; phosphate buffer 0.05 M + 0.1 M KCl, pH 7.4; potential: −50 mV vs. Ag/AgCl; incubation time: 120 s. Measurements were carried out in triplicate.

Figure 6.

Lineweaver-Burk diagram for bis(2-chloroethyl)amine. Amperometric measurements of different concentrations of bis(2-chloroethyl)amine: plot a (black line): 2.5 mM; plot b (green line): 1 mM; plot c (red line): 0 mM. Choline oxidase concentration: 180 mU/mL; choline concentration: 0.5 mM; phosphate buffer 0.05 M + 0.1 M KCl, pH 7.4; potential: −50 mV vs. Ag/AgCl; incubation time: 120 s. Measurements were carried out in triplicate.

Figure 7.

(A) Study of percentage inhibition vs. incubation time. Choline oxidase concentration: 180 mU/mL; choline concentration: 0.5 mM; 2-chloroethyl ethyl sulfide concentration: 0.01 mM; Phosphate buffer 0.05 M + 0.1 M KCl, pH 7.4; potential: −50 mV vs. Ag/AgCl; (B) Study of percentage inhibition vs enzymatic units. Choline concentration: 0.5 mM; 2-chloroethyl ethyl sulfide concentration: 0.01 mM; Phosphate buffer 0.05 M + 0.1 M KCl pH 7.4; potential: −50 mV vs. Ag/AgCl; incubation time: 120 s. Measurements were carried out in triplicate.

Figure 8.

(A) Study of percentage inhibition vs. incubation time. Choline oxidase concentration: 180 mU/mL; choline concentration: 0.5 mM; 2-chloroethyl phenyl sulfide concentration: 0.68 mM; Phosphate buffer 0.05 M + 0.1 M KCl, pH 7.4; potential: −50 mV vs. Ag/AgCl; (B) Study of percentage inhibition vs. enzymatic units. Choline concentration: 0.5 mM; 2-chloroethyl phenyl sulfide concentration: 0.68 mM; Phosphate buffer 0.05 M + 0.1 M KCl, pH 7.4; potential: −50 mV vs. Ag/AgCl; incubation time: 120 s. Measurements were carried out in triplicate.

Scheme 1.

Mustard agents.