Botulinum Neurotoxin Serotypes Detected by Electrochemical Impedance Spectroscopy

Abstract

Botulinum neurotoxin is one of the deadliest biological toxins known to mankind and is able to cause the debilitating disease botulism. The rapid detection of the different serotypes of botulinum neurotoxin is essential for both diagnosis of botulism and identifying the presence of toxin in potential cases of terrorism and food contamination. The modes of action of botulinum neurotoxins are well-established in literature and differ for each serotype. The toxins are known to specifically cleave portions of the SNARE proteins SNAP-25 or VAMP; an interaction that can be monitored by electrochemical impedance spectroscopy. This study presents a SNAP-25 and a VAMP biosensors for detecting the activity of five botulinum neurotoxin serotypes (A–E) using electrochemical impedance spectroscopy. The biosensors are able to detect concentrations of toxins as low as 25 fg/mL, in a short time-frame compared with the current standard methods of detection. Both biosensors show greater specificity for their compatible serotypes compared with incompatible serotypes and denatured toxins.

Rapid Detection of Botulinum Neurotoxins—A Review

“…The main advantage being that they allow for a high sensitivity to be achieved in relation to surface interactions and changes [94]. Some of the areas in which these methodologies are being utilised include sensors for various biomarkers [95,96,97] and toxins [98], along with pathogens [99,100]; this is not only due to the high levels of sensitivity, but also because these techniques yield rapid detection times compared to the mouse bio-assay [101]. Both cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) have been utilised in the development of BoNT sensors [94,101].…”

“…Some of the areas in which these methodologies are being utilised include sensors for various biomarkers [95,96,97] and toxins [98], along with pathogens [99,100]; this is not only due to the high levels of sensitivity, but also because these techniques yield rapid detection times compared to the mouse bio-assay [101]. Both cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) have been utilised in the development of BoNT sensors [94,101]. Briefly, in CV a charge can be measured that relates to the number of species on a working electrode that are able to be oxidised, as well as the number of electrons involved in the oxidation [102].…”

“…An electrochemical impedance spectroscopy detection method was developed utilising the ferri/ferrocyanide redox probe to measure the impedance that occurred at the surface of a SNAP-25 and VAMP modified gold electrode [101]. Both proteins were used and tested due to their ability to detect different serotypes of BoNT, (Table 2).…”

“…Upon incubation with the toxin the proteins immobilised on the electrode surface would become cleaved leading to the loss of a small fragment, ~9 amino acids (Figure 6). This leads to a measurable decrease in the Rct, allowing for the quantification of the concentration of BoNT present [101].…”

“…The EIS detection method was able to distinguish the presence of active BoNT to a sensitivity level of 25 fg/mL with slight variation between serotypes [101]. The ability to distinguish active toxin is a key component of MBA replacement assays, as it is the primary objective required in the pharmaceutical industry.…”