# Rapid, label-free surface plasmon resonance discrimination between Bothrops and Crotalus venoms using clinical antivenom as the capture reagent

## TL;DR

A new biosensor can quickly and accurately detect Bothrops snake venom without cross-reacting with Crotalus venom, helping improve snakebite treatment.

## Contribution

A label-free SPR biosensor using clinical antivenom for rapid, specific detection of Bothrops venom with no cross-reactivity to Crotalus venom.

## Key findings

- The biosensor detected Bothrops venom at concentrations as low as 0.848 μgmL−1.
- No cross-reactivity was observed between Bothrops antivenom and Crotalus venom.
- Venom detection occurred within 14 minutes, showing rapid diagnostic capability.

## Abstract

Snake envenoming is recognized as a global health problem, affecting thousands of people every year. One of the main challenges in addressing this issue is the correct identification and treatment of these envenomations, particularly in locations where people do not have easy access to hospitals. In Brazil, the genus Bothrops is responsible for the majority of envenomations, followed by Crotalus. This study reports a simple methodology for detecting crude venom from snakes of the genus Bothrops, through interaction with their corresponding antibodies, using a high-sensitivity optical biosensor. The protocol consists of adding antibodies (present in the commercial antivenom) to the sensor surface, followed by the addition of Crotalus venom (nonspecific), and then Bothrops venom (specific), resulting in changes in the refractive index, to evaluate cross reactions between them. Different concentrations of raw venom from snakes of the genus Bothrops and Crotalus were tested, starting with a concentration of 6.784 μgmL−1 and progressing until reaching the minimum detectable concentration. The binding capacity of venom to antivenom was investigated at two concentrations of antivenom: 5 μgmL−1 and 50 μgmL−1. Both antivenom and snake venoms were solubilized in phosphate-buffered saline (PBS). The results show an accurate detection of the antigen of interest (Bothrops venom), tested at different concentrations. The biosensor was able to detect venom up to a concentration of 0.848 μgmL−1. In addition, no interference from nonspecific binding between the Bothrops antivenom and Crotalus venom was detected. The detection of specific venom (Bothrops) occurred in a satisfactory time (up to 14 minutes). The results provide evidence that the biosensor and the methodology employed can be considered a diagnostic model under development, which can help health workers to better identify and treat envenomated people.

This study was conducted to address the critical need for rapid and accurate snakebite diagnosis by developing a specific optical biosensor methodology capable of distinguishing genera Bothrops from Crotalus venom, by using thus clinical antivenom facilitating timely and effective antivenom treatment. We developed a label-free Surface Plasmon Resonance (SPR) biosensor using clinical Bothrops antivenom (BAV) to detect Bothrops venom, finding that the device could rapidly and specifically identify the venom at concentrations as low as 0.848 μgmL−1 with no cross-reactivity to Crotalus venom. These findings indicate that the developed SPR biosensor serves as a rapid, highly sensitive, and reusable diagnostic tool capable of accurately distinguishing Bothrops from Crotalus envenomation using BAV, thereby offering a promising solution for improving clinical decision-making and treatment effectiveness in resource-limited settings.