Researchers create biosensor to detect pesticide

The basic principle of a biological sensor to identify a highly toxic pesticide in water and food was used to devise a rapid test for dengue fever.

By Elton Alisson

Agência FAPESP – Researchers at the Universidade de São Paulo’s São Carlos Physics Institute (IFSC-USP), in collaboration with colleagues from the Universidade Federal do Mato Grosso (UFMT), have created a biological sensor that detects the presence of a highly toxic pesticide in water, soil or food in just minutes. The pesticide in question is methamidophos, which is being banned in Brazil but is still used for several crops in the country.

Developed under the auspices of the National Institute of Science and Technology on Organic Electronics (INEO)—one of the National Institutes of Science and Technology (INCTs) funded by FAPESP and the National Council of Scientific and Technological Development (CNPq)—the sensor could be adapted for the detection of other types of pesticides, according to the researchers. The basic principle behind the device has also led to the development of a rapid test to detect the dengue fever virus.

“We chose methamidophos to be detected by the sensor because, although it has been banned in several countries, there are indications of use of this extremely toxic pesticide in the Mato Grosso State,” said Nirton Cristi Silva Vieira, a post-doctoral student at IFSC (FAPESP fellow) and one of the mentors in the pesticide biosensor project and the rapid dengue test.

Vieira explained that methamidophos is used mainly in soybean crops to kill caterpillars and weevils that attack the oilseed. The pesticide easily penetrates the soil and water table, contaminating water and food and affecting the central nervous system by inhibiting the action of acetylcholinesterase, an enzyme that promotes connections (synapses) between neurons.

In humans, in addition to affecting neurological functions, methamidophos can cause damage to the immunological, reproductive and endocrinological systems and can lead to death.

In partnership with Francisco Eduardo Gontijo Guimarães, an IFSC professor and his mentor during his own doctoral studies, Vieira was a mentor of Izabela Gutierrez de Arruda during her master’s work at UFMT. Arruda’s research was focused on developing a rapid and portable test to detect the presence of methamidophos utilizing acetylcholinesterase. To this end, the researchers developed a pH sensor that measures protons (H+ ions). The sensor consists of a glass sheet comprising silicon oxide on a nanometric scale, on which acetylcholinesterase is immobilized, maintaining high activity.

When the sensor is placed in a solution— such as soybean or tomato extract—that contains a low concentration of methamidophos, the activity of acetylcholinesterase is inhibited and the enzyme produces fewer protons than it would in the absence of the pesticide.

A small device (also developed by the researchers) measures this difference in the quantity of protons produced by the enzyme in the sensor when exposed to different concentrations of the pesticide.

Similar to a glucose monitor used by diabetics, the device indicates the enzyme’s level of activity and, consequently, the methamidophos contamination rate of the sample, based on tension standards. These standards are measured by the researchers at different concentrations of acetylcholine, a substance that acts as a neurotransmitter and is similar to the pesticide.

“As we introduced the sensor into solutions with different concentrations of pesticide, the activity of acetylcholinesterase, measured in terms of potential differences, varied, and we managed to quantify it,” explained Vieira.

Other applications

According to Vieira, the sensor could be adapted to detect other categories of pesticides in the classes of carbamates and organophosphates—the same family that methamidophos is in—that also inhibit the activity of acetylcholinesterase.

For this detection to be feasible, however, measuring the activity of different concentrations of each pesticide would be necessary to prevent one from occulting another. “The standard electric signal in other categories of pesticides could vary because the inhibition of the action mechanism of acetylcholinesterase by each pesticide is different. For this reason, the sensor must be recalibrated to detect them,” said Vieira.

She said that the biosensor has sparked the interest of a biotechnology company in Minas Gerais for manufacturing and commercialization. The estimated cost of the device—including the sensor and the gauge—will be between R$ 100 and R$ 200 per unit.

The most expensive component, according to the researchers, is acetylcholinesterase. In an attempt to replace this enzyme, scientists will attempt to obtain another type of enzyme from fruits, such as avocado and banana, with properties similar to those of acetylcholinesterase.

“We currently buy a purified form of the enzyme, which is very expensive. The idea is to obtain the raw extract of an enzyme similar to acetylcholinesterase from fruit to measure the concentrations of pesticide,” said Vieira.

Currently, according to the researchers, analyses of pesticide contamination in the Mato Gross State are sent to São Paulo or Rio de Janeiro and take days to be processed.

The investigators emphasize that a biosensor will help to reduce the cost and time required to obtain results to just a few minutes. “In order to analyze contaminated soil samples, for example, just mix them with water to soak the earth, leaving the sensor immersed for 15 minutes in the solution containing the dissolved pesticide, and place it on the gauge to obtain a contamination rate,” Vieira explained.

The idea of developing the sensor emerged during an encounter between researchers at IFSC and their UFMT colleagues at INEO and resulted in the Mato Grosso university’s first patent request in its 40 years of existence.

“At one of the annual INEO meetings, we contacted a group of UFMT researchers who had the idea of developing a pesticide sensor because Mato Grosso is the largest grain-producing state in Brazil and currently uses methamidophos in its crops,” Guimarães said. “At the time, Vieira was doing research on biosensors, and we decided to initiate a collaborative project with the UFMT group, led by Professor Romildo Jerônimo Ramos, to develop this pesticide biosensor.”

Dengue virus detection

During his ongoing postdoctoral studies, Vieira intends to develop biosensors that use antibodies instead of enzymes, such as those in the methamidophos biosensor, to detect protein markers of dengue virus contamination and the onset of acute myocardial infarction.

In partnership with a Brazilian biotechnology company, Vieira, along with IFSC master’s student Alessandra Figueiredo and Professor Guimarães, developed a system to detect the NS1 protein secreted by the virus in the first days of infection. “This protein marks the presence of the dengue fever virus and, consequently, the beginning of infection,” he said.

According to Vieira, the majority of existing sensors focus on detecting dengue fever infection indirectly, using an immobilized antibody that is linked to the NS1 protein and needs a secondary antibody, which is generally marked with other molecules.

Based on the same biosensor principle as for detection of methamidophos, a sensor developed by the researchers promises to detect the presence of NS1 protein precisely and directly. “The sensor for detection of dengue fever infection is in the patenting process. We still have not found a final product,” said Vieira.