Sensor detects dengue before the first symptoms appear

Researchers at the University of São Paulo, working in partnership with a biotechnology company, have developed a device that instantly detects a protein secreted by all four types of the virus that cause the disease.

By Elton Alisson

Agência FAPESP – Researchers at the São Carlos Institute of Physics of the University of São Paulo (IFSC-USP) and DNApta Biotecnologia, a company based in São José do Rio Preto, São Paulo State, Brazil, have developed a biosensor capable of detecting dengue before the first symptoms of the disease appear.

Created as part of a master’s degree project undertaken by graduate student Alessandra Figueiredo along with postdoctoral work performed by Nirton Cristi Silva Vieira with a scholarship from FAPESP, the device was developed under the aegis of the Nanomedicine & Nanotoxicology Group at IFSC-USP, led by Professor Valtencir Zucolotto, in collaboration with Professor Francisco Guimarães of the National Institute of Science and Technology on Organic Electronics (INEO), one of the National Science & Technology Institutes (INCTs) funded by FAPESP and the National Council for Scientific & Technological Development (CNPq). The biosensor was described in an article in Nature Publishing Group’s Scientific Reports.

“The biosensor is capable of diagnosing dengue faster, at a lower cost and more easily than the laboratory tests that exist today,” said Vieira, a postdoctoral scholar at IFSC-USP and one of the authors of the article.

The biosensor technology is based on the electrical detection of non-structural protein 1 (NS1), which is secreted by all four dengue viruses (DEN1, DEN2, DEN3 and DEN4). The protein is found at detectable concentrations from the second to ninth days after onset of the disease in the blood of people with both primary and secondary infections (first and additional dengue infections, respectively). This makes NS1 an excellent biomarker of dengue virus infection, Vieira told Agência FAPESP.

“The advantage of using NS1 to detect dengue is that the disease can be diagnosed sooner, as early as the second or third day after infection,” said Vieira. “On average, the symptoms of dengue don’t start to appear until the sixth day after the patient is bitten by the Aedes aegypti mosquito.”

NS1 secreted by the dengue virus is typically detected using antibodies such as immunoglobulin G (IgG), which is obtained either via the fusion of B lymphocytes from the spleens of immunized animals with myeloma cells (B-cell tumor line) or by extraction from the blood of mammals inoculated with NS1.

Both processes have drawbacks. The B-cell fusion process is very expensive. The problem with obtaining antibodies from NS1 antibodies in mammalian blood is that the quantity is very small. “The process is extremely low-yield,” Vieira said.

To increase the production of NS1 antibodies, DNApta Biotecnologia developed a technique whereby recombinant NS1 from all four dengue virus serotypes is produced in E. coli bacteria and then injected into laying hens. Recombinant proteins are artificially produced in the laboratory from cloned genes.

Using this technique, the biotech company can obtain large amounts of IgY, the immunoglobulin from the yolks of the eggs laid by hens inoculated with recombinant NS1, as a substitute for IgG, the immunoglobulin extracted from mammalian blood.

“Hens are great producers of antibodies. A very large quantity of IgY can be obtained from the yolks of eggs laid by hens inoculated with NS1,” said Sérgio Moraes Aoki, the scientific director of DNApta.

The company supplied recombinant NS1 from dengue virus and hen egg yolk immunoglobulin (IgY) to the researchers at IFSC-USP to develop the dengue biosensor. The company shares the patent on the device with the USP Innovation Agency, the university’s technology transfer office.

“This is the first time IgY has been used as a biological recognition element in a biosensor designed to recognize NS1 protein,” Aoki said.

Sensor composition

The biosensor developed by the research group consists of a nanometer-scale gold electrode with a sample of IgY immobilized on the top and a reference electrode with constant electrical potential.

When the electrode with immobilized IgY comes into contact with NS1, its electrical potential changes relative to the reference electrode because the connection between the protein and the antibody produces an electrical signal.

The electrical signal is “read” by a computer using custom software, which presents the findings within 30 minutes at most. Test results can be accessed in real time via a smartphone or laptop.

“The higher the concentration of NS1 in contact with the electrode with immobilized IgY, the greater the difference in electrical potential,” Vieira explained. To assess the biosensor’s effectiveness the researchers performed tests with samples of NS1 at concentrations of 0.01-10 micrograms per milliliter (μg/mL), covering the range of NS1 concentrations found in the blood of patients diagnosed with dengue.

The test results showed that the device was capable of detecting the presence of NS1 at a minimum concentration of 0.09 μg/mL.

“The average concentration of NS1 in the blood of people infected by the dengue virus is 2 micrograms per milliliter. The biosensor succeeded in detecting much lower concentrations than this,” Vieira said.

The researchers have gained initial approval from the Ethics Committee of the Federal University of São Carlos (UFSCar) to test the biosensor using blood samples from dengue-infected subjects, starting within a few months after final approval.

“We’ve already developed a prototype sensor,” Vieira said. “The advantage is that IgY, the immunoglobulin obtained from egg yolk used by the sensor, is much cheaper than other antibodies, so the device can be mass-produced.”

New biosensors

In addition to the dengue biosensor developed in partnership with the IFSC-USP research group, DNApta plans to develop new biosensors to detect NS1 protein from the dengue virus using other technological platforms, through the “Development of electrochemical biosensors for detecting dengue virus NS1 protein” project, which is supported by FAPESP’s Innovative Research in Small Business (PIPE) program.

“This next generation of electrochemical biosensors we plan to develop will again use IgY from the egg yolks of laying hens inoculated with NS1, but in this case [the bionsensors] will have carbon electrodes,” Aoki said.

The biotech company also aims to develop portable dengue detection devices that are similar to the portable blood glucose meters used by diabetics but are also capable of being connected to a smartphone or laptop and transmitting data in real time to a center for epidemiological monitoring of the disease, Aoki explained. “We plan to extend this idea to the detection of other diseases besides dengue,” he said.

According to the researchers, the laboratory tests most widely used today to diagnose dengue do not reliably detect the virus in the first few days after infection, when the symptoms of dengue are similar to those of other infectious diseases.

Complete blood count (CBC), erythrocyte sedimentation rate (ESR) and platelet count, for example, are insufficient to confirm a dengue diagnosis.

The tests most widely used by local clinics and health centers, based on serological detection of antibodies such as IgG and IgM, can be performed only from the sixth day of infection onward because the human organism does not produce specific antibodies that combat NS1 before then.

However, existing molecular methods, such as enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), are expensive, entail multiple stages and require specially trained professionals to perform them, according to the researchers.

“We expect the type of dengue test we’re developing to have several advantages over the conventional tests used today,” Aoki said.

The research findings are described in the paper “Electrical detection of dengue biomarker using egg yolk immunoglobulin as the biological recognition element” (doi: 10.1038/srep07865), by Figueiredo et al., which can be read in Scientific Reports at www.nature.com/srep/2015/150119/srep07865/full/srep07865.html.