The epidemiological statistics for mosquito-borne diseases are staggering. In Brazil, according to the Ministry of Health’s Surveillance Department (SVS-MS), about 1.5 million cases of dengue were reported in 2016, as well as some 272,000 cases of chikungunya fever and 215,000 cases of Zika fever. It also says 143,000 cases of malaria were notified in 2015.
Strategies to defeat these epidemics include prevention by combating the various species of mosquito that transmit the viruses concerned, development of vaccines, epidemiological surveillance with rapid diagnosis of the sick, and clinical and outpatient treatment.
In the sphere of epidemiological surveillance, research groups in Brazilian universities are developing low-cost biosensors to speed up diagnosis. For example, the Biopol group in the Chemistry and Biochemistry & Biology Departments of the Federal University of Paraná (UFPR) in Curitiba are developing an immunochip to detect dengue.
“Dengue is detected indirectly in our sensor,” said Cleverton Pirich, a PhD student in biochemistry and one of the authors of the study. “What it detects is not the virus but an antigen that’s characteristic of infection by dengue. This is done by means of antibodies immobilized on the biosensor, which rapidly detect the presence of the antigen in a blood sample, indirectly diagnosing infection.”
The immunochip can detect the presence of molecules of the antigen (NS1) for dengue in blood serum, quickly providing a positive or negative result. The results of the study were published in the journal Biosensors and Bioelectronics.
This is made possible by using an electrochemical property called the piezoelectric effect – the ability of certain materials, especially quartz crystals, to generate a voltage when subjected to mechanical stress or vibration.
Piezoelectric sensors are devices that use the piezoelectric effect to measure changes in pressure, acceleration, temperature, strain or force by converting them to an electrical charge.
To validate the results and guarantee the efficiency of the immunochip developed in Curitiba, Pirich and his supervisor, Professor Maria Rita Sierakowski, had recourse to Roberto Manuel Torresi, a professor in the University of São Paulo’s Chemistry Institute (IQ–USP) who certified the functioning of the quartz crystal microbalance with energy dissipation monitoring (QCM-D) and their interpretation of the results.
“A microbalance uses the reverse piezoelectric effect. In the specific case of the new immunochip, an electrical charge is applied to the crystal and the frequency of this signal changes when molecules of the dengue antigen NS1 present in a sample are deposited on the crystal,” Torresi said.
Like the immunochip, the QCM-D also uses the reverse piezoelectric effect. However, its precision is far greater and it can also detect clock changes.
“The microbalance in our lab is far more sophisticated than the others in Brazil,” said Torresi, also one of the authors of the article published in Biosensors and Bioelectronics and principal investigator for the Thematic Project “Optimization of the physicochemical properties of nano-structured materials for applications in molecular recognition, catalysis and energy conversion/storage”.
IQ–USP’s microbalance (QCM-D), as well as the Biopol group’s two devices (a QCM purchased with the support of CAPES, the Ministry of Education’s Office for Faculty Development, and a QCM-D purchased with the support of FINEP, the Brazilian Innovation Agency) confirmed the presence of NS1 in all the blood samples contaminated by addition of the dengue antigen, thus validating detection via the immunochip.
“The immunochip was developed to detect molecules of the dengue antigen in any material in a liquid medium, but the principle can be used to detect other diseases, and in environmental or public health applications to detect contaminants in water, food or elsewhere in the environment,” Sierakowski said.
The substrate for the immunochip is an imported quartz crystal on which the other components are deposited in thin layers. The first layer on top of the crystal is gold, and the next is a polyethylenimine film.
The third and last layer is a thin film of oxidized nanocrystals derived from chemical treatment of cellulose industrial waste and prepared so as to react chemically in the presence of the dengue antigen. The chemical reaction leads to a change in the response of the nanocrystals, which is reflected in turn by a change in the patterns of frequency and energy dissipation.
It is the precise measurement of such changes in frequency and energy dissipation patterns that indicates whether the dengue antigen is present and hence whether the patient from whom the blood sample was taken has been infected by dengue virus.
The process may appear lengthy, but after development of the biosensor the response is practically immediate. Drops from the sample are placed on the biosensor: the presence of the NS1 antigen can be determined using no more than 0.03 microgram per milliliter.
“What matters most to patients in terms of diagnosis isn’t knowing how many molecules of the antigen there are in the sample but knowing whether they’re infected and if so, starting the right treatment as soon as possible,” Pirich said. “The aim is a qualitative diagnosis, a positive or negative result. Our proposal paves the way for the development of simpler and more affordable equipment to fulfill this purpose.”
“As discussed and shown by our study, if an immunochip of this type is developed and commercialized it could be a real-time diagnostic tool capable of providing results in approximately 15 minutes.”
The article “Piezoelectric immunochip coated with thin films of bacterial cellulose nanocrystals for dengue detection” (doi: http://dx.doi.org/10.1016/j.bios.2017.01.068) by Cleverton Luiz Pirich, Rilton Alves de Freitas, Roberto Manuel Torresi, Guilherme Fadel Picheth and Maria Rita Sierakowski can be retrieved from: sciencedirect.com/science/article/pii/S0956566317300684.