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Advances in diagnostics

New sensors developed in Brazil for clinical analysis of dengue are more sensitive and able to provide earlier detection

01-01Raul AguiarTwo recently developed sensors may lead to faster and cheaper methods of clinical analysis and diagnosis of diseases. In San Carlos, a team at the University of São Paulo (USP) has perfected a type of chemical transducer, known as a contactless conductivity detection system (C4D), making it 10,000 times more sensitive. This advancement is comparable to the best existing methods for clinical and chemical analysis with microfluidic systems using microchips. In Rio de Janeiro, researchers at the Pontifical Catholic University (PUC-Rio), in partnership with researchers at the Federal University of Pernambuco (UFPE), created a fiber optic sensor to diagnose dengue.

Renato Souza Lima, a chemist at USP’s Institute of Chemistry of São Carlos (IQSC) and the National Nanotechnology Laboratory (LNNano) of the Brazilian Center for Research in Energy and Materials, located in Campinas, says that in the last decade microfluidic devices have been frequently used as an analytical tool in diverse areas, such as heavy metal analysis, quality control of food and beverages, and biological applications in medicine. Microchips with the C4D system have other advantages, such as ease of miniaturization and their universality as a detector. “That makes this technique an ideal choice for a wide variety of chemical and biochemical analyses,” says Lima.

Despite its advantages, the C4D does, however, have an important limitation when compared to classical electrochemical techniques such as amperometry and voltammetry: its low sensitivity. “These two types of analyses are thousands of times more sensitive than contactless detection (C4D),” explains Emanuel Carrilho, a USP IQSC professor and Lima’s adviser for his doctorate. “That’s why our goal was to increase the device’s efficiency by expanding the coverage area of the electrodes (responsible for detecting the substances under analysis) and reducing the thickness of the dielectric (electrical insulator that covers the electrodes). And so, what we did was transform the diagnostic device, which was less sensitive, into a system 10,000 times more efficient.”

To achieve this result, the São Carlos researchers modified the machine’s architecture by changing the location of the electrodes. Typically, the C4D microchips are composed of a glass slide with microchannels, through which the fluid to be analyzed flows, and another flat slide, which serves as a “lid,” and on which two electrodes are installed. In this configuration, the electrodes remain outside the microchannels, which record on another glass slide. Thus, the only way to increase the device’s sensitivity would be to increase the detection area of the electrodes, which is impractical. “Our solution was to put them inside the microchannels, in the form of a concentric ring,” Carrilho says. To prevent the electrode from coming into contact with the substance to be analyzed, which is typical of the C4D detector, it is separated from the substance by a thin 200-nanometer layer of silicon dioxide that covers the electrode.

062-065_Diagnosticos_216-01To do a clinical analysis of blood or urine, for example, a drop of the material is induced to pass through the channels, where the electrode detects the presence of substances of interest, whether endogenous, such as glucose or uric acid, for example, or exogenous, such as drugs or pollutants. This is done indirectly, because the sensor (microchip) measures the electrical conductivity of the microfluidic sample. “This conductivity changes from substance to substance and from concentration to concentration of the same substance,” explains Carrilho. “Any substance that changes the conductivity of the solution filling the channel can be detected.”

Optics on dengue
The sensor developed by the PUC-Rio and UFPE teams, in turn, is based on localized surface plasmon resonance (LSPR), an optical phenomenon that occurs when light interacts with metal nanoparticles, inducing a collective excitation of electrons. LSPR allows certain wavelengths (colors) to be absorbed. Isabel Cristina Carvalho, a physicist and head of the PUC-Rio Department of Physics Optoelectronics Laboratory, and one of the project’s coordinators, explains that the device is made with a thin gold film, 6 nanometers in thickness, that is deposited on the tip of an optical fiber, then heated for four minutes at 600ºC, which transforms it into gold nanoparticles.

“The NS1 antibody of the protein of the same name excreted by the virus is placed on one end of the fiber on top of the gold nanoparticles” says Rosa Dutra, a professor at PUC-Rio. “The other end is connected to a coupler, from which two other optical fibers extend, one of which is connected to a source of white light and the other to a spectrometer that detects the reflected signal at the end of the fiber containing the nanoparticles and the anti-NS1 antibodies,” says Carvalho. In the test, if the solution does not contain the antigen, the wavelength measured by the spectrometer does not undergo modification. On the other hand, if the signal being measured does vary in color, this will determine the various concentrations of the NS1 antigen.

Alexandre Camara, a PhD student of Carvalho’s, explains how all this works together. “The LSPR effect, due to the nanoparticles immobilized with anti-NS1 antibodies on the optical fiber tip, is affected by the external environment, that is, the presence or absence of the NS1 antigen. The sensor’s response is highly dependent on this external environment, and any change in this factor causes the color absorbed through the medium to change and the signal being monitored to change. “We do not detect the dengue virus directly, but the presence or absence of the protein (NS1) excreted by the virus. In the disease’s acute phase, this protein has a higher value, which is an early indicator of the severity of the disease.”

062-065_Diagnosticos_216-02Paula Gouvêa, a physicist with the Fiber Optic Sensors Laboratory (LSFO) at PUC-Rio and also one of the project’s leaders, says that the dengue sensor originated with another sensor, previously created by her group. “This is an adaptation of one we began to develop in 2007,” she recalls. “At that time we were collaborating with LSFO, the Optoelectronics Laboratory, and the Royal Institute of Technology, based in Sweden, to develop a fiber optic sensor using gold nanoparticles.” In 2011, UFPE’s Renato Araújo saw a presentation on the device by Gouvêa, and got the idea to adapt it to detect dengue.

And so the collaboration between the PUC-Rio and UFPE groups started in 2012. “It began with the experimental work done by two students: Alexandre Camara and Ana Carolina Dias,” Gouvêa says. At that stage of adapting the sensor to detect dengue, the study was conducted at the two universities. “Camara learned the technique at UFPE in Recife and brought it to Rio.” For now, tests have only been conducted on solutions made in the laboratory with dengue antigens. The next step will be to conduct in-vivo measurements with blood samples from infected patients. “What we have done so far is a proof of the concept of the new sensor, which is not a prototype,” says Araújo. “Like ours, there are a few methods that have been demonstrated in the laboratory that could be used to diagnose dengue. Transforming a result such as ours into a marketable product still requires a number of additional steps, including an economic evaluation of various production techniques.”

No symptoms
The results obtained in tests of the new device proved to be very promising. One of its biggest advantages is that it allows dengue to be detected from the first day of infection, when the patient has not yet begun to experience symptoms of the disease. This is very useful, because early diagnosis can prevent the deaths of patients who do not receive proper treatment in a timely manner, and avert more serious problems, such as those caused by hemorrhagic dengue. “Another advantage of our sensor is the fact that even a very tiny sample can be measured,” adds Camara. “The short time required for the test (in 20 minutes you can have a diagnosis) and expected low cost of production also make it attractive.” Dutra says that the sensor can also be portable and used in laboratories.

The work was funded through a partnership between the Brazilian Federal Agency for the Support and Evaluation of Graduate Education (Capes) and the Swedish Foundation for International Cooperation in Research and Higher Education, which supports joint studies between Brazil and Sweden. The research also had funding from the two universities, the National Council for Scientific and Technological Development (CNPq) and the Rio de Janeiro Research Foundation (Faperj). An article about the project was published in the journal Optics Express.
The chemical sensor for clinical analysis developed by the USP São Carlos team is also still not ready. “It is patented but needs further development,” says Carrilho. It is about to go from the university to a technology-based company prior to reaching the market. A São Carlos company called ParteCurae Analysis showed interest in a transfer of the technology.” According to Carrilho, there are only two small manufacturers with C4D microchips on the market, so the improvements developed by the researchers on this type of sensor could make it more competitive. The research was supported by FAPESP, through a doctoral research grant to Lima, and by the Brazilian Innovation Agency (FINEP) and resulted in an article published in the journal ChemComm of the Royal Society of Chemistry.

Ultrasensitive electrochemical microfluidic systems (No. 2010/08559-9); Grant Mechanism Doctoral research grant; Principal investigator Emanuel Carrilho-IQ-USP; Grant Recipient Renato Souza Lima; Investment R$ 88,808.87 (FAPESP).

Scientific articles
LIMA, R.S. et al. Highly sensitive contactless conductivity microchips based on concentric electrodes for flow analysis. Chemical Communications. Published online on October 9, 2013.
CAMARA, A.R. et al. Dengue immunoassay with an LSPR fiber optic sensor. Optics Express. V. 21, No. 22, p. 27023-31. November 2013.