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Chemistry

Early cancer diagnosis

New biosensors should be able to detect pancreatic and ovarian tumors more quickly

Electronic device developed at the USP São Carlos Institute of Physics: it holds a layer of antibodies that recognize the antigen associated with pancreatic cancer

Eduardo CesarElectronic device developed at the USP São Carlos Institute of Physics: it holds a layer of antibodies that recognize the antigen associated with pancreatic cancerEduardo Cesar

According to estimates by the National Cancer Institute (INCA), Brazil is expected to have 600,000 new cases of cancer in 2016.  Since early diagnosis is one of the key weapons in fighting the disease, two research teams from São Paulo have developed a new device to detect early stage tumors.  One group of researchers from the São Carlos Institute of Physics at the University of São Paulo (IFSC-USP), in collaboration with the Barretos Cancer Hospital in inland São Paulo State, has created a biosensor to detect pancreatic cancer.  Another group of researchers, this one out of the Center for Research and Development of Functional Materials, (CDMF), one of the FAPESP-funded Research, Innovation and Dissemination Centers (RIDC) in Araraquara, has developed a biosensor capable of detecting ovarian tumors as well as Hepatitis C, another disease prevalent in Brazil.  Both devices are in the prototype phase and are awaiting approval from the Brazilian Health Surveillance Agency (ANVISA) for use in hospitals, clinical analysis laboratories and physicians’ offices.

A biosensor is a device that incorporates a biological recognition element, such as an enzyme, antibody or antigen, used to selectively measure specific substances related to cancer and other diseases found in blood samples.  The two groups working independently to investigate biosensors would both like to create portable devices–similar to today’s glucose meters used to measure blood glucose levels–to take a reading of the test results and indicate whether or not the patient has cancer (see infographic).

One of the best-known methods for diagnosing cancer—already implemented on a large scale—is the ELISA Test, which is a blood test based on the specific interaction between antigens and antibodies.  But unlike ELISA tests in which detection occurs through the use of reagents and enzyme reactions, biosensors measure molecular interactions between antigens and antibodies with no need for intermediary enzymes.    While it takes1.5 to 2 hours to obtain results through ELISA tests, biosensors could reveal results in a mere 30 minutes. Another advantage of biosensors is that they use one quarter of the amount of blood while delivering higher sensitivity—the newest generation of biosensors are 1,000 times more sensitive than ELISA tests.

“The goal of our work is to create a simpler and less expensive method,” explains physicist Andrey Soares, a doctoral candidate in the Bernhard Gross Polymer Group at IFSC, responsible for creating the biodevice that detects pancreatic cancer. “The scientific literature includes several reports of biosensors that utilize electrochemical or optical techniques to detect cancer.  Ours is based on electrical measurements.”  The study was conducted under the direction of IFSC Professor Osvaldo Novais de Oliveira Junior, with participation from researchers at the Barretos Cancer Hospital.  Analysis of the data collected by the biosensor employed computational methods for visualization developed by Fernando Vieira Paulovich and Maria Cristina Ferreira de Oliveira, professors at the USP São Carlos Institute of Mathematical Sciences and Computation (ICMC).

The electronic device developed at USP consists of two thin films:  one that contains chitosan (a polysaccharide removed from the shell of a shrimp) and concanavalin A (a protein extracted from jack bean seeds), and another that has an active layer of antibodies that can detect the CA19-9 antigen.  Elevated concentrations of this antigen are found in individuals who suffer from pancreatic cancer.  These two nanometric-scale films rest on an electrode (material that conducts electricity) imprinted on a strip, similar to the kind used on quick tests of glycemic index.  “By placing a sample of the patient’s blood on the biosensor, we can see that it interacts with the active layer of antibodies, generating an electric signal that allows us to determine whether there is an excessive amount of CA19-9 in the material collected,” Soares says.

Oliveira explains that one of the main challenges in producing a biosensor is preserving the function of the biomolecules that serve as the active elements of the device.  In order to do this, researchers employ matrices made of materials that help preserve the activity of the biomolecules.  “In our biosensor, the role of the matrix is played by the chitosan and concanavalin A, two low-cost materials that can be obtained from natural sources.  Concanavalin A interacts with the chitosan, forming a thin, stable film on the surface of the electrode,” says physicist Oliveira, elected president of the Brazilian Materials Research Society (SBPMat) in January 2016.  “Stability is important for mechanically immobilizing the active layer, allowing us to build devices that achieve both high sensitivity and selectivity.”  The study is funded by FAPESP in partnership with the Barretos Cancer Hospital. “Up to now, our tests have been conducted using cancer cells produced in the laboratory.  The next step will be to conduct tests that use actual patient blood samples,” says Andrey Soares, adding that the required number of patient samples has not yet been determined.

076-079_Biossensor_242At the CDMF, housed in the Chemistry Institute of the São Paulo State University in Araraquara (IQ-Unesp), research for development of a biosensor to detect ovarian cancer and Hepatitis C was led by Professor Maria Aparecida Zaghete Bertochi, with collaboration from master’s degree candidate João Paulo de Campos da Costa and two doctoral candidates: Gisane Gasparotto and Glenda Biasotto. Professor Paulo Inácio da Costa at the Unesp School of Pharmaceutical Sciences and researcher Talita Mazon at the Renato Archer Center for Information Technology (CTI) also contributed to the study.

The biosensor has an architecture similar to the USP device: an active layer of biomolecules, a stabilizing matrix – in this case, one that consists of cystamine and glutaraldehyde – and a sensing electrode, responsible for converting the signal generated by the chemical interaction between the biomolecules and markers of the target diseases into an electrical signal. “Our biosensor is an analytical device that converts the immunochemical, biochemical or biological response into a measurable signal. It is disposable and the method of electrochemical measurement it uses results in less expensive diagnoses compared with current systems,” explains electrical engineer Campos da Costa. The biosensor now yields results within one hour but researchers are working on modifications that will reduce this response time to 10 minutes. ELISA and other current tests used to detect hepatitis confirm a positive result based on the presence of virus proteins. On the whole they take more than two hours.

For now, the biosensor is capable of providing individual diagnoses of ovarian cancer and Hepatitis C – in other words, they diagnose one disease at a time.  The goal is to perfect the device so that more diseases can be detected at the same time.  In the case of ovarian cancer, the device enables the detection of a high-molecular-weight glycoprotein known as CA 125, which is associated with the appearance of this particular cancer. Studies indicate that 90% of women who present elevated concentrations of this glycoprotein in their blood go on to develop the disease. “A monoclonal antibody was attached to the surface of the sensing electrode so that, in the presence of the CA 125 antigen, it would bind specifically to this glycoprotein and cause interference in the device’s electrical current,” explains Campos da Costa.

To diagnose the viral infections associated with Hepatitis C, the same sensor enables detection of specific antibodies for a protein found in the virus.  “If the particular protein is present in the blood, the bond between it and the antibody incubated on the electrode produces a signal that alters the electrode’s electrical potential.  A computer application interprets this signal and the diagnosis is made,” says the Unesp researcher.  The Ministry of Health estimates that 1.4 to 1.7 million people in Brazil may have come in contact with the virus that causes Hepatitis C and a portion of this contingent will actually develop the chronic infection.

The Unesp researchers soon plan to file a patent application for the biodevice with the Brazilian Industrial Property Institute (INPI). The project to develop portable equipment for biosensor readings is already underway in cooperation with the division of Signal Processing and Instrumentation under the Graduate Electrical Engineering Program at USP São Carlos. When everything is ready, researchers at Unesp are considering setting up a company to manufacture and sell the device.

According to Emanuel Carrilho, a professor at the Chemistry Institute of USP São Carlos and member of the National Institute of Bioanalytical Science and Technology (INCT Bio), there has been a strong focus in recent years on a line of research into the development of biosensors for detecting cancer. “Today’s greatest challenge is identifying the molecules that indicate early stage cancer,” Carrilho says.  “Once we actually have these predictive biomarkers, the biosensors will be important tools for early diagnosis of the disease.”

Projects
1. CDMF – Center for Research and Development of Functional Materials (nº 2013/07296-2); Grant Mechanism Research, Innovation and Dissemination Centers (RIDC); Principal Investigator Elson Longo (Unesp); Investment R$20,965,210.37 (over five years).
2. Nanostructured films from biologically-relevant materials (nº 2013/14262-7); Grant Mechanism Thematic Project; Principal Investigator Osvaldo Novais de Oliveira Junior (USP); Investment R$2,539,907.03.
3. Development of ZnO films and nanostructures for application in sensors and nanogenerators (nº 2011/19561-7); Grant Mechanism Scholarships in Brazil – Regular – Doctoral; Grant Recipient Glenda Biasotto (IQ-Unesp); Principal Investigator: Maria Aparecida Zaghete Bertochi (IQ-Unesp); Investment: R$31,239.12.
4. Development of ZnO films and nanostructures for energy extraction: nanopower generators and piezotronics (nº 2012/11979-5); Grant Mechanism Regular Research Grant; Principal Investigator Maria Aparecida Zaghete Bertochi (Unesp); Investment R$296,813.67.

Scientific article
SOARES, A.C. et al. Controlled film architectures to detect a biomarker for pancreatic cancer using impedance spectroscopy. ACS Applied Materials & Interfaces. V. 7, No. 46, p. 25930-7. November 2015.