Unprecedented research by Unicamp's Institute of Chemistry (IQ) allowed the manufacture of a highly sensitive device, the size of a fifty-cent coin with 64 integrated sensors, capable of early identification of breast cancer, the most common among women, with 8,2 million annual deaths worldwide, according to a report released in 2013 by the International Agency for Research on Cancer (Iarc), a body linked to the World Health Organization (WHO).

The device, developed by researcher Cecília de Carvalho Castro e Silva and professor Lauro Tatsuo Kubota, can detect in a very simple way and in a few minutes the presence of a protein that indicates the emergence of a breast tumor, still in its pre- development, before the appearance of the nodule. The examination with the device, which has not yet been tested on living beings, will allow the detection of cancer through a single drop of blood.

Researcher Cecília de Carvalho Castro e Silva explains that the biosensor is capable of identifying the HER2 protein (Human Epidermal Growth Factor Receptor 2, its acronym in English) which, in abnormal quantities, is expressed in 25% to 30% of breast cancer cases. The HER2 protein thus becomes, according to the scholar, an important biomarker for breast cancer.

“Studies show that there are cells developing in the breast tissue before the tumor appears. Therefore, before a lump appears, it would be possible to detect breast cancer early. Traditional methods use breast examination and mammography. During a physical examination, a woman can only identify cancer when the nodule is one centimeter or larger. On mammography it is possible to detect nodules of up to four millimeters. In these cases, the cancer is already present and, often, it can be too late”, observes Cecília Castro e Silva.

Still according to her, the objective was to develop an analytical tool so that patients or risk groups could monitor, month by month, the levels of this protein. “Many studies show that, six months before the patient begins to develop the tumor, HER2 levels in the blood serum increase, going from what would be a normal level of 12 nanograms per milliliter, until reaching the stage of 15 nanograms per milliliter or more. ”, he relates.

The device capable of carrying out this detection was designed as a type of field-effect transistor based on graphene modified with gold nanoparticles. The researcher explains that the electrical conductivity in these devices can be modulated through interaction with chemical and biological species. 

The ultrasensitivity of this biosensor was possible thanks to three factors, according to the author of the work: the type of graphene used; the incorporation of gold nanoparticles; and the oriented immobilization of antibodies on graphene. Antibodies, proteins produced by the immune system to identify and neutralize bacteria, viruses or tumor cells, were used as receptor elements of the device.

“The biosensor has a graphene sheet with gold nanoparticles. These particles are immobilized, and this is what allowed an even higher level of detection. After these gold particles were immobilized on the graphene, we immobilized the antibodies. Antibodies specifically recognize this HER2 protein. Therefore, when these antibodies interact with this protein, there are changes in conductivity values. And we were able to associate the concentration of this biomarker with changes in the device’s conductivity”, he reveals.

Using these three strategies, the device with 64 sensors is capable of identifying a quantity of HER2 protein of up to 500 femtograms per milliliter, that is, a volume ten times greater than fifteen grams per milliliter. In addition to ultrasensitivity, the method developed provides for the large-scale development of the biosensor.

The device was developed as part of Cecília Castro e Silva's doctoral thesis, recently defended at the IQ Postgraduate Program. The study, supervised by professor Lauro Tatsuo Kubota, from the Unit's Analytical Chemistry Department, is part of a line of research aimed at creating electrochemical sensors for detecting species of biological, pharmaceutical and environmental interest.

Part of the experiments were carried out at Rutgers University, in New Jersey, in the United States, through a sandwich doctorate between the two institutions. In New Jersey, in the laboratory coordinated by Professor Manish Chhowalla, the researcher carried out the synthesis and characterization of graphene.

“We synthesized it via chemical vapor deposition process. Using this technique, it is possible to synthesize this monolayer of carbon atoms measuring up to four inches”, points out Cecília Castro e Silva, who has a degree in chemistry from the State University of Maringá.

After a year in the United States, funded by the federal Science without Borders program, Cecília and professor Lauro Kubota began working on the manufacture of the device and the processes involving the area of ​​microelectronics. At this stage, there was the collaboration of technicians and researchers from the Center for Semiconductor Components (CCS) at Unicamp, coordinated by professor José Alexandre Diniz, from the Faculty of Electrical and Computer Engineering (FEEC). 

“An important point to highlight is that to develop this device we learned how the transistor works and manufactures. And this is not a trivial task for a chemist. We did not simply ask an electrical engineer to develop the transistor, but with his help, we developed the device. This is because we wanted to learn how to make a transistor, understand how it works and use its principle according to our objectives. We are now able to configure the transistor in the way we believe is best for detecting. This is a very relevant gain”, says advisor Lauro Kubota.

In addition to the partnerships, the research was developed within the scope of the National Institutes of Science and Technology Program (INCTs) in Bioanalytics, coordinated by professor Lauro Kubota. The INCTs are centers of excellence created by the Ministry of Science, Technology and Innovation (MCTI), with funding from the National Council for Scientific and Technological Development (CNPq) and the São Paulo State Research Support Foundation (Fapesp). Unicamp has nine INCTs to date. 

POTENTIALITIES

The device has a series of potentialities, according to advisor Lauro Kubota. “We could, for example, in each of the 64 biosensors, develop procedures for recognizing markers for different types of disease. Therefore, with a simple drop of blood, it would be possible, in the future, to make a complete diagnosis of the individual”, he predicts. 

At this point, Cecília Castro e Silva informs that the next step of the research is to work towards mobilizing different types of antibodies to recognize different biomarkers for breast cancer. “We would have an extremely accurate test, capable of detecting, for example, whether that woman is expressing other types of biomarkers for breast cancer, not just the one originating from the HER2 protein. Therefore, more women could be diagnosed by the test.”

Furthermore, the miniaturization of the device means it can be produced on a large scale at a relatively low cost. The researcher highlights that the biosensor can be manufactured on plastic substrates, which would further reduce the cost, making it, at the same time, disposable, due to the type of sample used. 

“We could even make this flexible substrate biocompatible. This will allow, for example, that in the future these devices can be bioimplantable, providing continuous monitoring, both in women undergoing treatment and in risk groups”, he adds.

Publication

Tese: “Development of graphene-based field effect transistor biosensors (GraFETs) decorated with gold nanoparticles applied in the ultrasensitive detection of breast cancer biomarkers”

Author: Cecilia de Carvalho Castro e Silva

Advisor: Lauro Tatsuo Kubota

Units: Institute of Chemistry (IQ) and Faculty of Electrical and Computer Engineering (FEEC)

Financing: Fapesp and CNPq