Obstacle course for portable biosensors

The Covid-19 pandemic triggered a race among researchers worldwide to develop new methods for detecting the Sars-CoV-2 virus, the cause of the disease. The goal was to create solutions as cheap and fast as tests sold in pharmacies, but with the same sensitivity as the best laboratory examination available at the time. Dozens of low-cost portable devices capable of detecting the virus in a few minutes, even in low concentrations, were described in scientific articles and released to the press. Most of them, however, did not reach the market. Transforming prototypes into commercial products remains the main challenge for researchers developing portable or wearable biosensors. In addition to rapidly identifying infections by viruses and bacteria, these devices promise to facilitate the diagnosis and monitoring of a series of diseases, replacing blood tests with rapid analyses of tears, saliva, or sweat in some applications. They could also allow for earlier diagnoses and personalized treatments. “Bringing what works on the bench to the real world is often a very challenging step for researchers, but I feel that the academic community is moving in this direction,” evaluates physicist Laís Brazaca, from the Institute of Chemistry at the University of São Paulo (IQSC-USP). She is the first author of a review article on wearable sensors for health, published in June in the journal ACS Applied Materials & Interfaces. Brazaca leads a FAPESP-funded project whose goal is to develop wearable biosensors to monitor proteins in sweat associated with diseases. Most devices that work adhered to the skin detect only electrolytes (sodium, chlorine ions, and others) and metabolites (small molecules, such as glucose and lactate) in sweat. “My idea is to develop devices that collect and analyze larger molecules, such as proteins,” she says. Blood transports a wide variety of these molecules, but only a few can cross blood vessels and reach the sweat. Among them are clusterin and dermicidin. In the blood, high concentrations of clusterin are associated with Alzheimer's disease, while elevated levels of dermicidin have been linked to breast cancer. One of the project's objectives is to verify whether these relationships also occur in sweat, which may depend on many factors of the organism. There is also the fact that the concentrations of proteins measured by the sensor may differ from the actual amounts present in the sweat. Finally, it is necessary to solve the difficulty of integrating a kind of miniature chemistry laboratory, called a microfluidic circuit, into a flexible device, along with a microelectronic circuit capable of recording and wirelessly transmitting the data to a computer or cell phone. To overcome these obstacles, Brazaca collaborates with the chemists Emanuel Carrilho, her colleague at IQSC-USP; Khaled Abdelazim, from Harvard University; and Juliane Sempionatto, from Rice University (USA), who is also a nanoengineer. Recently, Sempionatto's group developed prototypes of wearable biosensors to measure electrolytes and metabolites in sweat. Another project of hers seeks to monitor hormones related to women's health. “There is an enormous need for tools that allow tracking hormonal changes from adolescence to menopause,” she explains. The prototypes of both projects still need further work to reach the market. “The field is maturing,” says Sempionatto, also a coauthor of the review. “The real challenge is to transform data reading into reliable and useful clinical information for a medical decision.” From the space to the shelf The development of wearable sensors began in the 1980s to remotely monitor the vital signs of astronauts during space missions. These first instruments gave rise to so-called physical sensors, capable of recording small pressure variations associated with the functioning of the heart, lungs, and nervous system, for example. Many of these measurements can now be made by cell phones and smartwatches. The first wearable biological sensors emerged in the late 1990s. These devices consist of a surface capable of harboring a biological component—an antibody, an enzyme, a piece of DNA, or even an entire cell—that reacts in the presence of a specific molecule produced by the human body, by viruses or bacteria. This reaction generates a measurable signal, such as a color change, in colorimetric biosensors, or a change in the electrical properties of the material in electrochemical ones. In the 1980s, the development of wearable sensors began to remotely monitor the vital signs of astronauts in space missions In the late 1990s, the first wearable biological sensors arrived, which react in the presence of a specific molecule produced by the human body, by viruses or bacteria Today: Rapid tests for Covid-19, HIV, and pregnancy are the main colorimetric biosensors, where the reaction generates a color change, available on the market Among electrochemical ones, the only ones already common are glucose meters 60 thousand scientific articles on wearable biosensors for health have been published since 2005, according to a review led by physicist Laís Brazaca, from IQSC-USP Currently, rapid tests for Covid-19, HIV, and pregnancy, among others, are the main colorimetric biosensors on the market. Among electrochemical ones, the only ones already common are glucose meters. In general, they analyze a drop of blood obtained by a finger prick, while the most advanced ones work on a patch applied to the arm, monitoring glucose for several days through micro-needles. These products represent a small fraction of the prototypes already developed. The review led by Brazaca accounted for 60 thousand scientific articles on wearable biosensors for health published since 2005. New materials have made the devices more flexible and compatible with the skin and other parts of the body. “The great variety of possible materials and applications has made the field attractive for basic and applied research, but consolidation into products requires focus on commercially viable applications,” considers physicist Osvaldo Novais de Oliveira Jr., from the Institute of Physics at USP (IFSC-USP), also a coauthor of the review. With support from FAPESP, Oliveira develops biosensors for health and environmental monitoring. His group has already created devices that identify genes associated with head and neck cancer, tested with samples from patients from the Hospital de Amor in Barretos (SP). The main difficulty of biosensors is to maintain their operation for long enough outside the laboratory. Their biological components are fragile molecules that, after being immobilized on the sensor by chemical bonds, need to remain active under conditions very different from those found in the human body. To extend their lifespan, researchers have resorted to more stable synthetic versions and protective layers. Still, many do not last more than a few weeks. An example is the electrochemical biosensor integrated into glasses, developed in Joseph Wang's laboratory at the University of California in San Diego. Sempionatto was Wang's doctoral student, while Brazaca interned in the laboratory in 2017, during her doctorate, under the guidance of materials engineer Valtencir Zucolotto, from IFSC-USP. Together, they collaborated to create the first wearable biosensor capable of monitoring alcohol in tears, as well as vitamins and glucose. The enzymes used for detection had to be prepared at most one day before use. In addition to the stability of the biological components, the synthesis of the nanomaterials used in the sensor interface involves chemical reactions whose control can be challenging. “Reproducibility is very difficult,” states chemist Juliana Cancino-Bernardi, from the Faculty of Philosophy, Science and Letters at Ribeirão Preto University of São Paulo (FFCLRP-USP). According to her, these materials have inevitable size variations during manufacturing, which can alter their electronic properties. This means that biosensors produced by the same process can generate different electrical signals in response to the same concentration of the target substance. “To generate reliable data over days, weeks, or months, the sensor's behavior needs to be very stable or, at least, predictable,” Sempionatto points out. Real Samples Another problem is that most studies test their prototypes only under idealized laboratory conditions. Some proceed to analyze synthetic fluids—artificial versions of sweat, saliva, or blood with known composition. Rarer are those that use real fluid samples from patients. Only a small fraction reaches clinical tests. “A difference of our group has always been to use real samples,” states chemist William Reis de Araújo, who coordinates a laboratory in the area at the State University of Campinas (Unicamp) and also interned in Wang's laboratory. At the height of the pandemic, in 2020, the researcher from Unicamp initiated a remote collaboration with Cesar de la Fuente-Nunez' group from the University of Pennsylvania (USA), who specializes in antimicrobial compounds. Two students of Araújo went to his laboratory to develop biosensors based on the ACE2 protein, a receptor present on the surface of human cells and used by the SARS-CoV-2 virus to invade them. The strategy proved correct because, although new variants of the virus have made many antibody-based biosensors obsolete, they continued to bind to ACE2. One of the devices was a modified graphite microelectronic circuit, laser-printed on paper. Each disposable unit cost less than R$ 25. After receiving a sample of saliva or nasal swab with a cotton swab, the sensor was inserted into a small commercial device called a potentiostat, connected to a cell phone or computer—the result came in four minutes. In 2021, the team validated the biosensor in 321 patients at the university hospital. Two years later, they reduced its cost to about R$ 17. Despite estimating that millions of units could be manufactured per week during the pandemic, the patent has not yet been licensed to a company. In 2022, a global outbreak of monkeypox, better known as mpox (viral disease transmitted through contact with fluids of infected people or animals), reached Campinas, with 96 cases registered. The episode led Araújo to create the first electrochemical biosensor to detect the virus in blood samples, saliva, or lesion secretions. Two years later, his team improved the biosensor, initially made of paper, in the form of a flexible fabric that can be incorporated into dressings. This work integrates a project by Araújo funded by FAPESP to create disposable biosensors for detecting viral and bacterial infections. Other devices could differentiate dengue, zika, and chikungunya, as well as identify the species of bacteria responsible for an infection. One of the completed prototypes combines four tests on a tongue depressor—the stick used by doctors to facilitate examination of the mouth and throat. Invisible Steps Rice University Juliane Sempionatto's team (first on the left), at Rice University, tests biosensors on the skin of a volunteer's forearm (second on the left). The device measures sweat electrolytes and transmits its data to a cell phone (third on the left)Rice University Araújo emphasizes that although the goal is to create increasingly cheaper tests, the basic research necessary to develop a prototype requires inputs and equipment costing around R$ 100 thousand. According to Brazaca's review, the investment to bring a new device to the market can exceed hundreds of millions of dollars, which may discourage investment in projects. For Sempionatto, funding agencies need to better support less visible but decisive stages, such as reproducibility studies, collection standardization, clinical validation, usability, quality control, and regulatory planning. “These studies, less attractive in scientific novelty, are what determine whether a technology can be used outside the laboratory.” “And companies need to participate earlier in the process, helping to define cost, manufacturing, user experience, and payment methods through the health system,” she says. “Doctors and patients must also be involved from the beginning. Development works best when all areas work together continuously.” This transition between the academic and commercial world is known as the valley of death, explains Araújo. To overcome it, he considers programs such as Innovative Research in Small Enterprises (PIPE), from FAPESP, essential, which fund studies in micro, small, and medium-sized enterprises. “PIPE stimulated the creation of companies from our laboratory,” says Brazaca. Her colleague Carrilho encouraged students to start businesses after working, from 2007 to 2009, as visiting professors at Harvard University, in George Whitesides' group, known for creating technologies that led to companies. In 2012, three of Carrilho's students accepted the challenge and founded Partecurae Analysis, focused on rapid diagnostic tests for animal health. Four years later, another group created Solve Biotechnology, dedicated to human health. Both received initial funding from PIPE. The two companies merged in 2020, giving rise to Prothera Biotechnology. During the pandemic, the company developed a rapid test for Covid-19, similar to those available on the market, which was commercialized in the São Carlos region. “I was overjoyed when I saw our product in the pharmacy,” recalls researcher Regiane Travensolo, co-founder of Partecurae and Prothera. The manufacturing of the tests, however, was only possible thanks to a partnership with an investor, which ended due to legal issues in 2023. Since then, Prothera has restructured its business model and began offering consulting to companies and researchers interested in entrepreneurship in the biotechnology field. Monitoring Pollutants Technologies similar to those of health biosensors are being adapted to monitor substances difficult to detect in the environment. These devices can contribute to food conservation, precision agriculture, and the control of so-called emerging pollutants—agrochemicals, pharmaceuticals, hormones, microplastics, and other invisible contaminants that accumulate in rivers and lakes. Last year, Araújo's group adapted their technique for manufacturing paper electrodes to create a small waterproof sensor that can be attached to leaves and other parts of plants to detect the herbicide paraquat, prohibited by the National Health Surveillance Agency (Anvisa) in 2020 due to its health risks. The device works through a chemical reaction between the electrodes and the herbicide molecules and transmits its results wirelessly to an application. To detect water contaminated with paraquat, chemist Cibely Martin, from the State University of Paulista (Unesp) of Ilha Solteira, developed nanomaterials that increase the performance of electrochemical sensors and allow for detection through spectroscopy. “They identify a family of compounds and their concentration, while spectroscopy provides the fingerprint of each molecule,” explains Martin, who coordinates a project funded by FAPESP. The paraquat molecules are so similar to those of other herbicides that electrochemistry alone cannot distinguish them. Only the combination of the two techniques allows them to differentiate them and make the detection more selective. At the Federal University of ABC (UFABC), the biophysicist Wanius Garcia leads a project funded by FAPESP to develop nanosensors for detecting antibiotics and insecticides in environmental samples, such as soil and water bodies, or in biological matrices, such as blood. The idea arose after his group, in collaboration with the team of biochemist Claudener Teixeira, from the Federal University of Cariri (UFCA), showed that certain proteins of the lectin family bind to antibiotic molecules of the aminoglycoside class, such as gentamicin, used in the treatment of severe infections. The researchers showed that this interaction alters the light absorption by silver halide nanoparticles coated with lectins, altering their optical properties. Garcia has been applying the same principle with enzymes called esterases to detect insecticides. “We have already demonstrated the sensitivity of the technique, but we still need to verify its specificity to know if other molecules can interfere with the detection,” he explains. According to him, in the future the entire procedure can be integrated into a portable device—for example, a cell phone—allowing for rapid analyses and direct readings on the screen.