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Mimetism in the laboratory

Synthetic substances for use in biosensors imitate the behavior of biological compounds

MIGUEL BOYAYANElectrodes with graphite, gold and carbon increase the biosensor’s stabilityMIGUEL BOYAYAN

Butterflies, moths, chameleons, and many other animal species, at the slightest sign of the approach of the enemy, change color or shape to resemble the surrounding environment and confuse their predators. This capacity for imitation, known as mimetism, inspired researchers from the State University of Campinas (Unicamp) to create synthetic compounds that reproduce, with advantages, the activity of antibodies, enzymes, cells, receivers and other biological components that are fundamental for the functioning of biosensors, like the one that measures the glucose level in diabetics, the portable glycosometer sold in a pharmacies.

Named biomimetic systems, the synthetic compounds have the objective of recognizing ions or molecules of the substances analyzed by the biosensors, expanding and facilitating the dissemination of this kind of chemical analysis. “Another objective is to assure stability (maintenance of activity) over long periods for these devices, avoiding one of the critical points that prevents the marketing of these products for a vast range of applications”, points out Professor Lauro Tatsuo Kubota, from Unicamp’s Chemistry Institute, who heads up the research with biosensors, within a theme project coordinated by Professor Elias Ayres Guidetti Zagatto, from the Center for Nuclear Energy in Agriculture (Cena), of the University of São Paulo (USP), in Piracicaba.

The researchers’ intention is to create devices that emit chemical responses, translated by the physical components that make up the biosensor, like electrodes, optical fibers, and polymer conductors. The choice of enzymes, antibodies or cells is guided by the substance that the device was programmed to identify in waters, beverages, foodstuffs, or in blood or urine tests. As many of these biological components are not stable for a long period, Kubota decided to replace them by a stable synthetic substance, based on copper and iron, placed in a form of monolayer on the biosensor’s surface. The choice of the two elements occurred because the active part of the enzymes to be replaced is made up of these metals.

Stability magnified
The new material proved to be efficient regarding its sensitiveness and selectiveness, and preserved for more than one year the stability of the biosensor, a difference that opens the market doors to the product. Up until now, the researchers from Unicamp have already developed different synthetic compounds, of environmental, pharmaceutical and medicinal interest, which have resulted in three requests for patents. The prospect is for the new biosensors to cost far less than those used at present.

The results achieved by the researchers from Unicamp and Cena, presented at an international congress in Dusseldorf, Germany, in 2002, attracted the attention of researchers from Europe and the United States, who proposed partnerships. Kubota does not rule out collaborations in other lines of research, but says that in the case of biomimetics his team is now far ahead. Some companies have also been in touch with the researchers, but no commercial partnership has been entered into so far, because it is still necessary to invest a bit more to get the device in the final format for marketing.

Besides the research that involves the synthetic compounds, the team from Unicamp is also working on another front: the study of new materials for stabilizing the biological components, which has proved to be very promising. A proof of this is that they have already developed a biosensor to assess the level of alcohol in the blood, based on graphite, silicon modified with niobium, and methylene blue as a coloring, which has achieved a precision of 99% and remains stable for at least three months. A far longer period than that reached by works described in the literature, which have come to one week at the most. As to the breathalyzers existing on the market, they are not totally precise and only succeed in detecting alcohol in high concentrations.

Compared with the traditional methods, in which reagents need to be added to the sample, to produce a color or fluorescence that makes it possible to get a reading, analysis with a biosensor is simple, quick and economical. All that it takes is to put a sample into the device and carry out the measurement, which consists of converting the chemical reaction into measurable energy, such as electricity. This current can also be translated by a processor, with the result appearing in a display that shows the concentration of the substance sought for. In this system, the quantities of antibodies or enzymes used are minimal, which means a reduction in costs, pollutant waste, and time in analysis. Kubota explains that the whole process is done in a very selective manner. “Among the various substances present in the sample, only the one desired is identified.”

Phytotherapic control
Besides the glycosometer, there are other biosensors on sale in the market, which assess urea (the working of the kidneys) and lactate (to assess the reaction of the muscles after physical exercise). “We are working with the idea of joining various devices into a single item, to determine lactate, creatinine, glucose, and many other parameters”, says the researcher. A single drop of blood will be sufficient to carry out a check-up in a portable minilaboratory.

The applications under study extend over a vast range, including the analysis of medicines during the manufacturing process and of the antioxidant properties of phytotherapics, and the monitoring of the level of stress, from an analysis of the activity of the glutathione peroxidase enzyme, which acts in the organism’s defense system in the combat against free radicals.

As to the system that analyzes medicines in the form of capsules and pills, the researches are advanced and will result in a patent request. This assessment is usually done by sampling in a liquid medium, with the addition of several reagents, which generates a reasonable quantity of waste. Not to mention that the results are not known immediately. “The way we are proposing, it is possible to analyze samples in real time directly in the production system, without generating any waste”, Kubota says. In this way, instead of the control being carried out by sampling, the entire production is encompassed. In the case of any problem being detected in the batch being manufactured, just a few pills would be discarded, not the whole batch.

Another biosensor being developed at Unicamp’s IQ with a great potential for immediate application is a device that analyzes the active ingredients of plant extracts from an infusion. Although controlling the quality of this kind of medicine has grown a lot in Brazil, Kubota points out that it is important to carry out a more accurate analysis, to assess whether the properties proclaimed really remain in the commercial product, because the preservation of the active principles of plants depends on several factors, such as climate, soil, and the time of planting and picking.

The researcher started to work in the area of sensors and biosensors ever since he was hired by Unicamp, in 1994. Since that year, he has now worked on various researches funded by FAPESP, one of which involved a Thematic Project, the Study and Development of New Detection Systems for Analytical Applications, concluded in 2001. The idea of taking part in another thematic project, this time with partners from different areas, had the objective of swapping knowledge in order to try to arrive at a consensus on what the best system of analysis is, for determining and quantifying biological and environmental samples.

Brazilian contribution
While Kubota is working on biosensors, another researcher from Unicamp’s IQ, Professor Marco Aurélio Zezzi Arruda, who is also a participant in the same project, is dedicating himself to studying natural and synthetic materials that are candidates for increasing the sensitivity of the analysis methods, with minimum waste generated. Among the natural ones, there are rice husks, vegetable sponges (loofahs) and vermicompost, produced by some kinds of worm after digesting materials like sand, soil, and organic matter. They showed promising results in detecting small concentrations of metals, such as copper, zinc, lead, and cadmium, with low operational costs.

The natural materials are placed in small quantities (20 to 25 milligrams) into tiny columns (tubes) of polyethylene. These tubes are inserted into flow injection analysis (FIA) systems, and the whole set is coupled to the technique of atomic absorption spectrometry, which is based on the absorption of radiation coming from lamps that are specific for each element to be determined. The absorption is proportional to the concentration of the metal present in the sample. Accordingly, it is possible to quantify metals present in foodstuffs, beverages, inorganic materials, biological matter, waters and effluents (domestic waste, or waste released by industrial production). Arruda explains that the natural materials have succeeded in pre-concentrating metals in small quantities, increasing the sensitivity of the analytic method. The next stage of his research provides for the use of these materials in biosensors, to assess whether they really do improve the efficiency of the electrochemical methods. Besides the synthetic compounds developed by Kubota, the natural materials are also being assessed by Professor Zagatto, who is working to perfect the flow injection analysis systems, applied in a pioneering manner in Cena’s Analytic Chemistry laboratories in the 1970’s by Professor Henrique Bergamin Filho (1932-1996) and currently adopted in laboratories all over the world. Zagatto, who used to be part of the team led by Bergamin, says that this contribution has made it possible to automate the process of analyses and the miniaturization of these systems.

The technology consists of injecting the sample into capillary tubes, which run like water inside closed pipes. During transport, it is dispersed, and may also undergo dilutions, which makes it possible to carry out different stages associated with the analytic method, such as the addition of reagents, separation, and concentration. It all works as if the analytic system were a laboratory sealed off from contamination. The sample processed passes through a meter that monitors the product of the chemical reactions involved. The result of the analysis is obtained in a few seconds, the time needed for the sample to run through the capillaries, and the rate of sampling is high, generally over one hundred samples an hour.

Zagatto points out that exploring this technology has revealed a new field of knowledge for chemical analysis, such as the monitoring of chemically unstable species, that is, of products that decompose in a short time. Before this, the main reagents for chemical analyses on sale were the fruit of the selection of raw materials, kept as a precious industrial secret, that have been produced since the 18th century with the objective of forming a stable compound at the end. Stability was indispensable for the success of the analytic method, since the coloring produced has to be stable and reflect the concentration to be determined.

It so happens that, in the course of some applications, the sample becomes turbid, but in flow analysis this deterioration in no way endangers the results, since the sample is analyzed a few seconds after its insertion into the analyzer. Furthermore, the samples and the reagents are to be found in the region of microliters, which means a saving in raw material and low generation of waste, even in the case of toxic products. Until the end of October this year, the date forecast for the conclusion of the thematic project, there is still plenty of work ahead, but the results already achieved with the biomimetic compounds and natural materials show that they are now ready to be used in several kinds of chemical analysis.

The project
Employment of New Materials and Different Reactional Environments for Improvement in Sensitiveness and Selectiveness in the Analysis of Biological and Environmental Samples (nº 99/12124-7); Modality
Thematic Project; Coordinator Elias Ayres Guidetti Zagatto – Center for Nuclear Energy in Agriculture (Cena)/USP; Investment R$ 221,063.00 and US$ 163,470.00