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Medicine

Paper laboratory

Cellulose and wax: a new clinical testing model

Eduardo CesarUrine test with four evaluation parametersEduardo Cesar

A small, stamp-sized paper square that works like a minilaboratory of clinical analyses, is to be used this year in Santa Luzia do Itanhy, in the state of Sergipe, to evaluate the basic state of the health of children and adolescents in this town with little more than 10 thousand inhabitants. Called microfluidic paper-based analytical device or µPADs, these analytical systems were developed by a research group led by professor George Whitesides from Harvard University in the United States, with the participation of professor Emanuel Carrilho, from the Chemistry Institute of São Carlos of the University of São Paulo (USP) and a member of INCT Bioanalitica, the National Institute of Bioanalitic Science and Technology. The tests are conducted using a small volume of liquids, such as a drop of blood or of urine. This liquid runs through microchannels engraved on a porous piece of paper with a commercial wax jet printer.

After printing, the sheet is warmed up to 150o C for two minutes, melting the wax and enabling the formation of the microchannels – the paths along which the analyzed liquids will run. At the end of these printed channel, reagents such as enzymes and antibodies are placed. These change colors depending on the substance tested. The wax operates like a hydrophobic barrier. In other words, it keeps liquids such as urine and blood from spreading randomly on the paper, which is rendered impermeable with a polymer. The test is simple and highly practical. All that is required is to place the liquid to be tested at the entrance of the device printed on the paper. The liquid promptly runs through the microchannels and, in contact with the reagent, changes color. The result takes 30 minutes at most, a long enough time span in which to achieve a chemical reaction and color change. Still, this amount of time is, for instance, far shorter than that required in the traditional Elisa test, the first blood test to check for the HIV virus. Elisa takes two to three hours to yield a result. “The analysis on paper is only visual,” notes Carrilho.

Multiple diagnoses
At present, there are devices that can already be used to test the level of glucose, a parameter of diabetes, and of the total protein in urine, which is fundamental to find out how the kidney function is performing or whether there is a urinary tract infection. However, the paper minilaboratory might come to be used to diagnose malaria, tuberculosis, Aids and other diseases. “Many of the chemical reactions conducted in labs can be transferred to this paper,” says Carrilho, who worked at Whitesides’ laboratory for two years thanks to a FAPESP New Frontiers grant for the development of bioanalytic systems. He also signed four scientific articles published between 2008 and 2010 in the journal Analytical Chemistry, a fortnightly periodical from the American Chemical Society, on the development and application of the microfluidic paper devices, plus an article in Angewandte Chemie, one of the most prestigious chemistry journals.

Since then, the construction of the devices has progressed and the group led by Carrilho has contributed to this. At present, they plan, among other targets, to work on a test to assess the presence of gluten in food or beverages. Information on the presence or absence of gluten in product labels is crucial for those who suffer from celiac disease, which consists of being permanently intolerant to gluten, a protein found in wheat, oats, barley and malt, among other cereals.

There is also work being done on the paper device to detect heavy metals, an indication of pollution, and for water and food quality tests. Since returning to Brazil, Carilho has focused on developing new analytical systems that can be adapted to Brazilian conditions, by simplifying certain stages of the production process. This has led to a patent request for the microfluidic system. For the time being, no partnership has materialized for the transfer of this technology, but several conversations have been held with investors and some of the development work is being conducted jointly with WAMA Diagnósticos, a company headquartered in São Carlos that focuses on fast clinical tests.

The first bioanalytic assay created by Whitesides’ group at Harvard was an analysis of urine to measure glucose and proteins. Based on a central channel, looking like a stylized tree trunk, three branches or test zones opened out. The assays were based on known chemical and enzymatic reactions of urine analysis. The results showed that independent assays could be carried out simultaneously on the different areas of the same test system, with no cross-contamination of the reagents. In other words, several clinical analyses can be achieved at the same time with results that are similar to those from a traditional laboratory, without using a large number of samples, test tubes or refrigerators and without having to wait a fair amount of time “at least one day” for the results.

The design of the device may vary in terms of the number of branches that come out of the central trunk and of the closure at their ends, where the reagents are placed. Carrilho’s group is currently working on a system with five branches, two are for repeating each test and the central one is to quantify creatinine, a normalization parameter.

EDUARDO CESARMulti-layered arrangement enables different liquids to pass without mixingEDUARDO CESAR

Besides being quick, another advantage of this microsystem is the low cost of the materials used. In the case of the reagents, which color the samples and that are expensive, only tiny amounts are needed, in the order of 0.1 microliter (one liter divided by one million) of reagent per test. “With a single sheet of paper it is possible to produce 120 devices, at the printing cost of 5 cents of one US dollar,” says Carrilho. The manufacturing is also quite simple and does not require complex equipment.

At present, the researcher’s group is preparing one thousand devices for the tests that are to be conducted in Santa Luzia do Itanhy. All the homes will be visited by agents of the Federal Government’s Family Health Program (PSF). The initiative is part of a pilot project that is being implemented in that town by IPTI, the Technology Innovation Research Institute, a multidisciplinary study center associated with the PSF program. At a later stage, the devices will be made at the IPTI research center itself, the technology being transferred to the community.

Accessible testing
The idea is to introduce the analyses minilaboratory to other Brazilian towns. When any doubts arise as to the test results, a simple mobile phone photo can be sent to a specialist, who will provide a diagnosis after analyzing the image. The answer may arrive in the form of a text message on the original mobile, along with a prescription for the right treatment. “Telemedicine is a new way of making diagnosis even cheaper,” says Carrilho. It was precisely the notion of introducing these tests into poor countries, simply and at a low cost, that led to the development of the first microfluidic device on paper, as part of a research line that professor Whitesides has named Simple Solutions. The objective is to introduce the simple diagnosis tool in remote locations in Africa or of other developing nations, where there are no physicians or clinics. The research was financed by the Melinda and Bill Gates Foundation, which donated approximately US$10 million for five years.

The countless possibilities of this innovative bioanalysis platform are still being tested by the research groups involved in this project. They include the study of new paper devices, methods for the production of the devices and the development of new components, such as electrodes, valves, filters, mixers and surfacing that might expand the number of tested substances and consequently of the tests that can be conducted. The researchers are also focusing on the development of new methods for the stabilization of the reagents stored in the devices, so that they can be distributed without refrigeration.

The project
Development of microfluidic tools to enable the development and production of bioanalytic systems for chemistry, biology, biochemistry and medicine (nº 2006/02007-9); Type Grant for Work Abroad – New Frontiers; Supervisor George Whitesides – Harvard University; Grant holder Emanuel Carrilho – USP; Investment R$81,929.01 (FAPESP)

Scientific articles
CHENG, C.-M. et al. Paper-based Elisa. Angewandte Chemie International Edition. v. 49, p. 4.771-74. 2010.
MARTINEZ, A.W. et al. Diagnostics for the developing world: Microfluidic paper-based analytical devices. Analytical Chemistry. v. 82, p. 3-10. 2010.
CARRILHO, E. et al. Understanding wax printing: A simple micropatterning process for paper-based microfluidics. Analytical Chemistry. v. 81, p. 7.091-95. 2009.

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