Capillary-Driven Toner-Based Microfluidic Devices for Clinical ...

Technical Note pubs.acs.org/ac

Capillary-Driven Toner-Based Microfluidic Devices for Clinical Diagnostics with Colorimetric Detection Fabrício Ribeiro de Souza,† Guilherme Liberato Alves,† and Wendell Karlos Tomazelli Coltro*,†,‡ †

Instituto de Química, Universidade Federal de Goiás, Campus Samambaia, P.O. Box 131, 74001-970, Goiânia, GO, Brazil Instituto Nacional de Ciência e Tecnologia de Bioanalítica, 13083-970, Campinas, SP, Brazil

‡

S Supporting Information *

ABSTRACT: The fabrication of toner-based microfluidic devices to perform clinical diagnostics with capillary action and colorimetric detection is described in this report. Test zones and microfluidic channels were drawn in a graphic software package and laser printed on a polyester film. The printed layout and its mirror image were aligned with an intermediary cut-through polyester film and then thermally laminated together at 150 °C at 60 cm/min to obtain a channel with ca. 100-μm depth. Colorimetric assays for glucose, protein, and cholesterol were successfully performed using a desktop scanner. The limit of detection (LD) values found for protein, cholesterol, and glucose were 8, 0.2, and 0.3 mg/mL, respectively. The relative standard deviation (RSD) values for an interdevices comparison were 6%, 1%, and 3% for protein, cholesterol, and glucose, respectively. Bioassays were successfully performed on toner-based devices stored at different temperatures during five consecutive days without loss of activity. Likewise paper chips, toner-based microfluidic devices also integrate the current generation of disposable devices.26 The fabrication technology of the latter does not require a photolithographic step, plasma activation, or thermal treatment. Toner-based devices are drawn in graphic software and directly printed on a polyester film surface with an office laser printer. The printed channels are usually sealed against a mirrored image or a blank polyester using a laminator.26,27 Polyestertoner (PT) devices have been successfully explored to a wide range of applications including microchip electrophoresis,27−29 electrospray tips,27 micromixers,30 preconcentrators,31 solidphase extraction and PCR amplification,32 and, more recently, DNA analysis.33 The purpose of this technical note is to report the fabrication of capillary-driven toner-based microfluidic devices to perform clinical diagnostics with colorimetric detection. Glucose, cholesterol, and protein assays were chosen to demonstrate the feasibility of the proposed devices. These analytes have been selected due to their clinical relevance and association with diseases commonly found in developing and developed countries. The raised levels in glucose and cholesterol may lead the patients to have some diseases including diabetes, heart disease, and stroke, among other conditions.34 Besides glucose and cholesterol, the total serum protein assay is an useful diagnostic to assess the clinical conditions of the patient.

The development of microfluidic devices for clinical diagnostics has exhibited an impressive growth in the last years. Recent reports have focused on the fabrication of low cost devices to be used in point-of-care (POC) diagnostics in developing countries, where the availability of funds is limited.1−6 In this scenario, the availability of non- or minimally instrumented microfluidic devices is well-suited to be used in these locations.7 Capillary-driven microfluidic devices can meet these requirements because they are efficient, fast, and, most importantly, they do not need external power equipment.8−12 The spontaneous motion of a liquid inside microchannels toward functionalized surfaces or reaction chambers is one of the key advantages to obtaining a noninstrumented device for a rapid clinical diagnostics. For this reason, capillary-driven microfluidics have received special attention mainly because they can be combined with emerging platforms, including paper and toner-based devices, for bioanalytical applications or POC diagnostics.10,12 Paper-based microfluidic devices have appeared as a very promising platform for POC diagnostics in the last five years.2,13−25 The paper substrate is inexpensive and lightweight, and it is available in different forms.16 Paper-based devices can be produced by a wide range of methods including photolithography,13,14 inkjet printing,15,24 plasma oxidation,18 wax printing,19 flexographic printing23,24 and wax screen-printing.25 Examples of glucose, cholesterol, lactate, uric acid, and proteins assays with colorimetric detection have been successfully reported on paper-based microfluidic devices.2,13,16−20,22−24 © 2012 American Chemical Society

Received: August 30, 2012 Accepted: October 17, 2012 Published: October 17, 2012 9002

dx.doi.org/10.1021/ac302506k | Anal. Chem. 2012, 84, 9002−9007

Analytical Chemistry

Technical Note

100-μm deep. Figure 1 depicts the simplified fabrication process in 3D and cross-section views as well as the example of device layout for diagnostics used as proof-of-concept. Bioassays and Colorimetric Detection. During the preparation of toner-based devices for performing bioassays, a paste made of cellulose powder and water (1:4 m/m) was added to each test zone and was allowed to dry at room temperature during 30 min. Afterward, reagents for bioassays were spotted on the dry test zones to carry out the colorimetric measurements. Further details about all bioassays are available in the Supporting Information (SI). Standard or artificial samples (40 μL) were added to the central zone to distribute the sample inside microchannels by capillary action. The robustness of toner-based devices was investigated during five consecutive days at different storage temperatures. The scanner mode of a DeskJet multifunction printer (Hewlett-Packard, model F4280) was used to capture the resulting images. After capturing images, they were converted to a 8-bit grayscale in CorelPhoto-Paint. The arithmetic mean of pixel intensity within each test zone was used to quantify the colorimetric measurement.

Depending on the level of protein in serum, the diagnostics can indicate liver or kidney diseases.34 All analytes were successfully detected in artificial samples of human serum.

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EXPERIMENTAL SECTION Materials and Reagents. Cellulose powder, glucose oxidase (181 U/mg), D-glucose, horseradish peroxidase (73 U/mg), 4-aminoantipyrine, cholesterol esterase (300 U), cholesterol oxidase (204 U), potassium iodide, methylene blue, coomassie brilliant blue dye, piperazine-N, W-bis-2ethanesulfonic acid (PIPES), sodium monohydrogen phosphate, and sodium dihydrogen phosphate were acquired from Sigma Aldrich Co. (Saint Louis, MO, USA). The artificial serum sample and standards of total protein (40 mg/mL) and cholesterol (2.0 mg/mL) were obtained from Doles Reagentes (Goiânia, GO, Brazil). Polyester sheets (A4 size, model CG 3300) and toner cartridge (model CB435A) were purchased from 3M (São Paulo, SP, Brazil) and Hewlett-Packard (Palo Alto, CA, USA), respectively. All chemicals were used as received without further purification. Fabrication of Toner-Based Microfluidic Devices. Toner-based microfluidic devices were fabricated by a directprinting process.27−29 First, the desirable layout and its mirror image were drawn in Corel Draw software and printed on a polyester film using a laser printer with 1200-dpi resolution (Hewlett-Packard model 1102w). A cut-through polyester film was inserted between both printed images in order to enhance the channel depth and ensure the fluidic transport by capillary action. A polyester piece was cut at a local printing service (Ordones Laser Ltda, Goiânia, GO, Brazil) using a CO2 laser ablation machine. Before laminating, the zones in the upper piece of transparency were perforated (see Figure 1A) with a paper punch in order to access the microfluidic channels. The three polyester pieces were thermally laminated at 150 °C under a rate of 60 cm/min. The layout of the proposed device (35 mm × 35 mm) consisted of four test zones interconnected by microfluidic channels and one central inlet zone to sample distribution. All channels were 10-mm long, 1-mm wide, and ca.

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RESULTS AND DISCUSSION Capillary-Driven Microfluidics. The goal of this report is to communicate the results related to clinical assays performed on minimally instrumented toner-based devices. The use of capillary action to provide the distribution of sample inside microchannels is advantageous once no external power equipment (pumps, high-voltage power supplies, for example) to manipulate the fluid is required. On toner-based devices, the microfluidic structure is defined by polyester sheets and toner walls. The use of a cut-through polyester placed in the middle of two printed layouts increases the channel depth to ca. 110 μm. This strategy has assured the spontaneous capillary transport of fluid inside channels in association with the hydrostatic pressure.35 In comparison with paper-based microfluidic devices, where the sample is distributed by a lateral-flow, toner-based devices do not require any additional step to change the hydrophobic nature of channels.13,22 Due to its simplicity, the proposed device is quite attractive for applications in locations with limited resources. The effect of the added volume to the sample inlet zone as well as the influence of the channel width on the flow rate magnitude was evaluated. As it can be seen in Figure 2A, the higher the sample volume, the greater flow rate magnitude. These results are expected once the capillary pressure is directly proportional to the sample volume.8,9 Channels with width ranging from 0.5 to 1.0 mm were produced in order to investigate the effect of their dimensions on the flow rate magnitude. Figure 2B shows that lower flow rates were found to wider channels. According to a mathematical model reported by Zimmermann and co-workers,9 the flow in a microchannel can be estimated through the capillary pressure divided by the flow resistance. In addition, the flow resistance is dependent on the channel width. On the basis of their model,9 the wider the channels, the higher the flow resistance. Consequently, wider channels promote lower flow rates. Our results are in agreement with previous reports about capillary-driven microfluidic systems.8,10,11 The flow rate was determined by using a contactless conductivity detector for microfluidic devices previously published.36 The flow magnitude was based on the time for a

Figure 1. Simplified fabrication process of toner-based microfluidic devices for clinical assay in (A) 3D and (B) cross-section views and (C) layout of a typical device used to demonstrate the capability of performing colorimetric assays. 9003



Technical Note

hydrophobic nature of the microfluidic structure. The strategy adopted to produce this hybrid device has exhibited advantages including reproducibility, specificity and capability of performing by the first time bioassays with clinically relevant analytes on the proposed platform. Before running all assays simultaneously in a single device, each bioassay was performed separately in the four test zones of the each device (Figure 3A−C). This study shows that good

Figure 2. Effect of (A) sample volume and (B) channel width on the flow rate induced by capillary action on toner-based microfluidic devices. In part A, channel width was 0.5 mm; in B, the sample volume added to central zone was 10 μL.

Figure 3. Scanned images regarded to (A) cholesterol, (B) glucose, and (C) total protein assays performed separately in four test zones on a single device. Image D shows the simultaneous assays of all analytes with a standard mixture.

methylene blue aqueous solution (1 mg/mL) to fill the distance between the injection point and the detector.

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COLORIMETRIC ASSAYS Colorimetric detection of analytes is usually based on an enzymatic reaction causing a color conversion.24 In this current report, a desktop scanner was used to capture the resulting images of all assays on toner-based devices. Preliminary tests were performed with a cell phone camera and a portable optical microscope, however, interference due to the external light was often found. In our first devices, the main problem observed was the poor reproducibility of colorimetric assays. When the reagents were spotted on each test zone, part of this solution flowed through the channel due to the hydrostatic pressure and the hydrophilic nature of polyester surface. Consequently, it was noticed the development of color in the entrance of microfluidic channels during the running of bioassays. In order to estimate the hydrophobic nature, angle contact measurements were taken according to a procedure detailed elsewhere37 and compared to native PDMS and glass surfaces. The contact angles (see Figure S-1, available in the SI) for polyester, PDMS, and glass were 60 ± 2°, 88 ± 1°, and 42 ± 1°, respectively. These data reveal that the polyester surface is more hydrophilic than the surface of native PDMS and more hydrophobic than the surface of glass. To circumvent the problem mentioned above, it was added a cellulose paste into each test zone to ensure the presence of all reagents only in the test zones. For all assays, a 10 μL aliquot of the cellulose paste was placed at each zone. After drying, the cellulose-based test zones act as detection chambers for the bioassays. When compared with paper-based devices,13,16,22 the hybrid device composed of polyester, toner, and cellulose does not require any chemical or physical treatment to change the

reproducibility can be achieved for all bioassays on toner-based microfluidic chips. The relative standard deviation (RSD) values for the mean pixel intensity in all three assays ranged from 2% to 5%. Table S-1 (available in the SI) shows the values achieved for all zones related to each bioassay. As shown in Figure 3, protein, cholesterol, and glucose gave blue, red, and yellow colors, respectively. For glucose assays, higher concentrations provided brown colors. Once each assay demonstrated the reproducibility, a standard mixture containing protein (10.0 mg/mL), cholesterol (1.0 mg/mL), and glucose (1.8 mg/mL) was added to the central inlet zone in a tonerbased microfluidic device. Figure 3D shows a typical example of simultaneous detection of all analytes in a single device. All images were recorded after 10 min of reaction. Calibration curves were performed in order to determine the linear concentration range and the limits of detections (LD) for each assay. The LD values for all bioassays were calculated based on the ratio between three times the standard deviation for the control test and the angular coefficient of each analytical curve. The equations extracted from each linear regression are depicted in Figure S-2 (see the SI). The glucose assay was linear in the concentration range between 0 and 2.2 mg/mL with a LD of 0.3 mg/mL. This range is appropriate for clinical use once levels of glucose in serum below 0.5 mg/mL and above 1.3 mg/mL may be an indicative of disease.34 According to the values found on the proposed devices, the level of glucose could be quantified in other biological fluids such as urine samples. As reported by Martinez et al., urine is the most representative biological fluid and the detection of glucose at levels above 1.4 mM (0.25 mg/mL) in urine can also be an indicative of disease or physiological disorder.16 9004



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The cholesterol assay was linear for concentrations ranging from 0 to 2.0 mg/mL, with a LD of 0.2 mg/mL. This range represents the regular levels of cholesterol in serum; however, it does not comprise the full range of concentrations of cholesterol detected in clinical diagnostics (1.0−4.0 mg/mL). Levels of cholesterol above 2.4 mg/mL may lead to a high risk factor for heart disease.34 We did not run tests with concentrations of cholesterol above 2.0 mg/mL; however, the quantitative analysis of higher levels can be performed by diluting the sample. Lastly, total protein assay presented a linear correlation for a concentration range between 0 and 40 mg/mL with a LD of 8 mg/mL. This range is helpful on assessing certain clinical conditions that can rapidly evolve and worsen. The level of total serum proteins below 30 mg/mL represents a clinical condition of hypoproteinemia.34 This may occur due to an inadequate production of protein or to the loss of protein in the urine (albuminuria) caused by liver and kidney diseases, respectively.16,34 This assay can be useful to investigate and distinguish different renal diseases; however, a most complete diagnostic using specific proteins (albumin and globulin, for example) and other colorimetric indicators have to be performed.16 Robustness of the Devices. As discussed earlier, the use of cellulose powder has substantially improved the reproducibility of bioassays on toner-based microfluidic devices. As it can be seen in Figure S-3 and Table S-2 (see the SI), the assays performed on three different devices (device-to-device comparison) exhibited a great reproducibility. The RSD values for protein, cholesterol, and glucose assays were 6%, 1%, and 3%, respectively. For protein tests, a single device has been used during five consecutive assays (data not shown). On the other hand, enzymatic assays were performed just once per device. The lifetime of the devices has been evaluated over five consecutive days at different storage temperatures. In this study, thirty devices were prepared according to the experimental procedure and kept at 10, 25, and 40 °C. Ten devices were evaluated at each storage temperature for studying the effect of presence of trehalose on the stability of the enzymatic activity. Stored chips were tested every day with a standard mixture of protein, glucose, and cholesterol. As it can be seen in Figure 4, the mean intensity for bioassays at all storage temperatures decreased quickly without the presence of trehalose. Once the behavior for all assays was similar (except proteins which do not require enzyme), just the data for glucose are shown. The decay for the mean pixel intensities ranged from 45 to 65%. The shelf life for these assays without trehalose has been estimated to be around 3 days. The addition of trehalose has extended the lifetime of toner-based devices for all storage temperatures. In the presence of this disaccharide, the RSD values for 10, 25, and 40 °C were, respectively, 13%, 2%, and 6% over five consecutive days. These data allow concluding that the trehalose assures the stability at different storage temperatures during five consecutive days without loss of activity. These results are in agreement with recent papers related to bioassays on paper-based microfluidic devices.16,22 Semiquantitative Analysis in Serum Samples. Capillary-driven toner-based microfluidics devices were investigated for simultaneous analysis of glucose, cholesterol, and total protein in an artificial serum sample. This clinical sample was used to determine the accuracy of the proposed method. Toner-based devices were able to detect all analytes before and

Figure 4. Effects of the storage temperature on the stability of enzymatic assays. Data shown in A, B, and C exhibit the results for glucose assay at 10, 25, and 40 °C, respectively. The symbols ● and ■ represent the data without and with trehalose, respectively.

after three serial dilutions (1/5, 1/10, and 1/100 in v/v proportion). Scanned images as well as the mean pixel intensity recorded for each assay with the diluted solutions are shown in Figures S-4 and S-5 (available in the SI). On the basis of the semiquantitative analysis of the serum sample, the values found for glucose, cholesterol, and total proteins were 0.8, 1.2, and 47.8 mg/mL, respectively. As it can be seen in Table 1, all Table 1. Comparison between the Certified Data and Values Calculated with Our Method analytes

certified values (mg/mL)

our proposed method (mg/mL)

glucose cholesterol protein

0.6−0.8 1.1−1.4 39.2−49.8

0.8 1.2 47.8

values found with toner-based devices are in agreement with the concentration ranges provided by the supplier. The concentrations were determined in the artificial serum sample based on analytical curves depicted in Figure S-2 (SI). Future work will focus on the study of other clinically relevant assays including bovine serum albumin, lactate, uric acid, and triglycerides.

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CONCLUSIONS It has been demonstrated that toner-based microfluidic devices can be used to perform clinical assays with capillary action and colorimetric measurements. Detection of glucose, protein, and cholesterol has been successfully achieved in an artificial serum sample in therapeutical concentration range. The values found on toner-based microfluidic device were similar to those provided by supplier. One of the main advantages is that toner-based technology does not need either photolithographic steps or thermal treatment. In the proposed device, a cut-through polyester layer added in the middle of printed layouts was prepared using a laser ablation machine. Although this step requires sophisti9005



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(3) Rivet, C.; Lee, H.; Hirsch, A.; Hamilton, S.; Lu, H. Chem. Eng. Sci. 2011, 66, 1490−1507. (4) Chin, C. D.; Laksanasopin, T.; Cheung, Y. K.; Steinmiller, D.; Linder, V.; Parsa, H.; Wang, J.; Moore, H.; Rouse, R.; Umviligihozo, G.; Karita, E.; Mwanbarangwe, L.; Branstein, S. L.; van de Wijgertm, J.; Sahabo, R.; Justman, J. E.; El-sadr, W.; Sia, S. K. Nat. Med. 2011, 17, 1015−1019. (5) Govindarajan, A. V.; Ramachandran, S.; Vigil, G. D.; Yager, P.; Böhringer, K. F. Lab Chip 2012, 12, 174−181. (6) Mao, X.; Huang, T. J. Lab Chip 2012, 12, 1412−1416. (7) Weigl, B.; Domingo, G.; LaBarre, P.; Gerlach, J. Lab Chip 2008, 8, 1999−2014. (8) Juncker, D.; Schmid, H.; Drechsler, U.; Wolf, H.; Wolf, M.; Michel, B.; de Rooij, N.; Delamarche, E. Anal. Chem. 2002, 74, 6139− 6144. (9) Zimmermann, M.; Shmid, H.; Hunziker, P.; Delamarche, E. Lab Chip 2007, 7, 119−125. (10) Gervais, L.; Delamarche, E. Lab Chip 2009, 9, 3330−3337. (11) Yang, D.; Krasowska, M.; Priest, C.; Popescu, M. N.; Ralston, J. J. Phys. Chem. C 2011, 115, 18761−18769. (12) Hitzbleck, M.; Avrain, L.; Smekens, V.; Lovchik, R. D.; Mertens, P.; Delamarche, E. Lab Chip 2012, 12, 1972−1978. (13) Martinez, A. W.; Phillips, S. T.; Butte, M. J.; Whitesides, G. M. Angew. Chem., Int. Ed. 2007, 46, 1318−1320. (14) Martinez, A. W.; Phillips, S. T.; Wiley, B. J.; Gupta, M.; Whitesides, G. M. Lab Chip 2008, 8, 2146−2150. (15) Abe, K.; Suzuki, K.; Citterio, D. Anal. Chem. 2008, 80, 6928− 6934. (16) Martinez, A. W.; Phillips, S. T.; Carrilho, E.; Thomas, S. W.; Sindi, H.; Whitesides, G. M. Anal. Chem. 2008, 80, 3699−3707. (17) Zhao, W.; van den Berg, A. Lab Chip 2008, 8, 1988−1991. (18) Li, X.; Tian, J.; Nguyen, T.; Sen, W. Anal. Chem. 2008, 80, 9131−9134. (19) Carrilho, E.; Martinez, A. W.; Whitesides, G. M. Anal. Chem. 2009, 81, 7091−7095. (20) Ellerbee, A. K.; Phillips, S. T.; Siegel, A. C.; Mirica, K. A.; Martinez, A. W.; Striehl, P.; Prentiss, M.; Whitesides, G. M. Anal. Chem. 2009, 81, 8447−8452. (21) Pelton, R. Trends Anal. Chem. 2009, 28, 925−942. (22) Dungchai, W.; Chailapakul, O.; Henry, C. S. Anal. Chim. Acta 2010, 674, 227−233. (23) Olkkonen, J.; Lehtinen, K.; Erho, T. Anal. Chem. 2010, 82, 10246−10250. (24) Mäaẗ tänem, A.; Fors, D.; Wang, S.; Valtakari, D.; Ihalainen, P.; Peltonen, J. Sensor Actuat. B−Chem. 2011, 160, 1404−1412. (25) Dungchai, W.; Chailapakul, O.; Henry, C. S. Analyst 2011, 136, 77−82. (26) Coltro, W. K. T.; de Jesus, D. P.; da Silva, J. A. F.; do Lago, C. L.; Carrilho, E. Electrophoresis 2010, 31, 2487−2498. (27) do Lago, C. L.; da Silva, H. D. T.; Neves, C. A.; Brito-Neto, J. G. A.; da Silva, J. A. F. Anal. Chem. 2003, 75, 3853−3858. (28) Coltro, W. K. T.; da Silva, J. A. F.; Carrilho, E. Electrophoresis 2008, 29, 2260−2265. (29) Coltro, W. K. T.; Lunte, S. M.; Carrilho, E. Electrophoresis 2008, 29, 4928−4937. (30) Liu, A. L.; He, F. Y.; Wang, K.; Zhou, T.; Lu, Y.; Xia, X. H. Lab Chip 2005, 5, 974−978. (31) Yu, H.; Lu, Y.; Zhou, Y. G.; Wang, F. B.; He, F. Y.; Xia, X. H. Lab Chip 2008, 8, 1496−1501. (32) Duarte, G. R. M.; Price, C. W.; Augustine, B. H.; Carrilho, E.; Landers, J. P. Anal. Chem. 2011, 83, 5182−5189. (33) Duarte, G. R. M.; Coltro, W. K. T.; Borba, J. C.; Price, C. W.; Carrilho, E.; Landers, J. P. Analyst 2012, 137, 2692−2698. (34) McPherson, R. A.; Pincus, M. R. Henry’s Clinical Diagnosis and Management by Laboratory Methods, 22nd ed.; Elsevier Saunders: Philadelphia, 2011. (35) Saito, R. M.; Coltro, W. K. T.; de Jesus, D. P. Electrophoresis 2012, 33, 2614−2623.

cated equipment limited to a few research groups, it is important to point out that thirty five pieces were obtained in a single polyester sheet (A4 size) within five minutes. The price of each cut-through polyester piece has been estimated to be ca. 0.10 cents. Even with the capability of mass production in a short time period, the polyester piece can also be easily cut using a scalpel. This tool has been used in the fabrication of our first devices; however, the lack of uniformity along the channel, the poor device-to-device reproducibility, and the limitation to produce more complex microfluidic geometries were some inconveniences found. Figure S-6 (see the SI) depicts two optical micrographies comparing the cut performed by a CO2 laser and a scalpel. Taking into account the considerations above, the cost of a complete device is estimated to be ca. 0.15 cents (including the costs of polyester film, toner layer, and also the cut-through polyester). This low cost stimulates the use of the proposed devices for POC diagnostics in places with limited resources. In addition, this attractive feature suggests discarding of the toner chip after its use. The definition of PT channels with toner and three polyester surfaces assures a high aspect-to-ratio microfluidic structure which allows sample handling with capillary transport. Furthermore, some additional advantages have been found including the excellent zone-to-zone (RSD ≤ 5%) and deviceto-device (RSD < 8%) reproducibility. We hope this work along with paper technology2 and fluidic batteries38recently reportedcan be helpful to revolutionize the healthcare systems for the POC diagnostics.

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ASSOCIATED CONTENT

S Supporting Information *

Details about the bioassays preparation, images, and data about the zone-to-zone and device-to-device comparison and semiquantitative analysis. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: + 55 62 3521 1097. Fax: + 55 62 3521 1167. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project has been supported by Conselho Nacional de ́ Desenvolvimento Cientifico e Tecnológico (CNPq)grant No. 477067/2010-7. The authors gratefully acknowledge the research fellowship granted from CNPq to W.K.T.C. and scholarships granted from CAPES to F.R.d.S. and from CNPq to G.L.A. Professors D. Damasceno and K. F. F. Silva are thanked for their helpful assistance in graphic designs and dye donation (comassie brilliant blue G), respectively. We also recognize the GEM (Grupo de Eletroforese e Microssistemas) from UNICAMP, for using their facilities (CO2 laser) to produce the first devices, and Dr. M. Hathcock, for english revision.

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REFERENCES

(1) Yager, P.; Edwards, T.; Fu, E.; Helton, K.; Nelson, K.; Tam, M. R.; Weigl, B. H. Nature 2006, 442, 412−418. (2) Martinez, A. W.; Phillips, S. T.; Whitesides, G. M.; Carrilho, E. Anal. Chem. 2010, 82, 3−10. 9006



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(36) Coltro, W. K. T.; da Silva, J. A. F.; Carrilho, E. Anal. Methods 2011, 3, 168−172. (37) Piccin, E.; Coltro, W. K. T.; da Silva, J. A. F.; Neto, S. C.; Mazo, L. H.; Carrilho, E. J. Chromatogr. A 2007, 1173, 151−158. (38) Thom, N. K.; Yeung, K.; Pillion, M. B.; Phillips, S. T. Lab Chip 2012, 12, 1768−1770.

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