Low-Cost Fabrication of Paper-Based Microfluidic

Jul 19, 2012 - photolithography in 2007,11 a series of creative methods of patterning paper have been reported, ... are capable of creating permanent ...
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Low-Cost Fabrication of Paper-Based Microfluidic Devices by OneStep Plotting Jinfang Nie, Yun Zhang,* Liwen Lin, Caibin Zhou, Shuhuai Li, Lianming Zhang, and Jianping Li College of Chemistry and Bioengineering, Guilin University of Technology, Guilin 541004, China S Supporting Information *

ABSTRACT: In this technical note, we describe a facile method for one-step fabrication of paper-based microfluidic devices, by simply using commercially available permanent markers and metal templates with specific patterns. The fabrication process involves only a single step of plotting pattern in paper; it can be typically finished within 1 min. The ink marks formed in the patterned paper will act as the hydrophobic barriers to define the hydrophilic flow paths or separate test zones. Various paper devices can be created by using different templates with corresponding patterns. Transparent adhesive tape-sandwiched devices could protect their assay surfaces from potential contamination. In the proof-of-concept experiments, circular paper test zones (∼3 mm diameter) were fabricated for colorimetric and quantification detection of prostate-specific antigen (PSA) as a model target, based on dotimmunogold staining assays coupled with gold enhancement amplification. Several serum specimens were additionally evaluated with this new approach and the results were compared with the commercial chemiluminescence immunoassay, validating its feasibility of practical applications. Such a one-step plotting method for paper patterning does not require any specialized equipments and skills, is quite inexpensive and rapid, and thus holds great potential to find wide applications especially in remote regions and resource-limited environments such as small laboratories and private clinics.

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paper stamp-based printing,25 plasma etching,26 laser treatment,27 cutting,28 and mechanical plotting.29 The need remains, nevertheless, for more simple fabrication techniques that are sufficiently inexpensive to be widely applied by nontechnical personnel, particularly in remote regions, small laboratories, and private clinics. In this technical note, we report a one-step plotting method of patterning paper that has many of these merits, by using commercially available permanent markers and metal templates with specific patterns. Permanent markers are porous pens that are capable of creating permanent writing on a variety of surfaces from plastic to metal to stone. Generally the ink used is water resistant and consists of a colorant, a solvent (typically ethanol), and a hydrophobic resin. Herein, the inexpensive permanent markers are used to directly plot (create) paper devices with the aid of iron templates with designed patterns that are fabricated by a traditional laser cutting technique. During the plotting of patterns in paper, the ink can penetrate through the paper body. After a rapid evaporation of the solvent, the resins remaining in the marks in paper will form the hydrophobic barriers to direct the flow paths or independent test zones. The fabrication process is schematically shown in Figure 1.

apid, simple, and sensitive measurement of proteins is a crucial need in many areas in modern biomedical research such as clinical diagnostics, drug discovery, and biodefense.1−3 In particular, clinical measurement of collections of cancer biomarkers show great promise for meeting highly reliable predictions for early cancer detection.4 Modern commercial immunoassays, including the Immulite,5 Beckman Coulter Access,6 Nichols Advantage,7 and optimized enzyme-linked immunosorbent assay (ELISA)8 provide sensitive and selective analysis of various disease biomarkers in serum, but these analytical systems are difficult to implement in resource-poor regions as they are expensive and large, require well-trained medical personnel, and need considerable volumes of reagents and samples. In recent years, there is an increasing interest in the development of paper-based microfluidic devices that are created by patterning hydrophobic materials in hydrophilic paper. These systems can combine some of the capabilities of traditional microfluidic devices (e.g., small volumes of fluids required for each bioassay) with the low cost, simplicity, and portability of paper-strip tests. They are considered to be the ideal bioassay platforms to develop point-of-care diagnostic devices for use in less-industrialized countries, in remote settings, or even in home care services.9,10 Since Martinez et al. pioneered the field by patterning chromatography paper via photolithography in 2007,11 a series of creative methods of patterning paper have been reported, such as wax printing,12−20 inject printing,19,21 laser printing,20,22 flexographic printing,23,24 © 2012 American Chemical Society

Received: December 31, 2011 Accepted: July 19, 2012 Published: July 19, 2012 6331

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Technical Note

power of the laser were filled to be 2 ms, 150 Hz, and 160 W, respectively. The patterns were predesigned on a personal computer using CorelDraw X6. The iron slice used herein for template fabrication is one example of practice, but other kinds of slice-shaped metal materials such as stainless steel should be suitable as well. Fabrication of Paper-Based Microfluidic Devices. The fabrication process is schematically shown in Figure 1. The metal (iron) template with a specific pattern was placed on a piece of chromatography paper. A permanent marker was then used to directly plot the pattern of the template on the paper surface. The resultant patterned paper was subsequently left at room temperature to evaporate the solvent in ink marks. The plotting time mainly depended on the complexity of the device’s pattern. Note that it is necessary to avoid the exposure of the mark-patterned paper in organic reagents (e.g., acetone) that can dissolve the resin in hydrophobic marks. Number- and color-coded devices for multianalyte analysis were fabricated by marking the different test regions of the corresponding patterned paper with different numbers and colors, respectively. Contamination-resistant devices were additionally created by sandwiching the patterned paper with two pieces of transparent adhesive tape. Gold-Enhanced Immunogold Staining Assays for PSA Detection. To demonstrate the use of ink mark-patterned paper in the detection of PSA model target, a set of 3 mm diameter circular test zones were prepared and used for running dot-immunogold staining assays coupled with gold enhancement amplification. The top and bottom faces of the test zones are open to the atmosphere. The procedures for the immunoassays on the patterned paper are similar as the sandwich ELISA format. In a typical PSA-detection experiment, to immobilize mAb on the patterned paper, a 3 μL solution of the protein in PBS (0.1 g/L) was first spotted on a test zone and allowed to air-dry for 10 min under ambient conditions. The nonspecific adsorption sites in paper were subsequently blocked by incubating the test zone in 3 μL of a mixture of 0.05% (v/v) Tween-20 and 5% (w/v) BSA in PBS and allowing the zone to air-dry for another 10 min under ambient conditions. Then, 3 μL of sample solution containing prefilled concentration of PSA in PBS was added on the patterned paper, and the PSA-bound zone was allowed to air-dry for 10 min under ambient conditions. Next, a 3 μL solution containing GNP-pAb in PBS was added to the test zone and allowed to incubate for 3 min. Finally, the GNP-bound zone was incubated in 3 μL of gold enhancer solution for 3 min to realize the enlargement of gold nanoparticle labels for colorimetric or quantitative analysis. Note that the test zone was washed three times before each new reagent was introduced, by adding PBS containing 0.05% (v/v) Tween-20 to the top of the zone while bringing its bottom in contact with the blotting paper. Control experiments for PBS (without PSA analyte) or comparison experiments for BSA and HSA (as analytes) were carried out in the same way. Quantitative Analysis. To achieve relative levels of PSA on test zones, the intensity of staining image of each zone was captured by a desktop scanner (Uniscan A3, China) and further measured using the grayscale function with the blue channel option of the ACDSee image processing software. The staining intensities of sample (Is) and PBS (control, Ic) were measured; the relative intensity was defined as (Is − Ic). Of note, each of the staining intensity was given out with the average value of five different positions of the same staining result.

Figure 1. Schematic of the one-step plotting method for patterning paper with the use of a permanent marker and a template with a specific pattern.

Different templates were used to create various paper devices such as independent test zones (or arrays). Coded devices for multiplexed bioassays could be fabricated by labeling the elements of test regions with different numbers or colors. In addition, the patterned paper was sandwiched with two pieces of transparent adhesive tape to provide contamination-resistant devices. The analytical capability of the as-prepared paper devices was further demonstrated by running dot-immunogold staining assays (coupled with the gold enhancement amplification) for colorimetric and quantification detection of a protein target, namely, prostate-specific antigen (PSA) in serum samples. PSA is chosen as the initial target for the study because it is one of the most valuable tumor markers that have been used successfully for diagnosis, screening, and postsurgical management of prostate cancer patients.1,8 Moreover, PSA is also explored as a screening target for breast cancer.30,31

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EXPERIMENTAL SECTION Reagents and Materials. Prostate-specific antigen (PSA) was purchased from the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China). PSA monoclonal antibody (mAb) was obtained from Tianjian Biotechnology Company (Tianjin, China). PSA polyclonal antibody (pAb) was purchased from Shanhai Xinran Biology Technology Company (Shanghai, China). The clinical serum specimens from patients with prostate disease were obtained from the Third Xiangya Hospital (Changsha, China). The PSA chemiluminescent immunoassay kit was purchased from the Autobio Company (Zhenzhou, China). Bovine serum albumin (BSA) and human serum albumin (HSA) were the products of the Dingguo Biological Product Company (Beijing, China). All other reagents were of analytical grade. Phosphate-buffered saline (PBS, 10 mmol/L, pH 7) solution was prepared with Na2HPO4 and KH2PO4. The gold enhancer solution was a 1:1 (v/v) mixture of 0.01% HAuCl4 and 0.04 mol/L NH2OH·HCl prepared before use.32 The preparation of gold nanoparticles (GNPs) with an average diameter of approximately 15 nm and the GNP-pAb conjugates was accomplished according to a previously reported method.33 All solutions were prepared with deionized water (specific resistivity >18 MΩ cm). Chromatography paper was purchased from the Shanghai Shuoguang Electronic Technology Company (Shanghai, China). Permanent markers were the products of the Shanghai Lemei Stationery Company (Shanghai, China). A piece of iron slice was used to fabricate the metal templates with specific patterns on a JQMX3015-550 laser cutting machine that was from Wuhan Chutian Laser Group Co., Ltd. (Wuhan, China). The duration and frequency of the laser pulses and the average 6332

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Figure 2. (A) The front side images of the patterned paper before (left) and after (right) the ink-free hydrophilic zones were wetted with red aqueous solutions. (B) The back side image of the patterned paper.

Figure 3. Shape- (A), number- (B), and color-coded (C) paper-based microfluidic devices capable of performing multianalyte bioassays with minimized operator error and reading error.

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RESULTS AND DISCUSSION In this work, we propose a one-step plotting strategy for paper patterning (Figure 1). This novel method, which makes the use of quite inexpensive permanent markers and metal (iron) templates with designed patterns, only involves one step of plotting the patterns of ink marks in paper. In comparison with the existing methods for the fabrication of paper-based microfluidic devices (see Table S1 in Supporting Information), the proposed technique requires no experience, large equipment, additional power supply, or specialized skills. The truly facile fabrication process can be accomplished by any nontechnical personnel. These advantages would make it able to be implemented especially in remote regions and resourcelimited environments such as small laboratories and private clinics. Figure 2 shows the comparisons of the front side and back side images of the ink mark-patterned paper, from which one can see that the darkness of the back side was similar to that of the front side. This indicated that the ink had penetrated through the paper body during the plotting process. After the evaporation of the solvent, the resins remaining in the marks in paper will form the hydrophobic barriers that could direct the flowing of aqueous solutions (Figure 2A, right). That is, the ink-free hydrophilic independent test zones or channels were defined by the hydrophobic resin-containing regions. It was experimentally found that the evaporation of the solvent in ink marks was so rapid that the mark-patterned paper was ready for use once the plotting process was completed. Actually, a typical paper device (i.e., 3 cm × 3 cm) could be fabricated within 1 min. Of course, it should be pointed out that the fabrication time mainly depends on the complexity of the device’s pattern. For instance, it would take 6−10 min to create a 96-microzone paper plate (see Figure S1 in the Supporting Information). It is very important to avoid an operator or reading error in paper-based multiplexed bioassays. Generally, the errorminimizing method is based on the shape coding where different test regions are designed with multiple shapes (Figure 3A), but it offers a low coding capacity with a very limited number (typically three) of distinguishable shape codes in a single device. Alternatively, two strategies based on numbercoding and color-coding are proposed in this work to minimize

operator or reading error by marking different test regions with different numbers (Figure 3B) and colors (Figure 3C), respectively. Although the number-coding method was initially reported by Fenton et al.,28 the design of separating the codes from the assay zones described herein should be more suitable for bioassays since it could avoid an additional step of preventing the spreading of aqueous agent or sample solutions from the assay area to the number codes. The number of the distinguishable number or color codes is mainly dependent on the areas of each test region and the whole device used. To protect the assay surface from potential contamination, the device could be further sandwiched with two pieces of transparent adhesive tape (Figure 4).

Figure 4. Contamination-resistant paper-based microfluidic device that is sandwiched with transparent adhesive tape.

To demonstrate the use of ink mark-patterned paper in the detection of PSA model target, a set of 3 mm diameter circular paper test zones were fabricated and used for carrying out sandwich-type dot-immunogold staining assays based on the gold enhancement amplification (Figure 5A). This system was chosen for several reasons: (1) the colorimetric readout can allow untrained personnel to provide rapid, reliable answers at reduced cost without the need for a specialized detector;34,35 (2) as compared with conventional silver staining method for the enlargement of GNP labels, the gold enhancing strategy adopted herein benefits from shorter reaction time and higher assay sensitivity;36 and (3) each of the used test zones was ∼3 mm in diameter and required only ∼3 μL of reagents (i.e., PSA mAbs, GNP-pAbs, and gold enhancer solution) and sample to fill due to its small volume. 6333

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Figure 5. (A) Schematic of the colorimetric detection of PSA based on sandwich dot-immunogold staining assays coupled with gold enhancement. (B) The staining results obtained from nine samples. (C) The calibration curve describing the relationship between the relative intensity of the color developed in the test zones shown in part B and the logarithm values of PSA concentrations.

The main experimental parameters have been optimized, including the concentration and incubation time for the immobilization of primary antibody, the reaction time for the two steps of immunoreactions, and the reaction time for the enlargement of GNP labels (see Figure S2 in the Supporting Information). Under the optimized conditions, a set of samples containing PSA concentrations in the range 0.05−100 μg/L were detected with the gold-enhanced immunngold staining assay method. It is clearly observed that that the intensity of the staining color that developed in each test zone is proportional to the amount of PSA (Figure 5B). When the PSA concentration is 0.05) between the results of the two methods. Moreover, one can find from Table S2 in the Supporting Information that the developed immunogold

Figure 6. Correlation of the analytical results for 10 human serum specimens (without dilutions) separately obtained by the proposed gold-enhanced immunogold staining assay and by the CLIA method, corresponding to a regression equation of y = 1.037x − 0.04606 and a correlation coefficient of 0.9961 (P > 0.05). Each data point represents the average value of 5 (staining assay) and 3 (CLIA) repetitive experiments.

staining method provides a comparable detection limit but requires lower volumes of reagents and samples, shorter assay time, and a cheaper detection device in comparison with the CLIA. These results indicate that this novel technique may hold great promise as a viable alternative tool for the determination of PSA in real clinical serum samples.

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CONCLUSIONS We have developed a one-step plotting method for the fabrication of paper-based microfluidic devices with the use of permanent markers and metal (iron) templates with specific patterns. The proposed strategy offers the main advantages over the conventional techniques in that it does not require any specialized skills or equipment and is less expensive and easier to perform. The as-prepared paper devices (i.e., test microzones) were demonstrated to implement low-volume, rapid gold-enhanced dot-immunogold staining assays of PSA model analyte with high sensitivity and selectivity. Such a truly lowcost, simple, and efficient paper micropatterning technology thus represents a powerful addition to the existing arsenal of the fabrication of paper devices especially in remote regions and resource-limited environments (e.g., small laboratories and private clinics) for point-of-care bioapplications. 6334

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Technical Note

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86 773 5896453. Fax: +86 773 5896839. E-mail: zy@ glite.edu.cn. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grant Nos. 21105017 and 21165007) and the Doctoral Fund of Guilin University of Technology (Grant Nos. 002401003320 and 002401003337).

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