Equipment-Free Quantitative Measurement for Microfluidic Paper

In this article, we describe MTWP (movable-type wax printing), a facile method for the ... wax and polymer) in hydrophilic paper to offer complex micr...
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Equipment-Free Quantitative Measurement for Microfluidic PaperBased Analytical Devices Fabricated Using the Principles of MovableType Printing Yun Zhang,* Caibin Zhou, Jinfang Nie,* Shangwang Le, Qun Qin, Fang Liu, Yuping Li, and Jianping Li College of Chemistry and Bioengineering, Guilin University of Technology, Guilin 541004, China S Supporting Information *

ABSTRACT: Microfluidic paper-based analytical devices (μPADs) are a growing class of low-cost chemo/biosensing technologies designed for point-of-use applications. In this article, we describe MTWP (movable-type wax printing), a facile method for the fabrication of μPADs. MTWP is inspired by the Chinese movable-type printing and requires only a hot plate and homemade small iron movable components. It is able to pattern various wax microstructures in paper via a simple adjustment of the number, patterning forms or types of the metal movable components. This inexpensive and versatile method may thus hold great potential for producing wax-patterned μPADs by untrained operators at minimized cost in developing countries. In addition, two novel equipment-free assay methods are further developed to render μPAD measurements straightforward and quantitative. They use the flow-through time of a detection reagent in a three-dimensional paper device and the number of colored detection microzones in a 24-zone paper device as the detection motifs. The timing method is based on the selective wettability change of paper from hydrophilic to hydrophobic that is mediated by enzymatic reactions. The counting method is carried out on the basis of oxidation−reduction reactions of a colored substance, namely iodine. Their utility is demonstrated with quantitative detection of hydrogen peroxide as a model analyte. These methods require only a timer or a cell phone with a timing function and the abilities of seeing color and of counting for quantitative μPAD measurement, thus making them simple, cost-efficient, and useful sensor technologies for a great diversity of point-of-need applications especially in resource-poor settings.

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common limitation of the need for tools that are expensive and rare in laboratories of the developing world, such as lithography equipment, wax printers, plasma oxidizers, and CO2 lasers. Moreover, trained personnel are required to use and maintain these tools. Recently, some efforts have been alternatively dedicated to develop simple, low-cost fabrication methods, requiring minimal external instrumentation for implementation in developing countries. For instance, a wax-screen-printing method, which only utilized solid wax, patterned masks, and a hot plate, was reported by Dungchai et al. for the prototyping of μPADs.25 Songjaroen and co-workers proposed a dipping method for wax patterning in paper by dipping the paper that was sandwiched with a metal mask and a magnet slice into molten wax.26 We had reported a simple one-step hand-plotting method to create μPADs using commercially available permanent markers and metal templates with specific patterns.27 In these methods, nevertheless, each type of device has to be fabricated using a customized mask or template. Additionally, the fabrication resolutions might vary according to the skill of different technicians. Hence, the need still remains for more efficient fabrication techniques that can be

icrofluidic paper-based analytical devices (μPADs) have recently attracted increasing interest since Martinez and co-workers pioneered this field in 2007.1 Micro-PADs are generally created by patterning hydrophobic materials (typically wax and polymer) in hydrophilic paper to offer complex microfluidic functions. In comparison with the first-generation microfluidic devices fabricated with silicon, glass, and polymer materials, μPADs possess many attractive features that make them ideally suited to point-of-use and point-of-care assays for measuring the quantity of analytes of interest in a variety of environments that lack access to laboratory infrastructure. Key among these features include low cost of materials, ease of device fabrication, storage, transport, and use, the ability to modify paper with a variety of reagents, and the ability to function without the addition of an external mechanical or electrical fluid-driving pump. As a result, the last five years have witnessed fast progress in the development of various μPADs for broad applications such as medical diagnosis and environmental monitoring.2−6 A set of techniques have been reported for μPAD fabrication via hydrophilic−hydrophobic patterning of paper, mainly including photolithographic,1,7,8 wax printing,9−16 inkjet printing,17−20 plasma etching,21 knife shaping,22 and laser treating and cutting.23,24 These approaches can offer high fabrication speed and high resolution of hydrophobic barriers and microfluidic paths. However, they may still suffer from one © 2014 American Chemical Society

Received: September 22, 2013 Accepted: January 20, 2014 Published: January 20, 2014 2005

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Figure 1. Schematic of the MTWP method for fabricating a wax-patterned 24-zone μPAD.

implemented in the developing world for producing μPADs by untrained operators at minimized cost. Quantitative detection is also key to the device functionality. Measurements of signals on μPADs can be realized by using several techniques, with optical imaging (for colorimetric assays with a camera or a scanner)1,28−45 and electrochemistry46−54 being the most common types. Other methods established include chemiluminescence and electrochemiluminescence,55−61 absorbance and fluorescence,3062−64 Raman,65,66 and mass spectrometry.67 However, these measurements typically require external customized readers and specialized skills. They therefore can be expensive and complicated, especially in the context of extremely resource-poor environments such as remote villages and private clinics in the developing world. The World Health Organization has listed “equipment-free” as one of seven necessary attributes for ideal point-of-care diagnostic tests in these regions. To address these issues, a simple equipment-free approach was recently demonstrated by Cate et al. to render μPAD measurement quantitative using the distance of color development as a detection motif.68 Lewis and co-workers reported another two interesting methods for equipment-free quantification of target analyte in three-dimensional (3D) μPADs by measuring time and by directly counting the number of strips that turn color, respectively.69 Both of the two methods were based on the selective change in the wetting properties of paper from hydrophobic to hydrophilic. In this work, we describe two complementary strategies for equipment-free quantitative assays on μPADs that are fabricated using the principles of movable-type printing. Movable-type printing, one of the Four Great Inventions of Ancient China, was first introduced by Bi Sheng around A.D. 1040. It is a system of printing and typography that uses movable components to reproduce elements of a document (usually individual letters or punctuation) on the surface of paper. Inspired by its excellent flexibility, a new method is developed herein for simple, low-cost, and rapid fabrication of μPADs, which we call movable-type wax printing (MTWP). As schematically shown in Figure 1, the MTWP method involves three core operations: (1) Assembling of a set of homemade small iron movable components into a desirable complete pattern on an iron-supporting substrate when its magnetic field is “OFF”; (2) heating of the patterned components in molten wax being heated on a hot plate after the support’s magnetic field is adjusted to be “ON”; and (3) printing of the hot “stamp” consisting of the support and the patterned metal components (coated with a layer of molten wax) on the surface of the paper. The heat retained on the metal components allows the molten wax to maintain its mobility until it penetrates through the paper’s thickness to form hydrophobic barriers for directing flow paths. Different μPADs can be

created by simply adjusting the number, patterning forms, or types of the metal movable components. Operators are only required to possess the abilities of assembling/heating/printing these iron components. The heating temperature for the wax and patterned iron components was optimized. The fabrication adjustability, resolution, and reproducibility of the MTWP method were also investigated in detail. By using the resultant wax-patterned μPADs, two equipmentfree methods are then developed for the first time for quantifying the level of hydrogen peroxide (H2O2) as a model analyte. The first method is based on the measurement of time required for a detection reagent (i.e., water) to flow through a hydrophilic test region predeposited with horseradish peroxidase (HRP) and 3′3′5′5′-tetramethylbenzidine (TMB) in a single conduit within a 3D μPAD. In the presence of H2O2, catalytic reaction of TMB with HRP can produce hydrophobic products. This change in the wetting property of paper from hydrophilic to hydrophobic subsequently prolongs the flowthrough time of the detection reagent. The change level of paper wettability depends on the analyte concentration. Compared with the hydrophobic−hydrophilic-change-based timing method reported by Lewis et al.,69 the hydrophilic− hydrophobic-change-based timing method proposed herein demonstrated superior performance for the H2O2 detection in terms of linear detection range and limit of detection. The second method involves counting the number of detection microzones that turn color in response to the presence of H2O2 in a single piece of multizone wax-patterned paper. This method is based on the reduction reaction of colorless potassium iodide (KI) with H2O2 in an acidic environment that yields colored iodide (I2). Sodium hyposulfite (Na2S2O3) can further oxidize the I2 product into colorless I− ions again, but the color of excess I2 will remain if the Na2S2O3 is insufficient. Since each successive detection microzone in the patterned paper is predeposited with the same concentrations of KI and a weak acid and with increasing amounts of Na2S2O3, the number of the detection microzones that finally turn color depends on the H2O2 concentration. The results show that the two methods require no equipment other than a timer or a cell phone with a timing function, the ability to see color, and the ability to count for the quantification of H2O2 analyte in samples.

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EXPERIMENTAL SECTION Reagents and Apparatus. Horseradish peroxidase (HRP, RZ > 3, A > 300 units/mg) was obtained from Sangon Biological Engineering Co., Ltd. (Shanghai, China). 3′3′5′5′tetramethylbenzidine (TMB, single component precipitationtype) was the product of Beijing Biosubstrate Technologies Co., Ltd. (Beijing, China). Hydrogen peroxide (H2O2, 30%, w/ 2006

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analytical ability. Commercially available vacuum packaging technique may endow these devices with more satisfactory storage stability. Piece 3 paper microzone is also patterned filter paper. It contained no reagents and its white hydrophilic region served as the readout region. For the H2O2 assay, 4 μL of sample solution was spotted on piece 1 patterned NC. Ten minutes later, 2 μL of a 2 M solution of H2SO4 was added to stop the catalytic reaction, followed by air-drying under ambient conditions. Piece 1 paper microzone was then stacked successively with pieces 2 and 3 microzones to form a 3D μPAD by sandwiching them with two pieces of hollow iron slices that were held even tighter by two iron clamps. The resulting 3D device contained a single hydrophilic conduit that extended in the z direction from one end of the device to the other. After a water droplet of 3 μL was added to piece 1 microzone using a micropipet, a timer or a cell phone with timing function having a resolution of tens of milliseconds was used to record the time required for it to flow through the single conduit, beginning when water was added and terminating when the hydrophilic region in the piece 3 microzone was wetted fully with the red ink redissolved from the piece 2 microzone by water (see the video in the Supporting Information to get a better image of the situation ). The flow-through time of sample (Ts) and PBS (control, Tc) were measured; the relative flow-through time was defined as (Ts − Tc). Of note, each relative flow-through time was given out with the average value of five parallel experiments. Counting-Based Equipment-Free H2O2 Assay. The counting-based equipment-free assay method for the quantification of H2O2 uses a 24-zone patterned NC device fabricated by the MTWP, according to the pattern shown in Figure 1. This paper device contains 24 hydrophilic circular microzones as detection regions that are branched out of a central hydrophilic region (for sample addition). During the 24 paper microzones, nos. 1 and 2 microzones served as blank comparison regions and contained no reagents and only HAc, respectively. No. 3 microzone contained KI and HAc (each was predeposited from 4 μL of a 500 mM solution and dried under ambient conditions). Other 21 microzones were also predeposited with the two reagents of the same concentrations and Na2S2O3 with increasing amounts in each subsequent zone. The whole device was further sandwiched between two pieces of transparent adhesive tape, but the central region for sample addition was separately covered by another small piece of tape. The freshly prepared detection reagent-immobilized device was loaded in a seal bag and could be stored in a dark place under room temperature for at least 2 months without significant change in its analytical capability. More satisfactory storage stability could be expected if it was packaged using a vacuum technique. Prior to H2O2 assay, the small piece of tape on the device was solely torn off and then a 50 μL sample was added to the central hydrophilic region. After it was separated evenly into the 24 paper microzones, counting the number of microzones that turned color (yellow) enabled the quantification of H2O2 in the sample.

v) was from Shanghai Jingchun Reagent Co., Ltd. (Shanghai, China). Potassium iodide (KI), sodium hyposulfite (Na2S2O3), and acetic acid (HAc) were purchased from West Long Chemical Co., Ltd. (Guangzhou, China). All of the other reagents were of analytical grade. Unless otherwise specified, all solutions were prepared with deionized water (with a specific resistivity >18.2 MΩ cm) from an ultrapure water system (UPS-II-20L) that was provided by Chengdu Yue Chun Technology Co., Ltd. (Chengdu, China). White semirefined paraffin solid wax was obtained from Shuangdafeng Wax Company (Cangzhou, China). The wax is mainly made of a mixture of hydrophobic straight-chain alkanes (that fall within the 8 ≤ n ≤ 15 range) and has a melting point range of 58−60 °C and a density of ∼0.9 g/cm3. Filter paper made from pure cellulose was purchased from Hangzhou Xinhua Paper Industry Co., Ltd. (Hangzhou, China). Nitrocellulose (NC) membrane (0.2 μm pore size) was from Shanghai Sinopharm Group Co., Ltd. (Shanghai, China). Filter paper and NC used herein are two examples of practice for fabricating μPADs, but other kinds of paper substrates such as chromatography paper are suitable as well. All iron movable components with specific patterns were made using a JQMX3015−550 laser cutting machine that was provided from Wuhan Chutian Laser Group Co., Ltd. (Wuhan, China). The duration and frequency of laser pulses and the average power of laser were filled to be 2 ms, 150 Hz, and 160 W, respectively. The patterns of the components were predesigned on a personal computer using CorelDraw X6. Each component is 2 mm thick × 5 mm high. An iron supporting substrate with a built-in adjustable magnet also obtained from this company is used for assembling (patterning) these iron components. Fabrication of μPADs by Movable-Type Printing. The fabrication principle and process behind the MTWP method are schematically shown in Figure 1. A set of iron movable components were first assembled into a desirable complete pattern (e.g., 24-zone) on the iron support’s surface by adjusting the magnetic field (Figure 1, left). The patterned metal components were then heated for several minutes in molten wax being heated on a hot plate with an operating temperature relatively higher than the wax’s melting point range (i.e., 58−60 °C). The depth of the molten wax should be lower than the height of the components (i.e., 5 mm). The heated patterned iron components (coated with a layer of molten wax) and the iron support were then used synergistically as a “heavy stamp” (∼450 g) to print a corresponding pattern of wax microstrcutures into paper (Figure 1, right). Various waxpatterned μPADs were created by simply adjusting the number, patterning forms, or types of the iron movable components. Timing-Based Equipment-Free H 2O 2 Assay. The timing-based equipment-free assay method for the quantification of H2O2 uses a 3D device made up of 3 pieces of waxpatterned paper microzones. Each foursquare microzone has a side length of 5 cm and contains a 5 mm diameter circular hydrophilic region. Piece 1 microzone is a patterned NC membrane. It was predeposited with 4 μL of a 1:1 (v/v) mixture of HRP (50 μg/mL) and TMB (9 mg/mL) and dried under ambient conditions. Piece 2 paper microzone is patterned filter paper. It contained red ink that was predeposited from a 1 μL solution, followed by a drying treatment. The freshly prepared reagent-immobilized pieces 1 and 2 microzones were loaded in a sealed bag and could be stored at 4 °C for at least 3 month with no significant loss of

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RESULTS AND DISCUSSION Fabrication of μPADs by the Principles of Chinese Movable-Type Printing. In this work, a new kind of wax printing method, namely the MTWP is described initially for μPAD fabrication using the principles of Chinese movable-type printing (Figure 1). This method uses very cheap wax in addition to a low-cost hot plate. It requires only the abilities to

2007

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hydrophilic paper path and hydrophobic wax barrier were estimated to be ∼1.3 and 1.1 mm, respectively. Moreover, an average relative standard deviation less than 8% of measured diameters of 10 sets of 3 × 3 circular microzone arrays created by 10 different operators was achieved (data not shown), which demonstrated a good fabrication reproducibility of the MTWP method. The reason may be presumably due to the fact that the printing of the patterned iron components and iron support on the surface of the paper makes the use of their own weight rather than the pressure exerted by the operators. The stability study further showed that no shape changes were observed in the wax micropatterns in the as-prepared μPADs when they were kept in an oven of 50 and 45 °C for 2 h and 1 month, respectively. In other words, the solid wax microstructures in paper did not melt at the elevated temperatures and functioned well as hydrophobic barriers. As a result, the wax-patterned hydrophilic paper channels without shape changes maintained good water flow ability. The results suggested that these devices could be stored under 50 °C, which is sufficient for use in common nonlaboratory environments. Equipment-Free Quantification of H2O2 by Measuring Flow-Through Time. The first timing-based method for H2O2 quantification uses a 3D device consisting of 3 pieces of waxpatterned paper microzones for each assay run. This equipment-free method is based on the measurement of the water flow-through time that is altered by the analyte-introduced selective wettability change of the first paper microzone (piece 1) from hydrophilic to hydrophobic. Taking into account the device’s small thickness (several hundreds of micrometers), the paper type plays a key role in achieving measurable water flowthrough time. NC is a kind of widely applied polymer membrane that has uniform pore size, surface, and inner microstructures10 and is less hydrophilic than other types of paper such as filter paper. The initial studies showed that the NC membrane facilitated stable and reproducible water flowing in the device, leading to reproductive timing measurement. Since NC is expensive, it was only used to create the piece 1 microzone, while low-cost filter paper was chosen for fabricating the pieces 2 and 3 microzones. The detection principle and procedures of the timing-based method are schematically shown in Figure 3. Piece 1 paper microzone (patterned NC) contains HRP and TMB, while piece 2 microzone (patterned filter paper) contains predeposited red ink. Piece 3 microzone (patterned filter paper) with

assemble, heat, and print homemade iron movable components using an adjustable magnetic field for patterning wax microstructures in paper. Figure 2A displays the figure of an as-

Figure 2. Photographs of (A) a wax-patterned 24-zone μPAD after its hydrophilic regions were wetted with a red aqueous solution, (B) homologous 18-, 15-, 12-, and 9-zone μPADs fabricated by adjusting (reducing) the number of the iron components that were used for the 24-zone μPAD shown in (A), (C) different 12-zone devices created by changing the patterning forms of the iron components of the same number, and (D) other types of 16- and 8-zone μPADs and a 3 × 3 circular microzone array fabricated by varying the types of homologous iron movable components. Each of the scale bars is 5 mm.

prepared wax-patterned 24-zone μPAD (fabricated according to the pattern shown in Figure 1) after the adsorption of a hydrophilic red ink solution via capillary action. One can find from Figure 2A that this MTWP method is able to allow for the formation of well-defined wax patterns in paper. More importantly, the patterned wax microstructures across the thickness of the paper acted well as hydrophobic barriers to direct the flowing of the red solution in the 24-zone μPAD. MTWP has a good fabrication adjustability that is inherited from the movable-type printing technique. That is, a group of homologous iron movable components can be used to create diversified μPADs. For example, other four kinds of homologous μPADs, namely the 18-, 15-, 12-, and 9-zone μPADs (Figure 2B) were fabricated by simply adjusting (reducing) the number of the same group of iron components used for the 24-zone μPAD (Figure 1). Different 12-zone devices were also fabricated by changing the patterning forms of the iron components of the same number (Figure 2C). In addition, other types of μPADs such as 16- and 8-zone μPADs and a 3 × 3 circular microzone array could be produced using corresponding groups of homologous iron movable components (Figure 2D). Since wax wicking in porous paper media typically is affected by temperature, the effect of this variable for heating wax and patterned iron movable components was tested. It was experimentally found that the production of well-defined wax patterns in paper was independent of temperature over the range of 100−130 °C. The result may come from the fact that the heating temperature higher than the melting point range of the wax (i.e., 58−60 °C) can make the molten wax easy and rapid to penetrate through the paper body. The fabrication resolution of the wax-patterned μPAD in the chosen type of paper (filter paper) was further characterized. As shown in the Figure S1 of the Supporting Information, the smallest widths of

Figure 3. Schematic of the timing-based method for equipment-free quantification of H2O2 based on the enzymatic reaction-mediated wettability change of defined paper regions from hydrophilic to hydrophobic. 2008

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white hydrophilic region contains no reagents and serves as the readout region. After the reactions of the sample with the reagents in the piece 1 microzone, it is further stacked successively with pieces 2 and 3 to form a 3D μPAD that contains a single hydrophilic conduit extending in the z direction from one end of the device to the other. Piece 3 will become red after the detection reagent (water) redissolves the red ink in piece 2 microzone and in turn distributes it to the piece 3 microzone. The time that is required for water to flow through the single conduit in the 3D device is recorded using a low-cost timer or a cell phone with timing function. When no H2O2 exists in the sample, one can find that the water added to the piece 1 microzone can wet its whole circular hydrophilic region (Figure 4A, left). This result reflects the fact

Figure 5. The calibration curve describing the relationship between the relative flow-through time and the logarithm values of H2O2 concentrations. Each data point represents the average value of five repetitive experiments. The error bars reflect the standard deviations from the average values.

detection with no significant changes in linear concentration range and limit of detection (Figure S2 of the Supporting Information). In the hydrophobic−hydrophilic-change-based timing method reported by Lewis et al.,69 the limit of detection for H2O2 is 0.7 mM and the dynamic range is 0.7−100 mM. The reason for the better detection performance gained from the hydrophilic−hydrophobic-change-based timing method described herein may be that it is easier for H2O2 of low concentrations to induce the wettability change of paper from hydrophilic to hydrophobic, rather than vice versa. Equipment-Free Quantification of H2O2 by Counting. The second equipment-free assay method, which uses only a 24-zone μPAD, is also developed for more straightforward H2O2 determination. Its detection principle and procedures are schematically shown in Figure 6. The 24-zone paper device

Figure 4. Comparison results of the timing-based method carried out for (A) samples without and (B) with 0.3 mM H2O2. Each of the scale bars is 1 mm.

that the piece 1 microzone remains good hydrophilicity. As a consequence, it took only 2.50 s for water to flow through the single hydrophilic conduit in the 3D device (Figure 4A, right). On the other hand, in the presence of H2O2 in the sample, this analyte can react with HRP and TMB in the piece 1 microzone to yield a colored hydrophobic product. Since the product changes the wetting property of paper from hydrophilic to hydrophobic, the water added to the piece 1 microzone forms a hemispherical droplet in the central region (Figure 4B, left). In comparison with the hydrophilic paper, the hydrophobic paper allows less volume of water to wick from the piece 1 microzone to the piece 2 microzone per unit time. The total flow-through time of water in the 3D device is accordingly increased from 2.50 to 4.35 s (Figure 4B, right). The flow-through time of water positively relates to the hydrophobicity level of the paper in the piece 1 microzone that depends on the H 2 O 2 concentration. Thus, quantification of H2O2 in samples can be achieved by measuring the flow-through time of water. The main experimental factors for H2O2 determination were optimized, including the concentrations of HRP and TMB and the volumes of water and red ink (data not shown). Under the optimized conditions, a set of samples containing various H2O2 concentrations were assayed using the proposed timing method. The exponential relationship between the relative flow-through time and the logarithm values of H 2 O 2 concentrations is illustrated in Figure 5. As indicated, this timing assay was linearly sensitive to H2O2 in the concentration range of 10 μM−500 mM. The limit of detection for H2O2 was determined to be ∼9 μM, as defined by the concentration resulting in a relative flow-through time that was three times the standard deviation of the control. The average relative standard deviation of all tested concentrations was 8.6%. The reagent-immobilized paper devices that had been stored for 3 months were experimentally found to enable the H2O2

Figure 6. Schematic of the counting-based method for equipment-free quantification of H2O2 based on the oxidation−reduction reactions of I 2.

contains 24 hydrophilic microzones as detection regions that are branched out of a foursquare central hydrophilic region for sample addition. No. 1 paper microzone, which is defined according to the cut part of the device in a clockwise direction, contains no reagents, whereas HAc is predeposited in no. 2 microzone. The two microzones served as blank (comparison) regions. No. 3 paper microzone contains both KI and HAc. Nos. 4−24 paper microzones are also predeposited with the 2009

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two reagents of the same concentrations in addition to Na2S2O3 with increasing amounts in each subsequent microzone. After the sample is added to the central hydrophilic region, it is further separated evenly into the 24 detection microzones. Except for the nos. 1 and 2 microzones, colored I2 of the same concentration is produced in other 22 microzones via the reduction reaction of KI with H2O2 in the sample in the presence of HAc. Except for the no. 3 microzone, however, the I2 yielded can further react with Na2S2O3 that is predeposited in the remaining 22 microzones. If the amount of Na2S2O3 in the microzone is sufficient compared with that of the I2 yielded, colored I2 can be fully reduced to colorless I− ions finally. White detection regions can be observed in such paper microzones [i.e., the nos. 11−24 microzones (marked with “c” in Figure 6, right)]. On the contrary, in the absence of Na2S2O3 or in the presence of insufficient Na2S2O3, the I2 that remained results in the production of colored detection regions in the nos. 3−10 paper microzones (marked with “a” and “b” in Figure 6, right). For the nos. 3−24 paper microzones that contain KI and HAc, the number of the colored microzones positively relates to the levels of I2 yielded in the detection regions that depend on the analyte concentrations. Accordingly, quantifying H2O2 in samples can be realized by counting the number of the colored paper microzones in the 24-zone μPAD. Figure 7 shows two

Figure 8. The calibration curve describing the relationship between the number of colored paper microzones and the logarithm values of H2O2 concentrations. Each data point represents the average value of five repetitive experiments. The error bars reflect the standard deviations from the average values.

are additionally demonstrated to implement two new quantitative assays that operate by measuring the flow-through time of a detection reagent or by counting the number of colored paper detection microzones. The proposed strategies offer the main advantages over most conventional fabrication and detection techniques in that they do not require expensive equipment and specialized skills. They could thereby be expected to find broad point-of-use applications, especially including point-of-care diagnostic assays sought by the World Health Organization for use in developing countries. While H2O2 was used as a model analyte in the initial proofof-concept studies of the two timing- and counting-based assay methods, our future studies will focus on (1) the improvement of timing accuracy in distinguishing samples of different concentrations by seeking more efficient enzymatic reactions that are able to lengthen the water flow-through time per reaction, (2) the improvement of detection sensitivity by combing with new detection chemistries for additional signal amplification, and (3) the equipment-free quantitative assays of a variety of analytes of interest that use enzyme oxidases to produce H2O2 as the secondary analyte.

Figure 7. Photographs of two 24-zone μPADs that were used for assays of (A) 35 mM and (B) 200 mM H2O2, respectively, before (left) and after (right) postprocessing. Each of the scale bars is 5 mm. The postprocessing of each original photograph was conducted by using ACDSee 3.1 (an image analysis software), according to the procedures listed as follows: (1) conversing the original colored form into the negative form, (2) adjusting the color contrast ratio of the resultant image to be 100%, (3) adjusting the color level of the obtained image to be 30%, (4) adjusting the color contrast ratio of the resultant image to be 50%, and (5) conversing the form of the resultant image into the negative form.

examples of 24-zone devices that were exposed to different concentrations of H2O2. Figure 8 reveals a linear relationship between the number of colored paper microzones and the logarithm values of H2O2 concentrations. In this initial proofof-concept assay, the linear range is 0.65−300 mM H2O2 and the limit of detection is 0.65 mM (defined as the lowest value that was detected). The average relative standard deviation for all measured concentrations was 7.1%. It was further found that the reagent-loaded paper devices that had been stored for 2 months allowed for the H2O2 assay with no significant changes in the linear range as well as the detection limit (Figure S3 of the Supporting Information). For the hydrophobic−hydrophilic-change-based counting method reported by Lewis et al.,69 on the other hand, the limit of detection for H2O2 is 10 mM and the dynamic range is 10−100 mM.

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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 Authors

*E-mail: [email protected]. Tel: +86 773 5896453. Fax: +86 773 5896839. *E-mail: [email protected]. Notes

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The authors declare no competing financial interest.

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CONCLUSION Herein, we describe a new kind of wax printing method termed MTWP (movable-type wax printing) for simple, low-cost, and adjustable fabrication of μPADs. The as-prepared paper devices

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grants 21105017, 21205021, and 2010

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Analytical Chemistry

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21365009) and the Guangxi Natural Science Foundation (Grants 2012GXNSFBA053030 and 2012GXNSFBA053029).

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dx.doi.org/10.1021/ac403026c | Anal. Chem. 2014, 86, 2005−2012