Technical Note pubs.acs.org/ac
Method for Fabrication of Paper-Based Microfluidic Devices by Alkylsilane Self-Assembling and UV/O3‑Patterning Qiaohong He,* Cuicui Ma, Xianqiao Hu, and Hengwu Chen Institute of Microanalytical Systems, Department of Chemistry, Zhejiang University, Zijin’gang Campus, Hangzhou 310058, China S Supporting Information *
ABSTRACT: This work presents a novel and facile method for fabricating paper-based microfluidic devices by means of coupling of hydrophobic silane to paper fibers followed by deep UV-lithography. After filter paper being simply immersed in an octadecyltrichlorosilane (OTS) solution in n-hexane for 5 min, the hydrophilic paper became highly hydrophobic (water contact angle of about 125°) due to the hydrophobic OTS molecules were coupled to paper’s cellulose fibers. The hydrophobized paper was then exposed to deep UV-lights through a quartz mask that had the pattern of the to-beprepared channel network. Thus, the UV-exposed regions turned highly hydrophilic whereas the masked regions remained highly hydrophobic, generating hydrophilic channels, reservoirs and reaction zones that were well-defined by the hydrophobic regions. The resolution for hydrophilic channels was 233 ± 30 μm and that for between-channel hydrophobic barrier was 137 ± 21 μm. Contact angle measurement, X-ray photoelectron spectroscopy (XPS) and attenuated total reflectance Fourier transform-infrared (ATR-FT-IR) spectroscopy were employed to characterize the surface chemistry of the OTS-coated and UV/O3-treated paper, and the related mechanism was discussed. Colorimetric assays of nitrite are demonstrated with the developed paper-based microfluidic devices.
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pattern.18 Alternatively, AKD solution was directly printed onto the paper by using an inkjet printer.19,20 Such prepared μPADs have very low cost due to the cheapness and availability of the AKD and can survive from bending/folding and solvent washing as well. Haller et al. patterned chromatography papers via photoinduced vapor-phase grafting of photocleavable poly(o-nitrobenzylmethacrylate) (PoNBMA) to the paper fibers, followed by UV-lithography of the PoNBMA coating through a photomask.21 Organosilane-based self-assembling monolayers (SAMs) are of great interest for chemically modifying surfaces of substrates because of the availability of various silane agents with different functionalities, low cost, and ease of coupling to substrates. In the past decades, the silane coupling technique has been widely used to functionize inorganic materials such as silica and alumina. Recently, this technique has been exploited to modify cellulose fibers of paper.22,23 Thus, various silane agents with hydrophobic functionalities (long chain hydrocarbons or fluorine-bearing hydrocarbons) were coupled to the paper fibers to hydrophobize paper sheets,24,25 and silane agents with amine moieties were attached to the paper fibers as solidcatalysts or as the arches for immobilizing of enzymes.26,27 To the best of our knowledge, however, no work on wettability-
aper-based microfluidic analytical devices (μPADs) have recently attracted great interest since Whitesides and coworkers first introduced this concept in 2007.1 Compared to the conventional microfluidic devices fabricated with silicon, glass, and polymer materials, μPADs possess attractive features such as inexpensive, biocompatible, easy-to-use, (especially without need of external fluid-driving pump), and easy-todispose. As a result, varieties of μPADs have been developed in the recent 5 years for medical diagnosis, food analysis, and environmental monitoring.2−5 Several techniques have been reported in the literature for fabrication of μPADs via hydrophilic−hydrophobic patterning of paper, including photolithgraphy,1,6−8 ink jet etching,9 wax printing and dipping,10−14 plotter printing,15 flexography printing,16 and hand plotting.17 In all the above-mentioned approaches, the patterned hydrophobic materials were physically deposited on the surface of paper fibers. Thus, the patterned hydrophobic materials might be attacked by organic solvents, which would be used during assay for elution of analyte or cleaning of channels, etc. In addition, the hydrophobic barriers formed via physical deposition of hydrophobic materials, such wax and photoresist, would be damaged due to bending and folding of paper. Recently, Shen’s group reported a novel approach for fabrication of μPADs by using alkyl ketene dimer (AKD), a paper-sizing agent to generate hydrophilic−hydrophobic contrast on paper sheets.18−20 In their work, the paper sheets were dipped in an AKD solution followed by plasma treatment through a stenciled metal mask to form a hydrophobic−hydrophilic © 2012 American Chemical Society
Received: October 28, 2012 Accepted: December 17, 2012 Published: December 17, 2012 1327
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hydrophobic patterns on filter paper by using silane agents involved two steps. First, the paper was entirely hydrophobized by coupling silane molecules with a suitable hydrophobic group onto the surface of the paper fibers via the condensation reaction between the silane and the hydroxyl moieties on paper fibers. Second, hydrophilic channels were constructed by selective decomposition of the hydrophobic groups of the silane molecules in the regions of to-be-constructed channels by UV-photolysis. Hydrophobization of the Paper via OTS-Coating. In previous works on hydrophobization of paper via the silane coupling reaction, most of the employed silanes were of alkoxyl types such as methoxylsilane or ethoxylsilane.23−27 Therefore, the coupling reactions should be carried out at elevated temperatures for dozens of minutes even several hours. In the present work, OTS was selected due to its hydrophobic property offered by the long-chain alkyl group (−C18H37), its high coupling-reactivity provided by its three Si−Cl bonds, and its commercial availability. As the OTS solution should penetrate into the paper before OTS molecules could react with the hydroxyl moieties of cellulose fibers, the coupling reaction between the OTS and cellulose fibers might be influenced by a period of immersing the paper in OTS solution and the OTS concentration in n-hexane solution as well. Thus, the optimal immersing time and OTS concentration were first investigated. Tests revealed that the coupling reaction could be completed in a very short period of immersing time. As shown in Figure 1a, curve I), the filter paper became strongly
patterning of paper sheets by using the silane coupling technique has been reported. In this paper, we report a novel and facile approach for the fabrication of μPADs via the hydrophobization of the filter paper by coupling octadecyltrichlorosilane (OTS) to paper fibers, followed by UV-lithography of the OTS coating. The silanization reaction between the OTS and filter paper and the feasibility of wettability-patterning of the OTS-coated paper (abbreviated as OTS-paper) via region-selective UV degrading of the coated OTS were investigated. The μPADs prepared with the developed approach were demonstrated for colorimetric assays of nitrite ions in food samples.
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EXPERIMENTAL SECTION Fabrication of the Paper-Based Microfluidic Device. Whatman No.1 filter paper was cut into appropriate sizes. The fabrication process for the μPAD is schematically shown in the Supporting Information, Figure S1. The paper sheet was immersed in 0.1% (v/v) OTS (Acros Organics, Springfield, NJ) solution in n-hexane at room temperature for 5 min, during which OTS molecules penetrated the paper and chemically coupled to cellulose fibers of the paper. After removed from the OTS solution, the paper sheet was rinsed sequentially with nhexane and ethanol and dried under nitrogen stream. The OTS-paper sheet was then covered with a quartz mask having the designed channel pattern, aligned and clamped. The assembly was then exposed to the UV-lights emitted from the mercury lamp equipped in a PL16-110 UV-cleaner (Sen Lights Corporation, Osaka, Japan) for 90 min. The UV-light power was measured 35 mW/cm at 254 nm. After withdrawing from the UV-cleaner, the μPAD is ready for use. Assay of Nitrite with Paper-Based Microfluidic Devices. A μPAD for the NO2− assay possessed a flowershaped channel network that was fabricated with the present method. During the assay, 5 μL of indicator solution, consisting of 50 mM sulfanilamide, 330 mM citric acid, and 10 mM n-(1napthyl) ethylenediamine in 80% methanol, was first pipetted into the central zone. As soon as the indicator solution fully penetrated into the detection zones, 0.25 μL portions of standard solutions or the prepared sample solution were individually pipetted into the detection zones for color development. After the spots on the device dried in air (∼5 min), the μPAD was scanned with a desktop scanner, and the collected image was converted to grayscale with Adobe Photoshop CS3. The calibration curve was constructed according to the measured gray intensities of the standards. Nitrite concentration in the sample solution was read against the calibration curve. A real sample of processed red cubilose (a traditional Chinese nutritious food and medicine as well) was analyzed for its nitrite contents. The sample solution was prepared with the procedure regulated by National Standardization Bureau of China.28 Safety Consideration. OTS will react with water (some violently) releasing flammable, toxic, or corrosive gases. Thus, the experiments of μPAD fabrication should be performed in a well-ventilated hood, while wearing protective gloves and goggles.
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Figure 1. Effects of OTS-treating time, OTS concentration (a), and UV/O3 exposing time of the OTS-paper (b) on the WCAs. (a, curve I) 0.1% OTS was used, (a, curve II) OTS treating time was kept for 5 min; n = 9.
RESULTS AND DISCUSSION Formation of Hydrophilic−Hydrophobic Pattern on Paper. In the present work, the generation of hydrophilic− 1328
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hydrophobic (WCAs in the range of 125−130°) after being immersed into a 0.1% (v/v) OTS solution for only 5 min. No substantial changes in WCAs were observed for the OTS-paper sheets when the OTS concentration was increased from 0.1% to 2% (v/v) under the constant treating time of 5 min (Figure 1a, curve II). If the concentration of OTS exceeded 0.5%, however, the OTS-paper became fragile after storage at ambient temperature for a few days. It could be most possibly ascribed to the corrosion effect of HCl molecules that were generated due to OTS hydrolysis.29 As treatment with a 0.1% OTS solution for 5 min could turn the paper from hydrophilic to hydrophobic while producing little negative effect on the paper strength, it was selected as the optimized conditions for the hydrophobization of the paper. Hydrophilization of OTS-Paper via UV/O3 Treatment. Region-selective hydrophilization of the OTS-paper is the key for the fabrication of μPADs via the silane coupling technique. It has been reported that the self-assembled monolayers (SAMs) of alkyl silanes formed on glass and silica surfaces can be degraded to hydrophilic by deep UV-light in combination with ozone.30,31 If the UV-degrading technique also works for the on-paper coated OTS, the hydrophilic− hydrophobic pattern of the OTS-paper would be straightforwardly realized via UV-lithography through a photomask. Thus, the effectiveness of the UV-degradation was tested by exposing the OTS-paper sheets to the deep UV-lights and the photogenerated ozone for varied times and characterized by measuring of WCAs on the UV/O3 exposed OTS-paper surface. As shown in Figure 1b, the WCAs dramatically decreased from 130 ± 3° to about 0° (water spread quickly on the UV/O3-treated OTS-paper) with the increase of the exposure time from 0 to about 90 min. Thus, the hydrophobic OTS-paper can be turned to highly hydrophilic by simply exposing to the UV/O3. The increase in hydrophilicity for the UV-exposed OTS-paper (abbreviated as UV-OTS-paper) surface can be attributed to the conversion of alkyl chains to hydrophilic moieties such as −CHO and −COR (refer to the section of Mechanism Studies). Wetability-Patterning of Paper. On the basis of the observations discussed in the above sections, a simple approach for the hydrophilic−hydrophobic pattern of the paper was established by OTS silanization of the paper followed by exposing the OTS-paper to UV/O3 through a mask. After an OTS-paper sheet was region-selectively exposed to the UV/O3, water spread quickly on the unmasked region while kept as a drop in the masked region (as shown in Figure 2a). Water penetration was well-defined within the UV/O3 exposed region so that the clear boundaries could be observed between the masked and unmasked regions (Figure 2b). If immersing one piece of such patterned paper in a dye solution, only the unmasked regions could be wetted and colored while the masked regions were kept dry as shown by Figure 2c. These results verified the feasibility of using the OTS-coating with cooperation of UV-lithography to fabricate μPADs. Resolution and Stability. The resolution of the prepared hydrophilic−hydrophobic pattern on filter paper was evaluated by using the method described in ref 6. As shown in Figure 3, the resolution for hydrophilic channels was 233 ± 30 μm and that for between-channel hydrophobic barrier was 137 ± 21 μm, and both are comparable to what Martinez et al. reported in ref 6 where the hydrophilic−hydrophobic contrast was achieved by spin-coating of resist on the paper surface followed by photolithography.
Figure 2. Hydrophilic−hydrophobic contrast on the UV/O3-patterned OTS-paper: (a) drop of dye solution (5 μL) was applied on unmasked region (left side) and masked region (right side), respectively; (b) after aliquots of 0.5 μL differently colored solutions being pipetted into hydrophilic reservoirs (3 mm in diameter); (c) after a pattern of hydrophilic lines with varied widths being immersed in a Rodamine B solution and drying.
Figure 3. The resolutions of hydrophilic−hydrophobic contrast on the μPADs prepared with the present method. Part a shows the resolution of the hydrophilic channels. The narrowest fully wetted hydrophilic channels was the third from the right, and it was 200 μm in designedwidth and measured to be 233 ± 30 μm. Part b shows the resolution of the hydrophobic barriers. The narrowest hydrophobic barrier capable of blocking aqueous dye solution was the third from the right, 200 μm in designed-width and measured 137 ± 21 μm.
The hydrophilic−hydrophobic pattern on the filter paper was very stable. After the patterned paper was stored at ambient temperature for 6 months or more, the hydrophilic−hydrophobic contrast remained the same as its initial status. Thus, the observed WCAs on the hydrophobic region before and after 1-, 2-, and 6-months storage were 129 ± 1, 128 ± 2, 126 ± 3, and 129 ± 2, respectively, and no significant change in the speed of water spreading in the hydrophilic region was observed before 1329
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Figure 4. Colorimetric assay of nitrite anions via Griess color-reaction by using μPAD: (a) scanned image of the color-developed μPAD and (b) calibration curve for nitrite ion (B, blank).
solutions occurred. In the present work, the Griess colorreaction was used for determination of nitrite ions. Figure 4a shows a scanned image of the color-developed μPAD where seven detection zones were for standard solutions and the last one was for the sample solution. On the basis of the color intensities of the seven zones for standard solutions, a typical calibration curve for nitrite ion was constructed as shown in Figure 4b. Thus, the dynamic linear range was in the range of 0.156−2.50 mM NO2−, within which a regression equation of y = 3.815x + 0.2353 (x and y represent NO2− concentration at millimolar and color intensity at 103 arbitrary unit, respectively) was obtained together with a correlation coefficient of 0.9965. The proposed method was validated by determination of NO2− in the red cubilose sample. The NO2− content in sample determined by the proposed μPAD method was (0.5440 ± 0.0102 g/kg), which agreed well with the concentration (0.5285 ± 0.0052 g/kg) (n = 3) determined by ion chromatography. This result demonstrates that the designed μPAD device can be applied for quick determination of NO2− content in food samples.
and after storage. Furthermore, the patterns could survive from being immersed in organic solvents such as ethanol, acetone, nhexane, and methylene chloride for at least 24 h. In addition, bending and folding of the patterned paper did not damage the patterns on the paper. Mechanism Studies. To understand the mechanism of hydrophobic and hydrophilic conversion of the paper caused by OTS coating and UV/O3-treatment, spectroscopic analyses were conducted for the paper sheets before and after OTScoupling and UV/O3-exposing. XPS analyses revealed that the spectrum for native paper only contained C1s and O1s peaks while the spectra for OTS-paper and UV-OTS-paper contained Si1p and Si2p double peaks in addition to the C1s and O1s peaks and that the C/O ratios of 2.2, 9.5, and 1 were observed for native paper, OTS-paper, and the UV-OTS-paper, respectively (see the Supporting Information, Figure S2). This indicated that OTS was coupled to the fiber surfaces of the paper after OTS-coating and that silicon atoms remained on the paper and oxygen-rich moieties were formed after UV/O3 treatment of the OTS-paper. ATR-FT-IR spectra further confirmed the deduction. Compared to the spectrum for native paper, two new strong absorption bands at ∼2911/cm and 2844/cm, which could be assigned to the C−H stretching of CH2 group in long hydrocarbon chain −C18H37, appeared in the IR spectrum for the OTS-paper, indicating OTS was attached to the paper after the silane coupling reaction (see the Supporting Information, Figure S3). After the OTS-paper subjected to UV/O3 exposure, relatively strong and overlapped absorption bands centered at ∼1700/cm, which was the characteristic absorption caused by CO stretching vibration of the carbonyl groups, appeared in company of the disappearance of ∼2911/cm and 2844/cm peaks. Thus, it can be concluded that the long alkyl chain of OTS molecules coupled to the paper fibers were decomposed to form polar oxygen-containing moieties such as ketones, aldehydes, and carboxylates. These analyses were similar to the reports for UV/O3 degrading of OTS-SAM formed on the glass surface.32 Assay of Nitrite in Food Sample. To demonstrate the feasibility of μPAD as a quantitative analysis device, a flowershaped μPAD with eight detection zones was fabricated for assay of nitrite in food samples. This design allowed the quantitative determination of analyte concentrations via measuring the surface color intensities in the detection zones, where the designed color reaction between the penetrating-in indicator reagent and pipetted standard solutions or sample
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CONCLUSIONS Filter paper can be hydrophobized by coupling of hydrophobic silane of OTS to the paper fibers at room temperature within several minutes, and hydrophobic−hydrophilic patterning of paper sheets can be achieved by exposing the OTS-paper to deep UV-lights through a photomask. The UV-exposed region becomes hydrophilic because the octadecyl hydrocarbon chain of the OTS is degraded to the oxygen-containing polar moieties under the coaction of deep UV-lights and the photogenerated ozone. On the basis of these observations, a facile approach for fabrication of μPADs has been established. The developed method features simplicity in operation, low in cost, and no need for a clean room and expensive equipment and, therefore, can be employed by analysts or biologists themselves to prepare μPADs in their own chemical laboratories. Owing to that, the OTS molecules are covalently bonded to the paper fibers rather than physically deposited on paper as photoresist or wax does, and such prepared μPADs are very stable. They can tolerate the attack of some organic solvents and, consequently, can be used in both aqueous and organic medium. They can survive from bending and folding without damage of the patterns as well. Since various silane agents with different functionalities are commercially available, the developed approach can be extended with a few modifications to prepared μPADs with 1330
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different functionalities for chemical analysis, bioassay, and cellbased research.
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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
*E-mail:
[email protected]. Phone: +86-571-88206773. Fax: +86-571-88273572. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work is funded by the National Natural Science Foundation of China (Project No. 20890020) and the National Science & Technology Supporting Program of China (Grant 2012BAI13B06)
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