Flexographically Printed Fluidic Structures in Paper - Analytical

Anal. Chem. 2010, 82, 10246–10250

Flexographically Printed Fluidic Structures in Paper Juuso Olkkonen,* Kaisa Lehtinen, and Tomi Erho VTT Technical Research Centre of Finland, P.O. Box 1000, 02044 VTT, Finland This Technical Note demonstrates a simple method based on flexographic printing of polystyrene to form liquid guiding boundaries and layers on paper substrates. The method allows formation of hydrophobic barrier structures that partially or completely penetrate through the substrate. This unique property enables one to form very thin fluidic channels on paper, leading to reduced sample volumes required in point-of-care diagnostic devices. The described method is compatible with roll-to-roll flexography units found in many printing houses, making it an ideal method for large-scale production of paper-based fluidic structures. The use of paper as an inexpensive platform for diagnostic devices has gained great interest during the recent year.1-7 The most attractive property of a porous paper substrate is that it supports capillary flow enabling sample transfer onto desired reaction spots in a controlled manner without any external pumps. The first method to form liquid guiding hydrophobic regions into paper was introduced by Martinez et al.8 In the method, paper is first soaked in photoresist and then exposed to UV light through a mask defining the shape of the hydrophobic regions. The development step removes the photoresist from the unexposed areas revealing the original paper substrate. Abe et al.9 have reported an inkjet-based technique, in which paper is first soaked in a 1.0 wt % solution of polystyrene in toluene, making the entire paper hydrophobic. Hydrophilic channel regions are formed by inkjet printing toluene on the desired regions. The printing is * To whom correspondence should be addressed. E-mail: [email protected]. (1) Pelton, R. Trends Anal. Chem. 2009, 28, 925–942. (2) Martinez, A. W.; Phillips, S. T.; Whitesides, G. M.; Carrilho, E. Anal. Chem. 2010, 82, 3–10. (3) Martinez, A. W.; Phillips, S. T.; Carrilho, E.; Thomas, S. W., III; Sindi, H.; Whitesides, G. M. Anal. Chem. 2008, 80, 3699–3707. (4) Martinez, A. W.; Phillips, S. T.; Whitesides, G. M. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 19606–19611. (5) Ellerbee, A. K.; Phillips, S. T.; Siegel, A. C.; Mirica, K. A.; Martinez, A. W.; Striehl, P.; Jain, N.; Prentiss, M.; Whitesides, G. M. Anal. Chem. 2009, 81, 8447–8452. (6) Carrilho, E.; Phillips, S. T.; Vella, S. J.; Martinez, A. W.; Whitesides, G. M. Anal. Chem. 2009, 81, 5990–5998. (7) Aikio, S.; Gronqvist, S.; Hakola, L.; Hurme, E.; Jussila, S.; Kaukoniemi, O.V.; Kopola, H.; Kansakoski, M.; Leinonen, M.; Lippo, S.; Mahlberg, R.; Peltonen, S.; Qvintus-Leino, P.; Rajamaki, T.; Ritschkoff, A.-C.; Smolander, M.; Vartiainen, J.; Viikari, L.; Vilkman, M. Bioactive Paper and Fibre Products: Patent and Literary Survey, VTT Working Papers: Espoo, Finland, 2006. (8) Martinez, A. W.; Phillips, S. T.; Butte, M. J.; Whitesides, G. M. Angew. Chem., Int. Ed. 2007, 46, 1318–1320. (9) Abe, K.; Suzuki, K.; Citterio, D. Anal. Chem. 2008, 80, 6928–6934.

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repeated 10-30 times to etch open the surface of the paper substrate. Bruzewicz et al.10 have used a modified desktop x,yplotter to generate hydrophobic barriers by printing a solution of hydrophobic polymer (polydimethylsiloxane; PDMS) dissolved in hexanes onto filter paper. Since the printed PDMS has to penetrate through the entire paper thickness and curing takes 1 h at 70 degrees, PDMS slightly spreads in lateral dimensions reducing the resolution of the method. The printed feature size of ∼1.0 mm was demonstrated. Li et al.11-13 have used alkyne ketene dimer (AKD) to make paper hydrophobic. AKD is typically cured in an oven at 100 °C for 5 min. AKD can be applied to selected areas by inkjet printing, or the entire paper can be hydrophobized. Then, selected regions are hydrophilized by a plasma treatment through a patterned metal film. Fenton et al.14 have fabricated paper- and nitrocellulose-based lateral flow devices that are shaped in two dimensions by a computer controlled knife. Lu et al.15 and Carrilho et al.16 have independently studied formation of hydrophobic barriers by wax printing. A patterned layer of wax is first printed on a paper surface and then enforced to penetrate through the entire thickness of paper by melting the wax on a hot plate. When the wax penetrates through the paper, it also spreads in lateral directions reducing sharpness of the hydrophobic boundaries. Except the knife cutting approach,14 the techniques presented so far in the literature for creating hydrophobic barrier structures into paper are multistep processes including process steps not found in typical printing houses and, thus, are not readily suited for high throughput mass production. In this paper, we present a method based on flexographic printing of polystyrene that enables direct roll-to-roll production of liquid guiding barrier structures into porous paper substrates in existing printing houses. All structures presented in this work were fabricated with the printing speed of 60 m/min. EXPERIMENTAL SECTION Materials. Polystyrene pellets (Mw of 290 000, Mn of 130 000, secondary standard), Triton X-100, glucose oxidase (from Aspergillus niger, Type X-S, G7141), glucose, and organic (10) Bruzewicz, D. A.; Reches, M.; Whitesides, G. M. Anal. Chem. 2008, 80, 3387–3392. (11) Li, X.; Tian, J.; Nguyen, T.; Shen, W. Anal. Chem. 2008, 80, 9131–9134. (12) Li, X.; Tian, J.; Shen, W. Cellulose 2010, 17, 649–659. (13) Li, X.; Tian, J.; Garnier, G.; Shen, W. Colloids Surf., B 2010, 76, 564–570. (14) Fenton, E. M.; Mascarenas, M. R.; Lopez, G. P.; Sibbett, S. S. ACS Appl. Mater. Interfaces 2009, 1, 124–129. (15) Lu, Y.; Shi, W.; Jiang, L.; Qin, J.; Lin, B. Electrophoresis 2009, 30, 1497– 1500. (16) Carrilho, E.; Martinez, A. W.; Whitesides, G. M. Anal. Chem. 2009, 81, 7091–7095. 10.1021/ac1027066  2010 American Chemical Society Published on Web 11/23/2010

Figure 1. (a) Schematic illustration of the flexography unit used in the study. (b) Relief patterns in the printing plate define the hydrophobic regions to be formed into paper.

solvents (toluene, xylene) were purchased from Sigma-Aldrich and used without any further purification. Cationic polyamide (CPAM) and polyethylene imine (PEI) were obtained from Kemira Oyj, Finland. Phenol red (pH indicator) was obtained from Fluka, and polyvinyl alcohol (PVA, Moviol 4-88) was from Clariant GmbH. Clean room paper (Contec C1) and chromatography paper (Whatmann grade 1 Chr) were purchased from VWR. The used clean room paper is hydroentangled nonwoven containing 60% cellulose and 40% polyester. Instruments. Hydrophobic regions into paper were formed by RK Flexiproof 100 unit (R K Print Coat Instruments Ltd., United Kingdom). A ceramic anilox roll having volume of 18 cm3/m2 was used in the printing tests. Printing plates were patterned on 1.7 mm thick Miraclon BF (RBCor, LLC) material by a photolithographic process in Espoon Painolaatta Oy, Finland. Widths of the formed fluidic channel in paper were measured from digital images taken by an Olympus BX60 microscope equipped with an Olympus C-2000 Z digital camera. Surface tensions of aqueous solutions were measured by a Kibron AquaPi tensiometer. Viscosities of printing inks were determined by an m-VROC viscometer (Rheosense, Inc.). Flexographic Printing of Hydrophobic Barrier Regions. A schematic picture of the used flexographic unit is shown in Figure 1a. A substrate paper (297 × 105 mm2) is fixed to an impression roll. The ink is applied into the ink reservoir by a pipet. The ink transfers onto an anilox roll covered by thousands of small cells. The volume of the cells on the anilox roll defines the amount of ink transferred onto the printing plate containing a relief pattern; see Figure 1b. The excess ink from the anilox roll is removed by a doctor blade. When the printing process starts, the anilox roll accelerates to the printing speed and rotates four times to distribute the ink. Then, the plate

and impression roll rotate through one revolution to transfer the ink onto the paper substrate. Solutions (2.5, 5, and 10 wt %) of polystyrene in toluene and alternatively in xylene were used as printing inks. In all tests, the printing speed was 60 m/min. Pressure between the plate roll and the impression roll was optimized to obtain sufficient ink penetration into paper. All printings were performed so that, after printing a single ink layer, more ink was added by a pipet into the ink reservoir. Thus, the time delay between two printings, when two layers are printed sequentially one upon the other, was 5-10 s. Typically, the anilox roll was washed after printing of 10-20 ink layers. Fabrication of a Glucose Indicator. When glucose reacts with glucose oxidase (GOx), the reaction produces hydrogen peroxide and gluconic acid.17 In this work, we detect the presence of glucose via gluconic acid to demonstrate the viability of paperbased fluidic structures in chemical analysis. A glucose indicator shown in Figure 5 was fabricated on chromatography paper. The channel structures were formed by printing a 5 wt % solution of polystyrene in xylene so that one uniform layer was printed on the backside and one patterned layer was printed on the front side. The indicator consists of five pairs of reaction spots, as illustrated in Figure 5, each pair having a different amount of GOx (c1-c4, R). GOx was mixed into a 2 wt % solution of PVA in deionized water. PVA was used to increase long-term stability of GOx.18 The GOx concentration in the solutions was varied so that c1 ) 100 mg/mL, c2 ) 0.4c1, c3 ) 0.1c1, and c4 ) 0.05c1. The reference spots containing only PVA solution without GOx are marked by R in Figure 5. GOx-PVA aliquots of 0.2 µL were applied into the reaction spots by a micropipet and allowed to dry at room temperature for several hours. We note that also GOx-PVA could have been flexographically printed into the reaction spots, but it would have required four separate printing steps or a printing plate in which the reaction spot regions are designed in such a way that the four desired amounts of GOx can be transferred in a single printing step. Then, an unpatterned layer of water-based ink containing 1.77 wt % phenol red and 4.91 wt % CPAM was flexographically printed with a 4 cm3/m2 anilox roll on top of the entire fluidic channel structure. CPAM was used to immobilize phenol red into the paper substrate. Finally, a uniform layer of 10 wt % solution of PEI in water was printed with a 4 cm3/m2 anilox roll to increase pH in the reactions spots and cause a color change from yellow to purple in the layer containing phenol red. When glucose solution is applied to reaction spots, the reaction between GOx and glucose produces gluconic acid that changes the color back to yellow. Determination of Channel Boundary Roughness. The roughness of formed fluidic channels in paper was determined by the following procedure: (1) Deionized water colored by blue food dye is applied into the channel and allowed to dry at room temperature. (2) The channel having length of 10 mm is imaged by a microscope using the transmission illumination mode and the magnification of 1.25. (3) The color RGB images are then read into Matlab R2010b (The MathWorks, Inc.). (4) The two boundaries between the fluid channel and the hydrophobic barrier region are resolved from the B color channel of the image. (5) Two lines that fit best to the longitudinal boundaries of the channel (17) Raba, J.; Mottola, H. A. Crit. Rev. Anal. Chem. 1995, 25, 1–42. (18) Boyd, S.; Letcher, K.; Yamazaki, H. Biotechnol. Tech. 1996, 10, 693–698.


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Figure 3. Cross-sectional image of a 1 mm wide fluidic channel in chromatography paper. The cross-section is perpendicular to the flow direction. Deionized water colored by blue food dye was applied into the channel. The channel was formed by flexographically printing 5 wt % polysterene in xylene so that one unpatterned layer is printed on the back side and one patterned layer is printed on the front side. Due to the polystyrene layer on the back side, blue deionized water does not penetrate through the entire paper thickness.

Figure 2. Effect of polystyrene concentration, the choice of organic solvent (toluene/xylene), and the number of printed layers (1-3) on the barrier properties of the printed layers in chromatography paper. Images were taken by a microscope using the transmission illumination mode. A 10 µL drop of blue food dye colored deionized water was applied to each channel having nominal width of 1 mm.

are determined by the least-squares fitting. (6) The perpendicular distances (di, i ) 1, 2, 3, ...) between the fitted boundary line and each point at the rough boundary are calculated. (7) The root-mean-square (rms) of the distances di is calculated to obtain the rms boundary roughness. RESULTS AND DISCUSSION Printing of Fluidic Channels. Liquid guiding barriers to define fluidic channels in paper were formed by flexographically printing 2.5, 5, and 10 wt % solutions of polystyrene in toluene and alternatively in xylene. Clean room and chromatography paper were used as printing substrates. Figure 2 shows how polystyrene concentration (2.5, 5, 10 wt %), the choice of organic solvent (xylene/toluene), and the number of printing layers (1-3) affect the barrier properties of the printed polystyrene layers in chromatography paper. In each case, 10 µL of blue food dye colored deionized water was applied onto a 1 mm wide channel. Figure S-1 (Supporting Information) shows the results when the barriers are formed into clean room paper. In the case of chromatographic paper, when the polystyrene concentration is 2.5 wt %, not enough polystyrene is transferred on paper to form hydrophobic barriers. When the polystyrene concentration is raised to 5 wt %, leakyfree channels are obtained with two printing layers when xylene is used as a solvent. With toluene, the channels are partially leaky. This is probably due to the fact that xylene exhibits a higher boiling point than toluene, providing more time for the ink to penetrate into paper before complete solvent evaporation. With the polystyrene concentration of 10 wt % and two printed layers, a well-defined channel is formed on the paper surface but not through the entire paper thickness. This is caused by the high ink viscosity that increases with both solvents from ∼6 to ∼25 10248


mPa · s (see Figure S-2, Supporting Information) when the polystyrene concentration is increased from 5 to 10 wt %. The high ink viscosity decreases the penetration depth due to slower ink movement. All in all, the results show that with both printing substrates at least two sequentially printed layers are required to obtain waterproof barriers. The number of patterned printed layers on the front side can be reduced by printing uniform polystyrene layers on the back side of the substrate. The hydrophobic layers on the back side provide a barrier layer to prevent fluid escape from the channel through the substrate film. It also prevents any foreign substances from the underlying support (e.g., from a table surface) to enter fluidic channels. In addition, the back side hydrophobic layers can reduce the effective thickness of the substrate supporting capillary flow. This enables construction of thin channels (e.g., 50 µm) on a relatively thick substrate (e.g., 200 µm). Since the mechanical rigidity of 200 µm thick substrate is much higher than that of a 50 µm thick substrate, the presented approach provides a unique way to produce thin channels without losing the mechanical rigidity required for easy handling. Thin channels also reduce the sample volume required, for instance, in multianalyte tests. When one layer of 5 wt % polystyrene-xylene ink is printed on the back side of clean room paper or chromatography paper, only one patterned layer of the same ink is required on the front side of the paper. A cross-sectional image, perpendicular to the flow direction, of a 1 mm wide fluidic channel fabricated on chromatography paper using the proposed method is shown in Figure 3. Clearly, the ink layer printed on the back side forms a hydrophobic barrier layer that prevents liquid flow along the entire paper thickness. Figure 4a,c shows top views of fluidic structures formed on chromatography and clean room paper. The structure of Figure 4a was filled with 12 µL of denionized water colored by red food dye. Nominal channel widths in Figure 4c are 0.25, 0.5, 1, and 2 mm. According to microscopic measurements, the actual channels widths are 0.4, 0.6, 1.2, and 2.15 mm, i.e., slightly wider than the gaps between the relief patterns in the printing plate. The channel boundaries are not completely sharp. The rms boundary roughness on chromatography paper was determined to vary between 20-30 µm in 10 measured samples. It should be noted that the boundary roughness depends strongly on the structural properties of the used paper substrate (i.e., uniformity, pore size, and fiber length). In the structures presented so far, the entire paper substrate outside the fluidic channels was made hydrophobic by printing

Figure 4. Fluidic structures formed into chromatography paper (a) and clean room paper (c) by flexography printing 5 wt % polystyrene in xylene so that one unpatterned layer is printed on the back side and one patterned layer is printed on the front side. Deionized water colored by red and blue food dye was applied into channels. Nominal channel widths are marked in millimeters. Microscope images of dashed boxes shown in (a) and (c) are shown in (b) and (d), respectively.

polystyrene. We also studied what is the minimum nominal barrier width that produces leaky-free fluidic barriers when barriers are printed on chromatography paper so that one 5 wt % polystyrenexylene layer is printed on the back side and one patterned layer is printed on the front side. As a test structure, we used a circular ring structure shown in Figure S-3, Supporting Information. According to this test, the boundary width should be at least 400 µm to obtain a leaky-free structure in a reproducible manner. In practice, this also means that the minimum width of the hydrophobic region between two adjacent channels has to be at least 400 µm. Reproducibility. We fabricated matrixes of fluidic channels, each matrix consisting of 12 structures similar to Figure 4a, on 40 chromatography paper sheets (300 × 100 mm2). A uniform layer of of 5 wt % polystyrene-xylene was printed on the back side of the paper, and a patterned layer was printed on the front side. All 480 of the fabricated fluidic structures were leak free. The nominal channel width in the patterns was 1 mm, while the measured channel widths varied between 1.0 and 1.2 mm. Barrier Properties of Polystyrene Layers for Different Fluids. When barrier structures are formed into paper by a photolithographic process using, e.g., SU-8 polymer, the paper substrate becomes not only hydrophobic but also pores in the paper structure are filled by SU-8. Thus, fluidic channels with SU8-based barriers can guide liquids having extremely low surface tension without any significant leaks. Printed polystyrene layers make the paper substrate only hydrophobic, and thus, liquids having low enough surface tension will escape from a channel. We added small amounts of nonionic surfactant Triton X-100 to deionized water and determined that, when the surface tension is higher than 35 mN/m, polystyrene barriers can hold the liquid in the channel. We also visually verified that human blood and phosphate-buffered saline solution do not leak out from the channels.

Figure 5. Color response of a glucose indicator on chromatography paper for glucose concentrations of 50, 25, 10, and 5 mM. Glucose was dissolved in deionized water. Sample drops of 20 µL were applied into the middle part of the channel structure. The indicator consists of five pairs of reactions spots, each pair having a different amount of glucose oxidase (GOx). GOx was applied into reaction spots in PVA-based solutions and then dried. The GOx concentration (c1-c4) in the solutions was varied so that c1 ) 100 mg/mL, c2 ) 0.4c1, c3 ) 0.1c1, c4 ) 0.05c1. The reference spots containing no GOx are marked by R.

Comparison to Other Fabrication Methods. The first method to produce fluidic structures into paper was based on the use of photoresist and UV patterning.8 The method allows the fabrication of very narrow channels on various substrates; channel widths down to 250 µm have been reported for chromatography paper.19 By inkjet printing of AKD, 300 µm wide channels have been formed into Whatmann filter paper (No. 4).13 The presented method allows formation of 500 µm wide channels with the boundary roughness of 30 µm on chromatography paper in a reproducible manner. Channels having the width of ∼550 µm and the rms boundary roughness of 57 µm have been produced with wax printing.16 As a difference to wax printing, ink spreading in the presented method is relatively minimal and it does not have to be taken into account when designing fluidic patterns. Glucose Indicator. To demonstrate feasibility of paper-based fluidic channels in chemical analysis, we fabricated a glucose indicator on chromatography paper. An example of the fabricated indicator is shown in Figure 5. Since the reaction speed between glucose and GOx depends on the amount of GOx available, the indicator consists of five pairs of reaction spots, each pair having a different amount of GOx. Figure 5 shows the color response for glucose concentrations of 50, 25, 10, and 5 mM after 10 min from the sample application. Clearly, the color formation from purple to yellow depends on the glucose concentration. By changing the time period after which the indicator is read, the optimal glucose concentration range of the indicator can be altered. (19) Martinez, A. W.; Phillips, S. T.; Wiley, B. J.; Gupta, M.; Whitesides, G. M. Lab Chip 2008, 8, 2146–2150.


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CONCLUSIONS A crucial part in fabrication of liquid guiding boundaries by means of printing methods is penetration of hydrophobizing inks through the entire depth of the paper substrate. A surprising result found in this study is that sufficient ink penetration can be obtained with high speed flexographic printing if several ink layers sequentially on top of each other. The ink penetration is promoted by the pressure between the printing plate and the paper substrate. In this study, we printed polystyrene to make paper hydrophobic. We note that the method is not limited to polystyrene, being equally well suited for other hydrophobizing agents, including alkyne ketene dimer, poly(methyl methacrylate), and cross-linked polyvinyl alcohol. Benefits of polystyrene are that it requires no heat treatment and it is biocompatible and widely used, e.g., in food industry and microtiter plates. We also found that with polystyrene it is sufficient to print one uniform layer on the back side of the paper and one patterned layer on the front side. With this approach, we were able to reduce the capillary thickness of the paper substrate, leading to reduced sample volumes required, e.g., in multianalyte tests. The barrier layer on the back side could be also patterned and contain, e.g., openings that could work as sample inlets and outlets in 3D fluidic structures constructed by stacking of patterned papers.

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A great advantage of flexographic printing is that also biomolecules and other reagents required, for example, in analytical and diagnostic tests can be transferred by it on paper substrates. This may enable the fabrication of complete analytical devices on paper substrates in a single roll-to-roll process. ACKNOWLEDGMENT This work was done in the BioAct2 project which was made possible by the financial support by Tekes-the Finnish Funding Agency for Technology and Innovation. The authors would also like to acknowledge the financial support of the following companies and the invaluable guidance of the representatives of these companies in the steering group of the project: UPMKymmene Oyj, BASF Oyj, Tervakoski Oyj, Orion Diagnostica Oy, Hansaprint Oy, and Oy Medix Biochemica Ab. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review June 21, 2010. Accepted November 8, 2010. AC1027066

Flexographically Printed Fluidic Structures in Paper - Analytical

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