Low-cost and rapid prototyping of microfluidic paper-based analytical devices by inkjet printing of permanent marker ink

Abstract

We described a low-cost method for rapid prototyping of a microfluidic paper-based analytical device by inkjet printing of permanent marker ink. After the evaporation of ink solvent is printed onto the paper, hydrophobic resin remains and forms hydrophobic barriers for microfluidic flow and analysis.

---

Introduction

Of growing interest in analytical chemistry is the development of micro analytical devices for point-of-care applications in clinical diagnostics, food safety testing, environmental monitoring and forensic analysis. The microfluidic paper-based analytical device (μPAD) is a newly developed technology, which utilizes the paper as the substrate to pattern the microfluidic channels delimited by the hydrophobic barriers.¹ Compared to the microfluidic analytical devices fabricated on the materials of glass, silicon and polymers, this technique has advantages of low cost, easy-to-use, ease of fabrication, portability, disposability and the ability to function without external fluid driving pumps.² These features are very attractive for performing point-of-care testing in clinical diagnostics,^3–5 food safety testing,^6–8 environmental protection^9,10 and chemical education,¹¹ particularly in those less-industrialized regions and resource-limited settings.

Various methods have been widely used for the fabrication of μPADs, including photolithography,^1,7 laser etching^12,13 and plasma treatment.¹⁴ The common limitations of these methods, however, are the time-consuming fabrication processes and the require expensive instruments such as lithographic equipment, laser and plasma oxidizer, which may pose challenges for the fabrication and applications of μPADs in the developing world and resource-limited regions. Moreover, trained personnel are required for the operation and maintenance of these expensive instruments, thereby further raising the cost of making μPADs. These limitations could be addressed by using a metal template having a specific design to deposit the hydrophobic material on hydrophilic paper substrate to form the hydrophobic barrier.^2,15–17 This strategy shows various advantages, including low cost, simplicity and high speed, in fabrication of the μPADs. Although the metal masks could be used repeatedly and no expensive instruments are required for fabrication of μPADS, the metal masks have to be fabricated by expensive instruments such as linear cutting machines or laser cutting machines. To address this limitation, we recently described a novel method for fabrication of μPADs by direct printing of trimethoxyoctadecylsilane solution using a paper mask instead of a metal one.¹⁸ Because the paper mask could be fabricated by cutting with a common knife and scissor, the expensive cutting machine for fabrication of the metal mask is therefore not obligatory. More recently, we developed a method for fabrication of μPADs by selective wet etching of hydrophobic filter paper using a paper mask.¹⁹ Alternatively, Mu et al.²⁰ described a simple and low-cost method for fabrication of μPADs by a craft punch patterning (CPP) method. In their work, only a metal mold (about 2 US %) and filter paper were used to fabricate the μPADs. The common limitation of using a metal mask or a paper mask demonstrated in these methods, however, is the inability for mass production of μPADs. In addition, the design and pattern of fabricated μPADs is determined by the pattern in the mold or mask. Therefore, the pattern could not be easily changed and adjusted. Using a printing technique, digitally designed patterns could be easily printed onto paper substrates for mass production of μPADs.^21–25 For example, wax printers have been widely used to deposit wax onto the paper substrate to generate hydrophobic barrier.^21,22 Unfortunately, this method uses an expensive wax printer, which is usually not available and affordable for the common laboratories in the less-industrialized regions. Recently, an inkjet printing method has become a simple and cost-effective alternative to wax printing for patterning microstructure on the filter paper. Bruzewicz et al.²³ described low-cost inkjet printing of poly(dimethylsiloxane) (PDMS) dissolved in hexane to define a microchannel in paper. Later, Shen's group²⁴ fabricated μPADs by inkjet printing of alkenyl ketene dimer (AKD) prepared in heptane onto filter paper. Alternatively, Abe et al.²⁵ printed organic solvent (toluene) to dissolve the hydrophobic poly(styrene) layer, which was formerly obtained by soaking the paper in a poly(styrene) solution prepared with toluene. The inkjet printing strategy possesses the advantages of low-cost, simplicity, fast and mass prototyping over other fabrication methods reported thus far. However, the cartridges and printers may be easily attacked and damaged by the organic solvents, mainly due to the strong dissolving power of the organic solvents (hexane, heptane and toluene) used in those printing solutions.

In this work, we described a novel, rapid and low-cost strategy for fabrication of μPADs by inkjet printing of permanent marker ink onto filter paper. The permanent marker ink generally consists of a colorant, a hydrophobic resin and a solvent (usually ethanol).² After the evaporation of the solvent from the filter paper, the hydrophobic resin remains in the filter cellulose to form hydrophobic barriers. Being free of any expensive instruments and reagents, this method uses only a cheap inkjet printer (Canon ip2780), filter paper and permanent marker ink, which are available and affordable worldwide to prototype the μPADs. Thus, this method could be used to fabricate μPADs by untrained personnel with minimum cost. This feature could be very attractive to the fabrication and application of μPADs in those less-industrialized regions and resource-limited settings. More importantly, instead of using the organic solvents having strong dissolving power (hexane, heptane and toluene), ethanol is used as the solvent in the permanent marker ink. Therefore, the cartridge and printers are not easily attacked and damaged by the solvent. A glucose assay was performed on the fabricated μPADs with a good performance, demonstrating its potential as a reliable quantitative analysis device.

Experimental

Chemicals and reagents

Five brands of permanent marker ink were used to print onto the filter paper, including Chenqi, Aidi, Baoke, Mapai and Wuqiannian. All chemicals used were of analytical grade unless mentioned otherwise, and demineralized water was used throughout. For glucose assay, chemicals and reagents were prepared as a reference.¹⁸ Specifically, a 200 mM phosphate buffer solution was prepared by combining 21.85 g Na₂HPO₄·12H₂O and 6.08 g NaH₂PO₄·2H₂O in 300 mL of H₂O, followed by adjustment of pH to 7.0 with 1.0 mol L⁻¹ NaOH or HCl solution and diluting to 500 mL. A potassium iodide solution of 6.0 mol L⁻¹ was prepared by dissolving 4.980 g potassium iodide in 5 mL of water. Glucose oxidase solution was prepared by dissolving 20 mg of glucose oxidase (Biological grade, Shanghai Jinsui Bio-Technology Co., Ltd. Shanghai, China) in 50 mL of buffer solution. Horseradish peroxidase solution was prepared by dissolving 13.4 mg of horseradish peroxidase (Biological grade, Shanghai Jinsui Bio-Technology Co., Ltd. Shanghai, China) in 50 mL of buffer solution. These two enzyme solutions were mixed in a ratio of 1 : 1 prior to use. A glucose stock standard solution (100 mmol L⁻¹) was prepared by dissolving 0.9915 g glucose in 20 mL of H₂O and was diluted to 50 mL. The serum samples were collected and prepared from chicken's blood. Briefly, 0.8 g of EDTA was added into a beaker followed by pouring into 100 mL of blood slowly and gently. After 10 mL of the sample was collected and centrifuged at 2000 rpm for 20 min, the serum sample was collected for further analysis.

Fabrication of μPADs

The pattern was designed on a personal computer with CorelDraw X3 software. The permanent marker ink was filtrated through a 0.45 μm filter membrane before use. The cartridge (Canon 815) was modified by removing the sponge in the cartridge, which is also available and affordable in the second-hand market. The permanent marker ink was then loaded into the cartridge with a plastic syringe. An inkjet printer (Canon Pixma ip2780) was used for double-sided printing of permanent marker ink onto the filter paper having a thickness of 0.24 mm (102, Hangzhou Xinhua Paper Limited, Hangzhou, China). The printed filter paper was then allowed to air dry for 15 min, allowing the evaporation of ethanol. Thus, the hydrophobic resin and the colorant were retained in the filter paper to form a visible hydrophobic barrier, which may be helpful to analytical applications.

Procedure for glucose assay

The flower-shaped μPAD fabricated for glucose assay consists of 6 detection zones, 6 channels and 1 central unit or reservoir. The glucose assay was based on the principle described previously.²⁶ The procedure for glucose assay consists of the following steps: first, 15 μL of potassium iodide solution was pipetted in the central unit of the μPAD and then allowed to air dry for 10 min; second, 0.5 μL of mixed enzyme solution was spotted onto the 6 detection zones respectively, followed by air drying for 8 min; next, 0.5 μL of the standard and sample solution were spotted onto the detection zones. The images of the colorimetric assay were captured with a digital camera (Canon IXUS9515, Japan) or a mobile phone and stored in JPEG format. The gray values of the detection zones were measured with the ImageJ software for quantitative analysis of glucose contents.

Results and discussions

Generation of hydrophilic–hydrophobic contrast

The permanent marker ink usually contains a hydrophobic resin, a solvent (ethanol) and a colorant. The ink solution was printed onto the filter paper using an inkjet printer and then allowed to penetrate completely across the thickness of the filter cellulose. After evaporation of the solvent from the filter paper, the hydrophobic resin and colorant retains in the filter cellulose, generating a hydrophobic barrier across the complete thickness of the paper. Fig. 1 shows that the aqueous liquid could be confined into the hydrophilic channel and zones delimited by the hydrophobic barrier.

Fig. 1 Image of a fabricated flower-shaped μPAD after dropping 25 μL of aqueous blue solution onto the central unit.

Effect of permanent marker inks

To investigate the water-resistant capability of permanent marker inks on the filter paper, circle zones were painted on the filter paper with a cotton swab penetrated with inks, followed by adding 2 μL of aqueous blue solution onto the circle zones. As shown in Fig. 2A, one ink brand (Baoke) could not be used as printing solution to form a hydrophobic barrier due to its inability to resist aqueous solution. The remaining four brands were then filtered through a 0.45 μm filter membrane to further investigate the water-resistance of these inks. As shown in Fig. 2B, the Chenqi brand could not resist the aqueous solutions effectively, indicating that this brand could not be printed onto the filter paper to form the hydrophobic barrier. The remaining three brands, which were filtered, were then printed onto the filter paper using an inkjet printer. Fig. 3 shows that Aidi ink could be printed onto the filter paper with a good resolution, whereas Mapai and Wuqiannian inks were printed onto the filter paper with a relative low resolution, and this may be due to the difference in viscosity and density of inks resulting from the variance in composition. Thus, Aidi brand was selected as the printing solution in this work.

Fig. 2 (A) Images of hydrophobic barriers formed with non-filtered permanent marker ink of Chenqi (a), Baoke (b), Wuqiannian (c), Aidi (d) and Mapai (e) brands. (B) Images of hydrophobic barriers formed with filtered permanent marker ink of Chenqi (a), Aidi (b), Mapai (c) and Wuqiannian (d) brands.

Fig. 3 Images of patterns printed with permanent marker ink of Mapai (A), Wuqiannian (B) and Aidi (C) brand.

Analytical application

To demonstrate the μPAD fabricated by this proposed method as a quantitative analysis device, the glucose content in chicken serum was detected as in the procedure described above. In this reaction, glucose is oxidized by glucose oxidase to produce hydrogen peroxide. The produced hydrogen peroxide is then reduced to water by horseradish peroxidase, along with the oxidation of iodide to iodine.^19,26 Fig. 4A shows the image of the glucose assay performed on the μPAD where 5 detection units are for the standard glucose solutions in a range of 3.0–20 mmol L⁻¹ and the remaining one is for the serum sample. The gray intensity in each detection zone was measured with ImageJ software by subtraction of the blank value. Data were imported into Origin (version 7.5) to obtain the linear correlation between the gray intensity, GI, and glucose concentration, C_glucose. The linear equation between the gray intensity and glucose concentration was GI = 4.7C_glucose + 1.1 (n = 3) with a correlation coefficient of 0.993 (Fig. 4B). The glucose content in the serum sample determined by this proposed method was 14.7 ± 2.0 mmol L⁻¹, which compared favorably with that (16.3 ± 2.2 mmol L⁻¹) (n = 3) determined by a standard method.²⁷ This result demonstrates that the μPAD fabricated by this presented strategy could be used as a reliable and robust quantitative analysis device in point-of-care clinical testing.

Fig. 4 (A) Image of glucose assay on the μPAD with varied glucose concentrations in a range of 3.0–20 mmol L⁻¹ (#1–5) and one serum sample (#6). (B) Gray value varies as a function of glucose concentration in a range of 3.0–20 mmol L⁻¹, obtained from three repeated experiments.

Conclusions

We developed a novel method for rapid fabrication of μPADs by inkjet printing of permanent marker ink onto filter paper. This method allows low-cost prototyping of μPADs in a simple and rapid way. The cost for fabrication of one μPAD is less than 0.02 RMB yuan using this presented method. Different from those inkjet printing methods reported previously, this method used ink dissolved in ethanol instead of organic solvents having strong dissolving power such as hexane, heptane and toluene, which may easily damage the inkjet printer and cartridge. In comparison with the methods of making μPADs by plotting with permanent marker ink using a marker pen,^2,28 our method allows mass production of μPADs in a rapid way. One limitation of our proposed method is that the ink residual should be removed from the cartridge if the ink is not going to be used for a long period of time, because the volume of ink solution may gradually decrease due to the evaporation of ethanol. Although glucose assay was performed on the fabricated μPADs to demonstrate the potential in bioassays, this method would also be very attractive to the development of micro analytical devices for point-of-care applications in clinical diagnostics, environmental testing, food safety monitoring and forensic analysis.

Acknowledgements

The authors thank Dr Meng Sun at University of Kansas for checking and polishing the writing of the paper. Financial support from the Guangdong Provincial Natural Science Foundation of China (Grant S2012040007274 and S2013010012046) and the Research Start-up Fund of Hanshan Normal University (Grant QD20120521) is gratefully acknowledged.

Notes and references

1. A. W. Martinez, S. T. Phillips, M. J. Butte and G. M. Whitesides, Angew. Chem., Int. Ed., 2007, 46, 1318.
2. J. F. Nie, Y. Zhang, L. W. Lin, C. B. Zhou, S. H. Li, L. M. Zhang and J. P. Li, Anal. Chem., 2012, 84, 6331.
3. L. M. Tian, J. J. Morrissey, R. Kattumenu, N. Gandra, E. D. Kharasch and S. Singamaneni, Anal. Chem., 2012, 84, 9928.
4. H. Noh and S. T. Phillips, Anal. Chem., 2010, 82, 8071.
5. Y. Jiang, C. C. Ma and X. Q. Hu, Progr. Chem., 2014, 26, 167.
6. J. C. Jokerst, J. A. Adkins, B. Bisha, M. M. Mentele, L. D. Goodridge and C. S. Henry, Anal. Chem., 2012, 84, 2900.
7. Q. H. He, C. C. Ma, X. Q. Hu and H. W. Chen, Anal. Chem., 2013, 85, 1327.
8. S. M. Z. Hossain, R. E. Luckham, M. J. McFadden and J. D. Brennan, Anal. Chem., 2009, 81, 9055.
9. M. M. Mentele, J. Cunningham, K. Koehler, J. Volckens and C. S. Henry, Anal. Chem., 2012, 84, 4474.
10. Y. Sameenoi, P. Panymeesamer, N. Supalakorn, K. Koehler, O. Chailapakul, C. S. Henry and J. Volckens, Environ. Sci. Technol., 2013, 47, 932.
11. L. F. Cai, Y. Y. Wu, C. X. Xu and Z. F. Chen, J. Chem. Educ., 2013, 90, 232.
12. G. Chitnis, Z. W. Ding, C. L. Chang, C. A. Savran and B. Ziaie, Lab Chip, 2011, 11, 1161.
13. C. L. Sones, I. Katis, P. J. W. He, B. Mills, M. F. Namiq, P. Shardlow, M. Ibsen and R. W. Eason, Lab Chip, 2014, 14, 4567–4574.
14. X. Li, J. F. Tian, T. Nguyen and W. Shen, Anal. Chem., 2008, 80, 9131.
15. W. Dungchai, O. Chailapakul and C. S. Henry, Analyst, 2011, 136, 77.
16. T. Songjaroen, W. Dungchai, O. Chailapakul, C. S. Henry and W. Laiwattanapaisal, Lab Chip, 2012, 12, 3392.
17. T. Nurak, N. Praphairaksit and O. Chailapakul, Talanta, 2013, 114, 291.
18. L. F. Cai, Y. Wang, Y. Y. Wu, C. X. Xu, M. H. Zhong, H. Y. Lai and J. S. Huang, Analyst, 2014, 139, 4593.
19. L. F. Cai, C. X. Xu, S. H. Lin, J. T. Luo, M. D. Wu and F. Yang, Biomicrofluidics, 2014, 8, 056504.
20. X. Mu, L. Zhang, S. Y. Chang, W. Cui and Z. Zheng, Anal. Chem., 2014, 86, 5338–5344.
21. Y. Lu, W. W. Shi, L. Jiang, J. H. Qin and B. C. Lin, Electrophoresis, 2009, 30, 1497.
22. E. Carrilho, A. W. Martinez and G. M. Whitesides, Anal. Chem., 2009, 81, 7091.
23. D. A. Bruzewicz, M. Reches and G. M. Whitesides, Anal. Chem., 2008, 80, 3387.
24. X. Li, J. F. Tian, G. Garnier and W. Shen, Colloids Surf., B, 2010, 76, 564.
25. K. Abe, K. Kotera, K. Suzuki and D. Citterio, Anal. Bioanal. Chem., 2010, 398, 885.
26. S. A. Klasner, A. K. Price, K. W. Hoeman, R. S. Wilson, K. J. Bell and C. T. Culbertson, Anal. Bioanal. Chem., 2010, 397, 1821.
27. Editing group for Experiment of Inorganic and Analytical Chemistry at Nanjing University, Experiment of Inorganic and Analytical Chemistry, Higher Education Press, 4th edn, 2006, p.159.
28. X. E. Fang, H. Chen, X. Y. Jiang and J. L. Kong, Anal. Chem., 2011, 83, 3596.

---