A Simple and Rapid Immunoassay Based on Microchip Electrophoresis Using a Reagent-Release Cartridge

Transcription

1 Short Communication A Simple and Rapid Immunoassay Based on Microchip Electrophoresis Using a Reagent-Release Cartridge Kenji SUEYOSHI *, Yuta MIYAHARA, Tatsuro ENDO, Hideaki HISAMOTO Graduate School of Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai , Japan Abstract In this article, we report a simple and rapid immunoassay based on microscale electrophoresis using a microchip equipped with a reagent-release at the sample reservoir. The consists of an outer tube, a paper filter retaining dried reagents and an antibody, and inner tubes to fix the paper filter. When a sample solution containing an antigen is introduced into the sample reservoir through the reagent-release, the reagents and the antibody retained on the filter dissolve immediately into the sample solution. The sample solution and reagents containing the antibody are then mixed, promoting rapid immunoreaction between the antigen and the antibody. After sample introduction into the reservoir, the antigen-antibody complex is rapidly separated from the free antibody by microchip zone electrophoresis. The immunoassays for bovine serum albumin and human IgG were easily performed merely by pouring the sample solution into the in the newly developed device followed by microchip electrophoresis, resulting in the rapid and clear separation of the immunocomplexes from the free antibodies. These results showed that the developed device was applicable to a rapid and simple immunoassay. Keywords: Immunoassay; Microchip electrophoresis; Reagent-release 1. Introduction Immunoassays are a type of protein assay based on the immunoreaction between an antibody and antigen. In various research fields such as biology, pharmacology, and life sciences, immunoassays are widely used to detect target molecules because of their high selectivity and the high binding constants of immunocomplexes [1-3]. Most immunoassays are conventionally carried out using a microplate. However, bulk-scale assays often require long analysis times and large amounts of samples/reagents. Moreover, labor-intensive manipulations are required to mix the solutions for the immunoreaction, separate the free antibody from the antigen-antibody complex (bound/free (B/F) separation) for selective assays, with low background signals. In enzyme-linked immunosorbent assay (ELISA) [4-5], further sensitive detection can be achieved by using an enzyme-labeled secondary antibody and its substrate, but it needs a longer analysis time and cumbersome procedures. To overcome these drawbacks, immunoassay methods based on microfluidic devices have been developed recently [6-15]. Microfluidic devices offer several advantages owing to miniaturization, such as rapid reactions due to a short length for diffusion, effective reactions due to high surface/volume ratio, and minimal consumption of samples and reagents. Using this concept, Sato et al. reported the microfluidic immunoassay using microbeads filled in the microchannel, shortening the total analysis time from 45 h to 35 min [6]. Wang et al. developed a rapid and ultrasensitive microfluidic solid-phase ELISA using an actuatable microwellpatterned microchip, realizing the extremely low detection limit of 21.8 am [13]. These microfluidic techniques enabled rapid immunoassays with minimal consumption of reagents and samples, although the difficulty in the fabrication of these devices remains a problem. Thus, the running costs of immunoassays have not been reduced markedly by the above-mentioned reported microfluidic assays. Our group has developed simple and rapid assays using reagent-release capillaries (RRCs) [18-30], in which the reagents are adsorbed onto the inner surface of the capillary as a soluble coating. When the sample solution is * Corresponding author: Kenji SUEYOSHI Received: 29 October 2015 Tel: ; Fax: Accepted: 10 December sueyoshi@chem.osakafu-u.ac.jp J-STAGE Advance Published: 17 December 2015 DOI: /jpchrom

2 spontaneously introduced into the capillary by a capillary action, the adsorbed reagents dissolve again and mix with the sample solution to give a fluorescent signal. In the case of the enzyme activity assays using RRCs [19-24], the relatively simple and rapid assays could be demonstrated merely by injecting the sample solution. However, the immunoassay using RRCs requires long analysis times and multiple exchanges of the solutions in the RRCs [27]. Immunoassay devices using hydrogels immobilizing fluorescent substrates have also been developed to simplify the experimental procedure [29,30]; however, the slow diffusion of the enzyme-labeled antibodies into the hydrogel results in long times. Hence, to develop a rapid and simple immunoassay with little consumption of reagents/solutions, we propose a different concept, which combines immunoreaction using reagent-release with the B/F separation based on microchip electrophoresis (MCE). MCE is electrophoretic separation that is carried out in microfluidic channels; it is an important separation technique in microfluidic assays owing to its advantages, such as rapid analysis, high resolution, and minimal consumption of samples [31,32]. Thus, MCE is considered to be suitable for analyses of biosamples; several B/F separation techniques have also been reported [33-35]. Koutny et al. reported the separation and quantification of free and bound labeled antigen in a competitive immunoassay within 30 s [33]. Hou et al. demonstrated rapid B/F separation within 10 s, using discontinuous polyacrylamide hydrogels filled in the microchannel [35]. However, almost all of the reports required pretreatments, such as mixing the sample solution with its antigen solution and incubation for the immunoreaction. Combinations of on-chip reactions and MCE analyses have also been reported [36]; however, complex channel design and voltage control are often required for these assays. In the concept proposed here, on the other hand, a reagent-release is simply connected to the sample reservoir of the microchip with a simple cross channel design. The consists of an outer tube, a paper filter retaining dried reagents, and inner tubes to fix the paper filter, so that it can be easily fabricated merely by assembling these parts, at low cost. In this study, we carried out a preliminary investigation on the fabrication of the proposed reagent-release and evaluated the microfluidic immunoassay based on MCE, using the developed. 2. Experimental 2.1. Materials and reagents Poly(dimethylsiloxane) (PDMS) prepolymer (SILPOT 184) and curing agent (SILPOT 184 CAT) were purchased from Dow Corning Toray Co., Ltd. (Tokyo, Japan). SU and SU-8 developer were purchased from Kayaku MicroChem Co., Ltd. (Tokyo, Japan). Paper filter (No.2) was purchased from ADVANTEC Co., Ltd. (Tokyo, Japan). Glass tube and teflon tube were purchased from AS ONE Co., Ltd. (Osaka, Japan). Cover glass was purchased from Matsunami Glass Ind., Ltd. (Osaka, Japan). Fluorescein was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Fluorescein isothiocyanate labeled bovine serum albumin (FITC-BSA), anti-bsa, FITC-antihuman IgG, human IgG, and poly(vinyl alcohol) (PVA) were all purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium dodecylsulfate (SDS) was purchased from Kishida Chemical Co., Ltd. (Osaka, Japan). Hydroxyethyl cellulose (HEC) was purchased from Sigma-Aldrich Japan (Tokyo, Japan). Sodium dihydrogen phosphate and disodium hydrogen phosphate were purchased from Wako Pure Chemical Industries (Osaka, Japan) Preparation of the dry reagent paper-in-tube type Filter papers retaining dried reagents (dry reagent paper) were prepared by impregnating fluorescent molecules by individually soaking filter papers in the reagent solution (0.2 mg/ml FITC-BSA in phosphate buffer for anti-bsa assay, or 0.2 mg/ml FITC-anti-human IgG (FITC-anti-IgG) (3) (2) assembled reagent-release PDMS/glass microchip R3 microchannel R2 R4 inner tubes (1) reagent-retained filter paper outer tube R2 R3 R1 R1 4 mm R4 reservoir Fig. 1. Fabrication of the dry reagent paper-in-tube type and the microchip for electrophoretic analysis. -30-

3 in phosphate buffer for human IgG assay). After drying the reagent-impregnated filter papers, they were cut into a circular shape (3 mm in diameter). The dry reagent paper-in-tube type s were assembled using two glass tubes (for inner tube: outside diameter, 3 mm; height, 7.5 mm) and a Teflon tube (for outer tube: inside diameter, 3 mm; height, 15 mm) to fix the filter papers retaining the dried reagents, as shown in Fig. 1a Fabrication of microfluidic device for electrophoretic analysis PDMS/glass microchips with a conventional crosschannel configuration were prepared by conventional soft lithography [9]. The width and depth of the channels in the fabricated microdevice were approximately 80 and 40 μm, respectively. The diameter and volume of the reservoirs were approximately 4 mm and 50 μl, respectively. To prevent protein adsorption onto the channel surface and suppress electroosmotic flow, channels were coated with PVA by the following procedure: briefly, the whole channels were flushed with 10 mm SDS solution to increase the hydrophilicity of the inner surface. The channels were then filled with 2% (w/v) PVA solution and dried by applying vacuum for 30 min, using a vacuum pump (GLD-100, ULVAC, Inc., Chigasaki, Japan). The assembled was finally installed inside the sample reservoir before the immunoassay Electrophoretic analyses of antigen-antibody reaction products The prepared microchips were filled with 30 mm phosphate buffer solution (ph 7.4) containing 2% HEC as a running buffer by capillary action. The containing a reagent-retained filter paper was then connected to the sample reservoir (Fig. 1b), after which 50 μl of a sample solution was introduced into the sample reservoir through the. After 5 min of incubation, the immunoreaction products were separated on the basis of molecular sieving electrophoresis by applying the programmed voltages to each reservoir, as shown in Table 1. SELFOC μ-fluorescence detector (Nippon Sheet Glass Co., Ltd., Tokyo, Japan) was used for detection. The excitation and emission wavelengths were 470 and 530 nm, respectively Immunoassay using the reagent-release The fixed with the filter paper retaining FITC-anti-human IgG, as mentioned above, was used to confirm the applicability of the developed device to immunoassays of model biocompounds. When the sample solution was poured into the installed in the sample reservoir, the reagents retained on the filter paper dissolved immediately and mixed with the sample solution. The resulting mixed solution then passed through the and reached the sample reservoir. Before the MCE analysis, the was removed from the reservoir. The programmed voltages were then applied to the reservoirs via platinum electrodes. The other experimental conditions were similar to those described above. Table 1. The programmed voltages for MCE analyses. Voltage / kv Time / s R1 R2 R3 R4 Loading Injection Separation FITC-BSA FITC-BSA Immuno complex Fig. 2. Evaluation of the prepared in MCE. Anti-BSA concentration: 0 μg/ml, 5 μg/ml. 3. Results and discussion 3.1. Electrophoretic separation of the antigen-antibody complex from the free antigen In the MCE analysis, after pouring 50 μl phosphate buffer into the in which the paper retaining FITC-BSA was assembled, a single peak was detected as shown in Fig. 2a. On the other hand, in the case of the sample solution containing anti-bsa, two peaks were successfully observed at high resolution, as shown in Fig. 2b. The peak area of the first peak decreased, whereas that of the second peak increased, upon increasing the concentration of anti-bsa in the sample solution. Thus, the first and second peaks were identified to be that of FITC-BSA and its immunocomplex, respectively. These results indicate that the proposed method of using this newly developed will provide a rapid and simple -31-

4 immunoassay, merely by microchip electrophoresis after pouring the sample solution into the Evaluation of the releasing and mixing efficiency of the developed To determine the efficiency of the release and mixing of the retained reagents from the s, the fixed with the fluorescein-retained filter paper was prepared and connected to the sample reservoir. After the buffer solution passed through the, it was removed from the reservoir. Fluorescein released from the filter paper was then electrokinetically introduced into the separation channel. After fluorescein reached the detection point, constant fluorescence intensity was observed for 300 s (Fig. 3a), which indicated that the retained reagents were released and mixed sufficiently upon penetration of the sample solution passing through the filter paper in the. The efficiency of the antigen-antibody reaction was also evaluated by measuring the peak area of the immunocomplex with varying incubation times for the immunoreaction of the model antigen, FITC-BSA. The obtained values of the immunocomplex peak area have been summarized in Fig. 3b, which clearly shows that the antigen-antibody reaction was completed within 5 min. These results indicate that the immunoreaction was efficient due to the rapid mixing of the released antigen with the antibody in the sample solution while the sample solution passed through the. Thus, it was confirmed that the fabricated device allows rapid immunoassay by the relatively simple pretreatment of merely pouring the sample solution into the prepared Application of the fabricated device to analysis of human IgG To confirm the applicability of the developed method to the immunoassay of model biocompounds, the sample solution containing human IgG was poured into the sample reservoir via the with the assembled FITC-anti-IgG-retained filter paper. As a result, the peaks of the free antibody and its immunocomplex were successfully separated by MCE (Fig. 4). The calculated peak area of the second peak increased linearly with increasing concentrations of human IgG in the sample solution ( μg/ml, y = 3.03x 3.16, R 2 = 0.988), which indicates that this method is applicable to the quantitative analysis of human IgG. Under these experimental conditions, limit of detection (LOD) of the immunocomplex was estimated to be 6.1 μg/ml. This LOD value is relatively higher than that obtained by conventional ELISA (ng/ml-pg/ml order), making it difficult to apply the proposed device for immunoassays of extremely low-concentration targets such as tumor markers for cancer. On the other hand, for the analysis of high-concentration Peak area (a.u.) Δ Time (s) Time (min) Fig. 3. Evaluation of the efficiency of reagent-release and immunoreaction. Electrokinetic injection of the mixed solution after dropping the sample solution into the. Applied voltages to R1, R2, R3, and R4 were floating, ground, floating, and 1.5 kv, respectively. Effect of incubation time on the immunoreaction. Experimental conditions were the same as Fig. 2, without incubation. Immunocomplex FITC-anti-IgG Fig. 4. Immunoassay of human IgG using the proposed method. intravital samples such as human IgG (ca mg/ml), the proposed device would enable us to easily measure analytes with a shorter analysis time, in comparison to conventional ELISA methods. Consequently, these results suggest that the proposed device would enable simple and rapid immunoassays with minimal amounts of samples and reagents. 4. Conclusion In this study, we developed and evaluated a novel electrophoresis microchip combined with a dry reagent paper-in-tube type containing reagent-retained filter paper. Using the device, the free antigen was rapidly separated from its immunocomplex by a simple procedure. The antigen-antibody reaction was completed within about 5 min, owing to the effective mixing achieved by using the -32-

5 . In the MCE analysis of human IgG, the peaks of the FITC-anti IgG and immunocomplex were also successfully separated and identified, which indicates that the proposed device is applicable to the quantitative analysis of human IgG. These results suggest that the proposed device enables us to conduct immunoassays based on MCE, with ease of handling, short analysis time, and minimal consumption of samples and reagents. Future application of online sample preconcentration methods to this newly developed device will enable highly sensitive immunoassays. Acknowledgment This work was partially supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan ( , , and ) and the Funding Program for Next Generation World-Leading Researchers (LR031) from the Government of Japan. References [1] Chait, E. M.; Ebersole, R. C. Anal. Chem. 1981, 53, 682A-692A. [2] Hage, D. S. Anal. Chem. 1995, 67, 455R-462R. [3] Hage, D. S. Anal. Chem. 1999, 71, 294R-304R. [4] Surugiu, I.; Svitel, J.; Ye, L.; Haupt, K.; Danielsson, B. Anal. Chem. 2001, 73, [5] Campanella, L.; Eremin, S.; Lelo, S. D.; Martini, M Tomassetti, E. Sens. Actuators B 2011, 156, [6] Sato, K.; Tokeshi, M.; Kimura, H.; Kitamori, T. Anal. Chem. 2001, 73, [7] Wang, J.; Ibáñez, A.; Chatrathi, M. P.; Escarpa, A. Anal. Chem. 2001, 73, [8] Oyama, Y.; Osaki, T.; Kamiya, K.; Kawano, R.; Honjoh, T.; Shibata, H.; Ide, T.; Takeuchi, S. Lab Chip 2012, 12, [9] Lin, R.; Skandarajah, A.; Gerver, R. E.; Neira, H. D.; Fletcher, D. A.; Herr, A. E. Lab Chip 2015, 15, [10] Yi, H.; Pan, J.-Z.; Shi, X.-T.; Fang, Q. Talanta 2013, 105, [11] Koh, C.-Y.; Schaff, U. Y.; Piccini, M. E.; Stanker, L. H.; Cheng, L. W.; Ravichandran, E.; Singh, B.-R.; Sommer, G. J.; Singh, A. K. Anal. Chem. 2015, 87, [12] Okada, H.; Hosokawa, K.; Maeda, M. Anal. Sci. 2011, 27, [13] Wang, T.; Zhang, M.; Dreher, D. D.; Zeng, Y. Lab Chip 2013, 13, [14] Li, Y.; Xuan, J.; Song, Y.; Wang, P.; Qin, L. Lab Chip 2015, 15, [15] Jung, W.; Han, J.; Kai, J.; Lim, J.-Y.; Sul, D.; Ahn, C. H. Lab Chip 2013, 13, [16] Hadd, A. G.; Raymond, D. E.; Halliwel, J. W.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1997, 69, [17] Wang, J.; Chatrathi, M. P.; Tian, B. Anal. Chem. 2001, 73, [18] Henares, T. G.; Funano, S.-i.; Sueyoshi, K.; Endo, T.; Hisamoto H. Anal. Sci. 2014, 30, [19] Henares, T. G.; Takaishi, M.; Yoshida, N.; Terabe, S.; Mizutani, F.; Sekizawa, R.; Hisamoto, H. Anal. Chem. 2007, 79, [20] Henares, T. G.; Mizutani, F.; Sekizawa, R.; Hisamoto, H. Anal. Bioanal. Chem. 2008, 391, [21] Kimura, Y.; Henares, T. G.; Funano, S.-i.; Endo, T.; Hisamoto, H. RSC Adv. 2012, 2, [22] Uchiyama, Y.; Okubo, F.; Akai, K.; Fujii, Y.; Henares, T. G.; Kawamura, K.; Yao, T.; Endo, T.; Hisamoto, H. Lab Chip 2012, 12, [23] Ishimoto, T.; Jigawa, K.; Henares, T. G.; Endo, T.; Hisamoto, H. Analyst 2013, 138, [24] Ishimoto, T.; Jigawa, K.; Henares, T. G.; Sueyoshi, K.; Endo, T.; Hisamoto, H. RSC Adv. 2014, 12, [25] Henares, T. G.; Maekawa, E.; Okubo, F.; Mizutani, F.; Yao, T.; Sekizawa, R.; Hisamoto, H. Anal. Sci. 2009, 25, [26] Henares, T. G.; Tsutsumi, E.; Yoshimura, H.; Kawamura, K.; Yao, T.; Hisamoto, H. Sens. Actuators B 2010, 149, [27] Tsutsumi, E.; Henares, T. G.; Funano, S-.I.; Kawamura, K.; Endo, T.; Hisamoto, H. Anal. Sci. 2012, 28, [28] Fujii, Y.; Henares, T. G.; Kawamura, K.; Endo, T.; Hisamoto, H. Lab Chip 2012, 12, [29] Wakayama, H.; Henares, T. G.; Jigawa, K.; Funano, S.-i.; Sueyoshi, K.; Endo, T.; Hisamoto, H. Lab Chip 2013, 13, [30] Funano, S.-i.; Sugahara, M.; Henares, T. G.; Sueyoshi, K.; Endo, T.; Hisamoto, H. Analyst 2015, 140, [31] Smith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A. K.; Gartner, F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk D. C. Anal. Biochem. 1985, 150, [32] Fister, J. C.; Jacobson, S. C.; Davis, L. M.; Ramsey, J. M. Anal. Chem. 1998, 70, [33] Koutny, L. B.; Schmalzing, D.; Taylor, T. A.; Fuchs, M. Anal. Chem. 1996, 68, [34] Chiem, N. H.; D. J. Harrison, Anal. Chem. 1997, 69, [35] Hou. C.; Herr, A. E. Anal. Chem. 2010, 82, [36] Chiem, N. H.; Harrison, D. J. Clin. Chem. 1998, 44:3,