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Letters to Analytical Chemistry Microfluidic Western Blot Wenying Pan, Wei Chen, and Xingyu Jiang* CAS Key Lab for Biological Effects of Nanomaterials and Nanosafety, National Center for NanoScience and Technology, 11 Beiyitiao, ZhongGuanCun, Beijing 100190 China We develop a novel method for Western blot based on microfluidics, incorporating the internal molecular weight marker, loading control, and antibody titration in the same protocol. Compared with the conventional method which could detect only one protein, the microfluidic Western blot could analyze at least 10 proteins simultaneously from a single sample, and it requires only about 1% of the amount of antibody used in conventional Western blot. With the rapid growth of our understanding of complex networks of signaling pathways and limited supplies of biological materials such as cells, there is an increasing need for methods capable of detecting multiple proteins from a single Western blot.1,2 To address this goal, many approaches have been demonstrated in the past decades. For example, membrane stripping allows multiple times of sequential probing of the same blot,3 but the signals could be weakened by successive stripping of antibodies. Fluorescent organic dyes or quantum dots can label secondary antibodies to enable multicolor detection of several proteins in a blot,2,4,5 but the number of proteins that can be detected is limited by the number of distinguishable optical channels. Surface-enhanced Raman scattering detection of multiple proteins directly on blotting membrane has also been reported;6 however, this technique may suffer from complications due to spectrum analysis. Microfluidic technology has been widely applied in the field of biological research, such as cell culture,7-11 single cell * To whom correspondence should be addressed. Fax: (+86) 10-6265-6765. E-mail:
[email protected]. (1) Towbin, H.; Staehelin, T.; Gordon, J. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 4350–4354. (2) Ornberg, R.; Harper, T.; Liu, H. Nat. Methods 2005, 2, 79–81. (3) Sadra, A.; Cinek, T.; Imboden, J. B. Anal. Biochem. 2000, 278, 235–237. (4) Kamiya, M.; Urano, Y.; Ebata, N.; Yamamoto, M.; Kosuge, J.; Nagano, T. Angew. Chem., Int. Ed. 2005, 44, 5439. (5) Bakalova, R.; Zhelev, Z.; Ohba, H.; Baba, Y. J. Am. Chem. Soc. 2005, 127, 9328–9329. (6) Han, X. X.; Jia, H. Y.; Wang, Y. F.; Lu, Z. C.; Wang, C. X.; Xu, W. Q.; Zhao, B.; Ozaki, Y. Anal. Chem. 2008, 80, 2799–2804. (7) Taylor, A. M.; Blurton-Jones, M.; Rhee, S. W.; Cribbs, D. H.; Cotman, C. W.; Jeon, N. L. Nat. Methods 2005, 2, 599–605. (8) Li, Y.; Yuan, B.; Ji, H.; Han, D.; Chen, S.; Tian, F.; Jiang, X. Angew. Chem., Int. Ed. 2007, 46, 1094–1096. (9) Gu, W.; Zhu, X.; Futai, N.; Cho, B. S.; Takayama, S. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 15861–15866. (10) Boedicker, J. Q.; Vincent, M. E.; Ismagilov, R. F. Angew. Chem., Int. Ed. 2009, 48, 5908–5911.
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detection,12-14 genetic analysis,15,16 and immunoassays.17-21 Several review papers on microfluidic bioanalysis discussed the necessity to miniaturize ELISA and other types of immunoassays for biological research and clinical diagnosis.22-25 Recently, Herr et al.26 attempted to introduce the concept of immunoblotting into microfluidics. However, the method they developed could detect only one protein from a single sample and could not reveal any information on the molecular weight of proteins. Here, we present a method that combines a microfluidic immunoassay with conventional protein blotting, which we call microfluidic Western blot (µWB), to analyze the expression and molecular weight of multiple proteins within one sample. This method consists of four main stages: (i) we use sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to separate proteins in cell lysates according to their molecular weights; (ii) we transfer proteins from the polyacrylamide gel to a polyvinylidene fluoride (PVDF) membrane by an electrotransfer system, which immobilizes a series of protein bands onto the membrane (Figure 1a); (iii) we place a microfluidic network (see Supporting Information, Fabrication of the Microfluidic Network) on the blotted membrane with the channels perpendicular to the (11) Mehta, G.; Lee, J.; Cha, W.; Tung, Y.; Linderman, J.; Takayama, S. Anal. Chem. 2009, 81, 3714–3722. (12) Koster, S.; Angile, F. E.; Duan, H.; Agresti, J. J.; Wintner, A.; Schmitz, C.; Rowat, A. C.; Merten, C. A.; Pisignano, D.; Griffiths, A. D.; Weitz, D. A. Lab Chip 2008, 8, 1110–1115. (13) Adams, A. A.; Okagbare, P. I.; Feng, J.; Hupert, M. L.; Patterson, D.; Gottert, J.; McCarley, R. L.; Nikitopoulos, D.; Murphy, M. C.; Soper, S. A. J. Am. Chem. Soc. 2008, 130, 8633–8641. (14) Allen, P. B.; Doepker, B. R.; Chiu, D. T. Anal. Chem. 2009, 81, 3784– 3791. (15) Dimov, I. K.; Garcia-Cordero, J. L.; O’Grady, J.; Poulsen, C. R.; Viguier, C.; Kent, L.; Daly, P.; Lincoln, B.; Maher, M.; O’Kennedy, R.; Smith, T. J.; Ricco, A. J.; Lee, L. P. Lab Chip 2008, 8, 2071–2078. (16) Wang, T. H.; Peng, Y.; Zhang, C.; Wong, P. K.; Ho, C. M. J. Am. Chem. Soc. 2005, 127, 5354–5359. (17) Liu, Y.; Yang, D.; Yu, T.; Jiang, X. Electrophoresis 2009, 30, 1–7. (18) Bernard, A.; Michel, B.; Delamarche, E. Anal. Chem. 2001, 73, 8–12. (19) Jiang, X. Y.; Ng, J. M. K.; Stroock, A. D.; Dertinger, S. K. W.; Whitesides, G. M. J. Am. Chem. Soc. 2003, 125, 5294–5295. (20) Yang, D. Y.; Niu, X.; Liu, Y. Y.; Wang, Y.; Gu, X.; Song, L. S.; Zhao, R.; Ma, L. Y.; Shao, Y. M.; Jiang, X. Y. Adv. Mater. 2008, 20, 4770–4775. (21) Murphy, B.; He, X.; Dandy, D.; Henry, C. Anal. Chem. 2008, 80, 444–450. (22) Whitesides, G. M. Nature 2006, 442, 368–373. (23) Yager, P.; Edwards, T.; Fu, E.; Helton, K.; Nelson, K.; Tam, M.; Weigl, B. Nature 2006, 442, 412–418. (24) Dittrich, P.; Tachikawa, K.; Manz, A. Anal. Chem. 2006, 78, 3887–3907. (25) Bange, A.; Halsall, H.; Heineman, W. Biosensors Bioelectron. 2005, 20, 2488– 2503. (26) He, M.; Herr, A. Anal. Chem. 2009, 81, 8177–8184. 10.1021/ac1000493 2010 American Chemical Society Published on Web 04/28/2010
Figure 1. Illustration of µWB. (a) Proteins are transferred from a polyacrylamide gel to PVDF membrane by electroblotting.(b) The µWB chip is assembled by incorporating a PDMS microfluidic network with the blotted PVDF membrane. The microfluidic channels are oriented perpendicular to the protein bands on the membrane. Antibodies for specific proteins are introduced in parallel microfluidic channels.
protein bands. The microchannels are sealed simply by pressing the polydimethylsiloxane (PDMS) microfluidic network gently against the membrane. There is no detectable leakage in the channel though the membrane is porous, because the PDMS chip seals conformally with the PVDF membrane; (iv) we detect multiple proteins simultaneously on the PVDF membrane by incubating different primary antibodies in parallel microfluidic channels (Figure 1b). These channels are 150 µm in width, 100 µm in height, and 3.5 cm in length; (v) we peel off the microfluidic network and incubate the whole PVDF membrane in fluorescent dye-labeled secondary antibody solution. The fluorescence signals can be recorded by a laser imager or a fluorescence microscope. Further details are described in the Supporting Information, Microfluidic Immunoassay. To show the utility of µWB, we detected seven proteins simultaneously in NIH-3T3 cells (see Supporting Information, Detection of Multiple Proteins by µWB). The target proteins included three cytoskeletal proteins (β-actin, β-tubulin, R-tubulin), two proteins related to metabolism (GAPDH, F1-ATPase), and two signal transduction proteins (Annexin II, pan-14-3-3). The fluorescence image of this experiment shows that the specificity of detection is high, with no detectable cross-reaction in neighboring channels (Figure 2a). Besides detecting the expression of multiple proteins on one chip, µWB is applicable to other applications of conventional Western blot, including loading control, molecular weight marker, and antibody titration. Loading controls, such as GAPDH, β-actin, and β-tubulin, are commonly used in Western blot to confirm that protein loading is uniform across the gel. Researchers usually need to perform two Western blots (by either cutting the membrane into two halves or by stripping the membrane after the first blot) to detect the target protein and the loading control separately. µWB allows simultaneous detection of both the protein of interest and the loading control in one experiment by introducing antibodies for loading control and target proteins in separate channels on the same chip. Furthermore, it is convenient to introduce the internal molecular weight marker into µWB. The molecular weight information of proteins is important to eliminate false positive signals and indicate post-translational modifications such as phosphorylation.27 However, the commonly used protein molecular weight marker (27) Pan, J. M.; Wang, Q.; Snell, W. J. Dev. Cell 2004, 6, 445–451.
Figure 2. Fluorescence image of µWB. (a) Detection of seven proteins in NIH-3T3 cells. Target proteins in each microchannel (from left to right): (1) β-actin; (2) β-tubulin; (3) pan-14-3-3; (4) F1-ATPase; (5) Annexin II; (6) R-tubulin; (7) GAPDH. (b) The fluorescence image with internal molecular weight marker in the first microchannel. The fluorescence signals in the first microchannel indicate (from top to down): R-tubulin, β-actin, GAPDH, and pan-14-3-3. The target proteins in the other three microchannels: (2) F1-ATPase; (3) Annexin II; (4) β-tubulin. Dotted lines indicate the areas of the membrane that were exposed to the solutions introduced by the microfluidic channels. Scale bar, 1 mm.
is a mixture of several recombinant proteins, which could not be visualized simultaneously with target proteins from a single image. Here, we show that µWB readily accommodates the internal molecular weight marker (Figure 2b). The internal molecular weight marker can be obtained by mixing several antibodies that target highly expressed cellular proteins, such as housekeeping proteins. The mixture of antibodies was introduced in one microchannel in parallel with target-specific primary antibodies in other microchannels on the same chip (see Supporting Information, Internal Molecular Weight Marker Experiment). The molecular mass of each protein signal can be determined by comparison with known proteins in the internal molecular weight marker. An important routine in Western blot is finding the optimal antibody dilution which gives the best staining with minimum background or nonspecific binding. As the optimal dilution is influenced by a number of factors such as the equilibrium constant between the antibody and the antigen and the concentration of antigen on the membrane, it is necessary to carry out antibody titration with serially diluted antibody solutions before most experiments. Antibody titration could be performed simultaneously in µWB with different dilutions of antibody incubated in different channels instead of laborious parallel experiments (Figure 3c). To compare µWB with conventional Western blot, Annexin II was detected by both conventional and microfluidic Western blot in NIH-3T3 cell lysates with total protein concentration ranging from 10 µg/well to 0.3125 µg/well (see Supporting Information, Comparison between Conventional Western Blot and µWB). To determine the optimal primary antibody concentration where binding saturation occurs, we performed antibody titration using a serially diluted antibody solution (Supporting Information, Figure S-2a). From the titration curve (Supporting Information, Figure S-2b,c), we found that the saturating concentration of primary antibody was approximately 4.2 nM (1:320 diluted) in conventional Western blot and 66.7 nM (1:20 diluted) in µWB. The required concentration of primary antibody was higher in µWB than in conventional Western blot when attaining equivalent signal Analytical Chemistry, Vol. 82, No. 10, May 15, 2010
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Figure 3. Comparison between µWB and conventional Western blot. (a) Sensitivity of both protocols is similar, determined by the analysis of Annexin II in 2-fold serial dilutions of NIH-3T3 cell lysates. The primary antibody was diluted to a 1:80 dilution in conventional Western blot (WB) and serially diluted (each dilution is 1/4, starting from 1:5) in µWB. (b) The linearity of µWB. Fluorescence intensity was plotted against the amount of total protein in NIH-3T3 cell samples loaded into the well of the gel, and a linear fit was performed. The correlation coefficient (R) was 0.993. (c) The antibody titration was performed in µWB with serial dilution of primary antibody incubated in different channels. Dotted lines in Figure 3c indicate the areas of the membrane that were exposed to the solutions introduced by the microfluidic channels. Scale bar, 1 mm.
intensity. We explained this phenomenon using the equilibrium reaction model in both conventional Western blot and µWB (details are described in Supporting Information, Supplementary Equations). Compared with conventional Western blot which consumes more than 1 mL of antibody solution in one experiment, µWB requires less than 1 µL of antibody solution in each microchannel. Even with higher required antibody concentration, µWB needs only about 1% of the amount of antibody used in conventional Western blot for the same purpose. The result in Figure 3a demonstrates that µWB offers similar sensitivity to the conventional method when saturating concentrations of the antibody (1: 20 diluted for µWB and 1:80 diluted for conventional Western blot) were used. For both methods, the limits of detection are around 1.25 µg of total protein per well. (The detection limit is defined as the concentration corresponding to a signal three times the noise level of background.) To investigate whether the changes of signal intensity reflect the relative changes in the expression of a specific protein, we (28) Justman, Q.; Serber, Z.; Ferrell, J., Jr.; El-Samad, H.; Shokat, K. Science 2009, 324, 509.
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plotted signal intensity against the amount of total protein loaded into the well of the gel and performed a linear fit. The correlation coefficient (R) is 0.977 using conventional Western blot (Supporting Information, Figure S-3) and 0.993 using µWB (Figure 3b). These results indicate that the fluorescence intensity increases approximately in proportion to the amount of target protein, which proves that µWB could be used to analyze protein expression semiquantitatively as conventional Western blot. The microfluidic Western blot is easy to apply in ordinary biological laboratories, because it is adaptable to commercial SDSPAGE and protein blotting systems. The microfluidic network can go with different sizes of PVDF membrane by choosing a suitable number of microchannels and channel length. We tested the maximal size of the membrane with commercially available membranes, which shows that the size of the membrane is not a limiting factor. We chose the channel width to be 150 µm in this system because wider channels cause bubbles in injection and narrower ones result in indistinguishable fluorescent signals. There is no limit for the channel length, but it should cover the range of molecular weights of target proteins. In conclusion, µWB allows the analysis of the expression of multiple proteins consuming only microliters of the antibody solution. This method has the potential for research in the fields of signaling transduction pathways,28 protein-interaction networks, and proteomics. ACKNOWLEDGMENT We thank Prof. W. Liang (The Institute of Biophysics, CAS), Prof. G. Nie and Prof. C. Chen (NCNST), and Prof. B. Yu (Tsinghua University) for technical assistance in performing western blot, Prof. D. Liu (NCNST) for the help with the Typhoon Imager, B.Yuan, D. Wang, and Y. Xie (NCNST) for providing NIH3T3 cells, and K. Sun for performing photolithography. This work is funded by HFSP, the National Science Foundation of China (20890020, 90813032), the Chinese Academy Sciences (KJCX2-YWM15) and the Ministry of Science and Technology (2007CB714502, 2008ZX10001-010). SUPPORTING INFORMATION AVAILABLE Description of the materials and methods used. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review January 8, 2010. Accepted April 26, 2010. AC1000493
