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 andTechnology, 11 Beiyitiao, ZhongGuanCun, Beijing 100190 China

We develop a novel method for Western blot based onmicrofluidics, incorporating the internal molecular weightmarker, loading control, and antibody titration in the sameprotocol. Compared with the conventional method whichcould detect only one protein, the microfluidic Westernblot could analyze at least 10 proteins simultaneouslyfrom a single sample, and it requires only about 1% ofthe amount of antibody used in conventional Western blot.

With the rapid growth of our understanding of complexnetworks of signaling pathways and limited supplies of biologicalmaterials such as cells, there is an increasing need for methodscapable of detecting multiple proteins from a single Westernblot.1,2 To address this goal, many approaches have beendemonstrated in the past decades. For example, membranestripping allows multiple times of sequential probing of the sameblot,3 but the signals could be weakened by successive strippingof antibodies. Fluorescent organic dyes or quantum dots can labelsecondary antibodies to enable multicolor detection of severalproteins in a blot,2,4,5 but the number of proteins that can bedetected is limited by the number of distinguishable opticalchannels. Surface-enhanced Raman scattering detection of multipleproteins directly on blotting membrane has also been reported;6

however, this technique may suffer from complications due tospectrum analysis.

Microfluidic technology has been widely applied in the fieldof biological research, such as cell culture,7-11 single cell

detection,12-14 genetic analysis,15,16 and immunoassays.17-21

Several review papers on microfluidic bioanalysis discussed thenecessity to miniaturize ELISA and other types of immunoassaysfor biological research and clinical diagnosis.22-25 Recently, Herret al.26 attempted to introduce the concept of immunoblotting intomicrofluidics. However, the method they developed could detectonly one protein from a single sample and could not reveal anyinformation on the molecular weight of proteins. Here, we presenta method that combines a microfluidic immunoassay with con-ventional protein blotting, which we call microfluidic Western blot(µWB), to analyze the expression and molecular weight of multipleproteins within one sample.

This method consists of four main stages: (i) we use sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)to separate proteins in cell lysates according to their molecularweights; (ii) we transfer proteins from the polyacrylamide gel toa polyvinylidene fluoride (PVDF) membrane by an electrotransfersystem, which immobilizes a series of protein bands onto themembrane (Figure 1a); (iii) we place a microfluidic network (seeSupporting Information, Fabrication of the Microfluidic Network)on the blotted membrane with the channels perpendicular to the

* To whom correspondence should be addressed. Fax: (+86) 10-6265-6765.E-mail: xingyujiang@nanoctr.cn.

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(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.

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(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.

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Anal. Chem. 2010, 82, 3974–3976

10.1021/ac1000493 2010 American Chemical Society3974 Analytical Chemistry, Vol. 82, No. 10, May 15, 2010Published on Web 04/28/2010

protein bands. The microchannels are sealed simply by pressingthe polydimethylsiloxane (PDMS) microfluidic network gentlyagainst the membrane. There is no detectable leakage in thechannel though the membrane is porous, because the PDMS chipseals conformally with the PVDF membrane; (iv) we detectmultiple proteins simultaneously on the PVDF membrane byincubating different primary antibodies in parallel microfluidicchannels (Figure 1b). These channels are 150 µm in width, 100µm in height, and 3.5 cm in length; (v) we peel off the microfluidicnetwork and incubate the whole PVDF membrane in fluorescentdye-labeled secondary antibody solution. The fluorescence signalscan 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 proteinssimultaneously in NIH-3T3 cells (see Supporting Information,Detection of Multiple Proteins by µWB). The target proteinsincluded three cytoskeletal proteins (�-actin, �-tubulin, R-tubulin),two proteins related to metabolism (GAPDH, F1-ATPase), and twosignal transduction proteins (Annexin II, pan-14-3-3). The fluo-rescence image of this experiment shows that the specificity ofdetection is high, with no detectable cross-reaction in neighboringchannels (Figure 2a). Besides detecting the expression of multipleproteins on one chip, µWB is applicable to other applications ofconventional Western blot, including loading control, molecularweight marker, and antibody titration.

Loading controls, such as GAPDH, �-actin, and �-tubulin, arecommonly used in Western blot to confirm that protein loadingis uniform across the gel. Researchers usually need to performtwo Western blots (by either cutting the membrane into two halvesor by stripping the membrane after the first blot) to detect thetarget protein and the loading control separately. µWB allowssimultaneous detection of both the protein of interest and theloading control in one experiment by introducing antibodies forloading control and target proteins in separate channels on thesame chip.

Furthermore, it is convenient to introduce the internal molec-ular weight marker into µWB. The molecular weight informationof proteins is important to eliminate false positive signals andindicate post-translational modifications such as phosphorylation.27

However, the commonly used protein molecular weight marker

is a mixture of several recombinant proteins, which could not bevisualized simultaneously with target proteins from a single image.Here, we show that µWB readily accommodates the internalmolecular weight marker (Figure 2b). The internal molecularweight marker can be obtained by mixing several antibodies thattarget highly expressed cellular proteins, such as housekeepingproteins. The mixture of antibodies was introduced in onemicrochannel in parallel with target-specific primary antibodiesin other microchannels on the same chip (see SupportingInformation, Internal Molecular Weight Marker Experiment). Themolecular mass of each protein signal can be determined bycomparison with known proteins in the internal molecular weightmarker.

An important routine in Western blot is finding the optimalantibody dilution which gives the best staining with minimumbackground or nonspecific binding. As the optimal dilution isinfluenced by a number of factors such as the equilibrium constantbetween the antibody and the antigen and the concentration ofantigen on the membrane, it is necessary to carry out antibodytitration with serially diluted antibody solutions before mostexperiments. Antibody titration could be performed simultaneouslyin µWB with different dilutions of antibody incubated in differentchannels instead of laborious parallel experiments (Figure 3c).

To compare µWB with conventional Western blot, Annexin IIwas detected by both conventional and microfluidic Western blotin NIH-3T3 cell lysates with total protein concentration rangingfrom 10 µg/well to 0.3125 µg/well (see Supporting Information,Comparison between Conventional Western Blot and µWB). Todetermine the optimal primary antibody concentration wherebinding saturation occurs, we performed antibody titration usinga serially diluted antibody solution (Supporting Information, FigureS-2a). From the titration curve (Supporting Information, FigureS-2b,c), we found that the saturating concentration of primaryantibody was approximately 4.2 nM (1:320 diluted) in conventionalWestern blot and 66.7 nM (1:20 diluted) in µWB. The requiredconcentration of primary antibody was higher in µWB than inconventional Western blot when attaining equivalent signal(27) Pan, J. M.; Wang, Q.; Snell, W. J. Dev. Cell 2004, 6, 445–451.

Figure 1. Illustration of µWB. (a) Proteins are transferred from apolyacrylamide gel to PVDF membrane by electroblotting.(b) The µWBchip is assembled by incorporating a PDMS microfluidic network withthe blotted PVDF membrane. The microfluidic channels are orientedperpendicular to the protein bands on the membrane. Antibodies forspecific proteins are introduced in parallel microfluidic channels.

Figure 2. Fluorescence image of µWB. (a) Detection of sevenproteins in NIH-3T3 cells. Target proteins in each microchannel (fromleft to right): (1) �-actin; (2) �-tubulin; (3) pan-14-3-3; (4) F1-ATPase;(5) Annexin II; (6) R-tubulin; (7) GAPDH. (b) The fluorescence imagewith internal molecular weight marker in the first microchannel. Thefluorescence signals in the first microchannel indicate (from top todown): R-tubulin, �-actin, GAPDH, and pan-14-3-3. The target proteinsin the other three microchannels: (2) F1-ATPase; (3) Annexin II; (4)�-tubulin. Dotted lines indicate the areas of the membrane that wereexposed to the solutions introduced by the microfluidic channels.Scale bar, 1 mm.

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intensity. We explained this phenomenon using the equilibriumreaction model in both conventional Western blot and µWB(details are described in Supporting Information, SupplementaryEquations).

Compared with conventional Western blot which consumesmore than 1 mL of antibody solution in one experiment, µWBrequires less than 1 µL of antibody solution in each microchannel.Even with higher required antibody concentration, µWB needsonly about 1% of the amount of antibody used in conventionalWestern blot for the same purpose. The result in Figure 3ademonstrates that µWB offers similar sensitivity to the conven-tional 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 around1.25 µg of total protein per well. (The detection limit is definedas the concentration corresponding to a signal three times thenoise level of background.)

To investigate whether the changes of signal intensity reflectthe relative changes in the expression of a specific protein, we

plotted signal intensity against the amount of total protein loadedinto the well of the gel and performed a linear fit. The correlationcoefficient (R) is 0.977 using conventional Western blot (Support-ing Information, Figure S-3) and 0.993 using µWB (Figure 3b).These results indicate that the fluorescence intensity increasesapproximately in proportion to the amount of target protein, whichproves that µWB could be used to analyze protein expressionsemiquantitatively as conventional Western blot.

The microfluidic Western blot is easy to apply in ordinarybiological laboratories, because it is adaptable to commercial SDS-PAGE and protein blotting systems. The microfluidic network cango with different sizes of PVDF membrane by choosing a suitablenumber of microchannels and channel length. We tested themaximal size of the membrane with commercially availablemembranes, which shows that the size of the membrane is not alimiting factor. We chose the channel width to be 150 µm in thissystem because wider channels cause bubbles in injection andnarrower ones result in indistinguishable fluorescent signals.There is no limit for the channel length, but it should cover therange of molecular weights of target proteins.

In conclusion, µWB allows the analysis of the expression ofmultiple proteins consuming only microliters of the antibodysolution. This method has the potential for research in the fieldsof signaling transduction pathways,28 protein-interaction networks,and proteomics.

ACKNOWLEDGMENTWe 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 performingwestern blot, Prof. D. Liu (NCNST) for the help with the TyphoonImager, B.Yuan, D. Wang, and Y. Xie (NCNST) for providing NIH-3T3 cells, and K. Sun for performing photolithography. This workis funded by HFSP, the National Science Foundation of China(20890020, 90813032), the Chinese Academy Sciences (KJCX2-YW-M15) and the Ministry of Science and Technology (2007CB714502,2008ZX10001-010).

SUPPORTING INFORMATION AVAILABLEDescription 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(28) Justman, Q.; Serber, Z.; Ferrell, J., Jr.; El-Samad, H.; Shokat, K. Science

2009, 324, 509.

Figure 3. Comparison between µWB and conventional Western blot.(a) Sensitivity of both protocols is similar, determined by the analysisof Annexin II in 2-fold serial dilutions of NIH-3T3 cell lysates. Theprimary antibody was diluted to a 1:80 dilution in conventional Westernblot (WB) and serially diluted (each dilution is 1/4, starting from 1:5)in µWB. (b) The linearity of µWB. Fluorescence intensity was plottedagainst the amount of total protein in NIH-3T3 cell samples loadedinto the well of the gel, and a linear fit was performed. The correlationcoefficient (R) was 0.993. (c) The antibody titration was performed inµWB with serial dilution of primary antibody incubated in differentchannels. Dotted lines in Figure 3c indicate the areas of themembrane that were exposed to the solutions introduced by themicrofluidic channels. Scale bar, 1 mm.

3976 Analytical Chemistry, Vol. 82, No. 10, May 15, 2010