Published online 28 October 2010; doi:10.1038/nprot.2010 · 2010-10-28 · far western blotting for...

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© 2010 Nature America, Inc. All rights reserved. PROTOCOL 1844 | VOL.5 NO.11 | 2010 | NATURE PROTOCOLS INTRODUCTION Microfluidic analysis of biological molecules (e.g., protein, DNA) has dramatically enhanced speed, efficiency and sensitivity over conventional benchtop bioanalysis and diagnostic methods 1–3 . Miniaturization afforded by microfluidic technology has inspired deep interest in portable, automated clinical diagnostics for early stage disease detection 4–6 . Perhaps more important than the small- form factor, the potential for streamlined integration of multiple functions and even multiple protein assays in a compact micro- fluidic architecture has opened the door to a new era of automation in life science laboratories. Over the past decade, development of functional components for sample preparation, separation, purification, sorting and mixing using micro- and nanofluidic technology has boomed 7 . Successfully integrated microfluidic tech- nologies boast precise control and rapid transport. Such tools have been designed for applications from cell culture assays to human point-of-care disease diagnostics 8 . Nevertheless, one of the most important areas in which fully integrated multistage microfluidic technologies are needed is the area of bioanalytical separations. Although single-stage microfluidic assays are commercially available, development of multistage microfluidic assays rivaling the per- formance of benchtop methods has lagged. In particular, a key suite of multistage biomolecular assays, ‘immunoblots’, would benefit tremendously from the automation, speed and quantitation capabili- ties afforded by fully integrated microfluidic technologies. Successful incorporation of biomolecular sieving matrices into microfluidic electrophoresis technology has yielded enhanced sepa- ration efficiency 9–13 . Among the various sieving structures explored, including micro/nanomachined structures and functional chemical materials 14,15 , polyacrylamide (PA) gel is notable. PA gels are a com- mon and powerful separation matrix for conventional benchtop electrophoretic separations of proteins, peptides, DNA and other biomolecules. PA gels are typically used in slab-gel formats includ- ing polyacrylamide gel electrophoresis (PAGE); two-dimensional (2D) PAGE; and western, Southern and northern blotting anal- yses 16–19 . The cross-linked 3D pore networks in PA gels support optimized, effective sieving performance with tunable pore size for a wide range of biological samples. Further, functionalization of PA gel with myriad chemical properties, including incorpo- ration of streptavidin, makes the material even more versatile 20 . The protocol detailed here exploits a major design advantage inher- ent to cross-linked PA gels: ready photopatterning of PA gels as a means of integrating dissimilar functionalities within one mono- lithic microdevice. Key to our protocol is the deliberate selection of materials and tools found in most biology laboratories, making both fabrication and use of the approach feasible for translation from our bioengineering laboratory to life science laboratories. In addition, we describe a protocol for glass chip regeneration and reuse after native western blotting assay completion, which makes the approach even more economically feasible. To the best of our knowledge, limited effort has been made to use microfluidic technology to streamline all steps needed to obtain mobility and binding-based identity information in one continu- ous assay 21 . To achieve this goal, we recently reported on automated microfluidic protein immunoblotting through high-resolution regional photopatterning of PA gel elements with multiple chemi- cal and physical properties 20,22 . Major design goals were to overcome limitations of conventional slab-gel immunoblotting, including multiple manual interventions, low throughput and substantial consumption of reagents 22 . A glass microfluidic chamber with sup- porting microfluidic channel networks was photopatterned with several different PA gel elements to integrate the functionalities of PAGE and subsequent antibody-based blotting. Owing to the favo- rable scaling of electrophoretic transport and our fully integrated chip design, our blotting assays complete in minutes, rather than in a day as typically required for conventional slab-gel methods. In addition, the approach described here is readily adaptable to a broad range of multistage assays. Important potential adapta- tions include several variations of blotting assays (i.e., separation of sample followed by affinity blotting): western blotting for proteins, far western blotting for protein interactions and complexes, Southern blotting for detection of specific DNA sequences, northern blotting for RNA, and eastern blotting for detection of protein glycosylation, among others 23 . The microfluidic chamber format is versatile and, for example, is currently being adapted by our group for the blotting of single electrophoretic protein separation against multiple affinity reagents. Demonstrated only for antibody blotting, the chamber patterning protocol described here may also allow users to pattern various blotting reagents including antibodies (e.g., full antibody, Automated microfluidic protein immunoblotting Mei He & Amy E Herr Department of Bioengineering, University of California, Berkeley, California, USA. Correspondence should be addressed to A.E.H. ([email protected]). Published online 28 October 2010; doi:10.1038/nprot.2010.142 This protocol describes regional photopatterning of polyacrylamide gels in glass microfluidic devices as a platform for seamless integration of multiple assay steps. The technology enables rapid, automated protein immunoblotting, demonstrated in this study for native western blotting. The fabrication procedure is straightforward and requires approximately 3 h from the start of gel photopatterning to completion of native protein western blotting, a substantial time savings over slab-gel immunoblotting. The assay itself requires less than 5 min. Importantly, all assay stages are programmably controlled by a high-voltage power supply and monitored by an epifluorescence microscope equipped with a charge-coupled device camera. Our approach overcomes severe limitations associated with conventional immunoblotting, including multiple steps requiring manual intervention, low throughput and substantial consumption of reagents. We also describe a simple chemical recycling protocol so that glass chips can be reused. The fabrication technique described forms the basis for a diverse suite of bioanalytical tools, including DNA/RNA blotting and multidimensional separations.

Transcript of Published online 28 October 2010; doi:10.1038/nprot.2010 · 2010-10-28 · far western blotting for...

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IntroDuctIonMicrofluidic analysis of biological molecules (e.g., protein, DNA) has dramatically enhanced speed, efficiency and sensitivity over conventional benchtop bioanalysis and diagnostic methods1–3. Miniaturization afforded by microfluidic technology has inspired deep interest in portable, automated clinical diagnostics for early stage disease detection4–6. Perhaps more important than the small-form factor, the potential for streamlined integration of multiple functions and even multiple protein assays in a compact micro-fluidic architecture has opened the door to a new era of automation in life science laboratories. Over the past decade, development of functional components for sample preparation, separation, purification, sorting and mixing using micro- and nanofluidic technology has boomed7. Successfully integrated microfluidic tech-nologies boast precise control and rapid transport. Such tools have been designed for applications from cell culture assays to human point-of-care disease diagnostics8. Nevertheless, one of the most important areas in which fully integrated multistage microfluidic technologies are needed is the area of bioanalytical separations. Although single-stage microfluidic assays are commercially available, development of multistage microfluidic assays rivaling the per-formance of benchtop methods has lagged. In particular, a key suite of multistage biomolecular assays, ‘immunoblots’, would benefit tremendously from the automation, speed and quantitation capabili-ties afforded by fully integrated microfluidic technologies.

Successful incorporation of biomolecular sieving matrices into microfluidic electrophoresis technology has yielded enhanced sepa-ration efficiency9–13. Among the various sieving structures explored, including micro/nanomachined structures and functional chemical materials14,15, polyacrylamide (PA) gel is notable. PA gels are a com-mon and powerful separation matrix for conventional benchtop electrophoretic separations of proteins, peptides, DNA and other biomolecules. PA gels are typically used in slab-gel formats includ-ing polyacrylamide gel electrophoresis (PAGE); two-dimensional (2D) PAGE; and western, Southern and northern blotting anal-yses16–19. The cross-linked 3D pore networks in PA gels support optimized, effective sieving performance with tunable pore size for a wide range of biological samples. Further, functionalization of PA gel with myriad chemical properties, including incorpo-ration of streptavidin, makes the material even more versatile20.

The protocol detailed here exploits a major design advantage inher-ent to cross-linked PA gels: ready photopatterning of PA gels as a means of integrating dissimilar functionalities within one mono-lithic microdevice. Key to our protocol is the deliberate selection of materials and tools found in most biology laboratories, making both fabrication and use of the approach feasible for translation from our bioengineering laboratory to life science laboratories. In addition, we describe a protocol for glass chip regeneration and reuse after native western blotting assay completion, which makes the approach even more economically feasible.

To the best of our knowledge, limited effort has been made to use microfluidic technology to streamline all steps needed to obtain mobility and binding-based identity information in one continu-ous assay21. To achieve this goal, we recently reported on automated microfluidic protein immunoblotting through high-resolution regional photopatterning of PA gel elements with multiple chemi-cal and physical properties20,22. Major design goals were to overcome limitations of conventional slab-gel immunoblotting, including multiple manual interventions, low throughput and substantial consumption of reagents22. A glass microfluidic chamber with sup-porting microfluidic channel networks was photopatterned with several different PA gel elements to integrate the functionalities of PAGE and subsequent antibody-based blotting. Owing to the favo-rable scaling of electrophoretic transport and our fully integrated chip design, our blotting assays complete in minutes, rather than in a day as typically required for conventional slab-gel methods. In addition, the approach described here is readily adaptable to a broad range of multistage assays. Important potential adapta-tions include several variations of blotting assays (i.e., separation of sample followed by affinity blotting): western blotting for proteins, far western blotting for protein interactions and complexes, Southern blotting for detection of specific DNA sequences, northern blotting for RNA, and eastern blotting for detection of protein glycosylation, among others23. The microfluidic chamber format is versatile and, for example, is currently being adapted by our group for the blotting of single electrophoretic protein separation against multiple affinity reagents. Demonstrated only for antibody blotting, the chamber patterning protocol described here may also allow users to pattern various blotting reagents including antibodies (e.g., full antibody,

Automated microfluidic protein immunoblottingMei He & Amy E Herr

Department of Bioengineering, University of California, Berkeley, California, USA. Correspondence should be addressed to A.E.H. ([email protected]).

Published online 28 October 2010; doi:10.1038/nprot.2010.142

this protocol describes regional photopatterning of polyacrylamide gels in glass microfluidic devices as a platform for seamless integration of multiple assay steps. the technology enables rapid, automated protein immunoblotting, demonstrated in this study for native western blotting. the fabrication procedure is straightforward and requires approximately 3 h from the start of gel photopatterning to completion of native protein western blotting, a substantial time savings over slab-gel immunoblotting. the assay itself requires less than 5 min. Importantly, all assay stages are programmably controlled by a high-voltage power supply and monitored by an epifluorescence microscope equipped with a charge-coupled device camera. our approach overcomes severe limitations associated with conventional immunoblotting, including multiple steps requiring manual intervention, low throughput and substantial consumption of reagents. We also describe a simple chemical recycling protocol so that glass chips can be reused. the fabrication technique described forms the basis for a diverse suite of bioanalytical tools, including Dna/rna blotting and multidimensional separations.

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Fab fragment), lectins or other affinity reagents23. With slight chan-nel network design adjustments, the approach can be adapted to support multiple concurrent separations of one or multiple sam-ples. This heterogeneous gel photopatterning approach is a flexible and powerful new tool that is applicable, more broadly, to multi-stage assays including 2D electrophoresis.

Comparison with micro/nanomachined sieving structuresRecently, micro/nanofluidic sieving structures have been produced by plasma etching or imprinting in a polydimethylsiloxane (PDMS) or silica substrate for protein and DNA separations24,25. One notable example is the anisotropically patterned nanosieve array or ‘ANA’ chip introduced to separate long DNA ladder samples using an entropy trap array26,27. Huang et al.28 devised a ‘DNA prism’ chip to rapidly and continuously separate long DNA fragments. Compared with PA gels, micro/nanomachined structures possess advantages, such as regular and precisely engineered geometries and mechanical robustness. However, fabrication of micro/nanomachined struc-tures requires access to and training on specialty equipment not otherwise needed by most life science researchers29,30. For example, a 1D feature in a nanomachined array may require deposition, photo-lithography, deep reactive ion etching and wet anisotropic potas-sium hydroxide (KOH) etching procedures, which can require up to 12 h and clean room access31. Chemical functionalization can also be labor intensive32. Consequently, we have developed a methodology on the basis of equipment, reagents and techniques that life scien-tists are likely already skilled at using. Further, the PA gel patterning technique is affordable and can be implemented so as not to require clean room access, associated user fees or the purchase of special microfabrication equipment. PA gels are versatile, both chemically and physically, and can be used broadly for protein preconcentra-tion, mixing, separation, purification and immunoprobing33–35.

The major limitations associated with PA gel structures include ensuring the physical robustness of the gel during electrophoresis and fabricating the structures so as to achieve repeatable pore-size distributions among like structures. As is the case with slab-gel technologies, PA gels can break down if operated at high electric potentials (i.e., above several thousand volts) or in the presence of harsh solvents. Users should also avoid using poor-quality acryla-mide reagents (e.g., containing acrylic acid above ~0.001% (wt/wt)) or contaminated buffers (e.g., those containing metals, nonbuffer ions or breakdown products), as fabrication and/or operation of the final PA gel can be compromised. Nevertheless, with well-con-trolled fabrication protocols, repeatable fabrication of nanoporous structures is possible, as has been demonstrated in laboratories for decades using chemically initiated slab-gel casting. For the pho-toinitiated PA gel microcasting detailed here, fabrication control can also be achieved using uniform ultraviolet (UV) excitation and standardized fabrication protocols. For example, we have recently used microscope objective-based photolithography to achieve high analyte migration reproducibility (3% run-to-run variation) for on-chip homogeneous electrophoretic immunoassays36.

Comparison with other functional chemical materialsPolymer beads and polymer monoliths are alternative chemical mate-rials commonly used in microfluidic devices for functional integra-tion. Polymer beads are attractive, as the wide range of commercially available sizes and flexible chemical properties have made beads ubiquitous in immunoassays, reactor beds and chromatography37.

Packing considerations are taken into account during chip design. Most on-chip packing techniques need frit or weir structures to trap beads in specific device regions. Packing can require high pres-sures for loading, which can be cumbersome when localizing beads in complex geometries (e.g., geometries beneficial to multistage assays)38. Polymer monoliths share many of the benefits of packed chromatographic beads, including high surface area and readily controlled surface chemistries. In addition, polymer monoliths present some of the same distinct advantages for microfluidic applications as those presented by PA gels, including amenabil-ity to photopatterning for localization of features. Namely, use of monoliths and gel structures obviates the need for frits or other retaining structures, as chemical anchoring to the device surface is facile. The porosity and surface chemistry of the monolith can be controlled by adjusting the composition of the precursor solu-tion39,40. Typically, polymer monoliths have a more porous structure (1–10 µm for pore diameter) than do PA gels (1–100 nm for pore diameter), which leads to much lower back pressure compared with PA gels. Thus, polymer monoliths (i.e., acrylate) are useful when pressure-driven or other bulk flows are desired. PA gels are appropriate when electrophoretic manipulation of biomolecules is desired41.

Comparison with conventional slab-gel immunoblottingConventional slab-gel western blotting has changed little since it was first introduced in 1979 (ref. 42). Although powerful, the slab-gel procedure is time consuming and labor intensive43. Variable transfer, blotting efficiency and reproducibility are all concerns for quanti-tative analysis. One very recent advance in protein blotting intro-duced a new high-throughput, yet low separation resolution (SR) format based on microarrays. The technique enables the analysis of hundreds of samples in one workflow, but it requires multiple man-ual handling steps and the use of disparate pieces of equipment44. To mitigate time and equipment requirements, we have introduced an alternative fully integrated on-chip automated immunoblotting technology that relies on regional photopatterning of PA gels within microfluidic networks. PA gel photopatterning with subsequent immunoblotting can now be completed in 3 h (< 5 min for the assay) using standard biological reagents and antibodies. In addi-tion, the microchannel network is designed to be compatible with charge-coupled device (CCD)-based full-field imaging and allows for fully programmable electrophoretic operation. The separation, transfer and in-gel blotting stages are monitored within one field of view. Thus, this imaging approach provides an ability to quantify sample blotting capture and losses directly, which is difficult with slab-gel blotting. Further improvements in detection sensitivity, multiplexing and readout are under way in our lab, including an additional performance goal of protein quantitation.

Experimental designFigure 1 outlines the five major steps comprising immunoblot chip fabrication and operation, including (i) chip design and fabrica-tion; (ii) PA gel photopatterning; (iii) PDMS fluidic interfacing fabrication; (iv) assay operation; and (v) glass chip recycling in preparation for subsequent, new PA gel photopatterning.

Glass chip design and fabrication. Commercial computer-aided design (CAD) software is available to design the glass chip. AutoCAD (2007) was used in this protocol to generate the

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immunoblot chip layout, which was then sent by e-mail to a com-mercial supplier to generate a chrome-glass photomask (Photo Sciences). Although glass microdevice processing is now well estab-lished, researchers who may not wish to invest time or resources in developing the capability for in-house glass processing can purchase glass microfluidic chips (standard and custom layouts) from sev-eral commercial vendors. These vendors provide some design and full fabrication services for both glass and, in some cases, polymer devices. Examples include Caliper Life Sciences (vendor used in this work for glass fabrication), as well as Micronit, Micronics, ALine, Microfluidic Chip Shop and Micralyne, among others. Alternately, researchers who are interested in in-house glass chip fabrication may choose to use well-established glass hydrofluoric acid (HF) etching and thermal bonding protocols45–47.

The immunoblot glass chip layout consists of a cross injector and a 1 mm × 1.5 mm rectangular chamber connected to liquid res-ervoirs through microchannel arrays. The rectangular chamber houses the separation and blotting gels (Fig. 2a,b). The size of the chamber was chosen to be compatible with CCD-based imaging on epifluorescence microscopy systems. Here 14 parallel side channels were used abutting the chamber; each channel was ~20 µm deep, ~10 µm wide and ~4 mm long (Fig. 2a; Supplementary Fig. 1). Microchannel arrays were designed to yield uniform electric fields over the large chamber area during separation and trans-fer steps in both the vertical and horizontal dimensions48,49. The channel arrays connect the microchamber to buffer reservoirs (as indicated in Fig. 2a, numbered 1–8) wherein voltages are applied (Step 36; Table 1). The resistance of the channel arrays (parallel) is designed to be larger than the sheet

resistance of the chamber49. Ideally, the smaller the dimensions of the side channels and the greater the number of channels in each array, the more uniform the resulting electrical field within the chamber. In practice, arrays of ~10 channels of the given dimensions provide sufficient control for this chip layout and application. The exact chip geometry is provided in Supplementary Figures 1–3.

PA gel photopatterning. Spatial control of PA gel properties pro-vides a design and fabrication tool important for integrating the assay steps needed to complete protein blotting. The PA gel regions (sample loading, PAGE separation and immunoblotting) were all co-located in one microfluidic chamber through our development and use of multistage photopatterning (Fig. 3). The PA gel pho-topatterning mask was designed using AutoCAD and printed on transparency film by a commercial high-resolution printing service (FineLine Imaging). As an alternative, a high-resolution commer-cial printer is sufficient to generate economical transparency mask patterns suitable for PA gel photopatterning (8,000 d.p.i. with dot diameters of 3 µm). Slits in the mask as narrow as 15 µm provide acceptable edge resolution on the resultant PA gel features.

Using the transparency film mask and an inverted epifluores-cence microscope (Olympus IX-50) equipped with a mercury lamp (100 W)50, we implemented PA gel photopatterning (Fig. 3b). Alignment between the transparency mask and the immunoblot chip relied on a manual adjust x–y translation stage on the inverted microscope, yielding an alignment precision of tens of micro-meters. Alternately, a fine programmable translation stage or even a photolithography stepper system can be used if higher spatial resolution is needed in the photopatterning. To achieve a desired UV intensity across the field of view (~13 mW cm − 2), we used a neutral density filter. It is important to note that the mercury lamp power diminishes with use, so the UV power should be monitored by a UV meter before each polymerization. The photopolymeriza-tion times were determined empirically based on the intensity of the UV light source, the composition of the acrylamide precursor solution and the desired pore size.

To provide the blotting function of the blotting region, we cross-linked streptavidin-acrylamide into the blotting gel matrix. This functionality was used to immobilize biotinylated anti-bodies (and other blotting reagents, e.g., lectin, aptamers, Fab

Glass chip

Glass chips

Glass chip recycling

Glass chip

Photomask

Puncher

Fluidic interface Automated blotting

Glasschip

×4 objective

UV light

PDMSsheet

PA gel dissolutionunder heating

PA gel photopatterning

Steps 1–4

Steps 5–26

Steps 27–32

Steps 40–43

Steps 33–39

Figure 1 | Schematic of the immunoblot protocol. Steps include glass chip fabrication, PA gel photopatterning, PDMS fluid interface fabrication, on-chip assay and chip recycling. Details for each step are found in the PROCEDURE section.

Sampleinjector

a b

Load & stack sample PAGE Transfer Blot

Chamber Channelarray

8

6

4

2 3

1

5Loading

i i i i i

Blotting

Separation

7

Figure 2 | Immunoblot chip design and operation. (a) Schematic design of the immunoblot chip for analysis of native proteins. The sample (2), sample waste (3), buffer (1, 4, 5, 6) and buffer waste (7, 8) reservoirs are indicated in sketch (not to scale). The microchamber houses a large-pore-size protein loading gel, smaller-pore-size protein separation gel and antibody-functionalized blotting gel. Colored dyes were used to visualize the different gel regions. (b) The automated multistage assay protocol is overlaid on micrographs of the microchamber showing the gel regions. The on-chip immunoblot protocol includes sample loading, PAGE separation, transfer and in gel blotting. The blue and yellow bands in each panel schematically represent unique protein peaks as they migrate in the direction of the electric current ‘i ’. Scale bar, 300 µm.

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fragments) for immunoidentification of native PAGE-resolved proteins. Immobilization of antibody can be achieved either during fabrication of the chip or after complete fabrication, which provides more flexibility for end users to use antibodies selected after chip fabrication is completed. During chip fabrica-tion, both streptavidin-acrylamide and biotinylated antibodies are included in the blotting gel precursor solution. Alternately, biotinylated antibodies can be electrophoresed (from reservoir 5 in Fig. 2a) across the streptavidin-decorated blotting gel after fabrication, resulting in immobilization of antibody in the blotting region. The two strategies are further detailed in our previous report21.

PDMS manifold fabrication. To facilitate macro-to-micro fluidic and electrical interfacing, as well as easy cleaning and storage of fully fabricated immunoblot chips in refrigerated solution, we used an inexpensive, rapidly fabricated, adapt-able PDMS gasket. PDMS adheres to glass sufficiently for the atmospheric operation of the immunoblot chip. As the gasket is temporarily attached to the immunoblot glass chip, the PDMS can be quickly detached for immunoblot chip storage followed by chemical recycling.

Preparation of samples. The proteins or antigens to be analyzed (Step 35) are fluorescently labeled in-house using an Alexa Fluor 488 protein labeling kit per the manufacturer’s instructions and purified using Bio-Gel P-6 columns. Antibodies are biotinylated using an EZ-Link Micro Sulfo-NHS-Biotinylation Kit. The level of biotin incorporation is measured by a 2-(4′-hydroxyazobenzene) benzoic acid (HABA) assay per the manufacturer’s instructions. Fluorescently labeled negative controls and full molecular weight ladders can be included in the sample mixture. Here we use Alexa Fluor 488–labeled BSA as a negative con-trol; this allows us to identify confounding effects such as nonspecific binding and/or size exclusion at any PA gel boundary. Our model sample, prostate-specific antigen (PSA), was extracted from human seminal plasma as described previously51. The PSA sample was fluorescently conjugated with Alexa Fluor 488 dye to produce a detect-able fluorescence signal. All samples are stored at 4 °C in the dark until use. A 5 µl PSA sample (~500 nM) is pipetted into the sample reservoir (Fig. 2a, reservoir 2) for the on-chip native protein blot.

Assay performance. The total native western blot assay is auto-mated using a programmable high-voltage power supply, which controls sample loading, vertical protein separation and lateral transfer to the blotting region for in situ target identification. We have used both custom in-house power supplies and commer-cially available supplies (e.g., LabSmith). To control the electric field fringing–induced sample dispersion common in chamber-like geometries, an array of field-control channels border the chamber. Optimization of the applied voltages is quickly achieved through simulation of the load and transfer electric fields using COMSOL Multiphysics software (version 3.5a; COMSOL AB). The simula-tion was conducted under 2D conductive media DC conditions, as described in our previous reports22. Empirical determination is also effective in determining the optimized voltage program for experimental operation.

The on-chip assay performance can be validated by conven-tional slab-gel native western blotting, which typically takes 1–2 d to complete and requires 5–8 µg of protein and antibody samples. Conventional standard western blotting protocols23 are available and should be followed to validate the on-chip assay.

Glass chip recycling. After immunoblot chip use, the PA gel can be easily dissolved from the glass channels and chamber. This allows reuse of the glass chip for subsequent PA gel photopattern-ing. We have found a mixture of perchloric acid and hydrogen peroxide under heating to be effective in dissolving all PA gel material. Further, we find no adverse effect on subsequent PA gel photopatterning in recycled chips. Extreme care should be taken during chip regeneration, as the regeneration solution is a strong oxidizer and highly corrosive. Handling, use and storage of the material should include important personal, exhaust and operational safety controls.

table 1 | Typical on-chip immunoblotting voltage and current control program.

Voltage and current program (operation durationa)

reservoir numbers as indicated in Figure 2a

1 2 3 4 5 6 7 8

1 Sample loading (60 s) 0 V 0 V 500 V 0 µA 0 µA 0 µA 0 µA 0 V

2 Injection and separation (30–60 s) 0 V 150 V 150 V 90 V 90 V 0 µA 0 µA 180 V

3 Transfer and blotting (30–60s) 0 µA 0 µA 0 µA 0 µA 0 µA 0 V 50 V 0 µAaDuration of each step presented is typical; actual durations will vary depending on sample analyzed.

Glass chipchamber

Photomask

a b

UV UV UV

Define antibody functionalizedblotting region

Define separationgel region

Define loading gel region withstacking interface

6%T, Ab 6%T 3%T

Figure 3 | Photopatterning of gels in the immunoblot chip chamber. (a) Epifluorescence microscope with a transparency film mask for high-resolution photopatterning. (b) Illustration of three-step regional photopatterning to unify the loading gel, PAGE separation gel and blotting gel into one microchamber for automated on-chip immunoblotting. %T indicates acrylamide monomer concentration in the gel precursor solution. ‘Ab’ indicates that biotinylated antibody is present.

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MaterIalsREAGENTS

2,2-Azobis[2-methyl-N-(2-hydroxyethyl) propionamide] (VA-086; Wako Chemicals)Acrylamide/bis-acrylamide (30%, 29:1; Sigma, cat. no. A2792) ! cautIon This material is highly toxic, carcinogenic and teratogenic. Avoid direct contact and always handle in a fume hood. As with any laboratory chemical, review and understand all Material Safety Data Sheet (MSDS) information.Streptavidin-acrylamide (Invitrogen, cat. no. S21379)Tris-glycine, premixed 10× native electrophoresis buffer (250 mM Tris, 192 mM glycine (pH 8.3); Bio-Rad, cat. no. 161-0734)Biotinylated anti-fPSA (AbD Serotec, cat. no. 7820-0217 for in-house biotinylation; R&D, cat. no. BAF1344 for commercial biotinylation)2-Hydroxyethyl cellulose in water, 5% (wt/vol) (HEC; Sigma, cat. no. 434973, average molecular weight ~720,000) crItIcal This material is widely used as a thickener and stabilizer and has varied average molecular weights. The high viscosity enables the solution to substantially decrease any residual liquid flow in the channel network. This reagent was chosen owing to: (i) low cost and ease of handling, (ii) optimal fluid viscosity without creating clogs in microchannels, (iii) inert and stable chemical properties and (iv) ease of removing the solution from chip reservoirs by simple buffer washing.Sylgard 184 silicone encapsulant, 0.5 kg (184 SIL ELAST KIT 0.5 kg; Dow Corning) crItIcal The higher-percentage-base cured PDMS (19:1 weight ratio) provides a compliant gasket with suitable annealing and adherence to the glass microdevices, as compared with common PDMS recipes (base to curing agent 10:1).Alexa Fluor 488 Protein Labeling Kit (Invitrogen, cat. no. A10235/A20181)Bio-Gel P-6 column (Bio-Rad, cat. no. 732-6222)EZ-Link Micro Sulfo-NHS-Biotinylation kit (Pierce, cat. no. 21925) crItIcal Use HABA assay (instructions can be found from Pierce, cat. no. 21425) to measure the level of biotin incorporation to ensure reproducible biotinylation.Sodium hydroxide (ACS grade; Sigma-Aldrich, cat. no. S5881) ! cautIon Corrosive, avoid direct contact.Methanol (ACS grade; Sigma-Aldrich, cat. no. 322415)Acetone (ACS grade; Sigma-Aldrich, cat. no. 650501)Perchloric acid (70% (wt/wt), ACS grade; Sigma-Aldrich, cat. no. 311421) ! cautIon Corrosive, always handle in an appropriate fume hood with personal protection equipment. Read MSDS carefully.Hydrogen peroxide (30% (wt/wt), ACS grade; Sigma-Aldrich, cat. no. H3410) ! cautIon Corrosive, always handle in a fume hood with personal protection equipment.Distilled water

Optional reagents for slab-gel western blottingPVDF/filter paper sandwiches (Invitrogen, cat. no. LC2005)Sponge pad for XCELL II blotting (Invitrogen, cat. no. EI9052)NOVEX HRP chromogenic substrate (TMB) (Invitrogen, cat. no. WP20004)HRP-conjugated anti-IgG (Invitrogen, cat. no. 62-6520)Prestained protein ladder (Bio-Rad, cat. no. 161-0374)Tris-glycine native sample buffer (2×; Invitrogen, cat. no. LC2673)Tris-glycine native running buffer (10×; Invitrogen, cat. no. LC2672)Western blotting transfer buffer (Invitrogen, cat. no. LC3675)Blocking buffer (Invitrogen, cat. no. W10132-B)Washing buffer (Invitrogen, cat. no. W10132-A)

EQUIPMENTEpifluorescence microscope system with 100-W mercury lamp housing (IX-70 and IX-50; Olympus)Peltier-cooled interline CCD camera, 1,392 × 1,040, with 12-bit image acquisition (CoolSNAP HQ2; Roper Scientific)UV ×4 objective (UPLANS-APO; Olympus, NA 0.18) crItIcal The UV objective should provide ~90% UV transmission in the excitation wave-length range of 330–375 nm for efficient photopolymerization.

••

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×10 Objective (Olympus, NA 0.3)Filter cube with excitation at 330–375 nm and emission at 580–620 nm for UV photopatterning. Filter cube with excitation at 460–490 nm and emis-sion at 505–535 nm for fluorescence detection (Olympus)High-voltage power supply equipped with platinum electrodes (custom made in-house) crItIcal The power supply should equip eight inde-pendent output ports in both current and voltage control modes, allowing the user to adjust the voltages continuously at each buffer reservoir for both programmed operation and empirical refinement of the applied potentials.Neutral density filter set (Omega), which is used to adjust the UV intensityUltraviolet light meter (MANNIX UV340; Mannix Instrument, 290–390 nm)Filtered mercury lamp (B100-AP; UVP, 300–380 nm) with cooling fanCorning hot plate with digital display (Corning, cat. no. 6795-600D)Bath sonicator (Bransonic 220; Branson Ultrasonics)Vacuum line and nitrogen gas lineComputer with software (WinView, ImageJ, OriginLab, COMSOL and AutoCAD)XCELL SureLock Mini Cell and Blot Model (Invitrogen, cat. no. EI9051)4–12% Tris-glycine mini gel (Invitrogen, cat. no. EC6035BOX)Mini gel power supply (Model 250/2.5; Bio-Rad)Molecular Imager ChemiDoc XRS + System with Quantity One software (Bio-Rad Laboratories)Teflon tweezers

REAGENT SETUP (prepare fresh before use)Blotting gel precursor solution In this study, we used 6%T, 3.3%C acrylamide/bis-acrylamide (29:1) as the blotting gel precursor, incubated with 4 µM streptavi-din-acrylamide, biotinylated anti-fPSA(1:4) and 1× Tris-glycine native buffer. crItIcal Notations %T and %C indicate the percentage of total acryla-mide (wt/vol) and cross-linker (wt/wt), respectively, which can be adjusted by mixing with buffer (e.g., 1× Tris-glycine) to define the gel pore size. crItIcal For optimized photoinitiation, 0.2% (wt/vol) VA-086 is used in the gel precursor mixture. Note that the photoinitiator VA-086 is soluble in water and is an attractive choice for this system because of its non-ionic groups, rapid photodecomposition and hydrophilicity. Store VA-086 in the refrigerator shielded from light. Prepare precursor solutions immediately before use, as VA-086 decomposes on exposure to light and water. crItIcal All solutions should be brought to room temperature (RT, 25 °C) before preparation of precursor solutions, as cold solutions have a higher capacity for dissolved oxygen than solutions at RT. ! cautIon Acrylamide and bis-acrylamide are highly toxic, and are potential human carcinogens and teratogens; avoid direct contact and always handle in a fume hood. As with any laboratory chemical including those used in this protocol, review and understand all MSDS information.Separation gel precursor solution The separation gel consists of 6%T acrylamide/bis-acrylamide (29:1), 1× Tris-glycine native buffer and 0.2% (wt/vol) VA-086. crItIcal All solutions should be brought to RT before preparation of precursor solutions, as cold solutions have a higher capac-ity for dissolved oxygen than solutions at RT. ! cautIon Acrylamide and bis-acrylamide are highly toxic, and are potential human carcinogens and teratogens; avoid direct contact and always handle them in a fume hood. As with any laboratory chemical, review and understand all MSDS information.Loading gel precursor solution The loading gel consists of 3%T acrylamide/bis-acrylamide (29:1), 1× Tris-glycine native buffer and 0.2% (wt/vol) VA-086. crItIcal All solutions should be brought to RT before preparation of precursor solutions, as cold solutions have a higher capacity for dissolved oxygen than solutions at RT. ! cautIon Acrylamide and bis-acrylamide are highly toxic, and are potential human carcinogens and teratogens; avoid direct contact and always handle in a fume hood. As with any laboratory chemical, review and understand all MSDS information.

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proceDureDesign and fabrication of immunoblot chips ● tIMInG 4–10 d1| Use AutoCAD software to draw the chip layout (see chip layout used in this work in supplementary Figs. 1–3). In all, 2–4 h are required to finish the drawing for intermediate to advanced users, whereas more time should be allotted for novice users (e.g., a few days) to complete tutorials on the basics of AutoCAD.

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2| Save the design in an appropriate format (GDXII or DXF) and submit to a commercial supplier such as Photo Sciences for chrome-gold mask printing. The turnaround time for the printing service is typically 2–3 d after the design is finalized with the vendor.

3| Submit the photopolymerization mask design for transparency film mask printing. The turnaround time for the printing service is typically 2–3 d.

4| Glass wet etch, drill and bond chips in a clean room using standard procedures46. Every user of a clean room needs proper training before accessing and using the facility. Typically, the fabrication takes 1–3 d, depending on the quantity of chips and experience level of the user. Alternately, the mask can be submitted to a commercial glass chip vendor for a 1- to 3-week turnaround on glass device fabrication.

pa gel regional photopatterning ● tIMInG 1.5–3 h5| Use a 0.1 M sodium hydroxide solution to wash the microchannels by applying vacuum to one reservoir and continuously replenishing the sodium hydroxide solution in the rest of the reservoirs. Use pipette tips that fit snuggly into liquid access holes to hold a larger volume of solution (~50 µl). Flush 10 min for new chips and 30 min for recycled chips. The sodium hydroxide solution will refresh the glass surface and provide uniform surface properties.

6| Use distilled water to flush the microchannel again for 20 min. Thereafter, dry the channels by purging with nitrogen gas. This step removes the excess sodium hydroxide.

7| Inspect the microchannels using bright-field microscopy. crItIcal step Only proceed if channels are free of debris or small particles. If debris is observed, repeat Steps 5–7.

8| Prepare the blotting gel precursor solution (containing photoinitiator, monomer, cross-linker, binding reagents and buffer solution, all at RT) and mix in a microcentrifuge tube as described in the REAGENT SETUP section. As is good laboratory practice, immediately label the blotting precursor tube. crItIcal step For antibody copolymerization patterning, incubation takes 1 h. Submerge the precursor vial in a sonicator bath for 2–3 min for better mixing. Right before photopolymerization, add 0.2% (wt/vol) VA-086 into the precursor solution. Keep the precursor vial in the dark to avoid degradation of the photoinitiator.

9| Prepare the separation gel precursor solution in a microcentrifuge tube as indicated in REAGENT SETUP. Label the separation precursor tube. crItIcal step Keep the precursor vial in the dark to avoid degradation of the photoinitiator.

10| Prepare the loading gel precursor solution in a micro-centrifuge tube as indicated in REAGENT SETUP. Label the loading precursor tube. crItIcal step Keep the precursor vial in the dark to avoid degradation of the photoinitiator.

11| Use a sharp needle to pierce a tiny hole in the closed cap of the microcentrifuge tube with the blotting precur-sor solution inside. Use good laboratory practices to avoid possible injury. Connect this microcentrifuge tube with vacuum as shown in Figure 4a. Place the evacuated microcentrifuge tube into a sonicator bath for 5 min. Agitate the tube by tapping on the sonicator bath walls, being careful not to introduce liquid from the sonication bath into the tube. The sonication aids removal of air bubbles or dissolved oxygen in solution. Evacuation of the tube accelerates the degassing. crItIcal step Carefully degas for at least 5 min with agitation and degassing until no bubbles are visually observed in the microcentrifuge tube or solution.

Vacuuma

c d

b

HEC drops

Vacuum

Precursor solution

Precursorsolution

Figure 4 | Gel precursor solution preparation. (a) Degassing in sonicator bath under house vacuum. (b) Loading gel precursor into microchannel by capillary force. (c) HEC drops applied on chip reservoirs with filled precursor solution. (d) Precursor exchange by gently applying vacuum to one reservoir and replenishing precursor solution in the rest of the reservoirs.

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12| Use a micropipette to transfer drops of blotting precursor solution into one reservoir of the microchip as shown in Figure 4b. The precursor solution fills the microchannel via capillary action (wicking). crItIcal step If there are trapped bubbles in the channels, gently use vacuum to suck the bubbles out while replenishing the precursor solution in the rest of the reservoirs.

13| Use 5% HEC drops to cover all the reservoirs filled with precursor solution as shown in Figure 4c. Place the chip on a flat surface in the dark for 5 min to allow flow in microchannels to equilibrate to quiescence. crItIcal step HEC is used to stop hydrodynamic flow in the microchannel, and not for other purposes, such as preventing the gel from drying. The 5% HEC drops are gently applied onto each reservoir to eliminate any existing hydrostatic head and yield quiescent flow conditions inside the channels, as is needed for high-resolution photopolymerization.

14| Turn on the microscope mercury lamp. Switch to the ×4 objective and filter cube with UV light excitation at 330–375 nm. Set up the transparency film mask on top of the microscope stage as shown in Figure 3a and tape it to the microscope stage. Apply on the neutral density filters necessary to achieve a UV intensity of approximately 13 mW cm − 2 through the film mask, measuring with a UV light meter. Thereafter, close the shutter until use. crItIcal step Measure the UV intensity right before photopolymerization to ensure reproducible patterning.

15| Place the previously prepared chip from Step 13 on top of the film mask. Using the microscope eyepiece, align the microchamber to the exposure window. Tape the chip to the film mask, allowing exposure of the right side of the chamber to UV light as indicated in Figure 3b. Set the timer for 8 min. Open the shutter and start the timer. Close the shutter when 8 min has elapsed. crItIcal step Ensure good chip/mask alignment so as to avoid polymerization in the injection channel connected with the microchamber. ! cautIon Do not look directly at the UV light, as it is harmful to eyes and skin. Use a UV shield or a cover box to cover the microscope stage.

16| During the 8-min exposure time, degas the separation precursor solution as instructed in Step 11.

17| After blotting gel photopolymerization, remove the chip from the stage and carefully wash the chip reservoirs with distilled water until no HEC debris remains. Thereafter, fill the reservoirs with the degassed separation gel precursor.

18| Connect reservoir 1 (in Fig. 2a) with vacuum and replenish the rest of the reservoirs with separation precursor solution as shown in Figure 4d. About 40 µl solution should be enough to exchange and fill the unpolymerized channel. ! cautIon Polymerized blotting gel should block fluid flow to reservoir 7 under suction. Use gentle vacuum to avoid damaging that polymerized gel region.

19| Repeat Step 13 to cover all the reservoirs with HEC drops and allow the chip to equilibrate to quiescent flow conditions for 5 min.

20| Repeat Step 15 for alignment. Align so as to expose the left side of the chamber to UV light as indicated in the second stage of Figure 3b. Tape the chip to the film mask. Set the timer for 5 min. Open the shutter and start the timer immediately. Close the shutter when 5 min has elapsed. ! cautIon Do not look directly at the UV light. It is harmful to the eyes and skin. Use a UV shield or a cover box to cover the microscope stage.

21| During the 5-min exposure time, degas the loading precursor solution as instructed in Step 11.

22| After separation gel photopolymerization, take the chip off the stage and wash away the HEC drops as mentioned in Step 17. Connect reservoir 3 with vacuum. Exchange the unpolymerized solution in the injection channel with loading precursor solution as shown in Figure 4d. ! cautIon The blotting and separation gels are polymerized in the microchamber, thus obviating fluid flow between all reservoirs except 1 and 2. Therefore, the loading precursor solution can only be exchanged through reservoirs 1 and 2.? troublesHootInG

23| Repeat Step 13 to cover all the reservoirs with HEC drops and allow the chip to equilibrate to quiescent flow conditions for 5 min.

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24| During the equilibration period, turn on the filtered mercury lamp to warm it up. Set the polymerization platform 7 cm above the lamp to achieve a UV intensity of approximately 10 mW cm − 2, as determined using the UV intensity meter.

25| Fix the chip on the polymerization platform, 7 cm over the lamp, (and conduct a flood exposure (i.e., no masking is used in this step), as shown in the third step in Figure 3b. Expose for 8 min. ! cautIon Use a UV-shielded facial mask and a cover box to shield the UV illumination. crItIcal step A cooling fan should be used during this step to eliminate undesired heating effects.

26| After flood exposure, inspect the chip under a microscope to ensure that no bubbles are formed in the channels (three gel regions are located in the microchamber as shown in Figure 2a). crItIcal step Inspection under a microscope after each polymerization step is necessary to ensure successful photopat-terning. Wash away the HEC drops and store the chip in buffer solution at 4 °C until use. Never let the gels dry out in the channels or chamber.? troublesHootInG

pDMs manifold fabrication ● tIMInG 2–3 h27| Place a disposable plastic cup on the scale and mix 19:1 weight ratio of base and curing agent. ! cautIon PDMS is a sticky substance. Use disposable, unpowdered gloves during handling. crItIcal step Avoid introducing dust and other particles into the PDMS.

28| Stir the PDMS mixture using a glass rod for 3 min as indicated in Figure 5a. Wipe away the PDMS residue on the glass rod for next use. After stirring, the mixture will contain air bubbles. These bubbles must be removed before the PDMS can be used to make a manifold.

29| Place the plastic cup containing the mixture into a larger and heavier container, such as a Pyrex beaker. Leave the PDMS in a desiccator connected to a vacuum line for 1 h of degassing. The PDMS is ready to be taken out of the vacuum when there are no bubbles remaining in the liquid. ! cautIon Release the vacuum valve on the desiccator slowly, as the sudden rush of air may knock over the cup and spill the PDMS material.? troublesHootInG

30| After degassing, take out the cup and pour the PDMS slowly into a glass Petri dish, as indicated in Figure 5a. Make a PDMS layer about 3–5 mm thick. Cover the Petri dish, avoiding bubble creation and introduction of dust into the PDMS. Place the Petri dish on a flat surface for self-curing at RT overnight. For faster curing, place the Petri dish on a hot plate at 100 °C for 1 h. pause poInt A 4-inch square PDMS sheet is sufficient to make more than five manifolds. Keep the fabricated PDMS sheet clean for next use.

31| Use a sharp knife to cut out a small square PDMS piece approximately the size of the microchip. Use a puncher as shown in Figure 5b to punch ~2-mm-diameter holes in the PDMS sheet. The punched holes should align with the glass chip reservoirs. ! cautIon To avoid contamination, use a clean plastic wrap to cover the PDMS during handling.

32| Align the PDMS manifold on the clean surface of the glass chip. Align holes with glass chip reservoirs. The PDMS surface will immediately anneal to the glass surface as shown in Figure 5c. Fill the manifold reservoirs with buffer solution immediately to avoid gels drying in the channels. Place the whole assembly on the microscope stage with the microchamber in the center of the field of view. crItIcal step Avoid any contamination on the PDMS manifold surface before adhering PDMS to the glass surface.? troublesHootInG

a

b c

Figure 5 | PDMS manifold fabrication. (a) Materials and reagents required to make PDMS sheet. (b) PDMS manifold with punched holes. (c) Annealing of PDMS manifold on glass chip surface. The punched holes align to glass chip reservoirs.

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on-chip immunoblotting ● tIMInG < 0.5 h33| Use a voltage-programmable power supply to control the electrophoretic operation. Insert the eight output electrical lines with Pt electrodes into the PDMS manifold reservoirs filled with buffer, as shown in Figure 6. For physical stabil-ity, fix the chip assembly on the microscope stage with tape. Fix each electrode using a mounting frame that is anchored on the microscope stage.

34| Switch the filter cube to enable FITC fluorescence detection. Turn on the CCD camera and high-voltage power supply. Input the voltage sequence program into the control software, including the sample loading step, separation step and transfer to blotting step. The voltages at each buffer reservoir can be adjusted continuously to generate different electric fields in the immunoblot chip. The suggested voltage applied on each reservoir can be found in table 1 for reference.

35| Load the sample to be analyzed (see Experimental design) into sample reservoir 2 by micropipetting while applying the sample loading voltage program as described in table 1. Use a ×10 objective and CCD camera with a ×0.63 magnifier to observe the microchamber and ensure that the loading is effective.? troublesHootInG

36| Adjust the camera field of view to allow imaging of both the PAGE separation axis and the blotting region (1,392 × 1,040 pixels). With the camera-control software (WinView), adjust the camera exposure to achieve optimal fluorescence signal (which is sample dependent) with a suggested maximum exposure time of ~400 ms. After sample loading, switch the voltage program to run the vertical PAGE separation (table 1, with typical durations indicated). crItIcal step Several PAGE separations can be run in the vertical direction, without lateral transfer to the blotting region, as a means of optimizing the PAGE separation conditions (e.g., electrical field strength, duration of separation, exposure times).

37| Start recording an image series for all assay steps at a frequency of two frames per second while switching the voltage program to the PAGE separation step. PAGE completion is achieved when the ladder and/or major species of interest are fully resolved. After PAGE completion (30–60 s), switch the voltage program to initiate lateral transfer of all proteins to the blotting region (table 1). As proteins migrate through the blotting region, target proteins that interact with immobilized antibodies are retained in the blotting region. Proteins that do not interact with the immobilized antibodies will migrate out of the chamber. Images collected at completion of PAGE and at completion of blotting are the most important for data analysis. Comparison of the final PAGE image to the image of proteins retained in the blotting region allows direct spatial mapping of PAGE peak position (i.e., electrophoretic mobility) to blotted peak position (i.e., known antibody binding partner).? troublesHootInG

38| Use ImageJ or similar software to extract fluorescence intensity (FL signal) from collected image sequences. Electro-pherograms can be generated by plotting time-resolved fluorescence at a single point in the field of view. Separation performance metrics (e.g., SR and FL signal response) can be quantified by nonlinear least-squares fitting of electropherograms using analysis software, such as OriginLab52.

39| After on-chip immunoblotting, replace the solutions in all reservoirs with fresh buffer and electrophoretically run the buffer through the device for 1 h to ensure that residual sample is retained in the device.! cautIon For best blotting results, reuse of the blotting region is not suggested because of potential contamination. The glass chip can be regenerated by following the subsequent recycling steps and used to fabricate a new immunoblot device.

Glass chip recycling ● tIMInG ~20 h40| Place the used immunoblot chip into a water tube as shown in Figure 7a. Chemical dissolution can be conducted right away or after accumulation of a number of used gel chips.

Figure 6 | Running an immunoblot assay. Pt electrodes submerged in PDMS gasket reservoirs. Eight input voltage lines fixed on the microscope stage for voltage program control of separation, horizontal dimension transfer and blot.

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41| Mix perchloric acid and hydrogen peroxide at a 2:1 volume ratio in a glass container. Using Teflon tweezers, place the chips into the glass container housing the dissolution mixture. crItIcal step Load the chips into solution one by one to ensure that each chip is thoroughly soaked. ! cautIon Always handle solution in a fume hood with personal protec-tive equipment, including face shield, lowered hood sash, impact-resistant chemical safety goggles, acid-resistant lab apron, lab coat and appropriate gloves (i.e., nitrile only—long gauntlet suggested). A perchloric acid spill kit should be nearby for emergencies.

42| Place the dissolution mixture container on a hot plate in a fume hood and incubate overnight at 75 °C, as shown in Figure 7b. The dissolution mixture can be reused for 1 week and has no observed impact on the glass channel surface and geometry. ! cautIon Keep the container cap loose, as this dissolution process produces gas that can build in the container and cause a pressurized explosion.? troublesHootInG

43| After dissolution, take the glass chips out with Teflon tweezers. Wash each glass chip thoroughly with water and repeat Steps 5 and 6 to clean the microchannel surface. The chips are ready for the next gel photopatterning.

? troublesHootInGTroubleshooting advice can be found in table 2.

table 2 | Troubleshooting table.

step problem possible reason solution

22 There are bubbles trapped at the outlets of channels or in chamber

Air bubbles were intro-duced when exchanging the precursor solution

Fill all reservoirs with precursor solution, then gently apply vacuum at one reservoir (e.g., reservoir 3) while replenishing precursor solution in all other reservoirs. Repeat with suction at reservoirs 1 and 2

Cannot exchange loading gel precursor solution in injection channel

Undesired polymerization formed at the junction

If the junction is clogged, use dissolution protocol to regenerate glass chip. Ensure good alignment of chip on the mask to avoid this undesired polymerization during patterning

26 Bubbles are visible in the chamber or channels after flood exposure and gel polymerization

Incomplete degassing during gel precursor solution preparation

Fabrication of the gels should commence in either a new chip or a chip regenerated by the described regeneration protocol. In the next fabrication cycle, carefully degas the solution with sonication and evacuation of the precursor vials. Ensure that the precursor solutions are at RT before preparation

29 PDMS mixture always spills out of the plastic cup once vacuum is applied

An abrupt, strong vacuum causes PDMS to ‘boil over’ the side of the cup

Gently establish vacuum by turning on the vacuum line by only a few turns, then turn vacuum off. Repeat this switching several times

a b

Figure 7 | Glass microfluidic chip regeneration. (a) Glass immunoblot chip stored in water tube. (b) Gel dissolution in solution of perchloric acid and hydrogen peroxide (2:1 volume ratio) at 75 °C for glass chip regeneration.

(continued)

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● tIMInGSteps 1–4, Design and fabrication of immunoblot chips: 4–10 dSteps 5–7, Glass channel surface treatment: 0.5–1 hSteps 8–13, Gel precursor solution preparation: 0.5–1 hSteps 14–26, Photopatterning of three functional gel regions: 0.5 hSteps 27–30, Fabrication of PDMS sheet: 2 hSteps 31 and 32, PDMS manifold preparation: 10 minSteps 33–39, On-chip immunoblotting: < 0.5 hSteps 40–43, Glass chip recycling: ~20 h

antIcIpateD resultsCompletion of the protocol yields rapid on-chip target protein immunoblotting, as shown in Figure 8. This automated native immunoblotting was completed within 5 min and identified free prostate–specific antigen (fPSA) in an extracted purified human seminal fluid sample. Results were validated by conventional slab-gel western blotting following a standard west-ern blotting protocol43 (Fig. 8c). In the on-chip protein blotting assay, mapping protein mobility (determined by PAGE) to blotting affinity (determined in blotting region) is critical to develop and complete a successful immunoblot. Therefore, two performance metrics are key to achieving the desired results. First, the PAGE SR (i.e., along the PAGE separation axis) must be optimized for a user’s sample and for measurement needs. Here we use a discontinuous or ‘stacking’ gel36 by fabricating a large pore size 3%T loading gel adjacent to a smaller pore size 6%T separation gel. The sample stacking (here in a homo-geneous buffer system) allows us to achieve an average SR = 1.5 in 30 s within 1 mm of separation length (even for native samples) (Fig. 8a). On the basis of the sample, you may need different separation gel architectures (including gradient gels) to achieve optimal separation performance. Second, conserving the PAGE separation resolution during electrophoretic trans-fer and blotting is critical. As described previously, arrays of field control channels are suggested to obtain a uniform electric field distribution in the microchamber used here (Fig. 8b).

By monitoring the separation, transfer and in-gel blotting stages for a prelabeled sample within one field of view, image analysis can be effectively used to assess transfer efficiency and reproducibility. This format provides the opportunity

32 PDMS manifold does not anneal to glass surface

PDMS surface was contaminated

Clean the PDMS manifold by soaking in a pure acetone solution with sonication for 3 min. Only use clean tweezers to handle manifold

Both glass and PDMS surfaces are not fully dry

Blow off the PDMS and glass surfaces with nitrogen gas for 5 min

35 Injected sample plug is tailing

Insufficient ‘pull back’ voltage was applied at reservoirs 2 and 3

Adjust voltages on reservoirs 1, 3 and 8 to obtain the desired injection plug shape

Injected sample plug does not move well into the separation chamber

‘Pull back’ voltage is greater than separation voltage applied on reservoir 8

Apply a pull back voltage on reservoirs 2 and 3 that ensures the pull back voltage is smaller than the separation voltage on reser-voir 8. Use CCD monitoring to assess injections and do not transfer peaks to blotting region until an ideal separation is achieved

37 Proteins are blocked at loading and separation gel interface

The pore size of the separation gel is too small relative to the protein sample

Adjust the %T in the precursor solution to be sufficient for the protein size range in your sample

Sample bands do not migrate in vertical direction and transfer in horizontal direction

The output Pt electrodes did not connect with reservoirs properly Pore size of the blotting gel is too small

Check if there is leakage between buffer reservoirs. Check for air bubbles in buffer reservoirs; bubbles can disrupt the electrical circuit. Adjust the %T in precursor of blotting gel to increase the gel pore size

42 PA gels are not dissolved The PA gels may have dried or bubbles may be trapped in the microchannel

If possible, carefully use high pressure to push bubbles out. Ensure each chip is thoroughly soaked and makes contact with dissolution solution. Increase the dissolution time

table 2 | Troubleshooting table (continued).

step problem possible reason solution

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to detect unlabeled antigen samples by running unlabeled samples together with fluorescently labeled protein standards, followed by post-blot introduction of labeled detection antibodies through side channels. Our design paradigm yields an adaptable analytical platform having relevance to all categories of immunoblotting, including western, Southern, northern and eastern blotting. The approach reported here forms the basis for a new approach to microfluidic protein assays.

11. Song, S., Singh, A.K., & Kirby, B.J. Electrophoretic concentration of proteins at laser-patterned nanoporous membranes in microchips. Anal. Chem. 76, 4589–4592 (2004).

12. Hatch, A.V., Herr, A.E., Throckmorton, D.J., Brennan, J.S. & Singh, A.K. Integrated preconcentration SDS-PAGE of proteins in microchips using photopatterned cross-linked polyacrylamide gels. Anal. Chem. 78, 4976–4984 (2006).

13. Herr, A.E. et al. Microfluidic immunoassays as rapid saliva-based clinical diagnostics. Proc. Natl Acad. Sci. USA 104, 5268–5273 (2007).

14. Peterson, D.S. Solid supports for micro analytical systems. Lab. Chip. 5, 132–139 (2005).

15. Svec, F. My favorite materials: porous polymer monoliths. J. Sep. Sci. 32, 3–9 (2009).

16. Schägger, H. Tricine-SDS-PAGE. Nat. Protoc. 1, 16–22 (2006).17. Wittig, I., Braun, H.P. & Schägger, H. Blue native PAGE. Nat. Protoc. 1,

418–428 (2006).18. Kinoshita, E., Kinoshita-Kikuta, E. & Koike, T. Separation and detection of

large phosphoproteins using Phos-tag SDS-PAGE. Nat. Protoc. 4, 1513–1521 (2009).

19. Stead, J.A. & McDowall, K.J. Two-dimensional gel electrophoresis for identifying proteins that bind DNA or RNA. Nat. Protoc. 2, 1839–1848 (2007).

20. He, M. & Herr, A.E. Microfluidic polyacrylamide gel electrophoresis with in-situ immunoblotting for native protein analysis. Anal. Chem. 81, 8177–8184 (2009).

21. Fan, A.C. et al. Nanofluidic proteomic assay for serial analysis of oncoprotein activation in clinical specimens. Nat. Med. 15, 566–571 (2009).

22. He, M. & Herr, A.E. Polyacrylamide gel photopatterning enables automated protein immunoblotting in a two-dimensional microdevice. J. Am. Chem. Soc. 132, 2512–2513 (2010).

23. Kurien, B.T. & Scofield, R.H. Protein Blotting and Detection: Methods and Protocols 1–33 (Humana Press, New York, USA, 2009).

24. Yamada, M., Mao, P., Fu, J. & Han, J. Rapid quantification of disease-marker proteins using continuous-flow immunoseparation in a nanosieve fluidic device. Anal. Chem. 81, 7067–7074 (2009).

25. Fu, J., Mao, P. & Han, J. Artificial molecular sieves and filters: a new paradigm for biomolecule separation. Trends Biotech. 26, 311–320 (2008).

26. Han, J. & Craighead, H.G. Separation of long DNA molecules in a microfabricated entropic trap array. Science 288, 1026–1029 (2000).

Sample load

11 s

1.0

mm

16 s 26 s

36 s 46 sSR = 1.52

Stack SeparationSeparate Transfer Blot & readout

Slab gel

1.0

mm

80 m

m

65 s

fPSA *

1

2

3

a b c

Figure 8 | Immunoblot assay results. (a) Fluorescent PSA sample loading, stacking and separation in immunoblot chip with minimum distortion (inverted grayscale CCD images, pixel size 6.45 µm × 6.45 µm, ×4 objective, NA 0.13). The ‘i ’ indicates the current flow direction. A 1-mm scale indication is provided. Representative images were collected at 11, 16 and 26 s after assay initiation. At PAGE completion (26 s), the separation resolution (SR) between the fastest two peaks is 1.52. (b) On-chip automated immunoblotting of native fPSA extracted from human seminal fluid. Inverted grayscale CCD images (pixel size 6.45 µm × 6.45 µm, ×10 objective NA 0.3) show the PA gel patterned chamber with three functional regions for separation, transfer and blot. Sample was labeled with Alexa Fluor 488 dye and used at a concentration of ~500 nM. The three representative images were collected at elapsed assay times of 36, 46 and 65 s. The dotted lines and arrows are visual aids to assist with spatial mapping of separation peak position to blotted peak position. A 1-mm scale indication is provided. (c) Gold standard slab-gel native immunoblotting confirms on-chip data (the fPSA band is indicated by an asterisk (*)). Left lane: PAGE slab gel separation (8-mm separation length). Right lane: PVDF membrane blot. Sample was labeled with Alexa Fluor 488 dye for native PAGE. The blot was detected by an HRP chromogenic method. Images were collected by Molecular Imager ChemiDoc XRS + System with filter (520DF30, 62 mm for fluorescent emission). Reprinted with permission from the Journal of the American Chemical Society; copyright 2010 American Chemical Society.

Note: Supplementary information is available via the HTML version of this article.

acknoWleDGMents We acknowledge financial support from the University of California, Berkeley and the QB3/Rogers Family Foundation Award. Facilities and equipment support from UC Berkeley’s Biomolecular Nanofabrication Center is also appreciated. PSA samples were a generous gift from D. Peehl, Stanford University. A.E.H. is an Alfred P. Sloan Foundation Research Fellow in chemistry.

autHor contrIbutIons M.H. designed chips and conducted experiments. M.H. and A.E.H. analyzed data and wrote the paper.

coMpetInG FInancIal Interests The authors declare no competing financial interests.

Published online at http://www.natureprotocols.com/. Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/.

1. Whitesides, G.M. The origins and the future of microfluidics. Nature 442, 368–373 (2006).

2. El-Ali, J.E., Sorger, P.K. & Jensen, K.F. Cells on chips. Nature 442, 403–411 (2006).

3. Craighead, H. Future lab-on-a-chip technologies for integrating individual molecules. Nature 442, 387–393 (2006).

4. Janasek, D., Franzke, J. & Manz, A. Scaling and the design of miniaturized chemical-analysis systems. Nature 442, 374–380 (2006).

5. Eijkel, J.C.T. & van den Berg, A. Nanofluidics: what is it and what can we expect from it? Microfluid. Nanofluid. 1, 249–267 (2005).

6. Yager, P. et al. Microfluidic diagnostic technologies for global public health. Nature 442, 412–418 (2006).

7. Thorsen, T., Maerkl, S.J. & Quake, S.R. Microfluidic large-scale integration. Science 298, 580–584 (2002).

8. Huang, B. et al. Counting low-copy number proteins in a single cell. Science 315, 81–84 (2007).

9. Liu, J., Yang, S., Lee, C.S. & DeVoe, D. Polyacrylamide gel plugs enabling 2-D microfluidic protein separations via isoelectric focusing and multiplexed sodium dodecyl sulfate gel electrophoresis. Electrophoresis 29, 2241–2250 (2008).

10. Das, C., Zhang, J., Denslow, N.D. & Fan, Z.H. Integration of isoelectric focusing with multi-channel gel electrophoresis by using microfluidic pseudo-valves. Lab. Chip. 7, 1806–1812 (2007).

Page 13: Published online 28 October 2010; doi:10.1038/nprot.2010 · 2010-10-28 · far western blotting for protein interactions and complexes, Southern blotting for detection of specific

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1856 | VOL.5 NO.11 | 2010 | nature protocols

27. Fu, J. et al. A patterned anisotropic nanofluidic sieving structure for continuous-flow separation of DNA and proteins. Nat. Nanotech. 2, 121–128 (2007).

28. Huang, L.R. et al. A DNA prism for high-speed continuous fractionation of large NDA molecules. Nat. Biotech. 20, 1048–1051 (2002).

29. Wirth, M.J. Separation media for microchips. Anal. Chem. 79, 800–808 (2007).

30. Zeng, Y., He, M. & Harrison, D.J. Microfluidic self-patterning of large-scale crystalline nanoarrays for high-throughput continuous DNA fractionation. Angew. Chem. Int. Ed. 47, 6388–6391 (2008).

31. Fu, J., Mao, P. & Han, J. Continuous-flow bioseparation using microfabricated anisotropic nanofluidic sieving structures. Nat. Protoc. 4, 1681–1698 (2009).

32. Xiong, L., Zhang, R. & Regnier, F.E. Potential of silica monolithic columns in peptide separations. J. Chromatogr. A 1030, 187–194 (2004).

33. Awada, C., Sato, T. & Takao, T. Affinity-trap polyacrylamide gel electrophoresis: a novel method of capturing specific proteins by electro-transfer. Anal. Chem. 82, 755–761 (2010).

34. Herr, A.E., Throckmorton, D.J., Davenport, A.A. & Singh, A.K. On-chip native gel electrophoresis-based immunoassays for tetanus antibody and toxin. Anal. Chem. 77, 585–590 (2005).

35. Herr, A.E. & Singh, A.K. Photopolymerized cross-linked polyacrylamide gels for on-chip protein sizing. Anal. Chem. 76, 4727–4733 (2004).

36. Hou, C. & Herr, A.E. Ultra-short separation length homogeneous electrophoretic immunoassays using on-chip discontinuous polyacrylamide gels. Anal. Chem. 82, 3343–3351 (2010).

37. Jemere, A.B., Oleschuk, R.D. & Harrison, D.J. Microchip-based capillary electrochromatography using packed beds. Electrophoresis 24, 3018–3025 (2003).

38. Oleschuk, R.D., Shultz-Lockyear, L.L., Ning, Y. & Harrison, D.J. Trapping of bead based reagents within microfluidic systems: on-chip solid-phase extraction and electrochromatography. Anal. Chem. 72, 585–590 (2000).

39. Yu, C., Xu, M., Svec, F. & Fréchet, J.M.J. Preparation of monolithic polymers with controlled porous properties for microfluidic chip

applications using photoinitiated free-radical polymerization. J. Polym. Sci. Part A Polym. Chem. 40, 755–769 (2002).

40. Le Gac, S.L., Carlier, J., Camart, J.C., Olivé, C.C. & Rolando, C. Monoliths for microfluidic devices in proteomics. J. Chromatogr. B 808, 3–14 (2004).

41. Song, S., Singh, A.K., Shepodd, T.J. & Kirby, B.J. Microchip dialysis of proteins using in situ photopatterned nanoporous polymer membranes. Anal. Chem. 76, 2367–2373 (2004).

42. Towbin, H., Staehelin, T. & Gordon, J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl Acad. Sci. USA 76, 4350–4354 (1979).

43. Wu, Y., Li, Q. & Chen, X.Z. Detecting protein-protein interactions by Far western blotting. Nat. Protoc. 2, 3278–3284 (2007).

44. Ciaccio, M.F., Wagner, J.P., Chuu, C.P., Lauffenburger, D.A., & Jones, R.B. Systems analysis of EGF receptor signaling dynamics with microwestern arrays. Nat. Methods 7, 148–155 (2010).

45. Kolli, M., Hamidouche, M., Bouaouadja, N. & Fantozzi, G., et al. HF etching effect on sandblasted soda-lime glass properties. J. Eur. Ceram. Soc. 29, 2697–2704 (2009).

46. He, Q. et al. Fabrication of 1D nanofluidic channels on glass substrate by wet etching and room-temperature bonding. Anal. Chim. Acta. 628, 1–8 (2008).

47. Iliescu, C., Chen, B. & Miao, J. On the wet etching of Pyrex glass. Sens. Actuators A 143, 154–161 (2008).

48. Lerch, M.A. & Jacobson, S.C. Electrokinetic fluid control in two-dimensional planar microfluidic devices. Anal. Chem. 79, 7485–7491 (2007).

49. Huang, L.R. et al. Generation of large-area tunable uniform electric fields in microfluidics arrays for rapid DNA separation. Tech. Dig. Int. El. Devices Meet. 1, 363–366 (2001).

50. Das, C., Fredrickson, C.K., Xia, Z. & Fan, Z.H. Device fabrication and integration with photodefinable microvalves for protein separation. Sensor Actuat. A Phys. 134, 271–277 (2007).

51. Sensabaugh, G.F. & Blake, E.T. Seminal plasma protein p30: simplified purification and evidence for identity with prostate-specific antigen. J. Urol. 144, 1523–1526 (1990).

52. Collins, T.J. ImageJ for microscopy. Biotechniques 43, S25–S30 (2007).