protocol

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. (aeh@berkeley.edu).

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.
© 2010 Nature America, Inc. All rights reserved.

INTRODUCTION
Microfluidic 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,

1844 | VOL.5 NO.11 | 2010 | nature protocols


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 structures
Recently, 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
© 2010 Nature America, Inc. All rights reserved.
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 materials
Polymer 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.
protocol
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 immunoblotting
Conventional 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 design
Figure 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
nature protocols | VOL.5 NO.11 | 2010 | 1845
 protocol
Glass chips resistance of the chamber49. Ideally, the smaller the dimensions of the
side channels and the greater the number of channels in each array,
Glass chip
the more uniform the resulting electrical field within the chamber.
In practice, arrays of ~10 channels of the given dimensions provide
Steps 1–4 sufficient control for this chip layout and application. The exact chip
geometry is provided in Supplementary Figures 1–3.
PA gel photopatterning Glass chip recycling
Glass chip
PA gel photopatterning. Spatial control of PA gel properties pro-
PA gel dissolution vides a design and fabrication tool important for integrating the
Photomask UV light under heating
assay steps needed to complete protein blotting. The PA gel regions
(sample loading, PAGE separation and immunoblotting) were all
Steps 5–26 ×4 objective Steps 40–43
co-located in one microfluidic chamber through our development
and use of multistage photopatterning (Fig. 3). The PA gel pho-
Puncher PDMS
sheet
topatterning mask was designed using AutoCAD and printed on
transparency film by a commercial high-resolution printing service
Glass
chip (FineLine Imaging). As an alternative, a high-resolution commer-
Steps 27–32 Steps 33–39
cial printer is sufficient to generate economical transparency mask
Fluidic interface Automated blotting
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
© 2010 Nature America, Inc. All rights reserved.

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.
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 a 1
2 3
chamber was chosen to be compatible with Sample
injector
CCD-based imaging on epifluorescence 4 5
Loading
microscopy systems. Here 14 parallel side
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
b
Load & stack sample PAGE Transfer Blot
Separation

Blotting

channels were used abutting the chamber;

each channel was ~20 µm deep, ~10 µm wide 6 7

and ~4 mm long (Fig. 2a; Supplementary i i i i i

Fig. 1). Microchannel arrays were designed Chamber

8 Channel
array
to yield uniform electric fields over the large
chamber area during separation and trans- Figure 2 | Immunoblot chip design and operation. (a) Schematic design of the immunoblot chip for
fer steps in both the vertical and horizontal analysis of native proteins. The sample (2), sample waste (3), buffer (1, 4, 5, 6) and buffer waste
dimensions48,49. The channel arrays connect (7, 8) reservoirs are indicated in sketch (not to scale). The microchamber houses a large-pore-size
the microchamber to buffer reservoirs (as 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
indicated in Fig. 2a, numbered 1–8) wherein
assay protocol is overlaid on micrographs of the microchamber showing the gel regions. The on-chip
voltages are applied (Step 36; Table 1). The immunoblot protocol includes sample loading, PAGE separation, transfer and in gel blotting. The blue
resistance of the channel arrays (parallel) and yellow bands in each panel schematically represent unique protein peaks as they migrate in the
is designed to be larger than the sheet direction of the electric current ‘i ’. Scale bar, 300 µm.

1846 | VOL.5 NO.11 | 2010 | nature protocols

 protocol

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

Voltage and current program (operation Reservoir numbers as indicated in Figure 2a

durationa) 1 2 3 4 5 6 7 8

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

2 Injection and separation (30–60 s) 0V 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 0V 50 V 0 µA

a
Duration of each step presented is typical; actual durations will vary depending on sample analyzed.

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,
© 2010 Nature America, Inc. All rights reserved.
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

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 a b
effects such as nonspecific binding and/or
size exclusion at any PA gel boundary. Our
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.
6%T, Ab 6%T 3%T
Glass chip
chamber

model sample, prostate-specific antigen

(PSA), was extracted from human seminal
plasma as described previously51. The PSA Photomask
UV UV UV
sample was fluorescently conjugated with
Define antibody functionalized Define separation Define loading gel region with
Alexa Fluor 488 dye to produce a detect- blotting region gel region stacking interface
able fluorescence signal. All samples are
Figure 3 | Photopatterning of gels in the immunoblot chip chamber. (a) Epifluorescence microscope
stored at 4 °C in the dark until use. A 5 µl with a transparency film mask for high-resolution photopatterning. (b) Illustration of three-step
PSA sample (~500 nM) is pipetted into the regional photopatterning to unify the loading gel, PAGE separation gel and blotting gel into
sample reservoir (Fig. 2a, reservoir 2) for one microchamber for automated on-chip immunoblotting. %T indicates acrylamide monomer
the on-chip native protein blot. concentration in the gel precursor solution. ‘Ab’ indicates that biotinylated antibody is present.

nature protocols | VOL.5 NO.11 | 2010 | 1847

 protocol
MATERIALS
REAGENTS
• 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
© 2010 Nature America, Inc. All rights reserved.
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 blotting
• PVDF/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)
EQUIPMENT
• Epifluorescence 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.
PROCEDURE
Design and fabrication of immunoblot chips ● TIMING 4–10 d
1| 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.
1848 | VOL.5 NO.11 | 2010 | nature protocols
• ×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 intensity
• Ultraviolet light meter (MANNIX UV340; Mannix Instrument, 290–390 nm)
• Filtered mercury lamp (B100-AP; UVP, 300–380 nm) with cooling fan
• Corning hot plate with digital display (Corning, cat. no. 6795-600D)
• Bath sonicator (Bransonic 220; Branson Ultrasonics)
• Vacuum line and nitrogen gas line
• Computer 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.
 protocol

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 h

5| 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.
© 2010 Nature America, Inc. All rights reserved.

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.
a Vacuum b Precursor solution

 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 c d Vacuum
Precursor
solution
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, HEC drops
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. Figure 4 | Gel precursor solution preparation. (a) Degassing in sonicator
bath under house vacuum. (b) Loading gel precursor into microchannel by
 CRITICAL STEP Carefully degas for at least 5 min with capillary force. (c) HEC drops applied on chip reservoirs with filled precursor
agitation and degassing until no bubbles are visually solution. (d) Precursor exchange by gently applying vacuum to one reservoir
observed in the microcentrifuge tube or solution. and replenishing precursor solution in the rest of the reservoirs.

nature protocols | VOL.5 NO.11 | 2010 | 1849

 protocol

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
© 2010 Nature America, Inc. All rights reserved.

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.

1850 | VOL.5 NO.11 | 2010 | nature protocols

 protocol

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 h

27| 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.
© 2010 Nature America, Inc. All rights reserved.

 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.
a
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 b c

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

nature protocols | VOL.5 NO.11 | 2010 | 1851

 protocol

On-chip immunoblotting ● TIMING  < 0.5 h

33| 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 Figure 6 | Running an immunoblot assay. Pt electrodes submerged in PDMS
supply. Input the voltage sequence program into the gasket reservoirs. Eight input voltage lines fixed on the microscope stage
control software, including the sample loading step, separation for voltage program control of separation, horizontal dimension transfer
step and transfer to blotting step. The voltages at each and blot.
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.
© 2010 Nature America, Inc. All rights reserved.

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 h

40| 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.

1852 | VOL.5 NO.11 | 2010 | nature protocols

 protocol

41| Mix perchloric acid and hydrogen

peroxide at a 2:1 volume ratio in a a b
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 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
emergencies.
© 2010 Nature America, Inc. All rights reserved.

glass chip regeneration.

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.

? TROUBLESHOOTING
Troubleshooting 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
Cannot exchange loading
gel precursor solution in
injection channel
26 Bubbles are visible in the
chamber or channels after
flood exposure and gel
polymerization
29 PDMS mixture always spills
out of the plastic cup once
vacuum is applied
Air bubbles were intro-
duced when exchanging
the precursor solution
Undesired polymerization
formed at the junction
Incomplete degassing
during gel precursor
solution preparation
An abrupt, strong vacuum
causes PDMS to ‘boil over’
the side of the cup
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
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
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
Gently establish vacuum by turning on the vacuum line by only
a few turns, then turn vacuum off. Repeat this switching several
times
(continued)

nature protocols | VOL.5 NO.11 | 2010 | 1853

 protocol

Table 2 | Troubleshooting table (continued).

Step Problem Possible reason Solution

32 PDMS manifold does not PDMS surface was
anneal to glass surface contaminated
Both glass and PDMS
surfaces are not fully dry
35 Injected sample plug is Insufficient ‘pull back’
tailing voltage was applied at
reservoirs 2 and 3
Clean the PDMS manifold by soaking in a pure acetone solution
with sonication for 3 min. Only use clean tweezers to handle
manifold
Blow off the PDMS and glass surfaces with nitrogen gas for 5 min
Adjust voltages on reservoirs 1, 3 and 8 to obtain the desired
injection plug shape

Injected sample plug does ‘Pull back’ voltage is Apply a pull back voltage on reservoirs 2 and 3 that ensures the
not move well into the greater than separation pull back voltage is smaller than the separation voltage on reser-
separation chamber voltage applied on voir 8. Use CCD monitoring to assess injections and do not transfer
reservoir 8 peaks to blotting region until an ideal separation is achieved

37 Proteins are blocked at The pore size of the Adjust the %T in the precursor solution to be sufficient for the
© 2010 Nature America, Inc. All rights reserved.

loading and separation gel separation gel is too small protein size range in your sample
interface relative to the protein 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 If possible, carefully use high pressure to push bubbles out. Ensure
or bubbles may be trapped each chip is thoroughly soaked and makes contact with dissolution
in the microchannel solution. Increase the dissolution time

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

ANTICIPATED RESULTS
Completion 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

1854 | VOL.5 NO.11 | 2010 | nature protocols

 protocol

a Sample load Stack Separation

b Separate Transfer Blot & readout
c Slab gel

11 s 16 s fPSA *

80 mm
26 s

1.0 mm
2
1.0 mm

SR = 1.52 36 s 46 s 65 s 3

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.
© 2010 Nature America, Inc. All rights reserved.

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.

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/. 17. Wittig, I., Braun, H.P. & Schägger, H. Blue native PAGE. Nat. Protoc. 1,
Reprints and permissions information is available online at http://npg.nature.
com/reprintsandpermissions/.
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