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Research Article
Microchip isoelectric focusing withmonolithic immobilized pH gradientmaterials for proteins separation
Monolithic immobilized pH gradient (M-IPG) materials were prepared in microchannles
by photoinitiated polymerization of acrylamide, glycidylmethacrylate and Bis, followed by
the attachment of focused Ampholine onto the surface of porous monoliths via epoxide
groups. With M-IPG materials as matrix, FITC-labeled ribonuclease B, myoglobin and
a-casein were well separated by microchip isoelectric focusing (mCIEF) without carrier
amphocytes (CAs) added in the buffer. Both chemical and pressure mobilization were
applied to drive focused zones for LIF detection. Our experimental results showed that
pressure mobilization was preferable with neglectable band broadening, and good peak
shape and high detection sensitivity were obtained. All these results demonstrate that
mCIEF with M-IPG materials is not only an efficient mode for protein enrichment and
separation but also attractive to couple with other CE modes to achieve multi-dimen-
sional separation or MS for further identification, without the interference of mobile
CAs.
Keywords:
Microchip isoelectric focusing / Monolithic immobilized pH gradient materials /Photopolymerization / Proteins DOI 10.1002/elps.200900209
1 Introduction
As Hjerten and Zhu [1] transferred IEF from the conven-
tional slab gel format to CE in 1985, CIEF has been regarded
as a powerful tool for protein analysis [2–4], which has
advantages of high peak capacity, high resolution and high
sensitivity. In the past decades, with the rapid development
of m-TAS techniques, microchip isoelectric focusing (mCIEF)
has been paid much attention and shown potentials of high
throughput analysis [5–9]. Furthermore, it has been used as
the first dimension of microfluidic chip-based multi-
dimensional separation [10–15].
In mCIEF, carrier ampholytes (CAs) are usually added in
the running buffer to establish a stable pH gradient.
However, CAs are high concentration salt mixture, and their
existence in the running buffer might lead to increased
current and Joule heat during focusing. In addition, they
might also interfere with further multi-dimensional
separation and the identification by MS. Therefore, the
establishment of stable pH gradient without mobile CAs in
the running buffer has been paid much attention. Several
methods, such as diffusion of OH� and H1 between anode
and cathode [16–17], Joule heat-induced temperature gradi-
ent [18–20] and spatially varied surface electric field [21],
have been developed. However, the stability of formed pH
gradient should be further improved.
The immobilization of pH gradient on matrix is another
efficient method to generate satisfactory pH gradient for IEF
separation, which was first reported as the solid gel strip
[22], formed by casting polyacrylamide gel matrix with
covalently bonded acrylamido buffers (Immobilines) of
different pK. Sommer et al. [23] applied conventional
immobilized pH gradient gels to microchips by the diffu-
sion of Immobilines across the separation channel prior to
gel photopolymerization, and several fluorescent pI markers
and proteins were separated without the addition of CAs.
However, the microscale casting procedure is complicated.
In our previous study, monolithic immobilized pH
gradient (M-IPG) materials were prepared in capillaries to
perform CIEF without CAs added in the buffer, and good
resolution was achieved for proteins and peptides separation
[24–26]. In this paper, based on our previous work, we
Yu Liang1,2
Yongzheng Cong1,2
Zhen Liang1
Lihua Zhang1
Yukui Zhang1
1National Chromatographic R. &A. Center, Dalian Institute ofChemical Physics, ChineseAcademy of Sciences, Dalian,P. R. China
2Graduate School of the ChineseAcademy of Sciences, Beijing,P. R. China
Received March 30, 2009Revised July 29, 2009Accepted September 2, 2009
Abbreviations: AAm, acrylamide; AIBN, azobisisobutyronitrile;
CAs, carrier ampholytes; lCIEF, microchip isoelectric focusing;
GMA, glycidylmethacrylate; M-IPG, monolithic immobilizedpH gradient
Correspondence: Professor Lihua Zhang, Key Laboratory ofSeparation Science for Analytical Chemistry, National Chroma-tographic R. & A. Center, Dalian Institute of Chemical Physics,Chinese Academy of Sciences, 457 Zhongshan Road, Dalian116023, P. R. ChinaE-mail: [email protected]:186-411-84379779
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com
Electrophoresis 2009, 30, 4034–40394034
proposed a simple method to prepare M-IPG materials in
microchannels by photoinitiated polymerization of acrylamide
(AAm), glycidylmethacrylate (GMA) and Bis, followed by the
attachment of focused Ampholines onto porous monolith
surface via epoxide groups. With such a matrix, several FITC-
labeled proteins were separated by mCIEF with high resolu-
tion, high detection sensitivity and high throughput.
2 Materials and methods
2.1 Reagents and instrumentation
DMSO was obtained from Shenyang Chemical Reagent
Plant (Shenyang, China). AIBN was purchased from The
Fourth Shanghai Reagent Plant (Shanghai, China) and
recrystallized in our laboratory. AAm and Bis were ordered
from Acros Organics (NJ, USA). 1,4-Butanediol, dodecanol
and GMA were from Fluka (St. Gallen, Switzerland).
3-methacryloxypropyltrimethoxysiliane (98%), Ampholine
(pH 3.5–10.0), ribonuclease B (from bovine pancreas),
myoglobin (from equine) and a-casein (from bovine milk,
70%) were purchased from Sigma (St. Louis, MO, USA);
SephacrylTM
S-200 was bought from Amersham Phamacia
Biotech (Uppsala, Sweden).
XX-15A/F UV lamps were purchased from Spectronics
(NY, USA). Type SG2506 glass plates were purchased from
Shaoguang Microelectronics (Changsha, China). Fused-silica
capillaries (75 mm id, 365 mm od) were purchased from
Ruifeng Chromatographic Device (Yongnian, China). Intel-
ligent eight-path-high-voltage electric device was purchased
form Shandong Normal University (Jinan, China). Microchip
electrophoresis system with an LIF detector was ordered from
Zhejiang University (Hangzhou, China).
2.2 Fluorescent labeling of proteins
Each kind of protein (about 1 mg) was, respectively,
dissolved in 300 mL of 0.02 M phosphate buffer (pH 8.0),
and then 5 mL of 20 mg/mL FITC dissolved in DMSO was
added. The mixture was incubated overnight at room
temperature with continuous stirring. The labeled proteins
were purified by SephacrylTM
S-200 before analysis.
2.3 Microchip fabrication
A simple straight microchannel, 100 mm wide and 4.5 cm
long, as shown in Fig. 1A, was fabricated with Type SG2506
glass plates by photolithography, wet chemical etching and
room-temperature bonding, as described by Fang et al [27].
Briefly, a design on a photomask with microchannels was
transferred onto a glass substrate with chromium and
AZ1805 photoresist coating by UV exposure. Then the
microchannels were etched in a well-stirred bath containing
dilute HF/HNO3. After reservoirs were drilled, etched
substrate and cover plate were washed sequentially with
acetone, detergent and water with high flow rate. Subse-
quently, the plates were dried by heating, followed by being
soaked in concentrated sulfuric acid for 8–12 h. Finally, the
plates were washed with water, bonded and dried at room
temperature
2.4 Pre-treatment of microchannels
Before photopolymerization, the inner wall of microchannels
was vinylized to ensure the covalent attachment of monolith.
Then the channel was washed by HCl (0.1 mol/L) for 30 min,
water for 30 min, NaOH (0.1 mol/L) for 2 h, water for 30 min
Figure 1. Photograph of microchip with M-IPG materials (A) andSEM of M-IPG materials in microchannels (B).
Electrophoresis 2009, 30, 4034–4039 Microfluidics and Miniaturization 4035
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com
and methanol for 30 min, respectively, followed by drying
with N2 in an oven at 701C for 2 h. Subsequently, the channel
was filled with 50% v/v 3-methacryloxypropyltrimethoxysi-
lane in methanol, and kept at room temperature in the dark
for 24 h. Finally, the vinylized channel was washed with
methanol and dried by N2.
2.5 Preparation of M-IPG materials in microchannels
M-IPG materials were prepared in microchannels by
photoinitiated polymerization with various reaction solution
compositions, as shown in Table 1. The mixture was first
purged with N2 for 1 min to remove dissolved O2. Then the
microchannel was completely filled with the polymerization
mixture and sealed with sealing tapes. The microchip was
subsequently covered with a mask, with only the channel
exposed to UV light for 20 min in a black box, equipped with
two 365 nm, 15 W UV lamps with an overall intensity of
3790 mW/cm2 at a reaction distance of 10 cm. The black box
was kept at room temperature, and to avoid undesired
thermal polymerization, a fan was equipped to dissipate
heat.
After polymerization, the monolith was first washed
with ethanol and water, and then filled with 10% w/v
Ampholine. Simultaneously, the anodic and cathodic
reservoirs were filled with 0.020 mol/L H3PO4 and NaOH,
respectively. With 1600 V voltage applied, Ampholines were
migrated in microchannel to form a stable pH gradient by
electric focusing. Until the current was kept stable, buffers
in both reservoirs were changed to water, and sealed with
sealing tapes before kept in an oven at 601C for 24 h to
immobilize CAs. Finally, M-IPG materials in microchannels
were washed by water for 1 h, and ready for separation, as
shown in the amplified zone in Fig. 1A.
2.6 Operation of lCIEF
After the microchannel with M-IPG materials was filled
with FITC-labeled proteins solution, the anodic and cathodic
reservoirs were filled with 0.020 mol/L H3PO4 and NaOH,
respectively. With 1600 V voltage applied, the focusing
was performed until the current was kept stable. The
focused sample zones were subsequently mobilized
either by chemical or pressure mobilization, and then
detected by an LIF detector. Chemical mobilization was
achieved by replacing NaOH (catholyte) with H3PO4
(anolyte), and 2400 V voltage was applied. Hydrodynamic
mobilization was performed by the manual pressure
pump, which was connected to the anodic reservoir via a
75 mm id fused-silica capillary, fixed by a home-made
interface.
3 Results and discussion
3.1 Optimization of polymerization solution
Photopolymerization technique has been widely used to
prepare monoliths in microchips [28–29], since the reaction
can be finished within a very short time even at room
temperature. Furthermore, the monoliths can be prepared
within any required position by using a photo mask.
In this study, to prepare monolithic matrix, AAm, GMA
and Bis were chosen as monomers, and binary porogens,
including dodecanol and 1,4-butanediol were selected, and
the reaction procedure is shown in Fig. 2A However, the
optimal composition for polymerization initiated by heat in
capillaries [14] was found not suitable to prepare M-IPG
materials in microchannels. Therefore, the optimization of
polymerization solution was performed.
The ratio of monomers has effects on both morphology
and functionality of monolithic matrix. The increase of
GMA is favorable to immobilize more Ampholines via epoxy
groups to establish stable pH gradient, while AAm was
helpful to improve the hydrophilicity of matrix, thus to
reduce the non-specific absorption of samples. In addition,
to improve the matrix rigidity, there should be enough Bis
in the polymer solution. After the systematic optimization,
the final ratio of GMA, AAm and Bis was chosen as
24.76:21.90:53.33 w/w.
The types and ratios of porogens have great effects on
the porous structure of monoliths. In our experiments,
1,4-butanediol was chosen to improve the homogeneity, and
a long-chain aliphatic alcohol, dodecanol, was selected to
improve the penetrability of monolithic matrix. Further-
more, DMSO was used to improve the solubility of mono-
mers and porogens. The effect of the ratio of 1,4-butanediol
to dodecanol on the porous structure of monoliths
was investigated. With the ratio shown in column a of
Table 1, translucent monoliths with poor permeability were
obtained. However, with more dodecanol added, the
permeability of monoliths was improved, but the homo-
geneity and rigidity of matrix became poor, as shown in
columns c and d. Therefore, the optimized weight ratio of
1,4-butanediol to dodecanol was chosen as 1:1 w/w, shown
in column b.
Table 1. Compositions of the polymerization solution and
characteristic of monolithic matrix
a b c d
Compositions (g) GMA 0.0230 0.0230 0.0230 0.0230
AAm 0.0260 0.0260 0.0260 0.0260
Bis 0.0560 0.0560 0.0560 0.0560
DMSO 0.4585 0.4585 0.4585 0.4585
1,4-Butanediol 0.3059 0.2292 0.2192 0.2092
Dodecanol 0.1523 0.2293 0.2393 0.2492
AIBN 0.0010 0.0010 0.0010 0.0010
Characteristics Permeability Poor Good Good Good
Homogeneity Good Good Good Poor
Rigidity Good Good Poor Poor
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3.2 Immobilization of Ampholine
According to our previous work, with monolithic poly
(GMA-co-AAm-co-Bis) as matrix, EOF generated from epoxy
groups could be ignored under electric field [14]. Therefore,
Ampholines were focused under electric field in micro-
channels with M-IPG materials to form a steady pH
gradient before immobilization. The focusing time of
Ampholines might affect the formation of pH gradient.
On the one hand, insufficient time might affect the linearity
of formed pH gradient; on the other hand, overlong time
might result in the obvious drift of pH gradient. In our
study, after ca. 5 min, the focusing current for Ampholines
stopped decreasing, and 5 min was chosen as CAs focusing
time.
As Ampholines were a complex mixtures of oligoamino
and oligacarboxylic acids, the focused Ampholines could be
bonded onto the monolith directly through the reaction
between the epoxy and amino or carboxyl groups, as shown
in Fig. 2B. Although high concentration of Ampholines
could also fasten the reaction and increase the immobilized
amount of Ampholines, it might also result in increased
current and Joule heat during focusing. Consequently,
10% w/v Ampholine was chosen to generate the immobi-
lized pH gradient via epoxy groups on monolithic matrix.
Although high temperature could accelerate the immobili-
zation, the diffusion of Ampholines happened simulta-
neously, which might degrade the formed pH gradient.
Therefore, the optimized temperature for immobilization
was selected as 601C. After the reaction for 24 h, the mate-
rials in microchannels were washed by water for 1 h to flush
out the unbound Ampholines.
Figure 1B shows the SEM image of internal morphol-
ogies of M-IPG materials prepared in microchannels, which
are tightly attached to the inner wall of microchip via the
covalent interaction between the polymer and the vinylized
surface. In addition, good homogeneity and large porous
structure are observed, which ensure uniformed pH gradi-
ent and decreased non-specific interaction between samples
and matrix during focusing.
3.3 Protein separation by lCIEF with M-IPG
materials
To perform mCIEF with M-IPG materials, the addition of
CAs in the running buffer is not necessary, as Ampholines
are attached onto the monolith surface, and the long chains
of Ampholines have the properties of ‘‘free’’ oligoamino and
oligacarboxylic acids within a small region around the
immobilized points, acting in the same role as free
Ampholines added in running buffer.
According to the theory of CIEF, high voltage could
improve the separation resolution. However, too high
voltage could lead to increased Joule heating, resulting in
bubbles formed in channels of mCIEF. After systematic
study, 1600 V was selected as the optimal voltage for the IEF
separation of FITC-labeled ribonuclease B, myoglobin and
a-casein.
After the microchannel with M-IPG materials was filled
FITC-labeled proteins, 1600 V focusing voltage was applied,
and proteins were focused according to their pIs. Within
2 min, the current stopped dropping, which indicated that the
isoelectric focusing of FITC-labeled proteins was completed.
Figure 2. Procedure for M-IPGmaterials preparation.
Electrophoresis 2009, 30, 4034–4039 Microfluidics and Miniaturization 4037
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com
Both chemical and pressure mobilization were applied
to mobilize the focused protein zones to the LIF detection
point. For chemical mobilization, after replacing NaOH
(catholyte) with H3PO4 (anolyte), the focused a-casein
migrated toward the cathodic reservoir under the voltage of
2400 V. When the detection point was set just behind the
focused zone, a sharp peak was detected by LIF (Fig. 3A).
However, when the detection point was set far behind the
focused zone, the detected peak was broad (Fig. 3B), which
indicates that, by chemical immobilization, the focused
zones were broadened seriously in microchannels with
M-IPG materials due to the relatively long migration time.
In addition, pressure mobilization, performed by the
manual pressure pump via a home-made interface, was
applied as well to drive the focused zones in microchannels
with M-IPG materials. As shown in Fig. 4, ribonuclease B,
myoglobin and a-casein were well separated by mCIEF with
M-IPG materials. The sharp peaks indicated that peak
broadening could be suppressed with the existence of
monolithic matrix, and pressure mobilization is preferable
for such a separation mode. The reproducibility was also
studied. As the focused sample zones were driven by
manual pressure pump, and it was difficult to apply repea-
table pressure, the reproducibility of the migration time of
each peak was not quite good. However, the peak shape was
of good reproducibility, and the resolution of proteins was
repeatable. In two consecutive runs, the resolution for
ribonuclease B and myoglobin was, respectively, 5.7 and 6.5,
and that for myoglobin and a-casein were, respectively, 7.0
and 7.5.
Compared with the traditional IEF, CIEF or mCIEF,
mCIEF with M-IPG materials has several advantages for
proteins separation. Besides high throughput, the avoidance
of moving CAs in buffer could decrease Joule heat generated
in microchannels during focusing. In addition, the mono-
lithic matrix in microchannels could prevent anolyte or
catholyte buffer from flowing into the channel, which could
keep a stable pH gradient and prevent gradient compres-
sion. Furthermore, the diffusion and the drift of focused
zones during mobilization could also be minimized due to
the existence of monolithic matrix. Most importantly, mCIEF
with M-IPG materials could be used as the first-dimensional
separation in chip-based multi-dimensional analysis or
mCIEF-MS, since compared with the traditional mCIEF with
CAs added in the buffer, the immobilized CAs could not
interfere with further separation and detection. When stored
in water, M-IPG materials prepared in microchannels could
Figure 3. Effect of detection point on chemical immobilization offocused FITC-labeled a-casein (0.026 mg/mL) by mCIEF experi-mental conditions: IEF conditions: catholyte: NaOH (20 mmol/L),anolyte: H3PO4 (20 mmol/L); separation voltage: 1.6 kV; focusingtime: 2 min; Chemical mobilization conditions: catholyte: H3PO4(20 mmol/L), anolyte: H3PO4 (20 mmol/L); chemical mobilizationvoltage: 2.4 kV; the distance of detection point and cathodereservoir: (A) 11 mm and (B) 4 mm.
Figure 4. Separation of ribonuclease B (0.0075 mg/mL), myoglo-bin (0.022 mg/mL) and a-casein (0.026 mg/mL) by mCIEF withpressure mobilization. Experimental conditions: catholyte:NaOH (20 mmol/L), anolyte: H3PO4 (20 mmol/L); separationvoltage: 1.6 kV; focusing time: 2 min; the distance of detectionpoint and cathodic reservoir was 4 mm. Pressure mobilizationwas performed by the manual pressure pump via a home-madeinterface (described in Section 2.6).
Electrophoresis 2009, 30, 4034–40394038 Y. Liang et al.
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com
be used for at least 20 days, and applied for the separation of
polar compounds for over 25 runs.
4 Concluding remarks
M-IPG materials were prepared in microchannles by photo
initiation, and successfully applied for microfluidic chip-
based CIEF without CAs in buffer. The experimental results
indicated that with pressure mobilization, proteins could be
separated with high resolution, high sensitivity and high
throughput. Without mobile CAs in buffer, such a separa-
tion mode is promising in further application for microchip-
based multi-dimensional CE separation and even the
hyphenation with MS.
The authors are grateful for the financial support fromNational Natural Science Foundation (20775080), NationalBasic Research Program of China (2007CB714503 and2007CB914100) and Knowledge Innovation Program ofChinese Academy of Sciences (KJCX2YW.H09).
The authors have declared no conflict of interest.
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