Dual Click reactions to micropattern proteins

Article abstract of DOI:10.1039/c1sm05991b

In the study described in this report orthogonal Click reactions were utilized to immobilize two different proteins on surfaces side-by-side and in multilayer constructs. Alkyne- and azide-functionalized poly(ethylene glycol) hydrogel features were fabricated. Copper-catalyzed Huisgen 1,3 dipolar cycloaddition and oxime chemistry were employed to conjugate an azide-functionalized ubiquitin and oxoamide-modified myoglobin, respectively. Multicomponent patterning was verified by fluorescence imaging.

Full text of DOI:10.1039/c1sm05991b

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PAPER
Dual Click reactions to micropattern proteins
ab
bc
ab
bc
ab
Rebecca M. Broyer, Eric Schopf, Christopher M. Kolodziej, Yong Chen and Heather D. Maynard*
Received 27th May 2011, Accepted 9th August 2011
DOI: 10.1039/c1sm05991b
In the study described in this report orthogonal Click reactions were utilized to immobilize two
different proteins on surfaces side-by-side and in multilayer constructs. Alkyne- and azide-
functionalized poly(ethylene glycol) hydrogel features were fabricated. Copper-catalyzed Huisgen 1,3
dipolar cycloaddition and oxime chemistry were employed to conjugate an azide-functionalized
ubiquitin and oxoamide-modified myoglobin, respectively. Multicomponent patterning was verified by
fluorescence imaging.
Introduction
Huisgen 1,3-dipolar cycloaddition reactions have also been
27
utilized to anchor biomolecules to surfaces. Azide functional-
ized peptides have been immobilized on alkynyl self assembled
28
Reactions such as the Cu(I) catalyzed Huisgen 1,3-dipolar
1,2
3
cycloaddition and oxime bond formation have been increas-
ingly used by researchers interested in melding biological and
synthetic molecules. So-called Click reactions are known to
proceed with high selectivity in benign solvents, tolerate a wide
range of functional groups, and produce products in high-
monolayers via Cu catalyzed triazole formation. Microwave
assisted azide/alkyne Click reactions have been reported to
29
produce carbohydrate/oligonucleotide chips. Green fluorescent
protein labeled with an azide at the C-terminus was immobilized
30
on alkyne functionalized agarose beads. In another report,
4,5
yields. A number of studies have utilized Huisgen 1,3-dipolar
2
00 nm patterns of poly(propargyl methacrylate) were produced
cycloaddition or oxime chemistry to create conjugates of
6–13
biomolecules such as sugars, DNA, proteins and even cells.
31
by e-beam lithography. An azide-functionalized coumarin dye
was used as a model system to Click to the patterns. The authors
then demonstrated immobilization of an azido-biotin via triazole
formation. These patterns were visualized with streptavidin-
coated quantum dots.
Herein, we report application of multiple Click reactions to
produce protein microarrays on surfaces. The proteins were
immobilized either side-by-side or in multilayer constructs using
both oxime chemistry and copper-catalyzed cycloaddition. To
our knowledge this is the first time dual Click reactions have been
used for this purpose.
Although proteins do not naturally contain the required
groups for oxime or copper catalyzed Click reactions, the
required moieties are readily added by a variety of methods.
Introduction of azides and alkynes into proteins has been
Oxime chemistry, the reaction between O-hydroxylamines and
ketones or aldehydes, has been shown to be effective for protein
32–36
37–39
accomplished by recombinant
and chemical techniques.
1
4–26
conjugation to surfaces.
For example, we prepared nano-
For example, recently, the diazo transfer reagent, imidazole-1-
40
sulfonyl azide hydrochloride was shown to successfully place
metre and micron-sized patterns by immobilizing a-oxoamide-
or levulinate-modified proteins onto patterns of aminooxy
azides at lysine side chains and the N-termini of horseradish
39
peroxidase and the red fluorescent protein DsRed. We
20,25
groups
Francis conjugated antibodies using oxime chemistry.
Meijer employed aniline-catalyzed oxime bond formation to
employed this strategy to produce azide-functionalized ubiquitin
for reaction with alkyne-functionalized surfaces. Aminooxy
groups or reactive carbonyls have also been incorporated into
26
ligate proteins to surfaces. Importantly, their results showed
that this method of site selective conjugation results in proteins
with significantly higher activities than non-specific coupling
using amidation chemistry. Retention of bioactivity of proteins
on surfaces is critical for application of these materials, and
typically activities are greatly reduced for proteins where the
immobilization chemistry is not site specific.
41
proteins. In this work, we exploited a transamination reaction
in particular that places a-oxoamide groups at the N-termini of
proteins to modify myoglobin for reaction with O-hydroxyl-
amine features.
Micron-sized features containing aminooxy or alkyne groups
were fabricated utilizing electron-beam (e-beam) lithography.
This system is ideal for biomolecule patterning because PEG is
a
Department of Chemistry and Biochemistry, University of California, Los
Angeles, California, 90095-1569, USA. E-mail: maynard@chem.ucla.edu
known to be protein resistant, which is important in producing
42–44
b
California Nanosystems Institute, University of California, Los Angeles,
California, 90095-1569, USA
patterns that minimize non-specific binding.
Furthermore,
c
different patterns and shapes, precise alignments and three
dimensional arrangements can be achieved using e-beam
Department of Mechanical and Aerospace Engineering, University of
California, Los Angeles, California, 90095-1569, USA
9
972 | Soft Matter, 2011, 7, 9972–9977
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2
5
lithography. We and others have demonstrated that e-beam
and silicon as substrate (substrate thickness: 1000mm, n1 ¼ 3.859,
k1 ¼ 0.016). The measurement was then repeated on the same
wafer after spin coating the polymer. The polymer layer was
fitted using values for the previously obtained silicon oxide
thickness and an additional chauchy layer model (n1 ¼ 1.45,
k1 ¼ 0.01). A minimum of 15 measurements were performed at 3
different locations and the values were then averaged.
lithography patterning of reactive polymers is effective for
25,45–48
fabricating patterns of proteins.
In this report we demon-
strate that iterative e-beam lithography patterning, coupled with
orthogonal Click reactions is effective to place two different
proteins on surfaces (Scheme 1).
Experimental
Materials
2
,5-Dioxopyrrolidin-1-yl pent-4-ynoate
8
-Arm amine terminated graft PEG was purchased from NOF
To a flame dried round bottom flask containing 100 mL of
freshly distilled methylene chloride (DCM) was added N-
hydroxysuccinimide (NHS, 1.0 g, 8.7 mmol, 1 eq), pentynoic acid
Corporation (Tokyo, Japan). 8-Arm aminooxy terminated PEG
25
was synthesized as previously described.
a-Ketoamide
41
myoglobin was synthesized as previously described. Alexa
Flour488 azide and all Alexa Flour secondary antibodies were
purchased from Invitrogen (Carlsbad, CA). Goat anti-
myoglobin antibody was purchased from Genetex (San Antonio,
TX). Mouse anti-Ubiquitin antibody was purchased from
Abcam (Cambridge, MA). All other reagents were purchased
from Sigma Aldrich (St. Louis, MO) or Fisher Biotech (Pitts-
burgh, PA) and utilized as received unless otherwise indicated.
(
0.94 g, 9.6 mmol, 1.1 eq), triethylamine (1.4 g, 14 mmol, 1.6 eq),
and 4-dimethylaminopyridine (DMAP) (0.11 g, 0.87 mmol, 0.1
eq). The resulting solution was stirred for 30 min before addition
of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, 1.8 g,
ꢀ
9
.6 mmol, 1.1 eq). The reaction was stirred for 4 h at 22 C, and
the solvent was removed in vacuo to give a crude product which
was subjected to column chromatography (100% DCM, R 0.4).
f
This product was further purified by recrystallization from 1 : 4
ethyl acetate : hexanes and sublimation at 0.2 Torr to obtain 2,
5
-dioxopyrrolidin-1-yl pent-4-ynoate as a white solid (0.69g, 40%
Instrumentation
1
yield). H NMR (500 MHz; CDCl ) d (ppm): 2.90–2.84 (bm, 5H,
3
1
13
H NMR, C NMR, and DEPT NMR were acquired on either
COCH
2
+ NHS), 2.62 (dt, 2H, J ¼ 7.5, 2.6 Hz, CH
2 2
CH C), 2.05
a Bruker ARX 500 MHz or 400 MHz spectrometer. Infrared
spectroscopy was recorded using a Perkin-Elmer SpectrumOne
Fourier Transfer IR (FTIR). UV-Visible (Vis) spectroscopy
analysis was performed with a BioMate 5 Spectrophotometer
13
t, 1H, J ¼ 2.6 Hz, CCH); C (400 MHz; CDCl
(
3
) d (ppm): 169.1,
13
67.2, 81.0, 70.2, 30.4, 25.7, 14.2; DEPT-135 C (400 MHz;
1
CDCl
3
) d (ppm): 69.9(-), 30.18(+), 25.5(+), 14.0(+) ppm. FTIR
ꢁ1
nmax/cm : 3304, 2948, 2258, 1815, 1787, 1734, 1430, 1415, 1371,
(
Thermo Spectronic Instruments). MALDI-TOF Mass spectra
1
203, 1085, 1067, 993, 906.
were acquired using an Applied Biosystems Voyager-DE-STR
MALDI-TOF instrument. AFM Images were obtained on
a Veeco Dimension 5000 Scanning Probe Microscope in tapping
mode equipped with a silicon AFM probe (BS-Tap300, Budg-
etSensors). For fluorescence visualization, a Zeiss Axiovert 200
fluorescent microscope equipped with an AxioCam MRm
monochrome camera was used. Pictures were acquired and
processed using AxioVision LE 4.1. Fluorescence intensity was
measured using NIH Image and signal to noise ratio was deter-
mined as (signal – background)/standard deviation of back-
ground. Ellipsometry was conducted on a Gaertner LSE stokes
ellipsometer. The spin coated polymer layer thicknesses were
measured with a HeNe laser at a single wavelenght of 632.8nm
and a 70 degree angle. The original Gaertner GEMP software
was used to fit the layer in the following manner: First the silicon
oxide on the piranah cleaned silicon wafer was measured and
fitted using the refractive index of Palik (n1 ¼.54264, k1 ¼ 0.00)
Synthesis of alkyne PEG
8-Arm amine-terminated PEG was added (20 mg mL ) to
ꢁ1
ꢁ
1
a solution of 2,5-dioxopyrrolidin-1-yl pent-4-ynoate (5 mg mL )
ꢀ
in CH
Cl
2 2
and allowed to react for 1 h at 23 C. The solvent was
removed in vacuo. The crude product was then re-dissolved in
Milli-Q water and purified by centrifugal filtration (3,000 MW
1
cutoff) followed by lyophilization to isolate the pure product. H
NMR (500 MHz, CDCl
1H), 3.69–3.58 (m, PEG peaks), 3.39 (m, CH
2H), 2.52 (dt, J ¼ 7.4 Hz, 2.6 Hz, CH CH C end group 2H), 2.38
3
) d (ppm): 6.48 (s, CONH end group
2
CH NH end group
2
2
2
(t, J ¼ 7.4 CH CH C end group 2H), 2.00 (t, J ¼ 2.6 Hz, CCH
2
2
end group 1H). Conversion ¼ 93% by comparison of the inte-
grations of the alkyne end group with the CH NH peak of the
2
2
unmodified PEG.
Scheme 1 Protein patterning by multiple Click reactions. Aminooxy and alkyne PEGs are patterned next to each other by e-beam lithography.
a-Ketoamide-myoglobin followed by azide-modified ubiquitin are then conjugated to the surface.
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Synthesis of imidazole-1-sulfonyl azide hydrochloride
Protein conjugation and pattern visualization
Synthesis of imidazole-1-sulfonyl chloride proceeded in 62%
Alkyne patterns. Patterns were incubated with azido-ubiquitin
ꢁ1
40
yield following a literature procudure. The product was isolated
1
as colorless needles. H NMR (500 MHz, D
(35 mg mL ) and 5 mM CuBr dissolved in 1 : 39 EtOH:Milli-Q
ꢀ
2
O) d (ppm): 9.34
water for 3 h at 23 C. After rinsing by gently squirting with PBS,
13
(
1H), 7.99 (1H), 7.57 (1H). C NMR (500 MHz, D O) d (ppm):
2
the bound protein was stained with a mouse anti-ubiquitin anti-
ꢁ1
ꢁ1
1
2
1
37.6, 123.1, 120.0 FTIR nmax/cm : 3104, 3059, 2499, 2425,
169,1913, 1582, 1508, 1425, 1298, 1229, 1189, 1159, 1137, 1103,
068, 982, 967.
body (50 mg mL in PBS) for 1h and AlexaFluorꢀ 488 anti-mouse
ꢁ1
secondary antibody (20 mg mL in PBS) for 30 min. Samples were
rinsed with PBS after each incubation step. Patterns were visual-
ized with fluorescence microscopy. Signal to noise 37 : 1.
Controls. As the first control, propargyl amine was first
Clicked to azido-ubiquitin and then the protein was incubated
with the alkyne surface using identical conjugation conditions as
above. As a second control, unmodified ubiquitin was subjected
to Click conditions with the alkyne patterns. In both cases, no
patterns were visualized.
Preparation of azide-ubiquitin
Ubiquitin was modified with two azides by reaction with aqueous
diazo transfer agent, imidazole-1-sulfonyl azide hydrochloride,
39
following a literature procedure (CAUTION!). To a solution of
ꢁ
1
ubiquitin in Milli-Q water (200 mL, 2.5 mg mL ) was added
ꢁ
1
imidazole-1-sulfonyl azide hydrochloride (85 mL, 2.0 mg mL ),
ꢁ1
K
2
CO
3
(100 mL, 2.0 mg mL ), and Cu(II)SO
4
ꢂH
2
O (25 mL,
ꢀ
Aminooxy patterns. The substrate was incubated with a solu-
ꢁ1
ꢁ1
1
.0 mg mL ). The solution was allowed to react for 24 h at 4 C.
tion of a-ketoamide myoglobin (10 mg mL in PBS, 25 mM, pH
6
Subsequent purification by centrifugal filtration (3,000 MWCO)
gave the azide-modified ubiquitin. The solution was diluted to
.5) for 1 h. After rinsing by gently squirting with PBS, the bound
protein was then stained with a goat anti-myoglobin antibody
ꢁ1
2
50 mL. Mass spectrum (MALDI-TOF) calcd for two transfers
based on 8635.51 observed for unreacted ubiquitin): 8687.51
(
10 mg mL in PBS) for 1h and AlexaFluorꢀ 568 anti-goat
(
ꢁ1
secondary antibody (20 mg mL in PBS) for 30 min. Samples
were rinsed with PBS after each incubation step. Patterns were
visualized using fluorescence microscopy. Signal-to-noise 33 : 1.
m/z; observed: 8695.02 m/z.
Pattern formation
Control. The protein was pre-incubated with O-methoxyamine
hydrochloride to consume the reactive carbonyl and then sub-
jected to identical staining procedures. Fluorescent patterns were
not observed.
A 1% or 2% (w/v) solution was prepared in methanol for the
aminooxy- and alkyne-functionalized polymers, respectively.
Silicon wafer chips were prepared by cleaning in Piranha (3 : 1
H SO :H O , CAUTION!) for 5 to 10 min, followed by a 10 s
2
4
2
2
Alkyne and aminooxy patterns. Patterns were first stained using
a-oxoamide modified myoglobin as described above, followed by
staining with azido-ubiquitin using the procedure described above.
rinse in Millipore H O, and blow drying with high pressure air.
2
1
5 ml of the polymer solution was then spincoated at 4000 rpm
for 20 s. The resulting aminooxy-PEG films were approximately
8 nm thick, and the alkyne-PEG films 40 nm thick by ellips-
4
ometry measurements. Patterning was accomplished by electron
beam exposure-induced crosslinking in a modified JEOL 5910
scanning electron microscope. Patterns were designed using a JC
Nabity e-beam lithographic system (Nanometre Pattern Gener-
ation System, version 9.0). An acceleration voltage of 30 kV was
used, with a beam current of approximately 5 pA. Aminooxy
Results and discussion
Poly(ethylene glycol) PEGs have been shown to cross-link to the
4
9
surfaces of either the native oxide of silicon or a PEG film when
45,48
exposed to focused electron beams.
This procedure results in
the formation of surface-bound hydrogels in the desired patterns.
ꢁ
2
patterns were written with a 40 mC cm area dose and alkyne
The crosslinking process is thought to occur via a radical
49
mechanism. End-functionalized polymers form features that
ꢁ2
patterns with a 20 mC cm area dose. The writing time was 6 s for
the aminooxy features 1 s for the alkyne features. Following
pattern writing, samples were removed from the vacuum and
immediately rinsed with methanol for 10 s followed by 10 s with
bear that functionality. In the work described herein, this process
was employed to produce aminooxy-functionalized and alkyne-
functionalized micropatterns. PEGs with chain ends bearing
aminooxy groups or alkynes were designed. The end groups
would result in features with the desired Click partners, and eight
arm stars were employed rather than linear polymers in order to
achieve a higher density of surface functionality. PEG was used
so that the polymer could be cross-linked to the surface and so
that nonspecific binding would be minimized.
2
Millipore H O.
Multiple patterns. For pattern alignment, gold alignment
reference marks were first fabricated on the silicon wafer by
25
conventional photolithography as described previously.
Following the procedure above, aminooxy polymer patterns
were generated. After pattern development, the alkyne end-
functionalized PEG was spincoated on top of the aminooxy
features and patterned as described above in the appropriate
position, according to the reference marks. AFM images were
taken, and the samples were used directly for conjugation of
proteins.
The aminooxy polymer was synthesized by a Mitunobu reac-
tion of 8-arm PEG with hydroxyphthalimide and subsequent
cleavage using hydrazine (Scheme 2). The 8-arm end function-
alized alkyne PEG was prepared by first synthesizing 2,5-
dioxopyrrolidin-1-yl pent-4-ynoate (NHS-alkyne). This was
achieved by EDC coupling of NHS to pentynoic acid to afford
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Fig. 1 Features prepared by electron beam were modified with proteins.
(
a) Myoglobin with a N-terminal a-oxoamide attached to aminooxy-
PEG triangle patterns. (b) Azido-ubiquitin immobilized to square alkyne
patterns. Proteins were visualized by primary and secondary antibody
staining. Scale bar ¼ 5 mm.
Scheme 2 Synthesis of aminooxy-8-arm PEG.
ratios of 33 : 1 for myoglobin and 37 : 1 for ubiquitin). In both
cases, fluorescence was clearly visualized on the patterns (Fig. 1).
Myoglobin was stained red and ubiquitin green, and red triangles
and green squares were observed by fluorescence microscopy
(
Fig. 1).
Patterns were subjected to controls in which the proteins were
first incubated with a small molecule that would saturate the
reactive group on the protein. In the case of the a-ketoamide-
myoglobin, the protein was reacted with O-methoxyamine
hydrochloride, and in the case of azido-Ub the protein was
Clicked with propargyl amine. Another control was carried out
in which unmodified ubiquitin was incubated with patterns of
Scheme 3 Synthesis of 8-arm alkyne PEG.
8
-arm alkyne-functionalized PEGs under identical Click condi-
the activated ester alkyne in 40% yield. Subsequent reaction with
amine-terminated 8-arm PEG (Scheme 3) resulted in 93%
1
conversion of the end groups as determined by H NMR.
tions. In all cases, patterns were not observed, suggesting that the
proteins reacted with the surfaces via Click chemistries as
designed.
The polymers were then subjected to e-beam patterning to
produce aminooxy-functionalized PEG triangle shapes and
alkyne-functionalized PEG squares. A 1% or 2% (w/v) solution
in methanol of the aminooxy- or alkyne-functionalized polymer,
respectively, was spincoated onto a silicon wafer with a native
oxide. Pattern formation was optimized for each polymer
We next exploited e-beam lithography to pattern both types of
polymers side-by-side (Scheme 1). Two different types of
patterns were produced. First 1 mm ꢃ 1 mm features (with 1 mm
spacing) were designed in which alternating squares of alkyne
and aminooxy PEGs were precisely aligned and used to immo-
bilize ubiquitin and myoglobin, respectively. Green and red
alternating squares were observed by fluorescence (Fig. 2a)
indicating that the proteins were patterned as expected. In the
second pattern, an alkyne representation was drawn from the
alkyne PEG and the letter ‘‘A’’ was fabricated from the amino-
oxy PEG. AFM showed that the desired shapes had been
ꢁ
2
individually and found to occur at doses of 20 mC cm for
ꢁ2
alkyne-functionalized PEG and 40 mC cm for aminooxy-
functionalized PEG. At lower doses the polymer was
insufficiently crosslinked for hydrogel formation, whereas at
significantly higher doses irradiation-induced side-reactions
could lead to chain scission and degradation of the patterned
hydrogels. The uncrosslinked polymer was rinsed away by
washing with methanol, followed by water.
Proteins were then modified for Click immobilization. In this
0
case, the myoglobin was subjected to pyridoxal 5 -phosphate to
41
install an N-terminal a-ketoamide. This PLP transamination
50
was introduced by Francis and coworkers. This same trans-
formation can also be undertaken using the Dixon and Fields
20,41,51
synthesis.
Ubiquitin was transformed using a recently
39
reported diazo transfer reaction. This method employs imid-
azole-1-sulfonyl azide hydrochloride to introduce azides at the
amine side chains of lysine residues and at the N-terminus of
proteins. To this end, imidazole-1-sulfonyl azide hydrochloride
was synthesized in 62% yield and incubated with ubiquitin.
Ubiquitin was modified with an average of two azides according
to MALDI-TOF analysis.
Fig. 2 Dual patterns of aminooxy-PEG and alkyne-PEG cross-linked
next to each other by e-beam lithography. Myoglobin was conjugated to
the aminooxy patterns and Ubiquitin was conjugated to the alkyne
patterns. Proteins (myoglobin, red and ubiquitin, green) were visualized
by primary and secondary antibody staining. (a) Micron patterns of 1 ꢃ 1
mm dots (1 mm spaces) of myoglobin on aminooxy PEG and ubiquitin on
alkyne PEG patterns arranged into alternating squares. (b) Alkyne and
Subsequent reaction of the oxo-myoglobin with aminooxy
patterns in slightly acidic pH, and azido-ubiquitin (azido-Ub)
with the alkyne patterns in the presence of Cu(I) were undertaken
‘‘A’’ shaped polymer patterns were observed in the AFM height image
(
Scheme 1) The proteins were visualized by fluorescence after
taken in tapping mode. (c) Patterns are visualized by fluorescence
staining with primary and secondary antibodies (signal-to-noise
imaging. Scale bars 5 mm.
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Soft Matter, 2011, 7, 9972–9977 | 9975

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prepared (Fig. 2b). Staining of these features revealed green
fluorescent ubiquitin and red fluorescent myoglobin on the
respective features as expected (Fig. 2c). Quantification of
adsorbed protein based on signal-to-noise ratios showed low
levels of cross-reactivity compared to the single protein experi-
ments (3 : 1 for myoglobin staining on alkyne features and 8 : 1
for ubiquitin staining on aminooxy features).
Click chemistry is an effective means to conjugate proteins to
surfaces and multiple, orthogonal Click reactions provide an
efficient route to surfaces where more than one type of biomol-
ecule is desired. We employed myoglobin and ubiquitin as model
systems. Yet the diazo transfer agent utilized herein, as well as
the transamination reaction employed, provide facile means to
modify proteins with Click partners; thus the number of possible
combinations of proteins that could be envisioned is large. In
addition, although two Click reactions were utilized, this
approach should be amenable to other Click reactions such as
thiol-ene, potentially increasing the number of different proteins
that could be simultaneously immobilized on a surface.
Since e-beam lithography has high alignment capabilities, we
next demonstrated that mutilayer patterns could be prepared.
These were achieved by precisely aligning the chip in order to
pattern four 1 ꢃ 1 mm or sixteen 500 ꢃ 500 nm features of
alkyne-PEG on top of 5 ꢃ 5 mm aminooxy-PEG boxes (Fig. 3a).
After protein immobilization and visualization (Fig. 3c and d),
the fluorescence overlay clearly showed the co-localization of the
fluorescence in the desired features (Fig. 3b), thus demonstrating
the ability to prepare multilayer constructs.
Conclusions
Herein, we described use of e-beam lithography to create
micropatterns of a protein reactive scaffold of 8-arm aminooxy
PEG and 8-arm alkyne PEG. A facile azide modification of
ubiquitin utilizing an aqueous diazo transfer agent allowed
immobilization of the azo-protein to alkyne PEG patterns via
a Huisgen 1,3-dipolar cycloaddition. Myoglobin that had been
subjected to a PLP transamination reaction was immobilized on
aminooxy-PEG features. Iterative conjugation of the proteins to
surfaces either side-by-side or in multilayer constructs produced
multicomponent features. The proteins were subsequently visu-
alized by fluorescent antibody staining.
Taken together the data demonstrate that dual Click reactions
coupled with e-beam lithography is effective for producing
multicomponent protein arrays. The results also show that side-
by-side and multilayer constructs can be prepared. Several of the
positive features of e-beam lithography were exploited, namely
the ability to pattern different features and shapes, as well as high
alignment capability to produce three dimensional constructs.
While several shapes and configurations were studied here,
a myriad of designs are possible utilizing this technique. This
should be useful for applications in which topography and
chemistry of the system must be precisely spatially controlled. In
addition, since with e-beam lithography smaller features are
easily accessed, nanoscale patterns should be possible.
Acknowledgements
This research was supported by the National Science Foundation
(
CAREER Award CHE-0645793) and through SINAM (DMI-
327077). RMB thanks the Department of Chemistry and
0
Biochemistry at the University of California, Los Angeles, for
a Departmental Fellowship. The authors thank Rock Mancini
for synthesizing 2,5-dioxopyrrolidin-1-yl pent-4-ynoate,
Professor Bruce Dunn for use of his ellipsometer, and Uland Lau
for performing ellipsometry measurements on the unpatterned
PEG films.
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Fig. 3 Dual multilayer Click protein patterns. Aminooxy PEG was first
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