Diels-Alder reaction for the selective immobilization of protein to electroactive self-assembled monolayers

Full text of DOI:10.1021/ja983529t

4286
J. Am. Chem. Soc. 1999, 121, 4286-4287
Diels-Alder Reaction for the Selective
in current over consecutive cycles (although with different rates),
but the structurally related cyclopentane and cyclopentene had
no effect over the same number of scans. Immersion of a
monolayer presenting HQ groups in electrolyte containing cp (15
mM) for 20 min had no effect on the voltammograms, demon-
strating that only the Q underwent reaction with cp. Grazing angle
FTIR spectroscopy further supported the reaction of Q with cp.
A monolayer presenting Q groups showed a carbonyl stretching
Immobilization of Protein to Electroactive
Self-Assembled Monolayers
Muhammad N. Yousaf and Milan Mrksich*
Department of Chemistry, The UniVersity of Chicago
Chicago, Illinois 60637
ReceiVed October 5, 1998
-1
mode at 1663 cm . After reaction with cp, this band was absent,
-1
and a band at 1653 cm corresponding to the carbonyl stretching
mode of the D-A adduct was observed.
The immobilization of biologically active molecules is impor-
tant for preparing substrates used in diagnostic assays, high-
throughput drug discovery, and attached cell culture. A wide
1
Figure 3 shows a plot for the loss in peak current for the
reduction of Q versus time for the data shown in Figure 2. Because
the concentration of cp was much greater than that of immobilized
Q, the data could be fit to an exponential decay to obtain a pseudo-
first-order rate constant, k′:
variety of methods are available, and the choice for a particular
application depends on the relative importance of many factors,
including selectivity and efficiency of the coupling reaction, the
stability of the resulting complex, and the suitability for preparing
patterned arrays of ligands. No single method meets all of these
criteria. The complex of streptavidin with biotin is overall the
best available method, but it has the limitations that the complex
is large and intrusive, and nonspecific adsorption of the protein
-k′t
I ) I + (I - I ) exp
t
f
o
f
where I
and I is the residual nonfaradaic current. The excellent fit of the
t o
is the peak current at time t, I is the initial peak current
2
can be problematic. Many chemical methods, including the
f
condensation of amines with activated carboxylic acids or with
aldehydes, are convenient and applicable to most molecules but
are limited by a lack of selectivity.3 Others, including the
coordination of Ni(II) complexes with oligo(histidine) motifs, have
excellent selectivity but often lack long-term stability.3 In this
paper we use self-assembled monolayers (SAMs) that present a
quinone group to demonstrate that the Diels-Alder (D-A)
reaction of this group with cyclopentadiene (cp) is an excellent
method for immobilization. This design also permits the reactivity
of the quinone to be modulated either chemically or electrochemi-
cally, by way of reduction to the hydroquinone which does not
participate in the D-A reaction.
experimental data to this equation indicates that the Q groups
are sufficiently isolated on the monolayer that the reactivity is
independent of the extent of reaction. We next repeated this
experiment with concentrations of cp ranging from 0.76 to 58
mM and in all cases found that the loss in peak current was
described well by an exponential decay and that the reaction
always proceeded to completion. The pseudo-first-order rate
constants increased linearly with the concentration of diene: the
slope of the best-fit line (Figure 3, inset) provided a second-order
a,b
c
-
1
-1
rate constant of kDA ) 0.26 M s , after adjustment for the
fraction of time that Q was present. It is striking that this
interfacial reaction is kinetically well-behaved.8
We first investigated the kinetic behavior for the D-A reaction
of cp with quinone attached to a monolayer (Figure 1). Cyclic
voltammetry of a mixed SAM presenting hydroquinone (HQ) and
We used the association of streptavidin to immobilized biotin
as a model system with which to demonstrate the D-A-mediated
9
immobilization of protein. To avoid problems with nonspecific
4
hydroxyl groups (øΗQ ) 0.25) showed that the HQ undergoes
protein adsorption, we instead used monolayers prepared from
oxidation at 220 mV to give the quinone (Q) and reduction at
alkanethiolates terminated in tetra(ethylene glycol) groups (1),
150 mV.5 Voltammograms over 50 consecutive cycles were
,6
10
-
which resist entirely the adsorption of protein, and an extended
linker to the HQ (2).11 Monolayers having ø
) 0.05 were
indistinguishable and showed the oxidation was reversible. When
cp was added to the electrolyte, consecutive voltammograms
showed a decrease in the peak current for both reduction and
oxidation (Figure 2). Several observations confirm that the loss
2
immersed in an aqueous solution of 1,4-benzoquinone for 10 min
to oxidize the HQ groups to the Q.12 These substrates were then
treated with a conjugate of cp and biotin (3, 10 mM in 1:1 THF:
7
in current was due to the D-A reaction of cp with Q. The
2
H O) for 2 h to immobilize biotin. We used surface plasmon
addition of other dienes (including cyclohexadiene and 1-cyclo-
pentadienyl methyl acetate) to the electrolyte gave similar losses
resonance (SPR) spectroscopy to characterize the binding of
streptavidin to the immobilized biotin.13 Figure 4 shows that
streptavidin associated with a monolayer to which biotin was
coupled and that this association was irreversible. When strepta-
vidin was presaturated with biotin (0.8 mM), the binding of the
*
To whom correspondence should be addressed.
(
1) Schuna, M.; Heller, R. A.; Theriault, T. P.; Konrad, K.; Lachenmeier,
E.; Davis, R. W. Trends Biotechnol. 1998, 16, 301-306. Mrksich, M. Curr.
Opin. Colloid Interface Sci. 1997, 2, 83-88. Hermanson, G. T. Bioconjugate
Techniques; Academic Press: New York, 1996.
(8) The solution reaction of 1-methyl-benzoquinone with cp in 1:1 THF:
(
2) Green, N. M. Methods Enzymol. 1990, 184, 51-67.
2
H O proceeded at least 300 times faster than did the interfacial reaction. We
(
3) (a) Wilbur, D. S.; Hamlin, D. K.; Pathare, P. M.; Weerawarna, S. A.
are exploring the basis for the difference in rates.
Bioconjugate Chem. 1997, 8, 572-584. (b) Horton, R. C.; Herne, T. M.; Myles,
D. C. J. Am. Chem. Soc. 1997, 119, 12980-12981. (c) Sigal, G. B.; Bamdad,
C.; Barberis, A. A.; Strominger, J.; Whitesides, G. M. Anal. Chem. 1996, 98,
(9) Spinke, J.; Liley, M.; Guder, H. J.; Angermaier, L.; Knoll, W. Langmuir
1993, 9, 1821-1825. Spinke, J.; Liley, M.; Schmitt, F. J.; Guder, H. J.;
Angermaier, L.; Knoll, W. J. Chem. Phys. 1993, 99, 7012-7019.
(10) Mrksich, M.; Whitesides, G. M. In Chemistry and Biological Ap-
plications of Polyethylene Glycol; ACS Symposium Series 680, American
Chemical Society: Washington, DC, 1997; p 361 and references therein.
(11) Alkanethiols 1 and 2 were synthesized in three and six steps,
respectively. Conjugate 3 was synthesized in seven steps. All intermediates
4
90-497.
(
4) øΗQ is the fraction of alkanethiolates in the monolayer that present HQ
and is determined by integrating the waves in cyclic voltammograms of the
monolayer.
(5) For previous reports of the electrochemistry of SAMs presenting HQ
1
groups, see: Hickman, J. J.; Ofer, D.; Laibinis, P. E.; Whitesides, G. M.;
Wrighton, M. S. Science 1991, 252, 688-691. Ye, S.; Yashiro, A.; Sato, Y.;
Uosaki, K. J. Chem. Soc., Faraday Trans. 1996, 92, 3813-3821.
gave satisfactory H NMR spectra. Details will be described in a subsequent
full report.
(12) We used a chemical oxidant because the instrument for SPR is not
compatible with electrochemistry. The ability to use chemical oxidants and
reductants to interconvert the HQ and Q also expands the utility of this method
to nonconducting substrates.
(
6) Cyclic voltammetry was performed with a Bioanalytical Systems CV-
5
0W potentiostat using a cell with the gold/SAM as the working electrode,
platinum wire as the counter electrode, and Ag/AgCl as the reference electrode.
(7) The tautomerization of the D-A adduct, which would yield a redox-
(13) For examples of the use of SPR to measure the association of proteins
with monolayers, see: Mrksich, M.; Grunwell, J. R.; Whitesides, G. M. J.
Am. Chem. Soc. 1995, 117, 12009-12010. Houseman, B. T.; Mrksich, M.
Angew. Chem., Int. Ed. 1999, 38, 782-785.
active quinone, requires strongly acidic or basic conditions and does not
proceed under the conditions employed here. See: Meinwald, J.; Wiley: G.
A. J. Am. Chem. Soc. 1958, 80, 3667-3670.
1
0.1021/ja983529t CCC: $18.00 © 1999 American Chemical Society
Published on Web 04/14/1999

Communications to the Editor
J. Am. Chem. Soc., Vol. 121, No. 17, 1999 4287
Figure 1. Reversible electrochemical oxidation of a monolayer that presents the HQ group affords a monolayer that presents the Q group, which can
in turn react with cp to give the D-A adduct. The figure shows only one possible product. Other products can arise through sigmatropic isomerization
of the cp prior to D-A reaction.
Figure 3. A plot of the peak current for reduction of immobilized Q
versus time (I is the peak current at time t and I is the peak current for
t o
the first scan). The loss of current is due to conversion of the Q to the
D-A adduct, which is not redox active. The data were fit to an
exponential decay (see text) to provide a pseudo-first-order rate constant
Figure 2. Consecutive cyclic voltammograms of a monolayer presenting
HQ groups (øΗQ ) 0.25) and hydroxyl groups in 1:1 THF:H O containing
Na PO (2mM), NaCl (75 mM), and cp (7.6 mM) at a scan rate of 25
mV/s.
2
2
4
(k′). The inset shows the relationship of k′ and concentration of cp. The
slope of the best-fit line gives the second-order rate constant (kDA).
protein to the surface was prevented and demonstrated that the
association was biospecific. Furthermore, streptavidin did not
immobilize to monolayers that presented either Q or HQ groups
mixed with glycol groups.14
The D-A reaction described here provides an attractive and
flexible method for bio-immobilization and is especially well-
suited for tailoring monolayers with peptides, carbohydrates, and
other low-molecular weight ligands.15 Because the reaction is
kinetically well-behaved, this method can be used to quantitatively
attach groups in low densities (<1%) where the direct determi-
nation of density is not straightforward. This method also allows
for the sequential immobilization of several ligands to a common
substrate, with independent control over the density of each ligand.
This chemistry further makes possible a class of dynamic
substrates for attached cell culture, wherein the immobilization
of biologically active ligands can be turned on at discrete times.
Figure 4. Data from SPR for the immobilization of streptavidin to a
monolayer presenting tetra(ethylene glycol) groups and biotin (see text
for details). A solution of PBS was flowed over the monolayer for 5 min
and replaced with a solution of streptavidin (5 µg/mL) for 10 min and
then with PBS for 10 min. The addition of biotin to the buffer containing
streptavidin prevented immobilization (biotin/EG
did not immobilize to monolayers presenting quinone and glycol groups
(Q/EG ) or glycol groups alone (EG ). The data are reported as the shift
4
+ biotin). Streptavidin
4
4
in resonance angle (θ). An increase in θ of 0.10° corresponds to a density
2
of adsorbed protein of 1 ng/mm .
16
range of tailored substrates for studies in cell biology and
applications in biotechnology.
This and related electroactive substrates will provide an exciting
(14) The magnitude of change in θ when streptavidin is flowed over the
Acknowledgment. We are grateful for support provided by the Searle
Scholars Program, the Camille and Henry Dreyfus Foundation (New
Faculty Award), and the National Science Foundation (CHE-9709039).
This work used facilities of the MRSEC supported by the National Science
Foundation (DMR-9400379).
monolayer presenting quinone and glycol groups is greater than that observed
with monolayers presenting only glycol groups and shows that the protein
adsorbs to the mixed monolayer. This adsorption does not result in im-
mobilization of streptavidin, since it is weak and rapidly reversible when the
protein-containing solution is replaced with buffer.
(15) SAMs are currently the best available class of model substrates for
studies in biointerfacial science. For reviews, see: Mrksich, M. Cell. Mol.
Life Sci. 1998, 54, 653-58. Mrksich, M.; Whitesides, G. M. Annu. ReV.
Biophys. Biomol. Struct. 1996, 25, 55-78.
JA983529T
(16) Hodneland, C. H.; Mrksich, M. Langmuir 1997, 13, 6001-6003.