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Direct Observation of Streptavidin Specifically Adsorbed on Biotin-Functionalized Self-Assembled Monolayers with the Scanning Tunneling Microscope.

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Direct Observation of Streptavidin Specifically
Adsorbed on Biotin-FunctionahzedSelf-Assembled
Monolayers with the Scanning Tunneling
Microscope**
By Lukas Haussling, Bruno Michel,* Helmut Ringsdorf,*
and Heinrich Rohrer
Molecular recognition, i.e. the specific and selective interaction between host and guest molecules, is of critical importance not only in biology, but also in materials science for the
molecular architecture of organized materials.['1
Scanning tunneling microscopy (STM) and related methods[2] facilitate direct investigation of adsorbed molespectrum of organic molecules can be
c u l e ~ . [ ~A
- ~broad
]
bound by functionalized chemisorbed monolayers to conducting surfaces for subsequent study with the STM.
Binding of biotin (vitamin H) by the bacterial protein
streptavidin is a model case of molecular recognition for the
following reasons: 1) The binding constant (K, =
Lmol-') is extremely high and very specific. 2) Biotin
can easily be functionalized and its binding properties are
only slightly influenced by this process.['] 3) Streptavidin has
four binding sites for biotin situated on two opposite sides of
the tetrameric protein. It can thus be used to link two different functions in the new molecular complex.
Since two-dimensional crystallization of fluorescencemarked streptavidin was recently achieved'" on biotinylated
lipid monolayers, we became interested in the analogous
experiment on so-called "self-assembled monolayers" (see
Scheme 1). Nuzzo and AlZara19a1as well as Sagidgblshowed
that such films adsorbed from isotropic solutions onto solid
supports are indeed monolayers with nearly perfect two di-
mensional organization. It was shown for the chemisorption
of thiols and disulfides onto gold surfaced"] that these selfassembled monolayers are well suited to functionalize metallic surfaces on a larger scale.
In order to perform the recognition reaction between
biotin and streptavidin on functionalized gold surfaces, we
synthesized the thiols 1 and 3, and disulfide 2.f"I 11-Mercaptoundecanol 1 was chemisorbed from a solution onto gold
(1 11),[13] and the contact angle measured (Table 1). Figure 1
shows the STM picture of a chemisorbed monolayer of 1 on
gold (1 11): a smooth surface with randomly distributed depression~['~]
(bare gold shows none of these depressions).
N
HS-OH
H
S
H
o
H
s
-HS-OH
HS-OH
H
S
HS%
- HA
HS-OH
HS-OH
HS-OH
H
S
O
H
S
-
N
0
N
N
N
N
N
0
Fig. 1. Monolayer of 1chemisorbed from a 2.1 x
M solution in ethanol on
gold (111); imaging conditions: tungsten tip, 200 mV and 100 PA. a) Three-dimensional image with simulated illumination. b) Top view of a) in gray-scale
scheme with a vertical dimension of 24 8,from black to white. c) Cross-section
along line AB with an additional monolayer of gold from C to D.
H
S
HS-OH
N
N
I
N
N
N
N
N
-
.-
O
-)H
N
6
N
N
N
Scheme I . Surface recognition on a mixed monolayer of 1 and 3 by streptavidin.
[*] Dr. B. Michel, Dr. H. Rohrer
[*'I
IBM Research Division, Zurich Research Laboratory
Saumerstrasse 4,CH-8803 Riischlikon (Switzerland)
Prof. H. Ringsdorf, Dipl. Chem. L. Haussling
Institut Organische Chemie der Universitat
J. J.-Becher-Weg20, W-6500 Mainz (FRG)
We thank Mrs. G . Bender for her enthusiastic help with the syntheses, and
Mr. H.-G. Bat2 (Boehringer, Mannheim) for a gift of streptavidin
Angew. Chem. Inl. Ed. Engl. 30 (1991) No. 5
The smooth parts cover approximately 95 % of the surface
(by STM analysis). The new organic surface exhibits the
same terraced structure (e.g., step height and sharpness) as
the uncovered gold films. The depressions have lateral dimensions of up to 35 A and a maximum depth of l l A (cf.
Fig. 1 b and c). We conclude that the gold substrate is covered by a densely packed monolayer of 1 except for the
depressions. These are either empty or contain loosely
packed molecules which yield laterally to the scanning tip.
We note further that the observed 95 % coverage also corresponds to the ratio of the area requirement of perpendicular
x
~uperlattice''~~
of sulparaffin chains to that of a
phur atoms on gold (11I), i.e., densely packed sulfur atoms
on the gold surface and a consequent loose packing of a
certain percentage of the hydrocarbon chains.
Following the same procedure, the bisbiotin disulfide 2
was adsorbed (see Table 1). The biotin-functionalized gold
surface was then dipped into the protein
still no
proteins were detectable with the STM, even after an incubation period of 12 hours.
Weber et aI.["I showed that the reason for the high binding constant of biotin to streptavidin is that the biotin molecule is completely engulfed by the protein. Therefore, streptavidin cannot bind tightly to a monolayer of 2 chemisorbed
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on gold, because of steric hindrance and limited mobility of
the densely packed biotin molecules. This steric hindrance
can be overcome by “diluting” the biotin functionalities on
the gold surface by means of coadsorption of an unfunctionalized thiol or disulfide. The mobility can be increased by the
introduction of a long hydrophilic spacer as with mercaptobiotin 3 (Scheme 1).
HS -OH
Y
1
o“c
NH
H H
d - ,
H
H
0
3
A N ? O
H H
-0
0
4
Table 1. Contact angles of water with chemisorbed thiols on gold(l11) surfaces
21.
Compound
Adsorption time [h]
1
14
5
18
2
3
21
24
113 2:l
113 50:l
Gold (111)
[a] adv
-
= advancing,
re
=
erdr[“I [a1
42
66
52
60
35
70-80
8,.
[“I [a]
7
25
20
receding
1
0 VCH
protein as well as areas covered with several layers; true
monolayer coverage was rarely found. The mobilities of both
the chemisorbed biotin and proteins docked to it are apparently rather small, causing lateral and vertical disorder of the
proteins.
18
8
38-50
Compound 3 was chemisorbed from pure solution and
mixtures with mercaptoundecanol 1 on gold (111) surfaces.
The monolayer of pure 3 is wetted sufficiently by water
(Table 1); a periodicity of 10 8, in one direction and 20 8, in
the perpendicular direction is visible in the STM (not
shown). Figure 2 shows a mixed monolayer obtained by
coadsorption from a solution of 1 and 3 (2:l). This monolayer exhibits marked depressions with lateral dimensions of
120 8, and a depth of approximately 3 8,. We associate this
with a difference in length of the two components, the depth
of the depressions indicating that the protruding spacer with
the biotin lies flat on the densely packed alkane layer. Occasionally, we observe a periodicity of 7.5 x 10 8, which is in
agreement with the length and width of the protruding hydrophilic part of 3. This, however, requires a much larger
ratio of 113 in the adsorbed monolayer than in the original
solution (i.e. 3 adsorbs slower than 1).
A mixed monolayer chemisorbed from a solution of 1 and
3 in the ratio 50:l was incubated with a buffered protein
solutionr161for 72 h at room temperature, subsequently removed and rinsed thoroughly with diluted buffer. The individual proteins are visible in the STM image (Fig. 3), es ecially in the profile along AB (Fig. 3 b). The width of 45 is
in accordance with values in the literature (42 x 42 x 56 A)[”]
(lattice constant 80 8,),11*1 We observed regions free of
570
Fig. 2. Chemisorbed monolayer of 3 and 1 (1 :2) on gold (111); imaging conditions as in Figure 1. a) Top view in gray-scale scheme with a vertical dimension
of 54 k, from black to white. b) Three-dimensional image of the zone marked
in a) with simulated illumination. c) Cross-section along line AB; B has an
additional monolayer of gold.
Verlagsgesellsehaft mbH, W-6940 Weinheim. 1991
Fig. 3. Streptavidin docked onto a chemisorbed monolayer consisting of 3 and
1 (150); imaging conditions as in Figure 1. a) Top view in gray-scale scheme
with a vertical dimension of 140 A from black to white. b) Cross-section along
line AB.
To demonstrate the effect of steric hindrance, lateral and
especially vertical mobility in the underlying self-assembled
monolayers, we spread the bisbiotin disulfide 2 on buffer
solution116]in a Langmuir trough. Figure 4 shows the pressure area diagram of this compound. We incubated these
monolayers with streptavidin at two surface pressures for
30-60 minutes at room temperature. The film of oriented
bisbiotinylated disulfide 2 and bound streptavidin was then
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- IrnN m-'I
0
100
A-
Fig. 4. Pressure area diagram of 2 at 20°C on water. A
cule, l7 = surface pressure.
= area
in 8, per mole-
transferred to the gold support by the Langmuir-Schafer
technique. Figure 5a shows the STM picture of the layer
obtained after incubating a monolayer of 2 for 30 minutes
with streptavidin at 1 mN m - l in the liquid analogous
phase. It shows terraces of about 3 8, in height caused by
steps in the gold substrate, and in the background a large
protein aggregate several hundred angstroms high (Fig. 5 b).
phase) during an incubation time of one hour. Here, however, small patches of proteins show tetragonal symmetry,
similar to the two-dimensional structures found with biotinylated lipid monolayers at the air-water interface.[*- *I
To compare the epifluorescence microscopy experiments
of Blankenburg et a1.t8. with the recognition reaction at
self-assembled monolayers on solid surfaces, we spread the
biotin lipid 4 on a buffer solution[16]and compressed it to
4 mN m- (solid analogue packing). Compound 4 forms
solid analogue packed monolayers and thus can be more
easily imaged by scanning tunneling microscopy. In addition
it could be shown by parallel monolayer experiments, that
this biotin lipid leads to many small protein domains at the
gas water interface, which are also more suitable for STM
experiments.
The subphase below this monolayer was then exchanged
by a solution of streptavidin (1 p ~ in) the same buffer. After
an hour at room temperature, this film was transferred to a
gold (111) surface by the Langmuir-Schafer technique and
investigated with STM. Figure 6 a shows the typical structure of the streptavidin aggregates, which have a two fold
symmetry, stretched shape and characteristic grooves in the
short sides. Most aggregates are 100-120 8, thick, corresponding to two layers of streptavidin; only few of the
protein aggregates are true monolayers, as demonstrated by
the trace of the line AB in Figure 6 b corresponding to two
double layered protein aggregates on the left and a single
layer protein aggregate on the right. Figure 6c displays a
close-up of the groove of such a protein aggregate. The
cross-section CD in Figure 6 d displays top left three protein
Fig. 5. Compound 2 spread over NaCI/Tris-HCl (each 10 mM), pH 7, incubated with streptavidin and transferred to gold (1 11); imaging conditions: gold tip,
200 mV and 100 PA. a) Three-dimensional image of an area with a streptavidin
aggregate grown in the liquid analogous phase at 1 mN m-', with simulated
illumination. b) Cross-section along line AB; the steps in the right-hand side of
the image correspond to monoatomic gold steps. c) Top view with streptavidin
docked on a monolayer of 2 in the gas analogous phase with a vertical dimension of 50 8, from black to white. d) Cross-section along line CD.
This needle-shaped type of aggregation has not been observed previously,[s, "1 but the protein concentration used in
this experiment was much higher than before. Figure 5c
shows the STM image of a monolayer composed of 2 and
streptavidin, which was kept at 0 mN rn-l (gas analogous
Angew. Chem. In(. Ed. Engl. 30 (1991) No. 5
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Fig. 6 . Biotin lipid 4 spread over NaCI/Tris-HCI solution (each 10 mM), pH 7.
exchanged with a subphase of streptavidin and transferred to gold (1 11); imaging conditions as in Figure 1. a) Top view of streptavidin aggregates in gray
scale scheme, the vertical dimension is 100 8, from black to white. b) Cross-section along line AB. c) Close up of a top view of a groove area of one of the
aggregates in a), shown in grayscale scheme; the vertical dimension in 55 8,
from black to white. d) Cross section along line CD. e) Three dimensional image
with simulated illumination of the edge of a multilayered protein aggregate. f)
Cross-section along line EF.
Verlagsgesellschafi mbH, W-6940 Weinheim. 155f
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molecules next to each other with lateral dimensions of
about 40 A. On the right are two protein molecules with a
spacing in between, this time corresponding to the 80 8, lattice constant found in monolayer experiments.[''] The overall irregularity in the packing points to the fact that the
protein aggregates are not perfectly ordered. In Figures 6e
and the cross-section 6f, part of a 400-500 8, thick aggregate of streptavidin is shown. This thickness corresponds to
about 8 layers of streptavidin, four of which are visible in the
image (Fig. 6 f). Such multilayered streptavidin aggregates at
lipid monolayers have not been observed previously. This
may be due to the unusually high concentration of streptavidin used in the incubation solution.
Chemisorbed self-assembled monolayers, e.g. of mercaptoundecanol 1, possess sufficient adhesion to the substrate to
allow the tunneling tip to scan over them without significant
displacement of the molecules. This is in contrast to monolayers of fatty acids or lipids, whose mobility on solids is too
high to resist repulsive or attractive tip interaction. We confirmed the surface coverage postulated from contact angle
measurement^,[^* even though the microstructure of the
layer is quite heterogeneous. Unlike Langmuir monolayers
on water, however, chemisorbed monolayers on solids exhibit only limited lateral and no vertical mobility.['91 Only
after three days incubation could we detect proteins docked
directly to the functionalized solid, whereas the same process
on a monolayer formed in a Langmuir trough took less than
one hour.
Nevertheless, upon imaging with the STM, the insufficient
adhesion of such transferred Langmuir-Blodgett monolayers
poses a problem. Good adhesion can be obtained by compounds of type 2, which can be spread on a liquid surface and
whose transferred monolayers are chemically bound by the
solid support. However, the fact that imaging of protein
aggregates on solid surfaces is possible at all is very promising for investigations of functionalized solid surfaces.
Received: October 8, 1990.
Revised version: February 1, 1991 [ Z 4235 IE]
German version: Angew. Chem. 103 (1990) 568
filtered off, recrystallized from ethanol, subjected to acid hydrolysis, and
recrystallized from n-hexane to yield 1 (95 %). 2: The Bunte salt of 1 l-bromoundecanol was converted into the symmetric disullide following the
description by Milligan and Swan [22j (98% yield). The disulfide was
isolated and esterified with a small surplus of a-biotin, following the method of Steglich et al. (231. The reaction mixture was separated chromatographically to produce pure 2 with a yield of 13%. 3: The activated ester
of 11-hromoundecanoic acid was treated with a surplus of 1,s-diamino3,6-dioxaoctane (German Texaco) and unchanged amine was removed.
The intermediate product was treated with biotin-active ester and the
product was isolated. The bromide was converted into the corresponding
Bunte Salt and subjected to acid hydrolysis. Flash chromatography
(CHCI,/CH,OH 1:l) yielded 3 (3% yield pure according to NMR spectrum).
[12] To characterize the gold-coated substrates quickly and easily, the contact
angle of a drop of water was measured at the three-phase line between air,
water, and solid. Liquid was pumped into the drops with a micrometerdriven glass syringe. We measured the constantly advancing or receding
contact angles. The contact angles were measured with the contact angle
microscope GI (Kruss Inc., Hamburg, FRG).
[13] As substrate for chemisorption and the STM measurements, epitaxially
grown gold on freshly cleaved mica was used. The mica was cleaved in air
and rinsed immediately with millipore water. 2500 8,of gold wasevaporated onto it at a pressure of < lo-' mbar. The gold was evaporated at a rate
of 5 A/s onto the mica, which was heated to 300°C before and during the
evaporation [21]. The surface shows (111) crystal faces which are atomically flat over regions of > 1000 A.
[14] The STM investigations were performed under normal conditions in air
with a Bioscope STM [20]. The images shown were recorded at a tunneling
current (f,)of lOOpA and a tunneling voltage ( V , tip to sample) of
200 mV. Using both electrochemically etched gold and also tungsten
tips, we obtained essentially the same results.
[15] R. G. Nuzzo, E. M. Korenic, L. H. Dubois, J. Chem. Phys. 93 (1990) 767.
[16] 1 0 - 1 0 0 p ~streptavidin in lOmM Tris-HCI, 10 mM NaCI, pH 7.4.
[17] P. C. Weber, D. H. Ohlendorf, J. J. Wendoloski, F. R. Salemme, Science
(Washington DC) 243 (1989) 85.
[18] a) M. Ahlers, R. Blankenburg, D. W. Grainger, P. Meller, H. Ringsdorf,
C. Salesse, Thin SolidFilms 180 (1989) 93; b) S. A. Darst, M. Ahlers, P. H.
Meller, E. W. Kubalek, R. Blankenburg, H. 0. Rihi, H. Ringsdorf, R. D.
Kornberg, Biophys. J., in press.
[19] K. L. Wolf Physik und Chemie der Grenzflachen, Vol. 2, Springer, Berlin
1959, p. 143.
I201 B. Michel, G. Travaglini, J. Microsc. (Oxford) 151 (1988) 681.
[21] V. M. Hallmark, S. Chiang, J. F. Raholt, J. D. Swalen, R. J. Wilson, Phys.
Rev. Lerf. 59 (1987) 2879.
[22] B. Milligan, J. Swan, J. Chem. SOC.1962 (1962) 2172.
1231 W. Steglich, B. Neises, Angew. Chem. 90 (1978) 556; Angew. Chem. Inf.Ed.
Engl. 17 (1978) 522.
+
CAS Registry numbers:
1,73768-94-2; 2,132722-88-4; 3,132722-89-5;4,122567-71-9; ll-hromoundecanol, 1611-56-9; 1I-hromoundecanoic acid, 2834-05-1 ; 1,8-diamino-3,6-dioxaoctane. 929-59-9; streptavidin, 9013-20-1.
[l] a) J. M. Lehn, Angew. Chem. 100 (1988) 91; Angew. Chem. Inr. Ed. Engl.
27 (1988) 89; h) H. Ringsdorf, B. Schlarb, J. Venzmer, ibid. 100 (1988) 117
and 27 (1988) 113.
[2] R. J. Behm, N. Garcia, H. Rohrer (Eds.): Scanning Tunneling Microscopy
( S T M ) and Related Merhods (NATO ASI Series E 184 (1991)).
[3] G. Travaglini, H. Rohrer, M. Amrein, H. Gross, Surf: Sci. 181 (1987) 514.
[4] J. S. Foster, J. E. Frommer, Narure 333 (1987) 542.
[5] a) M. Amrein, A. Stasiak, H. Gross, E. Stoll, G. Travaglini, Science
(Washington, DC) 240 (1988) 5124; b) M. Amrein, R. Diirr, A. Stasiak, H.
Gross, G. Travaglini, ibid. 243 (1989) 1708.
161 a) D. P. E. Smith, H. Horher, C. Gerher, G. Binnig, Science ( Washingron
D C ) 245 (1989) 43; h) B. Michel, G. Travaglini, H. Rohrer, C. Joachim, M.
Amrein, Z . Phys. B 76 (1989) 99.
[7] M. Wilcheck, E. Bayer, Trends Biochem. Sci. 14 (1989), 408; Anal.
Biochem. 171 (1988) 1.
[S] R. Blankenburg, P. Meller, H. Ringsdorf, C. Salesse, Biochemistry 28
(1989) 8214.
[9] a) R. G. Nuzzo, D. L. Allara, J. Am. Chem. Soc. 105 (1983) 4481: b) J.
Sagiv. ibid. 102 (1980) 92.
[lo] a) C. D. Bain, G. M. Whitesides, Science (Washingron D C ) 240(1988) 62:
b) C . D. Bain, J. Evall, G. M. Whithesides, J. Am. Chem. SOC.111 (1989)
7155; c) ibid. 1 1 1 (1989) 7164; d) C. D. Bain, G. M. Whitesides, Angew.
Chem. Adv. Maler. 101 (1989) 522: Angew. Chem. Inl. Ed. Engl. Adv.
Muter. 28 (1989) 506; Adv. Marer. 4 (1989) 110.
[ll] Compound 4 has been described in [8]. Compounds 1-3 were synthesized
as follows: 1: 11-hromoundecanol (Merck) was converted with sodium
thiosulfate into the corresponding Bunte salt. The crystalline product was
572
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Veriagsgesellschafi mbH. W-6940 Weinheim, 1991
The High-Yield Synthesis and Characterization
of the First Porphyrin-C yclam Dinucleating Ligand
and Its Iron(rrI)/Copper(Ir) Complex **
By Yironique Bulach, Dominique Mandon *
and Raymond Weiss*
Access to homo- or heterobimetallic porphyrin complexes
by simple and high-yield pathways is of special interest, not
only in the field of synthetic analogues of active sites in
biological processes, but in many other different areas where
metalloporphyrins are involved, such as catalysis, redox processes, mixed-valence chemistry, or even selective cleavage of
DNA." -41
[*I Prof. Dr. R. Weiss, Dipl.-Chem. V. Bulach, Dr. D. Mandon
Lahoratoire de Cristallochimie et Chimie Structurale
URA CNRS 424
Institut Le Bel, Universite Louis Pasteur
4, Rue Blaise Pascal, F-67070 Strasbourg Cedex (France)
[**I This work was supported by the Centre National de la Recherche Scientifique. We are indebted to Dr. E. Billand Prof. A . X . Trautwein (Medizinische Universitat Liibeck, Germany) for the ESR measurements and spectrum simulation; we thank Dr. M . Momenreau for helpful discussions.
R. W thanks the Alexander von Humholdt Foundation for financial support.
+
0570-0833/9t/O505-0572$3.50 .25/0
Angew. Chem. Inr. Ed. Engl. 30 (1991) No. 5
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