perceived by the naked eye. There is also no need to use analytical
instruments or labeled lectins. By using this expedient methodol-
ogy, a broad range of proteins can be rapidly evaluated for their
abilities to interact with the protein of interest in real time. The
method is easily generalized for studying other protein–protein
interactions simply by capping GNPs with corresponding cognate
substrates, rendering high-throughput screening of protein–protein
interactions possible. It should also be straightforward to use this
methodology to screen molecular libraries containing potential
drugs to disrupt cell–cell adhesion mediated by lectins. Further
work in this direction and the application of this colorimetric assay
in proteomic analysis are underway.
The authors gratefully acknowledge the National Science
Council and Ministry of Education, Taiwan for financial support
and Prof. Sunney Chan for suggestions.
Notes and references
1 Y. C. Cao, R. Jin, J.-M. C. Nam, S. Thaxton and C. A. Mirkin, J. Am.
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2 I. Remy and S. W. Michnick, Proc. Natl. Acad. Sci. USA, 1999, 96,
5394–5399.
Fig. 5 (a) SPR sensorgrams for binding of BS-I to the immobilized
ConA in PBS buffer at 25 uC. (b) The change in the response signal is
plotted against the concentration of BS-I.
3 e520 for 13-nm and 50-nm diameter gold nanoparticles are 2.7 6 108
and 1.5 6 1010 M21 cm21, respectively. For details see R. Jin, G. Wu,
Z. Li, C. A. Mirkin and G. C. Schatz, J. Am. Chem. Soc., 2003, 125,
1643–1654.
4 C. M. Niemeyer, Angew. Chem., Int. Ed., 2001, 40, 4128–4158.
5 R. Elghanian, J. J. Storhoff, R. C. Mucic, R. L. Letsinger and
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An independent determination of the binding constant between
ConA and BS-I was obtained from Biacore surface plasmon
resonance (SPR) biosensor.15 ConA was immobilized to a CM5
sensor chip through amine coupling and BS-I solution was injected
over the immobilized Con A. The resultant SPR sensorgrams are
shown in Fig. 5. As the concentration of BS-I increases, the
response is proportionally increased. The binding constant
assuming 1 : 1 stoichiometry determined from SPR data is 1.9 6
106, which is weaker than that determined by the Man-GNPs
methodology. The disparity in the binding magnitude can be
largely attributed to the intrinsic nature of these two methodol-
ogies. In the case of the SPR measurement, immobilizing one of
the interacting proteins on the chip blocks one side of the proteins.
As a result, only one side of the proteins is exposed for interacting
with the binding partners. In contrast, proteins of interest can
interact with each other in any direction in the case of the Man-
GNP system and the local density of ConA is probably higher
than it is on the CM5 sensor chip, rendering 1 : 2 binding possible.
It is reasonable that the cooperative binding strength of divalent
(1 : 2) binding is much greater than monovalent (1 : 1) binding.
The concentration of BS-I used in these studies also reflects the
binding strength. Only nanomolar quantities of BS-I were required
to influence the Man-GNP absorbance, whereas micromolar
amounts of BS-I were needed in the Biacore studies.
9 J. T. Dulaney, Anal. Biochem., 1979, 99, 254–267.
10 Six lectins are bandeiraea simplicifolia lectin I (BS-I), soybean agglutinin
(SBA), maackia amurensis (MAL), erythrina cristagalli lectin (ECL),
wheat germ agglutinin (WGA), bandeiraea simplicifolia lectin II (BS-II).
Three other proteins are RNaseA, trypsin inhibitor, bovine serum
albumin (BSA).
11 Spectroscopic data for the mannopyranoside thiol derivative used in the
preparation of Man-GNP: 1H-NMR (400 MHz, CD3OD, 25 uC, TMS):
d 5 8.02 (bs, 1 H, NH), 4.81 (d, J 5 1.5 Hz, 1 H), 3.88–3.80 (m, 3 H),
3.74–3.52 (m, 13 H), 3.39–3.32 (m, 2 H), 2.68 (t, J 5 7.3 Hz, 2 H), 2.49
(t, J 5 7.3 Hz, 1 H, SH), 2.20 (t, J 5 7.5 Hz, 2 H), 1.70–1.56 (m, 4 H),
1.42–1.26 (m, 12 H); 13C-NMR (CD3OD, 100 MHz): d 5 175.5, 101.4,
74.4, 72.4, 72.0, 71.5, 71.3, 71.2, 70.6, 68.5, 67.6, 62.9 ,40.5, 39.9, 37.2,
35.4, 30.8, 30.7, 30.6, 30.4, 29.7, 29.7, 27.3; IR (KBr) 3416, 3323, 2925,
2859, 1646, 1553, 1467, 1135, 1062 cm21; FAB-MS : m/z 512.3 (M+ + 1),
534.2 (M + Na+); FAB-HRMS calcd for C23H45NO9S (M+ + 1)
512.2894, found 512.2885.
12 Different sizes of Man-GNPs were prepared. It turns out that 32-nm
Man-GNPs give the best optical signals in our studies. Transmission
electron microscopic images of synthesized 32-nm Man-GNP alone and
in the presence of ConA are included in the ESI{.
13 J. Su and M. Mrksich, Angew. Chem., Int. Ed., 2002, 41, 4715–4718.
14 K. A. Connors, Binding Constants, The Measurement of Molecular
Complex Stability, Wiley-Interscience, New York, 1987.
We have shown convincingly that protein–protein interactions
can be evaluated qualitatively as well as quantitatively by using
gold nanoparticle-based competitive colorimetric assays. The
methodology does not require any special protein modifications
and is very sensitive; only nanomolar concentrations of proteins
are required. Moreover, color changes are conspicuous and readily
15 R. L. Rich, L. R. Hoth, K. F. Geoghegan, T. A. Brown, P. K. LeMotte,
S. P. Simons, P. Hensley and D. G. Myszka, Proc. Natl. Acad. Sci.
USA, 2002, 99, 8562–8567.
This journal is ß The Royal Society of Chemistry 2005
Chem. Commun., 2005, 4273–4275 | 4275