enzymatic reaction known as ‘‘rolling circle amplification’’ where
dNTPs (dATP, dTTP, dGTP and dCTP) are converted to a long
Notes and references
1
(a) R. Elghanian, J. J. Storhoff, R. C. Mucic, R. L. Letsinger and
C. A. Mirkin, Science, 1997, 277, 1078; (b) J. Liu and Y. Lu, J. Am.
Chem. Soc., 2003, 125, 6642; (c) J. Liu and Y. Lu, Angew. Chem., Int.
Ed., 2005, 45, 90; (d) V. Pavlov, Y. Xiao, B. Shlyahovsky and I. Willner,
J. Am. Chem. Soc., 2004, 126, 11768; (e) C. Tsai, T. Yu and C. Chen,
Chem. Commun., 2005, 4273; (f) C. Huang, Y. Huang, Z. Cao, W. Tan
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J. Jiang, Z. He, Z. Zhang, G. Shen and R. Yu, Nucleic Acids Res., 2005,
15
single-stranded DNA (ssDNA) by phi29 DNA polymerase. We
anticipated that dNTPs would bind AuNP more effectively than
the long ssDNA product because of the steric hindrance and large
8
b
secondary structures formed in long ssDNA, and that dNTPs
would stabilize AuNP more effectively than the long ssDNA. Since
dNTPs and the long ssDNA product both stabilize AuNPs
3
2
2
3, e168; (h) J. M. Nam, K. J. Jang and J. T. Groves, Nat. Protocols.,
007, 2, 1438; (i) R. Baron, B. Willner and I. Willner, Chem. Commun.,
007, 323; (j) Z. Wu, S. Zhang, M. Guo, C. Chen, G. Shen and R. Yu,
(
although to different extents), the assay was performed at a
specific salt concentration where dNTP/AuNP is stable whereas
16
long ssDNA/AuNP would aggregate. The results are shown in
Fig. S5 in ESI.{ Briefly, the AuNP solution mixed with the
reaction mixture that either excluded or contained DNA
polymerase was red and blue in color, respectively. The red-to-
blue color change indicated that AuNP aggregation occurred as
dNTPs were made into long ssDNA by phi29 DNA polymerase.
In conclusion, we have demonstrated simple and rapid enzyme-
sensing assays using the principle of non-crosslinking AuNP
aggregation. The use of AuNP is advantageous, particularly for
the enzymatic reactions that cannot be easily monitored by
traditional spectroscopic techniques, due to its simplicity and the
colorimetric detection nature. Moreover, compared to the cross-
linking AuNP aggregation, this non-crosslinking aggregation
induced by a change of the electrophoretic properties of AuNPs
is generally faster (the AuNP test assay in the present study was
Anal. Chim. Acta, 2007, 584, 122.
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Int. Ed., 2007, 46, 3468.
4 C. Guarise, L. Pasquato, V. De Filippis and P. Scrimin, Proc. Natl.
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3
5
6
126, 10076.
7
8
E. A. Hauser, Colloidal phenomena: an Introduction to the Science of
Colloids, McGraw-Hill, New York–London, 1939.
(a) W. Zhao, W. Chiuman, M. A. Brook and Y. Li, ChemBioChem,
2007, 8, 727; (b) H. Li and L. Rothberg, Proc. Natl. Acad. Sci. USA,
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Chem. Commun., 2006, 2, 3780.
9 R. F. Weaver, Molecular Biology, McGraw-Hill College, Boston, MA,
8
completed in 1 min). In the non-crosslinking system, the aggrega-
2002.
1
0 (a) J. J. Storhoff, R. Elghanian, C. A. Mirkin and R. L. Letsinger,
Langmuir, 2002, 18, 6666; (b) A. Gourishankar, S. Shuka, K. N. Ganesh
and M. Sastry, J. Am. Chem. Soc., 2004, 126, 13186; (c) H. Kimura-
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tion is driven by the London/van der Waals attractive force
8
c
between the nanoparticles. Once the electrostatic repulsion is
significantly reduced due to the loss (or screen) of surface charges,
the attractive forces dominate, leading to a rapid aggregation. In
contrast, in the crosslinking system, the aggregation is mainly
driven by random collisions between nanoparticles with relatively
1766.
8
c
slow Brownian motion. Furthermore, this approach is con-
ceptually simpler than the crosslinking system and, in principle,
can be applied to any target molecules which can affect the
electrophoretic properties of AuNPs, whereas in the crosslinking
aggregation process, the crosslinker has to carry at least two
binding tags in order to bring AuNP into close proximity. We
speculate that our assay can be adapted to other charged sub-
strates such as DNA oligonucleotides, amino acids and peptides,
and that similar assays could be easily developed to detect and
quantify other enzymes including ligases, endonucleases, proteases,
etc. Nevertheless, nonspecific aggregation of bare AuNP could be
a potential issue for the use of this assay in more complex matrices
11 The initial purpose of this dilution is to minimize the effect of local salt
concentration increase on the AuNP stability when reaction sample
solution is added into AuNP solution. However, our subsequent study
showed that the undiluted reaction solution could also be directly added
to AuNP solution without causing significant AuNP stability change
(see ESI,{ Fig. S2).
1
2 The lower CIAP concentration (e.g. 0.05 units/20 mL) could also be
detectable, but a longer reaction time (e.g. 12 h) was required to ensure
enough ATP had been converted into adenosine to get a quick color
change (1 min) in the subsequent AuNP test.
3 Control experiments where the AuNP stability with or without CIAP
were accessed by gradually adding NaCl solution (1 M) to AuNP
solution showed that CIAP did not stabilize AuNP either under studied
conditions.
14 I. R. Gibbons, M. P. Cosson, J. A. Evans, B. H. Gibbons, B. Houck,
K. H. Martinson, W. S. Sale and W. Y. Tang, Proc. Natl. Acad. Sci.
USA, 1978, 75, 2220.
5 (a) A. Fire and S. Xu, Proc. Natl. Acad. Sci. USA, 1995, 92, 4641; (b)
D. Liu, S. L. Daubendiek, M. A. Zillman, K. Ryan and E. T. Kool,
J. Am. Chem. Soc., 1996, 118, 1587.
6 Theoretically, there is a possibility that AuNPs are bridged by the RCA
product (or H-bonds between DNA molecule-adsorbed AuNPs).
However, we think this is a minor contribution to AuNP aggregation
in our assay given the fact that this type of inter-particle crosslinking
process is known as a relatively slow process (for example, see ref. 1a
and 8c). Considering the AuNP aggregation (or color change) in our
assay is completed in 1 min, we believe this aggregation is induced by the
loss (or screen) of surface charges.
1
(rather than pure buffers) and therefore a pre-purification step
might be required in some specific cases. Finally, since the
interaction between nucleobases and AuNP is quite complicated
1
1
0
and has been the subject of extensive debate, we hope this work
will provide insight into the nature of the interaction between
DNA (or nucleotides, nucleosides) and AuNPs.
1
The authors wish to thank the Natural Sciences and
Engineering Research Council of Canada (NSERC) and Sentinel
Bioactive Paper Network for financial support. Y. L. holds a
Canada Research Chair.
This journal is ß The Royal Society of Chemistry 2007
Chem. Commun., 2007, 3729–3731 | 3731