Analytical Chemistry Chemical Cytometry Quantitates Superoxide

Figure 1. Extinction spectra of solutions of (A) 0.48 nM Tween 20-
AuNPs and (B) 0.37 nM Tween 20-modified bare AuNPs (a) before
and (b, c) after the addition of (b) 1 µMHg 2+ and (c) 1 µMAg + . Tween
20-AuNPs are prepared in 20 mM phosphate at pH 12.0. The
incubation time is 5 min.
to reduce Ag + onto the Au surface. 38,39 The formation of Agcoated
AuNPs enables Tween 20 to be removed from the NP
surface, thereby inducing aggregation of the AuNPs in a highionic-strength
solution.
Evidence for the Formation of Hg-Au Alloys and Ag
Shells. To test the hypothesis mentioned above, we monitored
the extinction spectra of Tween 20-AuNPs in the absence and
presence of Hg 2+ and Ag + . Curve a in Figure 1A shows that the
SPR wavelength of Tween 20-AuNPs appears at 520 nm, indicating
that they are well dispersed in 20 mM phosphate solution at pH
12.0. In other words, Tween 20 molecules can indeed protect
citrate-capped AuNPs against a high-ionic-strength solution. 40 After
separately adding 1.0 µMHg 2+ (curve b) and 1.0 µMAg + (curve
c), we observed a decrease in the strength of SPR band at 520
nm and the formation of a new red-shift band. These changes
were characteristic of AuNP aggregation. The Hg 2+ - and Ag + -
(38) Xie, W.; Su, L.; Donfack, P.; Shen, A.; Zhou, X.; Sackmann, M.; Materny,
A.; Hu, J. Chem. Commun. 2009, 5263–5265.
(39) Xia, H.; Bai, S.; Hartmann, J.; Wang, D. Langmuir 2009.
(40) Shen, C.-C.; Tseng, W.-L.; Hsieh, M.-M. J. Chromatogr., A 2009, 1216,
288–293.
induced NP aggregation was nearly complete after 5 min (SI
Figure S2). Obviously, the deposition of Hg and Ag onto the Au
surface enables Tween 20 to be removed, thereby driving NP
aggregation. We ruled out the possibility that coordination
between Tween 20 and metal ions induces the NP aggregation
because Tween 20 does not contain any functional group to
interact with Hg 2+ and Ag + . To ensure the role of citrate ions
in the reduction of Hg 2+ and Ag + , the extinction spectra of bare
AuNPs modified with Tween 20 were examined under the same
conditions. The addition of Hg 2+ and Ag + to this type of AuNP
resulted in a rare shift in the SPR wavelength (Figure 1B),
clearly indicating that citrate ions are indispensable for detecting
Hg 2+ and Ag + using Tween 20-AuNPs. On the other hand, we
prepared Hg-Au alloy- and Ag-coated AuNPs and modified
them with Tween 20. Compared to Tween 20-AuNPs, a blue
shift in SPR and a decrease in SPR intensity were observed
for Tween 20-modified Hg-Au alloy-coated AuNPs (SI Figure
S3). This is attributed to the formation of Hg-Au alloy on the
surface of the AuNPs. A similar phenomenon was reported when
citrate-capped AuNPs were exposed in the presence of Hg vapor. 41
Moreover, this kind of NPs was found to be unstable in a highionic-strength
solution. This result reflects that Tween 20 molecules
were not attached to the surface of Hg-Au alloy, thereby
incapable of protecting NPs against a high-ionic strength solution.
SI Figure S4 shows that the deposition of Ag on the surface of
the AuNPs resulted in an increase SPR intensity relative to Tween
20-AuNPs. 42,43 Similarly, Tween 20-modified Ag-coated AuNPs
were aggregated in a high-ionic-strength solution because Tween
20 molecules were not adsorbed on the surface of Ag shell.
To provide further evidence for the formation of Hg-Au alloys
and Ag shells, we used ICP-MS to quantitatively determine the
composition of the precipitates, which were obtained by five cycles
of centrifugation of a solution of Tween 20-AuNPs and metal ions.
When a series of concentrations (0-1 µM) of Hg 2+ were present
in a solution of 0.48 nM Tween 20-AuNPs, the molar ratio of
Hg to Au in the precipitates gradually increased with increasing
Hg 2+ concentration (Figure 2A). A similar phenomenon was seen
in the case of Ag + (Figure 2B). If Hg-Au alloys and Ag shells
did not form on the Au surface, the molar ratios of Hg to Au and
Ag to Au should remain constant under these conditions. Under
identical treatment conditions (five centrifugation/washing cycles),
the composition of precipitates was also determined by EDX
analysis. The Hg content in the precipitates increased with
increasing Hg 2+ concentration (Figure 2C). We observed a
similar phenomenon when different concentrations of Ag + were
present in a solution of Tween 20-AuNPs (Figure 2D). These
results are in agreement with those obtained by ICP-MS. These
findings strongly support the idea that Hg 2+ - and Ag + -induced
aggregation of Tween 20-AuNPs is indeed the result of the
formation of Hg-Au alloys and Ag on the Au surface.
Effect of Surfactant Chain Length, NP Concentration, and
Ionic Strength. We next explored the effect of surfactant chain
length on the metal-ion-induced aggregation of the AuNPs. The
(41) Morris, T.; Copeland, H.; McLinden, E.; Wilson, S.; Szulczewski, G.
Langmuir 2002, 18, 7261–7264.
(42) Anandan, S.; Grieser, F.; Ashokkumar, M. J. Phys. Chem. C 2008, 112,
15102–15105.
(43) Guerrini, L.; Garcia-Ramos, J. V.; Domingo, C.; Sanchez-Cortes, S. J. Raman
Spectrosc. 2010, 41, 508–515.
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