Analytical Chemistry Chemical Cytometry Quantitates Superoxide

Figure 3. AFM images of 14.51 nm Fe3O4 MNP (A) and 32.82 nm Fe3O4 MNP (B) modified SPR gold substrate. The concentration of Fe3O4
MNPs is 1.6 nM, and the assembly time is 10 min.
coverage of Fe3O4 MNPs on SPR gold film could be reached
at a higher concentration due to the rapid diffusion adsorption.
It should be pointed out that the SPR angle shift resulting from
the adsorption of Fe3O4 MNPs is much higher than that of the
value resulting from the adsorption of most biomolecules under
the same concentration. 40,41 It means Fe3O4 MNPs greatly
enhance the signal of SPR spectroscopy. Importantly, the SPR
angle shift could be further increased when 32.82 nm Fe3O4
MNPs are used. Figure 2B shows the SPR angle shift curves
resulting from the adsorption of 32.82 nm Fe3O4 MNPs with the
same concentration sequence as 14.51 nm Fe3O4 MNPs. With
the increase of Fe3O4 MNP concentrations from 0.016 to 1.6
nM, the SPR angle shifts increase from 79.22 to 2479.79 m°.
To further understand the size effect of Fe3O4 MNPs on the
SPR response, the relation between SPR angle shifts and the
concentrations of Fe3O4 MNPs with the two sizes are compared
in Figure 2C. Obviously, much larger angle shifts are observed
for 32.82 nm Fe3O4 MNPs because of its larger moleculer
weight. Besides that, the affinity constants and surface coverage
for both kinds of Fe3O4 MNPs could also be obtained from
Figure 2C. From the data of Figure 2C, we can see that the
plasmon resonance shifts with the increasing Fe3O4 MNP
concentration and a simple Langmuir isotherm 42 could be used
to fit the data. The Langmuir equation used to fit the data is
given by
KA [C] Fe3O4 ∆λ ) ∆λmax1 + KA [C] Fe3O4 Where ∆λ is the angle shift caused by the adsorption of Fe3O4
MNPs, ∆λmax is the angle shift which will be observed at
saturation, KA is the apparent equilibrium affinity constant, and
[C]Fe3O4 is the concentration of Fe3O4 MNPs. Using the above
equation, KA values for 14.51 and 32.82 nm Fe3O4 MNPs are
(40) Mullett, W. M.; Lai, E. P. C.; Yeung, J. M. Methods 2000, 22, 77–91.
(41) Hoa, X. D.; Kirk, A. G.; Tabrizian, M. Biosens. Bioelectron. 2007, 23, 151–
160.
(42) Lee, H. J.; Wark, A. W.; Corn, R. M. Langmuir 2006, 22, 5241–5250.
6786 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010
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calculated to be around 7.14 × 10 8 and 2.63 × 10 9 M -1 ,
respectively, which are similar to the value of NPs as found in
literature. 43 The saturation responses ∆λmax of 14.51 and 32.82
nm are calculated to be 2165 and 3134 0 m, respectively. It can
be seen that 32.82 nm Fe3O4 MNPs have slightly higher affinity
for adsorption on the sensing surface due to their larger
molecular weight; therefore, they show larger angle shifts as
well as higher saturation response (∆λmax) compared to 14.51
nm Fe3O4 MNPs. In our following experiments, we, therefore,
use 32.82 nm Fe3O4 MNPs. The ratio (∆λ)/(∆λmax) gives the
fraction of surface coverage, and if the bulk concentration of
[C]Fe3O4 is equal to (1)/(KA), half of the surface sites will be
occupied ((∆λ)/(∆λmax) ) 0.5). On the basis of the experimental
results and the fitting parameters, we calculate that 80%
of the coverage ((∆λ)/(∆λmax) ) 0.8) will be obtained when
32.82 nm Fe3O4 MNPs with a concentration of 1.6 nM are used.
This concentration will be used in our following experiments
because the changes in SPR angle shift (∆λ) will be very small
even if we further increase the concentration of Fe3O4 MNPs.
To demonstrate that a dense monolayer of MNPs could be
formed on a SPR substrate within 10 min using a 1.6 nM Fe3O4
MNP solution as assembly solution, AFM is used to evaluate
the surface morphology of MNP modified SPR gold substrate.
Figure 3A,B shows the AFM images of 14.51 nm Fe3O4 MNPs
and 32.82 nm Fe3O4 MNPs on SPR gold substrate, respectively.
As seen from the AFM figures, the nanoislands are formed
after MNPs are assembled. The diameter of nanoislands in
Figure 3A is smaller than that of the value in Figure 3B due to
different diameter of MNPs. However, the dense layer of MNPs
on SPR gold substrate has been observed from Figure 3 for both
kinds of Fe3O4 MNPs at the present experimental condition,
which indicates the adsorption of Fe3O4 MNPs on SPR gold
substrate is fast and the present experimental condition is
suitable for further experiments.
(43) Liao, W. S.; Chen, X.; Yang, T. L.; Castellana, E. T.; Chen, J. X.; Cremer,
P. S. Biointerphases 2009, 4, 80–85.