Analytical Chemistry Chemical Cytometry Quantitates Superoxide

mL of Fe3O4 MNP solution under stirring for 0.5 h to activate
the carboxyl group on the surface of Fe3O4 MNPs. After that,
100 µL of 37.7 µM amino-modified antiadenosine aptamer was
added in this solution, and they were allowed to react for 2 h
to immobilize aptamer on the surface of Fe3O4 MNPs. After
that, 1 M ethanolamine was added for 1 h to block the
unreacted carboxyl groups. Then, this solution was centrifuged
at 14 000g at room temperature for 25 min twice to remove
the free amino-aptamer. At last, the Fe3O4 MNPs were
dispersed in 5 mL of pH 8.0 PBS buffer and stored at 4 °C.
In Situ SPR Measurement. The SPR experiments were done
using Eco Chemie Autolab SPR systems (Brinkmann Instruments,
New York). 29,30 It works with a laser diode fixed at a wavelength
of 670 nm, using a vibrating mirror to modulate the angle of
incidence of the p-polarized light beam on the SPR substrate. The
instrument was equipped with a cuvette. A gold sensor disk (25
mm in diameter) was mounted on the hemicylindrical lens (with
index-matching oil) to form the base of the cuvette. The cuvette
could contain sample with adjustable volume from 10 to 1000 µL.
An O-ring (3 mm inner diameter) between the cuvette and disk
prevents leakage. An autosampler (Eco Chemie) with a controllable
aspirating-dispensing-mixing pipet was used to add
samples into the cuvette and provide constant mixture by aspiration
and dispensing during measurements. This experimental
arrangement maintains a homogeneous solution and reproducible
hydrodynamic conditions. The injection rate and mixing rate for
all samples were 10 and 40 µL/s, respectively, with the total
volume for all samples dispensed in the SPR cell equal to 40 µL.
This setup allows us to measure the SPR angle shift in millidegrees
(m°) as a response unit to quantify the binding amount of
macromolecules to the sensor surface.
Details of the experiment are as follows. The SPR gold film
was initially immersed into the thiolated ss-DNA solution for 12 h
in order to assemble the monolayer of ss-DNA. Then, the modified
gold film was thoroughly rinsed with 50 mM Tris-HCl buffer
and water to remove the weakly adsorbed ss-DNA. Then, the ss-
DNA modified SPR gold film was immersed in 100 µM 6-mercaptohexanol
for 1htoblock the uncovered gold surface. This gold
film was used as a sensing surface to detect the amount of aptamer
possessing ss-DNA structure on Fe 3O4 MNP-aptamer conjugates,
which can be adjusted by the concentration of adenosine
added to the Fe3O4 MNP-aptamer conjugate solution. The
detection procedure is made up of two steps. First, Fe3O4
MNP-aptamer conjugate solution was mixed with different
concentrations of adenosine for 30 min. After that, adenosine
bound with Fe3O4 MNP-aptamer conjugates were separated
and enriched by centrifuging at 14 000g for 1 h twice. The
precipitation was dispersed in PBS again. The resulting solution
was injected into the SPR cell, and the SPR angle-time curve
was recorded. In order to reduce the disturbance of DNA
denaturation that resulted from the regenerating process for
the detecting results, we change a new substrate after each
detection. The modification of each gold substrate is carried
out under the same experimental condition. To confirm the
reproducibility of the detection result, different concentrations
(29) Wang, J. L.; Zhou, H. S. Anal. Chem. 2008, 80, 7174–7178.
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6784 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010
of the MNPs and adenosine are repeatedly detected for three
times.
RESULTS AND DISCUSSION
Characterization of Fe3O4 MNPs. Although soluble Fe3O4
MNPs could be easily synthesized by coprecipitation of aqueous
Fe 2+ /Fe 3+ salt solutions with the addition of a base under
an inert atmosphere at room temperature or at elevated
temperature, the size, shape, and composition of the MNPs
very much depends on the type of salts used (e.g., chlorides,
sulfates, nitrates), the Fe 2+ /Fe 3+ ratio, the reaction temperature,
the pH value, and ionic strength of the media. 31-33 Furthermore,
the Fe3O4 MNPs are also easy to aggregate. Inspired by the
synthesis of high-quality semiconductor nanocrystals and
oxides in nonaqueous media by thermal decomposition, 34-36
monodisperse Fe3O4 MNPs with controlled size has essentially
been synthesized through thermal decomposition of ion
compounds in high-boiling organic solvents. 37,38 Here, we
synthesize Fe3O4 MNPs by the pyrolysis of iron carboxylate in
an organic phase. By changing the ratio of FeO(OH) and oleic
acid, two kinds of Fe3O4 MNPs are prepared. Figure 1A,B
provides the TEM images of the prepared Fe3O4 MNPs and their
size distribution. The average size of Fe3O4 MNPs derived from
Figure 1A,B are 14.51 nm (n ) 300 particles) and 32.82 nm
(n ) 138 particles), respectively. Importantly, both kinds of Fe3O4
MNP size distributions are narrow, which indicates the
prepared Fe3O4 MNPs are monodisperse. After transferring
Fe3O4 MNPs from organic reagent to water solution by the
amphiphilic polymer, Fe3O4 MNPs show a good stability in both
PBS and Tris buffer due to the large hydrodynamic size of
polymer (data not shown), which are all beneficial for the
acquisition of accurate and repeated SPR analytical results.
SPR Response and Concentration Dependence of Fe3O4
MNPs. The basis of a particle-enhanced bioassay is that biomolecular
interaction events lead to particle immobilization, i.e., more
immobilized proteins yield higher particle coverage. 39 Currently,
most SPR instruments are able to quantitatively detect the
concentration of biomolecules through calculating the SPR angle
shift enhanced by the binding of nanoparticle. Considering our
synthesized Fe3O4 MNPs are protected by negative polymer,
2-mercaptoethyamine is used on SPR Au substrates for the
adsorption of Fe3O4 MNPs. The thiol group binds to the Au
surface, leaving the amine group free to bind with carboxyl
group in soluble Fe3O4 MNPs by electrostatic interaction.
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