Analytical Chemistry Chemical Cytometry Quantitates Superoxide
Analytical Chemistry Chemical Cytometry Quantitates Superoxide
Analytical Chemistry Chemical Cytometry Quantitates Superoxide
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Figure 4. Schematic representation of the SPR biosensor for the detection of the small molecules.<br />
Fe3O4 MNP Enhanced SPR Sensing for the Detection<br />
of Small Molecules. To further demonstrate the practicability<br />
of the amplification effect of Fe3O4 MNPs in an enhancing SPRbased<br />
bioassay, a novel SPR sensor based on indirect competitive<br />
inhibition assay (ICIA) for the detection of adenosine is<br />
constructed. The principle of this SPR sensor is shown in<br />
Figure 4. The partial complementary thiolated ss-DNA of antiadenosine<br />
aptamer is first immobilized on SPR gold film as a<br />
sensing surface. When Fe3O4 MNP-antiadenosine aptamer<br />
conjugate solution is added to the SPR cell in the absence of<br />
adenosine, Fe3O4 MNP-antiadenosine aptamer conjugates will<br />
be adsorbed to the SPR sensor by the DNA hybridization<br />
reaction and result in a huge change of SPR signal due to the<br />
amplification effect of Fe3O4 MNPs. However, the change of<br />
SPR signal will decrease after Fe3O4 MNP-antiadenosine<br />
aptamer conjugates bind with adenosine. This is because<br />
adenosine reacts with antiadenosine aptamer in Fe3O4 MNPantiadenosine<br />
aptamer conjugates and changes its structure<br />
from ss-DNA to tertiary structure, which cannot hybridize with<br />
its partial complementary ss-DNA immobilized on the SPR gold<br />
surface. Thus, the change of SPR signal will decrease with the<br />
increase of the number of Fe3O4 MNP-antiadenosine aptamer<br />
conjugates possessing tertiary structure, which is proportional<br />
to the concentration of adenosine.<br />
The essential prerequisite of this detection is to prepare Fe3O4<br />
MNP-antiadenosine aptamer conjugates. For this purpose,<br />
EDC and NHS are used as a carboxyl activating agent for the<br />
coupling of primary amines in aptamer and carboxyl groups<br />
in polymer coated MNPs. To demonstrate that MNPs have<br />
been labeled by an aptamer successfully, FT-IR spectroscopy<br />
is used to evaluate the surface modification of MNPs before<br />
and after aptamer labeling. Figure 5 shows the IR absorbance<br />
spectra of Fe3O4 MNPs before and after the aptamer label. For<br />
the polymer coated Fe3O4 MNPs, the absorbance peak near<br />
1710 cm -1 is assigned to υ (CdO) (stretch vibration of<br />
carbonyl) and amide I, peaks at 1550 cm -1 are assigned to<br />
amide II, and the peaks at 2854 and 2925 cm -1 are assigned to<br />
υs (C-H2) and υas (C-H2) (symmetric and asymmetric stretch<br />
vibrations of C-H2), respectively. In the region of 3200-3570<br />
Figure 5. FT-IR of polymer coated Fe3O4 MNPs before and after<br />
aptamer labeling.<br />
cm -1 , a peak due to the O-H stretch is expected. Several new<br />
peaks are observed after MNPs are labeled by the aptamer.<br />
The peaks at 1048 and 1211 cm -1 are attributed to stretching<br />
vibrations of the PO 2- in the aptamer. It should be pointed out<br />
that we are not able to confirm the presence of DNA-related<br />
peaks at 1550 cm -1 because the peak overlaps with amide II.<br />
After Fe3O4 MNP-antiadenosine aptamer conjugates are<br />
prepared successfully, the SPR detection is carried out<br />
according to the principle described in Figure 4. Figure 6A<br />
shows the SPR angle-time curves of the separation products<br />
obtained after Fe3O4 MNP-antiadenosine aptamer conjugates<br />
are reacted with different concentrations of adenosine for<br />
30 min. In the absence of adenosine, Fe3O4MNP-antiadenosine<br />
aptamer conjugates in solution directly hybridize with ss-<br />
DNA immobilized on SPR gold film, resulting in the largest<br />
SPR angle shift (∼1082.94 m°). This angle shift is much<br />
larger than that of the angle shift resulting from the binding<br />
of ss-DNA or most of the protein. The SPR angle shift<br />
resulting from the binding of Fe3O4 MNP-antiadenosine<br />
aptamer conjugates decreases (∼820.14 m°) after Fe3O4<br />
MNP-antiadenosine aptamer conjugates are reacted with<br />
<strong>Analytical</strong> <strong>Chemistry</strong>, Vol. 82, No. 16, August 15, 2010<br />
6787