Analytical Chemistry Chemical Cytometry Quantitates Superoxide

Numerous references had demonstrated nanoparticles (NPs)
could greatly enhance the sensitivity of SPR spectroscopy due to
the large molecular weight of nanoparticles in spite of the fact
that the size of nanoparticles is so small that it could not be
observed by other techniques. Several kinds of NPs including Au
NPs, 16-20 SiO2 NPs, 21 Pd NPs, 22 and Pt NPs 23 had been applied
to increase the SPR sensitivity for detecting all kinds of
biomolecules. However, the application of MNPs in the SPR
field is still limited. Considering the high refractive index and
the high molecular weight of MNPs, 24 it is possible to design
an excellent SPR biosensor using MNPs as an amplification
reagent. Once the amplifying effect of MNPs for a SPR signal
is demonstrated, it can then be proved that SPR will be a
powerful candidate for detecting MNP-based separation products.
There are very few works that have been done to study the
SPR response of MNPs, and most of them focus on utilizing
commercial strepavidin-conjugated MNPs for signal amplification.
25,26 Obviously, biotin needs to be attached on a SPR substrate
surface for the further binding of strepavidin-conjugated MNPs,
which limits the extensive application of MNPs in the SPR field.
To further understand the SPR response of MNPs and extend
the application of SPR in detecting MNP labeled biomolecules
and their separation product, in this work, we study the SPR
response of the carboxyl group modified Fe3O4 MNPs by
nonspecifically adsorbing the Fe3O4 MNPs on amino group
modified SPR gold substrate. The carboxyl groups on Fe3O4
MNPs allow the MNPs to be easily functionalized by all kinds
of biomolecules for extensive applications. Our results demonstrate
that the monolayer adsorption of Fe3O4 MNPs could
result in a big SPR angle shift with a low optical loss. On the
basis of the amplification effect of Fe3O4 MNPs, we further
demonstrate SPR spectroscopy can be used to sensitively detect
Fe3O4 MNP-enriched small molecules by an indirect competitive
inhibition assay (ICIA). In this case, Fe3O4 MNPs labeled
by antiadenosine aptamer are used both as the enrichment
reagent of adenosine and the amplification reagent of SPR
spectroscopy.
EXPERIMENTAL SECTION
Materials. Adenosine, uridine, cytidine, guanosine, ethanolamine,
6-mercaptohexan-1-ol (MCH), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDC), N-hydroxysuccinimide
(NHS), thrombin, FeO(OH), oleic acid, 1-octadecene, acetone,
(16) Golub, E.; Pelossof, G.; Freeman, R.; Zhang, H.; Willner, I. Anal. Chem.
2009, 81, 9291–9298.
(17) Riskin, M.; Tel-Vered, R.; Lioubashevski, O.; Willner, I. J. Am. Chem. Soc.
2009, 131, 7368–7378.
(18) Lioubashevski, O.; Chegel, V. I.; Patolsky, F.; Katz, E.; Willner, I. J. Am.
Chem. Soc. 2004, 126, 7133–7143.
(19) Zayats, M.; Pogorelova, S. P.; Kharitonov, A. B.; Lioubashevski, O.; Katz,
E.; Willner, I. Chem.sEur. J. 2003, 9, 6108–6114.
(20) Wang, J. L.; Munir, A.; Zhou, H. S. Talanta 2009, 79, 72–76.
(21) Luckarift, H. R.; Balasubramanian, S.; Paliwal, S.; Johnson, G. R.; Simonian,
A. L. Colloids Surf., B 2007, 58, 28–33.
(22) Lin, K. Q.; Lu, Y. H.; Chen, J. X.; Zheng, R. S.; Wang, P.; Ming, H. Opt.
Express 2008, 16, 18599–18604.
(23) Beccati, D.; Halkes, K. M.; Batema, G. D.; Guillena, G.; de Souza, A. C.;
van Koten, G.; Kamerling, J. P. ChemBioChem 2005, 6, 1196–1203.
(24) Grigoriev, D.; Gorin, D.; Sukhorukov, G. B.; Yashchenok, A.; Maltseva, E.;
Moehwald, H. Langmuir 2007, 23, 12388–12396.
(25) Teramura, Y.; Arima, Y.; Iwata, H. Anal. Biochem. 2006, 357, 208–215.
(26) Soelberg, S. D.; Stevens, R. C.; Limaye, A. P.; Furlong, C. E. Anal. Chem.
2009, 81, 2357–2363.
chloroform, poly(maleic anhydride-alt-1-octadecene) (molecular
weight: 30 000-50 000) and 2-(2-aminoethoxy)-ethanol were purchased
from Sigma and used as received. Sodium hydrogen
phosphate heptahydrate, potassium dihydrogen phosphate, and
sodium chloride were ordered from Alfa Aesar. All DNA molecules
were obtained from Integrated DNA Technologies (IDT). The
sequence of the adenosine-binding aptamer was 5′-NH 2-C6-AGA
GAA CCT GGG GGA GTA TTG CGG AGG AAG GT-3′
(aptamer), the sequence of its partial complementary strand
was 5′-SH-C6-ACC TTC CTC CGC-3′ (ss-DNA). DNA solutions
were prepared by dissolving DNA in 50 mM, pH 8.0 Tris-HCl
buffer including 138 mM NaCl. Different concentrations of
adenosine and 1 mM uridine, cytidine, and guanosine were all
prepared in the Tris-HCl buffer. All glassware used in the
experiment was cleaned in a bath of freshly prepared 3:1 HCl/
HNO3 (aqua regia) and rinsed thoroughly in H2O prior to use.
(Caution: Aqua regia solution is dangerous and should be
handled with care.)
Synthesis of Monodisperse Fe3O4 MNPs. Monodisperse
Fe3O4 MNPs were synthesized by the pyrolysis of iron carboxylate
in the organic phase. 27 In brief, a mixture of FeO(OH),
oleic acid, and 1-octadecene was refluxed at 320 °C for1h
under a nitrogen atmosphere. During this process, the solution
changed its color from turbid black to black. The resulting
MNPs were precipitated with acetone and collected by centrifuge
at 4000g. After that, Fe3O4 MNPs were further purified
by repeated extraction of the precipitate with CHCl3/acetone
(1:10) until a powder of Fe3O4 MNPs was obtained. The powder
of Fe3O4 MNPs was stored at room temperature for further
application.
Forming Soluble Fe3O4 MNPs by Phase Transfer. Fe3O4
MNPs were transferred to a PBS solution according to Yu’s
work with minor modifications. 28 Carboxy group modified
amphiphilic polymers was first prepared by mixing poly(maleic
anhydride-alt-1-octadecene) with 2-(2-aminoethoxy)-ethanol (molar
ratio 1:120) in chloroform overnight. Then, the monodisperse
Fe3O4 MNPs (purified and dispersed in chloroform) were
dispersed in the carboxy group modified amphiphilic polymer
solution, and the mixture was stirred overnight at room
temperature (molar ratio of Fe3O4/polymer was 1:10). After
that, PBS buffer (pH 8.0, 10 mM) was added to the chloroform
solution of the complexes with at least a 1/1 volume ratio;
chloroform was then gradually removed by rotary evaporation
at 35 °C and water-soluble carboxy group modified Fe3O4
MNPs were obtained in a clear and dark purple solution. This
transfer process had a 100% efficiency, and no residue was
observed. The original concentrations of ∼14.51 and ∼32.82
nm soluble Fe3O4 MNPs analyzed by atomic absorption
spectroscopy are 205.4 and 16.2 nM, respectively, which will
be used to prepare other concentrations of Fe3O4 MNP
solutions by dilution.
Synthesis of Fe3O4 MNP-Aptamer Conjugates. The
monodisperse and soluble Fe3O4 MNPs with ∼32.82 nm were
diluted into pH 8.0 PBS buffer with a final concentration of 1.6
nM. Then, 1 mg of EDC and 1 mg of NHS were added to 5
(27) Yu, W. W.; Falkner, J. C.; Yavuz, C. T.; Colvin, V. L. Chem. Commun. 2004,
20, 2306–2307.
(28) Yu, W. W.; Chang, E.; Sayes, C. M.; Drezek, R.; Colvin, V. L. Nanotechnology
2006, 17, 4483–4487.
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