Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Anal. Chem. 2010, 82, 6782–6789 Magnetic Nanoparticle Enhanced Surface Plasmon Resonance Sensing and Its Application for the Ultrasensitive Detection of Magnetic Nanoparticle-Enriched Small Molecules Jianlong Wang, Ahsan Munir, Zanzan Zhu, and H. Susan Zhou* Department of Chemical Engineering, Worcester Polytechnic Institute, 100 Institute Road, Worcester, Massachusetts 01609 Magnetic nanoparticles (MNPs) have been frequently used in bioseparation, but their applicability in bioassays is limited due to their extremely small size so that sensitive detection is difficult to achieve using a general technique. Here, we present an amplification technique using MNPs for an enhanced surface plasmon resonance (SPR) bioassay. The amplification effect of carboxyl group modified Fe3O4 MNPs of two sizes on SPR spectroscopy is first demonstrated by assembling MNPs on amino group modified SPR gold substrate. To further evaluate the feasibility of the use of Fe3O4 MNPs in enhancing a SPR bioassay, a novel SPR sensor based on an indirect competitive inhibition assay (ICIA) is developed for detecting adenosine by employing Fe3O4 MNP-antiadenosine aptamer conjugates as the amplification reagent. The results confirm that Fe3O4 MNPs can be used as a powerful amplification agent to provide a sensitive approach to detect adenosine by SPR within the range of 10-10 000 nM, which is much superior to the detection result obtained by a general SPR sensor. Importantly, the present detection methodology could be easily extended to detect other biomolecules of interest by changing the corresponding aptamer in Fe3O4 MNP-aptamer conjugates. This novel technique not only explores the possibility of the use of SPR spectroscopy in a highly sensitive detection of an MNP-based separation product but also offers a new direction in the use of Fe3O4 MNPs as an amplification agent to design high performance SPR biosensors. Over the past few decades, magnetic nanoparticles (MNPs) have been receiving increasing attention due to their unprecedented advantages such as higher surface-to-volume ratio for chemical binding, minimum disturbance to attached biomolecules, faster binding rates, higher miscibility, and higher specificity. 1,2 These characteristics of MNPs render them easier labeling by biomolecules, as well as easier binding with its target analytes. * Corresponding author. Tel.: 508-831-5275. Fax: 508-831-5936. E-mail: szhou@wpi.edu. (1) Pankhurst, Q. A.; Connoliy, J.; Johns, S. K.; Dobson, J. J. Appl. Phys. 2003, 36, 167–181. (2) Gijs, M. A. M. Microfluid. Nanofluid. 2004, 1, 22–40. 6782 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 Up to now, all kinds of biomolecules including DNA and RNA, 3 protein and peptides, 4 cell and virus 5-7 have been separated and concentrated under an external magnet by the use of MNPs as carriers. Compared with the springing up of MNPs in bioseparation, the application of MNPs in bioassays is limited because their size is too small to be detected by general techniques developed for detecting magnetic microbeads. In order to extend the application of MNPs in bioassays, some novel techniques had been used to detect MNPs, such as the electrochemical method, 8 IR spectroscopy, 9 fluorescence spectroscopy, 10 magnetic atomic force microscopy (AFM), 11 magnetic resonance imaging (MRI), 12 bio-bar-code, 13 etc. However, these methods either need labeling MNPs by electroactive probes or fluorescence molecules or need expensive experiment setups, thereby limiting them to be used on a benchtop scale and cannot be used for simple, in situ, and cost-effective detection of real samples. Here, we investigate the application of surface plasmon resonance (SPR) spectroscopy for fast, ultrasensitive, and in situ detection of the MNP-enriched biomolecules. SPR being a surfacesensitive characterization method not only can be used for analyzing the kinetic data including the equilibrium constant and the association and dissociation parameters between biomolecules by simulating SPR kinetic curves but also can be used in situ to detect the concentrations of biomolecules with high sensitivity and selectivity. 14,15 The surface plasmon used in SPR spectroscopy is highly sensitive to changes in the effective refractive index or the thickness of the test medium in the vicinity of the metal surface, especially for the molecules with high mass change. (3) Obata, K.; Tajima, H.; Yohda, M.; Matsunaga, T. Pharmacogenomics 2002, 3, 697–708. (4) Safarik, I.; Safarikova, M. BioMag. Res. Technol. 2004, 2, 7. (5) Pamme, N. Lab Chip 2006, 6, 24–38. (6) Zakhireh, J.; Gomez, R.; Esserman, L. Eur. J. Cancer 2008, 44, 2742–2752. (7) Safarik, I.; Safarikova, M. J. Chromatogr., B 1999, 722, 33–53. (8) Hsing, I. M.; Xu, Y.; Zhao, W. T. Electroanalysis 2007, 19, 755–768. (9) Ravindranath, S. P.; Mauer, L.; DebRoy, C.; Irudayaraj, J. Anal. Chem. 2009, 81, 2840–2846. (10) Song, Y. J.; Zhao, C.; Ren, J. S.; Qu, X. G. Chem. Commun. 2009, 1975– 1977. (11) Arakaki, A.; Hideshima, S.; Nakagawa, T.; Niwa, D.; Tanaka, T.; Matsunaga, T. Biotechnol. Bioeng. 2004, 88, 543–546. (12) Perez, J. M.; Josephson, L.; O’Loughlin, T.; Hogemann, D.; Weissleder, R. Nat. Biotechnol. 2002, 20, 816–820. (13) Li, Y.; Hong Cu, Y. T.; Luo, D. Nat. Biotechnol. 2005, 23, 885–889. (14) Li, X.; Wei, X. L.; Husson, S. M. Biomacromolecules 2004, 5, 869–876. (15) Li, X.; Husson, S. M. Biosens. Bioelectron. 2006, 22, 336–348. 10.1021/ac100812c © 2010 American Chemical Society Published on Web 07/20/2010
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. Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6783
Page 1 and 2: Anal. Chem. 2010, 82, 6745-6750 Let
Page 3 and 4: purchased from Invitrogen-Molecular
Page 5 and 6: Figure 3. Electropherograms of TPP-
Page 7 and 8: Anal. Chem. 2010, 82, 6751-6755 Res
Page 9 and 10: (pH 8.0) with cysteine and cystamin
Page 11 and 12: Figure 4. Ion mobility mass spectra
Page 13 and 14: GOx glucose + O298 gluconic acid +
Page 15 and 16: coater at 2000 rpm for 20 s, and th
Page 17 and 18: Figure 5. Comparison of cyclic volt
Page 19 and 20: in channels with either no grooves
Page 21 and 22: indicators of atmospheric processin
Page 23 and 24: Figure 1. GC/MS total ion chromatog
Page 25 and 26: Table 2. Concentrations and Stable
Page 27 and 28: on a substrate are preferred. 20-24
Page 29 and 30: Figure 4. SERS analysis of NAADP co
Page 31 and 32: Anal. Chem. 2010, 82, 6775-6781 Hig
Page 33 and 34: tion, 2 µL of proprionaldehyde wer
Page 35 and 36: Figure 4. Analysis of 2a by HPLC-MS
Page 37: eaction of 1a with PBH can be condu
Page 41 and 42: Figure 1. TEM images of the prepare
Page 43 and 44: Figure 4. Schematic representation
Page 45 and 46: esult in a big SPR signal change wi
Page 47 and 48: also reduces chemical noise, which
Page 49 and 50: Table 1. Extraction Yields, Liquid
Page 51 and 52: scanning of AMPP amides of the anal
Page 53 and 54: Anal. Chem. 2010, 82, 6797-6806 δ
Page 55 and 56: Figure 1. Schematic view of the pre
Page 57 and 58: from the specimen and enclosed in a
Page 59 and 60: Figure 4. (A) IRMS mass-44 chromato
Page 61 and 62: Table 3. Ambient Measurement Result
Page 63 and 64: Anal. Chem. 2010, 82, 6807-6813 Dir
Page 65 and 66: Polymerase (1 U per sample). Reacti
Page 67 and 68: of which was constant for all ampli
Page 69 and 70: analytically useful signals at less
Page 71 and 72: carbon black and RP-C18 for the ext
Page 73 and 74: solution in an equal volume, and 1
Page 75 and 76: Table 2. Concentrations and Ratios
Page 77 and 78: Anal. Chem. 2010, 82, 6821-6829 Mac
Page 79 and 80: mg/mL protein, followed by separati
Page 81 and 82: Figure 2. Productivity of SEQUST an
Page 83 and 84: Figure 4. High-resolution MS/MS spe
Page 85 and 86: Figure 6. Characterization of PSMs
Page 87 and 88: containing T-T mismatches. 23 Based
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Figure 1. Extinction spectra of sol
Page 91 and 92:
Figure 3. (A) The value of Ex650 nm
Page 93 and 94:
Figure 5. Extinction spectra and co
Page 95 and 96:
wished to explore the dehydration o
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constant medium for separation; we
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Figure 2. Standards of (Pi)n, n ) 1
Page 101 and 102:
Figure 5. Quantitative calibration
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Anal. Chem. 2010, 82, 6847-6853 Met
Page 105 and 106:
Figure 1. 226 Ra spectrum by liquid
Page 107 and 108:
Table 1. Counting Properties and De
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Table 3. Analysis of 226 Ra in Sedi
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mercial microarray scanner and fabr
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Figure 2. Optical transmission meas
Page 115 and 116:
Figure 5. Volcano plots detailing t
Page 117 and 118:
data as well. The 41 genes in Table
Page 119 and 120:
corresponding compound if its chemi
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Figure 1. Schematic illustration of
Page 123 and 124:
Table 1. Absolute Quantification Re
Page 125 and 126:
ment. Therefore, the long-time drea
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Figure 1. Chemically actuated micro
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Figure 2. Influence of a surfactant
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Figure 5. Device to eject and mix s
Page 133 and 134:
Anal. Chem. 2010, 82, 6877-6886 Imm
Page 135 and 136:
NaCl, phosphate buffer saline (PBS)
Page 137 and 138:
Figure 2. Product ion mass spectra
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-70 °C resolved the problem, givin
Page 141 and 142:
Table 1. Intraday Precision and Acc
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Anal. Chem. 2010, 82, 6887-6894 Fer
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Figure 1. Infrared spectra of (A) u
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Figure 3. Cyclic voltammograms obta
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Figure 6. Calculated charge from ch
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Anal. Chem. 2010, 82, 6895-6903 Ele
Page 153 and 154:
Table 1. Chemical Structure, pKa Va
Page 155 and 156:
pH with a tilted baseline (Figure 1
Page 157 and 158:
Table 2. Linearity and Detection Li
Page 159 and 160:
ascorbic acid (AA), uric acid (UA),
Page 161 and 162:
the multielement capabilities, the
Page 163 and 164:
RESULTS AND DISCUSSION Sulfur Detec
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Table 2. Molecular Properties and C
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Anal. Chem. 2010, 82, 6911-6918 Dir
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dilution and hybridization buffer.
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solution under appropriate incubati
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Figure 4. Standardization curve for
Page 175 and 176:
Anal. Chem. 2010, 82, 6919-6925 Ele
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the ×10 objective, to have a large
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Figure 2. With a suitable removal o
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the NB signal in a much better foot
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Scheme 1. Reactions of Selenium Rea
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Figure 2. (a) ESI-MS spectrum showi
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Figure 4. (a) ESI-MS spectrum showi
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Anal. Chem. 2010, 82, 6933-6939 Dif
Page 191 and 192:
trode 28 by a finite element using
Page 193 and 194:
Figure 4. Comparison between simula
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Figure 6. Comparison between simula
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educed in the vicinity of double bo
Page 199 and 200:
Figure 2. Normalized product ion ab
Page 201 and 202:
Figure 4. EID (a) and IRMPD (b) of
Page 203 and 204:
Anal. Chem. 2010, 82, 6947-6957 Ide
Page 205 and 206:
Figure 1. Schematic flowchart showi
Page 207 and 208:
difference, ppm compound Table 1. I
Page 209 and 210:
isoforms, its successful use, in th
Page 211 and 212:
Figure 5. Extracted ion current ESI
Page 213 and 214:
m/z 1172.935 was observed for Ser14
Page 215 and 216:
linked products via affinity tags.
Page 217 and 218:
Scheme 2. Fragmentation Mechanism o
Page 219 and 220:
Figure 1. (A) ESI-LTQ-CID-MS 2 prod
Page 221 and 222:
Figure 3. (A) ESI-LTQ-CID-MS 2 prod
Page 223 and 224:
Figure 5. (A) MALDI-TOF/TOF product
Page 225 and 226:
Anal. Chem. 2010, 82, 6969-6975 Ana
Page 227 and 228:
Figure 3. Equilibrium response as a
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Figure 6. The average measured resp
Page 231 and 232:
for the fill time, and we find that
Page 233 and 234:
nucleotide tails. 3-5 Thus, the amo
Page 235 and 236:
allow the use of higher aptamer con
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quent ligation of the aptamers afte
Page 239 and 240:
Anal. Chem. 2010, 82, 6983-6990 Imp
Page 241 and 242:
Figure 2. Configuration editor wind
Page 243 and 244:
Figure 4. Dependencies between volu
Page 245 and 246:
Since the time for liquid expulsion
Page 247 and 248:
Anal. Chem. 2010, 82, 6991-6999 Int
Page 249 and 250:
Figure 1. Representation of the Car
Page 251 and 252:
Table 2. Capillary Electrophoresis
Page 253 and 254:
Figure 4. (A) Typical raw electroph
Page 255 and 256:
field. DNA profiles were delivered
Page 257 and 258:
Another approach to handle this lim
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(principal components, PCs) in whic
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Figure 5. Relevant score plots and
Page 263 and 264:
CONCLUSIONS In this paper we have i
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electroactive-species loaded liposo
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Figure 2. Qdot-based FLFTS response
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Figure 6. Fluorescence imaging of Q
Page 271 and 272:
Anal. Chem. 2010, 82, 7015-7020 Sel
Page 273 and 274:
Table 1. Effect of 1 D-LC Condition
Page 275 and 276:
Figure 3. Peak-production rate vers
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Anal. Chem. 2010, 82, 7021-7026 Cel
Page 279 and 280:
luciferase (T7 control vector) was
Page 281 and 282:
Figure 3. Inhibitory effects of luc
Page 283 and 284:
Anal. Chem. 2010, 82, 7027-7034 Pat
Page 285 and 286:
Electrochemistry. Electrochemical m
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Figure 2. SEM images of Au substrat
Page 289 and 290:
Table 3. Film Thickness Measurement
Page 291 and 292:
Anal. Chem. 2010, 82, 7035-7043 Qua
Page 293 and 294:
Figure 1. (A) FL spectra of BSPOTPE
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Figure 4. (A) Variation in the FL i
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Figure 6. (A) FL spectra of BSPOTPE
Page 299 and 300:
Scheme 1. Proposed Mechanism for Fl
Page 301 and 302:
one or more drawbacks including poo
Page 303 and 304:
Figure 2. Free fluorophores do not
Page 305 and 306:
Anal. Chem. 2010, 82, 7049-7052 Dev
Page 307 and 308:
Figure 2. Comparative analysis of d
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Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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