Magnetic Nanoparticle Enhanced Surface Plasmon Resonance

Jul 20, 2010 - Magnetic nanoparticles (MNPs) have been frequently used in bioseparation, but their applicability in bioassays is limited due to their ...
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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: [email protected]. (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.

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Up to now, all kinds of biomolecules including DNA and RNA,3 protein and peptides,4 cell and virus5-7 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 NPs23 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′-NH2-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 for 1 h 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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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 ssDNA modified SPR gold film was immersed in 100 µM 6-mercaptohexanol for 1 h to block the uncovered gold surface. This gold film was used as a sensing surface to detect the amount of aptamer possessing ss-DNA structure on Fe3O4 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. (30) Wang, J. L.; Munir, A.; Li, Z. H.; Zhou, H. S. Biosens. Bioelectron. 2009, 25, 124–129.

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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 Fe2+/Fe3+ 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 Fe2+/Fe3+ 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. (31) Lu, A. H.; Salabas, E. L.; Schuth, F. Angew. Chem., Int. Ed. 2007, 46, 1222– 1244. (32) Kang, Y. S.; Risbud, S.; Rabolt, J. F.; Stroeve, P. Chem. Mater. 1996, 8, 2209–2211. (33) Massart, R. IEEE Trans. Magn. 1981, 17, 1247–1248. (34) Murray, C. B.; Norris, D. J.; Bawendi, M. G. J. Am. Chem. Soc. 1993, 115, 8706–8715. (35) Peng, X.; Wickham, J.; Alivisatos, A. P. J. Am. Chem. Soc. 1998, 120, 5343– 5344. (36) O’Brien, S.; Brus, L.; Murray, C. B. J. Am. Chem. Soc. 2001, 123, 12085– 12086. (37) Sun, S.; Zeng, H.; Robinson, D. B.; Raoux, S.; Rice, P. M.; Wang, S. X.; Li, G. J. Am. Chem. Soc. 2004, 126, 273–279. (38) Redl, F. X.; Black, C. T.; Papaefthymiou, G. C.; Sandstrom, R. L.; Yin, M.; Zeng, H.; Murray, C. B.; O’Brien, S. P. J. Am. Chem. Soc. 2004, 126, 14583– 14599. (39) Lyon, L. A.; Pena, D. J.; Natan, M. J. J. Phys. Chem. B 1999, 103, 5826– 5831.

Figure 1. TEM images of the prepared Fe3O4 MNPs: (A) 14.51 nm and (B) 32.82 nm.

Figure 2. Variation of SPR angle-time curves with the concentration of Fe3O4 MNPs: (A) 14.51 nm; (B) 32.82 nm. (C) Comparison of the variation of SPR angle shift with the concentration of Fe3O4 MNPs of 14.51 and 32.82 nm, respectively.

Figure 2 illustrates the changes that occur in the SPR angle shift as a function of the concentration for 14.51 nm (Figure 2A) and 32.82 nm (Figure 2B) Fe3O4 MNPs. In the case of 14.51 nm

Fe3O4 MNPs (Figure 2A), the SPR angle shifts gradually increase from 6.4 to 1111.06 m° with the increase of Fe3O4 MNP concentrations from 0.016 to 1.6 nM. It is evident that a higher Analytical Chemistry, Vol. 82, No. 16, August 15, 2010

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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 isotherm42 could be used to fit the data. The Langmuir equation used to fit the data is given by

∆λ ) ∆λmax

KA[C]Fe3O4 1 + KA[C]Fe3O4

(1)

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.

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calculated to be around 7.14 × 108 and 2.63 × 109 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 0m, 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.

Figure 4. Schematic representation of the SPR biosensor for the detection of the small molecules.

Fe3O4 MNP Enhanced SPR Sensing for the Detection of Small Molecules. To further demonstrate the practicability of the amplification effect of Fe3O4 MNPs in an enhancing SPRbased bioassay, a novel SPR sensor based on indirect competitive inhibition assay (ICIA) for the detection of adenosine is constructed. The principle of this SPR sensor is shown in Figure 4. The partial complementary thiolated ss-DNA of antiadenosine aptamer is first immobilized on SPR gold film as a sensing surface. When Fe3O4 MNP-antiadenosine aptamer conjugate solution is added to the SPR cell in the absence of adenosine, Fe3O4 MNP-antiadenosine aptamer conjugates will be adsorbed to the SPR sensor by the DNA hybridization reaction and result in a huge change of SPR signal due to the amplification effect of Fe3O4 MNPs. However, the change of SPR signal will decrease after Fe3O4 MNP-antiadenosine aptamer conjugates bind with adenosine. This is because adenosine reacts with antiadenosine aptamer in Fe3O4 MNPantiadenosine aptamer conjugates and changes its structure from ss-DNA to tertiary structure, which cannot hybridize with its partial complementary ss-DNA immobilized on the SPR gold surface. Thus, the change of SPR signal will decrease with the increase of the number of Fe3O4 MNP-antiadenosine aptamer conjugates possessing tertiary structure, which is proportional to the concentration of adenosine. The essential prerequisite of this detection is to prepare Fe3O4 MNP-antiadenosine aptamer conjugates. For this purpose, EDC and NHS are used as a carboxyl activating agent for the coupling of primary amines in aptamer and carboxyl groups in polymer coated MNPs. To demonstrate that MNPs have been labeled by an aptamer successfully, FT-IR spectroscopy is used to evaluate the surface modification of MNPs before and after aptamer labeling. Figure 5 shows the IR absorbance spectra of Fe3O4 MNPs before and after the aptamer label. For the polymer coated Fe3O4 MNPs, the absorbance peak near 1710 cm-1 is assigned to υ (CdO) (stretch vibration of carbonyl) and amide I, peaks at 1550 cm-1 are assigned to amide II, and the peaks at 2854 and 2925 cm-1 are assigned to υs (C-H2) and υas (C-H2) (symmetric and asymmetric stretch vibrations of C-H2), respectively. In the region of 3200-3570

Figure 5. FT-IR of polymer coated Fe3O4 MNPs before and after aptamer labeling.

cm-1, a peak due to the O-H stretch is expected. Several new peaks are observed after MNPs are labeled by the aptamer. The peaks at 1048 and 1211 cm-1 are attributed to stretching vibrations of the PO2- in the aptamer. It should be pointed out that we are not able to confirm the presence of DNA-related peaks at 1550 cm-1 because the peak overlaps with amide II. After Fe3O4 MNP-antiadenosine aptamer conjugates are prepared successfully, the SPR detection is carried out according to the principle described in Figure 4. Figure 6A shows the SPR angle-time curves of the separation products obtained after Fe3O4 MNP-antiadenosine aptamer conjugates are reacted with different concentrations of adenosine for 30 min. In the absence of adenosine, Fe3O4 MNP-antiadenosine aptamer conjugates in solution directly hybridize with ssDNA immobilized on SPR gold film, resulting in the largest SPR angle shift (∼1082.94 m°). This angle shift is much larger than that of the angle shift resulting from the binding of ss-DNA or most of the protein. The SPR angle shift resulting from the binding of Fe3O4 MNP-antiadenosine aptamer conjugates decreases (∼820.14 m°) after Fe3O4 MNP-antiadenosine aptamer conjugates are reacted with Analytical Chemistry, Vol. 82, No. 16, August 15, 2010

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Figure 6. (A) SPR angle-time curves of the separation products obtained after Fe3O4 MNP-antiadenosine aptamer conjugates are reacted with different concentrations of adenosine for 30 min. Inset: The linear relationship between the logarithms of adenosine concentrations and the SPR angle shift resulting from the binding of Fe3O4 MNP-antiadenosine aptamer conjugates. (B) SPR angle-time curves of 1 µM antiadenosine aptamer without adenosine (red line) and after react with 1 mM adenosine for 30 min (black line).

10 nM adenosine because parts of aptamers on Fe3O4 MNPs react with adenosine and form its tertiary structure, which cannot hybridize with its complementary ss-DNA. With the further increase of the concentration of adenosine added to the Fe3O4 MNP-antiadenosine aptamer conjugate solution, the SPR angle shift continuously decreases until most of the antiadenosine aptamer binds with adenosine. By analyzing the change of SPR angle shift with the concentrations of adenosine, a good linear relationship between the logarithms of adenosine concentrations and the SPR angle shift is obtained with a range of 10-1 × 104 nM (shown in the insert of Figure 6A). This detection result is comparable with most other aptasensors44 and is a little lower than that of detection results from SPR aptasensor which utilize Au NPs as an amplification reagent20,29 but much superior to the detection results obtained by a general SPR sensor based on a molecularly imprinted technique.45,46 It should be pointed out that the SPR angle shift only decreases to ∼107.33 m° even when the aptamer reacts with 1 mM adenosine. This nonspecific adsorption may come from the stereohindrance effect of aptamer possessing different structures. After adenosine is added to Fe3O4 MNP-aptamer conjugates, most free-coiled aptamers react with adenosine and form its tertiary structure. However, the formation of a tertiary structure of an aptamer may inhibit the further reaction between its adjacent free-coiled aptamer and adenosine. To clearly show the amplification effect of Fe3O4 MNPs, as a comparison, the SPR response resulting from the binding of aptamer without MNP is also studied. The black line in Figure 6B is the SPR angle shift resulting from the binding of 1 µM antiadenosine aptamer after antiadenosine aptamer reactes with 1 mM adenosine for 0.5 h. Only a 24.6 m° SPR angle shift is observed. Although the SPR angle shift resulting from the binding of antiadenosine aptamer could increase to 65.7 m° (Red line in Figure 6B) in the absence of adenosine, this value is still much lower than that of the (44) Wang, Y. L.; Wei, H.; Li, B. L.; Ren, W.; Guo, S. J.; Dong, S. J.; Wang, E. K. Chem. Commun. 2007, 5220–5222. (45) Taniwaki, K.; Hyakutake, A.; Aoki, T.; Yoshikawa, M.; Guiver, M. D.; Robertson, G. P. Anal. Chim. Acta 2003, 489, 191–198. (46) Yoshikawa, M.; Guiver, M. D.; Robertson, G. P. J. Mol. Struct. 2005, 739, 41–46.

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Figure 7. SPR angle-time curves of the separation products obtained after Fe3O4 MNP-antiadenosine aptamer conjugates are reacted with different analytes for 30 min.

value resulting from the binding of Fe3O4 MNP-antiadenosine aptamer conjugates (1082.94 m°). Besides sensitivity, the specification of aptamer promises the selectivity of the present SPR sensor for adenosine. Figure 7 exhibits SPR angle-time curves of the separation products obtained after Fe3O4 MNP-antiadenosine aptamer conjugates are reacted with different analytes for 30 min. It could be easily seen that the addition of Fe3O4 MNP-antiadenosine aptamer conjugates which are reacted with 1 × 106 nM cytidine, guanosine, and uridine result in big SPR angle shifts. However, The SPR angle shift only increases a little after Fe3O4 MNP-antiadenosine aptamer conjugates are reacted with 1 × 106 nM adenosine. These results not only demonstrate that Fe3O4 MNPs could greatly enhance the SPR signal but also, more importantly, give us an important indication that SPR spectroscopy could be an excellent candidate for detecting an MNP-based separation product. CONCLUSION In summary, the SPR response of the carboxyl group modified Fe3O4 MNPs of two different sizes onto an amino group modified SPR gold substrate has been studied. The results show the monolayer adsorption of Fe3O4 MNPs could

result in a big SPR signal change with a low optical loss. To evaluate the practicability of the use of Fe3O4 MNPs in enhancing the SPR signal for biosensing, a novel SPR sensor based on ICIA for the detection of adenosine is constructed using Fe3O4 MNP-antiadenosine aptamer conjugates as the amplification reagents. The experimental results demonstrate that the SPR sensor possesses a good sensitivity and a high selectivity for adenosine. Importantly, by changing the kind of biomolecules labeled by MNPs, the present detection method will be able to explore new applications of SPR spectroscopy for the detection of a large variety of MNPbased separation products. At the same time, the technique demonstrated in this work could also offer a new direction

in designing high performance SPR biosensors for sensitive and selective detection of small molecules by the amplification effect of MNPs. ACKNOWLEDGMENT This work is supported by National Science Foundation (EEC0823974). Authors thank Prof. Camesano Terri and her graduate student Sena Ada for assistance with AFM characterization.

Received for review March 10, 2010. Accepted July 8, 2010. AC100812C

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