In Situ Surface Plasmon Resonance Investigation of the Assembly

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In Situ Surface Plasmon Resonance Investigation of the Assembly Process of Multiwalled Carbon Nanotubes on an Alkanethiol Self-Assembled Monolayer for Efficient Protein Immobilization and Detection Weihua Hu, Zhisong Lu, Yingshuai Liu, and Chang Ming Li* School of Chemical & Biomedical Engineering, Center for Advanced Bionanosystems, Nanyang Technological University, 70 Nanyang Drive, Singapore 637457 Received December 21, 2009. Revised Manuscript Received February 18, 2010 In situ surface plasmon resonance (SPR) was used to study the assembly process of multiwalled carbon nanotubes (MWCNTs) quantitatively on an alkanethiol self-assembled monolayer (SAM) surface, showing that MWCNTs can follow the Langmuir adsorption kinetics to assemble spontaneously whereas the assembly temperature has an essential influence on the assembly kinetics and the surface distribution of MWCNTs. To further in situ investigate protein attachment on the MWCNT surface and its sensing application quantitatively, goat IgG was immobilized by three strategies: direct adsorption, covalent binding, and 1-pyrenebutanoic acid, succinimidyl ester (PBSE)-assisted attachment, of which the covalent binding approach provides the best protein loading capacity. The SPR label-free detection of anti-goat IgG demonstrates excellent performance with high sensitivity, good specificity, and rapid response in comparison to that with a plain substrate without MWCNT assembly reported in our previous work. This is contributed by the 3D MWCNT assembly matrix providing a high probe immobilization capability and superb accessibility for the target to enhance its sensing performance significantly.

Introduction Carbon nanotubes (CNTs) have fueled enormous interest in different research areas because of their unique geometrical, mechanical, physical, and chemical properties. In particular, in recent years CNTs have been extensively investigated as carriers for loading various biomolecules, such as protein, DNA, RNA, and other bioactive components for intracellular transport, drug delivery, bioimaging, and biosensing applications.1-7 Numerous biosensors utilizing the physicochemical properties and superior bio/electrocatalytic performance of CNTs have been successfully constructed for the sensitive detection of small-molecular-weight species of interest. However, the immunosensors using CNTs as protein carriers are still in their nascent stage.8 The main challenge is to assemble CNTs stably on the transducer surface in a proper fashion that allows for the convenient immobilization of receptor biomolecules and offers excellent accessibility for targets. To assemble CNTs on substrates, a simple way is physical adsorption by evaporating solution or dip-in coating, but it is *Corresponding author. Tel: þ65 67904485. Fax: þ65 67911761. E-mail: [email protected].

(1) Kam, N. W. S.; Dai, H. J. Am. Chem. Soc. 2005, 127, 6021. (2) Jia, N.; Lian, Q.; Shen, H.; Wang, C.; Li, X.; Yang, Z. Nano Lett. 2007, 7, 2976. (3) You, Y. Z.; Hong, C. Y.; Pan, C. Y. J. Phys. Chem. C 2007, 111, 16161. (4) Welsher, K.; Liu, Z.; Daranciang, D.; Dai, H. Nano Lett. 2008, 8, 586. (5) Besteman, K.; Lee, J. O.; Wiertz, F. G. M.; Heering, H. A.; Dekker, C. Nano Lett. 2003, 3, 727. (6) Ju, S. Y.; Papadimitrakopoulos, F. J. Am. Chem. Soc. 2008, 130, 655. (7) ShiKam, N. W.; Jessop, T. C.; Wender, P. A.; Dai, H. J. Am. Chem. Soc. 2004, 126, 6850. (8) Veetil, J. V.; Ye, K. Biotechnol. Prog. 2007, 23, 517. (9) Male, K. B.; Hrapovic, S.; Santini, J. M.; Luong, J. H. T. Anal. Chem. 2007, 79, 7831. (10) Erdem, A.; Papakonstantinou, P.; Murphy, H. Anal. Chem. 2006, 78, 6656. (11) Liu, G.; Lin, Y. Anal. Chem. 2006, 78, 835. (12) Joshi, K. A.; Prouza, M.; Kum, M.; Wang, J.; Tang, J.; Haddon, R.; Chen, W.; Mulchandani, A. Anal. Chem. 2006, 78, 331. (13) Zhang, M.; Liu, K.; Xiang, L.; Lin, Y.; Su, L.; Mao, L. Anal. Chem. 2007, 79, 6559.

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limited by weak stability.9-14 The stability could be enhanced if casting CNTs onto the substrate with binders.15-17 However, the binders often completely block the access of proteins in the solution. Decorating CNTs with polymers18,19 or thiols6,20-22 provides a promising alternative to tethering CNTs on a solid substrate, but the process is tedious. Most importantly, it is hard to obtain a density-controllable homogeneous CNT film with a uniform distribution for the above-discussed approaches. Intensive efforts have been focused on the direct assembly of CNTs or other nanowires on self-assembled monolayer (SAM) modified surfaces.23-29 Compatible with nanofabrication techniques such as dip-pen nanolithography, this method allows the large-scale assembly of nanotubes into well-defined functional (14) Yang, Y.; Zhu, Y.; Chen, Q.; Liu, Y.; Zeng, Y.; Xu, F. Small 2009, 5, 351. (15) Hrapovic, S.; Majid, E.; Liu, Y.; Male, K.; Luong, J. H. T. Anal. Chem. 2006, 78, 5504. (16) Tsai, Y. C.; Li, S. C.; Chen, J. M. Langmuir 2005, 21, 3653. (17) Valentini, F.; Amine, A.; Orlanducci, S.; Terranova, M. L.; Palleschi, G. Anal. Chem. 2003, 75, 5413. (18) Paloniemi, H.; Lukkarinen, M.; Aaritalo, T.; Areva, S.; Leiro, J.; Heinonen, M.; Haapakka, K.; Lukkari, J. Langmuir 2006, 22, 74. (19) Ding, Y. J.; Liu, J.; Jin, X. Y.; Lu, H. X.; Shen, G. L.; Yu, R. Q. Analyst 2008, 133, 184. (20) Profumo, A.; Fagnoni, M.; Merli, D.; Quartarone, E.; Protti, S.; Dondi, D.; Albini, A. Anal. Chem. 2006, 78, 4194. (21) Yu, X.; Munge, B.; Patel, V.; Jensen, G.; Bhirde, A.; Gong, J. D.; Kim, S. N.; Gillespie, J.; Gutkind, J. S.; Papadimitrakopoulos, F.; Rusling, J. F. J. Am. Chem. Soc. 2006, 128, 11199. (22) Gooding, J. J.; Wibowo, R.; Liu, J. Q.; Yang, W.; Losic, D.; Orbons, S.; Mearns, F. J.; Shapter, J. G.; Hibbert, D. B. J. Am. Chem. Soc. 2003, 125, 9006. (23) Huang, Y.; Duan, X.; Wei, Q.; Lieber, C. M. Science 2001, 291, 630. (24) Rao, S. G.; Huang, L.; Setyawan, W.; Hong, S. Nature 2003, 425, 36. (25) Hannon, J. B.; Afzali, A.; Klinke, C.; Avouris, P. Langmuir 2005, 21, 8569. (26) Im, J.; Kang, J.; Lee, M.; Kim, B.; Hong, S. J. Phys. Chem. B 2006, 110, 12839. (27) Su, L.; Gao, F.; Mao, L. Anal. Chem. 2006, 78, 2651. (28) Liu, J.; Casavant, M. J.; Cox, M.; Walters, D. A.; Boul, P.; Lu, W.; Rimberg, A. J.; Smith, K. A.; Colbert, D. T.; Smalley, R. E. Chem. Phys. Lett. 1999, 303, 125. (29) Wang, Y.; Maspoch, D.; Zou, S.; Schatz, G. C.; Smalley, R. E.; Mirkin, C. A. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 2026.

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Scheme 1. Schematic Illustration (Not to Scale) of an MWCNT Assembly on OCT-SAM and Sequential Protein Immobilization Using Three Strategiesa

a

Method I: direct adsorption. Method II: covalent binding. Method III: using PBSE as the linker.

networks. However, information concerning the assembly kinetics and stability is still scarce although it is critical for practical applications of CNT assemblies. To study and gain deeper insight into assembly in surface science systematically, in situ investigations are required. The attachment of bioreceptors to CNTs is another challenging task for CNT-based immunosensors. Via carbodiimide chemistry, the covalent attachment of proteins can be conveniently achieved by forming stable amide bonds6,30 through the carboxyl groups on CNTs. The noncovalent immobilization of biomolecules on CNTs by direct physical adsorption,1,7,22,31 electrostatic self-assembly using the layer-by-layer method,2,11 or employing special anchor molecules such as pyrenyl5,32 and phospholipid4,33 may be a good alternative because it can retain the superior electronic properties of CNTs. Nevertheless, the quantitative loading capacities of CNTs using different immobilization strategies are still not available currently. In this work, the assembly process of MWCNTs on the alkanethiol SAM at different temperatures was studied in situ using a surface plasmon resonance (SPR) technique for the first time. Goat IgG, as a model probe protein, was immobilized on the MWCNT assembly via direct adsorption, covalent attachment, or using 1-pyrenebutanoic acid, succinimidyl ester (PBSE) as the linker (Scheme 1). The respective protein loading capacities of these three methods were evaluated on the basis of SPR measurements. SPR detection of the target was also conducted to demonstrate the excellent performance of the resulting MWCNT matrix for immunosensing.

Experimental Section Chemicals and Reagents. n-Octadecanethiol (OCT, CH3(CH2)17SH), phosphate-buffered saline (PBS), bovine serum albumin (BSA), goat IgG (purified immunoglobulin, technical grade), anti-goat IgG (whole molecule, developed in rabbit), anti-bovine IgG (clone BG-18, mouse ascites fluid), 1-ethyl3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and N-hydroxysulfosuccinimide (sulfo-NHS) were purchased from Sigma-Aldrich. PBSE was purchased from AnaSpec Inc. (San Jose, CA). MWCNTs were purchased from Shenzhen Nanotech Co. Ltd. (China) and were refluxed in 2.6 M nitric acid for 2 h, followed by centrifugation, resuspension, filtration, and drying. All chemicals are of analytical or higher grade and used as received. All protein solutions were prepared by dilution with 0.01 M PBS (pH 7.4) and stored at 4 °C before use. EDC and sulfo-NHS were freshly dissolved in 0.01 M PBS, pH 5.5 before use. Deionized water used in this work was from a Millipore Milli-Q water purification system. SPR Equipment and Measurements. SPR measurements were performed with an Autolab SPRINGLE system (Echo Chemie B.V., Netherlands) equipped with a 670 nm monochromatic p-polarized light resource, as described previously.34-36 The SPR angle (θSPR, where minimum reflectivity obtained) was recorded in situ at a frequency of 1 Hz. For the investigation of the temperature-dependent MWCNT assembly, the temperature is controlled by flowing constant-temperature water from a water bath through a channel surrounding the SPR chamber and prism. Prior to each experiment, the SPR cell and gold chip were thoroughly cleaned with an ultrasonic cleaner with deionized water and acetone each for 5 min.

Assembly of MWCNTs on a SAM-Modified Au Surface.

(30) Huang, W.; Taylor, S.; Fu, K.; Lin, Y.; Zhang, D.; Hanks, T. W.; Rao, A. M.; Sun, Y. P. Nano Lett. 2002, 2, 311. (31) Fadel, T. R.; Steenblock, E. R.; Stern, E.; Li, N.; Wang, X.; Haller, G. L.; Pfefferle, L. D.; Fahmy, T. M. Nano Lett. 2008, 8, 2070. (32) Chen, R. J.; Zhang, Y.; Wang, D.; Dai, H. J. Am. Chem. Soc. 2001, 123, 3838. (33) Liu, Z.; Cai, W.; He, L.; Nakayama, N.; Chen, K.; Sun, X.; Chen, X.; Dai, H. Nat. Nano 2007, 2, 47.

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To form a compact SAM on the Au surface, 200 μL of 1 mM OCT ethanol solution was injected into the SPR chamber to incubate with the precleaned Au disk for 24 h, followed by (34) Hu, W.; Li, C. M.; Cui, X.; Dong, H.; Zhou, Q. Langmuir 2007, 23, 2761. (35) Dong, H.; Cao, X.; Li, C. M.; Hu, W. Biosens. Bioelectron. 2008, 23, 1055. (36) Hu, W.; Li, C. M.; Dong, H. Anal. Chim. Acta 2008, 630, 67.

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Hu et al. where 100 μg/mL goat IgG was directly added to the OCT-SAM surface and incubated for 2 h. SPR detection of the target protein, anti-goat IgG, was conducted on the sensing surface prepared by the covalent attachment method. Before detection, 2 mg/mL BSA solution was injected into the chamber to block the surface from nonspecific adsorption for 1 h and then rinsed with 0.01 M PBS. After a stable baseline in 0.01 M PBS, 100 μL of an anti-goat IgG solution with a certain concentration was added to the chamber and incubated for 1 h. To examine the detection specificity, 10 μg/mL anti-bovine IgG in 0.01 M PBS was used as well.

Figure 1. SPR responses (solid lines) and fitting curves (open circles) of MWCNTs assembling from an ethanol suspension onto an OCT-SAM surface at 4 (a), 25 (b), and 55 °C (c). Baselines are obtained in absolute ethanol. The right inset shows SPR plots before (black) and after (red) MWCNT assembly. The bottom inset shows the SPR response of the MWCNT assembly in 0.01 M PBS. intensive rinsing with ethanol. Then 200 μL of 0.2 mg/mL MWCNT ethanol solution was added to the chamber for a specific time, followed by intensive rinsing with ethanol. For low- or high-temperature (4 or 55 °C) assembly, the MWCNT solution was allowed to balance at the same temperature for 5 min prior to addition to the chamber. To examine the stability of adsorbed MWCNTs, 200 μL of 0.01 M PBS was injected to incubate with the MWCNT assembly for 24 h. Except for the MWCNT assembly, other experiments were conducted at room temperature.

Atomic Force Microscopy (AFM) and Fourier Transform Infrared (FTIR) Measurements. AFM was employed to characterize the surface distribution of the MWCNT assembly on SAM-modified Au. After the assembly was prepared by the same method described above, the SPR gold chip was removed from the prism and thoroughly dried in a gentle stream of N2 gas. AFM measurements were conducted on a Nanoman AFM (Veeco Metrology Group) in tapping mode at ambient temperature. FTIR spectra of MWCNTs and PBSE-modified MWCNTs were collected with a Spectrum GX (Perkin-Elmer, Wellesley, MA) via the KBr pellet method from 4000 to 400 cm-1 with 1 cm-1 resolution for 32 scans.

Immobilization of Goat IgG and the Detection of Antigoat IgG. Three strategies were employed to immobilize goat

IgG on the MWCNT assembly (corresponding to 900 mo angle increase), as shown in Scheme 1. The first method is direct adsorption, in which 100 μL of 100 μg/mL goat IgG solution was added to the SPR chamber and incubated with the MWCNT assembly for 2 h. The second method involves the traditional EDC/NHS method to covalently attach goat IgG. In detail, 100 μL of freshly prepared EDC (2 mM)/sulfo-NHS (5 mM) solution was injected into the SPR chamber to activate the carboxyl groups on MWCNTs by 0.5 h of incubation. After intensive washing with 0.01 M PBS and a stable SPR baseline, 100 μL of 100 μg/mL goat IgG was added and incubated for 2 h. The attached amount of goat IgG was determined after rinsing in 0.01 M PBS. The third strategy relies on PBSE as the anchor molecule. The MWCNT assembly was incubated with 1 mM PBSE in ethanol for 1 h, followed by washing with ethanol and PBS. Then 100 μL of 100 μg/mL goat IgG was added and incubated for 1 h. To understand the role of MWCNTs in protein immobilization, one control experiment was carried out for comparison, 8388 DOI: 10.1021/la9048105

Results and Discussion Assembly Behavior of MWCNTs on the OCT-SAM Surface. The assembly process of MWCNTs on OCT-SAM at different temperatures was monitored in situ and investigated by the SPR technique, as shown by the solid lines in Figure 1. The results have a good reproducibility of less than 5% relative standard deviation (RSD) and show that after the addition of the MWCNT ethanol solution the MWCNTs spontaneously assemble on the OCT-modified gold surface, resulting in a rapid increase in the SPR angle. The adsorbed amount of MWCNTs increases with adsorption time until a plateau value to achieve maximal coverage is reached. The results clearly reveal that the higher assembly temperature induces a faster adsorption rate. The SPR angle change is proportional to the number of adsorbed MWCNTs, and the in-situ-measured SPR results were used to study the kinetics of the assembly process. Generally, the adsorption of a substance at a liquid/solid interface can be described by Langmuir adsorption kinetics34 RðtÞ ¼ Req ð1 -e -ðka Cþkd Þt Þ

ð1Þ

where R(t), Req, ka, and kd represent the time-dependent surface density (or concentration) of MWCNTs, the equilibrium value for R at a certain bulk concentration C, the association rate constant, and the dissociation rate constant for the adsorption, respectively. Figure 1 also depicts the calculated fitting curves based on eq 1, which are in good agreement with the experimental curves (squared correlation factor R2 = 0.99641, 0.99682, and 0.98057 for curves a-c, respectively), suggesting that the adsorption is governed by the adsorption kinetics described in eq 1. The temperature displays an essential effect on the spontaneous assembly process. With increasing temperature from 4 to 55 °C, the assembly process takes place at a higher rate and reaches equilibrium more quickly. It is understandable that a higher temperature can accelerate the assembly process because the chaotic motion (Brownian movement) of MWCNTs in ethanol solution becomes stronger to have a higher probability of accessing the SAM surface and finally depositing on it. In fact, the effect of the environmental temperature on the reaction rate constants (ka and kd in this case) can be estimated by the empirical Arrhenius equation k ¼ Ae -Ea =RT where A, R, and Ea are the pre-exponential factor, the gas constant, and the activation energy for the adsorption (or desorption) process, respectively. Higher T results in larger k (i.e., a higher reaction rate). This is consistent with the temperaturedependent SPR responses shown in Figure 1. It is also confirmed by the fitting value for kaC þ kd (equal to 0.00929, 0.01228, and 0.02702 for 4, 25, and 55 °C, respectively). Langmuir 2010, 26(11), 8386–8391

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Figure 2. AFM images of MWCNTs assembled on an OCT-SAM surface at 4 (a), 25 (b), and 55 °C (c).

Figure 3. AFM image of the MWCNTs assembled on an OCTSAM surface corresponding to a 900 mo angle increase.

Both ka and kd change with temperature. Thus, when ΔEa > 0 (ΔEa = Eaa - Ead, the difference between the activation energies of the adsorption and desorption process), the equilibrium constant K (equal to ka/kd) will increase with increasing temperature; otherwise, K decreases. Theoretically, adsorption at a solid/liquid or solid/gas interface is an exothermic process (i.e., ΔEa < 0). Thus, a larger K should be obtained at a lower temperature. The SPR angular spectra recorded before (blank) and after (red) MWCNT assembly (inset in Figure 1) illustrate that after MWCNT assembly both the minimal reflectivity (resonance depth) and curve shape (resonance width) increase in addition to the 1200 mo angle increase. The changes could be ascribed to the nonzero imaginary dielectric constant (εim) of MWCNTs, which induces a decrease in the photon-plasmon coupling efficiency and a broadening of the angular spectrum.37 The stability of the MWCNT assembly in aqueous solution was investigated in 0.01 M PBS by SPR. Over 24 h, the SPR angle demonstrates a stable curve without any decline (inset in blue square of Figure 1), implying excellent stability of the adsorbed MWCNTs. Compared with the drop-dry method, this method apparently produces a much more stable MWCNT assembly. Morphology of Assembled MWCNTs. The morphology of the assembled MWCNTs was investigated by AFM. As shown in Figure 2b,c, the MWCNTs assembled at 25 and 55 °C uniformly (37) Kang, X.; Jin, Y.; Cheng, G.; Dong, S. Langmuir 2002, 18, 1713.

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distribute over a large area. The corresponding section analyses show that the height of most MWCNTs is below 80 nm, close to the diameter of the MWCNTs, which implies that the adsorbed MWCNTs are a monolayer assembly with mostly individual tubes rather than bundles. It has been reported that individual MWCNTs exist in the solution and possess a larger diffusion coefficient (i.e., stronger Brownian motion at a same temperature) compared with the bundles.26 Therefore, the individual MWCNTs have a higher probability of landing on the SAM surface. Very interestingly, however, for that assembled at 4 °C, many bundles on the surface and poor distribution homogeneity can be seen (Figure 2a), very likely resulting from the aggregation of MWCNTs at low temperature. As discussed above, when the temperature decreases, the intensity of the Brownian motion of MWCNTs decreases accordingly, thus causing the aggregation of MWCNTs to form bundles by the van der Waals force during assembly. Protein Immobilization on MWCNT Assembly. The surface density of an attached biomolecule probe is a critical parameter for biosensing applications. Until now, the protein immobilization on MWCNTs has not been quantitatively studied. In this work, as an example, goat IgG immobilization on the MWCNT assembly via three approaches (Scheme 1) was conducted and further quantitatively characterized by in situ SPR measurements to compare their immobilization capabilities. According to SPR plots in the inset of Figure 1, the MWCNT assembly induces an increase in the minimal reflectivity from ca. 5 to 25% and the SPR spectrum changes from a sharp dip to a broad shape. These can reduce the sensitivity of the sequential SPR measurements and lead to an underestimate of the real immobilized proteins when protein attachment takes place spatially far from the gold/solution interface.38 Therefore, to ensure accurate in situ measurements of sequential protein bindings, the assembled amount of MWCNTs was controlled to be equal (corresponding to a 900 mo angle change) by adjustment of the assembly time at 25 °C. A representative AFM image of the MWCNT assembly corresponding to a 900 mo SPR angle increase is displayed in Figure 3, showing an individual, stretched MWCNT feature on the OCT-SAM surface. (38) Liedberg, B.; Lundstrom, I.; Stenberg, E. Sens. Actuators, B 1993, 11, 63.

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Figure 4. SPR responses (a) and angle changes (b, n = 3) for goat IgG immobilization. (1) Direct adsorption onto OCT-SAM; (2) direct adsorption onto the MWCNT assembly; (3) covalent binding on the MWCNT assembly using the EDC/NHS method; and (4) binding on the MWCNT assembly using PBSE as the linker. The SPR response for sequential BSA incubation is shown on curve 3 in part a.

Figure 5. FTIR spectra of pristine MWCNTs (a) and PBSEmodified MWCNTs (b).

The real-time SPR responses to goat IgG attachment and the angle changes (Δθ, increase in the SPR angles) after immobilization are shown in Figure 4a,b, respectively. The number of attached proteins could be quantitatively calculated on the basis of the SPR angle change at a ratio of 120 mo for 1 ng/mm2.35,36 For comparison, the direct adsorption of goat IgG on the OCT-SAM surface without the MWCNT assembly is examined (curve 1), showing a very high value (ca. 487 ( 34 mo equal to a surface concentration of ca. 4.058 ng/mm2), possibly because of the strong hydrophobic attraction between goat IgG and the OCT surface. After the assembly of MWCNTs on the OCT surface, a much higher angle change (ca. 629 ( 45 mo equal to 5.242 ( 0.375 ng/mm2) is obtained after protein adsorption (curve 2). This is because the whole MWCNT 3D cylindrical surface could provide a larger surface area for protein adsorption than that of OCTSAM alone. In the PBSE-based protein attachment approach, the MWCNT is functionalized via π stacking between the aromatic pyrenyl group of PBSE and the sidewalls of MWCNTs, followed by a reaction of the succinimidyl ester group of PBSE with the amine groups of proteins.32 The characteristic peaks of PBSE are 8390 DOI: 10.1021/la9048105

observed on the FTIR spectrum of PBSE-treated MWCNTs (Figure 5b), explicitly confirming the PBSE modification of MWCNTs. The SPR angle reaches a plateau after 1 h of incubation in goat IgG solution with an angle increase of ca. 780 mo (curve 4 in Figure 4a, Δθ = 802 ( 52 mo in triplicate experiments equal to 6.683 ( 0.433 ng/mm2), which is larger than that of protein attachment through the direct absorption approach even though both approaches utilize the MWCNT sidewall. This could be ascribed to the more efficient protein attachment through the succinimidyl ester group of PBSE, which preattaches to the MWCNT sidewall with higher affinity than the hydrophobic attraction. The protein covalent attachment lies on the carboxyl groups introduced by the acidic oxidation at the open ends and defect sites of MWCNTs, which is verified by the FTIR spectrum in Figure 5a. The immobilization of proteins on the OCT/MWCNT assembly via EDC/NHS activation results in the largest angle change of 814 ( 58 mo (6.783 ( 0.483 ng/mm2), demonstrating its superiority to other two approaches. In this approach, it is very likely that both physical adsorption and covalent binding occur simultaneously for protein attachment, in which the activated carboxyl groups provide additional protein binding sites besides direct adsorption onto the MWCNT sidewall, thus delivering the best protein loading capacity on the OCT/MWCT surface. Protein Detection on MWCNT Assembly. After blocking with concentrated BSA solution (resulting in a ca. 70 mo angle increase for 1 h of incubation, as shown on curve 3 in Figure 4a), the SPR detection of anti-goat IgG was preformed to evaluate the sensing performance of the MWCNT assembly as a supporting matrix. (The covalent approach for goat IgG attachment was used herein.) Encouragingly, the SPR angle responses rapidly and sensitively to the anti-goat IgG in solution (Figure 6). These positive responses explicitly suggest good biological activity of the attached probe proteins. The inset of Figure 6 illustrates a measurable, stable angle increase even when the concentration is down to 0.2 μg/mL. In contrast to our previous work on a planar surface,36 the detection limit of this immunosensor is substantially lower. Owing to the small diffusion coefficients of proteins, the immune interaction is often limited by the slow mass transport of the target rather than the antibody-antigen binding kinetics.39,40 In this work, although MWCNTs are lying down on Langmuir 2010, 26(11), 8386–8391

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Figure 6. Representative SPR responses for the detection of antigoat IgG with concentrations of 10.0 (a), 5.0 (b), 2.0 (c), 1.0 (d), 0.5 (e), and 0 (f, negative control containing 10 μg/mL anti-bovine IgG) μg/mL. (Inset) Detailed SPR responses for 0.5 (e), 0.2 (g), and 0 (f, negative control containing 10 μg/mL anti-bovine IgG) μg/mL detection.

the OCT-SAM surface, they allow rapid mass transport of target proteins from bulk solution to the attached probes because of the unique access paths and short distance. Benefiting from the high concentration of the attached probe protein and the rapid mass transport of the target protein, the 3D MWCNT assembly results in significant immunosensing performance enhancement of not only the sensitivity but also the response time in comparison with the planar-surface-based systems. One control-detection experiment is conducted to check the detection specificity. As shown as curve f in the inset of Figure 6, no evident angle change is measured for even 10 μg/mL antibovine IgG in 0.01 M PBS, showing good specificity for anti-goat IgG to which the blocking step with high-concentration BSA solution in the preparation procedure makes a contribution. The binding amount of the target proteins is proportional to the bulk concentration of target proteins at a certain reaction time.36 The calibration curve for the detection of anti-goat IgG is obtained by plotting the angle increases after 2000 s of incubation time against the corresponding concentrations (Figure 7), exhibiting a good linear relationship. One may wonder whether the slopes of the in situ SPR responses in the initial time frame versus the concentrations are a better calibration curve. However, the in situ angle responses in a short time are too unstable with large variations to have well-defined slopes. Experimental results show (39) Driskell, J. D.; Uhlenkamp, J. M.; Lipert, R. J.; Porter, M. D. Anal. Chem. 2007, 79, 4141. (40) Sadana, A.; Sii, D. J. Colloid Interface Sci. 1992, 151, 166.

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Figure 7. Calibration curve (n = 3) for the detection of anti-goat IgG in 0.01 M PBS.

that 2000 s measurements have the best reproducibility and the lowest detection limit. The standard deviations in triplicate experiments are depicted on data points as well, unveiling good reproducibility of SPR detection (3.8% RSD for 10 μg/mL detection, n = 3). Compared with our previous work, the detection limit is substantially lower, owing to the 3D structure of the MWCNT assembly and, in turn, the excellent target accessibility provided during the detection.

Conclusions In situ SPR studies of MWCNT assembly on OCT-SAM and its protein immobilization were conducted, indicating that the MWCNTs can spontaneously assemble in a 3D structure following the Langmuir adsorption kinetics and the assembly temperature has a great influence on both the assembly kinetics and the surface distribution. The surface concentrations of the attached goat IgG on the MWCNT matrix are 5.242 ( 0.375, 6.683 ( 0.433, and 6.783 ( 0.483 ng/mm2 by using direct adsorption, PBSE-assisted immobilization, and EDC/NHS activation-based attachment, respectively. In comparison to the planar-substratebased detection platform, the SPR detection of anti-goat IgG on the probe-immobilized 3D MWCNT assembly demonstrates a significantly enhanced immunosensor with a higher sensitivity and a faster response time because of the superior assembly nanostructure for high probe concentration and good target accessibility. Acknowledgment. This work was financially supported by the Center of Advanced Bionanosystems, Nanyang Technological University.

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