Article pubs.acs.org/ac
Nanoparticle-Enhanced Surface Plasmon Resonance Detection of Proteins at Attomolar Concentrations: Comparing Different Nanoparticle Shapes and Sizes Min Jeong Kwon,† Jaeyoung Lee,‡ Alastair W. Wark,*,§ and Hye Jin Lee*,† †
Department of Chemistry and Green-Nano Materials Research Center, Kyungpook National University, 1370 Sankyuk-dong, Buk-gu, Daegu-city, 702-701, Republic of Korea ‡ Ertl Center for Electrochemistry and Catalysis/RISE & School of Environmental Science and Engineering, Gwangju Institute of Science and Technology, Gwangju 500-712, South Korea § Centre for Molecular Nanometrology, WestCHEM, Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow, U.K. G1 1XL S Supporting Information *
ABSTRACT: The application of biofunctionalized nanoparticles possessing various shapes and sizes for the enhanced surface plasmon resonance (SPR) detection of a protein biomarker at attomolar concentrations is described. Three different gold nanoparticle shapes (cubic cages, rods and quasi-spherical) with each possessing at least one dimension in the 40−50 nm range were systematically compared. Each nanoparticle (NP) was covalently functionalized with an antibody (anti-thrombin) and used as part of a sandwich assay in conjunction with a Au SPR chip modified with a DNA-aptamer probe specific to thrombin. The concentration of each NPantibody conjugate solution was first optimized prior to establishing that the quasi-spherical nanoparticles resulted in the greatest enhancement in sensitivity with the detection of thrombin at concentrations as low as 1 aM. When nanorod and nanocage antibody conjugates were instead used, the minimum target concentrations detected were 10 aM (rods) and 1 fM (cages). This is a significant improvement (>103) on previous NP-enhanced SPR studies utilizing smaller (∼15 nm) gold NP conjugates and is attributed to the functionalization of both the NP and chip surfaces resulting in low nonspecific adsorption as well as a combination of density increases and plasmonic coupling inducing large shifts in the local refractive index at the chip surface upon nanoparticle adsorption.
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INTRODUCTION Surface plasmon resonance (SPR) measurements of biomolecular interactions on the surface of thin gold films have emerged as one of the leading techniques for the fast, in situ detection of a wide range of biological targets.1−3 This optical technique is based on detecting small changes in the refractive index that are associated with the specific adsorption of the analyte onto chip areas where probe molecules have been immobilized. A key advantage of SPR is that the target biomolecule can be directly detected in real-time without prior fluorescent or enzymatic labeling.1−3 However, with detection limits typically in the low nanomolar range, recent technological developments have most often focused on the use of sandwich assays alongside enzymatic4 and nanoparticle-based5,6 strategies as well as combinations of both approaches7 to further enhance the SPR response. The majority of nanoparticle-enhanced SPR studies reported to date have centered on the use of DNA-functionalized gold nanoparticles ∼10−15 nm in diameter due to the wide availability of established procedures for the preparation of these conjugates.6−11 For example, detection limits ranging © 2012 American Chemical Society
from picomolar to low femtomolar levels (corresponding to a 103−106 improvement in sensitivity) have been reported by several groups utilizing a simple sandwich assay format with quasi-spherical nanoparticles (NPs) conjugated to a secondary DNA probe alongside a variety of SPR configurations.6,8−11 It is a logical next step that increasing the size (i.e., > 15 nm) of the nanoparticle secondary probe will result in a larger refractive index change following specific adsorption onto the SPR chip. However, there are very few literature reports exploring larger nanoparticle sizes12 or nonspherical particle shapes for the enhanced planar SPR detection of either nucleic acids or proteins.13−15 This is most likely due to the increased difficulty in biofunctionalizing larger particles while maintaining good colloidal stability in the saline conditions required for studying bioaffinity interactions. Consequently, protein functionalization based on electrostatic adsorption works more reliably for smaller nanoparticles. In addition, Mirkin et al.16 demonstrated Received: November 11, 2011 Accepted: January 6, 2012 Published: January 6, 2012 1702
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that the effective binding constant of DNA-modified NPs varies by several orders of magnitude depending on the NP size. Ultrasensitive SPR measurements also require the detection of very small changes in fractional surface coverage and the prevention of nonspecific adsorption onto the sensing surface is increasingly difficult to achieve with larger and poorer functionalized particles. In this paper, we compare the amplification performance of three different nanoparticle shapes (nanocages, nanorods, and quasi-spherical nanoparticles) for the enhanced SPR detection of thrombin. In addition to increasing the nanoparticle size, with each type used here possessing at least one dimension at ∼40−50 nm, changing the shape also introduces the possibility of adjusting the localized plasmon resonance properties of the nanoparticle over a much larger wavelength range. Gold nanospheres have a typical λmax of 520 to 550 nm, while the plasmon resonances of nanocages and especially nanorods extend further across the vis-NIR range.17,18 SPR-chip based measurements are usually performed at wavelengths ranging from ∼630 to 900 nm depending on the excitation light source used since the measurement performance becomes progressively poorer as the excitation wavelength approaches the gold interband transition region (∼500 nm). Recent work by both ourselves13 and other groups14,15 have demonstrated the use of nanorod-bioconjugates to significantly amplify the SPR response. However, the relative importance of plasmonic coupling between the nanorod and gold film substrate in addition to surface localized changes in density is not well understood. In an attempt to clearly demonstrate the remarkable improvements in sensitivity that can be achieved, we describe here a systematic study utilizing three different gold nanoparticle types which differ in shape and plasmonic profile, size, and also density. Thrombin was chosen as a model target protein in combination with a DNA aptamer probe covalently attached to the SPR-chip surface, while each nanoparticle was functionalized with an antibody also specific to thrombin to create a surface sandwich complex.
Figure 1. Schematics showing (a) the antibody functionalization of various gold nanoparticle shapes via the formation of a 11mercaptoundecanoic acid (MUA) monolayer on the nanoparticle surface followed by covalent linking to an antibody using EDC/NHSS cross-linking chemistry. (b) The attachment of a 5′-end aminemodified DNA aptamer onto a monolayer of either prolinker B only or a mixed layer of prolinker B and PEG created on the surface of the Au SPR chip. (c) Surface sandwich assay for thrombin (Th) using the aptamer-modified SPR chip in conjunction with different anti-Th coated gold nanoparticles.
HEPES buffer (pH 7.4) and a flow rate of 5 μL/min were used throughout for biomolecular interaction studies. Figure 1b highlights the surface attachment chemistry used for tethering DNA aptamers specific to thrombin onto a planar gold surface for SPR measurements. Bare gold chips were soaked in 3 mM prolinker B in chloroform for 6 h at room temperature followed by exposure to a 1 mM solution of 5′-amine modified Thaptamer (5′- H2N-AGT CCG TGG TAG GGC AGG TTG GGG TGA C(T)15-3′) for 4 h. Experiments were also performed using a mixed monolayer of prolinker B and thiolmodified PEG molecules to investigate possible further reductions in nonspecific adsorption. A minimum 1 h reaction time under a continuous flow rate of 5 μL/min was applied at sub-pM thrombin (Th) concentrations to ensure that a steadystate coverage of the Th/aptamer chip surface had been achieved. A relatively slow Th desorption rate (kd = ∼3.5 nM)20 from the aptamer surface helped minimize target desorption during a buffer rinse step before exposing the chip to antibody-nanoparticle conjugates. Further details on the procedures used are provided in the Supporting Information.
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EXPERIMENTAL SECTION Due to space considerations, the chemicals and protocols used are described extensively in the Supporting Information. Also included are details on the synthesis of the gold nanocages,17 nanorods,18 and quasi-spherical nanoparticles.19 Biofunctionalization of Au Nanoparticles with Different Shapes. A similar approach was used for the covalent immobilization of antibodies onto each of the three gold nanoparticle types used. An overview of the linking chemistry is shown in Figure 1a. Further details describing the procedures used to form a carboxylic acid terminated C11 alkanethiol (MUA) monolayer on the surface of all three nanoparticle types followed by EDC/NHSS chemistry to covalently attach either anti-thrombin or an anti-BSA control can also be found in the Supporting Information. Once prepared, the Au NPantibody conjugates were stored in 20 mM N-(2hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES) buffer containing 150 mM NaCl, 2 mM CaCl2 (pH 7.4). The formation of the antibody-nanoparticle conjugate was supported by UV−vis spectroscopy (Shimadzu UV-1800) and transmission electron microscopy (TEM) analysis (Hitachi H7600 and H-7100). Creation of Aptamer Biochips with Prolinker B for SPR Measurements. A Biacore 3000 was employed for all in situ surface bioaffinity detection measurements. Twenty mM
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RESULTS AND DISCUSSION Detection Methodology. A schematic of the approach used for the preparation of antibody functionalized nanoparticles is shown in Figure 1a alongside the preparation of the aptamer chip for SPR measurements (Figure 1b) and the sandwich assay format used for the enhanced detection of thrombin (Figure 1c). Prior to performing SPR measurements, stock solutions of differently shaped nanoparticles were prepared: i) gold nanocages, 42 (±3) nm, were created via galvanic displacement using Ag nanocube substrates; ii) gold nanorods with an average aspect ratio of 2.5 (length: 50 (±5) nm, width: 20 (±3) nm); and iii) gold quasi-spherical particles with an average diameter of 45 (±5) nm. Extinction spectra and TEM images for each of the stock solutions are shown in Figure 2 (see also Figures S1 and S2 in the Supporting Information for 1703
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Figure 2. UV−vis spectra of (a) gold nanocages (λmax = 638 nm, dotted line) and anti-Th coated gold nanocages (λmax = 646 nm, solid line), (b) gold nanorods (λmax = 644 nm, dotted line) and anti-Th coated gold nanorods (λmax = 653 nm, solid line), and (c) gold quasi-spherical nanoparticles (λmax = 531 nm, dotted line) and anti-Th coated gold quasi-spherical nanoparticles (λmax = 535 nm, solid line). Representative TEM images are also shown for each anti-Th nanoparticle conjugate.
interaction.23,24 SPR experiments using instead a control aptamer sequence showed no significant change in signal at nanomolar concentrations of thrombin. The surface could be regenerated by washing with HEPES buffer (pH 12.5) for 30 min followed by rinsing with 8 M Urea for 30 min, DI water for 30 min, and 20 mM HEPES buffer (pH 12.5) for 30 min. The chip surface was finally continually rinsed with 20 mM HEPES buffer (pH 7.4) for 30 min. The background SPR signal was compared to that obtained on the first use of the chip, and, if necessary, further washing was performed until a similar background signal was restored. The aptamer chip could be reused a minimum of five times. Nanoparticle-Enhanced SPR Sensing. Prior to directly comparing the SPR signal amplification performance of each nanoparticle-antibody conjugate type, the first step was to optimize the concentration of antibody-coated nanoparticles. This was done by performing a series of repeat measurements where the concentration of the protein target first exposed to the aptamer chip surface was maintained at a fixed value. Following a reaction time of at least 1 h at a constant flow rate of 5 μL/min to allow a steady-state surface coverage of thrombin to be established, buffer solution was then briefly injected followed by the antibody-NP conjugate. Figure 3
additional TEM images). All three shapes have at least one dimension that is around 40−50 nm in size. In addition, the plasmon resonances of the cages (Figure 2a) and rods (Figure 2b) significantly overlap with λmax values at ∼640 nm, while the quasi-spherical particles have a weak overlap at this wavelength with a lower λmax at 531 nm. As indicated in Figure 1a, the same approach was used to functionalize all three nanoparticle types with the formation of a carboxylic acid terminated alkanethiol layer followed by the use of EDC/NHSS cross-linking chemistry to covalently attach anti-thrombin (or anti-BSA control) molecules to the nanoparticle surface. In addition to being a robust and reproducible method, this was also a deliberate strategy to try and achieve similar binding affinity and nonspecific adsorption behaviors for each nanoparticle shape. Also, attempts to functionalize conventional Au NPs (d = 50 nm) prepared by citrate reduction via the same route were unsuccessful and one of the reasons for using CTAB stabilized quasi-spherical nanoparticles instead. Comparison of the UV−vis spectra in Figure 2a-c before and after antibody conjugation show a red-shift varying from 4 to 9 nm which also highlights differences in sensitivity between the various nanoparticle shapes to changes in their immediate environment associated with the formation of additional molecular layers. Once prepared, each NP-antibody conjugate was used within 2 days. An outline of the simple two-step approach used to covalently attach the thrombin-specific DNA aptamer to the SPR chip surface is shown in Figure 1b. This is based on a heterobifunctional cross-linker, prolinker B, which is a calix[4]arene derivative featuring both thiol-groups for self-assembly onto the gold surface and a crown-ether moiety that enables direct coupling to the amine group attached to the 5′-end of the aptamer sequence. A similar approach has recently been demonstrated for the surface immobilization of protein molecules onto thin gold films.21 Conventional SPR measurements monitoring the adsorption of thrombin without NP amplification was first performed where it was established that a target concentration of 1 nM could be detected. This performance is comparable to more well-established surface attachment strategies utilizing alkanethiols (e.g., MUAM) and bifunctional cross-linkers indicating that a similar probe surface density is achieved (i.e., ∼1012 molecules/cm2).7 A plot of fractional surface coverage versus thrombin concentration is shown in the Supporting Information (Figure S3) and from which a Langmuir adsorption coefficient value of 2.2 (±0.3) × 107 M−1 was calculated. This value is in good agreement with previous studies monitoring the aptamer-thrombin interaction.22 In addition, an adsorption coefficient of about 107−108 M−1 has been previously reported for the antibody-thrombin
Figure 3. A series of SPR sensorgrams obtained at different concentrations of anti-Th coated quasi-spherical nanoparticles. The thrombin target concentration was fixed at 10 aM prior to nanoparticle injection. The NP-conjugate concentrations were systematically varied with the corresponding λmax extinction values and calculated NP concentrations: (i) = 0.8 (0.1 nM), (ii) = 1.0 (0.13 nM), (iii) = 1.2 (0.16 nM), (iv) = 1.4 (0.18 nM), and (v) = 1.6 (0.21 nM). The nanoparticle concentration used for all subsequent measurements is indicated as a boldface line. Similar nanocage and nanorod data sets are presented in the Supporting Information, Figure S4.
shows a series of SPR response curves where the concentrations of the quasi-spherical NP bioconjugates were systematically varied at a fixed target thrombin concentration of 10 aM. Similar measurements were also performed for the nanocage and nanorod-antibody conjugates except the concentrations of 1704
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Figure 4. Representative SPR sensorgrams for the nanoparticle-enhanced detection of thrombin utilizing an aptamer-coated chip and subsequent signal amplification with anti-thrombin coated Au (a) nanocages, (b) nanorods, and (c) quasi-spherical nanoparticles. The concentration of each nanoparticle solution was fixed at the optimal values established in Figure 3, while thrombin concentrations ranging from 1 aM to 1 pM were selected depending on the NP-antibody conjugate used. Also shown in each data series, (a)−(c), are two control experiments; NC1 is associated with the nonspecific adsorption of the corresponding anti-Th NP conjugate in the absence of thrombin, while NC2 represents the nonspecific adsorption of the corresponding anti-BSA coated nanoparticles (same NP concentration) at chosen Th target concentrations of 10 fM, 100 aM, and 10 aM for (a), (b), and (c), respectively.
then nanospheres. This is likely due to differences in both the binding strength of each NP-antibody conjugate as well as the SPR signal-enhancing ability of each NP-type. When preparing the conjugates, a ∼100-fold molar excess of antibodies compared to NPs was used to ensure a suitably high fractional surface coverage; however, the relative importance of the NP shape itself in changing the conjugate binding strength is unknown. For this study, our focus was the maximum signal enhancement that could be achieved. Also shown in Figure 4 for each NP-antibody data series are two control measurements. The first, labeled NC1, demonstrates a very low level of nonspecific adsorption of nanoparticles onto a freshly prepared aptamer chip surface that has not been first exposed to target protein; such a low background signal is essential for performing ultrasensitive detection measurements. A second negative control (NC2) represents the adsorption of the corresponding anti-BSA coated nanoparticles (same NP concentration) onto the thrombin-specific aptamer functionalized surfaces in the presence of 10 fM, 100 aM, and 10 aM thrombin for (a), (b), and (c), respectively. Comparing the SPR signal changes following the injection of buffer solution for both control measurements shows a similar response and indicates a comparable level of nanoparticle nonspecific adsorption both in the presence and absence of thrombin as well as helping to further confirm the robustness of the surface chemistry used for attaching bioactive proteins onto the NP surface. In addition to performing control measurements, we also investigated whether replacing the probe monolayer on the SPR chip surface with a mixed layer of PEG-thiol and prolinker B prior to aptamer conjugation could further improve the sensing performance. The use of PEG molecules to reduce the nonspecific adsorption of proteins is well-established, and, based on previous work,28 a PEG MW of 1500 was used along with a PEG-SH:prolinker B stoichiometric ratio of 1:9. Next, the experiments described previously in Figure 4 at different thrombin concentrations were repeated again using the PEGmodified chip surface (see Supporting Information, Figure S5). Figure 5 plots the measured changes in the SPR response at selected thrombin concentrations for both aptamer only and PEG-modified chip surfaces. Here, the normalized SPR response was calculated by averaging the overall signal obtained after exposure to each NP-antibody conjugate in the two detection channels and then subtracting the corresponding
thrombin used were instead 10 fM and 100 aM, respectively (data shown in Supporting Information, Figure S4), which were chosen based on preliminary measurements with the overall improvement in detection performance discussed further below. The concentration of each NP-antibody conjugate stock solution was systematically varied in relation to its extinction value with the curves labeled (i)−(v) in Figure 3 correlating to OD values of 0.8, 1.0, 1.2, 1.4, and 1.6 at the plasmon peak. The corresponding NP concentrations are listed in the figure caption and were based on extinction coefficients of 6.06 × 109, 4.5 × 109, and 7.66 × 109 M−1·cm−1 for cages, rods, and quasi-spherical samples25−27 respectively at λmax values of 646, 653, and 535 nm. For each SPR response in Figure 3 and Figure S4, the initial increase is due to a combination of nanoparticle surface adsorption (specific and nonspecific) and changes in the refractive index of the bulk solution. The latter will continue to cause a temporary increase in signal at higher NP concentrations; however, when the SPR response curves are compared in the latter region of each plot where the colloid solution has been subsequently displaced by buffer solution it can be seen that above a certain nanoparticle concentration no further improvement in sensitivity was obtained. Consequently, all subsequent SPR measurements reported here were performed with stock NP-conjugate solutions having extinctions of 1.4 (cages), 1.4 (rods), and 1.6 (quasi-spherical shapes), which are represented as a boldface line in each of the three data series. A series of repeat experiments were then performed where the concentration of the thrombin target was systematically varied. Representative SPR plots are shown in Figure 4 for three data series where the target concentration ranged from 1 fM to 1 pM (cages), 10 aM to 10 fM (rods), and 1 aM to 1 fM (quasi-spherical NPs). Comparison of each data series clearly shows that the greatest signal amplification occurs for antithrombin coated quasi-spherical nanoparticle conjugates where target concentrations as low as 1 aM could be directly detected. For the nanorod bioconjugates, a slightly higher concentration of 10 aM was measured, while the nanocage bioconjugates were the poorest performing with successful measurements limited to concentrations greater than 0.5 fM. Comparison between each data series also shows significant differences in the kinetic curve shapes with the nanocages taking the longest time to reach a steady-state response followed by the nanorods and 1705
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quasi-spherical particle response was more than double that of the nanorod sample when comparing the overall signal changes following the injection of buffer-only solution. To gain further insight into the signal-enhancing role of the nanoparticle, our approach was designed to minimize a number of experimental parameters that could contribute to variation in sensing performance. These include adopting a uniform approach for functionalizing each NP type and the development of a robust methodology to prepare the aptamer SPR chips which result in very low nonspecific adsorption and good measurement reproducibility. Possible differences in the antibody attachment efficiency resulting in variations in fractional surface coverage between nanoparticle types were also taken into consideration by first optimizing the NPconjugate concentration. In addition, it can be assumed that the spacing between the immobilized NP and gold film surface will be similar in each measurement. Previous work involving SPR angle shift-based measurements has demonstrated that the NPenhanced SPR response significantly varies with separation distance.29−32 Also, all submonolayer changes in the surface film thickness remain within the ∼200 nm probe depth extending into the bulk water phase defined by the surface plasmonic field.33 Consequently, the differences in signal enhancement between each nanoparticle type must originate from the changes each adsorbed NP induces in the real and imaginary components of the refractive index of the thin film at the chip/ solution interface. The presence of the high-density gold NPs will affect the real component value, while near-field plasmonic coupling between the nanoparticle and gold film substrate will primarily affect the imaginary component. Plasmonic coupling will occur when there is sufficient overlap between the wavelength used to excite plasmons in the gold thin film (in this case a 760 nm LED light source) and the local SPR profile of an individual nanoparticle. The extinction plots in Figure 2 indicate that a stronger overlap exists for the cage and rod shapes compared to the quasi-spherical nanoparticles. However, it has been shown that the spectrum of individual gold nanoparticles will undergo a significant red-shift, on the order of 100 nm, when placed close to a gold metallic film with the shift also dependent on the surface-particle separation distance.31,32 Thus, plasmonic coupling is likely to occur to various extents for all three nanoparticle types. The poorer sensing performance of the nanocage bioconjugates can also be attributed to a lower material density compared to the rod and quasi-spherical particles. The fact that the extinction of the cages more strongly overlaps with the instrument excitation wavelength than the quasi-spherical nanoparticles also suggests that increases in surface film density are more dominant than plasmonic coupling effects. Furthermore, since the average volume of the gold nanorods used is about 3 times lower than that of the quasi-spherical particles, the rods will also induce a smaller change in the real component of the refractive index when compared with the quasi-spherical particles on a 1:1 basis. Finally, with each nanoparticle shape having at least one size dimension in common (∼40−50 nm) it is reasonable to assume that there will be relatively small differences in the surface area coverage of the SPR chip occupied by each NP type. Further work is required combining both modeling and measuring the spectral changes of each nanoparticle type when in the vicinity of a planar gold film to further resolve the relative importance of near-field plasmonic coupling toward changes in
Figure 5. Plot comparing measured SPR responses at selected thrombin concentrations for two different chip surfaces featuring either the aptamer only (filled circles) or a 1:9 mixed PEG/prolinker B monolayer (open circles). The SPR response obtained from the two detection channels were averaged and then normalized by subtracting the average of the signal obtained from the two control channels (NC1 and NC2 described in Figure 4) on the same chip. Symbols of (⧫, ◊), (■, □), and (●, ○) represent the respective data points for the nanocages, nanorods, and quasi-spherical NP measurements, and all other conditions were the same as those in Figure 4 and Figure S5.
average change in the two neighboring reference channels (NC1 and NC2) on the same chip surface. Furthermore, the error bars on each data point reflect a typical ±10% variation in the normalized SPR response when performing repeat measurements using both freshly prepared and regenerated aptamer chips. Each of the plots in Figure 5 clearly shows a reduction in the measured SPR response for the PEG-modified SPR chip surface. The results of additional control measurements for both chip surface formats are shown in the Supporting Information (Figures S6 and S7) where it can be seen that PEG modification reduces the nonspecific signal. However, the improvement in background noise does not compensate for much larger losses in sensitivity, which drops by an average of about 34%, 57%, and 62% for the nanocages, nanorods, and quasi-spherical nanoparticles, respectively (see Figure S7). This can be explained by a significant reduction in the surface density of aptamer probe molecules. Comparing Different NP-Conjugates. The differences in sensing performance between each type of nanoparticle are further highlighted in Figure 6. In each measurement, the
Figure 6. A comparison of three different nanoparticle-enhanced SPR measurements at a fixed thrombin concentration of 1 fM. The same nanoparticle concentrations as in Figure 4 were used with the SPR chip featuring an aptamer only monolayer with no PEG treatment.
thrombin target concentration was fixed at 1 fM and the relative changes in the SPR response compared when solutions of the three different anti-thrombin nanoparticle conjugates were injected. It can be clearly seen that very little change in SPR signal occurred upon the injection of nanocages, while the 1706
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ACKNOWLEDGMENTS This research was supported by National Research Foundation of Korea Grant funded by the Korean Government (20110004823) and (2009-0076851).
the local refractive index. However, the experimental approach described here establishes that the mass/volume ratio of the nanoparticle plays a more prominent role in enhancing the planar SPR signal than the coupling of the incident excitation light with the NPs localized SPR. We have clearly shown that achieving good control over the nanoparticle size and shape as well as the surface chemistries of both the chip sensor and nanoparticle can lead to very large improvements in SPR sensitivity.
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CONCLUSIONS
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ASSOCIATED CONTENT
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REFERENCES
(1) Yakes, B. J.; Prezioso, S.; Haughey, S. A.; Campbell, K.; Elliott, C. T.; DeGrasse, S. L. Sens. Actuators, B 2011, 156, 805−811. (2) Homola, J. Chem. Rev. 2008, 108, 462−493. (3) Grant, C. F.; Kanda, V.; Kitov, P.; Bundle, D. R.; McDermott, M. T. Langmuir 2008, 24, 14125−14132. (4) Li, Y.; Lee, H. J.; Corn, R. M. Anal. Chem. 2007, 79, 1082−1088. (5) Bolduc, O. R.; Masson, J.-F. Anal. Chem. 2011, 83, 8057−8062. (6) D’Agata, R.; Corradini, R.; Grasso, G.; Marchelli, R.; Spoto, G. ChemBioChem 2008, 9, 2067−2070. (7) Fang, S.; Lee, H. J.; Wark, A. W.; Corn, R. M. J. Am. Chem. Soc. 2006, 128, 14044−14046. (8) Taylor, J. D.; Linman, M. J.; Wilkop, T.; Cheng, Q. Anal. Chem. 2009, 81, 1146−1153. (9) Ito, M.; Nakamura, F.; Baba, A.; Tamada, K.; Ushijima, H.; Lau, K. H. A.; Manna, A.; Knoll, W. J. Phys. Chem. C 2007, 111, 11653− 11662. (10) Yao, X.; Li, X.; Toledo, F.; Zurita-Lopez, C.; Gutova, M.; Momand, J.; Zhou, F. Anal. Biochem. 2006, 354, 220−228. (11) Hayashida, M.; Yamaguchi, A.; Misawa, H. Jpn. J. Appl. Phys. 2005, 44, L1544−L1546. (12) Wang, S.; Shan, X.; Patel, U.; Huang, X.; Lu, J.; Li, J.; Tao, N. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 16028−16032. (13) Sim, H. R.; Wark, A. W.; Lee, H. J. Analyst 2010, 135, 2528− 2532. (14) Sendroiu, I. E.; Warner, M. E.; Corn, R. M. Langmuir 2009, 25, 11282−11284. (15) Yu, C.; Irudayaraj, J. Biophys. J. 2007, 93, 3684−3692. (16) Hurst, S. J.; Hill, H. D.; Mirkin, C. A. J. Am. Chem. Soc. 2008, 130, 12192−12200. (17) Skrabalak, S. E.; Au, L.; Li, X.; Xia, Y. Nat. Protoc. 2007, 2, 2182−2190. (18) Sau, T. K.; Murphy, C. J. Langmuir 2004, 20, 6414−6420. (19) Millstone, J. E.; Park, S.; Shuford, K. L.; Qin, L.; Schatz, G. C.; Mirkin, C. A. J. Am. Chem. Soc. 2005, 127, 5312−5313. (20) Hasegawa, H.; Taira, K.; Sode, K.; Ikebukuro, K. Sensors 2008, 8, 1090−1098. (21) Oh, S. W.; Moon, J. D.; Lim, H. J.; Park, S. Y.; Kim, T.; Park, J. B.; Han, M. H.; Snyder, M.; Choi, E. Y. FASEB J. 2005, 19, 1335− 1337. (22) Lin, S. F.; Ding, T. J.; Liu, J. T.; Lee, C. C.; Yang, T. H.; Chen, W. Y.; Chang, J. Y. Sensors 2011, 11, 8953−8965. (23) Schlecht, U.; Malave, A.; Gronewold, T.; Tewes, M.; Lohndorf, M. Anal. Chim. Acta 2006, 573−574, 65−68. (24) Baerga-Ortiz, A.; Bergqvist, S.; Mandell, J. G.; Komives, E. A. Protein Sci. 2004, 13, 166−176. (25) Liu, X.; Atwater, M.; Wang, J.; Huo, Q. Colloids Surf., B 2007, 58, 3−7. (26) Liao, H.; Hafner, J. H. Chem. Mater. 2005, 17, 4636−4641. (27) Jain, P. K.; Lee, K. S.; El-Sayed, I. H.; El-Sayed, M. A. J. Phys. Chem. B 2006, 110, 7238−7248. (28) Kim, S.; Lee, J.; Lee, S. J.; Lee, H. J. Talanta 2010, 81, 1755− 1759. (29) Golden, M. S.; Bjonnes, A. C.; Georgiadis, R. M. J. Phys. Chem. C 2010, 114, 8837−8843. (30) Mock, J. J.; Hill, R. T.; Degiron, A.; Zauscher, S.; Chilkoti, A.; Smith, D. R. Nano Lett. 2008, 8, 2245−2252. (31) He, L.; Smith, E. A.; Natan, M. J.; Keating, C. D. J. Phys. Chem. B 2004, 108, 10973−10980. (32) Driskell, J. D.; Lipert, R. J.; Porter, M. D. J. Phys. Chem. B 2006, 110, 17444−17451. (33) Raether, H. Surface Plasmons on Smooth and Rough Surfaces and on Gratings; Springer-Verlag: Berlin, 1988.
In this paper we have demonstrated that low attomolar concentrations of an unmodified protein biomarker can be detected as part of a simple sandwich assay using antibody functionalized nanoparticles in combination with SPR measurements of an aptamer-modified gold chip surface. The enhancement in sensitivity obtained has resulted in one of the most sensitive surface-based protein bioaffinity measurements reported to date. Conventional SPR measurements (without NP amplification) typically have a detection limit of >0.5 nM depending on the SPR sensing configuration. In addition, previous investigations involving nanoparticle-enhanced SPR measurements using a similar sandwich assay have been mostly limited to the use of smaller sized (200 nM). This could be overcome through systematically adjusting the concentration of the NP-antibody conjugate. Further work is now underway to transfer this approach to other detection targets including working directly with genomic samples as the sandwich assay approach is relatively straightforward compared to more complicated multistep signal amplification processes.
S Supporting Information *
Experimental details and Figures S1−S7. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected] (H.J.L.),
[email protected]. uk (A.W.W.). 1707
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