Article pubs.acs.org/ac
Single Gold Nanoparticle Localized Surface Plasmon Resonance Spectral Imaging for Quantifying Binding Constant of Carbohydrate−Protein Interaction Xiaojun Liu, Qingquan Zhang, Yang Tu, Wenfeng Zhao, and Hongwei Gai* School of Chemistry and Chemical Engineering, Jiangsu Key Laboratory of Green Synthesis for Functional Materials, Jiangsu Normal University, Xuzhou, Jiangsu 221116, China
ABSTRACT: Quantifying carbohydrate−protein (ligand−receptor) interactions is important to understand diverse biological processes and to develop new diagnostic and therapeutic methods. We develop an approach to quantitatively study carbohydrate−protein interactions by Au nanoparticle localized surface plasmon resonance (LSPR) peak position shift at the single particles level. Unlike the previous techniques for single particle LSPR spectral imaging, only the first-order streak of an individual nanoparticle is needed to extract a LSPR spectrum, which has great potential to increase throughput to 500 single particle spectra in each frame. LSPR peak shift of protein modified single Au nanoparticles is found to be a function of its ligand concentration, which can be used to fit the binding constants of the interactions. The moderate interactions of Antithrombin III (AT III) and heparins including low molecular weight heparin (LMWH) are determined as well as the strong interaction of transferrin and antitransferrin and the weak interaction of bovine serum album (BSA) and heparin. The measured binding constants of transferrin to antitransferrin, heparin and LMWH to AT III, and BSA to heparin are (3.0 ± 0.6) × 109 M−1, (3.1 ± 0.3) × 106 M−1, (8.0 ± 0.5) × 105 M−1, and (5.1 ± 0.1) × 103 M−1, respectively, which are in good agreement with the reported values.
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signal amplification, simplification of separation, and analogy to multivalent binding at cell surfaces.13−23 For example, Au nanoparticles were used in QCM, which resulted in a 16 time increase in detection signal.19 Another interesting development is that change in intensity of Au nanoparticle localized surface plasmon resonance (LSPR) is exploited to assess carbohydrate−protein interactions in a real-time, biocompatibility, and label-free way.24 In fact, LSPR peak position of Au nanoparticles is highly sensitive to local refractive index change caused by the binding of target molecules to Au nanoparticles. This unique property has been exploited for biosensors which can be classed into ensemble and single particle level, depending on the number of Au nanoparticles being monitored. Over the bulk counterpart, the advantages of single Au nanoparticle sensors have been summarized in a couple of
arbohydrate−protein interactions are involved in diverse biological processes, and specific carbohydrate−protein interactions play a vital role in some biological events like cell adhesion, cell differentiation, bacteria and virus infections, and cancer metastasis.1−3 At the cell surface, carbohydrate−protein interactions usually occur in multivalent form to enhance affinity, and many effectors determine whether specific carbohydrate−protein interaction happens and whether it is strong enough to trigger relevant events. Characterization of carbohydrate−protein interactions will lead to a better understanding of those biological processes and promote development of new diagnostic and therapeutic methods. Many techniques have been developed to evaluate multivalent carbohydrate−protein interactions, such as capillary electrophoresis, chromatography, NMR, ELISA, quartz crystal microbalance (QCM), surface-plasmon resonance (imaging), isothermal titration calorimetry, and glycan microarrays.4−12 Recently, nanoparticles, particularly Au nanoparticles, are introduced into some of these techniques for the purpose of © XXXX American Chemical Society
Received: August 10, 2013 Accepted: November 15, 2013
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reviews, including high sensitivity, high throughput, heterogeneity free, and low-dose reagent.25,26 A LSPR spectrum of a single Au nanoparticle with a diameter larger than 40 nm can be easily obtained through taking images by a dark-field microscope equipped with a charge coupled device (CCD) camera. The key is how to convert an image of single Au nanoparticle into a LSPR spectrum. Two kinds of methods have been developed for the task. One is to place a commercial spectrometer at the conjugate image plane in order to record a single particle’s spectrum.25,27−29 This approach needs to distinguish different particles prior to spectral monitoring, and only a limited number of nanoparticles are processed per minute. Another called transmission grating based spectral imaging is developed to enhance spectral throughput,30,31 by which we have investigated the spectra of single dyes and single quantum dots (QD) and have developed superlocalization spectral imaging for QDs.32−34 The principal is that a scattering light spot of a single nanoparticle is dispersed into a zerothorder spot and a first-order streak by a transmission grating inserted in front of CCD. The wavelength is directly proportional to the distance between the two orders. In the previous transmission grating based spectral imaging work,30−34 the images of the zeroth-order spot and the first-order streak of a single particle have to be simultaneously captured in the same frame to calibrate the origin of the wavelength. This undoubtedly deteriorates the imaging throughput. To address the issue, we utilize the cutoff edge of bandpass filters to calibrate and convert the first-order streak of a single nanoparticle into the spectrum. Using this technique, we demonstrate a method for studying carbohydrate−protein interactions by Au nanoparticle LSPR peak shift. Protein functionalized Au nanoparticles are incubated with its ligand at varying concentrations. The LSPR peak shift of the protein functionalized Au nanoparticles is a function of the initial concentration of the ligand and can be used to derive a binding constant through nonlinear fitting under the condition that the ligand is in large excess. This approach is verified by the moderate interactions between antithrombin III (ATIII) and heparins as well as the weak interaction between bovine serum album (BSA) and heparin and the strong interaction between antitransferrin and transferrin. To the best of our knowledge, there have been no reports of evaluating molecular interactions through the peak position shift of Au nanoparticle LSPR at both ensemble and single particle levels.
Park, IL). Rabbit antitransferrin was a kind gift from Sangon Biotech Co., Ltd. (Shanghai, China). Preparation of Protein Functionalized Au Nanoparticles. Prior to protein modification, Au nanoparticles were immobilized onto a glass slide. In brief, the glass slides were sequentially immersed into potassium dichromate/ sulphuric acid solution for 30 min at 70 °C, ultrasonically washed with ultrapure water 3 times, dried for 2 h at 120 °C, and placed into a vacuum desiccator along with an uncapped vial containing MPTS. The desiccator was vacuumed and kept in darkness for at least 2 h. The glass slide coated with thiol groups was reversibly sealed with a 5 mm thick PDMS square (30 mm × 30 mm) onto which a hole about 5 mm in diameter had been drilled at the center. The PDMS layer was ultrasonically washed with ethanol 3 times and blow-dried right before sealing. An aliquot of 5 μL of Au nanoparticle solution (30 time dilution) was added into each well, and the precipitation of Au nanoparticles was allowed to continue for 15 min. In order to coat Au nanoparticles with carboxyl group, the immobilized Au nanoparticles were washed with 25 mM MES buffer (pH 5.5) twice and incubated with 1 mM DDA for 5 min followed by the washing with ultrapure water. The Au nanoparticles were incubated with 20 μL of 1 mg/mL protein in 100 mM MES buffer for 30 min, and 3 μL of freshly prepared 10 mg/mL EDC in cold MES buffer was added into the mixture, with the exception of antitransferrin solution. A portion of 10 μL of antitransferrin solution was diluted to 450 μL with the same MES buffer, and the process was followed with centrifugation in a 100 kDa tube in order to remove bovine serum albumin. This was repeated 3 times before the antitransferrin solution was added into the PDMS hole. The interaction between the protein and the Au nanoparticles proceeded for 2 h at room temperature or overnight at 4 °C. Finally, the assembly was extensively washed and filled with ultrapure water and stored in a sealed box at 4 °C until use. Scattering Spectrum of the Bulk Au Nanoparticle Solution. The scattering spectrum of the bulk Au nanoparticle solution was measured in synchronous scan mode on a fluorescence spectrophotometer (Hitachi, F-4500). The scan wavelength is from 200.0 to 900.0 nm. The slit width and Δλ were set as 10.0 and 0.0 nm, respectively. Single Au Nanoparticle LSPR Spectral Imaging. Prior to observation under dark-field microscopy, the liquids were drained out of the hole, and the PDMS square was removed. Immediately, an aliquot (2 μL) of 20 mM phosphate buffer (pH 7.4) or heparin solution in the same phosphate buffer was deposited on the glass slide and sealed with a coverslip. The observation was conducted on an inverted fluorescent microscope (Olympus IX71) equipped with a 100× oil immersion objective with numerical aperture of 0.6 to 1.3 (Olympus, Tokyo, Japan) and an Evolve 512 electron-multiplied chargecoupled device from Photometrics (EMCCD; Tucson, USA). All filters (536/20 nm and 593/20 nm filters) were purchased from Semrock (Rochester, NY). The temperature, the gain, and the exposure time of the camera were maintained at −80 °C, 16, and 0.3 s, respectively. To achieve a single Au nanoparticle LSPR spectral image, a transmission grating with 70 lines per mm (Edmund Scientific, Barrington, NJ) was placed in front of the EMCCD camera, and the dispersed scattering light from a single Au nanoparticle under each filter and no filter was recorded. Image J and Origin software were used to process data.
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EXPERIMENTAL SECTION Chemical and Material. Gold nanoparticles of 70 nm in diameter were purchased from Nano Partz Inc. (Loveland, CO). The number of Au nanoparticles is 1.6 × 1010 per mL. Sylgard 184 PDMS oligomer and curing agent were from Dow Corning (Midland, MI). 4,4′-Dithiodibutyric acid (DDA), 2(N-morpholin o) et hanesulfonoic acid (MES), (3mercaptopropyl)triethoxysilance (MPTS), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), epidermal growth factor (EGF), BSA, and transferrin were obtained from Sigma (St. Louis, MO). Heparin with molecular weight of 15.3 kDa and low molecular weight heparin (LMWH) with molecular weight of 8.0 kDa were a kind gift from Aglyco Biotechnology Inc. (Beijing, China). Antithrombin III (AT III) was bought from Dongfeng Biotechnology (Shanghai, China). Tico brand of glass slides and other chemicals were from the local suppliers. Coverslips were from Fisher Scientific (Hanover B
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of the unpaired first-order streaks indicated by the dash squares in Figure 1B cannot be obtained due to lack of the zeroth-order spots. The percentage of unpaired first-order streaks is dependent on the size of the field of view, the density of Au nanoparticle distribution, and particularly the grating constant (d) and the distance (S). Under our conditions, 20% to 40% of the first-order streaks are usually unpaired. Sometimes, it is hard to match the zeroth-orders and their corresponding firstorders by the positions. Moreover, to accurately convert the unit of L from pixel into wavelength, a narrow band light source such as laser has to be introduced into the imaging system before measuring the spectra of single molecules or single nanoparticles.31 This is certainly a little more complicated for nonoptical experts. Here, we present a simple method to obtain an individual particle spectrum only by the first-order streak. The principal is illustrated in Figure 2. Figure 2A shows the first-order streak images of the single Au nanoparticles in the exactly same area consecutively taken under 536/20 and 593/ 20 nm bandpass filters and no filter (from top to bottom). The pixels’ intensities of the Au nanoparticle LSPR images in the dash square are extracted with Image J and drawn next in Figure 2B. The plots that are converted from the images taken under the bandpass filters have sharp changes in intensity indicated by the triangles. The changes are caused by the dramatically narrow transition between the maximal and minimal transmission ratio of the bandpass filters. The wavelength, which their lateral positions of the start, the middle, and the end of the sharp changes correspond to, should be equal to the wavelengths of the maximal, the half, and the minimal transmission ratios of the cutoff edge of the used bandpass filter as shown in Figure 2C. Using the middles of the sharp changes as calibration points, the pixels’ intensity spectrum taken under no filter in the bottom of Figure 2B is converted into the LSPR spectrum next in Figure 2C. To calibrate the scale accurately, two sets of bandpass filters are used. The peak wavelength (553.0 nm) is in good agreement with the one (553.8 nm, figure not shown) determined by the distance between the first-order spot and the second-order streak. The LSPR peak distribution of randomly selected more than 50 particles is plotted in Figure 3A. The mean value of the single particle LSPR peak wavelengths is determined to be 549.4 ± 0.4 nm using Gaussian fitting which agrees with the peak wavelength (551.8 ± 0.2 nm) of the scattering spectrum of the bulk Au nanoparticle solution in Figure 3B and the spectrum provided by the manufacturer as well. The interferences among nanoparticles may cause the ensemble spectrum to broaden compared to the spectra distribution of single nanoparticles. This method has great potential to increase analysis throughput since the zeroth-order spots are no longer needed. Theoretically, more than 500 distinguished streaks can be obtained in a single frame, considering that a first-order streak image occupies 58 × 4 pixels and the CCD device has a 512 × 512 pixel imaging area. Thus, the spectral throughput is calculated to be more than 1000 per minute since all the 3 frames have been taken within 30 s. Affinity Calculation. The equilibrium expression for the association reaction involving protein (P, receptor) immobilized onto Au nanoparticles and carbohydrate (C, ligand) is described by the following equation
RESULTS AND DISCUSSION Single Au Nanoparticle LSPR Imaging. The schematic of transmission grating based single particle spectral imaging is shown in Figure 1A. The scattering wavelength of single
Figure 1. (A) The scheme of transmission grating based spectral imaging dark-field microscopy. (B) The typical zero-order spot and first-order streak images of individual Au nanoparticles. Single triangle and double triangles point to the zero-order spots and the first-order streaks, respectively. The zero-order spot and the first-order streak pointed to by the same color triangles are pairs and belong to an individual Au nanoparticle. The squares in the dashed line mark the unpaired first-order streaks. The scale bar is 10 μm.
nanoparticle (λ) follows the equation, λ = L × d/S, (S, the distance between the grating and the EMCCD chip; d, the grating constant; L, the distance between the zeroth-order spot and the first-order streak). In previous work,32−34 an essential step is to accurately determine the value of L. Thus, the zerothorder spot and the first-order streak of a single nanoparticle have to appear in the same frame and then make a pair in order to determine the origin of the spectrum. Matching the zerothorder spots with the first-order streaks is performed by comparing their relative positions. In brief, if the graph drawn by connecting the center of the zeroth-order spots is the same as the one by the first-order streaks, the zeroth-order spots and the first-order streaks are paired. The first-order streaks that can not find the corresponding zeroth-order spots are unpaired. In Figure 1B, the pairs, the spots, and their corresponding streaks of single nanoparticles are labeled with a single triangle and double triangles in the same color, respectively. The spectrum
P + nC ⇌ PCn C
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Figure 2. Calibration of single Au nanoparticle LSPR spectral imaging by bandpass filters. (A) The representative first-order spectral images of individual Au nanoparticles in a same area consecutively taken under 536/20 and 593/20 nm filters and no filter (from top to bottom). The three images have been taken within 30 s. (B) The corresponding pixels’ intensity of the first-order streak of the same individual particle marked in panel A. The triangles in different colors indicate the start, the middle, and the end of the sharp intensity change of the LSPR image under filter. (C) The transmission spectrum of the bandpass filter and the scattering spectrum of the bulk Au nanoparticle solution in the same row of panel A and the obtained single particle LSPR spectrum are drawn in the red, blue, and black line, respectively. The triangles in different colors indicate the maximal, the middle, and the minimal transmission of the cutoff edge of the filter. The wavelengths indicated by the same color triangles in panels B and C are equivalent.
complex and the free protein are equal to the value of [APC]∗q and [AP]∗p, respectively. Here, [APC] and [AP] represent the equilibrium concentrations of the carbohydrate bound Au nanoparticles and the unbound Au nanoparticles, respectively. Therefore, the eq 2 can be re-expressed and simplified as the eqs 3 and 4 K=
[APC] × q [AP] × p × [C]n
K=α
Hence, the binding constant between the protein and the carbohydrate (K) is
[PCn] [P][C]n
(4)
where α is equal to the value of q/p. The LSPR peak position of Au nanoparticles is sensitive to the local surrounding media’s refractive index change caused by the binding of the target molecules to the Au nanoparticles. The protein functionalized Au nanoparticles are incubated with a series of carbohydrate solutions at varying concentrations. Thus, there exists a kinetic equilibrium between the protein functionalized Au nanoparticles and the carbohydrate, which resulted in a rapid reversible conversion between the bound and the unbound Au nanoparticles. Assuming the observed LSPR peak shift of the Au nanoparticles is proportional to the number of the bound Au nanoparticles, the observed (apparent) LSPR peak wavelength of the Au nanoparticles is
Figure 3. (A) The LSPR spectra distribution of single Au nanoparticles. The dashed line is the Gaussian fit of the histogram. (B) The synchronous scattering spectrum of the bulk Au nanoparticles solution in real line. The upper column distribution is rescaled in the dashed line.
K=
[APC] [AP][C]n
(3)
λ=
(2)
where PCn, P, C, and n are the equilibrium concentrations of the carbohydrate−protein complex, the free protein, the free carbohydrate, and the number of the binding sites on the protein. Since the protein is immobilized on the Au nanoparticles, the equilibrium concentration of the protein is related to the Au nanoparticle concentration. Assuming the number of proteins onto the carbohydrate bound Au nanoparticles and the unbound Au nanoparticles is q and p, the equilibrium concentrations of the carbohydrate−protein
[APC] [AP] λAPC + λAP [APC] + [AP] [APC] + [AP]
(5)
where λ, λAPC, and λAP are the apparent LSPR peak wavelength of the Au nanoparticles, the LSPR peak wavelength of the bound Au nanoparticles, and the unbound Au nanoparticles, respectively. A combination of the eqs 4 and 5 gives ⎛ ⎞ [C]n λ − λAP = (λAPC − λAP)⎜ n⎟ ⎝ α /K + [C] ⎠ D
(6)
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Figure 4. (A) Relationship between transferrin concentration and LSPR peak shift of antitransferrin functionalized Au nanoparticles. (B) Relationship between heparin concentration and LSPR peak shift of bovine serum albumin functionalized Au nanoparticles. (C) Relationship between heparin concentration and LSPR peak shift of EGF functionalized Au nanoparticles. (D) Relationship between heparin concentration and LSPR peak shift of AT III functionalized Au nanoparticles. (E) Relationship between LMWH concentration and LSPR peak shift of AT III functionalized Au nanoparticles. The real line is the model fitting curve according to eq 8. The calculated binding constants (K), the number of binding sites on the protein (n), and the Δλmax are listed in the figures.
concentrations and Δλmax (Δλmax= λAPC − λAP) is the difference in the LSPR peak wavelength between the bound Au nanoparticles and the unbound Au nanoparticles, in other words, the maximum of LSPR peak shift in the presence of the carbohydrate at infinite concentration. The eq 7 is a transform of the Hill equation.35 In the case that the carbohydrate is in large excess, the equilibrium concentration of the carbohydrate
The eq 6 can be rewritten ⎛ ⎞ [C]n Δλ = Δλmax ⎜ n⎟ ⎝ α /K + [C] ⎠
(7)
where Δλ (Δλ =λ − λAP) is the LSPR peak shift of the Au nanoparticles in the presence of the carbohydrate at certain E
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instance, the maximum of LSPR peak red shift of the AT III functionalized Au nanoparticles is 11.5 and 8.8 nm in the presence of heparin and LMWH, respectively. On the other hand, the red shifts are determined by the affinity strength. It is supported by the fact that the BSA functionalized Au nanoparticle LSPR peaks shift far more slightly than the AT III functionalized Au nanoparticle in the presence of heparin.
is approximately equal to the initial concentration, and the eq 6 can be described as ⎞ ⎛ [C0]n Δλ = Δλmax ⎜ n⎟ ⎝ α /K + [C0] ⎠
(8)
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where [C0] is the initial concentration of the carbohydrate. As seen in the eq 8, the LSPR peak shift of the protein functionalized Au nanoparticles is a function of the initial concentration of the carbohydrate and can be used to derive the binding constants for the carbohydrate−protein interactions by nonlinear fitting. Determination of Binding Constants. To test our theory, the strong binding of antibody to antigen such as antitransferrin to transferrin was quantitatively studied. The Au nanoparticles coated with antitransferrin were incubated with 2 μL of transferrin solution at a certain concentration and imaged with dark-field microscopy. For each concentration, more than 100 Au nanoparticles were randomly selected. The mean value of these single particle LSPR peak wavelengths, being equal to the peak wavelength of the bulk Au nanoparticle solution scattering spectrum, was obtained by the above-described procedures. Using the LSPR peak of the antitransferrin functionalized Au nanoparticles without the presence of transferrin as reference, the red shift of the antitransferrin functionalized Au nanoparticle LSPR peak is observed in the presence of transferrin and plotted against transferrin concentration in Figure 4A. With an increase in transferrin concentration, the red shift of the antitransferrin functionalized Au nanoparticle LSPR peak becomes bigger until saturated, which indicates the binding of transferrin to the antitransferrin functionalized Au nanoparticles. The weak binding of BSA to heparin was assessed as well. The relationship between heparin concentration and LSPR peak shift of the BSA functionalized Au nanoparticles is illustrated in Figure 4B. By nonlinear fitting of Δλ against [C0] in Figure 4A,B according to the eq 8, the binding constants of antitransferrin to transferrin and BSA to heparin are found to be (3.0 ± 0.6) × 109 M−1 and (5.1 ± 0.1) × 103 M−1, respectively, which are in agreement with the reported values (∼1010 M−1 and (1.24 ± 0.05) × 103 M−1).37,38 This indicates the applicable range of our theory is wide. In order to exclude the possibility that the red shifts of the receptor functionalized Au nanoparticle LSPR peak are from nonspecific binding, EGF that had been known not to have affinity to heparins is used as control.36 As shown in Figure 4C, the LSPR peaks of the EGF functionalized Au nanoparticles do not shift before and after the nanoparticles were incubated with heparin solutions, which implies that the bindings of ligand to the receptor functionalized Au nanoparticles are specific. Being a major inhibitor of coagulation enzymes, AT III has affinity to heparin, which enhances the activity of AT III several hundred-fold.39,40 Here, the bindings of AT III to heparins including LMWH are evaluated in Figure 4D,E. The curves have similar trends to the above bindings. The binding constants of AT III to heparin and LMWH are found to be (3.1 ± 0.3) × 106 M−1 and (8.0 ± 0.5) × 105 M−1, respectively, and agree well with the reported values in the literature (6.3 × 106 M−1 and 3.2 × 105 M−1).5 It is necessary to mention that the red shift of the receptor functionalized nanoparticle LSPR peak seems dependent on the molecular size of the ligand and the affinity strength of the ligand to the receptor together. On the one hand, the LSPR red shift is determined by the ligand size when the ligands have similar affinity to the receptor. For
CONCLUSIONS A method for evaluating carbohydrate−protein (ligand− receptor) interactions by LSPR peak shift of single Au nanoparticles is developed. The throughput limitation of the transmission grating based single particle spectral imaging, the zeroth-order spot of a single nanoparticle being in the same frame with its first-order streak, has been broken through calibrating the first streak by the cutoff edge of the inserted band-pass filters. The method has been used to successfully and quantitatively determine the interactions between receptors and ligands, including the strong interaction between transferring and antitransferrin, the moderate interactions between AT III and heparins, and the weak interaction between BSA and heparin. The method provides the opportunity to assess interactions in true equilibrium since the total concentrations of the receptor and the ligand are not disturbed by our means during the whole measurement. Moreover, the precise value of the receptor concentration is not required to be known as long as the ligand is in excess.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors are grateful to the Natural Science Foundation of China (NSFC, 21075033), Qinglan Project of Jiangsu Province, and Priority Academic Program Development of Jiangsu Higher Education Institutions.
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REFERENCES
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Single Gold Nanoparticle Localized Surface Plasmon Resonance Spectral Imaging for Quantifying Binding Constant of Carbohydrate−Protein Interaction Xiaojun Liu, Qingquan Zhang, Yang Tu, Wenfeng Zhao, and Hongwei Gai* School of Chemistry and Chemical Engineering, Jiangsu Key Laboratory of Green Synthesis for Functional Materials, Jiangsu Normal University, Xuzhou, Jiangsu 221116, China
ABSTRACT: Quantifying carbohydrate−protein (ligand−receptor) interactions is important to understand diverse biological processes and to develop new diagnostic and therapeutic methods. We develop an approach to quantitatively study carbohydrate−protein interactions by Au nanoparticle localized surface plasmon resonance (LSPR) peak position shift at the single particles level. Unlike the previous techniques for single particle LSPR spectral imaging, only the first-order streak of an individual nanoparticle is needed to extract a LSPR spectrum, which has great potential to increase throughput to 500 single particle spectra in each frame. LSPR peak shift of protein modified single Au nanoparticles is found to be a function of its ligand concentration, which can be used to fit the binding constants of the interactions. The moderate interactions of Antithrombin III (AT III) and heparins including low molecular weight heparin (LMWH) are determined as well as the strong interaction of transferrin and antitransferrin and the weak interaction of bovine serum album (BSA) and heparin. The measured binding constants of transferrin to antitransferrin, heparin and LMWH to AT III, and BSA to heparin are (3.0 ± 0.6) × 109 M−1, (3.1 ± 0.3) × 106 M−1, (8.0 ± 0.5) × 105 M−1, and (5.1 ± 0.1) × 103 M−1, respectively, which are in good agreement with the reported values.
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signal amplification, simplification of separation, and analogy to multivalent binding at cell surfaces.13−23 For example, Au nanoparticles were used in QCM, which resulted in a 16 time increase in detection signal.19 Another interesting development is that change in intensity of Au nanoparticle localized surface plasmon resonance (LSPR) is exploited to assess carbohydrate−protein interactions in a real-time, biocompatibility, and label-free way.24 In fact, LSPR peak position of Au nanoparticles is highly sensitive to local refractive index change caused by the binding of target molecules to Au nanoparticles. This unique property has been exploited for biosensors which can be classed into ensemble and single particle level, depending on the number of Au nanoparticles being monitored. Over the bulk counterpart, the advantages of single Au nanoparticle sensors have been summarized in a couple of
arbohydrate−protein interactions are involved in diverse biological processes, and specific carbohydrate−protein interactions play a vital role in some biological events like cell adhesion, cell differentiation, bacteria and virus infections, and cancer metastasis.1−3 At the cell surface, carbohydrate−protein interactions usually occur in multivalent form to enhance affinity, and many effectors determine whether specific carbohydrate−protein interaction happens and whether it is strong enough to trigger relevant events. Characterization of carbohydrate−protein interactions will lead to a better understanding of those biological processes and promote development of new diagnostic and therapeutic methods. Many techniques have been developed to evaluate multivalent carbohydrate−protein interactions, such as capillary electrophoresis, chromatography, NMR, ELISA, quartz crystal microbalance (QCM), surface-plasmon resonance (imaging), isothermal titration calorimetry, and glycan microarrays.4−12 Recently, nanoparticles, particularly Au nanoparticles, are introduced into some of these techniques for the purpose of © XXXX American Chemical Society
Received: August 10, 2013 Accepted: November 15, 2013
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reviews, including high sensitivity, high throughput, heterogeneity free, and low-dose reagent.25,26 A LSPR spectrum of a single Au nanoparticle with a diameter larger than 40 nm can be easily obtained through taking images by a dark-field microscope equipped with a charge coupled device (CCD) camera. The key is how to convert an image of single Au nanoparticle into a LSPR spectrum. Two kinds of methods have been developed for the task. One is to place a commercial spectrometer at the conjugate image plane in order to record a single particle’s spectrum.25,27−29 This approach needs to distinguish different particles prior to spectral monitoring, and only a limited number of nanoparticles are processed per minute. Another called transmission grating based spectral imaging is developed to enhance spectral throughput,30,31 by which we have investigated the spectra of single dyes and single quantum dots (QD) and have developed superlocalization spectral imaging for QDs.32−34 The principal is that a scattering light spot of a single nanoparticle is dispersed into a zerothorder spot and a first-order streak by a transmission grating inserted in front of CCD. The wavelength is directly proportional to the distance between the two orders. In the previous transmission grating based spectral imaging work,30−34 the images of the zeroth-order spot and the first-order streak of a single particle have to be simultaneously captured in the same frame to calibrate the origin of the wavelength. This undoubtedly deteriorates the imaging throughput. To address the issue, we utilize the cutoff edge of bandpass filters to calibrate and convert the first-order streak of a single nanoparticle into the spectrum. Using this technique, we demonstrate a method for studying carbohydrate−protein interactions by Au nanoparticle LSPR peak shift. Protein functionalized Au nanoparticles are incubated with its ligand at varying concentrations. The LSPR peak shift of the protein functionalized Au nanoparticles is a function of the initial concentration of the ligand and can be used to derive a binding constant through nonlinear fitting under the condition that the ligand is in large excess. This approach is verified by the moderate interactions between antithrombin III (ATIII) and heparins as well as the weak interaction between bovine serum album (BSA) and heparin and the strong interaction between antitransferrin and transferrin. To the best of our knowledge, there have been no reports of evaluating molecular interactions through the peak position shift of Au nanoparticle LSPR at both ensemble and single particle levels.
Park, IL). Rabbit antitransferrin was a kind gift from Sangon Biotech Co., Ltd. (Shanghai, China). Preparation of Protein Functionalized Au Nanoparticles. Prior to protein modification, Au nanoparticles were immobilized onto a glass slide. In brief, the glass slides were sequentially immersed into potassium dichromate/ sulphuric acid solution for 30 min at 70 °C, ultrasonically washed with ultrapure water 3 times, dried for 2 h at 120 °C, and placed into a vacuum desiccator along with an uncapped vial containing MPTS. The desiccator was vacuumed and kept in darkness for at least 2 h. The glass slide coated with thiol groups was reversibly sealed with a 5 mm thick PDMS square (30 mm × 30 mm) onto which a hole about 5 mm in diameter had been drilled at the center. The PDMS layer was ultrasonically washed with ethanol 3 times and blow-dried right before sealing. An aliquot of 5 μL of Au nanoparticle solution (30 time dilution) was added into each well, and the precipitation of Au nanoparticles was allowed to continue for 15 min. In order to coat Au nanoparticles with carboxyl group, the immobilized Au nanoparticles were washed with 25 mM MES buffer (pH 5.5) twice and incubated with 1 mM DDA for 5 min followed by the washing with ultrapure water. The Au nanoparticles were incubated with 20 μL of 1 mg/mL protein in 100 mM MES buffer for 30 min, and 3 μL of freshly prepared 10 mg/mL EDC in cold MES buffer was added into the mixture, with the exception of antitransferrin solution. A portion of 10 μL of antitransferrin solution was diluted to 450 μL with the same MES buffer, and the process was followed with centrifugation in a 100 kDa tube in order to remove bovine serum albumin. This was repeated 3 times before the antitransferrin solution was added into the PDMS hole. The interaction between the protein and the Au nanoparticles proceeded for 2 h at room temperature or overnight at 4 °C. Finally, the assembly was extensively washed and filled with ultrapure water and stored in a sealed box at 4 °C until use. Scattering Spectrum of the Bulk Au Nanoparticle Solution. The scattering spectrum of the bulk Au nanoparticle solution was measured in synchronous scan mode on a fluorescence spectrophotometer (Hitachi, F-4500). The scan wavelength is from 200.0 to 900.0 nm. The slit width and Δλ were set as 10.0 and 0.0 nm, respectively. Single Au Nanoparticle LSPR Spectral Imaging. Prior to observation under dark-field microscopy, the liquids were drained out of the hole, and the PDMS square was removed. Immediately, an aliquot (2 μL) of 20 mM phosphate buffer (pH 7.4) or heparin solution in the same phosphate buffer was deposited on the glass slide and sealed with a coverslip. The observation was conducted on an inverted fluorescent microscope (Olympus IX71) equipped with a 100× oil immersion objective with numerical aperture of 0.6 to 1.3 (Olympus, Tokyo, Japan) and an Evolve 512 electron-multiplied chargecoupled device from Photometrics (EMCCD; Tucson, USA). All filters (536/20 nm and 593/20 nm filters) were purchased from Semrock (Rochester, NY). The temperature, the gain, and the exposure time of the camera were maintained at −80 °C, 16, and 0.3 s, respectively. To achieve a single Au nanoparticle LSPR spectral image, a transmission grating with 70 lines per mm (Edmund Scientific, Barrington, NJ) was placed in front of the EMCCD camera, and the dispersed scattering light from a single Au nanoparticle under each filter and no filter was recorded. Image J and Origin software were used to process data.
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EXPERIMENTAL SECTION Chemical and Material. Gold nanoparticles of 70 nm in diameter were purchased from Nano Partz Inc. (Loveland, CO). The number of Au nanoparticles is 1.6 × 1010 per mL. Sylgard 184 PDMS oligomer and curing agent were from Dow Corning (Midland, MI). 4,4′-Dithiodibutyric acid (DDA), 2(N-morpholin o) et hanesulfonoic acid (MES), (3mercaptopropyl)triethoxysilance (MPTS), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), epidermal growth factor (EGF), BSA, and transferrin were obtained from Sigma (St. Louis, MO). Heparin with molecular weight of 15.3 kDa and low molecular weight heparin (LMWH) with molecular weight of 8.0 kDa were a kind gift from Aglyco Biotechnology Inc. (Beijing, China). Antithrombin III (AT III) was bought from Dongfeng Biotechnology (Shanghai, China). Tico brand of glass slides and other chemicals were from the local suppliers. Coverslips were from Fisher Scientific (Hanover B
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of the unpaired first-order streaks indicated by the dash squares in Figure 1B cannot be obtained due to lack of the zeroth-order spots. The percentage of unpaired first-order streaks is dependent on the size of the field of view, the density of Au nanoparticle distribution, and particularly the grating constant (d) and the distance (S). Under our conditions, 20% to 40% of the first-order streaks are usually unpaired. Sometimes, it is hard to match the zeroth-orders and their corresponding firstorders by the positions. Moreover, to accurately convert the unit of L from pixel into wavelength, a narrow band light source such as laser has to be introduced into the imaging system before measuring the spectra of single molecules or single nanoparticles.31 This is certainly a little more complicated for nonoptical experts. Here, we present a simple method to obtain an individual particle spectrum only by the first-order streak. The principal is illustrated in Figure 2. Figure 2A shows the first-order streak images of the single Au nanoparticles in the exactly same area consecutively taken under 536/20 and 593/ 20 nm bandpass filters and no filter (from top to bottom). The pixels’ intensities of the Au nanoparticle LSPR images in the dash square are extracted with Image J and drawn next in Figure 2B. The plots that are converted from the images taken under the bandpass filters have sharp changes in intensity indicated by the triangles. The changes are caused by the dramatically narrow transition between the maximal and minimal transmission ratio of the bandpass filters. The wavelength, which their lateral positions of the start, the middle, and the end of the sharp changes correspond to, should be equal to the wavelengths of the maximal, the half, and the minimal transmission ratios of the cutoff edge of the used bandpass filter as shown in Figure 2C. Using the middles of the sharp changes as calibration points, the pixels’ intensity spectrum taken under no filter in the bottom of Figure 2B is converted into the LSPR spectrum next in Figure 2C. To calibrate the scale accurately, two sets of bandpass filters are used. The peak wavelength (553.0 nm) is in good agreement with the one (553.8 nm, figure not shown) determined by the distance between the first-order spot and the second-order streak. The LSPR peak distribution of randomly selected more than 50 particles is plotted in Figure 3A. The mean value of the single particle LSPR peak wavelengths is determined to be 549.4 ± 0.4 nm using Gaussian fitting which agrees with the peak wavelength (551.8 ± 0.2 nm) of the scattering spectrum of the bulk Au nanoparticle solution in Figure 3B and the spectrum provided by the manufacturer as well. The interferences among nanoparticles may cause the ensemble spectrum to broaden compared to the spectra distribution of single nanoparticles. This method has great potential to increase analysis throughput since the zeroth-order spots are no longer needed. Theoretically, more than 500 distinguished streaks can be obtained in a single frame, considering that a first-order streak image occupies 58 × 4 pixels and the CCD device has a 512 × 512 pixel imaging area. Thus, the spectral throughput is calculated to be more than 1000 per minute since all the 3 frames have been taken within 30 s. Affinity Calculation. The equilibrium expression for the association reaction involving protein (P, receptor) immobilized onto Au nanoparticles and carbohydrate (C, ligand) is described by the following equation
RESULTS AND DISCUSSION Single Au Nanoparticle LSPR Imaging. The schematic of transmission grating based single particle spectral imaging is shown in Figure 1A. The scattering wavelength of single
Figure 1. (A) The scheme of transmission grating based spectral imaging dark-field microscopy. (B) The typical zero-order spot and first-order streak images of individual Au nanoparticles. Single triangle and double triangles point to the zero-order spots and the first-order streaks, respectively. The zero-order spot and the first-order streak pointed to by the same color triangles are pairs and belong to an individual Au nanoparticle. The squares in the dashed line mark the unpaired first-order streaks. The scale bar is 10 μm.
nanoparticle (λ) follows the equation, λ = L × d/S, (S, the distance between the grating and the EMCCD chip; d, the grating constant; L, the distance between the zeroth-order spot and the first-order streak). In previous work,32−34 an essential step is to accurately determine the value of L. Thus, the zerothorder spot and the first-order streak of a single nanoparticle have to appear in the same frame and then make a pair in order to determine the origin of the spectrum. Matching the zerothorder spots with the first-order streaks is performed by comparing their relative positions. In brief, if the graph drawn by connecting the center of the zeroth-order spots is the same as the one by the first-order streaks, the zeroth-order spots and the first-order streaks are paired. The first-order streaks that can not find the corresponding zeroth-order spots are unpaired. In Figure 1B, the pairs, the spots, and their corresponding streaks of single nanoparticles are labeled with a single triangle and double triangles in the same color, respectively. The spectrum
P + nC ⇌ PCn C
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Figure 2. Calibration of single Au nanoparticle LSPR spectral imaging by bandpass filters. (A) The representative first-order spectral images of individual Au nanoparticles in a same area consecutively taken under 536/20 and 593/20 nm filters and no filter (from top to bottom). The three images have been taken within 30 s. (B) The corresponding pixels’ intensity of the first-order streak of the same individual particle marked in panel A. The triangles in different colors indicate the start, the middle, and the end of the sharp intensity change of the LSPR image under filter. (C) The transmission spectrum of the bandpass filter and the scattering spectrum of the bulk Au nanoparticle solution in the same row of panel A and the obtained single particle LSPR spectrum are drawn in the red, blue, and black line, respectively. The triangles in different colors indicate the maximal, the middle, and the minimal transmission of the cutoff edge of the filter. The wavelengths indicated by the same color triangles in panels B and C are equivalent.
complex and the free protein are equal to the value of [APC]∗q and [AP]∗p, respectively. Here, [APC] and [AP] represent the equilibrium concentrations of the carbohydrate bound Au nanoparticles and the unbound Au nanoparticles, respectively. Therefore, the eq 2 can be re-expressed and simplified as the eqs 3 and 4 K=
[APC] × q [AP] × p × [C]n
K=α
Hence, the binding constant between the protein and the carbohydrate (K) is
[PCn] [P][C]n
(4)
where α is equal to the value of q/p. The LSPR peak position of Au nanoparticles is sensitive to the local surrounding media’s refractive index change caused by the binding of the target molecules to the Au nanoparticles. The protein functionalized Au nanoparticles are incubated with a series of carbohydrate solutions at varying concentrations. Thus, there exists a kinetic equilibrium between the protein functionalized Au nanoparticles and the carbohydrate, which resulted in a rapid reversible conversion between the bound and the unbound Au nanoparticles. Assuming the observed LSPR peak shift of the Au nanoparticles is proportional to the number of the bound Au nanoparticles, the observed (apparent) LSPR peak wavelength of the Au nanoparticles is
Figure 3. (A) The LSPR spectra distribution of single Au nanoparticles. The dashed line is the Gaussian fit of the histogram. (B) The synchronous scattering spectrum of the bulk Au nanoparticles solution in real line. The upper column distribution is rescaled in the dashed line.
K=
[APC] [AP][C]n
(3)
λ=
(2)
where PCn, P, C, and n are the equilibrium concentrations of the carbohydrate−protein complex, the free protein, the free carbohydrate, and the number of the binding sites on the protein. Since the protein is immobilized on the Au nanoparticles, the equilibrium concentration of the protein is related to the Au nanoparticle concentration. Assuming the number of proteins onto the carbohydrate bound Au nanoparticles and the unbound Au nanoparticles is q and p, the equilibrium concentrations of the carbohydrate−protein
[APC] [AP] λAPC + λAP [APC] + [AP] [APC] + [AP]
(5)
where λ, λAPC, and λAP are the apparent LSPR peak wavelength of the Au nanoparticles, the LSPR peak wavelength of the bound Au nanoparticles, and the unbound Au nanoparticles, respectively. A combination of the eqs 4 and 5 gives ⎛ ⎞ [C]n λ − λAP = (λAPC − λAP)⎜ n⎟ ⎝ α /K + [C] ⎠ D
(6)
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Figure 4. (A) Relationship between transferrin concentration and LSPR peak shift of antitransferrin functionalized Au nanoparticles. (B) Relationship between heparin concentration and LSPR peak shift of bovine serum albumin functionalized Au nanoparticles. (C) Relationship between heparin concentration and LSPR peak shift of EGF functionalized Au nanoparticles. (D) Relationship between heparin concentration and LSPR peak shift of AT III functionalized Au nanoparticles. (E) Relationship between LMWH concentration and LSPR peak shift of AT III functionalized Au nanoparticles. The real line is the model fitting curve according to eq 8. The calculated binding constants (K), the number of binding sites on the protein (n), and the Δλmax are listed in the figures.
concentrations and Δλmax (Δλmax= λAPC − λAP) is the difference in the LSPR peak wavelength between the bound Au nanoparticles and the unbound Au nanoparticles, in other words, the maximum of LSPR peak shift in the presence of the carbohydrate at infinite concentration. The eq 7 is a transform of the Hill equation.35 In the case that the carbohydrate is in large excess, the equilibrium concentration of the carbohydrate
The eq 6 can be rewritten ⎛ ⎞ [C]n Δλ = Δλmax ⎜ n⎟ ⎝ α /K + [C] ⎠
(7)
where Δλ (Δλ =λ − λAP) is the LSPR peak shift of the Au nanoparticles in the presence of the carbohydrate at certain E
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instance, the maximum of LSPR peak red shift of the AT III functionalized Au nanoparticles is 11.5 and 8.8 nm in the presence of heparin and LMWH, respectively. On the other hand, the red shifts are determined by the affinity strength. It is supported by the fact that the BSA functionalized Au nanoparticle LSPR peaks shift far more slightly than the AT III functionalized Au nanoparticle in the presence of heparin.
is approximately equal to the initial concentration, and the eq 6 can be described as ⎞ ⎛ [C0]n Δλ = Δλmax ⎜ n⎟ ⎝ α /K + [C0] ⎠
(8)
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where [C0] is the initial concentration of the carbohydrate. As seen in the eq 8, the LSPR peak shift of the protein functionalized Au nanoparticles is a function of the initial concentration of the carbohydrate and can be used to derive the binding constants for the carbohydrate−protein interactions by nonlinear fitting. Determination of Binding Constants. To test our theory, the strong binding of antibody to antigen such as antitransferrin to transferrin was quantitatively studied. The Au nanoparticles coated with antitransferrin were incubated with 2 μL of transferrin solution at a certain concentration and imaged with dark-field microscopy. For each concentration, more than 100 Au nanoparticles were randomly selected. The mean value of these single particle LSPR peak wavelengths, being equal to the peak wavelength of the bulk Au nanoparticle solution scattering spectrum, was obtained by the above-described procedures. Using the LSPR peak of the antitransferrin functionalized Au nanoparticles without the presence of transferrin as reference, the red shift of the antitransferrin functionalized Au nanoparticle LSPR peak is observed in the presence of transferrin and plotted against transferrin concentration in Figure 4A. With an increase in transferrin concentration, the red shift of the antitransferrin functionalized Au nanoparticle LSPR peak becomes bigger until saturated, which indicates the binding of transferrin to the antitransferrin functionalized Au nanoparticles. The weak binding of BSA to heparin was assessed as well. The relationship between heparin concentration and LSPR peak shift of the BSA functionalized Au nanoparticles is illustrated in Figure 4B. By nonlinear fitting of Δλ against [C0] in Figure 4A,B according to the eq 8, the binding constants of antitransferrin to transferrin and BSA to heparin are found to be (3.0 ± 0.6) × 109 M−1 and (5.1 ± 0.1) × 103 M−1, respectively, which are in agreement with the reported values (∼1010 M−1 and (1.24 ± 0.05) × 103 M−1).37,38 This indicates the applicable range of our theory is wide. In order to exclude the possibility that the red shifts of the receptor functionalized Au nanoparticle LSPR peak are from nonspecific binding, EGF that had been known not to have affinity to heparins is used as control.36 As shown in Figure 4C, the LSPR peaks of the EGF functionalized Au nanoparticles do not shift before and after the nanoparticles were incubated with heparin solutions, which implies that the bindings of ligand to the receptor functionalized Au nanoparticles are specific. Being a major inhibitor of coagulation enzymes, AT III has affinity to heparin, which enhances the activity of AT III several hundred-fold.39,40 Here, the bindings of AT III to heparins including LMWH are evaluated in Figure 4D,E. The curves have similar trends to the above bindings. The binding constants of AT III to heparin and LMWH are found to be (3.1 ± 0.3) × 106 M−1 and (8.0 ± 0.5) × 105 M−1, respectively, and agree well with the reported values in the literature (6.3 × 106 M−1 and 3.2 × 105 M−1).5 It is necessary to mention that the red shift of the receptor functionalized nanoparticle LSPR peak seems dependent on the molecular size of the ligand and the affinity strength of the ligand to the receptor together. On the one hand, the LSPR red shift is determined by the ligand size when the ligands have similar affinity to the receptor. For
CONCLUSIONS A method for evaluating carbohydrate−protein (ligand− receptor) interactions by LSPR peak shift of single Au nanoparticles is developed. The throughput limitation of the transmission grating based single particle spectral imaging, the zeroth-order spot of a single nanoparticle being in the same frame with its first-order streak, has been broken through calibrating the first streak by the cutoff edge of the inserted band-pass filters. The method has been used to successfully and quantitatively determine the interactions between receptors and ligands, including the strong interaction between transferring and antitransferrin, the moderate interactions between AT III and heparins, and the weak interaction between BSA and heparin. The method provides the opportunity to assess interactions in true equilibrium since the total concentrations of the receptor and the ligand are not disturbed by our means during the whole measurement. Moreover, the precise value of the receptor concentration is not required to be known as long as the ligand is in excess.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors are grateful to the Natural Science Foundation of China (NSFC, 21075033), Qinglan Project of Jiangsu Province, and Priority Academic Program Development of Jiangsu Higher Education Institutions.
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