J. Phys. Chem. B 2009, 113, 14405–14412
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Low Density Lipoprotein Detection Based on Antibody Immobilized Self-Assembled Monolayer: Investigations of Kinetic and Thermodynamic Properties Zimple Matharu,†,| Amay Jairaj Bandodkar,‡ G. Sumana,† Pratima R. Solanki,† E. M. I. Mala Ekanayake,§ Keiichi Kaneto,§ Vinay Gupta,| and B. D. Malhotra*,†,⊥ Department of Science and Technology Centre on Biomolecular Electronics, National Physical Laboratory, Dr. K. S. Krishnan Marg, New Delhi-110012, India, Department of Applied Chemistry, Institute of Technology, Banaras Hindu UniVersity, Varanasi- 221005, India, Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology Kitakyushu, 808-0196, Japan, Department of Physics and Astrophysics, UniVersity of Delhi, Delhi-110007, India, and Centre for NanoBioEngineering and Spintronics, Chungnam National UniVersity, Daejeon, 305-764, Korea ReceiVed: April 21, 2009; ReVised Manuscript ReceiVed: September 21, 2009
Human plasma low density lipoprotein (LDL) immunosensor based on surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) was fabricated by immobilizing antiapolipoprotein B (AAB) onto selfassembled monolayer (SAM) of 4-aminothiophenol (ATP). The AAB/ATP/Au immunosensor can detect LDL up to 0.252 µM (84 mg/dL) and 0.360 µM (120 mg/dL) with QCM and SPR, respectively. The SPR and QCM measurements were further utilized to study the reaction kinetics of the AAB-LDL interaction. The adsorption process involved was explored using Langmuir adsorption isotherm and Freundlich adsorption models. The thermodynamic parameters such as change in Gibb’s free energy (∆Gads), change in enthalpy (∆Hads), and change in entropy (∆Sads) determined at 283, 298, and 308 K revealed that the AAB-LDL interaction is endothermic in nature and is governed by entropy. Kinetic, thermodynamic, and sticking probability studies disclosed that desorption of the water molecules from the active sites of AAB and LDL plays a key role in the interaction process and increase in temperature favors binding of LDL with the AAB/ ATP/Au immunosensor. Thus, the studies were utilized to unravel the most important subprocess involved in the adsorption of LDL onto AAB-modified ATP/Au surface that may help in the fabrication of LDL immunosensors with better efficiency. LDL Cholesterol (mg/mL) ) Total Cholesterol HDL Cholesterol - 0.2X (Triglycerides) (1)
Introduction Cholesterol is an essential structural constituent of cell membranes. It is insoluble in water and is carried in plasma by a series of proteins containing micelles known as lipoproteins categorized as very low density lipoprotein (VLDL), low density lipoprotein (LDL), intermediate density lipoprotein (IDL), and high density lipoprotein (HDL).1 Among these, LDL is one of the cholesterol-rich lipoproteins that transports cholesterol to the arteries. LDL is a large protein and is composed of lipid (75-80%) and a single protein molecule, apolipoprotein B-100 (20-25%) that can be recognized by specific surface receptor proteins on cells that need to take up cholesterol. Cholesterolenriched low-density lipoproteins (LDL) in plasma promote the deposition of plasma lipids in the artery wall and thereby elicit the formation of atherosclerotic plaques. A high LDL cholesterol level may increase chances of developing coronary heart disease. Hence its estimation in blood has become clinically very important.2,3 LDL concentration is presently quantified in most clinical laboratories by an indirect method using Friedewald equation4 * To whom correspondence should be addressed. Tel.: +91-11-45609152. E-mail:
[email protected]. † National Physical Laboratory. | University of Delhi. ‡ Banaras Hindu University. § Kyushu Institute of Technology Kitakyushu. ⊥ Chungnam National University.
LDL can be quantitatively estimated by ultracentrifugation5 and NMR spectroscopy6 and its size can be determined by gradient-gel electrophoresis.7 However, for general clinical use these methods require additional steps and are time-consuming. Therefore availability of a simple detection system with high sensitivity is desirable. In this context, biosensors offer several benefits over conventional diagnostic tools as they include simplicity of use, specificity for the target analyte, speed, capability for continuous monitoring and multiplexing, together with the potentiality of coupling to low-cost, portable instrumentation.8-14 Attempts have recently been made toward the fabrication of sensors for LDL detection. Snellings et al. have fabricated an acoustic wave biosensor for detection of lipoprotein fractions using dextran sulfate (DS) modified self-assembled monolayer (SAM) of 11-mercapto-1-undecanol on gold (Au) surface.15 It has been found that DS coating is more selective to LDL fraction as compared to other lipoprotein fractions. However, the authors could not achieve reproducibility for coating DS onto a similar surface and the sensor lacked specificity. Therefore, rapid and highly specific interaction based sensor is urgently required for LDL detection. In this context, Matharu et al. have fabricated LDL sensor using specific LDL-heparin interaction by surface plasmon resonance (SPR) technique.16 Jie et al. have used apolipoprotein B-LDL interaction for the fabrication of CdS nanocrystal based electroluminescence biosensor for LDL detection.17 Yan et al. have used a highly specific antibody,
10.1021/jp903661r CCC: $40.75 2009 American Chemical Society Published on Web 10/07/2009
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antiapolipoprotein B, that acts as a LDL receptor and fabricated an electrochemical biosensor by adsorption of antiapolipoprotein B on gold nanoparticles-AgCl-polyaniline-modified glassy carbon electrode.18 However, no efforts have yet been made to understand the mechanism of LDL binding to antiapolipoprotein B and the parameters that govern the binding. An understanding of the mechanism governing the binding of LDL to antiapolipoprotein B is essential to manipulate the LDL adsorption on the immunosensor surface for the fabrication of LDL biosensors with improved characteristics. Although the binding mechanism of proteins is complex, the guiding rules and the plausible driving mechanism of protein adsorption can be predicted by kinetic and thermodynamic analysis of the interaction phenomenon. In this context, SPR and quartz crystal microbalance (QCM) are powerful techniques for in-depth online study of kinetics of target protein-receptor binding for label-free detection.19,20 SPR technique is very specific to the interface region and can probe the adsorption of molecules at the interface. The primary attraction of an SPR-based sensor is its capacity for highly specific detection of small molecules with a low detection limit being realized for a wide variety of analytes in complex matrices.20 The main advantage of SPR system lies in its application for determination of both the affinity and kinetics of interaction between two or more biomolecules, antibody-antigen interactions21 and detection of nucleic acid hybridization.22 Whereas in QCM based biosensors, change in mass based on Sauerbrey’s equation23,24 due to the immobilized biolayer is utilized for transduction. In the present manuscript, we report results of studies relating to the immobilization of antiapolipoprotein B monoclonal antibody (AAB) onto SAM of 4-aminothiophenol (ATP) via EDC-NHS chemistry for LDL detection. The detection of LDL was carried out using SPR and QCM techniques. The interaction of LDL with the AAB/ATP/Au immunosensor was described by both Langmuir and Freundlich adsorption models. The adsorption behavior of LDL onto AAB was investigated by probing the kinetic and thermodynamic properties of AAB-LDL interaction. On the basis of these studies, efforts were made to delineate the most important subprocess involved in the LDL adsorption on the AAB surface. Manipulation of this subprocess may help in fabricating LDL immunosensors with higher efficiency. Experimental Section Material and Procedures. Chemicals and Reagents. LDL (MW 3500 kDa), bovine serum albumin (BSA), N-hydroxysuccinimide (NHS), N-ethyl-N-(3-dimethylaminopropyl carbodimide) (EDC), ATP, and apolipoprotein B-100 (AB) (MW 515 kDa) were procured from Sigma-Aldrich (U.S.A.). Antiapolipoprotein B mouse monoclonal antibody (AAB), HDL, and VLDL were procured from Merck Biosciences. All other chemicals were of analytical grade and were used without further purification. The 50 nm thick Au-coated BK-7 glass plates (24 mm diameter) were purchased from Autolab, The Netherlands. Solution Preparation. Lyophilized powder (5 mg) of LDL was reconstituted with 1 mL of deionized water (18.2 MΩcm) to make a solution containing 150 mM NaCl of pH 7.4 and 0.01% EDTA. AAB solution (1 mg/mL) was prepared in 50 mM phosphate buffer saline solution of pH 7.4 containing 150 mM NaCl (PBS). Solutions of BSA (2 mg/mL), LDL, and AB were prepared in PBS. Formation and Modification of 4-Aminothiophenol (ATP) Self-Assembled Monolayer (SAM) on Gold (Au) Surface. Prior to SAM formation, Au plates were cleaned with acetone, ethanol, and with copious amount of deionized water. Further,
Matharu et al. SCHEME 1: Schematic Showing Stepwise Preparation of AAB/ATP/Au Immunosensor and LDL Binding
Au plates were treated with piranha solution (H2SO4/H2O2; 7:3) for about 5 min followed by rinsing with deionized water and later with acetone and ethanol. The precleaned Au plates were then immersed into a 2 mM solution of ATP in ethanol overnight at room temperature for SAM formation after which the plates were sonicated in ethanol for about 10 min and were then rinsed with ethanol and acetone followed by deionized water to remove any unbound ATP molecules. AAB was immobilized onto the ATP/Au surface using EDC-NHS chemistry25 in which EDC works as a coupling agent and NHS works as an activator. EDCNHS activates the -COOH group of AAB which subsequently reacts with the -NH2 group of ATP to produce a stable amide bond. For immobilization of AAB, the ATP/Au plate was incubated in the 1 mg/mL solution of AAB containing 0.2 M EDC and 0.05 M NHS for 4 h followed by washing with PBS containing 0.05% tween-20 to remove any unbound AAB. BSA has been extensively used for blocking the nonspecific adsorption of LDL.2,17,18 A control experiment performed for LDL attachment onto the BSA modified ATP surface provided a negligible change in the SPR angle indicating negligible binding of LDL with BSA-modified surface (see Supporting Information, Figure 2S). Hence, the prepared AAB/ATP/Au immunosensor was immersed in BSA for 1 h to block the nonspecific sites for LDL adsorption. The prepared AAB/ATP/Au immunosensor was exposed to different concentrations of LDL. The entire process was monitored by in-situ SPR and QCM measurements at 298 K. The SPR and QCM studies were further utilized to study the reaction kinetics of the AAB-LDL interaction. Morphological characterization of ATP/Au, AAB/ATP/Au, and LDL/AAB/ ATP/Au surfaces was carried out using scanning electron microscopy (Leo 440 model). Scheme 1 shows stepwise preparation of AAB/ATP/Au immunosensor and LDL binding. SPR Instrumentation and Measurements. The SPR measurements were carried out on SPR instrument (Autolab, Eco Chemie, The Netherlands) having Kretschmann configuration.16 In these studies, the SPR signal was recorded as a function of LDL concentration for the AAB/ATP/Au immunosensor. The
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Figure 1. (a) Au crystal along-with the QCM holder. (b) Setup for in-situ QCM measurement.
Figure 2. SEM images of (i) ATP/Au, (ii) AAB/ATP/Au, (iii) LDL/AAB/ATP/Au surfaces, and (iv) LDL/AAB/ATP/Au at higher magnification.
SPR signal was first recorded with running solution (PBS) to stabilize the AAB/ATP/Au immunosensor and to set the baseline. Subsequently, different concentrations of LDL were run onto the AAB/ATP/Au immunosensor surface. During each run, the first 300 s represents the baseline after which the sample (LDL solution) was introduced and allowed to interact with the AAB/ATP/Au immunosensor for the next 2 h (association phase). On the completion of the association phase, the extra solution was discarded and the AAB/ATP/Au immunosensor was washed with PBS (dissociation phase) and the SPR signal was recorded. The increase in the value of SPR angle after the complete process (net increase in the final baseline from the initial baseline) corresponds to the amount of bound analyte since other processing conditions were kept constant. The AAB/ ATP/Au immunosensor was regenerated with 0.2 M glycine solution of pH 2.4 between two consecutive binding phases for different LDL concentrations. QCM Instrumentation and Measurements. QCM studies were performed using Research Quartz Crystal Microbalance (RQCM) from Maxtek, Inc., composed of the Au crystal (resonance frequency ) 5 MHz, diameter ∼1.3 cm), oscillator unit, frequency counter, voltage supply, and PC interface
connection for signal output visualization. The AAB/ATPmodified gold crystal was placed in the holder and positioned by an O-ring so that only one side was exposed to the LDL solution. The crystal holder was fitted with a custom-made cell (1 mL) as shown in Figure 1. During the whole experiment, the cell was filled with 1 mL PBS as background solution. After obtaining the baseline, 50 µL of each concentration of LDL was injected in the cell and left for about 2 h for AAB-LDL binding. The AAB/ATP/Au immunosensor was regenerated by the procedure described earlier in the SPR instrumentation and measurements section. Results and Discussion Scanning Electron Microscopy (SEM) Studies. The surface morphologies of ATP/Au, AAB/ATP/Au, and LDL/AAB/ATP/ Au surfaces were studied using SEM. Figure 2i shows SEM picture of ATP/Au surface in which a homogeneous granular morphology could be seen. A uniform fibrous structure with some aggregation was observed after immobilization of AAB onto ATP/Au surface (Figure 2ii). This may be due to the fact that during immobilization of AAB onto ATP/Au surface via EDC-NHS, there is a possibility that the activated -COOH group
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of one AAB molecule may react with -NH2 group of the other AAB molecule and may lead to aggregation. However, it has been found that better immobilization and sensing characteristics can be achieved with EDC-NHS26 as it is a zero-length cross-linker and thus provides a direct linkage between two molecules with no intervening linker or spacer. Apart from EDC-NHS, other crosslinkers such as glutaraldehyde,27,28 glyoxal,29,30 dimethyl pimelimidate (DMP)31 and sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl1,3′-dithiopropionate (SASD)32 have also been used for covalent immobilization of biomolecules. These cross-linkers may perhaps limit the aggregation of the biomolecules. However, these along with many other cross-linkers involve the amino group of protein for bioconjugation. Because the paratopes of the antibody are present in the N-terminal region, the use of these cross-linkers might affect the activity of the antibody. Also, most of these cross-linkers act as a spacer between the biomolecule and the matrix and can thus affect the sensitivity and range of detection while using interface sensitive techniques like SPR and QCM. Figure 2iii,iv shows the SEM pictures of the LDL/AAB/ATP/ Au surface. The morphological change in the SEM images after incubation of LDL indicates the binding of LDL to the AAB/ ATP/Au surface. Response Studies of AAB/ATP/Au Immunosensor. SPR Studies. The performance of the AAB/ATP/Au immunosensor was studied at room temperature (∼298 K). Figure 3a shows the characteristic SPR sensograms obtained for the AAB/ATP/ Au immunosensor with various LDL concentrations at 298 K. The time dependent increment in the resonance angle during association clearly indicated the successful binding of LDL onto the AAB/ATP/Au surface. The inset shows the calibration curve [angle change (∆m°) vs LDL concentration] revealing linearity of the SPR immunosensor toward LDL in the range 0.072 µM (24 mg/dL) to 0.360 µM (120 mg/dL) with regression coefficient (R) of 0.999 and standard deviation (SD) as 2.45. The change in the SPR angle was found to increase even beyond 0.360 µM. Thus the prepared AAB/ATP/Au immunosensor can be used to quantify LDL in the physiological range (
