Article pubs.acs.org/JPCC
Electrochemical Impedance Analysis of Adsorption and Enzyme Kinetics of Calf Intestine Alkaline Phosphatase on SAM-Modified Gold Electrode S. Shrikrishnan, K. Sankaran,† and V. Lakshminarayanan* Raman Research Institute, C. V. Raman Avenue, Sadashivanagar, Bangalore-560080, India S Supporting Information *
ABSTRACT: In this work, we have explored the potential of electrochemical impedance technique to follow the kinetics of nonspecific adsorption of enzymes on to a self-assembled monolayer (SAM) of 3-mercapto benzoic acid (3-MBZ) formed on gold electrodes. We have also studied kinetics of enzyme− substrate reactions on the immobilized surface using this technique. During the adsorption process, the surface coverage is measured using the imaginary component of the impedance at a given frequency, which was found to follow Langmuir adsorption kinetics. The adsorbed enzyme was then allowed to interact with different concentrations of its substrate and the resulting reaction was followed in real time. Changes in the imaginary component of the impedance at various substrate concentrations have been found to follow Michelis−Menten kinetics. The results show that electrochemical impedance spectroscopy (EIS) is a powerful technique that can be used to follow enzymatic reactions on surfaces in real time. Our results also suggest that this technique has the potential to emerge as an effective immunosensor tool that can be utilized for a large range of enzyme systems.
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electrode surface brought about during these processes.15,16 Katz and Willner17 have extensively reviewed a multitude of studies based on this principle. The EIS uses a very small amplitude sinusoidal voltage (about 10 mV rms) to measure the impedance components at a given potential. The current response of the system to such small AC potential perturbations provides information on the impedance in a way that is related to the properties of the interfacial processes under investigation. This arises due to the physical structure of the material or chemical processes within it or a combination of both. Consequently, EIS is frequently used as a nondestructive technique for providing accurate and reliable information regarding the surface conditions such as adsorption and desorption processes at electrode surfaces. EIS allows such complex biorecognition events to be probed in a simple, sensitive, label-free, and mediator-free strategy.18−21 It was earlier shown by our group that the EIS can be used as a rapid and sensitive technique to study the adsorption kinetics of alkanethiols on the gold surface.22 In this work, we investigated the process of nonspecific protein adsorption onto SAM formed by carboxylic acid terminated thiol, viz., 3-mercapto benzoic acid (3-MBZ) on gold surfaces. Single-frequency impedance values were obtained and analyzed for various protein concentrations in different buffers and pH. Changes in the imaginary component of the
INTRODUCTION The adsorption of proteins on self-assembled monolayer (SAM)-modified metallic surfaces is of considerable interest in recent times owing to their potential applications in analyzing the activity of certain biomolecules and fabricating appropriate biosensors. There are several techniques that have been used to study the adsorption of proteins on SAMmodified gold and the reaction kinetics of these immobilized biocatalysts. Among them, some of the notable techniques are quartz crystal microbalance (QCMB),1−3 contact angle measurements,4 ellipsometry,4 reflectance absorption infrared spectroscopy,5 surface plasmon resonance (SPR),6−8 atomic force microscopy,8 and scanning tunneling microscopy. Enzyme assays can be performed by antibody-dependent fluorescence/luminescence, spectrophotometry, radiometry, chromatography, capillary electrophoresis,9 and isothermal titration calorimetry (ITC).10,11 While these methods provide very good means to study the enzyme adsorption phenomena for the purposes of specialized research at a laboratory scale, they have certain inherent limitations for the development of simple commercially viable devices. A broadly applicable, rapid, label-free, and simple technique should facilitate analysis of immobilized enzymes. Electrochemical techniques have been widely employed to study various chemical and biological phenomena on surfaces and to develop sensors.12−14 Among these, electrochemical impedance spectroscopy (EIS) has emerged as a powerful tool to study these biomolecular interactions by detecting changes in capacitance and interfacial electron transfer resistance at the © 2012 American Chemical Society
Received: March 22, 2012 Revised: July 9, 2012 Published: July 10, 2012 16030
dx.doi.org/10.1021/jp3027463 | J. Phys. Chem. C 2012, 116, 16030−16037
The Journal of Physical Chemistry C
Article
0.3 M HClO4 and measuring the charge consumed for the reduction of gold oxide formed during the forward cycle. Electrochemical Instrumentation. Cyclic voltammetry and chronoamperometry studies were carried out using an EG&G potentiostat (model 263A), which was interfaced using a GPIB card (National Instruments). For electrochemical impedance studies, the potentiostat was used along with an EG&G 5210 lock-in-amplifier controlled by Power Sine software. Real and imaginary components of the current were manually recorded using Agilent 54621A Digital Cathode Ray Oscilloscope (CRO) and Keithley 2000 Multimeter. Microcal Origin 6.0 software was used for graphical analysis and curve fitting. All impedance studies were carried out by applying an AC input signal of 10 mV rms at various frequencies. Electrochemical Cell. The three-electrode cell had a platinum foil of large surface area as a counter electrode, a saturated calomel reference electrode, and the modified gold disk electrode as a working electrode. Typically, 10 mL of the indicated buffer solution is used as the supporting electrolyte. Care was taken to ensure the distance between each electrode remained fairly constant for all the experiments. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) of ALP and Its Activity. SDS-PAGE is used to separate proteins as detergent complexes through a porous matrix on the basis of molecular size. The dye Coomassie Brilliant Blue is used to stain the proteins after the run. It is employed to estimate the amount of proteins present in a sample containing a mixture of proteins. In this study, the electrophoresis was carried out using a 12% gel with a 30% acrylamide−bisacrylamide mix (to provide porous polymer matrix). Protein molecular weight marker (Cat#SM0431 Fermentas) with 7 proteins ranging from 116 kDa (beta galactosidase) to 14.4 kDa (lysozyme) was used to determine the molecular weights and the amount of various proteins in the sample. The gel was analyzed using ImageJ image processing and gel analysis software. ALP activity was measured in the sample by incubating 200 ng of the sample in 100 μL of various concentrations of its substrate pNPP for 15 min at 37 °C. The quantity of product (p-nitrophenol) formed was determined by measuring the absorbance at 405 nm in a microplate reader. The adsorbed ALP was incubated for a prolonged period of 1565 min until the absorbance reaches its highest value at this wavelength. Self-Assembled Monolayer Formation. The SAM of 3MBZ was formed by placing the cleaned gold disk electrode in 20 mM ethanolic solution of the thiol for 12 h and subsequently rinsing with ethanol and finally in Millipore water. Enzyme Adsorption onto SAM. The commercial calf intestinal alkaline phosphatase (SRL) was analyzed using SDSPAGE to determine the purity of the enzyme. From the gel shown in Figure S1 (Supporting Information), it was estimated that 100 μg of the sample contained 3.2 μg of the enzyme. Other bands that appeared were taken as contaminant proteins. ALP monomer constitutes 44% of all the proteinaceous substances present. The enzyme was adsorbed on the SAM-modified gold electrodes by placing it in 0.1 mM solutions of ALP for 3 h. The protein-coated electrodes were then rinsed in Millipore water. The adsorption of enzyme was confirmed by its activity and by comparing impedance spectroscopy plots prior to and after the adsorption. Calculation of Surface Coverage. The adsorption of proteins onto SAM-modified gold causes the interfacial
impedance have been measured as a function of time, which was found to follow the Langmuir adsorption kinetics. Further, the adsorption of the enzyme alkaline phosphatase (ALP) was confirmed by allowing the immobilized enzyme to react with different concentrations of its substrates and following the reaction using impedance spectroscopy and single-frequency impedance analysis in real time. The impedance changes taking place at various substrate concentrations have been found to follow Michelis−Menten kinetics. Enzyme linked immunosorbent assay (ELISA) is a wellestablished technique to determine the quantity of proteinaceous substances and is widely used in molecular biology, diagnostics, immunology, and various other research fields. Here, the primary purpose is to determine the presence and measure the quantity of an antibody or antigen in a sample. An unknown amount of antigen in the sample is nonspecifically adsorbed on the surface of a well in a polystyrene microtiter plate. Then, an antibody specific to the surface immobilized antigen is allowed to bind with it. The antibody is usually genetically modified such that it is linked to either a fluorescing enzyme or an enzyme that can catalyze a reaction resulting in a change of color. Various modifications to the procedure have been made to improve the sensitivity of the system, but the technique remains quite complicated and time-consuming requiring highly skilled personnel for effective implementation. Electrochemical impedance spectroscopy technique has been frequently used to develop immuno-sensors23,24 that have the potential to outperform ELISA, but these sensors require the electrodes to be modified with monolayers of binding ligands (antibodies, single-stranded DNA, etc.) specific to the biomolecules being studied. We believe that the method for following nonspecific protein adsorption onto monolayermodified electrodes and their enzyme kinetics described in this article can be utilized to construct a simple automated device capable of immunosensing akin to ELISA and also broaden its scope by facilitating the study of a wider range of enzyme systems.
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EXPERIMENTAL SECTION Chemicals. All of the chemicals used in this study were analytical grade (AR) reagents: substrate buffer (pH 9.4, sodium carbonate, sodium hydrogen carbonate, and magnesium chloride), calf intestine alkaline phosphate (SRL and Sigma Aldrich), jack beans urease (SRL), aspergillusniger glucose oxidase (Fluka), pig liver esterase (Sigma Aldrich), 10 mM, phosphate buffers (pH 4.5, 5.2, and 7.2), para-nitro phenyl phosphate (pNPP), para-nitrophenyl acetate (pNPA), urea, Ldextrose, dimethyl sulphoxide, 3-mercaptobenzoic acid (3MBZ), carbonate buffer (pH 9.2), and citrate buffer (pH 4.5). Electrode Pretreatment. All electrochemical experiments were carried out using a gold disk working electrode of geometric area 0.008 cm2 fabricated by sealing a gold wire of 1 mm diameter in soda lime soft glass. Immediately before use, the polycrystalline gold disk electrode was polished with emery paper of grade 800 and 1500, followed by polishing in aqueous alumina slurries of 1 μm, 0.3 μm, and, finally, 0.05 μm grade, ultrasonicated in water to remove alumina particles for 1 min, and then cleaned with dil. Aqua regia (3:1:4 mixture of conc. HCl, conc. HNO3, and water) for one minute before each experiment. Finally, it was rinsed in distilled water thoroughly, followed by rinsing in Millipore water and ethanol before SAM formation. The true surface area of the gold disk electrode was measured by the standard technique of cycling the potential in 16031
dx.doi.org/10.1021/jp3027463 | J. Phys. Chem. C 2012, 116, 16030−16037
The Journal of Physical Chemistry C
Article
Figure 1. Impedance spectroscopy analysis of ALP adsorption onto SAM-modified gold in 10 mM phosphate buffer solution. (A) Nyquist plot of (a) bare gold, (b) SAM-modified gold, and (c) ALP adsorbed SAM-modified gold. (B) Phase angle Bode plot of (a) bare gold, (b) SAM-modified gold, and (c) ALP adsorbed SAM-modified gold. A peak phase angle of 83.5° is seen at 11.72 Hz for the ALP adsorbed SAM-modified gold.
where Z0 is the value of the imaginary component of the impedance for the 3-MBZ SAM-modified gold, Zt is the value of the imaginary component of the impedance at any time t during adsorption process, and Zf is the value of the imaginary component of the impedance at the maximum coverage of protein.
capacitance of the electrode to change since various charged species are associated with these macromolecules. The variations in the interfacial capacitance during the surface processes are reflected in the changes in the values of the imaginary component of the impedance measured on the SAMmodified electrodes.22 Proteins also have complex structural characteristics and configurations that facilitate ions to pass through them. Even if a continuous layer of proteins is formed, complete blocking of ionic flow within the porous charged film cannot be realized. Consequently, these surface processes also cause the changes in the imaginary component of the impedance. In this study, the electrode−solution interface is modeled as a simplified Randles equivalent circuit containing the charge transfer resistance and double layer capacitance in parallel. This is justified because the charged macromolecular film is not an ideal capacitor but has an associated resistive component due to ionic flow through it, thereby making it a resistive-capacitive film. We consider this model adequate enough to follow the interfacial changes during the adsorption process. The impedance (Z) for an RC circuit with R and C in parallel is given by Z(jω) =
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RESULTS AND DISCUSSION Protein ALP Adsorption to 3-MBZ SAM. The effect of the adsorption of ALP on gold modified with 3-MBZ SAM was followed from the imaginary component of impedance in various buffers of pH 4.5. At this pH value, the net charge on the protein is positive, which facilitates the adsorption of the protein onto the negatively charged carboxyl group of the 3MBZ. The impedance spectrum of the bare gold electrode, SAMmodified gold electrode, and after protein adsorption were recorded and shown in Figure 1A and the corresponding phase angle as a function of frequency in Figure 1B. It can be seen that there is a large increase in the imaginary component of the impedance after the 3-MBZ SAM formation (Figure 1A(b)). This is due to the lowering of the interfacial capacitance due to the formation of the SAM to which the imaginary component of the impedance is inversely related.22 The subsequent adsorption of proteins on to the SAM-modified surface, however, causes the imaginary component of the impedance to decrease (Figure 1A(c)). This trend is due to the increased ionic charges at the interface brought about by the adsorption of the macromolecular protein, which effectively increases the interfacial capacitance. A periodic recording of the impedance over various frequency ranges during the protein adsorption process was done to determine the optimum single frequency at which the impedance could be followed in real time. This value was arrived at on the basis of the extent of change in the impedance value, higher phase angle, least drift or fluctuations in the value, and speed of data acquisition. On the basis of these criteria, a frequency of 11.72 Hz was selected, which provided reproducible data in all subsequent studies. The 3-MBZ SAM-modified gold electrode was dipped in an ALP solution and the variation in impedance was followed in real time. This was done using various concentrations of ALP in four types of buffer solutions: 10 mM phosphate, 100 mM phosphate, 1 mM citrate, and 10 mM citrate buffers all maintaining a pH of 4.5. Though phosphate buffer does not effectively control the pH of the solution, this buffer system was
jωR2C R − 1 + ω 2R2C 2 1 + ω 2R2C 2
The imaginary component of the impedance (Z″) from this equation is given by Z″ =
ωR2C 1 + ω 2R2C 2
where ω is the frequency of the applied AC signal in Radians, and R and C are the interfacial resistance and capacitance, respectively. It is known that several factors such as microroughness of the electrode, ionic permeation through the film, and adsorption/ desorption of the electrolyte species influence the precise measurement of interfacial capacitance. Therefore, for the study of the enzyme system of interest in this work, we have assumed that the imaginary component of the impedance represents the dynamic processes occurring at the interface brought about by the adsorption of proteins. We have used the formula modified from the one adopted to follow the kinetics of adsorption of alkanethiols on gold by Subramaniam et al.22 for the calculation of the surface coverage (θt) at any instance of time, viz., θ(t ) = [(Z0 − Zt )/(Z0 − Zf )] 16032
dx.doi.org/10.1021/jp3027463 | J. Phys. Chem. C 2012, 116, 16030−16037
The Journal of Physical Chemistry C
Article
Figure 2. Surface coverage (θ) vs time (min) curves for ALP adsorption onto SAM-modified gold in (A) 10 mM phosphate buffer at ALP concentrations of (a) 100 μg/mL, (b) 80 μg/mL, (c) 60 μg/mL, (d) 50 μg/mL, (e) 45 μg/mL, (f) 40 μg/mL, andd (g) 10 μg/mL and in (B) 1 mM citrate buffer at ALP concentrations of (a) 50 μg/mL, (b) 30 μg/mL, and (c) 20 μg/mL.
chosen since, at a later stage, the enzymatic reactions of the immobilized enzymes were carried out and studied in phosphate buffers at more optimal pH values. It was considered appropriate to study both adsorption process on the electrode surface and kinetics of the immobilized enzymes using the same ionic species in the solution. Citrate buffers, which act as good buffers at pH 4.5, were used in this study to verify whether a similar trend is observed as that in the phosphate buffer system. The general trend observed was that the imaginary component of the impedance decreases rapidly and stabilizes near a final value within a certain duration ranging from a few minutes to an hour depending on the concentration of the enzyme. This value remains almost constant, which was confirmed by measuring the imaginary component of the impedance after 12 h. To compare the plots and measure the rate constants, the surface coverage of the adsorbed protein on 3-MBZ-modified gold is plotted as a function of time of adsorption as shown in Figure 2A,B for the phosphate and citrate buffers, respectively. The adsorption process was primarily followed in a 10 mM phosphate buffer for various concentrations of ALP. From the figure, it is seen that, at concentrations above 45 mg/mL, the gold surface rapidly reaches a point of maximum coverage leaving very little change in the imaginary component of the impedance after this stage. Figure 2B shows a similar response to the adsorption process of ALP on SAM-modified gold in 1 mM citrate buffer. The plots obtained for different systems are fitted with three commonly used models for adsorption,16 viz., the Langmuir model,
Figure 3. Langmuir adsorption isotherms fitted to coverage (θ) vs time (min) plots of ALP adsorption on 3-MBZ SAM-modified gold in 10 mM phosphate buffer (pH 4.5) at a concentration of 100 μg/mL.
concentrations did not display as clear a trend as was found with the Langmuir model. Table 1 presents the rate constant values obtained when the plots were fitted with the Langmuir model. As expected, the Table 1. Rate Constant for Adsorption of ALP onto 3-MBZ SAM-Modified Gold for Various Concentrations of the Enzyme in Different Buffers Using the Langmuir Adsorption Model buffer 10 mM phosphate buffer (pH 4.5) 3-MBZ
θ(t ) = [1 − e−kt ] the diffusion controlled Langmuir model,
100 mM phosphate buffer (pH 4.5) 3-MBZ
1/2
θ(t ) = [1 − e−kt ] 10 mM citrate buffer (pH 4.5) 3-MBZ
and the purely diffusion controlled adsorption model θ(t ) = kt 1/2
1 mM citrate buffer (pH 4.5) 3-MBZ
where k is the respective rate constants and t is the time. Of the three, the best fit is obtained with the Langmuir model with very low χ2 values (
