Electrochemical analysis of gold-coated magnetic nanoparticles for detecting immunological interaction
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- Pham, T.T. & Sim, S.J. J Nanopart Res (2010) 12: 227. doi:10.1007/s11051-009-9600-7
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An electrochemical impedance immunosensor was developed for detecting the immunological interaction between human immunoglobulin (IgG) and protein A from Staphylococcus aureus based on the immobilization of human IgG on the surface of modified gold-coated magnetic nanoparticles. The nanoparticles with an Au shell and Fe oxide cores were functionalized by a self-assembled monolayer of 11-mercaptoundecanoic acid. The electrochemical analysis was conducted on the modified magnetic carbon paste electrodes with the nanoparticles. The magnetic nanoparticles were attached to the surface of the magnetic carbon paste electrodes via magnetic force. The cyclic voltammetry technique and electrochemical impedance spectroscopy measurements of the magnetic carbon paste electrodes coated with magnetic nanoparticles–human IgG complex showed changes in its alternating current (AC) response both after the modification of the surface of the electrode and the addition of protein A. The immunological interaction between human IgG on the surface of the modified magnetic carbon paste electrodes and protein A in the solution could be successfully monitored.
KeywordsGold-coated magnetic nanoparticlesElectrochemical impedance spectroscopyCyclic voltammetryImmunological interactionNanomedicine
Typically, an immunoassay is based on the specific interaction between an antigen and an antibody with a complementary three-dimensional structure. Electrochemical biosensing has been developing to take advantage of nanoscale materials. Nanoparticles (NPs) have been widely used as labels in affinity biosensors or to enhance the detection signals for electrochemical immunosensors (Chen et al. 2006; Merkoci 2007; Tang et al. 2005, 2006a, b). Many important achievements have been reported in the area of amperometric and voltammetric electrochemical nanobiosensors (Pumera et al. 2007; Wu et al. 2007; Zhou et al. 2003, 2006). Besides, electrochemical impedance spectroscopy (EIS) has shown itself to be a sensitive technique for use in immunosensors, and the overall system provides information about the interface, its structure, and the reactions that occur (Bouafsoun et al. 2007; Navratilova and Skaladal 2004). Gold nanoparticles have been applied to enhance the immobilization of biological reagents on the surface of glass carbon electrodes (Huang et al. 2006). Magnetic nanoparticles modified with biological ligands, including proteins and nucleic acids, supported on a carbon paste electrode have been used in electrochemical detection (Zhang et al. 2007; Zhu et al. 2006).
Protein A from Staphylococcus aureus and immunoglobulin from animals has been widely applied to model immunological interactions (Briand et al. 2006; Schwartz et al. 2007; Touhami et al. 2007). In this study, electrochemical impedance techniques in combination with cyclic voltammetry (CV) were used for the purpose of detecting the layer-by-layer structure of a modified magnetic carbon paste electrode (MCPE). Gold-coated magnetic nanoparticles with sizes ranging from 20 to 70 nm were immobilized on the surface of the electrodes. The immunological reaction between human IgG immobilized on the nanoparticles and protein A from S. aureus was successfully conducted and analyzed. We report a novel method of using gold-coated magnetic nanoparticles to provide enhanced immunosensing in electrochemical applications.
Reagents and instruments
FITC–protein A (Lot no. 139 5427) was obtained from Invitrogen, Carlsbad, USA. Human IgG, whole molecule (Lot no. 75 722), was obtained from Jackson Human Research Lab Inc. (USA). 11-Mercaptoundecanoic acid (11-MUA) 95% from Aldrich was used to fabricate the self-assembled monolayer (SAM) on the nanoparticles. N-hydroxysuccinimide (NHS) and N-ethyl-N-(3-diethylaminopropyl) carbodiimide (EDC) were obtained from Sigma-Aldrich.
Phosphate buffer saline (PBS) whose pH was adjusted to 7.2 was used as the electrolyte in the measuring system and for the preparation of 5 mM of both K3[Fe(CN)6] and K4[Fe(CN)6]. Deionized water (>18 MΩ cm−1) was obtained from a Water Softener (Human Corporation, South Korea) purification system.
Cyclic voltammetry and EIS measurements were conducted using a Parstat 2263 system (Princeton Applied Research) with a commercial software program for the alternating current (AC) measurements. Field-emission scanning electron micrographs (FE-SEMs) of the magnetic nanoparticles were obtained with a JSM-7000F (celebrating voltage 0.5–30 kV) (JEOL, Tokyo, Japan). A UV/Vis spectrophotometer, model DU 730 (Beckman Coulter, USA), was used to measure the absorption of the nanoparticles. The fluorescence intensity was determined using a Spectramax Gemini Microplate Spectrofluorometer (Molecular Device, USA).
Preparation of magnetic nanoparticles
Magnetic cores of Fe3O4 were synthesized by the co-precipitation of Fe salts in strong alkaline solution, as reported elsewhere (Kang et al. 1996; Pham et al. 2008). Briefly, a mixture of 4.595 g of Fe(III) chloride and 1.71 g of Fe(II) chloride dissolved in an acidic solution was stirred under a nitrogen-protection environment and a solution of 2 M NaOH was added, resulting in a pale yellow solution which changed to brown and finally to dark black. The magnetic cores were washed continuously with water and diluted tetramethylammonium hydroxide (TMOH) solution and then stabilized in 0.1 M TMOH solution for further use. The magnetic cores were next coated with gold (Au) following the previously reported procedure in our article (Pham et al. 2008). One milliliter of 0.212 mM N(CH3)4-stabilized, oxidized Fe3O4 stock solution was diluted with 50 mL of 0.01 M sodium citrate and stirred for 30 min to exchange the absorbed OH− with citrate ions. Five milliliter of oxidized Fe3O4 from the working magnetic-core solution was used for the Au coating reaction in a total volume of 40 mL of 0.01 M sodium citrate. The solution of magnetic cores and sodium citrate was stirred and heated to boiling point and then 1 mL of a solution of 10 nM HAuCl4 was immediately added to the mixture. The heating was continued for 15 min after the addition of the Au3+ salts and the stirring was continued while the solution cooled to room temperature. The synthesized, gold-coated magnetic nanoparticles were stabilized by citrate ions.
Fabrication of nanoparticle–protein conjugates
The citrate-stabilized, gold-coated magnetic nanoparticle solution was standardized with respect to its absorbance at 528 nm. 11-MUA in ethanolic solution was added to the nanoparticle solution so that the final working concentration of 11-MUA had an absorbance of 2 at 528 nm. The solution was kept in dark-glass bottles with gentle shaking for 3 h to fabricate the SAM on the surface of the nanoparticles. The nanoparticles functionalized with 11-MUA were kept in darkness to prevent their aggregation prior to their further use.
The 11-MUA–nanoparticles were washed by centrifugation with diluted PBS before being activated with a 0.01 M mixed solution of EDC/NHS. Then, the nanoparticle solution was added to human IgG solution in PBS at pH 7.4. The solution was then gently agitated overnight in darkness. The nanoparticle–protein IgG conjugates were washed in PBS at pH 7.4 by centrifugation and then kept at 4 °C for further use.
Preparation of electrode
Solid magnetic carbon paste electrodes were fabricated following the procedure described in previous reports (Tang et al. 2006a, b; Zhang et al. 2007; Zhu et al. 2006). A mixture of melted paraffin (800 g) and graphite powder (1,000 mg) was blended thoroughly to get a homogeneous paste. A permanent magnet (diameter 7.85 mm, length 7.85 mm) was embedded into a polytetrafluoroethylene (PTFE) tube (aperture of 8 mm). The paste was stuffed into the PTFE tube to immobilize the magnet and the tube was left to harden for 1 day. The MCPEs were polished thoroughly with fine sandpapers to get a smooth surface. Finally, the electrodes were washed by ultrasonicating them in both distilled water and ethanol for 5 min each.
The magnetic nanoparticle–protein IgG conjugates suspended in PBS at pH 7.4 were dripped onto the surface of the MCPEs. The surface was dried, washed with PBS to remove any unbound nanoparticle and then kept at 4 °C for incubation with protein A or electrochemical measurement. The magnetic nanoparticles were firmly attached to the surface of the magnetic electrode due to the magnetic force.
Preparation of nanoparticles–protein conjugates for measuring fluorescence
Solutions of gold-coated magnetic nanoparticles were standardized to an absorbance of 1 at 528 nm. The nanoparticles were functionalized with 11-MUA so that the concentration of the working 11-MUA solution was 2 mM, and the incubation time used to make the SAM of 11-MUA was 5 h with gentle agitation. The functionalized magnetic nanoparticles were first washed with 0.1 M Tris solution pH 6.0 and then with PBS pH 7.4. The coupling reagent, EDC/NHS (ratio 1:1), with a working concentration of 0.01 M was used to induce the formation of amide bonds between the carboxyl groups of 11-MUA and human IgG. A 0.5-mg/mL IgG solution was used to immobilize human IgG onto the SAM of 11-MUA. Next, the conjugates were washed with PBS to discard any unbound IgG. The nanoparticle–IgG conjugates were stored at 4 °C under moist conditions. The immunological interaction was investigated with concentrations of protein A ranging from 2 to 1,190 nM. The nanoparticle solutions were then washed with PBS to remove excessive protein A by magnetic separation and centrifugation. A 200-μL aliquot from each solution was applied to a Microplate Spectrofluorometer. The concentration of FITC–protein A was determined according to the fluorescence intensity measured.
Electrochemical cell and procedure
All electrochemical experiments were performed at room temperature, and 1 mM PBS pH 7.4 containing 5 mM of the redox couple, K3[Fe(CN)6/K4[Fe(CN)6 was used as analytic solution. The exposed coating area was 0.25024 cm2. CV and EIS measurements were conducted using a Parstat 2263 system with a commercial software program for the AC measurements. A graphite counter was used with a saturated calomel electrode as the reference. Prior to the tests, the samples were kept in the solution for 1 h in order to stabilize the open-circuit potential. The applied potential scans were from −200 to 800 mV. The obtained current, I, is approximated on the μA scale with an optimal scan rate of 10 mV/s. In addition, for the EIS test, the amplitude of the sinusoidal perturbation was 10 mV. The frequency range was from 100 kHz to 10 mHz.
Results and discussion
Investigating immobilization of human IgG on the magnetic nanoparticles and immunological interaction of FITC–protein A and IgG through fluorescence intensity
Electrochemical characterization of modified electrodes
However, there is a remarkable change in the peak height and the peak-to-peak separation when the layer of SAM–human IgG conjugates is immobilized onto the nanoparticles (line 3). The thiol monolayer has been reported to decrease the peak values, and CV techniques were employed to confirm the presence of the monolayer of sulfur derivatives on the gold electrode (Bouafsoun et al. 2007). As shown in Fig. 3b, the layer of protein A bound to the layer of human IgG through their immunological interaction does not produce any substantial change in the shape of the CV curve or the peak height compared to that of the human IgG layer. However, the current obtained at the vertex between the charge and discharge phases of the CV curves increases as the incubated concentration of protein A decreases. This indicates the formation of a high-density layer of protein A on the human IgG layer. Increasing the absorption time of the McAb on the gold electrode led to a decrease in the peak current of the CV curves (Jie et al. 1999). The layer of protein A may affect the charge on the surface of the electrode and the transmission of electrons. In our experiment, PBS at pH 7.4 was used as the electrochemical solution to reduce such effects of the protein, since the pI of the protein is about 7.
Impedance analysis of magnetic carbon paste electrodes (MCPEs) modified with gold-coated magnetic nanoparticles and protein human IgG
Electrochemical analysis of the modified MCPEs
Rs (Ω cm2)
Rpore (Ω cm2)
Rct (Ω cm2)
Q1 (S s.5/cm2)
Q2 (S s.5/cm2)
Electrochemical analysis of binding of protein A and IgG on the surface of the modified MCPEs
Concentration of protein A (nM)
Rs (Ω cm2)
Rpore (Ω cm2)
Rct (Ω cm2)
Q1 (S s.5/cm2)
Q2 (S s.5/cm2)
Similarly, the interaction between protein A and IgG leads to the formation of a layer of protein A and the impedance of the modified electrodes is interpreted in Fig. 4b as well as in the simulated circuit in Fig. 5b. Electrochemical analysis was performed with three concentrations of protein A, 2.38, 23.81, and 238.09 nM. The impedance of the electrodes after the binding of protein A was simulated with the same fitting curve as that used for the modeling of protein IgG with Rs, CPE1–Rpore, and CPE2–Rct. Table 2 displays the analytical results of the impedance of the modified electrodes. The solution resistance and CPE2 decrease when the concentration of protein A increases. However, regarding the variation of CPE1 and CPE2, as well as Rpore according to changes of protein A concentrations, there are two phases in the immunological interaction between protein A and human IgG. These results are in agreement with the fluorescence intensity data of protein A bound onto the nanoparticles discussed above (Fig. 2). A 10 times increase in the concentration of protein A from 2.38 to 23.81 nM leads to a decrease of CPE1 and CPE2, but an increase of Rpore and Rct. The change of impedance may be caused by the thickness of the protein layer, as well as by the local charge of the protein layer, which may affect the local electrons of the interface. On the other hand, a 10 times increase in the concentration of protein A from 23.81 to 238.09 nM has the opposite effect on the values of CPE1, Rpore, and Rct. CPE1 increases while Rpore and Rct decrease. This result can be explained by the three-dimensional binding between IgG and protein A. At a concentration of 238.09 nM, protein A can form a stable layer on the surface of the electrode and the layer of IgG is total covered, while at a concentration of 2.38 nM, protein A only partly covers the layer of human IgG and, consequently, part of the IgG layer might still be exposed to the electrolyte solution. Therefore, in this experiment, we succeeded in application of gold-coated magnetic nanoparticles to electrochemically analyze the immunological interaction between human IgG and protein A from S. aureus. To understand more about the electrochemical kinetics of the protein A–human IgG interaction, detailed observations should be made at each phase to determine the stability and affinity of this immunological binding.
Gold-coated magnetic nanoparticles deposited onto a MCPE via magnetic force were utilized to immobilize human IgG through chemical ligands. The SAM of alkanethiol (11-MUA) serves as a good platform to immobilize human IgG onto the gold surface of the nanoparticles. The immunological interaction between human IgG and protein A from S. aureus was able to be monitored and analyzed with CV techniques and EIS. Moreover, this experiment shows how gold-coated magnetic nanoparticles can be made use for EIS or CV biosensors. The minimum concentration of the analyzed target can be as low as 20 nM.
This work was supported by the Korea Science and Engineering Foundation (KOSEF) National Research Laboratory (NRL) Program grant funded by the Korea government (MEST) (grant no. R0A-2008-000-20078-0) of the Republic of Korea.