Mol Biol Rep (2014) 41:1659–1668 DOI 10.1007/s11033-013-3014-4
An electrochemical immunosensor for digoxin using core–shell gold coated magnetic nanoparticles as labels Anita Ahmadi • Hanieh Shirazi • Narges Pourbagher Abolfazl Akbarzadeh • Kobra Omidfar
•
Received: 24 July 2013 / Accepted: 30 December 2013 / Published online: 7 January 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract A simple, sensitive, and low-cost immunosensor was designed for the detection of digoxin through core– shell gold coated magnetic nanoparticles (Fe3O4-Au-NPs) as an electrochemical label. Having had such a large potential for a variety of applications, Fe3O4-Au-NPs have attracted a considerable attention and are actively investigated recently. Digoxin is a cardiac glycoside which, at high level, can indicate an increased risk of toxicity. This new competitive electrochemical immunosensor was developed based on antigen–antibody reaction employing antigen (Ag) labeled Fe3O4-Au-NPs and PVA modified screen-printed carbon electrode surface in order to detect the serum digoxin. The structures of Fe3O4-Au-NPs were studied by transmission electron microscopy, X-ray diffraction and Fourier transformed infrared spectroscopy. Cyclic voltammetry and differential pulse voltammetry (DPV) were employed to determine the physicochemical and electrochemical properties of immunosensor. DPV was employed for quantitative detection of digoxin in biological samples. The developed immunosensor was capable to detect digoxin in the range from 0.5 to 5 ng mL-1, with a
A. Ahmadi H. Shirazi N. Pourbagher K. Omidfar (&) Biosensor Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, P.O. Box 14395/1179, Tehran, Islamic Republic of Iran e-mail: [email protected] A. Ahmadi K. Omidfar Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Research Institute, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran A. Akbarzadeh Department of Medicinal Chemistry, Tabriz University of Medical Sciences, Tabriz, Islamic Republic of Iran
detection limit as low as 0.05 ng mL-1. The proposed method represented acceptable reproducibility, stability, and reliability for the rapid detection of digoxin in serum samples. Keywords Core–shell gold coated magnetic nanoparticles Immunosensor Digoxin
Introduction Digoxin is a cardiac glycoside, which is widely prescribed for the treatment of congestive heart failure and various disturbances of cardiac rhythm [1, 2]. The therapeutic use of digoxin in patients is based on its ability to increase myocardial contractions, by increasing the blood output in all patients with heart failure [3–5]. However, toxicity may be encountered at a concentration as low as 2.0 ng mL-1 [6]. Therefore, given its narrow therapeutic range of 0.8–1.9 ng mL-1, serum digoxin concentration measurement is necessary in digitalized patients [6, 7]. Despite the therapeutic and toxic concentrations overlap, measurement of digoxin levels helps to maintain effective concentrations, to diagnose and to prevent overdosage. Several methods such as high performance liquid chromatography (HPLC) and immunochemical assays have been used for detecting of digoxin in serum [2, 4, 6–10]. HPLC has good linearity, limit of detection, precision and specificity; however, it is time-consuming, require sample pre-treatment and demand expensive instruments. Compared with HPLC, immunoassays are the most common and effective methods for monitoring serum digoxin level, despite requiring a long reaction time, special equipment, reagent, and involve multiple steps. With the increasing demand for on-site (pre)-screening and diagnosis, more
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ultrasensitive approaches should be described for realizing the requirement of clinical analysis. Recently, electrochemical immunosensors have attracted a great deal of attention. They offer a number of distinct features such as combining the high specificity and sensitivity of traditional immunoassay methods with the low detection limits, fast analysis, simplicity, easy handling, low costs and low endogenous background using different types of electrodes [11–13]. Coupled with the development of such techniques, a great deal of effort has been devoted to constructing the robust, sensitive, selective and practical biosensing instrumentations as point of care (PoC) diagnostic tests in laboratories [13, 14]. In a previous study [7], we developed a semi-quantitative immunostrip assay for the measurement of digoxin in serum using antibody gold nanoparticles (Au-NPs) conjugate as label. However, these types of assay do not provide a sensitive and reliable method for the quantitative measurement of analyte in biological sample [7, 9]. Furthermore, up to now, two immunosensors have been designed for detection of digoxin in serum based on flowthrough fluorosensors. Several problems have been attributed to fluorometric assays as they non-specifically reduce or enhance the signal output. Fluorescence detection is liable to changes in pH, temperature, concentration of ion and detergent, drying, and the solid matrix, leading to light scattering, high background, quenching and bleaching issues. Worse still, such methods take too long and often require high cost, phase separation or even special technology [15, 16]. Thus, exploring a new trace label that is based on the electrochemical principle would be valuable. For successful development of user-friendly tests with sufficient sensitivity, the type of label is very important. Fortunately, nanomaterial-based signal amplification strategies hold great promise in realizing ultrasensitive detection due to their versatile properties. A large number of nanomaterials, including metal NPs and carbon-based nanostructures can be utilized as labels in order to obtain the amplified electrochemical detection signal [17–22]. A Nanospheres with magnetic cores (Fe3O4-NPs) and gold shell structures have recently received more attention thanks to combined functions of Fe3O4 and gold. Fe3O4NPs have a typical super-paramagnetic nature, providing a convenient way for separation, isolation, and purification of biological samples via an external magnetic field while the functional reagents are attached onto the surface of the particles [27]. In fact, the combination of Fe3O4-NPs with Au-NPs, as a core–shell, possesses both properties of AuNPs and magnetism; offering one of the most excellent electrochemical signals for the enhancement of sensitivity, reduction in background and separation strategy.
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Therefore, they could have been widely used in analytical detection and potential biomedical applications including immunoassay, magnetic resonance imaging (MRI) contrast enhancement, hyperthermia, drug delivery and catalysis [28–32]. Fe3O4-Au-NPs are considered as excellent candidates for bioconjugation such as antigen, antibody or cell, due to their large surface area, good biocompatibility and separation [27, 29, 33–39]. The present study proposes a new direct competitive electrochemical immunosensor, combining a disposable chip with the Fe3O4-Au-NPs tracing tag in order to further improvements in signal amplification, magnetism separation and also reduction in background signal that is another crucial point in assay development (Scheme 1). The disposable chip was prepared by immobilizing capture antibody, secondary antibody, on poly vinyl alcohol (PVA) modified screen-printed carbon electrode (SPCE) surface in order to detect the serum digoxin. The organic matrixes such as PVA could assist antibody immobilization and also served as a potential stabilizer for binding sites of the antibody structure. The electrochemical signal, from binding antigen (Ag)-Fe3O4-Au-NPs complex, could be produced after electro-oxidization of AuCl4- in HCl, followed by its reduction in differential pulse voltammetry (DPV) mode [40, 41]. The proposed method demonstrated the capability to detect digoxin within the range 0.5–5 ng mL-1, with the detection limit down to 0.05 ng mL-1. The excellent analytical performance of the immunosensor provided its potential application in clinical analysis of low abundance, low molecular weight analytes in biological fluids. The magnetic core shell Fe3O4-Au-NPs composite is not only an excellent electronic tracing tag to obtain detection signal, but also provides a controllable method to construct a sensitive and specific platform in ultrasensitive bioanalysis.
Materials and methods Materials FeCl36H2O (99.0 %), FeCl24H2O (99.0 %) and NH3, HCl (99.0 %) were purchased from Acros Organics (USA). Chloroauric acid (HAuCl4), trisodium citrate, sodium azide, vinyl alcohol, mouse monoclonal anti-digoxin antibody (clone DI-22) and secondary antibody were purchased from Sigma Chemical Company (St. Louis, MO, USA). Ammonia, NaOH and other chemicals were analytical grade. The analytical HCl was purchased from Merck (Merck, Spain). All other chemicals were analytical grade and used without further purification. Deionized water was used in all the experiments.
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Scheme 1 Schematic illustration of a direct competitive electrochemical immunosensor fabrication process: a VA-secondary antibody was dropped on the working electrode of SPCE, photopolymerized VA under UV exposure; b BSA blocking; c incubation of the patient/ standard samples with digoxinBSA-Fe3O4-Au-NPs; d the preoxidation of Au-NPs was performed at the constant potential of ?1.30 V in 1 M HCl, e and then, the current signal was recorded by the voltammetric modes
Synthesis of Fe3O4-NPs Fe3O4-NPs were synthesized by co-precipitation method, in the presence of ammonia as a reducing agent, with slight modification as previously reported [35]. In brief, ferric (III) chloride hexahydrate (7.568 g) and ferrous (II) chloride tetrahydrate (3.173 g) with 1.75:1 mol ratio were dissolved in 120 mL deoxygenated water and vigorously stirred in the presence of nitrogen atmosphere-protected solution at 90 °C. Afterwards, a mixture of freshly deoxygenated ammonium hydroxide (6.4 M, 100 mL) was quickly added to the solution in order to maintain the pH at 9.0–9.5. While the color of the solution changed from yellow to black, stirring was allowed for an additional 30 min at room temperature. The solution was then cooled down to room temperature. The Fe3O4-NPs were washed several times by water and stored in Tetramethylammonium hydroxide (TMOH, 0.1 M) at room temperature. Preparation of Fe3O4-Au-NPs In order to synthesis Fe3O4-Au-NPs, 0.5 g of the prepared Fe3O4-NPs was transferred into 25 mL sodium citrate (0.01 M). The mixture was then stirred for 30 min to exchange absorbed OH- with citrate ions and to construct the final working magnetic-core solution. Various dilutions of the above solution were used for the Au coating reaction in a total volume of 20 mL of 0.01 M sodium citrate. The reaction solutions containing magnetic cores and reducing agent were heated to 98 °C under vigorous
stirring. Due to during this process, magnetite particles would be oxidized partially or totally to maghemite before their being coated. While boiling, hydrogen tetrachloroaurate (III) hydrate solution (0.01 M, 250 lL) was injected as soon as possible, and the initially brown solution changed color to dark brown and then to final deep red characteristic gold colloids. The heating mantle was removed 15 min after injection, and the stirring was continued for an additional 15 min. The Fe3O4-Au-NPs were continually washed with water in order to remove the extra gold particles. A total of four dilutions of oxidized Fe3O4 solutions in total volume of 20 mL were prepared; the colorless solution initially observed gradually became red to pinkish red which corresponds to 0.1, 0.2, 0.3 and 1 mL of working oxidized Fe3O4 respectively. Finally, in order to remove the magnetic particles, which were not coated by gold, we used HCl (1 M) for 60 min. During this period supernatant color was changed to yellow and its pH reduced to 1. Thereafter, Fe3O4-Au-NPs were washed several times with water until the pH reached the neutral value. NPs were stored in the Tris buffer (pH 8.1) for further use. The stability of the NPs was evaluated after two weeks storage at 37 °C. Preparation of digoxin–protein conjugates Digoxin was conjugated to bovine serum albumin (BSA) using our previously described method. The efficiency of conjugation was evaluated spectrophotometrically in 83 % H2SO4 [7].
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Immobilization of antibody or antigen (digoxin-BSA) onto the Fe3O4-Au-NPs Two different types of competitive assays based on digoxin-BSA-Fe3O4-Au-NPs and anti-digoxin monoclonal antibody (mAb)-Fe3O4-Au-NPs conjugate, were developed in this study. In order to prepare digoxin-BSA-Fe3O4-Au-NPs conjugates, 16 lg mL-1 of digoxin-BSA were added to 1 mL of Tris–HCl buffer (pH 7.4, 0.05 M) containing 2 mg Fe3O4Au-NPs. The mixture was gently mixed for 60 min at 37 °C and subsequently 1 mg mL-1 of BSA solution was added to block the residual surface of the NPs. After blocking, the Fe3O4-Au-NPs were coupled with dig-BSA and then magnetically separated from free dig-BSA. The Ag-Fe3O4Au-NPs was rinsed with washing buffer (Tris buffer, pH 8.1, 0.05 M) three times, re-suspended in Tris buffer (pH 8.1) and stored at 4 °C. The optimal amount of antigen for immobilization on the surface of the Fe3O4-Au-NPs was determined by employing the enzyme linked immunosorbent assay (ELISA), according to the method described in our previous works [40, 42]. The appropriate concentration of digoxin monoclonal antibody (16 lg) was conjugated to Fe3O4-Au-NPs using the same procedure as described above. Fabrication of the immunosensors For successful construction of the immunosensors, 2 % PVA was selected as the best concentration for antibody immobilization, based on our previous report [40]. Vinyl alcohol was dissolved in acetate buffer (pH 4.5, 100 mM) at 70 °C for 30 min to provide 2 % (w/v) homogeneous vinyl alcohol solution. To obtain a colloidal suspension, 5 lL of vinyl alcohol including 100 ng of secondary antibody dropped on the surface of working electrode and under UV conditions (220–250 nm, 20 min), the vinyl alcohol was polymerized to give the PVA. To block the nonspecific binding sites, the electrode was immersed with phosphate buffer (pH 7.2, 0.1 M) containing 0.5 % BSA for 15 min at 37 °C and then washed by 0.05 % Tween 20 in phosphate buffer to remove the unbounded BSA. The modified electrode was stored at 4 °C before use. DigoxinBSA was also coated on to the electrode surface using the same procedure as described earlier. Electrochemical measurements In order to optimize the calibration curve, two experiments was applied in this study. First, 10 lL of standard or real sample solution and 100 ng of anti-digoxin antibody, accompanying the 10 lL of labeled antigen, was added
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simultaneously onto the modified electrode surface with immobilized secondary antibody and incubated for 30 min at 37 °C. Thereafter, unbound Fe3O4-Au-NPs were rinsed three times with washing buffer. Second, the standard or real sample solution and antidigoxin antibody were first incubated with the labeled antigen for 20 min at 37 °C and then dropped onto the modified electrode surface with immobilized secondary antibody and incubated again for 15 min at 37 °C. The unbound labeled antigens were also rinsed three times with washing buffer. The modified electrode was placed in the electrochemical cell and 50 lL of 1 M HCl was added to cover the entire three-electrode zone of the SPCE surface for electrochemical study. The background response was recorded with the same condition on the PVA modified electrode without secondary antibody. The modified electrode was first monitored by cyclic voltammetry (CV), at a potential rang of 0–1.0 V and the scan rate of 50 mV s-1. The electrochemical detection of digoxin was carried out by DPV mode. The preoxidation of Fe3O4-Au-NPs was achieved at a constant potential of 1.3 V for 30 s in 1 M HCl to generate AuCl4- and immediately reduction of AuCl4- to Au° by DPV, while scanning 0.1–0.4 V with a step potential of 4 mV, a pulse amplitude of 50 mV, and a pulse period of 20 ms. All the potential records were performed at room temperature. The cathodic current peak was selected as the analytical signals for electrochemical detection of digoxin. Finally, the second experiment was selected for the real samples analysis. In another experiment, a mixture of 10 lL of secondary antibody-Fe3O4-Au-NPs solution, 100 ng of anti-digoxin antibody and 10 lL of standard or real sample was dropped onto the modified electrode surface with immobilized digoxin-BSA. The assay was performed as described previously. In order to investigate the reproducibility of the assay, two different serum samples with low (0.5 ng mL-1) and high (5 ng mL-1) concentration were tested for digoxin five independent analytical runs [40, 41].
Results and discussion Characterization of Fe3O4-Au-NPs Magnetic seeds were synthesized using co-precipitation in the presence of NH4OH under controlled condition (pH 10) and N2 protection gas. The optimum mole Fe3?/Fe2? ratio used was 1.7:1. The sodium citrate was deployed in order to reduce Au3? in the presence of the generated particles. The XRD pattern of the Fe3O4-NPs and Fe3O4-Au-NPs are presented in Fig. 1. For Fe3O4-NPs, five characteristic peaks at 2h° = 29.84°, 35.32°, 42.80°, 56.88° and 62.68° were
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Fig. 1 XRD patterns of Fe3O4 (a) and Fe3O4-Au-NPs (b). The powder of Fe3O4 and Au-NPs are presented by the bars
observed marked by their indices (2 2 0), (3 1 1), (4 0 0), (5 1 1) and (4 4 0). The XRD of the Fe3O4-Au-NPs also showed the same peaks indicating the unchangeable pace of Fe3O4 during coating process. Furthermore, gold also has the other diffraction peaks at 2h° = 38.2°, 44.88°, 64.4°, and 77.6° which can be indexed to 111, 200, 220, and 311 lattice planes of gold in a cubic phase, respectively. TEM image of the prepared Fe3O4-Au-NPs is shown in Fig. 2a. Fe3O4-NPs surface morphology analysis demonstrated the agglomeration of many ultrafine particles with diameters lower than 8 nm. TEM analysis revealed that the average particle size was increased from 8 nm (Fig. 2a) to 16 nm after gold coating (Fig. 2b). The synthesized NPs were consistently
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Fig. 3 FTIR spectrum of Fe3O4, Au and Fe3O4-Au-NPs (nanoparticles not immobilized)
scattered in the sample and appeared to form spherical structures (Fig. 2). Figure 3 shows the FTIR spectra of Fe3O4-NPs, Au-NPs and Fe3O4-Au-NPs. In comparison with the naked magnetic nanoparticles, abundant absorption peaks in spectrum of the modified particle confirmed the successful coating of Au-NPs on the iron oxide surface. Bands at 580–682 cm-1 in the spectra are indicating the Fe–O bending. There is also N–H stretching and bending in 3,110 and 1,620 cm-1, respectively. The peak of N–H bending implied that the Fe3O4-NPs were successfully coated by gold. Au-NPs have strong plasmon resonance properties and show the absorption peak at about 530 nm
Fig. 2 TEM image of prepared Fe3O4 (a) and Fe3O4-Au-NPs (b)
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Fig. 4 UV–Vis spectra of Au (a), Fe3O4-Au-NPs (b), Fe3O4-Au-NPs after HCl treatment (c) and Fe3O4 (d). A sharp absorption peak can be observed for the Au-NPs at 520 nm. Fe3O4-Au-NPs after HCl treatment shown sharper absorption peak
(curve a in Fig. 4). The UV–Vis absorption spectra of the, Fe3O4-Au-NPs, HCl treated Fe3O4-Au-NPs and Fe3O4-NPs are shown in Fig. 4b–d. The spectra of the Fe3O4-Au-NPs with various ratios of prepared Au3? to Fe3O4 showed that 0.2 mL of Fe3O4 solution was appropriate for the final experiment. Optimization of the immunoassay conditions Considering both high sensitivity and wide linear range, the optimal concentrations of coating antibody, anti-digoxin antibody and the digoxin-BSA-Fe3O4-Au-NPs suspension, are crucial to this direct competitive immunoassay. Therefore, the effects of different quantities of digoxinBSA-Fe3O4-Au-NPs (5, 10, 15, 20 and 25 lL), coated secondary antibody (25, 50, 100,150 and 200 ng) and antidigoxin antibody (25, 50, 100, 150 and 200 ng) were assessed. According to reduction peak currents in cyclic voltammograms, proper amounts of digoxin-BSA-Fe3O4Au-NPs, coated secondary antibody, and anti-digoxin antibody for the assay were determined as 16 lL, 100 ng, and 100 ng, respectively (data not shown). Subsequently, the effect of temperature on the immunoreactions was studied by CV method. Briefly, the reaction of digoxin-BSA-Fe3O4-Au-NPs, coated secondary antibody, and anti-digoxin antibody was examined at room temperature and 37 °C. It could be seen that the strong current signal was achieved at 37 °C. Next, the storage stability of the digoxin-BSA-Fe3O4Au-NPs over a period of 2 weeks at 37 °C was scanned periodically using DPV [40]. The DPV peak potentials and currents maintained essentially unchanged for at least 1 week at 37 °C, and then started to decrease slowly, with 86 % of NPs activity being presented at day 14 (data not shown).
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Fig. 5 Cyclic voltammograms of the different modified electrode measured in 1 M HCl at the potential range of 0.0 to ?1 V and the scan rate of 50 mV s-1: a bare electrode, b PVA, c secondary antibody-PVA, d secondary antibody-PVA-Fe3O4-Au-NPs and e digoxin-Fe3O4-Au-NPs and anti-digoxin antibody on secondary antibody-PVA. Inset effects of potential between 1.2 and 1.4 V (a) and preoxidation time (b) on DPV response
Optimization of detection conditions The conditions for electro-oxidization of the captured AuNPs are important parameters. The low potential failed to oxidize Au to AuCl4- in HCl, whereas the high potential could destruct SPCE surface and led to poor stabilization. Figure 5a shows the effect of preoxidation potential on DPV response. With the preoxidation time of 40 s, the peak current increased with the increasing preoxidation potential from ?1.20 to ?1.40 V and reached the maximum value at ?1.40 V. Beyond ?1.40 V, the peak current decreased to a great extent. Hence, ?1.30 V was selected as the optimal preoxidation potential, at which the oxidation time was optimized. As shown in Fig. 5b, the DPV response reached the maximum value at the time of 30 s. The reduction of AuNPs signals were increased with increasing time, reached their highest level at 30 s and were stable thereafter. Longer time led to the diffusion of the formed AuCl4- away from electrode surface and decreased the current. Therefore, 30 s was chosen as the optimal preoxidation time, which showed a feasible ability to implement a rapid detection. Cyclic voltammetric analysis of the modified electrode The fabrication of the digoxin-immunosensor including bare electrode and various modified SPCEs was studied by cyclic voltammetry. As expected, no signal was observed in the cyclic voltammograms of bare, PVA modified electrodes and secondary antibody-PVA. Due to the nonconductive nature of the PVA, no oxidoreduction reaction in the electrode
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surface would happen (Fig. 5a–c), according to our previous report [40]. To detect the non-specific reaction, 20 lL of Fe3O4-AuNPs suspension was added onto the PVA-secondary antibody modified electrode and current response was monitored. A weak signal from the immunosensor for secondary antibody-PVA-Fe3O4-Au-NPs SPCE was recorded due to the non specific interaction between NPs and PVA (Fig. 5d). A stronger signal at 0.3 V was observed in the presence of digoxin-Fe3O4-Au-NPs and anti-digoxin antibody on secondary antibody-PVA modified electrode which was produced by preoxidation of bound Fe3O4-Au-NPs and subsequent reduction of AuCl4- in Fe3O4-Au-NPs complex in electrochemical assays (Fig. 5e). Immunoassay performance As expected from a competitive mechanism, when increasing the concentration of free digoxin in standard and real samples, the intensity of cathodic peak current was decreased. This could be the result of increased interaction between free digoxin and primary antibody, compared with the antigen which was immobilized on the surface of Fe3O4-Au-NPs and vice versa. In order to increase speed, sensitivity of assay and calibration curve determination, many forms of electrochemical potential modulation have been offered over the years. DPV is one of these pulse techniques which can be used for quantitative determinations of analyte in aqueous and nonaqueous sample. Serial concentrations of standard solutions were prepared by diluting the known concentrations of digoxin with normal serum samples to obtain the final concentrations of 0.5–5 ng mL-1; and the competitive analysis experiments were carried out in triplicates for each concentration by DPV.
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A mixture of 10 lL of serial concentration of standard solution and 100 ng of anti-digoxin antibody was first incubated with the 10 lL of labeled antigen for 20 min at 37 °C. Then after washing and magnetic separation of antibody-digoxin-Fe3O4-Au-NPs and free digoxin-Fe3O4-AuNPs from other components placed on the surface of the modified electrodes with immobilized secondary antibody and incubated again for 15 min at 37 °C. After holding the working electrode at a condition potential of ?1.3 V for 30 s in HCl, the DPV measurements were carried out between ?0.2 and ?0.5 V with a scan rate of 50 mV s-1 (Fig. 6). The current signal generated on reduction of the Fe3O4-Au-NPs tagged digoxin was recorded as an electrochemical response. An increase in peak current of DPV with diminution of digoxin concentration in standard samples was observed. The dose–response curve obtained from the digoxin-immunosensor was decreased from 0.5 to 5 ng mL-1 (Fig. 6). Under optimal conditions, the digoxin detection limit of 0.05 ng mL-1 was estimated. The proposed competitive immunosensor benefitted from the wider detection range and lower limit of detection, compared to other digoxin immunosensors [15, 16]. The present method enjoys the advantage of the introduction of PVA onto the SPCE surface, which not only adsorbed enormous antibody molecules, but also promised good stabilization. More importantly, compare to other labels such as enzyme Fe3O4-Au-NPs composite as a tracing tag is not only capable of amplified the electrochemical signal under low concentrations of digoxin, but also can produce better signal for the improvement of sensitivity, stability, reduction in background and separation strategy and make our immunosensor more sensitive, providing great potential in clinical application.
Fig. 6 Differential pulse voltammograms for the electrochemical detection of digoxin upon serial dilutions of antigen between 0.5 and 5 ng mL-1 from bottom to top in 1 M HCl at scan rate of 50 mV s-1. Inset dose/response curve of digoxin using the PVA modified SPCEs
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Specificity, reproducibility, and stability of the immunosensor In order to evaluate the specificity of the immunosensor, a number of digoxin-like compounds and other steroids were prepared in the same as digoxin standard. The specificity of the immunosensor was examined by testing the DPV responses to the interfering substances, such as Aldospirone, Cortisol, Digitoxin, Estradiol, Progesterone and Testosterone prepared in serum free digoxin sample. The immunosensor was separately incubated with all of the above compounds at a concentration well over the maximum levels encountered in the clinical samples. Then, the immunosensor was separately incubated with 0.5 ng mL-1 digoxin solution with and without interference. We found that our immunosensor exhibited good specificity for detection of digoxin given that there were no obvious changes in peak currents (Ip = 208 ± 5), following incubation of the immunosensor with the above substances. The inter-assay precision of the immunosensor was evaluated by using five chips. The coefficients of variation (CV) were 6.2 and 5.4 % respectively for 0.5 and 2 ng mL-1 digoxin, indicating acceptable precision and fabrication reproducibility. The protocol of Omidfar et al. [40, 42] was followed to test the stability of the immunosensor over time. After being stored in a dark, dry chamber for a period of 2 weeks at 37 °C, the immunosensor possessed acceptable stability, as 92 and 85 % of the initial DPV response for digoxin could still be remained at day 12 and 14, respectively (Fig. 7). Such decrease in stability of the digoxin-BSA-Fe3O4Au-NPs probably was caused by a reduction of antigen activity sites. Thus, the good stability indicates the fact that the antigen-Fe3O4-Au-NPs, as label, can provide a promising potential for fabricating the new kind of assay based on the direct electrochemical immunosensors. It should be noted that stability studies of the digoxine absorbed on the Au-NPs and the designed immunosensor have been carried out for 2 weeks at 37 °C, which is equivalent to about more than 6 months, at 4 °C.
Fig. 7 The stability of the NPs after 2 weeks storage at 37 °C
Table 1 Comparison of digoxin determination in serum by immunosensor and ELISA Sample
Immunosensor (ng mL-1)
ELISA (ng mL-1)
RSD (%)
1
0.53
0.5
1.96
2
0.95
1
2.06
3
1.95
1.9
1.04
4
1.56
1.6
1.02
5
1.25
1.15
4.16
6
0.85
0.91
3.4
7
1.25
1.23
0.8
8
1.84
1.8
1.09
9
1.75
1.8
1.12
10
1.15
1.1
0.88
immunosensor and revealed that the prepared immunosensor had significant correlation with ELISA method. Furthermore, the results showed that our immunosensor could be employed for clinical diagnosis of digoxin in real samples.
Reliability of immunosensor assay
Conclusion
The reliability of our immunosensor was also examined by performing the test in ten serum samples and comparing the results with those obtained via ELISA method (Table 1). An increase in the peak current of DPV with diminution of digoxin concentrations in real samples was observed. Table 1 also shows the relative standard deviations (RSDs) of the determination estimated from three repeated measurements for real samples. An obtained RSD less than 5 % was the indicator of acceptable accuracy of the
This contribution described a sensitive and low-cost immunoassay method was constructed by combining PVAmodified SPCE with a Fe3O4-Au-NPs composite as an electrochemical label. This one-step assay is based on the competitive immunochemical reaction between the free digoxin in samples and immobilized digoxin on the Fe3O4Au-NPs surface for the limited antibody sites in solution. The Fe3O4-Au-NPs composite has a large specific area and good biochemical activity without requiring coupling reagents, thanks to the presence of Au core on the surface
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of the Fe3O4-NPs. Core–shell magnetic Au-Fe3O4-NPs tracing tag showed a strong electroactivity for signal amplification, whereas the PVA-modified SPCE immunosensor showed a good performance in biocompatibility to stable attachment to the capture antibody and retain its specific immunorecognition. With this competitive-type immunoassay format, the immunosensor exhibited a wide linear range, low detection limit, good specificity, and acceptable accuracy for the detection of hapten. The designed composite provided an efficient tracing tag for biomolecule recognition and signal amplification in highly sensitive bioanalytical assays. Acknowledgments This research was supported by a grant from Endocrinology and Metabolism Research Center of Tehran University of Medical Sciences, Tehran, I.R. Iran.
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An electrochemical immunosensor for digoxin using core–shell gold coated magnetic nanoparticles as labels Anita Ahmadi • Hanieh Shirazi • Narges Pourbagher Abolfazl Akbarzadeh • Kobra Omidfar
•
Received: 24 July 2013 / Accepted: 30 December 2013 / Published online: 7 January 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract A simple, sensitive, and low-cost immunosensor was designed for the detection of digoxin through core– shell gold coated magnetic nanoparticles (Fe3O4-Au-NPs) as an electrochemical label. Having had such a large potential for a variety of applications, Fe3O4-Au-NPs have attracted a considerable attention and are actively investigated recently. Digoxin is a cardiac glycoside which, at high level, can indicate an increased risk of toxicity. This new competitive electrochemical immunosensor was developed based on antigen–antibody reaction employing antigen (Ag) labeled Fe3O4-Au-NPs and PVA modified screen-printed carbon electrode surface in order to detect the serum digoxin. The structures of Fe3O4-Au-NPs were studied by transmission electron microscopy, X-ray diffraction and Fourier transformed infrared spectroscopy. Cyclic voltammetry and differential pulse voltammetry (DPV) were employed to determine the physicochemical and electrochemical properties of immunosensor. DPV was employed for quantitative detection of digoxin in biological samples. The developed immunosensor was capable to detect digoxin in the range from 0.5 to 5 ng mL-1, with a
A. Ahmadi H. Shirazi N. Pourbagher K. Omidfar (&) Biosensor Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, P.O. Box 14395/1179, Tehran, Islamic Republic of Iran e-mail: [email protected] A. Ahmadi K. Omidfar Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Research Institute, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran A. Akbarzadeh Department of Medicinal Chemistry, Tabriz University of Medical Sciences, Tabriz, Islamic Republic of Iran
detection limit as low as 0.05 ng mL-1. The proposed method represented acceptable reproducibility, stability, and reliability for the rapid detection of digoxin in serum samples. Keywords Core–shell gold coated magnetic nanoparticles Immunosensor Digoxin
Introduction Digoxin is a cardiac glycoside, which is widely prescribed for the treatment of congestive heart failure and various disturbances of cardiac rhythm [1, 2]. The therapeutic use of digoxin in patients is based on its ability to increase myocardial contractions, by increasing the blood output in all patients with heart failure [3–5]. However, toxicity may be encountered at a concentration as low as 2.0 ng mL-1 [6]. Therefore, given its narrow therapeutic range of 0.8–1.9 ng mL-1, serum digoxin concentration measurement is necessary in digitalized patients [6, 7]. Despite the therapeutic and toxic concentrations overlap, measurement of digoxin levels helps to maintain effective concentrations, to diagnose and to prevent overdosage. Several methods such as high performance liquid chromatography (HPLC) and immunochemical assays have been used for detecting of digoxin in serum [2, 4, 6–10]. HPLC has good linearity, limit of detection, precision and specificity; however, it is time-consuming, require sample pre-treatment and demand expensive instruments. Compared with HPLC, immunoassays are the most common and effective methods for monitoring serum digoxin level, despite requiring a long reaction time, special equipment, reagent, and involve multiple steps. With the increasing demand for on-site (pre)-screening and diagnosis, more
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ultrasensitive approaches should be described for realizing the requirement of clinical analysis. Recently, electrochemical immunosensors have attracted a great deal of attention. They offer a number of distinct features such as combining the high specificity and sensitivity of traditional immunoassay methods with the low detection limits, fast analysis, simplicity, easy handling, low costs and low endogenous background using different types of electrodes [11–13]. Coupled with the development of such techniques, a great deal of effort has been devoted to constructing the robust, sensitive, selective and practical biosensing instrumentations as point of care (PoC) diagnostic tests in laboratories [13, 14]. In a previous study [7], we developed a semi-quantitative immunostrip assay for the measurement of digoxin in serum using antibody gold nanoparticles (Au-NPs) conjugate as label. However, these types of assay do not provide a sensitive and reliable method for the quantitative measurement of analyte in biological sample [7, 9]. Furthermore, up to now, two immunosensors have been designed for detection of digoxin in serum based on flowthrough fluorosensors. Several problems have been attributed to fluorometric assays as they non-specifically reduce or enhance the signal output. Fluorescence detection is liable to changes in pH, temperature, concentration of ion and detergent, drying, and the solid matrix, leading to light scattering, high background, quenching and bleaching issues. Worse still, such methods take too long and often require high cost, phase separation or even special technology [15, 16]. Thus, exploring a new trace label that is based on the electrochemical principle would be valuable. For successful development of user-friendly tests with sufficient sensitivity, the type of label is very important. Fortunately, nanomaterial-based signal amplification strategies hold great promise in realizing ultrasensitive detection due to their versatile properties. A large number of nanomaterials, including metal NPs and carbon-based nanostructures can be utilized as labels in order to obtain the amplified electrochemical detection signal [17–22]. A Nanospheres with magnetic cores (Fe3O4-NPs) and gold shell structures have recently received more attention thanks to combined functions of Fe3O4 and gold. Fe3O4NPs have a typical super-paramagnetic nature, providing a convenient way for separation, isolation, and purification of biological samples via an external magnetic field while the functional reagents are attached onto the surface of the particles [27]. In fact, the combination of Fe3O4-NPs with Au-NPs, as a core–shell, possesses both properties of AuNPs and magnetism; offering one of the most excellent electrochemical signals for the enhancement of sensitivity, reduction in background and separation strategy.
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Therefore, they could have been widely used in analytical detection and potential biomedical applications including immunoassay, magnetic resonance imaging (MRI) contrast enhancement, hyperthermia, drug delivery and catalysis [28–32]. Fe3O4-Au-NPs are considered as excellent candidates for bioconjugation such as antigen, antibody or cell, due to their large surface area, good biocompatibility and separation [27, 29, 33–39]. The present study proposes a new direct competitive electrochemical immunosensor, combining a disposable chip with the Fe3O4-Au-NPs tracing tag in order to further improvements in signal amplification, magnetism separation and also reduction in background signal that is another crucial point in assay development (Scheme 1). The disposable chip was prepared by immobilizing capture antibody, secondary antibody, on poly vinyl alcohol (PVA) modified screen-printed carbon electrode (SPCE) surface in order to detect the serum digoxin. The organic matrixes such as PVA could assist antibody immobilization and also served as a potential stabilizer for binding sites of the antibody structure. The electrochemical signal, from binding antigen (Ag)-Fe3O4-Au-NPs complex, could be produced after electro-oxidization of AuCl4- in HCl, followed by its reduction in differential pulse voltammetry (DPV) mode [40, 41]. The proposed method demonstrated the capability to detect digoxin within the range 0.5–5 ng mL-1, with the detection limit down to 0.05 ng mL-1. The excellent analytical performance of the immunosensor provided its potential application in clinical analysis of low abundance, low molecular weight analytes in biological fluids. The magnetic core shell Fe3O4-Au-NPs composite is not only an excellent electronic tracing tag to obtain detection signal, but also provides a controllable method to construct a sensitive and specific platform in ultrasensitive bioanalysis.
Materials and methods Materials FeCl36H2O (99.0 %), FeCl24H2O (99.0 %) and NH3, HCl (99.0 %) were purchased from Acros Organics (USA). Chloroauric acid (HAuCl4), trisodium citrate, sodium azide, vinyl alcohol, mouse monoclonal anti-digoxin antibody (clone DI-22) and secondary antibody were purchased from Sigma Chemical Company (St. Louis, MO, USA). Ammonia, NaOH and other chemicals were analytical grade. The analytical HCl was purchased from Merck (Merck, Spain). All other chemicals were analytical grade and used without further purification. Deionized water was used in all the experiments.
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Scheme 1 Schematic illustration of a direct competitive electrochemical immunosensor fabrication process: a VA-secondary antibody was dropped on the working electrode of SPCE, photopolymerized VA under UV exposure; b BSA blocking; c incubation of the patient/ standard samples with digoxinBSA-Fe3O4-Au-NPs; d the preoxidation of Au-NPs was performed at the constant potential of ?1.30 V in 1 M HCl, e and then, the current signal was recorded by the voltammetric modes
Synthesis of Fe3O4-NPs Fe3O4-NPs were synthesized by co-precipitation method, in the presence of ammonia as a reducing agent, with slight modification as previously reported [35]. In brief, ferric (III) chloride hexahydrate (7.568 g) and ferrous (II) chloride tetrahydrate (3.173 g) with 1.75:1 mol ratio were dissolved in 120 mL deoxygenated water and vigorously stirred in the presence of nitrogen atmosphere-protected solution at 90 °C. Afterwards, a mixture of freshly deoxygenated ammonium hydroxide (6.4 M, 100 mL) was quickly added to the solution in order to maintain the pH at 9.0–9.5. While the color of the solution changed from yellow to black, stirring was allowed for an additional 30 min at room temperature. The solution was then cooled down to room temperature. The Fe3O4-NPs were washed several times by water and stored in Tetramethylammonium hydroxide (TMOH, 0.1 M) at room temperature. Preparation of Fe3O4-Au-NPs In order to synthesis Fe3O4-Au-NPs, 0.5 g of the prepared Fe3O4-NPs was transferred into 25 mL sodium citrate (0.01 M). The mixture was then stirred for 30 min to exchange absorbed OH- with citrate ions and to construct the final working magnetic-core solution. Various dilutions of the above solution were used for the Au coating reaction in a total volume of 20 mL of 0.01 M sodium citrate. The reaction solutions containing magnetic cores and reducing agent were heated to 98 °C under vigorous
stirring. Due to during this process, magnetite particles would be oxidized partially or totally to maghemite before their being coated. While boiling, hydrogen tetrachloroaurate (III) hydrate solution (0.01 M, 250 lL) was injected as soon as possible, and the initially brown solution changed color to dark brown and then to final deep red characteristic gold colloids. The heating mantle was removed 15 min after injection, and the stirring was continued for an additional 15 min. The Fe3O4-Au-NPs were continually washed with water in order to remove the extra gold particles. A total of four dilutions of oxidized Fe3O4 solutions in total volume of 20 mL were prepared; the colorless solution initially observed gradually became red to pinkish red which corresponds to 0.1, 0.2, 0.3 and 1 mL of working oxidized Fe3O4 respectively. Finally, in order to remove the magnetic particles, which were not coated by gold, we used HCl (1 M) for 60 min. During this period supernatant color was changed to yellow and its pH reduced to 1. Thereafter, Fe3O4-Au-NPs were washed several times with water until the pH reached the neutral value. NPs were stored in the Tris buffer (pH 8.1) for further use. The stability of the NPs was evaluated after two weeks storage at 37 °C. Preparation of digoxin–protein conjugates Digoxin was conjugated to bovine serum albumin (BSA) using our previously described method. The efficiency of conjugation was evaluated spectrophotometrically in 83 % H2SO4 [7].
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Immobilization of antibody or antigen (digoxin-BSA) onto the Fe3O4-Au-NPs Two different types of competitive assays based on digoxin-BSA-Fe3O4-Au-NPs and anti-digoxin monoclonal antibody (mAb)-Fe3O4-Au-NPs conjugate, were developed in this study. In order to prepare digoxin-BSA-Fe3O4-Au-NPs conjugates, 16 lg mL-1 of digoxin-BSA were added to 1 mL of Tris–HCl buffer (pH 7.4, 0.05 M) containing 2 mg Fe3O4Au-NPs. The mixture was gently mixed for 60 min at 37 °C and subsequently 1 mg mL-1 of BSA solution was added to block the residual surface of the NPs. After blocking, the Fe3O4-Au-NPs were coupled with dig-BSA and then magnetically separated from free dig-BSA. The Ag-Fe3O4Au-NPs was rinsed with washing buffer (Tris buffer, pH 8.1, 0.05 M) three times, re-suspended in Tris buffer (pH 8.1) and stored at 4 °C. The optimal amount of antigen for immobilization on the surface of the Fe3O4-Au-NPs was determined by employing the enzyme linked immunosorbent assay (ELISA), according to the method described in our previous works [40, 42]. The appropriate concentration of digoxin monoclonal antibody (16 lg) was conjugated to Fe3O4-Au-NPs using the same procedure as described above. Fabrication of the immunosensors For successful construction of the immunosensors, 2 % PVA was selected as the best concentration for antibody immobilization, based on our previous report [40]. Vinyl alcohol was dissolved in acetate buffer (pH 4.5, 100 mM) at 70 °C for 30 min to provide 2 % (w/v) homogeneous vinyl alcohol solution. To obtain a colloidal suspension, 5 lL of vinyl alcohol including 100 ng of secondary antibody dropped on the surface of working electrode and under UV conditions (220–250 nm, 20 min), the vinyl alcohol was polymerized to give the PVA. To block the nonspecific binding sites, the electrode was immersed with phosphate buffer (pH 7.2, 0.1 M) containing 0.5 % BSA for 15 min at 37 °C and then washed by 0.05 % Tween 20 in phosphate buffer to remove the unbounded BSA. The modified electrode was stored at 4 °C before use. DigoxinBSA was also coated on to the electrode surface using the same procedure as described earlier. Electrochemical measurements In order to optimize the calibration curve, two experiments was applied in this study. First, 10 lL of standard or real sample solution and 100 ng of anti-digoxin antibody, accompanying the 10 lL of labeled antigen, was added
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simultaneously onto the modified electrode surface with immobilized secondary antibody and incubated for 30 min at 37 °C. Thereafter, unbound Fe3O4-Au-NPs were rinsed three times with washing buffer. Second, the standard or real sample solution and antidigoxin antibody were first incubated with the labeled antigen for 20 min at 37 °C and then dropped onto the modified electrode surface with immobilized secondary antibody and incubated again for 15 min at 37 °C. The unbound labeled antigens were also rinsed three times with washing buffer. The modified electrode was placed in the electrochemical cell and 50 lL of 1 M HCl was added to cover the entire three-electrode zone of the SPCE surface for electrochemical study. The background response was recorded with the same condition on the PVA modified electrode without secondary antibody. The modified electrode was first monitored by cyclic voltammetry (CV), at a potential rang of 0–1.0 V and the scan rate of 50 mV s-1. The electrochemical detection of digoxin was carried out by DPV mode. The preoxidation of Fe3O4-Au-NPs was achieved at a constant potential of 1.3 V for 30 s in 1 M HCl to generate AuCl4- and immediately reduction of AuCl4- to Au° by DPV, while scanning 0.1–0.4 V with a step potential of 4 mV, a pulse amplitude of 50 mV, and a pulse period of 20 ms. All the potential records were performed at room temperature. The cathodic current peak was selected as the analytical signals for electrochemical detection of digoxin. Finally, the second experiment was selected for the real samples analysis. In another experiment, a mixture of 10 lL of secondary antibody-Fe3O4-Au-NPs solution, 100 ng of anti-digoxin antibody and 10 lL of standard or real sample was dropped onto the modified electrode surface with immobilized digoxin-BSA. The assay was performed as described previously. In order to investigate the reproducibility of the assay, two different serum samples with low (0.5 ng mL-1) and high (5 ng mL-1) concentration were tested for digoxin five independent analytical runs [40, 41].
Results and discussion Characterization of Fe3O4-Au-NPs Magnetic seeds were synthesized using co-precipitation in the presence of NH4OH under controlled condition (pH 10) and N2 protection gas. The optimum mole Fe3?/Fe2? ratio used was 1.7:1. The sodium citrate was deployed in order to reduce Au3? in the presence of the generated particles. The XRD pattern of the Fe3O4-NPs and Fe3O4-Au-NPs are presented in Fig. 1. For Fe3O4-NPs, five characteristic peaks at 2h° = 29.84°, 35.32°, 42.80°, 56.88° and 62.68° were
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Fig. 1 XRD patterns of Fe3O4 (a) and Fe3O4-Au-NPs (b). The powder of Fe3O4 and Au-NPs are presented by the bars
observed marked by their indices (2 2 0), (3 1 1), (4 0 0), (5 1 1) and (4 4 0). The XRD of the Fe3O4-Au-NPs also showed the same peaks indicating the unchangeable pace of Fe3O4 during coating process. Furthermore, gold also has the other diffraction peaks at 2h° = 38.2°, 44.88°, 64.4°, and 77.6° which can be indexed to 111, 200, 220, and 311 lattice planes of gold in a cubic phase, respectively. TEM image of the prepared Fe3O4-Au-NPs is shown in Fig. 2a. Fe3O4-NPs surface morphology analysis demonstrated the agglomeration of many ultrafine particles with diameters lower than 8 nm. TEM analysis revealed that the average particle size was increased from 8 nm (Fig. 2a) to 16 nm after gold coating (Fig. 2b). The synthesized NPs were consistently
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Fig. 3 FTIR spectrum of Fe3O4, Au and Fe3O4-Au-NPs (nanoparticles not immobilized)
scattered in the sample and appeared to form spherical structures (Fig. 2). Figure 3 shows the FTIR spectra of Fe3O4-NPs, Au-NPs and Fe3O4-Au-NPs. In comparison with the naked magnetic nanoparticles, abundant absorption peaks in spectrum of the modified particle confirmed the successful coating of Au-NPs on the iron oxide surface. Bands at 580–682 cm-1 in the spectra are indicating the Fe–O bending. There is also N–H stretching and bending in 3,110 and 1,620 cm-1, respectively. The peak of N–H bending implied that the Fe3O4-NPs were successfully coated by gold. Au-NPs have strong plasmon resonance properties and show the absorption peak at about 530 nm
Fig. 2 TEM image of prepared Fe3O4 (a) and Fe3O4-Au-NPs (b)
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Fig. 4 UV–Vis spectra of Au (a), Fe3O4-Au-NPs (b), Fe3O4-Au-NPs after HCl treatment (c) and Fe3O4 (d). A sharp absorption peak can be observed for the Au-NPs at 520 nm. Fe3O4-Au-NPs after HCl treatment shown sharper absorption peak
(curve a in Fig. 4). The UV–Vis absorption spectra of the, Fe3O4-Au-NPs, HCl treated Fe3O4-Au-NPs and Fe3O4-NPs are shown in Fig. 4b–d. The spectra of the Fe3O4-Au-NPs with various ratios of prepared Au3? to Fe3O4 showed that 0.2 mL of Fe3O4 solution was appropriate for the final experiment. Optimization of the immunoassay conditions Considering both high sensitivity and wide linear range, the optimal concentrations of coating antibody, anti-digoxin antibody and the digoxin-BSA-Fe3O4-Au-NPs suspension, are crucial to this direct competitive immunoassay. Therefore, the effects of different quantities of digoxinBSA-Fe3O4-Au-NPs (5, 10, 15, 20 and 25 lL), coated secondary antibody (25, 50, 100,150 and 200 ng) and antidigoxin antibody (25, 50, 100, 150 and 200 ng) were assessed. According to reduction peak currents in cyclic voltammograms, proper amounts of digoxin-BSA-Fe3O4Au-NPs, coated secondary antibody, and anti-digoxin antibody for the assay were determined as 16 lL, 100 ng, and 100 ng, respectively (data not shown). Subsequently, the effect of temperature on the immunoreactions was studied by CV method. Briefly, the reaction of digoxin-BSA-Fe3O4-Au-NPs, coated secondary antibody, and anti-digoxin antibody was examined at room temperature and 37 °C. It could be seen that the strong current signal was achieved at 37 °C. Next, the storage stability of the digoxin-BSA-Fe3O4Au-NPs over a period of 2 weeks at 37 °C was scanned periodically using DPV [40]. The DPV peak potentials and currents maintained essentially unchanged for at least 1 week at 37 °C, and then started to decrease slowly, with 86 % of NPs activity being presented at day 14 (data not shown).
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Fig. 5 Cyclic voltammograms of the different modified electrode measured in 1 M HCl at the potential range of 0.0 to ?1 V and the scan rate of 50 mV s-1: a bare electrode, b PVA, c secondary antibody-PVA, d secondary antibody-PVA-Fe3O4-Au-NPs and e digoxin-Fe3O4-Au-NPs and anti-digoxin antibody on secondary antibody-PVA. Inset effects of potential between 1.2 and 1.4 V (a) and preoxidation time (b) on DPV response
Optimization of detection conditions The conditions for electro-oxidization of the captured AuNPs are important parameters. The low potential failed to oxidize Au to AuCl4- in HCl, whereas the high potential could destruct SPCE surface and led to poor stabilization. Figure 5a shows the effect of preoxidation potential on DPV response. With the preoxidation time of 40 s, the peak current increased with the increasing preoxidation potential from ?1.20 to ?1.40 V and reached the maximum value at ?1.40 V. Beyond ?1.40 V, the peak current decreased to a great extent. Hence, ?1.30 V was selected as the optimal preoxidation potential, at which the oxidation time was optimized. As shown in Fig. 5b, the DPV response reached the maximum value at the time of 30 s. The reduction of AuNPs signals were increased with increasing time, reached their highest level at 30 s and were stable thereafter. Longer time led to the diffusion of the formed AuCl4- away from electrode surface and decreased the current. Therefore, 30 s was chosen as the optimal preoxidation time, which showed a feasible ability to implement a rapid detection. Cyclic voltammetric analysis of the modified electrode The fabrication of the digoxin-immunosensor including bare electrode and various modified SPCEs was studied by cyclic voltammetry. As expected, no signal was observed in the cyclic voltammograms of bare, PVA modified electrodes and secondary antibody-PVA. Due to the nonconductive nature of the PVA, no oxidoreduction reaction in the electrode
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surface would happen (Fig. 5a–c), according to our previous report [40]. To detect the non-specific reaction, 20 lL of Fe3O4-AuNPs suspension was added onto the PVA-secondary antibody modified electrode and current response was monitored. A weak signal from the immunosensor for secondary antibody-PVA-Fe3O4-Au-NPs SPCE was recorded due to the non specific interaction between NPs and PVA (Fig. 5d). A stronger signal at 0.3 V was observed in the presence of digoxin-Fe3O4-Au-NPs and anti-digoxin antibody on secondary antibody-PVA modified electrode which was produced by preoxidation of bound Fe3O4-Au-NPs and subsequent reduction of AuCl4- in Fe3O4-Au-NPs complex in electrochemical assays (Fig. 5e). Immunoassay performance As expected from a competitive mechanism, when increasing the concentration of free digoxin in standard and real samples, the intensity of cathodic peak current was decreased. This could be the result of increased interaction between free digoxin and primary antibody, compared with the antigen which was immobilized on the surface of Fe3O4-Au-NPs and vice versa. In order to increase speed, sensitivity of assay and calibration curve determination, many forms of electrochemical potential modulation have been offered over the years. DPV is one of these pulse techniques which can be used for quantitative determinations of analyte in aqueous and nonaqueous sample. Serial concentrations of standard solutions were prepared by diluting the known concentrations of digoxin with normal serum samples to obtain the final concentrations of 0.5–5 ng mL-1; and the competitive analysis experiments were carried out in triplicates for each concentration by DPV.
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A mixture of 10 lL of serial concentration of standard solution and 100 ng of anti-digoxin antibody was first incubated with the 10 lL of labeled antigen for 20 min at 37 °C. Then after washing and magnetic separation of antibody-digoxin-Fe3O4-Au-NPs and free digoxin-Fe3O4-AuNPs from other components placed on the surface of the modified electrodes with immobilized secondary antibody and incubated again for 15 min at 37 °C. After holding the working electrode at a condition potential of ?1.3 V for 30 s in HCl, the DPV measurements were carried out between ?0.2 and ?0.5 V with a scan rate of 50 mV s-1 (Fig. 6). The current signal generated on reduction of the Fe3O4-Au-NPs tagged digoxin was recorded as an electrochemical response. An increase in peak current of DPV with diminution of digoxin concentration in standard samples was observed. The dose–response curve obtained from the digoxin-immunosensor was decreased from 0.5 to 5 ng mL-1 (Fig. 6). Under optimal conditions, the digoxin detection limit of 0.05 ng mL-1 was estimated. The proposed competitive immunosensor benefitted from the wider detection range and lower limit of detection, compared to other digoxin immunosensors [15, 16]. The present method enjoys the advantage of the introduction of PVA onto the SPCE surface, which not only adsorbed enormous antibody molecules, but also promised good stabilization. More importantly, compare to other labels such as enzyme Fe3O4-Au-NPs composite as a tracing tag is not only capable of amplified the electrochemical signal under low concentrations of digoxin, but also can produce better signal for the improvement of sensitivity, stability, reduction in background and separation strategy and make our immunosensor more sensitive, providing great potential in clinical application.
Fig. 6 Differential pulse voltammograms for the electrochemical detection of digoxin upon serial dilutions of antigen between 0.5 and 5 ng mL-1 from bottom to top in 1 M HCl at scan rate of 50 mV s-1. Inset dose/response curve of digoxin using the PVA modified SPCEs
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Specificity, reproducibility, and stability of the immunosensor In order to evaluate the specificity of the immunosensor, a number of digoxin-like compounds and other steroids were prepared in the same as digoxin standard. The specificity of the immunosensor was examined by testing the DPV responses to the interfering substances, such as Aldospirone, Cortisol, Digitoxin, Estradiol, Progesterone and Testosterone prepared in serum free digoxin sample. The immunosensor was separately incubated with all of the above compounds at a concentration well over the maximum levels encountered in the clinical samples. Then, the immunosensor was separately incubated with 0.5 ng mL-1 digoxin solution with and without interference. We found that our immunosensor exhibited good specificity for detection of digoxin given that there were no obvious changes in peak currents (Ip = 208 ± 5), following incubation of the immunosensor with the above substances. The inter-assay precision of the immunosensor was evaluated by using five chips. The coefficients of variation (CV) were 6.2 and 5.4 % respectively for 0.5 and 2 ng mL-1 digoxin, indicating acceptable precision and fabrication reproducibility. The protocol of Omidfar et al. [40, 42] was followed to test the stability of the immunosensor over time. After being stored in a dark, dry chamber for a period of 2 weeks at 37 °C, the immunosensor possessed acceptable stability, as 92 and 85 % of the initial DPV response for digoxin could still be remained at day 12 and 14, respectively (Fig. 7). Such decrease in stability of the digoxin-BSA-Fe3O4Au-NPs probably was caused by a reduction of antigen activity sites. Thus, the good stability indicates the fact that the antigen-Fe3O4-Au-NPs, as label, can provide a promising potential for fabricating the new kind of assay based on the direct electrochemical immunosensors. It should be noted that stability studies of the digoxine absorbed on the Au-NPs and the designed immunosensor have been carried out for 2 weeks at 37 °C, which is equivalent to about more than 6 months, at 4 °C.
Fig. 7 The stability of the NPs after 2 weeks storage at 37 °C
Table 1 Comparison of digoxin determination in serum by immunosensor and ELISA Sample
Immunosensor (ng mL-1)
ELISA (ng mL-1)
RSD (%)
1
0.53
0.5
1.96
2
0.95
1
2.06
3
1.95
1.9
1.04
4
1.56
1.6
1.02
5
1.25
1.15
4.16
6
0.85
0.91
3.4
7
1.25
1.23
0.8
8
1.84
1.8
1.09
9
1.75
1.8
1.12
10
1.15
1.1
0.88
immunosensor and revealed that the prepared immunosensor had significant correlation with ELISA method. Furthermore, the results showed that our immunosensor could be employed for clinical diagnosis of digoxin in real samples.
Reliability of immunosensor assay
Conclusion
The reliability of our immunosensor was also examined by performing the test in ten serum samples and comparing the results with those obtained via ELISA method (Table 1). An increase in the peak current of DPV with diminution of digoxin concentrations in real samples was observed. Table 1 also shows the relative standard deviations (RSDs) of the determination estimated from three repeated measurements for real samples. An obtained RSD less than 5 % was the indicator of acceptable accuracy of the
This contribution described a sensitive and low-cost immunoassay method was constructed by combining PVAmodified SPCE with a Fe3O4-Au-NPs composite as an electrochemical label. This one-step assay is based on the competitive immunochemical reaction between the free digoxin in samples and immobilized digoxin on the Fe3O4Au-NPs surface for the limited antibody sites in solution. The Fe3O4-Au-NPs composite has a large specific area and good biochemical activity without requiring coupling reagents, thanks to the presence of Au core on the surface
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of the Fe3O4-NPs. Core–shell magnetic Au-Fe3O4-NPs tracing tag showed a strong electroactivity for signal amplification, whereas the PVA-modified SPCE immunosensor showed a good performance in biocompatibility to stable attachment to the capture antibody and retain its specific immunorecognition. With this competitive-type immunoassay format, the immunosensor exhibited a wide linear range, low detection limit, good specificity, and acceptable accuracy for the detection of hapten. The designed composite provided an efficient tracing tag for biomolecule recognition and signal amplification in highly sensitive bioanalytical assays. Acknowledgments This research was supported by a grant from Endocrinology and Metabolism Research Center of Tehran University of Medical Sciences, Tehran, I.R. Iran.
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