A novel impedimetric disposable immunosensor for rapid detection of a potential cancer biomarker

International Journal of Biological Macromolecules 66 (2014) 273–280

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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

A novel impedimetric disposable immunosensor for rapid detection of a potential cancer biomarker Engin Asav a , Mustafa Kemal Sezgintürk b,∗ a b

Kırklareli University, School of Health, Kırklareli, Turkey Namık Kemal University, Faculty of Science, Chemistry Department, Biochemistry Division, Tekirda˘g, Turkey

a r t i c l e

i n f o

Article history: Received 21 December 2013 Received in revised form 11 February 2014 Accepted 13 February 2014 Available online 20 February 2014 Keywords: Cancer biomarkers Biosensor Human epidermal growth factor receptor Electrochemical impedance spectroscopy Single frequency impedance

a b s t r a c t A specific and sensitive biosensor was developed successfully for quantitative detection of human epidermal growth factor receptor by electrochemical impedance spectroscopy. Anti-human epidermal growth factor receptor antibody was covalently immobilized onto a screen-printed carbon electrode modified with a carbon nanotube. Immobilization steps were characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and scanning electron microscopy (SEM). After human epidermal growth factor receptor ligates with anti-human epidermal growth factor receptor immobilized onto an electrode surface, charge transfer resistance changes considerably. This electrochemical response was correlated with human epidermal growth factor receptor concentration. Under optimal conditions, the proposed biosensor could detect human epidermal growth factor receptor 2 fg/mL with a linear range from 2 to 14 fg/mL, showing high sensitivity. Kramers–Kronig Transform was performed on the experimental impedance data. Meanwhile, in a biosensor system, the Single Frequency Impedance technique was first used for characterization of interaction between human epidermal growth factor receptor and anti-human epidermal growth factor receptor. Eventually, the proposed biosensor was applied to artificial serum samples spiked with human epidermal growth factor receptor. © 2014 Elsevier B.V. All rights reserved.

1. Introduction A tumor marker is defined as “a substance that may be found in tumor tissue or released from a tumor into the blood or other body fluids.” The phrase tumor marker is often used interchangeably with biomarker. However, the definition of a biomarker is broader. Biomarkers include not only substances associated with or released from tumor tissue, but also physiological markers or markers visualized using imaging technology. Biomarkers may also be substances released by the body in response to the tumor but not by the tumor per se. For instance, the immune system may react to the tumor by producing substances that can be detected in the blood. These substances may indicate the presence of a tumor, but are not actually produced by the tumor cells. Additionally, the term biomarkers can be applied to blood cancers, which do not form solid tumors. From this point of view, four homologous receptor tyrosine kinases EGFR (HER-1), HER-2 (ErbB2), HER-3 (ErbB3), and HER4 (ErbB4) are members of the human epidermal growth

∗ Corresponding author. Tel.: +90 2822502605. E-mail addresses: [email protected], [email protected] (M.K. Sezgintürk). http://dx.doi.org/10.1016/j.ijbiomac.2014.02.032 0141-8130/© 2014 Elsevier B.V. All rights reserved.

factor receptor (HER) family, which are considered as transmembrane receptors [1]. The expression of HER-3 has been detected in normal human adult and fetal tissues using polyclonal antibodies [2]. The amplification of HER-3 gene and/or overexpression of its protein have been reported in numerous cancers including breast carcinomas [3,4], pancreatic cancers [5], gastric cancers [6], non-small-cell lung carcinoma [7,8], colorectal cancer [2], hepatocellular carcinoma [9]. Normal HER3 levels in a healthy person range from 0.06 to 2.55 ng/mL. Moreover, to indicate risk of cancer, the abnormal levels of HER3 should be increased up to 12 ng/mL [10]. Hence the identification of protein biomarkers such as HER-3 shows promise for the earlier diagnosis and treatment planning of cancer, as well as to help in screening for cancer using imaging systems [11]. In the last two decades, many researchers have tried to detect HER-3 in blood and tissue samples by using various methods such as immunohistochemical [12], confocal immunofluorescent microscopy [13], enzyme-linked immunosorbent assay (ELISA) [14] and Northern blot [15]. Among these methods, the electrochemical immunoassay techniques have attracted considerable interest due to their high sensitivity, inherent simplicity, portability and low cost [16]. Recently, biosensor systems for detection of potential tumor markers including carcinoembryonic antigen (CEA) [16],

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alpha-fetoprotein [17], cancer antigen 125 (CA125) [18], prostate specific antigen (PSA) [19] and both human chorionic gonadotropin (hCG) and the activated leukocyte cell adhesion molecule (ALCAM) [20] have been reported. As carbon nanotube-modified screen-printed electrodes have a well-defined appearance and stability, are disposable, and exhibit high electrochemical reactivity, they are widely utilized in various biosensor systems such as genosensors [21,22], immunosensors [23], and amperometric biosensors [24,25]. In this paper, inspired by the high electrical conductivity and protein-loading ability of carbon nanotubes, an electrochemical immunosensor for HER-3 was fabricated simply by immobilization of anti-HER-3 on the electrode. We described for the first time an anti-HER-3 immobilization on a carbon nanotube-modified screen-printed electrode. Anti-HER-3 antibody was bound to the carbon nanotube surface by EDC/NHS couple. The final biosensor developed was used for the quantitative detection of early cancer prediction and detection. 2. Experimental 2.1. Reagents and material All reagents were purchased from Sigma–Aldrich (St. Louis, MO, USA). HER-3 and anti-HER-3 were also purchased from (St. Louis, MO, USA). HER-3, anti-HER-3, 1-ethyl-3-(3-dimehylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS) and 0.1% BSA (bovine serum albumin) were prepared in a phosphate buffer (50 mM, pH 7). The solutions of HER-3, anti-HER-3 and 0.1% BSA were stored at −20 ◦ C until use. Synthetic serum solution was prepared by using 4.5 mM KCl, 5 mM CaCl2 , 4.7 mM (D +)-glucose, 2.5 mM urea, 0.1% human serum albumin, and 145 mM NaCl. A redox probe solution was prepared in 50 mM, pH 7, phosphate buffer which contained 0.1 M KCl, 5 mM Fe(CN)6 4− and 5 mM Fe(CN)6 3− . 2.2. Apparatus Electrochemical experiments were carried out by using a Gamry Potentiostate/Galvanostate, Reference 600 (Gamry Instruments, Warminster, USA) interfaced with a PC via an EChem Analyst containing physical electrochemistry, pulse voltammetry, and electrochemical impedance spectroscopy software (Gamry Instruments, Warminster, USA). Single-walled carbon nanotubemodified screen-printed electrodes (coded as 110SWCNT) consisting of a carbon working electrode (SPE) (4 mm diameter), a silver/silver chloride reference electrode, and a carbon counter electrode were obtained from Dropsens, S.L. (Oviedo, Spain). 2.3. Fabrication of the impedimetric immunosensors Firstly, 5 ␮L of H2 SO4 (1.0 mM in distilled water) was dripped onto a single-walled carbon nanotube-modified screen-printed electrode (SPE) for the constitution of carboxyl groups onto the wall of the carbon nanotubes. After a 1-h incubation, electrode was washed with ultra-pure water gently, and afterwards was dried by a pure argon stream. Then, a 1-ethyl-3-(3-dimehylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) couple was used for activation of these carboxyl groups. For this purpose, a 5 ␮L aliquot of the EDC (0.2 M)–NHS (0.05 M) mixture was dropped onto carboxyl groups formed on SPE and the electrode was left to incubate for an hour in a dark ambient. Subsequently, a 5 ␮L aliquot of anti-HER-3 (0.1 ␮g/␮L) was applied onto modified SPE and then allowed to incubate for 1 h in a moisture medium. Afterwards, the electrode was immersed in ultra-pure water to remove physically adsorbed anti-HER-3 molecules. Finally, 5 ␮L of

BSA (0.1%) was dripped onto the final electrode to block active carboxyl ends. The bare (unmodified) and modified electrodes were denoted as SPE, SPE/H2 SO4 , SPE/H2 SO4 /EDC-NHS, SPE/H2 SO4 /EDCNHS/anti-HER-3, SPE/H2 SO4 /EDC-NHS/anti-HER-3/BSA. 2.4. Experimental measurements Cyclic voltammetry was utilized to characterize the steps of electrode modification and immobilization. The applied potential was varied between −500 and 500 mV (step size: 20 mV, scan rate: 50 mV/s) in the presence of a 5 mM [Fe(CN)6 4− ]/[Fe(CN)6 3− ] (1:1) solution which served as a redox probe containing 0.1 M KCl. For electrochemical impedance studies, an alternating wave of 10 mV amplitude was applied to the electrode over the formal potential of the redox couple (0 V). The redox couple used for the impedance studies was the same as used in cyclic voltammetry studies. Impedance spectra were collected in the frequency range between 10,000 and 0.05 Hz. 2.5. Scanning electron microscopy (SEM) Structural observations of an electrode surface modified by immobilization and HER-3 binding were performed by field emission scanning electron microscopy (FEI-Quanta FEG 250 model, at 10,000-fold magnifications) at the Scientific and Technological Research Center of Namık Kemal University (NABI˙ LTEM). The acceleration voltage of 5 kV was used to acquire the SEM images. 3. Results and discussion 3.1. Immobilization of anti-HER-3 onto carbon nanotube-modified screen-printed carbon electrode The formation of carboxyl groups onto carbon nanotubes, the activation of these carboxyl groups, and the immobilization of antiHER-3 onto the modified electrode were characterized with the help of electrochemical impedance spectroscopy and cyclic voltammetry. A well-defined characteristic voltammogram of the redox couple, Fe(CN)6 3−/4− was observed on the bare SPE. Fig. 1A shows the cyclic voltammograms obtained for the immobilization steps of anti-Her-3. It is obvious from Fig. 1 that the formation of carboxyl groups onto the side walls of the carbon nanotubes did not alter the peak currents of the redox probe. The activation of these carboxyl groups resulted in an increase in anodic and cathodic currents compared to a bare electrode. This might have been caused by an increase in the electron-transfer rate of the redox probe. The EDC/NHS reaction included the formation of an intermediate active ester which was the product of condensation of carboxyl groups and NHS. This intermediate product exhibited excellent electrochemical activity. Furthermore, the carboxyl groups on the carbon nanotubes could be activated directly just via amines of EDC, but owing to the formation of a more stable intermediate, the reaction did not occur efficiently when compared to EDC/NHS. In the activation step, it was possible to make electrochemical measurements just by this relatively stable intermediate product. After the incubation of the modified electrode with anti-HER-3, both the cathodic and anodic peak currents decreased dramatically through hindering the effect of anti-HER-3 on the electron-transfer rate. The addition of BSA that blocked free active ends also caused a decrease in peak currents, due to its protein structure. That is to say, that modification of the electrode surface by an insulator protein made it more resistive to redox probe diffusion. After binding HER-3, a significant decrease was observed in the anodic and cathodic peak currents. This again was caused by reduction of the electron-transfer rate by HER-3 proteins.

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affect the electron-transfer resistance of the surface. In this case, the carboxyl groups did not cause an alteration in the diffusion of the redox probe to the electrode surface. Electron-transfer resistance (Rct ) values were decreased significantly in the activation step of these carboxyl groups onto the side walls of the nanotubes via the EDC–NHS couple, owing to the formation of an intermediate active ester, which might have helped the redox probe to diffuse onto the electrode surface. The incubation of the modified electrode (SPE/H2 SO4 /EDC-NHS) with anti-HER-3 resulted in a significant increase in Rct values. In this case, anti-HER-3 could probably be a barrier to the transfer of the redox probe to the electrode surface. Similarly, blocking of active free carboxyl groups by BSA increased the electrical resistivity of the electrode surface. Finally, the experiment results showed excellent agreement between cyclic voltammetry and electrochemical impedance spectroscopy studies. 3.2. SEM characterization studies

Fig. 1. Electrochemical characterization of the biosensor [(A) cyclic voltammograms of anti-HER-3 immobilization by covalently attachment, ---: bare SPE, ---: SPE/H2 SO4 , ---: SPE/H2 SO4 /EDC-NHS, -䊉-䊉-: SPE/H2 SO4 /EDCNHS/anti-HER-3, -+-+-: SPE/H2 SO4 /EDC-NHS/anti-HER-3/BSA. (B) Electrochemical impedance spectra of HER-3 immobilization steps, ---: bare SPE, ---: SPE/H2 SO4 , ---: SPE/H2 SO4 /EDC-NHS, -䊉-䊉-: SPE/H2 SO4 /EDC-NHS/anti-HER-3, -+-+-: SPE/H2 SO4 /EDC-NHS/anti-HER-3/BSA. Inset equivalent circuit model: equivalent circuit model applied to fit the impedance measurements. [Rs , the ohmic resistance of the electrolyte solution; Cdl , associated with the double layer capacitance; Rct , the electron-transfer resistance and W, the Warburg impedance].

The surface morphologies of the proposed biosensor layers were also studied by SEM (Fig. 2). Bare SPE electrode showed an almost homogenous surface due to the nature of screen-printed carbon electrode (Fig. 2A), whereas the SPE/H2 SO4 /EDC-NHS/anti-HER-3 electrode showed that anti-HER-3 molecules were covered by covalent attachment onto the surface (Fig. 2B) and the multi particle aggregates with a roughly globular structure due to binding of BSA (Fig. 2C). Also, Fig. 2D shows more aggregation because of interaction between HER-3 and anti-HER-3. These results were good agreement with those obtained from impedance characterization studies. 3.3. Optimization experiments of the HER-3 biosensor

Fig. 1B shows a typical Nyquist diagram of impedance spectra of the bare SPE, carboxyl group formation, the EDC/NHS activation process, anti-HER-3 modification immobilization and BSA blocking in the frequency range from 0.05 to 10,000 Hz. The semicircular portion observed at high frequencies corresponds to the electrontransfer limited process, whereas the linear part represents the diffusion limited process. Electrochemical impedance spectroscopy (EIS) is one of the most important techniques for characterization of electrode surfaces. Nyquist Plots of bare electrode SPE, SPE/H2 SO4 , SPE/H2 SO4 /EDC-NHS, SPE/H2 SO4 /EDC-NHS/anti-HER3, SPE/H2 SO4 /EDC-NHS/anti-HER-3/BSA electrodes are shown in Fig. 1B. In Nyquist plots, the complex impedance is displayed as the sum of the real and imaginary components (Zre and Zim ); the semi-circle diameter at higher frequencies corresponds to the electron-transfer resistance (Ret ), and the linear part at lower frequencies corresponds to the diffusion process (Warburg impedance). The diameter of the semicircle also exhibits the blocking behavior of the modified electrode after each modification step. The electrochemical impedance data were fitted with an equivalent circuit model by utilizing commercial software called Gamry Echem Analyst, shown in Fig. 1B. This equivalent circuit model consisted of resistive and capacitive elements, as well as a Warburg element. Rs was the resistance of the working solution, and the Cdl (constant phase element) was connected with the capacitance of the complex bioactive layer. Rct was related to the electron-transfer resistance through the electrode surface, and the Warburg impedance, Zw , described the normal diffusion to the electrode surface through the complex layer. The electron-transfer resistance (Rct ) values of these electrodes were found to be 322.8, 274.9, 124.8, 389.8 and 616.7 , respectively. In the formation step of carboxyl groups onto unmodified electrode (SPE), the impedance spectra also showed that carboxyl groups that formed on the electrode surface did not considerably

To assess the effective catalytic properties of the biosensor proposed in regard to the HER-3 biomarker, the optimization of the immobilization steps was extremely important. For this purpose, the parameters such as concentrations of H2 SO4 , EDC–NHS, and anti-HER-3, and duration of anti-HER-3 incubation were optimized. First of all, it was shown that impedimetric and voltammetric methods are powerful tools for studying the immobilization and the measurement steps through the biosensor preparation and HER3 analysis stages. Details of these studies are given in the sections below. The surface functional groups anchored on/within carbons were found to be responsible for the variety in physicochemical and catalytic properties of the materials considered [26–28]. Many researchers have focused on how to modify as well as to characterize the surface functional groups of carbon materials in order to improve or extend their practical applications [28–30]. Acid treatment has generally been used to oxidize the porous carbon surface; it enhances the acidic property, removes the mineral elements and improves the hydrophilic property of the surface. The acid used in this case should be oxidization in nature; nitric acid and sulfuric acid were the most selected [31,32]. Carboxylation of the side walls of carbon nanotubes, in fact, is a product of acid oxidation treatments. Also, this pretreatment step is applied routinely for many purposes including cleaning of carbon nanotubes and as a first step in surface functionalization procedures. The carboxylation method used in this experiment was to generate oxidation on the surface of the carbon nanotube by using a portion of sulfuric acid, and to damage the structures of carbon atoms on the five-membered ring and six-membered ring of graphitic sheets of the carbon nanotubes. In order to determine the effect of the H2 SO4 concentration on the biosensor response, different biosensor systems were constructed by using 0.1, 1.0 and 2.0 M H2 SO4 . It can be seen from the results (Fig. 3A) that both the peak currents and electron-transfer

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Fig. 2. SEM characterization of the biosensor surfaces. [(A) Bare SPE, (B) SPE/H2 SO4 /EDC-NHS/anti-HER-3, (C) SPE/H2 SO4 /EDC-NHS/anti-HER-3/BSA, (D) SPE/H2 SO4 /EDCNHS/anti-HER-3/BSA/HER-3.].

Fig. 3. Optimization studies. [A. The effect of sulfuric acid concentration on biosensor performance. Calibration graphs obtained by the biosensors fabricated by different sulfuric acid concentrations, ---: 0.1 M, ---: 1 M, ---: 2 M]. [B. The effect of EDC/NHS concentrations on immobilization performance. Calibration graphs obtained by the biosensors fabricated by different EDC/NHS concentrations, ---: 0.02 M EDC-0.005 M NHS, ---: 0.2 M EDC-0.05 M NHS, ---: 2 M EDC-0.5 M NHS]. [C. HER-3 calibration graphs for the biosensors constructed by the different anti-HER-3 concentrations. Different anti-HER-concentrations, -䊉-䊉-: 0.1 ng/␮L, ---: 0.4 ng/␮L, ---: 2 ng/␮L, ---: 10 ng/␮L, -x-x-: 20 ng/␮L]. [D. Optimization of anti-HER-3 binding period. The biosensors prepared by different anti-HER-3 incubation periods, ---: 30 min, ---: 60 min, ---: 120 min].

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resistance values were affected by the concentration of H2 SO4 . Electron-transfer resistances from the impedance spectra indicate that the carboxylation degree of the surface decreased by using 0.1 and 1.0 M H2 SO4 when compared to an unmodified electrode surface. Probably, the reason is because, by forming carboxyl groups (hereafter carbonyl group) on the surface, the resonance of the carbon partial positive charge allows the negatively charged redox probe to diffuse through the electrode surface. By this process, the electron-transfer resistance can be reduced. However the linearity of the calibration curve belonging to the biosensor built up with 1.0 M H2 SO4 was better than that of other biosensors. In the case of using 2.0 M H2 SO4 , probably higher concentrations of acid could cause damage to the electrode surfaces. Hence, electron-transfer resistance values were not decreased considerably; nevertheless, further studies revealed that the immobilization of anti-HER-3 was not effectively caused by the destruction of the electrode surface. As a result, 1 M H2 SO4 solution was used in all experiments for activation of the carbon nanotube surface. The next important parameter was the concentration of the EDC–NHS couple. Carbodiimides provide the most popular compounds for labeling or cross-linking to carboxylic acids. The most readily available and commonly used carbodiimide is the watersoluble EDC for aqueous cross-linking. NHS esters are reactive groups formed by EDC activation of carboxylate molecules. NHS ester-activated crosslinkers and labeling compounds react with primary amines to yield stable amide bonds [33]. Different concentrations of EDC–NHS couple were used in biosensor fabrication to clarify the influence of concentrations on biosensor responses. The experiments showed that the response of the biosensor was influenced by the concentrations of EDC/NHS. HER-3 calibration curves obtained by the impedance spectra related to these findings can be seen in Fig. 3B. The experiments revealed that an increase in EDC/NHS concentration gave rise to unfavorable results. Higher concentrations of EDC/NHS couple than 0.2 M/0.05 M probably caused random and dense cross-linking of anti-HER-3 onto the electrode surface because anti-HER-3 protein also contained multiple carboxyls and amines. However, lower concentrations of EDC/NHS couple than 0.2 M/0.05 M led to a decrease in biosensor signals. In this condition, the signals were decreased because of insufficient cross-linking activity of the EDC/NHS. This was an expected result because the low levels of cross-linking agent should not crosslink anti-HER-3 to the electrode surface. Therefore, 0.2 M/0.05 M was chosen as the optimum concentration of EDC/NHS couple. Then, the effect of anti-HER-3 concentration applied to the active electrode surface was investigated in detail. Experiments were carried out with different concentrations of anti-HER-3 i.e. 0.1–0.4–2–10–20 ng/␮L. HER-3 calibration graphs related to different anti-HER-3 concentrations can be seen in Fig. 3C. The biosensor signals were not strongly influenced by the antiHER-3 concentration. The interactions between an antibody and its antigen are relatively complicated. Presumably, here neither 0.1 nor 20 ng/␮L anti-HER-3 were fully ligated by HER-3 injected onto the anti-HER-3-modified surface. However, when the results are evaluated with regard to the R2 values, it can clearly be shown that to increase the concentration of anti-HER-3 impaired the R2 value. This negative effect was most likely caused by the formation of a complex protein matrix on the electrode surface. In fact, this was a benefit as regards the cost of a biosensor construction because using an amount of 0.1 ng/␮L anti-HER-3 should reduce the cost of the biosensor. Consequently, in further studies, the amount of 0.1 ng/␮L anti-HER-3 was used. The final optimization study was devoted to an explanation of the incubation period for anti-HER-3. The experiment results showed that the immobilization performance of anti-HER-3 was strongly dependent on the incubation

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period. HER-3 calibration curves from these experiments can be seen in Fig. 3D. The immobilization of anti-HER-3 increased the charge transfer resistance drastically. This result was expected because of the blocking behavior of the anti-HER-3 layer on the electrode surface for the redox probe [Fe(CN)6 ]3−/4− , which was reflected in the impedance spectroscopy as an increase in the diameter of the semicircle at high frequencies. However, the charge transfer resistances, Rct , decreased considerably with the increase in incubation times. These results can also be seen in Fig. 3D. At the end of the 120-min incubation period, antiHER-3 solution on the electrode surface had completely evaporated spontaneously although the electrode was incubated in a moisture medium. Probably, the immobilization performance of anti-HER3 was negatively affected due the long time for incubation. It was possible that oxygen in the air attacked the protein, and anti-HER3 was probably lost its hydrate layer during the long incubation period. So anti-HER-3 should be denaturated by this effect. A similar effect was obtained by incubating anti-HER-3 for 60 min. However, it seemed that the highest Rct value was obtained after 30 min of incubation. As a consequence, according to the results obtained, it was efficient to incubate anti-HER-3 for 30 min. 3.4. Analytical characteristics of HER-3 biosensor In this study, electrochemical impedance spectroscopy was mainly utilized to determine the diffusion rate of the redox probe associated with changes in the electron-transfer resistance related to HER-3 concentration. The charge transfer resistances related to the HER-3 concentrations were calculated with the help of the equivalent circuit model given in inset Fig. 1B. To obtain a calibration curve of the biosensor, the variation of the absolute impedance calculated by the following equation was used: Rct = Rct(anti−HER−3/HER−3) − Rct(anti−HER−3) where Rct(anti-HER-3/HER-3) is the value of the charge transfer resistance after anti-HER-3 is coupled to HER-3, while Rct(anti-HER-3) is the value of the charge transfer resistance when anti-HER-3 is immobilized on the electrode. As can be seen from Fig. 4A, peak currents decreased with the increase in the concentrations of HER-3 standard solutions. Fig. 4B shows the Nyquist plot evaluation of the biosensor for different HER-3 concentrations. It was observed that the semi-circle diameter in the Nyquist plots increased with increasing HER-3 concentration. Moreover, the low frequencies could be used for concentration-dependent measurements. A calibration graph for HER-3 was prepared with the help of the differences in charge transfer resistances after HER-3 binding. The increment in HER-3 concentration increased the charge transfer resistance, Rct , achieving a linear range between 2 and 14 fg/mL. The calibration curve is given in Fig. 4C. The methods developed for the determination of HER-3 are frequently based on immunohistochemical or ELISA (Enzyme-Linked Immunosorbent Assay) procedures. For instance, a commercially available HER3 ELISA kit employs an antibody specific for human HER3, coated on a plate. The analysis procedure of this technique contains timeconsuming steps. Moreover, the cost of an assay kit is extremely high (1000 euros). However, the cost of one SPE-based immunosensor developed is approximately 5 euros. The minimum detectable level of HER3 is reported as typically less than 4 pg/mL. This limit is higher than those of our immunosensor reported here. One of the most critical issues in constructing a biosensor is the avoidance of inconsequent biosensor signals related to insufficient reproducibility. For this reason, the reproducibility of an HER-3 biosensor using different working electrodes that was fabricated six times using the same set of procedures for HER-3 analysis yielded varying

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E. Asav, M.K. Sezgintürk / International Journal of Biological Macromolecules 66 (2014) 273–280 Table 1 Kreamers–Kronig Transform, reproducibility, and artificial serum sample analysis. Kramers–Kronig transforms for different layers of the biosensor Biosensor surfaces

Goodness of fit values

Bare SPE SPE/H2 SO4 SPE/H2 SO4 /EDC-NHS SPE/H2 SO4 /EDC-NHS/anti-HER-3 SPE/H2 SO4 /EDC-NHS/anti-HER-3/BSA SPE/H2 SO4 /EDC-NHS/anti-HER-3/BSA/HER-3

14.44 9.189 5.966 6.444 0.55 5.093

Reproducibility of the biosensor Biosensor numbers

R2

y

Linear ranges (fg/mL)

1 2 3 4 5 6

0.9829 0.9847 0.9897 0.9918 0.9840 0.9845

66.88x + 89.36 79.0x + 7.2 59.295x + 39.89 38.529x + 94.643 71.46x + 155.42 66.33x + 58.76

2–14 2–14 2–14 2–14 2–14 2–14

HER-3 detection in artificial serum samples HER-3 (fg/mL)

Fig. 4. Cyclic voltammograms (A), impedance spectrums (B) obtained for different concentrations of HER-3, and, HER-3 calibration curve obtained by the biosensor based on anti-HER-3 (C).

signals or charge transfer resistances. However, the linear detection ranges of six biosensors were the same as between 2 and 14 fg/mL. The results are given in Table 1. The biosensor described here also had excellent reproducibility. The results are again summarized in Table 1. The repeatability was also clarified. As is known, repeatability should be defined as the degree of single biosensor that could be used continually for a series of measurements with the same concentration of HER-3. The charge transfer resistances of the biosensor were investigated when the biosensor was consecutively exposed to a 2 fg/mL HER-3 standard solution on five occasions. The repeatability of the measurements was very good considering that the correlation coefficient on measurements was 5.34%, and the average value and standard deviation were calculated as 2.15 and ±0.115 fg/mL, respectively. Thus, the generating biosensor could be used repeatedly. To examine the specificity of the biosensors, the biosensors were used in the analysis of samples containing a number of metabolites such as some amino acids (Asp, Gly, Phe, Glu, Trp, and Lys, His), glucose, albumin, VEGF (vascular endothelial growth factor), hemoglobin, IgA, IgM, insulin, haptoglobin, and parathyroid hormone, all of which could exist in human blood. The impedance spectrums related to these experiments showed that these proteins and substances did not interfere with the biosensor based on anti-HER-3 because in the charge transfer values related to the substances tested there were no meaningful changes. In this study, Kramers–Kronig Transform was one of the most important parameters that was analyzed. The Kramers–Kronig (K–K)

Added

Found by the biosensor

% Recovery

% Relative difference

2 4

2.18 4.22

109 105.5

9 5.5

relations can be used to evaluate data quality. The K–K relations demand that causal, complex plane spectral data show dependence between magnitude and phase. The real part of a spectrum can be obtained by integration of the imaginary part and vice versa. The Kramers–Kronig relations are integral equations that constrain the real and imaginary components of complex quantities for systems that satisfy conditions of linearity, causality, and stability [34–36]. In principle, the Kramers–Kronig relations can be used to determine whether the impedance spectrum of a given system has been influenced by bias errors caused, for example, by instrumental artifacts or time-dependent phenomena. The direct integration of the Kramers–Kronig relations involves calculating one component of the impedance from the other, e.g., the real component of impedance from the measured imaginary component. The result is compared to the experimental values obtained [36]. In the presented study, by using Kramers–Kronig Transforms, the real part of the experimental data was used to calculate an imaginary part of a linear, stable, and causal circuit. Likewise, the imaginary portion of the experimental data was used to calculate the real part of a linear, stable, and causal circuit. According to this calculation, a value named “Goodness of Fit” was obtained. In addition, a plot of the relative errors (Z/Z) for the real and imaginary parts of the experimental data was also drawn. In the present HER-3 biosensor, for all steps including preparation of the biosensor and measurement, Kramers–Kronig Transforms were performed. The plots of the Kramers–Kronig Transforms are given in Fig. 5. The goodness of fit values of different biosensor surfaces are also given in Table 1. The Kramers–Kronig Transforms performed on the electrochemical impedance spectra related to the biosensor revealed that the experimental data agreed with the data obtained by the transform. Nevertheless, it was very important that the impedance spectra showed a linear, stable, and causal circuit. Finally, it is also important to say that the system was not influenced by unknown variables. Moreover, in this study, a new impedimetric technique was applied to the biosensor for the first time. In literature, electrochemical impedance spectroscopy is used for the immobilization and characterization of a biosensor. Also, it is utilized to

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Fig. 5. The plots of Kramers–Kronig transforms performed on different layers of the biosensor. [The circles show experimental results, the lines on the circles show Kramers–Kronig Transforms performed on the experimental data. A: bare SPE, B: SPE/H2 SO4 , C: SPE/H2 SO4 /EDC-NHS, D: SPE/H2 SO4 /EDC-NHS/anti-HER-3, E: SPE/H2 SO4 /EDC-NHS/anti-HER-3/BSA].

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carbon nanotubes, and Fe(CN)6 4− was used as the redox probe for electrochemical sensing. The biosensor demonstrated reduced cost, functional simplicity, and improved sensitivity and linear range. The detection limit of biosensor was 2 fg/mL. The Kramers–Kronig transforms revealed that the whole biosensor system was linear, stable, and formed a causal circuit. This meant that the biosensor system was not influenced by unknown variables. Moreover, single frequency impedance analyses were carried out to identify the binding characteristics of HER-3 and anti-HER-3, for the first time in such a biosensor system. Thus, it is highly recommended that single frequency impedance can be used for different biosensor applications such as for evaluation or to clarify slow, time-dependent changes in a biosensor surface. Finally, this biosensor was successfully applied to HER-3 analysis of artificial serum samples. Acknowledgments Support from TÜBI˙ TAK (The Scientific and Technological Research Council of Turkey, Project number: 109T 172) is greatly acknowledged. Also we are grateful to Namık Kemal University Scientific and Technological Research Center (NABI˙ LTEM). References

Fig. 6. Single frequency impedance measuremet.

analyze the charge transfer resistances of a modified surface. In order to characterize the binding of HER-3 to anti-HER-3 which had been immobilized onto an electrode surface, the “single frequency impedance” technique was performed successfully for the first time. Single frequency impedance measures the impedance at a fixed frequency versus time. Consequently, it should be possible to control the repeat time and the total time of the experiment. For this purpose, the potentiostate was set up at a fixed frequency of 5 Hz. The impedance was measured at this fixed frequency as a function of time and phase angle for 60 min. The result is shown in Fig. 6. As can clearly be seen from Fig. 6, the results were very informative about the binding of HER-3 to anti-HER-3. The significant shift in the phase angle was due to the interaction between HER-3 and anti-HER-3. In this experiment, the most important consideration was to take care to prevent environmental interference effects such as a possible electromagnetic wave, an inductive effect from the surrounding area, or the magnetic stirring of the reaction cell that could cause an unstable electrode surface. Eventually, from the results, it can be concluded that single frequency impedance can be used for biosensor evaluation, process monitoring, or to evaluate slow, time-dependent changes in a biosensor surface. Finally, the artificial serum samples were evaluated using the developed biosensor for the determination of HER-3. Results from three different measurements were averaged (Table 1). The results presented here show that HER-3 concentrations of the artificial serum samples analyzed by our proposed biosensor were consistent with those of the spiked amount of it in the serum solutions. 4. Conclusion A highly sensitive electrochemical immunosensor has been developed for the determination of HER-3, which is an important potential biomarker. The proposed biosensor was constructed by the immobilization of anti-HER-3 antibody on the surface of a screen-printed carbon electrode modified with single-walled

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