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Copper-doped titanium dioxide nanoparticles as dual-functional labels for fabrication of electrochemical immunosensors
Biosensors and Bioelectronics 59 (2014) 335–341
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Copper-doped titanium dioxide nanoparticles as dual-functional labels for fabrication of electrochemical immunosensors Sen Zhang a,b, Hongmin Ma a, Liangguo Yan b, Wei Cao a, Tao Yan a, Qin Wei a,n, Bin Du a a Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, PR China b School of Resources and Environment, University of Jinan, Jinan 250022, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 2 January 2014 Received in revised form 19 March 2014 Accepted 27 March 2014 Available online 5 April 2014
Constructions of versatile electroactive labels are key issues in the development of electrochemical immunosensors. In this study, copper-doped titanium dioxide nanoparticle (Cu@TiO2) was synthesized and used as labels for fabrication of sandwich-type electrochemical immunosensors on glassy carbon electrode (GCE). Due to the presence of copper ions, Cu@TiO2 shows a strong response current when coupled to an electrode. The prepared nanocomposite also shows high electrocatalytic activity towards reduction of hydrogen peroxide (H2O2). The dual functionality of Cu@TiO2 enables the fabrication of immunosensor using different detection modes, that is, square wave voltammetry (SWV) or chronoamperometry (CA). While Cu@TiO2 was used as labels of secondary antibodies (Ab2), carboxyl functionalized graphene oxide (CFGO) was used as electrode materials to immobilize primary antibodies (Ab1). Using human immunoglobulin G (IgG) as a model analyte, the immunosensor shows high sensitivity, acceptable stability and good reproducibility for both detection modes. Under optimal conditions, a linear range from 0.1 pg/mL to 100 ng/mL with a detection limit of 0.052 pg/mL was obtained for SWV analysis. For CA analysis, a wider linear range from 0.01 pg/mL to 100 ng/mL and a lower detection limit of 0.0043 pg/mL were obtained. The proposed metal ion-based enzyme-free and noble metal-free immunosensor may have promising applications in clinical diagnoses and many other fields. & 2014 Elsevier B.V. All rights reserved.
Keywords: Metal ions-based immunosensor Human immunoglobulin G Square wave voltammetry Chronoamperometry
1. Introduction Immunoassay has been considered as a significant analytical tool in clinical diagnoses, environmental monitoring, pharmaceutical chemistry and biochemical studies (Tang et al., 2007; Wang et al., 2004; Cui et al., 2007). Various immunoassay strategies, such as chemiluminescence, quartz crystal microbalance, surface plasmon resonance and electrochemical method, have been extensively developed for detection of biomarkers (Xu et al., 2011; Nakanishi et al., 1996; Oh et al., 2004; Tang et al., 2009). Among these techniques, electrochemical immunosensors with high sensitivity, short analytical time, low cost and simple pretreatment procedure have been a preferable approach for clinical immunoassays (Tang et al., 2005; Marquette and Blum, 2006; Jie et al., 2007). As antibodies and antigens are intrinsically unable to act as redox partners, a label is often conjugated to either the antibody or antigen to generate electrochemical signal in fabrication of an immunosensor. Based on the specific antigen–antibody
n
Corresponding author. Tel.: þ 86 531 827 678 72; fax: þ 86 531 827 659 69. E-mail address: [email protected] (Q. Wei).
http://dx.doi.org/10.1016/j.bios.2014.03.060 0956-5663/& 2014 Elsevier B.V. All rights reserved.
interaction, the signal is then quantitatively related to the content of the antibody or antigen in an analyzing sample (Liu et al., 2010). Due to the unique redox property in low potentials, metal ions have been used as labels to fabricate electrochemical immunosensors. Metal ions are usually loaded on some carriers, such as magnetic poly (styrene-acrylic acid) nanospheres (Zhang et al., 2012). Incorporating two or three metal ions with well-separated signals into one system, multiplex electrochemical immunosensors can be developed for simultaneous determination of several analytes (Feng et al., 2012; Zhang et al., 2014). The metal ionsbased immunosensors often apply SWV as detection mode (Wang et al., 1998), while CA is usually used for the immunosensors fabricated with catalytic labels (Guo et al., 2013; Wei et al., 2011; Tang et al., 2008). The metal ions-based immunosensors applying CA as detection mode is scarce. As a result, metal ions-based dual-functional electroactive labels that enable the fabrication of immunosensor using different detection modes were seldom reported. Marker proteins are of tremendous importance in diagnosing the early stage of a disease or pathological condition. As a common protein, IgG is usually chosen to be a model analyte. A variety of detection assays for IgG have been developed such as fluorescence
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(Ma et al., 2005; Tan et al., 2014; Luo et al., 2014), surface plasmon resonance (Deng et al., 2010; Fernández et al., 2012), chemiluminescence and colorimetric sensors (Li et al., 2005; Liu et al., 2009; Shi et al., 2011; Lai et al., 2012). Electrochemical detection of IgG was mainly based on enzyme-labeled immunoassay (Darain et al., 2003; Cui and Zhu, 2010; Yang et al., 2011; Liu et al., 2012; Cao et al., 2013). Whereas, the stringent condition of using enzyme was a limiting factor. In order to improve stability and sensitivity, some other enzyme-free immunosensors were developed employing noble metals as labels (Dequaire et al., 2000; Chu et al., 2005; Leng et al., 2010; Noh et al., 2011; Lai et al., 2013; Ding et al., 2013). However, the exorbitant price of noble metal is a shortcoming in the fabrication of immunosensor. Therefore, developing an enzyme-free and noble metal-free immunosensor is attractive and challenging. Titanium dioxide (TiO2) has widespread applications. It has attracted a lot of attention due to its potentials as photocatalyst (Yu et al., 2002), sensor material (Bao et al., 2008) and electrode of photovoltaic cell (Mor et al., 2005). TiO2 has also been used in clinical orthopedics and dentistry owing to its excellent biocompatibility (Lin et al., 2013a). Recently, TiO2 has been used as labels to incubate with antibody for the fabrication of immunosensor. Metal ions-doped titanium dioxides combine the advantages of TiO2 and the unique property of metal ions. In this work, copper ions were introduced onto the surface of TiO2 nanoparticles via an assembly process. The nanocomposite (Cu@TiO2) can generate strong electrochemical signal under SWV. Most notably, it shows prominent electrocatalytic activity towards the reduction of hydrogen peroxide (H2O2). Hence, Cu@TiO2 was used as dualfunctional labels to construct immunosensors. The chemical stability of Cu@TiO2 brings superiority over enzyme, which may require rigorous operating conditions. It also does not need the participation of noble metal. Carboxyl functionalized graphene oxide (CFGO) was also used for fabrication of immunosensors. The carboxylic groups at the edge of GO surface make it easier to immobilize primary antibody (Ab1). Using immunoglobulin G (IgG) as a model analyte, the performance of the immunosensor was investigated in both SWV and CA modes. High sensitivity, acceptable stability, and good reproducibility were achieved for both detection modes.
2. Experimental method 2.1. Materials Human IgG, IgG primary antibodies (Ab1) and IgG secondary antibodies (Ab2) were purchased from Beijing Dingguochangsheng Biotechnology Co., Ltd. (Beijing, China). Graphite, tetrabutoxytitanium, sodium hydroxide (NaOH), hydrochloric acid (HCl), copper hydroxide (Cu(OH)2), and other chemicals were obtained from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Phosphate buffer solution (PBS) (0.1 M, pH 4.5, pH 5.0, pH 7.4) was prepared by mixing stock solutions of KH2PO4 and Na2HPO4. Ultrapure water was used throughout the experiment. All the chemicals were of analytical reagent grade. All electrochemical behaviors were performed by a traditional three-electrode system. It contained a glassy carbon electrode (GCE, 4 mm diameter) as working electrode, a platinum wire electrode as auxiliary electrode and a calomel electrode as reference electrode. All electrochemical measurements were carried out using a CHI 760D electrochemical workstation (Shanghai Chenhua Instrument Co., Ltd., China). Transmission electron microscope (TEM) images were obtained from an H-800 microscope (Hitachi, Japan). Fourier transform infrared spectroscopy (FTIR) spectrum was obtained from VERTEX 70 (Germany). X-ray
powder diffraction (XRD) was gathered from a Bruker D8 Focus diffractometer (Germany) using CuKα radiation (40 kV, 30 mA) of wavelength 0.154 nm.
2.2. Preparation of CFGO Graphene oxide (GO) was synthesized through the modified Hummer's method (Hummers and Offeman, 1958). Carboxylation of GO was prepared according to the previous reports (Sun et al., 2012, 2008; Park and Jung, 2012; ). The details are shown in Supplementary information SI.
2.3. Preparation of Cu@TiO2 TiO2 was prepared according to the literature (Zhong et al., 2008; Jiang et al., 2003). Cu@TiO2 was prepared according to the literature (Li et al., 2008). The details were shown in Supplementary information SII.
2.4. Preparation of labels The labels were prepared as follows: Cu@TiO2 (1.0 mg) were mixed with 0.5 mL of 2.5% glutaraldehyde solution and stirred for 1 h. The products were then centrifuged and dispersed in 0.5 mL of pH 7.4 PBS. 5.0 μg of IgG Ab2 were added into Cu@TiO2 and shaken for 12 h. After centrifugation, the obtained bioconjugates (Cu@TiO2–Ab2) were further washed with PBS at least three times and resuspended in 1.0 mL PBS as the assay solution.
2.5. Modification of immunosensor GCE was polished carefully with alumina powder of 1.0, 0.3 and 0.05 μm respectively, to a mirror-like surface, then cleaned through sonication in ethanol for half a minute and dried in air. 5.0 μL of CFGO was added onto the GCE. After drying, the electrode was activated with 75 mmol/L EDC-15 mmol/L NHS for 1 h (Qian et al., 2002). By the covalent bonding of the carboxyl groups of CFGO and amino groups on the IgG Ab1, 5.0 μL 10 μg/mL of IgG Ab1 was immobilized on the electrode tightly (Lin et al., 2013b). Subsequently, incubation with 1% wt bovine serum albumin (BSA) solution for 1 h was used to eliminate nonspecific binding between the antigen and the electrode surface. Afterwards, IgG buffer solution with a varying concentration was added onto the electrode surface and incubated for 1 h, and the electrode was washed extensively to remove unbound IgG molecules. Finally, the prepared Cu@TiO2–Ab2 was dropped onto the electrode surface. After another 1 h, the surface was washed once more and the prepared immunosensor was stored at 4 1C. A schematic diagram of the stepwise procedure of the immunosensor is shown in Fig. 1.
2.6. Measurement procedure The PBS buffer solution (pH 4.5, pH 5.0) was used for all the electrochemical measurements. For SWV measurement of the immunosensor, it was scanned from 0.4 to 0.4 V with a potential step of 5 mV, a frequency of 25 Hz, and an amplitude of 25 mV. For amperometric measurement of the immunosensor, a detection potential of 0.4 V was selected. After the background current was stabilized, 5.0 mmol/L H2O2 was added to the buffer and the current change was recorded.
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Fig. 1. Schematic representation of the preparation of immunosensor.
Fig. 2. TEM (A) and FTIR (B) of CFGO, TEM of TiO2 (C) and Cu@TiO2 (D), XRD of Cu@TiO2 (E) and UV–visible spectroscopy (F) of (a) Cu@TiO2; (b) Ab2; and (c) Cu@TiO2–Ab2.
3. Results and discussion 3.1. Characterization of CFGO and Cu@TiO2 As shown in Fig. 2A, sheet-like shapes of CFGO endow large surface area to immobilize Ab1 (Wang et al., 2011). Specific
absorption peaks of hydroxyl (3425 cm 1), carbonyl (1721 cm 1) and epoxy (1052 cm 1) groups appeared on the FTIR spectrum of CFGO (Fig. 2B) indicating that different kinds of oxygencontaining functional groups existed on the CFGO surface (Sun et al., 2012). Peak bands at 1721 and 1390 cm 1 are attributed to carboxy asymmetric and symmetric telescopic vibrations of CFGO
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(Wang et al., 2012b). The result was inconsistent with that of the existing groups of CFGO nanosheet synthesized with the chemical oxidation (Stankovich et al., 2006). The as-prepared TiO2 nanospheres possess uniform size with an average diameter of 250 nm (Fig. 2C). It can be inferred that the rough surface of the TiO2 nanospheres present great opportunity for the loading of copper ions. Fig. 2D shows the TEM image of Cu@TiO2 powders. The nanosphere structure of TiO2 was broken under hightemperature calcination. Thus irregular shape of Cu@TiO2 was observed. Fig. 2E shows the XRD patterns of Cu@TiO2 powders after being treated at 450 1C. All the diffraction peaks matched well with those from the Jade PDF card (84-1286) for TiO2. The peaks at 2θ ¼35.41 and 38.71 were assigned to CuO (002) and CuO (111), respectively (Choi and Kang, 2007). These results indicate that the copper ion components exist on the external surface of TiO2. The Cu@TiO2 was also characterized with energy dispersive spectroscopy (EDS) (Supplementary information SIII, Fig. S1). The presence of copper ions on TiO2 was further confirmed. The Cu@TiO2–Ab2 bioconjugates were characterized by UV– visible spectroscopy (Fig. 2F). All samples were dispersed in ultrapure water and mixed thoroughly. After laying aside for 2 min at room temperature, the UV–visible spectroscopy was monitored from 220 nm to 700 nm. The strong absorption in the 240–350 nm region was assigned to the band–band transition of TiO2 (curve a) (Jacob et al., 2003). For pure Ab2 solution, there was only one major peak at approximately 280 nm (curve b). When Ab2 was adsorbed onto Cu@TiO2, the peak of Ab2 was conspicuous and the uptrend of peak in the 240–350 nm region was observed (curve c).
3.2. SWV and CA characteristics of expectant labels To demonstrate whether Cu@TiO2 could generate electrical signal and promote the reduction of H2O2, the following tests were employed. Fig. 3A displays the SWV response of 2.0 mg/mL Cu@TiO2 (curve a) and TiO2 (curve b) in PBS (pH 4.5). A cuspidal peak at potential 0 V is obviously observed, which is ascribed to the redox reaction of copper ions. TiO2 exhibits weak catalytic performance towards H2O2 while Cu@TiO2 has remarkable response (Fig. 3B). The great catalytic performance of Cu@TiO2 towards H2O2 could enhance sensitivity of the immunosensor. Therefore, Cu@TiO2 can be employed as expectant labels for the versatile immunosensor.
3.3. Optimization of experimental conditions All the steps involved in the immunoassay were optimized. We investigated the influence of pH and the concentrations of CFGO on the performance of the immunosensor under SWV and CA. Fig. 4A shows that the best SWV current response of the immunosensor was obtained at pH 4.5 and the best CA current response of the immunosensor was obtained at pH 5.0. Thus, pH 4.5 and pH 5.0 of the PBS buffer were selected as one of the optimized conditions for SWV and CA, respectively. Electrical signal increases with the increase of CFGO concentration from 0.5 to 2.0 mg/mL and reaches the maximum at 2 mg/mL, but decreases when the concentration further increases. The increase of CFGO film thickness may lead to an increase of interface electron transfer resistance, and the electron transfer becomes more difficult. Therefore, 2 mg/mL of CFGO is chosen for all subsequent experiments. 3.4. Electrochemical impedance spectroscopy (EIS) characterization of immunosensor EIS is one of the most powerful tools used to investigate the interface properties of surface-modified electrodes. It is well known that the high frequency region of the impedance plot shows a semicircle related to the redox probe FeðCNÞ6 3 =4 , followed by a Warburg line in the low frequency region which corresponds to the diffusion step of the over all process (Panagopoulou et al., 2010). Therefore EIS was carried out to illustrate that all the steps of fabricating the immunosensor were effective. Curve a in Fig. 5 shows EIS of the bare GCE. It possesses a very small semicircle domain implying a very low electron transfer resistance in the electrolyte solution. Curve b displayed larger interfacial electron transfer resistance than curve a, and indicated that the CFGO hindered electron transfer of the electrode. This was due to the covalent oxygenated functional groups in CFGO causing some loss in electrical conductivity (Mkhoyan et al., 2009; Chen et al., 2012). After assembly of Ab1, an increased semicircle at high frequency region was observed (curve c). The increase of Ret was caused by the nonconductive properties of antibody, which obstructed the electron transfer of the redox probe from solution to electrode surface. The immobilization of BSA and IgG at the Ab1/CFGO/GCE resulted in a dramatically increased diameter of the semicircle (curves d and e), indicating a higher electron transfer resistance at the electrode interface, due to a decrease in the
Fig. 3. (A) SWV of 2 mg/mL TiO2 (a) and Cu@TiO2 (b) in pH 4.5 PBS; (B) CA of 2 mg/mL TiO2 (a) and Cu@TiO2 (b) toward addition of 5.0 mmol/L H2O2 in pH 5.0 PBS. Inset: enlargement of TiO2 catalyzing H2O2.
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Fig. 4. Effect of (A) pH,and (B) the concentration of CFGO on the response of the immunosensor to 10 ng/mL IgG.
equation (Bard and Faulkner, 1980): E ¼ EΘ þ
Fig. 5. EIS of (a) GCE, (b) CFGO/GCE, (c) Ab1/CFGO/GCE, (d) BSA/Ab1/CFGO/GCE, (e) IgG/BSA/Ab1/CFGO/GCE and (f) Cu@TiO2–Ab2/IgG/BSA/Ab1/CFGO/GCE in 0.1 mol/L KCl solution containing 5.0 mmol/L FeðCNÞ6 3 =FeðCNÞ6 4 (1:1).
heterogeneous rate constant of FeðCNÞ6 4 =FeðCNÞ6 3 . After the formation of the immunocomplex between the Cu@TiO2–Ab2 and IgG/BSA/Ab1/CFGO/GCE, the electron transfer resistance increased again (curve f). This increase is attributed to the non-conducting properties of the protein, which presumably obstructs electron transfer at the electrochemical probe. The above results clearly confirm that the immunosensor had been fabricated successfully (Feng et al., 2013).
3.5. SWV and CA detection of IgG at immunosensor SWV was used to evaluate the performance of the immunosensor (Fig. 6A). The peak currents of SWV were enhanced with increasing concentration of analytes under optimized conditions. The currents changed linearly with the concentrations of IgG in the range from 0.1 pg/mL to 100 ng/mL with a detection limit of 0.052 pg/mL. The linear regression equations was i (μA)¼9.870þ2.165 log c (ng/mL) with a statistically significant correlation coefficient of 0.992. Fig. 6A inset shows the SWV response of 0 pg/mL, 0.1 pg/mL, 1 pg/mL, 0.01 ng/mL, 0.05 ng/mL, 0.1 ng/mL, 0.5 ng/mL, 1 ng/mL, 5 ng/mL, 10 ng/mL, 50 ng/mL, and 100 ng/mL (a–l, in order) of analyte concentrations. A shifting of the peaks was observed from the inset. The interesting phenomenon could be explained through the Nernst
αvo RT ln vOR nF αR
where the v's are stoichiometric coefficients, αi is the activity of species i, O and R are oxidized and reduced forms, respectively. This relation, the Nernst equation, furnishes the potential of the O/R electrode as a function of the activities of О and R. Therefore, the more labels containing more copper ions showed positive potential versus low concentration of analytes. CA was also used to evaluate the performance of the immunosensor on account of the catalyze reduction of Cu@TiO2 towards H2O2. Under the optimum conditions, the immunosensors using Cu@TiO2–Ab2 as labels were used to detect different concentrations of IgG in pH 5.0 PBS at 0.4 V. The relationship between the current response toward 5.0 mmol/L H2O2 and IgG concentration is shown in Fig. 6B. As can be seen, the catalytic current change was linear with the concentration of IgG in the range from 0.01 pg/mL to 100 ng/mL with a detection limit of 0.0043 pg/mL. Fig. 6B inset correspondingly shows the CA response of 0.01 pg/mL, 0.1 pg/mL, 1 pg/mL, 0.01 ng/mL, 0.1 ng/mL, 1 ng/mL, 10 ng/mL, and 100 ng/mL (a–h, in order) of analyte concentrations. Table S1 (Supplementary information SIV) illustrates that the immunosensor described here has a lower detection limit than previously described immunosensors (Cui and Zhu, 2010; Leng et al., 2010; Liu et al., 2012; Yang et al., 2011; Lai et al., 2013; Cao et al., 2013; Qian et al., 2010; Wang et al., 2012a; Yin et al., 2010; Ding et al., 2013; Noh et al., 2011). The low detection limit may be attributed to two factors. On one hand, the Cu@TiO2 nanoparticle has good biocompatibility and high electrocatalytic activity toward H2O2. On the other hand, a relatively large amount of Ab2 has been conjugated onto the Cu@TiO2 nanoparticle-based labels, which can greatly increase the probability of Ab2–antigen interactions thereby leading to higher sensitivity. Moreover, the reproducibility, selectivity, and stability of the immunosensor were also investigated. To evaluate the reproducibility of the immunosensor, a series of five electrodes were prepared for detection of 1 ng/mL IgG. The relative standard deviations (RSD) of the measurements for the five electrodes were 3.2% under SWV and 4.7% under CA. The results indicated that the precision and reproducibility of the proposed immunosensor was quite well. To further investigate the selectivity of the proposed immunoassay method for the wanted antigen–antibody interaction, the interference studies were performed using various concentrations of interfering agent such as glucose, α-fetoprotein antigen (AFP),
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Fig. 6. Calibration curve of the immunosensor under (A) SWV and (B) CA toward different concentrations of IgG. The insets correspondingly show the SWV response and CA response of different analyte concentrations.
carcinoma antigen 125 (CA 125) and carcinoembryonic antigen (CEA). No remarkable difference of currents was observed in comparison with the result obtained in the presence of only IgG. Both under SWV and CA, the current variation due to the interfering substances was less than 5.0% of that without interferences, indicating that the selectivity of the immunosensor was acceptable. The stability of immunosensors is also a key factor in their application and development. The stability of the immunosensor was examined by checking periodically its current response. When the immunosensor was prepared and not in use, it was stored in a refrigerator at 4 1C. The current response of the as-prepared immunosensor decreased 4.7% under SWV and 3.5% under CA after 7-day storage. Two weeks later, the current response of the immunosensor decreased to about 83% under SWV and 87% under CA of its initial value. The slow decrease in the current response may be due to the gradual denaturation of biomolecules.
labels for fabrication of immunosensor has many desirable merits including sensitivity, accuracy, and detection multiformity.
Acknowledgment This study was supported by the Natural Science Foundation of China (Nos. 21175057, 21375047, and 21377046), the Science and Technology Plan Project of Jinan (No. 201307010) and Qin Wei thanks the Special Foundation for Taishan Scholar Professorship of Shandong Province and University of Jinan (No. ts20130937).
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.03.060. Reference
3.6. Analysis of spiking serum samples In order to investigate the possibility of the immunosensor to be applied for practical analysis, the detection of IgG in human serum samples was performed using the proposed immunosensor with standard addition methods. 0.1 ng/mL, 0.5 ng/mL and 1 ng/mL of IgG solution were added into serum samples. The recovery of detection IgG under SWV was from 98.6% to 104% and the RSD was in the range of 4.98–5.59%. The recovery of detection IgG under CA was from 90% to 102% and the RSD was in the range of 4.39–6.82% (Supplementary information SV, Table S2). The facts showed that the developed immunoassay could be applied to the clinical determination of the IgG levels in serum samples.
4. Conclusion In this investigation, a novel versatile immunosensor based on Cu@TiO2 nanoparticles which incubated with IgG Ab2 has been introduced for electrochemical detection of IgG in a sandwich-type immunoassay format. The electrochemical signal is generated by redox reaction of copper ions using SWV technology, and also produced by catalytic reaction towards H2O2 using CA technology. The highlight of the developed immunosensor is the multiformity of applicable analytical method. It can be applied in enzyme-free and noble metal-free detection of analytes. In summary, Cu@TiO2–Ab2 as
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Copper-doped titanium dioxide nanoparticles as dual-functional labels for fabrication of electrochemical immunosensors Sen Zhang a,b, Hongmin Ma a, Liangguo Yan b, Wei Cao a, Tao Yan a, Qin Wei a,n, Bin Du a a Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, PR China b School of Resources and Environment, University of Jinan, Jinan 250022, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 2 January 2014 Received in revised form 19 March 2014 Accepted 27 March 2014 Available online 5 April 2014
Constructions of versatile electroactive labels are key issues in the development of electrochemical immunosensors. In this study, copper-doped titanium dioxide nanoparticle (Cu@TiO2) was synthesized and used as labels for fabrication of sandwich-type electrochemical immunosensors on glassy carbon electrode (GCE). Due to the presence of copper ions, Cu@TiO2 shows a strong response current when coupled to an electrode. The prepared nanocomposite also shows high electrocatalytic activity towards reduction of hydrogen peroxide (H2O2). The dual functionality of Cu@TiO2 enables the fabrication of immunosensor using different detection modes, that is, square wave voltammetry (SWV) or chronoamperometry (CA). While Cu@TiO2 was used as labels of secondary antibodies (Ab2), carboxyl functionalized graphene oxide (CFGO) was used as electrode materials to immobilize primary antibodies (Ab1). Using human immunoglobulin G (IgG) as a model analyte, the immunosensor shows high sensitivity, acceptable stability and good reproducibility for both detection modes. Under optimal conditions, a linear range from 0.1 pg/mL to 100 ng/mL with a detection limit of 0.052 pg/mL was obtained for SWV analysis. For CA analysis, a wider linear range from 0.01 pg/mL to 100 ng/mL and a lower detection limit of 0.0043 pg/mL were obtained. The proposed metal ion-based enzyme-free and noble metal-free immunosensor may have promising applications in clinical diagnoses and many other fields. & 2014 Elsevier B.V. All rights reserved.
Keywords: Metal ions-based immunosensor Human immunoglobulin G Square wave voltammetry Chronoamperometry
1. Introduction Immunoassay has been considered as a significant analytical tool in clinical diagnoses, environmental monitoring, pharmaceutical chemistry and biochemical studies (Tang et al., 2007; Wang et al., 2004; Cui et al., 2007). Various immunoassay strategies, such as chemiluminescence, quartz crystal microbalance, surface plasmon resonance and electrochemical method, have been extensively developed for detection of biomarkers (Xu et al., 2011; Nakanishi et al., 1996; Oh et al., 2004; Tang et al., 2009). Among these techniques, electrochemical immunosensors with high sensitivity, short analytical time, low cost and simple pretreatment procedure have been a preferable approach for clinical immunoassays (Tang et al., 2005; Marquette and Blum, 2006; Jie et al., 2007). As antibodies and antigens are intrinsically unable to act as redox partners, a label is often conjugated to either the antibody or antigen to generate electrochemical signal in fabrication of an immunosensor. Based on the specific antigen–antibody
n
Corresponding author. Tel.: þ 86 531 827 678 72; fax: þ 86 531 827 659 69. E-mail address: [email protected] (Q. Wei).
http://dx.doi.org/10.1016/j.bios.2014.03.060 0956-5663/& 2014 Elsevier B.V. All rights reserved.
interaction, the signal is then quantitatively related to the content of the antibody or antigen in an analyzing sample (Liu et al., 2010). Due to the unique redox property in low potentials, metal ions have been used as labels to fabricate electrochemical immunosensors. Metal ions are usually loaded on some carriers, such as magnetic poly (styrene-acrylic acid) nanospheres (Zhang et al., 2012). Incorporating two or three metal ions with well-separated signals into one system, multiplex electrochemical immunosensors can be developed for simultaneous determination of several analytes (Feng et al., 2012; Zhang et al., 2014). The metal ionsbased immunosensors often apply SWV as detection mode (Wang et al., 1998), while CA is usually used for the immunosensors fabricated with catalytic labels (Guo et al., 2013; Wei et al., 2011; Tang et al., 2008). The metal ions-based immunosensors applying CA as detection mode is scarce. As a result, metal ions-based dual-functional electroactive labels that enable the fabrication of immunosensor using different detection modes were seldom reported. Marker proteins are of tremendous importance in diagnosing the early stage of a disease or pathological condition. As a common protein, IgG is usually chosen to be a model analyte. A variety of detection assays for IgG have been developed such as fluorescence
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(Ma et al., 2005; Tan et al., 2014; Luo et al., 2014), surface plasmon resonance (Deng et al., 2010; Fernández et al., 2012), chemiluminescence and colorimetric sensors (Li et al., 2005; Liu et al., 2009; Shi et al., 2011; Lai et al., 2012). Electrochemical detection of IgG was mainly based on enzyme-labeled immunoassay (Darain et al., 2003; Cui and Zhu, 2010; Yang et al., 2011; Liu et al., 2012; Cao et al., 2013). Whereas, the stringent condition of using enzyme was a limiting factor. In order to improve stability and sensitivity, some other enzyme-free immunosensors were developed employing noble metals as labels (Dequaire et al., 2000; Chu et al., 2005; Leng et al., 2010; Noh et al., 2011; Lai et al., 2013; Ding et al., 2013). However, the exorbitant price of noble metal is a shortcoming in the fabrication of immunosensor. Therefore, developing an enzyme-free and noble metal-free immunosensor is attractive and challenging. Titanium dioxide (TiO2) has widespread applications. It has attracted a lot of attention due to its potentials as photocatalyst (Yu et al., 2002), sensor material (Bao et al., 2008) and electrode of photovoltaic cell (Mor et al., 2005). TiO2 has also been used in clinical orthopedics and dentistry owing to its excellent biocompatibility (Lin et al., 2013a). Recently, TiO2 has been used as labels to incubate with antibody for the fabrication of immunosensor. Metal ions-doped titanium dioxides combine the advantages of TiO2 and the unique property of metal ions. In this work, copper ions were introduced onto the surface of TiO2 nanoparticles via an assembly process. The nanocomposite (Cu@TiO2) can generate strong electrochemical signal under SWV. Most notably, it shows prominent electrocatalytic activity towards the reduction of hydrogen peroxide (H2O2). Hence, Cu@TiO2 was used as dualfunctional labels to construct immunosensors. The chemical stability of Cu@TiO2 brings superiority over enzyme, which may require rigorous operating conditions. It also does not need the participation of noble metal. Carboxyl functionalized graphene oxide (CFGO) was also used for fabrication of immunosensors. The carboxylic groups at the edge of GO surface make it easier to immobilize primary antibody (Ab1). Using immunoglobulin G (IgG) as a model analyte, the performance of the immunosensor was investigated in both SWV and CA modes. High sensitivity, acceptable stability, and good reproducibility were achieved for both detection modes.
2. Experimental method 2.1. Materials Human IgG, IgG primary antibodies (Ab1) and IgG secondary antibodies (Ab2) were purchased from Beijing Dingguochangsheng Biotechnology Co., Ltd. (Beijing, China). Graphite, tetrabutoxytitanium, sodium hydroxide (NaOH), hydrochloric acid (HCl), copper hydroxide (Cu(OH)2), and other chemicals were obtained from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Phosphate buffer solution (PBS) (0.1 M, pH 4.5, pH 5.0, pH 7.4) was prepared by mixing stock solutions of KH2PO4 and Na2HPO4. Ultrapure water was used throughout the experiment. All the chemicals were of analytical reagent grade. All electrochemical behaviors were performed by a traditional three-electrode system. It contained a glassy carbon electrode (GCE, 4 mm diameter) as working electrode, a platinum wire electrode as auxiliary electrode and a calomel electrode as reference electrode. All electrochemical measurements were carried out using a CHI 760D electrochemical workstation (Shanghai Chenhua Instrument Co., Ltd., China). Transmission electron microscope (TEM) images were obtained from an H-800 microscope (Hitachi, Japan). Fourier transform infrared spectroscopy (FTIR) spectrum was obtained from VERTEX 70 (Germany). X-ray
powder diffraction (XRD) was gathered from a Bruker D8 Focus diffractometer (Germany) using CuKα radiation (40 kV, 30 mA) of wavelength 0.154 nm.
2.2. Preparation of CFGO Graphene oxide (GO) was synthesized through the modified Hummer's method (Hummers and Offeman, 1958). Carboxylation of GO was prepared according to the previous reports (Sun et al., 2012, 2008; Park and Jung, 2012; ). The details are shown in Supplementary information SI.
2.3. Preparation of Cu@TiO2 TiO2 was prepared according to the literature (Zhong et al., 2008; Jiang et al., 2003). Cu@TiO2 was prepared according to the literature (Li et al., 2008). The details were shown in Supplementary information SII.
2.4. Preparation of labels The labels were prepared as follows: Cu@TiO2 (1.0 mg) were mixed with 0.5 mL of 2.5% glutaraldehyde solution and stirred for 1 h. The products were then centrifuged and dispersed in 0.5 mL of pH 7.4 PBS. 5.0 μg of IgG Ab2 were added into Cu@TiO2 and shaken for 12 h. After centrifugation, the obtained bioconjugates (Cu@TiO2–Ab2) were further washed with PBS at least three times and resuspended in 1.0 mL PBS as the assay solution.
2.5. Modification of immunosensor GCE was polished carefully with alumina powder of 1.0, 0.3 and 0.05 μm respectively, to a mirror-like surface, then cleaned through sonication in ethanol for half a minute and dried in air. 5.0 μL of CFGO was added onto the GCE. After drying, the electrode was activated with 75 mmol/L EDC-15 mmol/L NHS for 1 h (Qian et al., 2002). By the covalent bonding of the carboxyl groups of CFGO and amino groups on the IgG Ab1, 5.0 μL 10 μg/mL of IgG Ab1 was immobilized on the electrode tightly (Lin et al., 2013b). Subsequently, incubation with 1% wt bovine serum albumin (BSA) solution for 1 h was used to eliminate nonspecific binding between the antigen and the electrode surface. Afterwards, IgG buffer solution with a varying concentration was added onto the electrode surface and incubated for 1 h, and the electrode was washed extensively to remove unbound IgG molecules. Finally, the prepared Cu@TiO2–Ab2 was dropped onto the electrode surface. After another 1 h, the surface was washed once more and the prepared immunosensor was stored at 4 1C. A schematic diagram of the stepwise procedure of the immunosensor is shown in Fig. 1.
2.6. Measurement procedure The PBS buffer solution (pH 4.5, pH 5.0) was used for all the electrochemical measurements. For SWV measurement of the immunosensor, it was scanned from 0.4 to 0.4 V with a potential step of 5 mV, a frequency of 25 Hz, and an amplitude of 25 mV. For amperometric measurement of the immunosensor, a detection potential of 0.4 V was selected. After the background current was stabilized, 5.0 mmol/L H2O2 was added to the buffer and the current change was recorded.
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Fig. 1. Schematic representation of the preparation of immunosensor.
Fig. 2. TEM (A) and FTIR (B) of CFGO, TEM of TiO2 (C) and Cu@TiO2 (D), XRD of Cu@TiO2 (E) and UV–visible spectroscopy (F) of (a) Cu@TiO2; (b) Ab2; and (c) Cu@TiO2–Ab2.
3. Results and discussion 3.1. Characterization of CFGO and Cu@TiO2 As shown in Fig. 2A, sheet-like shapes of CFGO endow large surface area to immobilize Ab1 (Wang et al., 2011). Specific
absorption peaks of hydroxyl (3425 cm 1), carbonyl (1721 cm 1) and epoxy (1052 cm 1) groups appeared on the FTIR spectrum of CFGO (Fig. 2B) indicating that different kinds of oxygencontaining functional groups existed on the CFGO surface (Sun et al., 2012). Peak bands at 1721 and 1390 cm 1 are attributed to carboxy asymmetric and symmetric telescopic vibrations of CFGO
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(Wang et al., 2012b). The result was inconsistent with that of the existing groups of CFGO nanosheet synthesized with the chemical oxidation (Stankovich et al., 2006). The as-prepared TiO2 nanospheres possess uniform size with an average diameter of 250 nm (Fig. 2C). It can be inferred that the rough surface of the TiO2 nanospheres present great opportunity for the loading of copper ions. Fig. 2D shows the TEM image of Cu@TiO2 powders. The nanosphere structure of TiO2 was broken under hightemperature calcination. Thus irregular shape of Cu@TiO2 was observed. Fig. 2E shows the XRD patterns of Cu@TiO2 powders after being treated at 450 1C. All the diffraction peaks matched well with those from the Jade PDF card (84-1286) for TiO2. The peaks at 2θ ¼35.41 and 38.71 were assigned to CuO (002) and CuO (111), respectively (Choi and Kang, 2007). These results indicate that the copper ion components exist on the external surface of TiO2. The Cu@TiO2 was also characterized with energy dispersive spectroscopy (EDS) (Supplementary information SIII, Fig. S1). The presence of copper ions on TiO2 was further confirmed. The Cu@TiO2–Ab2 bioconjugates were characterized by UV– visible spectroscopy (Fig. 2F). All samples were dispersed in ultrapure water and mixed thoroughly. After laying aside for 2 min at room temperature, the UV–visible spectroscopy was monitored from 220 nm to 700 nm. The strong absorption in the 240–350 nm region was assigned to the band–band transition of TiO2 (curve a) (Jacob et al., 2003). For pure Ab2 solution, there was only one major peak at approximately 280 nm (curve b). When Ab2 was adsorbed onto Cu@TiO2, the peak of Ab2 was conspicuous and the uptrend of peak in the 240–350 nm region was observed (curve c).
3.2. SWV and CA characteristics of expectant labels To demonstrate whether Cu@TiO2 could generate electrical signal and promote the reduction of H2O2, the following tests were employed. Fig. 3A displays the SWV response of 2.0 mg/mL Cu@TiO2 (curve a) and TiO2 (curve b) in PBS (pH 4.5). A cuspidal peak at potential 0 V is obviously observed, which is ascribed to the redox reaction of copper ions. TiO2 exhibits weak catalytic performance towards H2O2 while Cu@TiO2 has remarkable response (Fig. 3B). The great catalytic performance of Cu@TiO2 towards H2O2 could enhance sensitivity of the immunosensor. Therefore, Cu@TiO2 can be employed as expectant labels for the versatile immunosensor.
3.3. Optimization of experimental conditions All the steps involved in the immunoassay were optimized. We investigated the influence of pH and the concentrations of CFGO on the performance of the immunosensor under SWV and CA. Fig. 4A shows that the best SWV current response of the immunosensor was obtained at pH 4.5 and the best CA current response of the immunosensor was obtained at pH 5.0. Thus, pH 4.5 and pH 5.0 of the PBS buffer were selected as one of the optimized conditions for SWV and CA, respectively. Electrical signal increases with the increase of CFGO concentration from 0.5 to 2.0 mg/mL and reaches the maximum at 2 mg/mL, but decreases when the concentration further increases. The increase of CFGO film thickness may lead to an increase of interface electron transfer resistance, and the electron transfer becomes more difficult. Therefore, 2 mg/mL of CFGO is chosen for all subsequent experiments. 3.4. Electrochemical impedance spectroscopy (EIS) characterization of immunosensor EIS is one of the most powerful tools used to investigate the interface properties of surface-modified electrodes. It is well known that the high frequency region of the impedance plot shows a semicircle related to the redox probe FeðCNÞ6 3 =4 , followed by a Warburg line in the low frequency region which corresponds to the diffusion step of the over all process (Panagopoulou et al., 2010). Therefore EIS was carried out to illustrate that all the steps of fabricating the immunosensor were effective. Curve a in Fig. 5 shows EIS of the bare GCE. It possesses a very small semicircle domain implying a very low electron transfer resistance in the electrolyte solution. Curve b displayed larger interfacial electron transfer resistance than curve a, and indicated that the CFGO hindered electron transfer of the electrode. This was due to the covalent oxygenated functional groups in CFGO causing some loss in electrical conductivity (Mkhoyan et al., 2009; Chen et al., 2012). After assembly of Ab1, an increased semicircle at high frequency region was observed (curve c). The increase of Ret was caused by the nonconductive properties of antibody, which obstructed the electron transfer of the redox probe from solution to electrode surface. The immobilization of BSA and IgG at the Ab1/CFGO/GCE resulted in a dramatically increased diameter of the semicircle (curves d and e), indicating a higher electron transfer resistance at the electrode interface, due to a decrease in the
Fig. 3. (A) SWV of 2 mg/mL TiO2 (a) and Cu@TiO2 (b) in pH 4.5 PBS; (B) CA of 2 mg/mL TiO2 (a) and Cu@TiO2 (b) toward addition of 5.0 mmol/L H2O2 in pH 5.0 PBS. Inset: enlargement of TiO2 catalyzing H2O2.
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Fig. 4. Effect of (A) pH,and (B) the concentration of CFGO on the response of the immunosensor to 10 ng/mL IgG.
equation (Bard and Faulkner, 1980): E ¼ EΘ þ
Fig. 5. EIS of (a) GCE, (b) CFGO/GCE, (c) Ab1/CFGO/GCE, (d) BSA/Ab1/CFGO/GCE, (e) IgG/BSA/Ab1/CFGO/GCE and (f) Cu@TiO2–Ab2/IgG/BSA/Ab1/CFGO/GCE in 0.1 mol/L KCl solution containing 5.0 mmol/L FeðCNÞ6 3 =FeðCNÞ6 4 (1:1).
heterogeneous rate constant of FeðCNÞ6 4 =FeðCNÞ6 3 . After the formation of the immunocomplex between the Cu@TiO2–Ab2 and IgG/BSA/Ab1/CFGO/GCE, the electron transfer resistance increased again (curve f). This increase is attributed to the non-conducting properties of the protein, which presumably obstructs electron transfer at the electrochemical probe. The above results clearly confirm that the immunosensor had been fabricated successfully (Feng et al., 2013).
3.5. SWV and CA detection of IgG at immunosensor SWV was used to evaluate the performance of the immunosensor (Fig. 6A). The peak currents of SWV were enhanced with increasing concentration of analytes under optimized conditions. The currents changed linearly with the concentrations of IgG in the range from 0.1 pg/mL to 100 ng/mL with a detection limit of 0.052 pg/mL. The linear regression equations was i (μA)¼9.870þ2.165 log c (ng/mL) with a statistically significant correlation coefficient of 0.992. Fig. 6A inset shows the SWV response of 0 pg/mL, 0.1 pg/mL, 1 pg/mL, 0.01 ng/mL, 0.05 ng/mL, 0.1 ng/mL, 0.5 ng/mL, 1 ng/mL, 5 ng/mL, 10 ng/mL, 50 ng/mL, and 100 ng/mL (a–l, in order) of analyte concentrations. A shifting of the peaks was observed from the inset. The interesting phenomenon could be explained through the Nernst
αvo RT ln vOR nF αR
where the v's are stoichiometric coefficients, αi is the activity of species i, O and R are oxidized and reduced forms, respectively. This relation, the Nernst equation, furnishes the potential of the O/R electrode as a function of the activities of О and R. Therefore, the more labels containing more copper ions showed positive potential versus low concentration of analytes. CA was also used to evaluate the performance of the immunosensor on account of the catalyze reduction of Cu@TiO2 towards H2O2. Under the optimum conditions, the immunosensors using Cu@TiO2–Ab2 as labels were used to detect different concentrations of IgG in pH 5.0 PBS at 0.4 V. The relationship between the current response toward 5.0 mmol/L H2O2 and IgG concentration is shown in Fig. 6B. As can be seen, the catalytic current change was linear with the concentration of IgG in the range from 0.01 pg/mL to 100 ng/mL with a detection limit of 0.0043 pg/mL. Fig. 6B inset correspondingly shows the CA response of 0.01 pg/mL, 0.1 pg/mL, 1 pg/mL, 0.01 ng/mL, 0.1 ng/mL, 1 ng/mL, 10 ng/mL, and 100 ng/mL (a–h, in order) of analyte concentrations. Table S1 (Supplementary information SIV) illustrates that the immunosensor described here has a lower detection limit than previously described immunosensors (Cui and Zhu, 2010; Leng et al., 2010; Liu et al., 2012; Yang et al., 2011; Lai et al., 2013; Cao et al., 2013; Qian et al., 2010; Wang et al., 2012a; Yin et al., 2010; Ding et al., 2013; Noh et al., 2011). The low detection limit may be attributed to two factors. On one hand, the Cu@TiO2 nanoparticle has good biocompatibility and high electrocatalytic activity toward H2O2. On the other hand, a relatively large amount of Ab2 has been conjugated onto the Cu@TiO2 nanoparticle-based labels, which can greatly increase the probability of Ab2–antigen interactions thereby leading to higher sensitivity. Moreover, the reproducibility, selectivity, and stability of the immunosensor were also investigated. To evaluate the reproducibility of the immunosensor, a series of five electrodes were prepared for detection of 1 ng/mL IgG. The relative standard deviations (RSD) of the measurements for the five electrodes were 3.2% under SWV and 4.7% under CA. The results indicated that the precision and reproducibility of the proposed immunosensor was quite well. To further investigate the selectivity of the proposed immunoassay method for the wanted antigen–antibody interaction, the interference studies were performed using various concentrations of interfering agent such as glucose, α-fetoprotein antigen (AFP),
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Fig. 6. Calibration curve of the immunosensor under (A) SWV and (B) CA toward different concentrations of IgG. The insets correspondingly show the SWV response and CA response of different analyte concentrations.
carcinoma antigen 125 (CA 125) and carcinoembryonic antigen (CEA). No remarkable difference of currents was observed in comparison with the result obtained in the presence of only IgG. Both under SWV and CA, the current variation due to the interfering substances was less than 5.0% of that without interferences, indicating that the selectivity of the immunosensor was acceptable. The stability of immunosensors is also a key factor in their application and development. The stability of the immunosensor was examined by checking periodically its current response. When the immunosensor was prepared and not in use, it was stored in a refrigerator at 4 1C. The current response of the as-prepared immunosensor decreased 4.7% under SWV and 3.5% under CA after 7-day storage. Two weeks later, the current response of the immunosensor decreased to about 83% under SWV and 87% under CA of its initial value. The slow decrease in the current response may be due to the gradual denaturation of biomolecules.
labels for fabrication of immunosensor has many desirable merits including sensitivity, accuracy, and detection multiformity.
Acknowledgment This study was supported by the Natural Science Foundation of China (Nos. 21175057, 21375047, and 21377046), the Science and Technology Plan Project of Jinan (No. 201307010) and Qin Wei thanks the Special Foundation for Taishan Scholar Professorship of Shandong Province and University of Jinan (No. ts20130937).
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.03.060. Reference
3.6. Analysis of spiking serum samples In order to investigate the possibility of the immunosensor to be applied for practical analysis, the detection of IgG in human serum samples was performed using the proposed immunosensor with standard addition methods. 0.1 ng/mL, 0.5 ng/mL and 1 ng/mL of IgG solution were added into serum samples. The recovery of detection IgG under SWV was from 98.6% to 104% and the RSD was in the range of 4.98–5.59%. The recovery of detection IgG under CA was from 90% to 102% and the RSD was in the range of 4.39–6.82% (Supplementary information SV, Table S2). The facts showed that the developed immunoassay could be applied to the clinical determination of the IgG levels in serum samples.
4. Conclusion In this investigation, a novel versatile immunosensor based on Cu@TiO2 nanoparticles which incubated with IgG Ab2 has been introduced for electrochemical detection of IgG in a sandwich-type immunoassay format. The electrochemical signal is generated by redox reaction of copper ions using SWV technology, and also produced by catalytic reaction towards H2O2 using CA technology. The highlight of the developed immunosensor is the multiformity of applicable analytical method. It can be applied in enzyme-free and noble metal-free detection of analytes. In summary, Cu@TiO2–Ab2 as
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