Biosensors and Bioelectronics 66 (2015) 468–473
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A one-step electrochemiluminescence immunosensor preparation for ultrasensitive detection of carbohydrate antigen 19-9 based on multifunctionalized graphene oxide Yuhong Sha a, Zhiyong Guo a,n, Beibei Chen a, Sui Wang a, Guoping Ge a, Bin Qiu b, Xiaohua Jiang c a Faculty of Materials Science and Chemical Engineering, The State Key Laboratory Base of Novel Functional Materials and Preparation Science, Ningbo University, Ningbo 315211, PR China b Ministry of Education Key Laboratory of Analysis and Detection for Food Safety (Fuzhou University), Fujian Provincial Key Laboratory of Analysis and Detection for Food Safety, Department of Chemistry, Fuzhou University, Fuzhou 350002, PR China c School of Applied Chemistry and Biological Technology, Shenzhen Polytechnic, Shenzhen 518055, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 7 October 2014 Received in revised form 1 December 2014 Accepted 2 December 2014 Available online 3 December 2014
A one-step electrochemiluminescence (ECL) immunosensor for ultrasensitive detection of carbohydrate antigen 19-9 (CA19-9) was developed based on multi-functionalized graphene oxide (GO), which was prepared with N-(4-aminobutyl)-N-ethylisoluminol (ABEI) and CA19-9 antibody (anti-CA19-9) chemically bound to the surface of magnetic GO (nanoFe3O4@GO). ABEI and anti-CA19-9 acted as the electrochemiluminophore and the capture device for CA19-9 respectively. NanoFe3O4@GO enabled all the ABEI immobilized molecules electrochemically active due to its good conductivity, and brought multifunctionalized GO attracted on the surface of magnetic glass carbon electrode through magnetism. Thus the ECL immunosensor could be prepared through a one-step process that facilitates ultrasensitive detection of CA19-9. Under optimal conditions, the ECL intensity of the immunosensor decreased proportionally to the logarithmic concentrations of CA19-9 in the range of 0.001–5 U/mL with a detection limit of 0.0005 U/mL. This one-step ECL immunosensor showed good performance in specificity, stability, reproducibility, regeneration and application. It opened a new avenue to apply multi-functionalized bionanomaterials in ECL immunoassay. & 2014 Elsevier B.V. All rights reserved.
Keywords: Electrochemiluminescence immunosensor Carbohydrate antigen 19-9 Multi-functionalized graphene oxide N-(4-aminobutyl)-N-ethylisoluminol One-step preparation
1. Introduction Carbohydrate antigen 19-9 (CA19-9), a Lewis antigen of the cell surface associated mucin 1 (MUC1) protein with an average molecular weight of 1000 KDa, is one of the most important carbohydrate tumor markers (Gui et al., 2013). Concentration of CA19-9 in serum plays an important role in clinical diagnoses of pancreatic (Parikh et al., 2014), colorectal (Narita et al., 2014), gastric (Xiao et al., 2014), urothelial carcinomas (Jha et al., 2013), etc. The methods currently available for the determination of CA19-9 mainly include radioimmunoassay (Bekci et al., 2009; Ching and Rhodes, 1989; Cho and Kyung, 2014; Mu et al., 2003), electrochemical immunoassay (Tang et al., 2013; Wang et al., 2013, 2014; Zhang et al., 2014), chemiluminescence immunoassay (Jiang et al., 2004; Lin et al., 2004; Shi et al., 2014) and enzyme-linked immunoabsorbent assay (Grote et al., 2008; Heidari et al., 2014; Li n
Corresponding author. E-mail address: [email protected] (Z. Guo).
http://dx.doi.org/10.1016/j.bios.2014.12.013 0956-5663/& 2014 Elsevier B.V. All rights reserved.
et al., 2007; Li and Zhang, 2009). However, use of these methods still poses one or more of the following constraints: (1) experimental procedures are complex; (2) radioactive or toxic markers are needed, and (3) detection time is long. To eliminate the abovementioned constraints and readily apply to immunoassay uncompromisingly, lateral flow devices and specialized lab-chips (Gervais and Delamarche, 2009; Jönsson et al., 2008; Mohammed et al., 2014) were developed, allowing assays to be performed within minutes, consume microlitre volumes of sample and have the required detection sensitivities for clinically relevant biomarker targets. Additionally, some of these formats (Jönsson et al., 2008; Mohammed et al., 2014) could operate with portable and inexpensive ancillary instrumentation, reducing both cost and complexity of the assay in practical conditions. Even so, the detection sensitivity still needs some improvement, because the concentration of some biomarker targets in saliva and urine is not as high as that in serum, necessitating improvements in sensitivity, especially when less sample quantities are available. Therefore, a simple, sensitive and reliable method for detecting low levels of CA19-9 and other tumor markers is still promising and has
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potential applications in the clinical settings. Graphene oxide (GO) is a layered compound that can be synthesized by oxidation of natural graphite. Owing to the large surface area, good electrical conductivity and presence of oxygenated functional groups such as epoxide, hydroxyl, carboxyl groups, ketones and 6-membered lactol rings (Wu et al., 2013), GO can easily load magnetic iron oxide nanoparticles and form nanoFe3O4@GO, which has promising applications in a variety of fields such as biomedicine, magnetic energy storage, magnetic fluids, catalysis, and environmental remediation (Frey et al., 2009; Lu et al., 2009). Obviously, multi-functionalized GO materials, which are obtained from further functionalization of nanoFe3O4@GO with antibodies, enzymes, DNAs, aptamers and/or various labels and so on, have more potential applications, and are attracting more and more interests (Teymourian et al., 2013). However, only a few reports are available on the application of multi-functionalized GO for building immunosensors. Electrochemiluminescence (ECL) is an important and promising method in designing immunosensors because of high sensitivity, wide dynamic range, simplified optical setup, and very low background signal (Richter, 2004; Yang et al., 2010), in which N-(4aminobutyl)-N-ethylisoluminol (ABEI), a derivative of luminol, is one of the most widely used systems. It contains amino group which enables itself as a favorable immunoassay marker (Yang et al., 2002). Based on the advantages of ECL and specific recognition of immunoassay, ECL immunoassay provides a fast, sensitive and selective method for determining disease-related proteins with short assay time and simplified optical setup (Sun et al., 2012). Recently, Liu et al. (2014) developed an universal ECL immunoassay platform based on gold nanoparticle dotted reduced graphene oxide composite, which accelerated electron transfer between the detection probe and the electrode, and increase the surface area of the working electrode to load greater amounts of the antibodies. For the detection of CA19-9, Gan et al. (2013) reported an ECL immunosensor using graphene/CdTe quantum dot bionanoconjugates as signal amplifiers; however, the preparation and detection procedures were relatively complex and time-consuming. To our best knowledge, no paper has been published on immunoassay of CA19-9 using multi-functionalized GO. In recent years, immunosensors developed for the diagnostic assay of biomolecules have attracted considerable interests (Lu et al., 2009). To achieve satisfactory sensitivity and selectivity, the preparation and detection procedures of the immunosensor are becoming more and more complicated and tediously long. Therefore, development of rapid, simple, and preferably one-step immunosensors are gaining momentum (Dawan et al., 2013; Gao et al., 2011; Huang et al., 2011; Ivnitski and Rishpon, 1996; Kuang et al., 2013; Xu et al., 2014). However, up to now, one-step ECL immunosensor has been scarcely reported. In this work, a one-step ECL immunosensor for ultrasensitive detection of CA19-9 based on multi-functionalized GO, with ABEI and CA19-9 antibody chemically bound to the surface of nanoFe3O4@GO is reported. The immunosensor was easily built through only one step, and showed excellent detection performances including sensitivity, stability, specificity and simplicity. Thus, this work provides a promising approach for the detection of tumor markers in clinical applications and other biosensor applications.
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Ltd. (Shanghai, China). Bovine serum albumin (BSA), N-(4-aminobutyl)-N-ethylisoluminol (ABEI), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ferric chloride hexahydrate (FeCl3 6H2O), ferrous chloride tetrahydrate (FeCl2 4H2O), hydrogen peroxide (H2O2, 30 wt%) and ammonium hydroxide (NH4OH, 25 wt%) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All other reagents are of analytical grade or above and used without further purification. Carbonate buffer solution (CBS) was prepared using Na2CO3 10H2O and NaHCO3, and the pH of which was 9.90 unless otherwise stated. Ultrapure water obtained from Millipore water purification system (Z 18 MΩ, Milli-Q, Millipore, Billerica, MA, USA) was used throughout the experiment. 2.2. Apparatus A laboratory-built ECL detection system was used as described previously (Guo et al., 2013). A three-electrode system containing a bare or modified magnetic glass carbon electrode (3 mm diameter, Gaossunion, Wuhan, China) as working electrode, a platinum wire electrode as counter electrode and an Ag/AgCl (3 mol/L KCl) electrode as reference electrode, was used. Electrochemical impedance spectroscopy (EIS) analysis was carried out using a CHI 660E Electrochemistry Workstation (Chenhua Instrument Company, Shanghai, China), which used the same three-electrode system as that of the ECL detection. The transmission electron microscope (TEM, FEI, Hillsboro, Oregon, USA) was used to characterize the morphology of graphene oxide and magnetic graphene oxide. 2.3. Synthesis of magnetic graphene oxide nanoFe3O4@GO Graphene oxide (GO) was prepared according to Hummer’s method (Hummers and Offeman, 1958), and the magnetic graphene oxide, nanoFe3O4@GO, was prepared as described previously (Kassaee et al., 2011) with some modifications. In a typical procedure, 40 mg GO was dispersed in 40 mL water and ultrasonicated for 30 min in a round-bottom flask. Then, 50 mL iron source solution containing 800 mg FeCl3 6H2O and 300 mg FeCl2 4H2O was added into the flask with vigorous stirring at room temperature. Subsequently, the solution was heated to 85 °C, ammonium hydroxide (25 wt%) was added dropwise to increase the pH to 10, and rapidly stirred for 45 min. Then, the resulted black composite, magnetic graphene oxide nanoFe3O4@GO, was extracted using a magnet and washed several times with water. As shown in Fig. 1A, a black homogeneous dispersion existed without an external magnetic field, and the black composite was attracted to the wall of the vial in a short time when a magnet was present, indicating that nanoFe3O4@GO obtained remained magnetic enough to meet the need of immunosensor preparation. TEM images of GO and nanoFe3O4@GO, presented in Fig. 1B, show that Fe3O4 nanoparticles were not so evenly distributed over the GO sheets, because the relatively monodispersed and disaggregated Fe3O4 nanoparticles were especially difficult to obtain due to the inherent magnetism. 2.4. Preparation of multi-functionalized graphene oxide anti-CA19-9/ABEI–nanoFe3O4@GO composite
2. Experimental 2.1. Reagents and materials Carbohydrate antigen 19-9 (CA19-9) and CA19-9 antibody (anti-CA19-9) were purchased from Shanghai Linc-Bio Science Co.,
The preparation procedure of multi-functionalized graphene oxide anti-CA19-9/ABEI–nanoFe3O4@GO composite is shown in Fig. 2A. Firstly, 200 mL of 1 mg/mL nanoFe3O4@GO was added into 200 mL of mixture solution containing 100 mg/mL EDC and 10 mg/mL NHS. After adjusting the pH to 5, the solution obtained
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ultrasonically in anhydrous ethanol and water in succession, and allowed to dry using N2. Then, 5 mL of multi-functionalized graphene oxide anti-CA19-9/ABEI–nanoFe3O4@GO composite solution was dropped on the electrode. Once the composite was attracted on the surface of the magnetic glass carbon electrode in a few minutes, the ECL immunosensor was one-step prepared very simply and ready for the detection (Fig. 2B). 2.6. ECL detection As shown in Fig. 2B, the ECL immunosensor was incubated in 5 mL of CA19-9 samples for 1 h at 37 °C and then washed carefully with water to remove unbound CA19-9. When a chronoamperometry (30 s pulse period, 0.25 s pulse width, 0 V initial potential, 1 V pulse potential) was applied to the working electrode in the electrolyte solution (0.05 mol/L CBS at pH 9.90 containing 1 mmol/L H2O2), an ECL signal was generated and recorded with the voltage of the photomultiplier at 700 V.
3. Results and discussion Fig. 1. (A) The photographs of nanoFe3O4@GO separation from aqueous solution under an external magnetic field. (B) TEM image of GO and nanoFe3O4@GO.
was shaken for 1 h at ambient temperature to form a stable active easter layer on the surface of GO, and the resulted black precipitate was thoroughly washed with water 3 times using a magnet. Then, 100 mL mixture solution containing 13.8 mg/mL ABEI and 0.5 ng/mL anti-CA19-9 was added and shaken for another 4 h. Finally, the resulting black composite was obtained after adding 100 mL of 2 wt % BSA and reacting for 1 h to block non-specific binding sites. The multi-functionalized graphene oxide anti-CA19-9/ABEI–nanoFe3O4 @GO composite was obtained after washing and reconstructing in 200 mL of water. In this construct, anti-CA19-9 could recognize and capture target CA19-9 and ABEI was the electrochemiluminophore; and nanoFe3O4 along with these compounds made the one-step preparation of the ECL immunosensor possible. 2.5. One-step preparation of the ECL immunosensor The bare magnetic glass carbon electrode was polished successively with 1.0, 0.3 and 0.05 mm alumina slurry, washed
3.1. Characterization of the ECL immunosensor 3.1.1. ECL behavior As depicted in Fig. 3A, no ECL response was observed on bare magnetic glass carbon electrode (curve a) and nanoFe3O4@GO coated electrode (curve b). In contrast, with ABEI–nanoFe3O4@GO, a strong ECL signal was observed (curve c), indicating that ABEI, the electrochemiluminophore, was successfully immobilized on GO. In the case of anti-CA19-9/ABEI–nanoFe3O4@GO coated electrode, the ECL intensity decreased (curve d) because anti-CA19-9 and BSA immobilization hindered the ECL signal emitted from the electrode surface and blocked the electron transfer on the electrode interface to inhibit the ECL reaction. When the immunosensor was incubated in a sample containing 0.5 U/mL CA199 which was linked to the surface of the electrode through antigen–antibody reaction, a significant reduction in the ECL intensity was observed (curve e). It is because the high molecular weight of CA19-9 (as high as 41000 kDa) and its huge molecular volume effectively hindered the ECL signal and blocked the electron transfer.
Fig. 2. The schematic diagram for (A) the preparation of multi-functionalized graphene oxide, and (B) the fabrication and application protocol of the ECL immunosensor using multi-functionalized graphene oxide.
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Fig. 3. (A) ECL signals obtained with (a) bare magnetic glass carbon electrode, (b) (a)þnanoFe3O4@GO, (c) (a)þ ABEI–nanoFe3O4@GO, (d) (a)þ anti-CA19-9/ ABEI–nanoFe3O4@GO, and (e) (d) þ0.5 U/mL CA19-9. Experimental condition: chronoamperometry; initial potential, 0 V; pulse potential, 1 V; pulse width, 0.25 s; pulse period, 30 s; H2O2, 1 mmol/L; CBS, 0.05 mol/L, and pH 9.90. (B) EIS of (a) bare magnetic glass carbon electrode, (b) (a) þnanoFe3O4@GO, (c) (a)þ ABEI–nanoFe3O4 @GO, (d) (a)þ anti-CA19-9/ABEI–nanoFe3O4@GO, and (e) (d) þ 0.05 U/mL CA19-9. Experimental conditions of EIS: magnetic glass carbon electrode in 0.05 mol/L PBS (5 mmol/L [Fe(CN)6]3 /4 , pH 7.4). The frequency range from 0.01 to 100,000 Hz with a signal amplitude of 5 mV.
3.1.2. EIS behavior EIS was used to monitor the process of electrode modification in 0.01 mol/L phosphate buffer solution containing 5 mmol/L Fe(CN)63 /4 . The curve of EIS shows a line at low AC modulation frequency and a semicircle at high AC modulation frequency, while the electron transfer resistance Ret corresponds to the latter. As shown in Fig. 3B, due to the good electrical conductivity of GO and nanoFe3O4, the diameter of semicircle decreased significantly with nanoFe3O4@GO assembly (curve b) when compared with the bare magnetic glass carbon electrode (curve a). When ABEI molecules were immobilized, Ret increased slightly (curve c). However, when the immunosensor was constructed, Ret increased remarkably (curve d) because anti-CA19-9 and BSA were subsequently conjugated and increased the difficulty in electron transfer. Finally, in presence of CA19-9 at the concentration of 0.05 U/mL, Ret increased significantly again (curve e) because the immobilization of antigen generated an insulating protein layer on the assembled surface and hindered the interfacial electron transfer further. 3.2. Optimization of experimental conditions To establish the optimal conditions, pH value of electrolyte solution, concentration of H2O2 in electrolyte solution, incubation temperature and incubation time were investigated by detecting CA19-9 solution at the concentration of 0.05 U/mL (Fig. S1). Based on an earlier work, 9.90 and 1 mmol/L were selected as the optimal pH of electrolyte solution and concentration of H2O2 in electrolyte solution (Du et al., 2014). Optimum incubation temperature and incubation time were observed to be 37 °C and 1 h, respectively.
Fig. 4. (A) ECL signals of the immunosensor in the presence of 0.5 U/mL CA19-9 in pH 9.90 CBS containing 1 mmol/L H2O2 under continuous chronoamperometry for 18 cycles. (B) ECL intensity from the immunosensor for the detection of CA19-9 at different concentrations (0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1 and 5 U/mL); and (C) The calibration curve obtained through plotting ECL intensity against the logarithmic concentrations of CA19-9 (U/mL).
3.3. Detection of CA19-9 using the ECL immunosensor As presented in Fig. 4A, the ECL signals of the immunosensor in the presence of 0.5 U/mL CA19-9, under consecutive pulse potential scans from 0 to 1 V for 18 cycles, were high and stable, suggesting that the immunosensor was suitable for ECL detection. As indicated in Fig. 4B, under the optimal conditions, the ECL intensity of the immunosensor (y) decreased linearly with the logarithmic concentrations of CA19-9 (x) over the range of 0.001– 5 U/mL, and the regression equation was y ¼1735.8–2602.8 log x (U/mL) with a correlation coefficient r of 0.9953 (Fig. 4C). According to the method previously reported for indirect detection involving signal decrease or quenching (Chen et al., 2014), the detection limit for CA19-9 was roughly estimated to be 0.0005 U/mL, which was around 30-fold lower than the detection limit of 0.016 U/mL reported previously (Shi et al., 2014). Compared with conventional methods, the sensitivity of this method is greatly improved due to following two reasons: (1) In conventional methods, at the most tens of labeling molecules could be labeled onto an antibody molecule to maintain the immunoactivity of antibody (Fung and Wong, 2001; Pei et al., 2013).
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Table 1 Recovery tests for CA19-9 in spiked human serum and urine samples (x¯ ± s , n¼ 3). Samples
Added (U/mL)
Found (U/mL)
RSD (%)
Recovery (%)
Serum 1 Serum 2 Serum 3 Urine 1 Urine 2 Urine 3
0.001 0.1 1 0.001 0.1 1
0.001137 0.000105 0.09757 0.0047 1.0157 0.056 0.00098 7 0.000103 0.10357 0.0056 0.995 7 0.058
9.3 4.8 5.5 10.3 5.4 5.8
113.0 97.5 101.5 98.0 103.5 99.5
In contrast to the conventional methods, much larger number of labeling molecules is involved in the proposed method, for example, thousands of electrochemiluminophore molecules were chemically bound to a GO sheet. (2) The nanoFe3O4@GO has a good electrical conductivity (Teymourian et al., 2013), thus it can significantly extend the outer Helmholtz plane (OHP) of the electrode. It means that all the ABEI molecules immobilized on GO were effective in the area between the OHP and the electrode. It can even be considered that all ABEI molecules were directly immobilized on the surface of the electrode. Therefore, all of them could effectively emit ECL signals. In contrast, in conventional methods, most of the ABEI molecules labeled were beyond OHP, and they could not emit ECL signals (Du et al., 2014). Therefore, it is understandable that the detection sensitivity was improved greatly. 3.4. Specificity, stability, reproducibility, and regeneration of the immunosensor In order to illustrate the possible interference of non-specific antigen, various species of interfering proteins including carcinoma antigen 50 (CA50), carcinoma antigen 125 (CA125), carcinoma antigen 15-3 (CA15-3) and carcinoma antigen 242 (CA242) at the concentration of 10 U/mL were used to further investigate the specificity of the proposed immunosensor. The ECL intensity of CA50, CA125, CA15-3 and CA242 were all about 11,000, nearly similar to that of the blank sample. In contrast, the value observed with 0.05 U/mL CA19-9 solution was 5000 and that of 0.001 U/mL was 9500, indicating that the CA50, CA125, CA15-3 and CA242 did not interfere. These results demonstrated that the immunosensor developed has an excellent specificity for the determination of CA19-9. The stability of the immunosensor proposed was investigated by checking its relative activity after storage at 4 °C in dark for one month. The ECL intensity for the detection of 0.05 U/mL CA19-9 was 93.5 74.7% (n ¼5) of the initial value, which was obtained when the immunosensor was constructed freshly. Results indicate that the developed immunosensor has acceptable stability upon storage. Reproducibility of the immunosensor is one of the most important problems in immunoassay. The reproducibility property of the immunosensor was evaluated by determining 0.05 U/mL CA199 with five equally prepared immunosensors. The relative standard deviation (RSD) of the measurements was 8.1%, indicating the excellent precision and reproducibility of the immunosensor. Regeneration of the immunosensor was examined by detecting 0.05 U/mL CA19-9 with a same immunosensor. To break the antibody–antigen linkage, the immunosensor was regenerated by dipping into 0.2 mol/L glycine–hydrochloric acid (Gly–HCl) buffer solution (pH¼2.8) for 8 min. An average recovery of 92.6% and an intra-assay RSD of 6.4% were obtained when the consecutive measurements were repeated ten times, suggesting that the immunosensor could be regenerated and used for at least ten times.
3.5. Application of the immunosensor in serum and urine samples The application and reliability of the proposed ECL immunosensor was investigated by determining spiked CA19-9 samples with different concentrations. The samples were prepared by adding standard CA19-9 solution to blank human serum and urine. As shown in Table 1, the results showed an acceptable recovery in the range of 97.5–113.0% and RSD in the range of 4.8– 10.5%, indicating that the proposed method is promising and has potential applications to the analysis of clinical samples.
4. Conclusions An effective ECL immunosensor was developed with a one-step preparation method and successfully used for the ultrasensitive determination of CA19-9 based on multi-functionalized graphene oxide anti-CA19-9/ABEI–nanoFe3O4@GO, which had four functions as follows: (1) ABEI acted as the electrochemiluminophore; (2) anti-CA19-9 acted as the capture device for CA19-9 through antigen–antibody immunoreactions; (3) nanoFe3O4@GO extended the OHP, enabled all ABEI molecules immobilized on GO effective in emitting ECL signals and thus improved the detection sensitivity; and (4) nanoFe3O4@GO provided the feasibility of one-step preparation. The ECL immunosensor developed afforded ultrasensitive detection of CA19-9 with satisfactory specificity, stability, reproducibility and regeneration. Therefore, this immunosensor is a promising tool for the detection of tumor marker in clinical applications and other biosensor applications.
Acknowledgements Financial supports from Natural Science Foundation of China (81273130), Zhejiang Provincial Natural Science Foundation of China (LY13B070013), Ningbo Science and Technology Bureau (2010A610164), Major Project of Fujian Province (2011N5008), and Special Funds for Strategic Emerging Industries of Shenzhen (2113K3070038) are gratefully acknowledged. This work was also sponsored by K.C. Wong Magna Fund in Ningbo University.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.12.013.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
A one-step electrochemiluminescence immunosensor preparation for ultrasensitive detection of carbohydrate antigen 19-9 based on multifunctionalized graphene oxide Yuhong Sha a, Zhiyong Guo a,n, Beibei Chen a, Sui Wang a, Guoping Ge a, Bin Qiu b, Xiaohua Jiang c a Faculty of Materials Science and Chemical Engineering, The State Key Laboratory Base of Novel Functional Materials and Preparation Science, Ningbo University, Ningbo 315211, PR China b Ministry of Education Key Laboratory of Analysis and Detection for Food Safety (Fuzhou University), Fujian Provincial Key Laboratory of Analysis and Detection for Food Safety, Department of Chemistry, Fuzhou University, Fuzhou 350002, PR China c School of Applied Chemistry and Biological Technology, Shenzhen Polytechnic, Shenzhen 518055, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 7 October 2014 Received in revised form 1 December 2014 Accepted 2 December 2014 Available online 3 December 2014
A one-step electrochemiluminescence (ECL) immunosensor for ultrasensitive detection of carbohydrate antigen 19-9 (CA19-9) was developed based on multi-functionalized graphene oxide (GO), which was prepared with N-(4-aminobutyl)-N-ethylisoluminol (ABEI) and CA19-9 antibody (anti-CA19-9) chemically bound to the surface of magnetic GO (nanoFe3O4@GO). ABEI and anti-CA19-9 acted as the electrochemiluminophore and the capture device for CA19-9 respectively. NanoFe3O4@GO enabled all the ABEI immobilized molecules electrochemically active due to its good conductivity, and brought multifunctionalized GO attracted on the surface of magnetic glass carbon electrode through magnetism. Thus the ECL immunosensor could be prepared through a one-step process that facilitates ultrasensitive detection of CA19-9. Under optimal conditions, the ECL intensity of the immunosensor decreased proportionally to the logarithmic concentrations of CA19-9 in the range of 0.001–5 U/mL with a detection limit of 0.0005 U/mL. This one-step ECL immunosensor showed good performance in specificity, stability, reproducibility, regeneration and application. It opened a new avenue to apply multi-functionalized bionanomaterials in ECL immunoassay. & 2014 Elsevier B.V. All rights reserved.
Keywords: Electrochemiluminescence immunosensor Carbohydrate antigen 19-9 Multi-functionalized graphene oxide N-(4-aminobutyl)-N-ethylisoluminol One-step preparation
1. Introduction Carbohydrate antigen 19-9 (CA19-9), a Lewis antigen of the cell surface associated mucin 1 (MUC1) protein with an average molecular weight of 1000 KDa, is one of the most important carbohydrate tumor markers (Gui et al., 2013). Concentration of CA19-9 in serum plays an important role in clinical diagnoses of pancreatic (Parikh et al., 2014), colorectal (Narita et al., 2014), gastric (Xiao et al., 2014), urothelial carcinomas (Jha et al., 2013), etc. The methods currently available for the determination of CA19-9 mainly include radioimmunoassay (Bekci et al., 2009; Ching and Rhodes, 1989; Cho and Kyung, 2014; Mu et al., 2003), electrochemical immunoassay (Tang et al., 2013; Wang et al., 2013, 2014; Zhang et al., 2014), chemiluminescence immunoassay (Jiang et al., 2004; Lin et al., 2004; Shi et al., 2014) and enzyme-linked immunoabsorbent assay (Grote et al., 2008; Heidari et al., 2014; Li n
Corresponding author. E-mail address: [email protected] (Z. Guo).
http://dx.doi.org/10.1016/j.bios.2014.12.013 0956-5663/& 2014 Elsevier B.V. All rights reserved.
et al., 2007; Li and Zhang, 2009). However, use of these methods still poses one or more of the following constraints: (1) experimental procedures are complex; (2) radioactive or toxic markers are needed, and (3) detection time is long. To eliminate the abovementioned constraints and readily apply to immunoassay uncompromisingly, lateral flow devices and specialized lab-chips (Gervais and Delamarche, 2009; Jönsson et al., 2008; Mohammed et al., 2014) were developed, allowing assays to be performed within minutes, consume microlitre volumes of sample and have the required detection sensitivities for clinically relevant biomarker targets. Additionally, some of these formats (Jönsson et al., 2008; Mohammed et al., 2014) could operate with portable and inexpensive ancillary instrumentation, reducing both cost and complexity of the assay in practical conditions. Even so, the detection sensitivity still needs some improvement, because the concentration of some biomarker targets in saliva and urine is not as high as that in serum, necessitating improvements in sensitivity, especially when less sample quantities are available. Therefore, a simple, sensitive and reliable method for detecting low levels of CA19-9 and other tumor markers is still promising and has
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potential applications in the clinical settings. Graphene oxide (GO) is a layered compound that can be synthesized by oxidation of natural graphite. Owing to the large surface area, good electrical conductivity and presence of oxygenated functional groups such as epoxide, hydroxyl, carboxyl groups, ketones and 6-membered lactol rings (Wu et al., 2013), GO can easily load magnetic iron oxide nanoparticles and form nanoFe3O4@GO, which has promising applications in a variety of fields such as biomedicine, magnetic energy storage, magnetic fluids, catalysis, and environmental remediation (Frey et al., 2009; Lu et al., 2009). Obviously, multi-functionalized GO materials, which are obtained from further functionalization of nanoFe3O4@GO with antibodies, enzymes, DNAs, aptamers and/or various labels and so on, have more potential applications, and are attracting more and more interests (Teymourian et al., 2013). However, only a few reports are available on the application of multi-functionalized GO for building immunosensors. Electrochemiluminescence (ECL) is an important and promising method in designing immunosensors because of high sensitivity, wide dynamic range, simplified optical setup, and very low background signal (Richter, 2004; Yang et al., 2010), in which N-(4aminobutyl)-N-ethylisoluminol (ABEI), a derivative of luminol, is one of the most widely used systems. It contains amino group which enables itself as a favorable immunoassay marker (Yang et al., 2002). Based on the advantages of ECL and specific recognition of immunoassay, ECL immunoassay provides a fast, sensitive and selective method for determining disease-related proteins with short assay time and simplified optical setup (Sun et al., 2012). Recently, Liu et al. (2014) developed an universal ECL immunoassay platform based on gold nanoparticle dotted reduced graphene oxide composite, which accelerated electron transfer between the detection probe and the electrode, and increase the surface area of the working electrode to load greater amounts of the antibodies. For the detection of CA19-9, Gan et al. (2013) reported an ECL immunosensor using graphene/CdTe quantum dot bionanoconjugates as signal amplifiers; however, the preparation and detection procedures were relatively complex and time-consuming. To our best knowledge, no paper has been published on immunoassay of CA19-9 using multi-functionalized GO. In recent years, immunosensors developed for the diagnostic assay of biomolecules have attracted considerable interests (Lu et al., 2009). To achieve satisfactory sensitivity and selectivity, the preparation and detection procedures of the immunosensor are becoming more and more complicated and tediously long. Therefore, development of rapid, simple, and preferably one-step immunosensors are gaining momentum (Dawan et al., 2013; Gao et al., 2011; Huang et al., 2011; Ivnitski and Rishpon, 1996; Kuang et al., 2013; Xu et al., 2014). However, up to now, one-step ECL immunosensor has been scarcely reported. In this work, a one-step ECL immunosensor for ultrasensitive detection of CA19-9 based on multi-functionalized GO, with ABEI and CA19-9 antibody chemically bound to the surface of nanoFe3O4@GO is reported. The immunosensor was easily built through only one step, and showed excellent detection performances including sensitivity, stability, specificity and simplicity. Thus, this work provides a promising approach for the detection of tumor markers in clinical applications and other biosensor applications.
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Ltd. (Shanghai, China). Bovine serum albumin (BSA), N-(4-aminobutyl)-N-ethylisoluminol (ABEI), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ferric chloride hexahydrate (FeCl3 6H2O), ferrous chloride tetrahydrate (FeCl2 4H2O), hydrogen peroxide (H2O2, 30 wt%) and ammonium hydroxide (NH4OH, 25 wt%) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All other reagents are of analytical grade or above and used without further purification. Carbonate buffer solution (CBS) was prepared using Na2CO3 10H2O and NaHCO3, and the pH of which was 9.90 unless otherwise stated. Ultrapure water obtained from Millipore water purification system (Z 18 MΩ, Milli-Q, Millipore, Billerica, MA, USA) was used throughout the experiment. 2.2. Apparatus A laboratory-built ECL detection system was used as described previously (Guo et al., 2013). A three-electrode system containing a bare or modified magnetic glass carbon electrode (3 mm diameter, Gaossunion, Wuhan, China) as working electrode, a platinum wire electrode as counter electrode and an Ag/AgCl (3 mol/L KCl) electrode as reference electrode, was used. Electrochemical impedance spectroscopy (EIS) analysis was carried out using a CHI 660E Electrochemistry Workstation (Chenhua Instrument Company, Shanghai, China), which used the same three-electrode system as that of the ECL detection. The transmission electron microscope (TEM, FEI, Hillsboro, Oregon, USA) was used to characterize the morphology of graphene oxide and magnetic graphene oxide. 2.3. Synthesis of magnetic graphene oxide nanoFe3O4@GO Graphene oxide (GO) was prepared according to Hummer’s method (Hummers and Offeman, 1958), and the magnetic graphene oxide, nanoFe3O4@GO, was prepared as described previously (Kassaee et al., 2011) with some modifications. In a typical procedure, 40 mg GO was dispersed in 40 mL water and ultrasonicated for 30 min in a round-bottom flask. Then, 50 mL iron source solution containing 800 mg FeCl3 6H2O and 300 mg FeCl2 4H2O was added into the flask with vigorous stirring at room temperature. Subsequently, the solution was heated to 85 °C, ammonium hydroxide (25 wt%) was added dropwise to increase the pH to 10, and rapidly stirred for 45 min. Then, the resulted black composite, magnetic graphene oxide nanoFe3O4@GO, was extracted using a magnet and washed several times with water. As shown in Fig. 1A, a black homogeneous dispersion existed without an external magnetic field, and the black composite was attracted to the wall of the vial in a short time when a magnet was present, indicating that nanoFe3O4@GO obtained remained magnetic enough to meet the need of immunosensor preparation. TEM images of GO and nanoFe3O4@GO, presented in Fig. 1B, show that Fe3O4 nanoparticles were not so evenly distributed over the GO sheets, because the relatively monodispersed and disaggregated Fe3O4 nanoparticles were especially difficult to obtain due to the inherent magnetism. 2.4. Preparation of multi-functionalized graphene oxide anti-CA19-9/ABEI–nanoFe3O4@GO composite
2. Experimental 2.1. Reagents and materials Carbohydrate antigen 19-9 (CA19-9) and CA19-9 antibody (anti-CA19-9) were purchased from Shanghai Linc-Bio Science Co.,
The preparation procedure of multi-functionalized graphene oxide anti-CA19-9/ABEI–nanoFe3O4@GO composite is shown in Fig. 2A. Firstly, 200 mL of 1 mg/mL nanoFe3O4@GO was added into 200 mL of mixture solution containing 100 mg/mL EDC and 10 mg/mL NHS. After adjusting the pH to 5, the solution obtained
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ultrasonically in anhydrous ethanol and water in succession, and allowed to dry using N2. Then, 5 mL of multi-functionalized graphene oxide anti-CA19-9/ABEI–nanoFe3O4@GO composite solution was dropped on the electrode. Once the composite was attracted on the surface of the magnetic glass carbon electrode in a few minutes, the ECL immunosensor was one-step prepared very simply and ready for the detection (Fig. 2B). 2.6. ECL detection As shown in Fig. 2B, the ECL immunosensor was incubated in 5 mL of CA19-9 samples for 1 h at 37 °C and then washed carefully with water to remove unbound CA19-9. When a chronoamperometry (30 s pulse period, 0.25 s pulse width, 0 V initial potential, 1 V pulse potential) was applied to the working electrode in the electrolyte solution (0.05 mol/L CBS at pH 9.90 containing 1 mmol/L H2O2), an ECL signal was generated and recorded with the voltage of the photomultiplier at 700 V.
3. Results and discussion Fig. 1. (A) The photographs of nanoFe3O4@GO separation from aqueous solution under an external magnetic field. (B) TEM image of GO and nanoFe3O4@GO.
was shaken for 1 h at ambient temperature to form a stable active easter layer on the surface of GO, and the resulted black precipitate was thoroughly washed with water 3 times using a magnet. Then, 100 mL mixture solution containing 13.8 mg/mL ABEI and 0.5 ng/mL anti-CA19-9 was added and shaken for another 4 h. Finally, the resulting black composite was obtained after adding 100 mL of 2 wt % BSA and reacting for 1 h to block non-specific binding sites. The multi-functionalized graphene oxide anti-CA19-9/ABEI–nanoFe3O4 @GO composite was obtained after washing and reconstructing in 200 mL of water. In this construct, anti-CA19-9 could recognize and capture target CA19-9 and ABEI was the electrochemiluminophore; and nanoFe3O4 along with these compounds made the one-step preparation of the ECL immunosensor possible. 2.5. One-step preparation of the ECL immunosensor The bare magnetic glass carbon electrode was polished successively with 1.0, 0.3 and 0.05 mm alumina slurry, washed
3.1. Characterization of the ECL immunosensor 3.1.1. ECL behavior As depicted in Fig. 3A, no ECL response was observed on bare magnetic glass carbon electrode (curve a) and nanoFe3O4@GO coated electrode (curve b). In contrast, with ABEI–nanoFe3O4@GO, a strong ECL signal was observed (curve c), indicating that ABEI, the electrochemiluminophore, was successfully immobilized on GO. In the case of anti-CA19-9/ABEI–nanoFe3O4@GO coated electrode, the ECL intensity decreased (curve d) because anti-CA19-9 and BSA immobilization hindered the ECL signal emitted from the electrode surface and blocked the electron transfer on the electrode interface to inhibit the ECL reaction. When the immunosensor was incubated in a sample containing 0.5 U/mL CA199 which was linked to the surface of the electrode through antigen–antibody reaction, a significant reduction in the ECL intensity was observed (curve e). It is because the high molecular weight of CA19-9 (as high as 41000 kDa) and its huge molecular volume effectively hindered the ECL signal and blocked the electron transfer.
Fig. 2. The schematic diagram for (A) the preparation of multi-functionalized graphene oxide, and (B) the fabrication and application protocol of the ECL immunosensor using multi-functionalized graphene oxide.
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Fig. 3. (A) ECL signals obtained with (a) bare magnetic glass carbon electrode, (b) (a)þnanoFe3O4@GO, (c) (a)þ ABEI–nanoFe3O4@GO, (d) (a)þ anti-CA19-9/ ABEI–nanoFe3O4@GO, and (e) (d) þ0.5 U/mL CA19-9. Experimental condition: chronoamperometry; initial potential, 0 V; pulse potential, 1 V; pulse width, 0.25 s; pulse period, 30 s; H2O2, 1 mmol/L; CBS, 0.05 mol/L, and pH 9.90. (B) EIS of (a) bare magnetic glass carbon electrode, (b) (a) þnanoFe3O4@GO, (c) (a)þ ABEI–nanoFe3O4 @GO, (d) (a)þ anti-CA19-9/ABEI–nanoFe3O4@GO, and (e) (d) þ 0.05 U/mL CA19-9. Experimental conditions of EIS: magnetic glass carbon electrode in 0.05 mol/L PBS (5 mmol/L [Fe(CN)6]3 /4 , pH 7.4). The frequency range from 0.01 to 100,000 Hz with a signal amplitude of 5 mV.
3.1.2. EIS behavior EIS was used to monitor the process of electrode modification in 0.01 mol/L phosphate buffer solution containing 5 mmol/L Fe(CN)63 /4 . The curve of EIS shows a line at low AC modulation frequency and a semicircle at high AC modulation frequency, while the electron transfer resistance Ret corresponds to the latter. As shown in Fig. 3B, due to the good electrical conductivity of GO and nanoFe3O4, the diameter of semicircle decreased significantly with nanoFe3O4@GO assembly (curve b) when compared with the bare magnetic glass carbon electrode (curve a). When ABEI molecules were immobilized, Ret increased slightly (curve c). However, when the immunosensor was constructed, Ret increased remarkably (curve d) because anti-CA19-9 and BSA were subsequently conjugated and increased the difficulty in electron transfer. Finally, in presence of CA19-9 at the concentration of 0.05 U/mL, Ret increased significantly again (curve e) because the immobilization of antigen generated an insulating protein layer on the assembled surface and hindered the interfacial electron transfer further. 3.2. Optimization of experimental conditions To establish the optimal conditions, pH value of electrolyte solution, concentration of H2O2 in electrolyte solution, incubation temperature and incubation time were investigated by detecting CA19-9 solution at the concentration of 0.05 U/mL (Fig. S1). Based on an earlier work, 9.90 and 1 mmol/L were selected as the optimal pH of electrolyte solution and concentration of H2O2 in electrolyte solution (Du et al., 2014). Optimum incubation temperature and incubation time were observed to be 37 °C and 1 h, respectively.
Fig. 4. (A) ECL signals of the immunosensor in the presence of 0.5 U/mL CA19-9 in pH 9.90 CBS containing 1 mmol/L H2O2 under continuous chronoamperometry for 18 cycles. (B) ECL intensity from the immunosensor for the detection of CA19-9 at different concentrations (0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1 and 5 U/mL); and (C) The calibration curve obtained through plotting ECL intensity against the logarithmic concentrations of CA19-9 (U/mL).
3.3. Detection of CA19-9 using the ECL immunosensor As presented in Fig. 4A, the ECL signals of the immunosensor in the presence of 0.5 U/mL CA19-9, under consecutive pulse potential scans from 0 to 1 V for 18 cycles, were high and stable, suggesting that the immunosensor was suitable for ECL detection. As indicated in Fig. 4B, under the optimal conditions, the ECL intensity of the immunosensor (y) decreased linearly with the logarithmic concentrations of CA19-9 (x) over the range of 0.001– 5 U/mL, and the regression equation was y ¼1735.8–2602.8 log x (U/mL) with a correlation coefficient r of 0.9953 (Fig. 4C). According to the method previously reported for indirect detection involving signal decrease or quenching (Chen et al., 2014), the detection limit for CA19-9 was roughly estimated to be 0.0005 U/mL, which was around 30-fold lower than the detection limit of 0.016 U/mL reported previously (Shi et al., 2014). Compared with conventional methods, the sensitivity of this method is greatly improved due to following two reasons: (1) In conventional methods, at the most tens of labeling molecules could be labeled onto an antibody molecule to maintain the immunoactivity of antibody (Fung and Wong, 2001; Pei et al., 2013).
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Table 1 Recovery tests for CA19-9 in spiked human serum and urine samples (x¯ ± s , n¼ 3). Samples
Added (U/mL)
Found (U/mL)
RSD (%)
Recovery (%)
Serum 1 Serum 2 Serum 3 Urine 1 Urine 2 Urine 3
0.001 0.1 1 0.001 0.1 1
0.001137 0.000105 0.09757 0.0047 1.0157 0.056 0.00098 7 0.000103 0.10357 0.0056 0.995 7 0.058
9.3 4.8 5.5 10.3 5.4 5.8
113.0 97.5 101.5 98.0 103.5 99.5
In contrast to the conventional methods, much larger number of labeling molecules is involved in the proposed method, for example, thousands of electrochemiluminophore molecules were chemically bound to a GO sheet. (2) The nanoFe3O4@GO has a good electrical conductivity (Teymourian et al., 2013), thus it can significantly extend the outer Helmholtz plane (OHP) of the electrode. It means that all the ABEI molecules immobilized on GO were effective in the area between the OHP and the electrode. It can even be considered that all ABEI molecules were directly immobilized on the surface of the electrode. Therefore, all of them could effectively emit ECL signals. In contrast, in conventional methods, most of the ABEI molecules labeled were beyond OHP, and they could not emit ECL signals (Du et al., 2014). Therefore, it is understandable that the detection sensitivity was improved greatly. 3.4. Specificity, stability, reproducibility, and regeneration of the immunosensor In order to illustrate the possible interference of non-specific antigen, various species of interfering proteins including carcinoma antigen 50 (CA50), carcinoma antigen 125 (CA125), carcinoma antigen 15-3 (CA15-3) and carcinoma antigen 242 (CA242) at the concentration of 10 U/mL were used to further investigate the specificity of the proposed immunosensor. The ECL intensity of CA50, CA125, CA15-3 and CA242 were all about 11,000, nearly similar to that of the blank sample. In contrast, the value observed with 0.05 U/mL CA19-9 solution was 5000 and that of 0.001 U/mL was 9500, indicating that the CA50, CA125, CA15-3 and CA242 did not interfere. These results demonstrated that the immunosensor developed has an excellent specificity for the determination of CA19-9. The stability of the immunosensor proposed was investigated by checking its relative activity after storage at 4 °C in dark for one month. The ECL intensity for the detection of 0.05 U/mL CA19-9 was 93.5 74.7% (n ¼5) of the initial value, which was obtained when the immunosensor was constructed freshly. Results indicate that the developed immunosensor has acceptable stability upon storage. Reproducibility of the immunosensor is one of the most important problems in immunoassay. The reproducibility property of the immunosensor was evaluated by determining 0.05 U/mL CA199 with five equally prepared immunosensors. The relative standard deviation (RSD) of the measurements was 8.1%, indicating the excellent precision and reproducibility of the immunosensor. Regeneration of the immunosensor was examined by detecting 0.05 U/mL CA19-9 with a same immunosensor. To break the antibody–antigen linkage, the immunosensor was regenerated by dipping into 0.2 mol/L glycine–hydrochloric acid (Gly–HCl) buffer solution (pH¼2.8) for 8 min. An average recovery of 92.6% and an intra-assay RSD of 6.4% were obtained when the consecutive measurements were repeated ten times, suggesting that the immunosensor could be regenerated and used for at least ten times.
3.5. Application of the immunosensor in serum and urine samples The application and reliability of the proposed ECL immunosensor was investigated by determining spiked CA19-9 samples with different concentrations. The samples were prepared by adding standard CA19-9 solution to blank human serum and urine. As shown in Table 1, the results showed an acceptable recovery in the range of 97.5–113.0% and RSD in the range of 4.8– 10.5%, indicating that the proposed method is promising and has potential applications to the analysis of clinical samples.
4. Conclusions An effective ECL immunosensor was developed with a one-step preparation method and successfully used for the ultrasensitive determination of CA19-9 based on multi-functionalized graphene oxide anti-CA19-9/ABEI–nanoFe3O4@GO, which had four functions as follows: (1) ABEI acted as the electrochemiluminophore; (2) anti-CA19-9 acted as the capture device for CA19-9 through antigen–antibody immunoreactions; (3) nanoFe3O4@GO extended the OHP, enabled all ABEI molecules immobilized on GO effective in emitting ECL signals and thus improved the detection sensitivity; and (4) nanoFe3O4@GO provided the feasibility of one-step preparation. The ECL immunosensor developed afforded ultrasensitive detection of CA19-9 with satisfactory specificity, stability, reproducibility and regeneration. Therefore, this immunosensor is a promising tool for the detection of tumor marker in clinical applications and other biosensor applications.
Acknowledgements Financial supports from Natural Science Foundation of China (81273130), Zhejiang Provincial Natural Science Foundation of China (LY13B070013), Ningbo Science and Technology Bureau (2010A610164), Major Project of Fujian Province (2011N5008), and Special Funds for Strategic Emerging Industries of Shenzhen (2113K3070038) are gratefully acknowledged. This work was also sponsored by K.C. Wong Magna Fund in Ningbo University.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.12.013.
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