Biosensors and Bioelectronics 65 (2015) 307–313
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
One-step synthesis of redox-active polymer/AU nanocomposites for electrochemical immunoassay of multiplexed tumor markers Zhimin Liu 1, Qinfeng Rong 1, Zhanfang Ma, Hongliang Han n Department of Chemistry, Capital Normal University, Beijing 100048, China
art ic l e i nf o
a b s t r a c t
Article history: Received 12 September 2014 Accepted 7 October 2014 Available online 30 October 2014
In this work, a simple and sensitive multiplexed immunoassay protocol for simultaneous electrochemical determination of alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) was designed using redox-active nanocomposites. As the redox-active species, the poly(o-phenylenediamine) (POPD)/Au nanocomposite and poly(vinyl ferrocene-2-aminothiophenol) (poly(VFc-ATP))/Au nanocomposite were obtained by one-step method which HAuCl4 was used as the oxidant. With Au nanoparticles (AuNPs), the nanocomposites were successful to immobilize labeled anti-CEA and anti-AFP as the immunosensing probes. The proposed electrochemical immunoassay enabled the simultaneous monitoring of AFP and CEA in a wide range of 0.01–100 ng mL 1. The detection limits was 0.006 ng mL 1 for CEA and 0.003 ng mL 1 for AFP (S/N¼3). The assay results of serum samples with the proposed method were well consistent with the reference values from standard ELISA method. And the negligible crossreactivity between the two analytes makes it possesses potential promise in clinical diagnosis. & 2014 Elsevier B.V. All rights reserved.
Keywords: Poly(o-phenylenediamine)/Au nanocomposite Poly(vinyl ferrocene-2-aminothiophenol)/ Au nanocomposite Electrochemical immunosensor Carcinoembryonic antigen Alpha-fetoprotein
1. Introduction The development of materials science has brought significant progress for simultaneous electrochemical determination of multiplex biomarkers (Wilson, 2005; Liu et al., 2014; Harper et al., 2007; Jia et al., 2014; Karimi-Maleh et al., 2013; Tavana et al., 2012; Ensafi et al., 2011). Compared with the traditional materials, nanomaterials have stimulated intense research over past decades due to their potential advantages for electrochemical bioassay, including the excellent biocompatibility, facile synthesis, flexible control over the size, high surface area, good conductivity, and easy modification for biomolecule (Rosi and Mirkin, 2005; Hao et al., 2010; Cosnier et al., 2008; Wang et al., 2008; Jeong et al., 2013; Li et al., 2011; Elyasi et al., 2013; Moradi et al., 2013). Various nanomaterials such as metal nanoparticles, quantum dots, graphene oxide, and magnetic nanoparticles, have been used for redox-active species loading (Cui et al., 2008; Kong et al., 2012; Gao et al., 2013; Feng et al., 2011; Yang et al., 2014). However, they can hardly be directly used to attach proteins or other biomacromolecules after the upload of signal tags, which is an issue for biosensor fabrication (Wei et al., 2010). Therefore, there is a growing demand to develop a nanomaterial which could easily load redox-active species and proteins, and it n
Corresponding author. E-mail addresses: [email protected] (Z. Ma), [email protected] (H. Han). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.bios.2014.10.012 0956-5663/& 2014 Elsevier B.V. All rights reserved.
becomes the most important thing in fabricating excellent electrochemical immunosensors. Conjugated polymer–nanoparticle composites, with many advantages such as facile synthesis, substantial solubility, processability, and adjustable moderate conductivity, have received much attention for the design and fabrication of electrochemical immunosensor (Yan et al., 2008; Chen et al., 2008; Devi et al., 2013; Jia et al., 2011; Zang et al., 2013). Generally, there are two main ways to prepare conjugated polymer nanocomposites (CPNC). The first approach is to immobilize nanoparticles on the surface of conjugated polymer through functional groups such as amine or thiols in conjugated polymer via covalent bonds (Pillalamarri et al., 2005; Baker et al., 2011). Whereas, it requires special functional groups for the covalently loading of nanoparticles and limits the application of this method. The second strategy is to modify conjugated polymer with nanoparticles noncovalently via π–π stacking, electrostatic interaction, van der Waals interactions, or hydrogen bonding (Ivanov et al., 2013; Mazeiko et al., 2013). The serious problem associated with the second approach is to remove the residual nanoparticles by tedious washing. To obtain redoxactive nanocomposites, the CPNC must load the redox-active species with the same procedure as mentioned above. In summary, the redox-active nanocomposites are usually prepared step by step, which face the the problems of the complicated process and poor stability. Furthermore, its multiple experimental steps and hard-to-control manipulation make it time-consuming and cost-expensive. Thus, it is still a challenge to explore new strategies for further improvement of the simplicity and stability.
308
Z. Liu et al. / Biosensors and Bioelectronics 65 (2015) 307–313
To resolve such problems, here we report a one-step approach to synthesize poly(o-phenylenediamine) (POPD)/Au nanocomposite and poly(vinyl ferrocene-2-aminothiophenol) (poly(VFcATP))/Au nanocomposite at room temperature by chemical polymerization. O-phenylenediamine (OPD), a kind of redox-active material, has a planar aromatic structure which can be initiated to be conjugated polymer by radical polymerization when the oxidant exists. Moreover, the amine groups of o-phenylenediamine have the reducing capacity which can be used to prepare metal nanoparticles. Vinyl ferrocene (VFc), an olefin derived from ferrocene, with the excellent redox activity, which can also be polymerized by radical polymerization via initiator. Although it can be used as the redox species, it is hard to be modified with biomolecule or nanoparticles due to the lack of modifiable groups and poor dissolvability. After copolymerize with 2-aminothiophenol (ATP), the amine and thiols groups could be introduced into the chain of the poly(vinyl ferrocene) for improving the biocompatibility and the capacity of loading metal nanoparticles or biomolecules. To initiate the polymerization of OPD, VFc and ATP, HAuCl4 was used as the oxidant, resulting in the formation of POPD/Au and poly(VFc-ATP)/Au nanocomposites, respectively. These conjugated polymer/Au nanocomposites integrated the advantages of excellent redox-activity, facile synthesis, good biocompatibility and stability by one-step method, which have not been reported. The Au nanoparticles (AuNPs) attached densely on the surface of the electroactive polymers in situ by the covalent bonds, which could not only be more stable, but also provide large amount of active sites for immobilizing capture antibodies. Thus, the redox-active polymer/Au nanocomposites were successfully used to fabricate a simple, and sensitive multiplexed electrochemical immunosensor for carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) detection. The electrochemical signals were simultaneously obtained at two peak potentials and the peak currents were dependent on the concentration of the corresponding analytes. The negligible cross-reactivity between the two analytes makes it possesses potential promise in clinical diagnosis.
electron microscopy (TEM) with a JEOL-100CX electron microscope under 80 kV accelerating voltage. The elemental analysis was studied by an Escalab 250 X-ray photoelectron spectroscope (Thermofisher, American) employing amonochromatic Al Kα radiation. The structure of the nanocomposites was investigated by Fourier transform infrared spectroscopy (FTIR). All electrochemical measurements were carried out on a CHI-832 electrochemical analyzer (Chenhua, Shanghai, China). A three-electrode electrochemical cell was composed of a modified gold electrode (GE, 4 mm in diameter) as the working electrode, a platinum wire and an Ag/AgCl electrode (saturation of KCl) as the auxiliary electrode and the reference electrode, respectively. 2.3. Preparation of POPD/AU nanocomposites The POPD/Au nanocomposites were synthesized by one-step method. In general, 2.32 mg OPD was added into 4.7 mL of the distilled water with stirring. After the OPD was dissolved, 0.3 mL 4% HAuCl4 was added into the above solution. Then the mixture was vigorously stirred at room temperature for 4 h. The resulting POPD/Au nanocomposites were subsequently centrifuged with ethanol and ultrapure water, and re-dispersed in ultrapure water for use. 2.4. Preparation of the poly(VFc-ATP)/AU nanocomposites The poly(VFc-ATP)/Au nanocomposites were also synthesized by copolymerization in one-step process. Briefly, VFc (0.0212 g) and ATP (2 μL, at room temperature) were mixed in 4.7 mL of distilled water with stirring. After 15 min, 0.3 mL 4% HAuCl4 was added into the above solution and vigorously stirred at room temperature for 4 h. The obtained poly(VFc-ATP)/Au nanocomposites were subsequently centrifuged with ethanol and ultrapure water, and re-dispersed in ultrapure water for use. 2.5. Preparation of the poly(2-aminothiophenol) (PATP) The PATP was synthesized according to our previous work (Liu and Ma, 2014).
2. Experimental section 2.6. Preparation of AuNPs 2.1. Reagents and chemicals Human immunoglobulin G (IgG), and albumin from bovine serum (BSA) were purchased from Chengwen Biological Company (Beijing, China). Mouse anti human monoclonal antibody to carcinoembryonic antigen (anti-CEA), mouse anti human alpha fetoprotein monoclonal antibody (anti-AFP), CEA and AFP were obtained from Shanghai Linc-Bio Science Co., Ltd. (Shanghai, China). Sodium borohydride (NaBH4, 98%) was purchased from Sigma (USA). Hydrogen tetrachloroaurate (III) hydrate (HAuCl4 XH2O), vinyl ferrocene (VFc), D-(þ )-glucose, ammonium peroxidisulfate (APS), ascorbic acid (AA), o-phenylenediamine (OPD), uric acid (UA), and 2-aminothiophenol (ATP) were achieved from Alfa Aesar (Tianjin, China). Clinical human serum Samples were obtained from the Capital Normal University Hospital (Beijing, China). KH2PO4 (99%), HCl, Na2HPO4 (99%), KCl, potassium ferricyanide (K3Fe(CN)6), and potassium ferrocyanide (K4Fe(CN)6) were obtained from Beijing Chemical Reagents Company (Beijing, China). All chemicals were of analytical grade and used without further purification. 2.2. Apparatus The distilled water (resistivity 418 MΩ) was used throughout. The morphology of nanocomposites was performed transmission
The AuNPs were synthesized according to the as-reported method (Sun and Ma, 2012). In general, 5 nm AuNPs were prepared at room temperature by adding 1 mL 1% sodium citrate solution to the 100 mL 0.01% HAuCl4 aqueous solution with stirring. After 1 min, 1.6 mL 0.075% NaBH4 (dissolved in 1% sodium citrate solution) was added. The mixture was kept stirring until its color turned to red. The AuNPs were stored at 4 °C for use. 2.7. Preparation of immunosensing probes The immunosensing probes were fabricated by immobilizing labeled anti-CEA and anti-AFP onto POPD/Au and poly(VFc-ATP)/ Au nanocomposites, respectively. Firstly, the labeled anti-CEA (100 μL, 1 mg mL 1) was mixed with the obtained POPD/Au nanocomposites in 1 mL 0.01 M phosphate buffer (PB, pH 7.3) with gently stirring for 12 h. After centrifugation, the obtained POPD/Au–anti-CEA was incubated in a solution of BSA (10%, w/w) for 2 h to block any possible remaining active sites to avoid any nonspecific absorption. Then, the BSA blocked POPD/Au–anti-CEA was re-dispersed in 1 mL 0.01 M PB (pH ¼ 7.3) and stored in 4 °C for use. The poly(VFc-ATP)/Au nanocomposites were immobilized the labeled anti-AFP with the same procedure as preparing POPD/ Au–anti-CEA above. The obtained poly(VFc-ATP)/Au-anti-AFP was re-dispersed in 1 mL 0.01 M PB (pH 7.3) and stored in 4 °C for use.
Z. Liu et al. / Biosensors and Bioelectronics 65 (2015) 307–313
2.8. Preparation of the immunosensor The GE was polished repeatedly using alumina powder and then thoroughly cleaned before use. The immunosensing matrix and fabrication process of immunosensor was the same as our previous work (Liu and Ma, 2013). Briefly, 5 μL 0.5 g L 1 brown solution of PATP was casted on the pretreated GE at room temperature for 8 h to adsorb the conducting long-chain polythiols. After that, the electrode was thoroughly rinsed with deionized water and dried under a stream of nitrogen gas for the immobilization of AuNPs. Next, the AuNPs coated electrode was incubated in a mixed solution of 200 μg mL 1 anti-CEA and anti-AFP solution at 4 °C overnight. Subsequently, excess antibodies were washed away with 0.01 M PB (pH 7.3), and 1% BSA solution was applied to block possible remaining active sites of the electrode. After washing with 0.01 M PB (pH 7.3), the immunosensor was stored at 4 °C for use. 2.9. Simultaneous measurement procedure To perform the immunoreaction and the electrochemical measurement, the immunosensor was first incubated in the mixture of CEA and AFP solution with various concentrations at 37 °C for 1 h. After that, it was incubated in the mixture of 1:1 diluted POPD/Auanti-CEA and poly(VFc-ATP)/Au-anti-AFP. The electrodes were washed with 0.01 M PB (pH 7.3) between the modified steps. Subsequently, differential pulse voltammetric (DPV) was performed to record the electrochemical responses for simultaneous
309
detection of CEA and AFP in phosphate buffered solution (0.1 M PBS, pH 6.98). The DPV measurement was carried out from 0.7 V to 0.8 V (vs. Ag/AgCl) with a pulse amplitude of 50 mV and a pulse width of 50 ms.
3. Results and discussion 3.1. Characterization of redox-active polymer/AU nanocomposites POPD/Au and poly(VFc-ATP)/Au nanocomposites were synthesized by chemical oxidative polymerization which HAuCl4 was used as the oxidant. The mechanism of polymerization as follows. Firstly, HAuCl4 initiates the formation of radical cation of o-phenylenediamine, 2-aminothiophenol and vinyl ferrocene. In the second step, coupling of radical cations occurs, with subsequent formation of the dimer and elimination of two protons. It is then oxidized to be the diradical dication which could couples with a monomer radical cation, resulting in propagation of the chain (Barbero et al., 1989; Tzou and Gregory, 1992). Finally, the resultant polymer (POPD and poly(VFc-ATP)) could be obtained after the chain termination. Accompanied with the formation of Au nanoparticles, the polymer/Au nanocomposites were obtained. FTIR was used to characterize the structure of POPD/Au and poly(VFc-ATP)/Au nanocomposites (Fig. S1 in supplementary information). The FTIR spectrum of POPD/Au shows the characteristic peak at 1475 cm 1 which assigned to the C8C stretching modes of benzenoid rings (Fig. S1A). The band corresponding to C‒
Fig. 1. Typical TEM images of (A) PODP/Au and (B) poly(VFc-ATP)/Au, the scale bar is 200 nm; and the XPS spectrum of (C) PODP/Au and (D) poly(VFc-ATP)/Au.
310
Z. Liu et al. / Biosensors and Bioelectronics 65 (2015) 307–313
N stretching vibration is observed at 1151 cm 1. For poly(VFcATP)/Au (Fig. S1B), the C8C stretching modes of benzenoid rings and quinonoid rings are appeared at 1475 and 1525 cm 1 (Zengin et al., 2002). Compared to the FTIR spectrum of pure PATP (Liu and Ma, 2013), the S3H stretching vibrations of poly(VFc-ATP)/Au at 2576 cm 1 was disappeared, revealing that the Au3S band was formed. Moreover, the characteristic peaks at 1056 and 1105 cm 1come from the cyclopentadienyl of Fc (Roginski et al., 1988). The C3Fe stretching vibration is observed at 482 cm 1. The results confirm that the copolymer of ATP and VFc was formed. The typical TEM images of POPD/Au nanocomposites are shown in Fig. 1A. It was observed that the surface of POPD was densely covered by AuNPs with the size of about 50 nm, which indicated that the efficient reduction of HAuCl4 by amine groups in OPD. And it was suitable to be used as the signal tags because of its excellent redox-activity and large amount of AuNPs to immobilize proteins. The detailed compositional analysis of POPD/Au nanocomposites were further characterized by XPS as showed in Fig. 1C. The XPS peaks of Au 4f, C 1s and N 1s core level regions could be obviously observed. The appearing of Au 4f doublet (85.1 eV and 88.7 eV, in Fig. S2A) was consistent with the Au0 state, revealing the presence of AuNPs on the surface of POPD. For poly(VFc-ATP)/Au, Fig. 1B displays that the nanocomposites were spheres with the size about 50 nm. Moreover, the AuNPs on the surface of copolymer was about 10 nm. To investigated the chemical composition of poly(VFc-ATP)/Au nanocomposites, XPS characterization was also employed. As shown in Fig.1D, the elements of Fe, C, Au, N, and S were included. The appearing of Au 4f doublet also indicated the existence of AuNPs on the surface of copolymer. The binding energies of Fe 2p3/2 and Fe 2p1/2 were observed at 708 and 721 eV (as shown in Fig. S2B), respectively, which assigned to VFc. The peaks of N 1s and S 1 s were appeared at 400 and 164 eV, respectively, showed the existing of ATP. Thus, these results indicated that POPD/Au and poly(VFc-ATP)/Au nanocomposites have been successfully prepared. 3.2. Principle and characterization of the multiplexed electrochemical immunoassay The schematic illustration of the stepwise immunosensor fabrication process is shown in Scheme 1. To fabricate stable and sensitive immunosensing matrix, a conducting polythiols polymer-PATP was used to immobilize on the GE though Au‒S bonds. It plays two important roles. Firstly, the multiple thiol groups of PATP provide tenacious attachment to GE surfaces due to the cooperative binding through multiple thiols bonds (Johnson and Levicky, 2003). Significantly, the remnant (unbound) thiols
moieties of PATP can provide reactive sites for modification with AuNPs to fix more antibodies. Secondly, the excellent conductivity can further enhance the sensitivity of immunosensors since the PATP acts as an effective mediator for electron transfer in redox (Luo and Do, 2004). As a result, the PATP and AuNPs could be utilized to build immunosensing platform. The immunosensing probes were fabricated by choosing excellent redox-active species. With two amine groups on the molecule, the OPD could be oxidized to be conjugated polymer with efficient redox-activity by one-step method which the HAuCl4 was used as the Oxidant. The remained amine groups could reduce the HAuCl4 to be AuNPs immobilized on the surface of AuNPs by covalent bond. For the VFc, it is not convenient to immobilize the biomolecule even if it has good redox-activity. But it could be copolymerized with the ATP to introduce the –SH and amine groups into the chain of the copolymer for the easy modification. By the chemical polymerization method, the HAuCl4 not only could oxidize the VFc and ATP to form copolymer, but also itself can be reduced to be AuNPs which attached on the copolymer by Au3S bond. Thus, The POPD/Au and poly(VFc-ATP)/Au nanocomposites integrate the advantages of simple preparation, easy modification, and excellent electrochemical signals. Subsequently, the immunosensing probes were successfully prepared based on immobilizing labeled anti-CEA and anti-AFP on POPD/Au and poly(VFc-ATP)/Au nanocomposites via AuNPs, respectively. With the sandwich-type assay format, the electrochemical signals were simultaneously obtained at the two different peak potentials. The peak currents were dependent on the concentration of the corresponding antigens, respectively. The DPV measurements were used to monitor the electrochemical behavior of the modification procedure after each step in 5 mM K3[Fe(CN)6]/K4[Fe(CN)6]-PBS (pH 6.98) in Fig. 2A. It was observed that the current of PATP modified electrode (curve b) was lower than that of a bare GE (curve a), which was attributed to the electron transfer ability of PATP is not as good as bare GE. In contrast, the adsorption of AuNPs led to an obvious increase of the peak current (curve c) owing to the formation of an excellent electron-transfer layer and the increase of the area of electrode. Subsequently, it was found that the current response further decreased after the electrode modified with anti-CEA and antiAFP (curve d). After the immunosensor was blocked with BSA (curve e) and incubated in a solution with 1 ng mL 1 CEA and AFP (curve f), the current responses were decreased gradually. This may originate from the insulating BSA, CEA and AFP protein layers on the electrode that retards the electron transfer. After that, the immunosensor was simultaneously incubated in the mixture of 1:1 diluted POPD/Au-anti-CEA and poly(VFc-ATP)/Au-anti-AFP in 0.01 M PB (pH 7.3) for 1 h, so that the two probes could be modified on the electrode. The two pairs of peaks at 0.43 V and 0.2 V were assigned to the redox-activity of PODP and poly (VFc-ATP), respectively, and monitored by the DPV in 0.1 M PBS (pH 6.98) as shown in Fig.2B. The peaks separation between two pairs of the peaks was about 630 mV (ΔEpc). Thus, a simultaneous detection of CEA and AFP was possible, according to the position of the corresponding peak. 3.3. Optimization of conditions for electrochemical detection
Scheme 1. The immunosensor.
fabrication
process
of
the
multiplexed
electrochemical
The pH value of detection solution is an important factor in the performance of immunosensor. It not only has a great influence on the activity of the antigens and antibodies, but also on the immunosensing matrix and redox-active species. As shown in Fig. S3. The current response increased rapidly with increasing pH value from 4.92 to 6.98 and then decreased as pH increase. That is because the activity of the immunoreaction between antibody and antigen could be seriously influenced by pH (Liu et al., 2014). The better the immunoreaction activity, the more redox-active probes
Z. Liu et al. / Biosensors and Bioelectronics 65 (2015) 307–313
311
Fig. 2. DPV (A) of the GE modified with (a) bare GE, (b) PATP, (c) PATP-AuNPs, (d) modified GE incubated with anti-CEA and anti-AFP, (e) blocked with 1% BSA, and (f) incubated with 1 ng mL 1 CEA and AFP after blocked with 1% BSA. All the experiments were performed in 5 mM K3[Fe(CN)6]/K4[Fe(CN)6]-PBS (pH 6.98); and DPV (B) the immunosensor incubated with excess PODP/Au-anti-CEA and poly(VFc-ATP)/Au-anti-AFP in PBS, pH 6.98.
could be modified on the electrode, resulting in the maximal current response. In general, the optimal reactivity of antibody and antigen was at neutral condition (Viswanathan et al., 2006; Chen et al., 2006; Li et al., 2009). Thus, pH 6.98 was optimal and corresponding PBS was used for the following measurements. 3.4. Evaluation of repeatability, cross-talk, cross-reactivity and selectivity As well known, repeatability is a key factor in the practical application for a successful immunosensor. In order to evaluate the repeatability of the immunoassay, two panel immunosensing experiments were carried out by the freshly prepared modified electrodes in five times. The relative standard deviation were 2.8% and 3.6% for 0.1 ng mL 1 CEA and AFP, 4.3% and 3.2% for 10 ng mL 1 CEA and AFP, respectively. The result indicated that the proposed immunosensor displayed good repeatability. To investigate the cross-reactivity between the analytes and noncognate antibodies, two different antibodies of CEA and AFP were immobilized on the electrode, and then immunoreacted with 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100 ng mL 1 of CEA and 1 ng mL 1 AFP (Fig. S4A), or 1 ng mL 1 CEA and 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100 ng mL 1 of AFP (Fig. S4B), respectively. Lastly it was incubated with the immunoprobes (POPD/Au–anti-CEA and poly(VFc-ATP)/ Au–anti-AFP) to carry out the sandwich-type immunoreactions. As expected, the results showed that the single detection of the corresponding analyte is good agreement with the simultaneous detection of two analytes (The DPV measurements are shown in Fig. S4.), indicating that the multiplex detection exhibited low interference with each other and the cross-reactivity between the
two analytes could be ignored. Additionally, no other substrate or mediator was required in the detection procedure, thus, no electrochemical cross-talk occurred. The results indicate that the multiplex detection exhibited low interference with each other and the cross-reactivity between the two analytes was negligible. In order to evaluate the selectivity of the immunosensor, the changes in the current responses of 1 ng mL 1 CEA and AFP induced by the 100 ng mL 1 interferences, such as UA, IgG, glucose, BSA, and AA were measured. As shown in Fig.S5, the interference was negligible, showing that the developed immunosensor with good selectivity. 3.5. Analytical performance of the multiplexed immunoassay Under optimal conditions, the sensitivity and dynamic range of the immunoassay were evaluated the multiplexed immunoassay were evaluated toward CEA and AFP. After incubation with various analyte levels and excess immunosensing probes, the DPV measurements of the modified electrodes were carried out in 0.1 M PBS (pH 6.98). As shown in Fig. 3A, the DPV peak currents of the multiplexed immunoassay increased as the CEA and AFP concentrations increased in the range of 0.01–100 ng mL 1 for both CEA (Fig.3B) and AFP (Fig.3C). The correlation coefficients were 0.9965 and 0.9986, respectively. The detection limits reached 0.006 ng mL 1 for CEA and 0.003 ng mL 1 for AFP at a signal-tonoise ratio of 3. Compared with other multiplex electrochemical immunosensors for the detection of CEA and AFP, this proposed immunosensor displayed a better performance than some earlier reported studies (as shown in Table 1).
Fig. 3. DPV responses (A) and calibration curves for different concentrations of (B) CEA and (C) AFP in PBS, pH 6.98.
312
Z. Liu et al. / Biosensors and Bioelectronics 65 (2015) 307–313
Table 1 Comparison of the analytical performance of some multiplex electrochemical immunosensors Linear range (ng mL 1)
Redox-active species
CGS-MB/CGS-PB FCA-MPS/HRP-MPS Hydroquinone Thi@HPtNP/Fc@HPtNP Fc/Thi AuNPs-PB/Thi@GS PODP/poly(VFc-ATP)
Detection limit (ng mL 1)
Refs.
CEA
AFP
CEA
AFP
0.5–80 0.5–45 25–150 0.5–50 0.01–50 0.6–80 0.01–100
0.5–50 1–90 10–200 0.3–45 0.01–50 0.6–80 0.01–100
0.05 0.2 1.2 0.08 0.01 0.12 0.006
0.1 0.5 1.0 0.05 0.01 0.08 0.003
Kong et al. (2013) Lin et al. (2011) Wilson (2005) Song et al. (2010) Lai et al. (2012) Feng et al. (2014) This work
Table 2 Assay results of clinical serum samples using the proposed and reference methods. Sample no.
1 2 3 4 5 6 7 8 9 10
Proposed method (ng mL 1)
ELISA (ng mL 1)
Relative error (%)
CEA
AFP
CEA
AFP
CEA
AFP
3.06 70.04 1.36 70.03 1.04 70.04 2.84 70.05 0.87 70.04 0.97 70.05 0.69 70.04 3.26 70.06 4.89 70.05 1.05 70.03
4.20 7 0.05 2.067 0.05 3.23 7 0.07 2.87 7 0.06 4.62 7 0.03 0.25 7 0.02 0.477 0.02 0.34 7 0.03 0.357 0.02 0.62 7 0.03
2.977 0.05 1.417 0.06 0.98 7 0.07 2.777 0.05 0.93 7 0.03 1.03 7 0.04 0.667 0.03 3.187 0.06 4.99 7 0.05 1.09 7 0.04
4.34 7 0.06 1.98 7 0.07 3.167 0.05 2.98 7 0.08 4.79 7 0.06 0.247 0.02 0.447 0.03 0.36 7 0.02 0.377 0.02 0.59 7 0.03
3.03 3.55 6.12 2.53 5.38 5.82 4.55 2.52 2.00 3.67
3.23 4.04 2.22 3.69 3.55 4.17 6.82 5.56 5.41 5.08
3.6. Application in analysis of serum samples In order to evaluate the effect of the present method to detect real samples, the immunoassay for clinical serum samples was investigated by analyzing five real samples in comparison with the enzyme-linked immunosorbent assay (ELISA) method. As shown in Table 2, the results obtained by proposed immunosensor are well consistent with the data determined by ELISA, showing that present method can be practically applied in clinical analysis for simultaneous determination of CEA and AFP.
4. Conclusion In this work, POPD/Au and poly(VFc-ATP)/Au) nanocomposites were synthesized by one-step method and used to fabricate a simple and sensitive electrochemical immunoassay for simultaneous detection of multiple biomarkers. As redox-active species, the POPD/Au and poly(VFc-ATP)/Au) nanocomposites integrated the advantages of excellent redox-activity, facile synthesis, easy modification, good biocompatibility and stability, which were successfully used as the immunosensing probes. The experimental results showed that the proposed immunosensor enabled simultaneous monitoring of CEA and AFP in a single run. In particular, the convenient operation and ultrahigh sensitivity of the proposed immunoassay method provided a promising potential in clinical applications.
Acknowledgments This research was financed by Grants from the National Natural Science Foundation of China (21273153), the Beijing Natural Science Foundation (2132008), the Project of Construction of
Innovative Teams and Teacher Career Development for Universities and Colleges Under Beijing Municipality (IDHT20140512), and the Research Base Construction Projects of Beijing Municipal Education Commission.
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.2014.10.012.
References Baker, C.O., Shedd, B., Tseng, R.J., Martinez-Morales, A.A., Ozkan, C.S., Ozkan, M., Yang, Y., Kaner, R.B., 2011. ACS Nano 5, 3469–3474. Barbero, C., Silber, J.J., Sereno, L., 1989. J. Electroanal. Chem. 236, 333–352. Chen, W., Lu, Z.S., Li, C.M., 2008. Anal. Chem. 80, 8485–8492. Chen, J., Tang, J.H., Yan, F., Ju, H.X., 2006. Biomaterials 27, 2313–2321. Cosnier, S., Lonescu, R.E., Holzinger, M., 2008. J. Mater. Chem. 18, 5129–5133. Cui, R., Huang, H., Yin, Z., Gao, D., Zhu, J.J., 2008. Biosens. Bioelectron. 23, 1666–1673. Devi, R., Relhan, S., Pundir, C.S., 2013. Sens. Actuators B 186, 17–26. Elyasi, M., Khalilzadeh, M.A., Karimi-Maleh, H., 2013. Food Chem. 141, 4311–4317. Ensafi, A.A., Karimi-Maleh, H., Mallakpour, S., Hatami, M., 2011. Sens. Actuators B 155, 464–472. Feng, D.X., Li, L.H., Han, X.W., Fang, X., Li, X.Z., Zhang, Y.Z., 2014. Sens. Actuators B 201, 360–368. Feng, L.Y., Chen, Y., Ren, J.S., Qu, X.G., 2011. Biomaterials 32, 2930–2937. Gao, Q., Han, J.M., Ma, Z.F., 2013. Biosens. Bioelectron. 49, 323–328. Hao, R., Xing, R.J., Xu, Z.C., Hou, Y.L., Gao, S., Sun, S.H., 2010. Adv. Mater. 22, 2729–2742. Harper, J.C., Polsky, R., Wheeler, D.R., Dirk, S.M., Brozik, S.M., 2007. Langmuir 23, 8285–8287. Ivanov, S., Tsakova, V., Bund, A., 2013. Electrochim. Acta 90, 157–165. Jeong, B.J., Akter, R., Han, O.H., Rhee, C.K., Rahman, M.A., 2013. Anal. Chem. 85, 1784–1791. Jia, X.L., Liu, Z.M., Liu, N., Ma, Z.F., 2014. Biosens. Bioelectron. 53, 160–166. Jia, W.Z., Su, L., Lei, Y., 2011. Biosens. Bioelectron. 30, 158–164. Johnson, P.A., Levicky, R., 2003. Langmuir 19, 10288–10294.
Z. Liu et al. / Biosensors and Bioelectronics 65 (2015) 307–313
Kong, F.Y., Li, X.R., Zhao, W.W., Xu, J.J., Chen, H.Y., 2012. Electrochem. Commun. 14, 59–62. Kong, F.Y., Xu, B.Y., Du, Y., Xu, J.J., Chen, H.Y., 2013. Chem. Commun. 49, 1052–1054. Karimi-Maleh, H., Biparva, P., Hatami, M., 2013. Biosens. Bioelectron. 48, 270–275. Lai, W.Q., Zhuang, J.Y., Tang, J., Chen, G.N., Tang, D.P., 2012. Microchim. Acta 178, 357–365. Li, Q.F., Zeng, L.X., Wang, J.C., Tang, D.P., Liu, B.Q., Chen, G.N., Wei, M.D., 2011. ACS Appl. Mater. Interfaces 3, 1366–1373. Li, X.H., Dai, L., Liu, Y., Chen, X.J., Yan, W., Jiang, L.P., Zhu, J.J., 2009. Adv. Funct. Mater. 19, 3120–3128. Lin, J.H., Wei, Z.J., Mao, C.M., 2011. Biosens. Bioelectron. 29, 40–45. Liu, N., Ma, Z.F., 2014. Biosens. Bioelectron. 51, 184–190. Liu, N., Liu, Z.M., Han, H.L., Ma, Z.F., 2014. J. Mater. Chem. B 2, 3292–3298. Liu, Z.M., Ma, Z.F., 2013. Biosens. Bioelectron. 46, 1–7. Luo, Y.C., Do, J.S., 2004. Biosens. Bioelectron. 20, 15–23. Mazeiko, V., Kausaite-Minkstimiene, A., Ramanaviciene, A., Balevicius, Z., Ramanavicius, A., 2013. Sens. Actuators B 189, 187–193. Moradi, R., Sebt, S.A., Karimi-Maleh, H., Sadeghi, R., Karimi, F., Bahari, A., Arabi, H., 2013. Phys.Chem. Chem. Phys. 15, 5888–5897. Pillalamarri, S.K., Blum, F.D., Tokuhiro, A.T., Bertino, M.F., 2005. Chem. Mater. 17, 5941–5944. Roginski, R.T., Shapley, J.R., Drickamer, H.G., 1988. J. Phys. Chem. 92, 4316–4319. Rosi, N.L., Mirkin, C.A., 2005. Chem. Rev. 105, 1547–1562.
313
Sun, X.B., Ma, Z.F., 2012. Biosens. Bioelectron. 35, 470–474. Song, Z.J., Yuan, R., Chai, Y.Q., Zhuo, Y., Jiang, W., Su, H.L., Che, X., Li, J.J., 2010. Chem. Commun. 46, 6750–6752. Tavana, T., Khalilzadeh, M.A., Karimi-Maleh, H., Ensafi, A.A., Beitollahi, H., Zareyee, D., 2012. J. Mol. Liq. 168, 69–74. Tzou, K., Gregory, R.V., 1992. Synth. Met. 47, 267–277. Viswanathan, S., Wu, L.C., Huang, M.R., Ho, J.A., 2006. Anal. Chem. 78, 1115–1121. Wang, S.P., Wu, Z.S., Qu, F.L., Zhang, S.B., Shen, G.L., Yu, R.Q., 2008. Biosens. Bioelectron. 24, 1020–1026. Wei, Q., Mao, K.X., Wu, D., Dsi, Y.X., Yang, J., Du, B., Yang, M.H., Li, H., 2010. Sens. Actuators B 149, 314–318. Wilson, M.S., 2005. Anal. Chem. 77, 1496–1502. Yan, W., Chen, X.J., Li, X.H., Feng, X.M., Zhu, J.J., 2008. J. Phys. Chem. B 112, 1275–1281. Yang, Z.H., Chai, Y.Q., Yuan, R., Zhuo, Y., Li, Y., Han, J., Liao, N., 2014. Sens. Actuators B 193, 461–466. Zang, S., Liu, Y.J., Lin, M.H., Kang, J.L., Sun, Y.M., Lei, H.T., 2013. Electrochim. Acta 90, 246–253. Zengin, H., Zhou, W., Jin, J., Czerw, R., Smith, J.D.W., Echegoyen, L., Carroll, D.L., Foulger, S.H., Ballato, J., 2002. Adv. Mater. 14, 1480–1483.
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
One-step synthesis of redox-active polymer/AU nanocomposites for electrochemical immunoassay of multiplexed tumor markers Zhimin Liu 1, Qinfeng Rong 1, Zhanfang Ma, Hongliang Han n Department of Chemistry, Capital Normal University, Beijing 100048, China
art ic l e i nf o
a b s t r a c t
Article history: Received 12 September 2014 Accepted 7 October 2014 Available online 30 October 2014
In this work, a simple and sensitive multiplexed immunoassay protocol for simultaneous electrochemical determination of alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) was designed using redox-active nanocomposites. As the redox-active species, the poly(o-phenylenediamine) (POPD)/Au nanocomposite and poly(vinyl ferrocene-2-aminothiophenol) (poly(VFc-ATP))/Au nanocomposite were obtained by one-step method which HAuCl4 was used as the oxidant. With Au nanoparticles (AuNPs), the nanocomposites were successful to immobilize labeled anti-CEA and anti-AFP as the immunosensing probes. The proposed electrochemical immunoassay enabled the simultaneous monitoring of AFP and CEA in a wide range of 0.01–100 ng mL 1. The detection limits was 0.006 ng mL 1 for CEA and 0.003 ng mL 1 for AFP (S/N¼3). The assay results of serum samples with the proposed method were well consistent with the reference values from standard ELISA method. And the negligible crossreactivity between the two analytes makes it possesses potential promise in clinical diagnosis. & 2014 Elsevier B.V. All rights reserved.
Keywords: Poly(o-phenylenediamine)/Au nanocomposite Poly(vinyl ferrocene-2-aminothiophenol)/ Au nanocomposite Electrochemical immunosensor Carcinoembryonic antigen Alpha-fetoprotein
1. Introduction The development of materials science has brought significant progress for simultaneous electrochemical determination of multiplex biomarkers (Wilson, 2005; Liu et al., 2014; Harper et al., 2007; Jia et al., 2014; Karimi-Maleh et al., 2013; Tavana et al., 2012; Ensafi et al., 2011). Compared with the traditional materials, nanomaterials have stimulated intense research over past decades due to their potential advantages for electrochemical bioassay, including the excellent biocompatibility, facile synthesis, flexible control over the size, high surface area, good conductivity, and easy modification for biomolecule (Rosi and Mirkin, 2005; Hao et al., 2010; Cosnier et al., 2008; Wang et al., 2008; Jeong et al., 2013; Li et al., 2011; Elyasi et al., 2013; Moradi et al., 2013). Various nanomaterials such as metal nanoparticles, quantum dots, graphene oxide, and magnetic nanoparticles, have been used for redox-active species loading (Cui et al., 2008; Kong et al., 2012; Gao et al., 2013; Feng et al., 2011; Yang et al., 2014). However, they can hardly be directly used to attach proteins or other biomacromolecules after the upload of signal tags, which is an issue for biosensor fabrication (Wei et al., 2010). Therefore, there is a growing demand to develop a nanomaterial which could easily load redox-active species and proteins, and it n
Corresponding author. E-mail addresses: [email protected] (Z. Ma), [email protected] (H. Han). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.bios.2014.10.012 0956-5663/& 2014 Elsevier B.V. All rights reserved.
becomes the most important thing in fabricating excellent electrochemical immunosensors. Conjugated polymer–nanoparticle composites, with many advantages such as facile synthesis, substantial solubility, processability, and adjustable moderate conductivity, have received much attention for the design and fabrication of electrochemical immunosensor (Yan et al., 2008; Chen et al., 2008; Devi et al., 2013; Jia et al., 2011; Zang et al., 2013). Generally, there are two main ways to prepare conjugated polymer nanocomposites (CPNC). The first approach is to immobilize nanoparticles on the surface of conjugated polymer through functional groups such as amine or thiols in conjugated polymer via covalent bonds (Pillalamarri et al., 2005; Baker et al., 2011). Whereas, it requires special functional groups for the covalently loading of nanoparticles and limits the application of this method. The second strategy is to modify conjugated polymer with nanoparticles noncovalently via π–π stacking, electrostatic interaction, van der Waals interactions, or hydrogen bonding (Ivanov et al., 2013; Mazeiko et al., 2013). The serious problem associated with the second approach is to remove the residual nanoparticles by tedious washing. To obtain redoxactive nanocomposites, the CPNC must load the redox-active species with the same procedure as mentioned above. In summary, the redox-active nanocomposites are usually prepared step by step, which face the the problems of the complicated process and poor stability. Furthermore, its multiple experimental steps and hard-to-control manipulation make it time-consuming and cost-expensive. Thus, it is still a challenge to explore new strategies for further improvement of the simplicity and stability.
308
Z. Liu et al. / Biosensors and Bioelectronics 65 (2015) 307–313
To resolve such problems, here we report a one-step approach to synthesize poly(o-phenylenediamine) (POPD)/Au nanocomposite and poly(vinyl ferrocene-2-aminothiophenol) (poly(VFcATP))/Au nanocomposite at room temperature by chemical polymerization. O-phenylenediamine (OPD), a kind of redox-active material, has a planar aromatic structure which can be initiated to be conjugated polymer by radical polymerization when the oxidant exists. Moreover, the amine groups of o-phenylenediamine have the reducing capacity which can be used to prepare metal nanoparticles. Vinyl ferrocene (VFc), an olefin derived from ferrocene, with the excellent redox activity, which can also be polymerized by radical polymerization via initiator. Although it can be used as the redox species, it is hard to be modified with biomolecule or nanoparticles due to the lack of modifiable groups and poor dissolvability. After copolymerize with 2-aminothiophenol (ATP), the amine and thiols groups could be introduced into the chain of the poly(vinyl ferrocene) for improving the biocompatibility and the capacity of loading metal nanoparticles or biomolecules. To initiate the polymerization of OPD, VFc and ATP, HAuCl4 was used as the oxidant, resulting in the formation of POPD/Au and poly(VFc-ATP)/Au nanocomposites, respectively. These conjugated polymer/Au nanocomposites integrated the advantages of excellent redox-activity, facile synthesis, good biocompatibility and stability by one-step method, which have not been reported. The Au nanoparticles (AuNPs) attached densely on the surface of the electroactive polymers in situ by the covalent bonds, which could not only be more stable, but also provide large amount of active sites for immobilizing capture antibodies. Thus, the redox-active polymer/Au nanocomposites were successfully used to fabricate a simple, and sensitive multiplexed electrochemical immunosensor for carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) detection. The electrochemical signals were simultaneously obtained at two peak potentials and the peak currents were dependent on the concentration of the corresponding analytes. The negligible cross-reactivity between the two analytes makes it possesses potential promise in clinical diagnosis.
electron microscopy (TEM) with a JEOL-100CX electron microscope under 80 kV accelerating voltage. The elemental analysis was studied by an Escalab 250 X-ray photoelectron spectroscope (Thermofisher, American) employing amonochromatic Al Kα radiation. The structure of the nanocomposites was investigated by Fourier transform infrared spectroscopy (FTIR). All electrochemical measurements were carried out on a CHI-832 electrochemical analyzer (Chenhua, Shanghai, China). A three-electrode electrochemical cell was composed of a modified gold electrode (GE, 4 mm in diameter) as the working electrode, a platinum wire and an Ag/AgCl electrode (saturation of KCl) as the auxiliary electrode and the reference electrode, respectively. 2.3. Preparation of POPD/AU nanocomposites The POPD/Au nanocomposites were synthesized by one-step method. In general, 2.32 mg OPD was added into 4.7 mL of the distilled water with stirring. After the OPD was dissolved, 0.3 mL 4% HAuCl4 was added into the above solution. Then the mixture was vigorously stirred at room temperature for 4 h. The resulting POPD/Au nanocomposites were subsequently centrifuged with ethanol and ultrapure water, and re-dispersed in ultrapure water for use. 2.4. Preparation of the poly(VFc-ATP)/AU nanocomposites The poly(VFc-ATP)/Au nanocomposites were also synthesized by copolymerization in one-step process. Briefly, VFc (0.0212 g) and ATP (2 μL, at room temperature) were mixed in 4.7 mL of distilled water with stirring. After 15 min, 0.3 mL 4% HAuCl4 was added into the above solution and vigorously stirred at room temperature for 4 h. The obtained poly(VFc-ATP)/Au nanocomposites were subsequently centrifuged with ethanol and ultrapure water, and re-dispersed in ultrapure water for use. 2.5. Preparation of the poly(2-aminothiophenol) (PATP) The PATP was synthesized according to our previous work (Liu and Ma, 2014).
2. Experimental section 2.6. Preparation of AuNPs 2.1. Reagents and chemicals Human immunoglobulin G (IgG), and albumin from bovine serum (BSA) were purchased from Chengwen Biological Company (Beijing, China). Mouse anti human monoclonal antibody to carcinoembryonic antigen (anti-CEA), mouse anti human alpha fetoprotein monoclonal antibody (anti-AFP), CEA and AFP were obtained from Shanghai Linc-Bio Science Co., Ltd. (Shanghai, China). Sodium borohydride (NaBH4, 98%) was purchased from Sigma (USA). Hydrogen tetrachloroaurate (III) hydrate (HAuCl4 XH2O), vinyl ferrocene (VFc), D-(þ )-glucose, ammonium peroxidisulfate (APS), ascorbic acid (AA), o-phenylenediamine (OPD), uric acid (UA), and 2-aminothiophenol (ATP) were achieved from Alfa Aesar (Tianjin, China). Clinical human serum Samples were obtained from the Capital Normal University Hospital (Beijing, China). KH2PO4 (99%), HCl, Na2HPO4 (99%), KCl, potassium ferricyanide (K3Fe(CN)6), and potassium ferrocyanide (K4Fe(CN)6) were obtained from Beijing Chemical Reagents Company (Beijing, China). All chemicals were of analytical grade and used without further purification. 2.2. Apparatus The distilled water (resistivity 418 MΩ) was used throughout. The morphology of nanocomposites was performed transmission
The AuNPs were synthesized according to the as-reported method (Sun and Ma, 2012). In general, 5 nm AuNPs were prepared at room temperature by adding 1 mL 1% sodium citrate solution to the 100 mL 0.01% HAuCl4 aqueous solution with stirring. After 1 min, 1.6 mL 0.075% NaBH4 (dissolved in 1% sodium citrate solution) was added. The mixture was kept stirring until its color turned to red. The AuNPs were stored at 4 °C for use. 2.7. Preparation of immunosensing probes The immunosensing probes were fabricated by immobilizing labeled anti-CEA and anti-AFP onto POPD/Au and poly(VFc-ATP)/ Au nanocomposites, respectively. Firstly, the labeled anti-CEA (100 μL, 1 mg mL 1) was mixed with the obtained POPD/Au nanocomposites in 1 mL 0.01 M phosphate buffer (PB, pH 7.3) with gently stirring for 12 h. After centrifugation, the obtained POPD/Au–anti-CEA was incubated in a solution of BSA (10%, w/w) for 2 h to block any possible remaining active sites to avoid any nonspecific absorption. Then, the BSA blocked POPD/Au–anti-CEA was re-dispersed in 1 mL 0.01 M PB (pH ¼ 7.3) and stored in 4 °C for use. The poly(VFc-ATP)/Au nanocomposites were immobilized the labeled anti-AFP with the same procedure as preparing POPD/ Au–anti-CEA above. The obtained poly(VFc-ATP)/Au-anti-AFP was re-dispersed in 1 mL 0.01 M PB (pH 7.3) and stored in 4 °C for use.
Z. Liu et al. / Biosensors and Bioelectronics 65 (2015) 307–313
2.8. Preparation of the immunosensor The GE was polished repeatedly using alumina powder and then thoroughly cleaned before use. The immunosensing matrix and fabrication process of immunosensor was the same as our previous work (Liu and Ma, 2013). Briefly, 5 μL 0.5 g L 1 brown solution of PATP was casted on the pretreated GE at room temperature for 8 h to adsorb the conducting long-chain polythiols. After that, the electrode was thoroughly rinsed with deionized water and dried under a stream of nitrogen gas for the immobilization of AuNPs. Next, the AuNPs coated electrode was incubated in a mixed solution of 200 μg mL 1 anti-CEA and anti-AFP solution at 4 °C overnight. Subsequently, excess antibodies were washed away with 0.01 M PB (pH 7.3), and 1% BSA solution was applied to block possible remaining active sites of the electrode. After washing with 0.01 M PB (pH 7.3), the immunosensor was stored at 4 °C for use. 2.9. Simultaneous measurement procedure To perform the immunoreaction and the electrochemical measurement, the immunosensor was first incubated in the mixture of CEA and AFP solution with various concentrations at 37 °C for 1 h. After that, it was incubated in the mixture of 1:1 diluted POPD/Auanti-CEA and poly(VFc-ATP)/Au-anti-AFP. The electrodes were washed with 0.01 M PB (pH 7.3) between the modified steps. Subsequently, differential pulse voltammetric (DPV) was performed to record the electrochemical responses for simultaneous
309
detection of CEA and AFP in phosphate buffered solution (0.1 M PBS, pH 6.98). The DPV measurement was carried out from 0.7 V to 0.8 V (vs. Ag/AgCl) with a pulse amplitude of 50 mV and a pulse width of 50 ms.
3. Results and discussion 3.1. Characterization of redox-active polymer/AU nanocomposites POPD/Au and poly(VFc-ATP)/Au nanocomposites were synthesized by chemical oxidative polymerization which HAuCl4 was used as the oxidant. The mechanism of polymerization as follows. Firstly, HAuCl4 initiates the formation of radical cation of o-phenylenediamine, 2-aminothiophenol and vinyl ferrocene. In the second step, coupling of radical cations occurs, with subsequent formation of the dimer and elimination of two protons. It is then oxidized to be the diradical dication which could couples with a monomer radical cation, resulting in propagation of the chain (Barbero et al., 1989; Tzou and Gregory, 1992). Finally, the resultant polymer (POPD and poly(VFc-ATP)) could be obtained after the chain termination. Accompanied with the formation of Au nanoparticles, the polymer/Au nanocomposites were obtained. FTIR was used to characterize the structure of POPD/Au and poly(VFc-ATP)/Au nanocomposites (Fig. S1 in supplementary information). The FTIR spectrum of POPD/Au shows the characteristic peak at 1475 cm 1 which assigned to the C8C stretching modes of benzenoid rings (Fig. S1A). The band corresponding to C‒
Fig. 1. Typical TEM images of (A) PODP/Au and (B) poly(VFc-ATP)/Au, the scale bar is 200 nm; and the XPS spectrum of (C) PODP/Au and (D) poly(VFc-ATP)/Au.
310
Z. Liu et al. / Biosensors and Bioelectronics 65 (2015) 307–313
N stretching vibration is observed at 1151 cm 1. For poly(VFcATP)/Au (Fig. S1B), the C8C stretching modes of benzenoid rings and quinonoid rings are appeared at 1475 and 1525 cm 1 (Zengin et al., 2002). Compared to the FTIR spectrum of pure PATP (Liu and Ma, 2013), the S3H stretching vibrations of poly(VFc-ATP)/Au at 2576 cm 1 was disappeared, revealing that the Au3S band was formed. Moreover, the characteristic peaks at 1056 and 1105 cm 1come from the cyclopentadienyl of Fc (Roginski et al., 1988). The C3Fe stretching vibration is observed at 482 cm 1. The results confirm that the copolymer of ATP and VFc was formed. The typical TEM images of POPD/Au nanocomposites are shown in Fig. 1A. It was observed that the surface of POPD was densely covered by AuNPs with the size of about 50 nm, which indicated that the efficient reduction of HAuCl4 by amine groups in OPD. And it was suitable to be used as the signal tags because of its excellent redox-activity and large amount of AuNPs to immobilize proteins. The detailed compositional analysis of POPD/Au nanocomposites were further characterized by XPS as showed in Fig. 1C. The XPS peaks of Au 4f, C 1s and N 1s core level regions could be obviously observed. The appearing of Au 4f doublet (85.1 eV and 88.7 eV, in Fig. S2A) was consistent with the Au0 state, revealing the presence of AuNPs on the surface of POPD. For poly(VFc-ATP)/Au, Fig. 1B displays that the nanocomposites were spheres with the size about 50 nm. Moreover, the AuNPs on the surface of copolymer was about 10 nm. To investigated the chemical composition of poly(VFc-ATP)/Au nanocomposites, XPS characterization was also employed. As shown in Fig.1D, the elements of Fe, C, Au, N, and S were included. The appearing of Au 4f doublet also indicated the existence of AuNPs on the surface of copolymer. The binding energies of Fe 2p3/2 and Fe 2p1/2 were observed at 708 and 721 eV (as shown in Fig. S2B), respectively, which assigned to VFc. The peaks of N 1s and S 1 s were appeared at 400 and 164 eV, respectively, showed the existing of ATP. Thus, these results indicated that POPD/Au and poly(VFc-ATP)/Au nanocomposites have been successfully prepared. 3.2. Principle and characterization of the multiplexed electrochemical immunoassay The schematic illustration of the stepwise immunosensor fabrication process is shown in Scheme 1. To fabricate stable and sensitive immunosensing matrix, a conducting polythiols polymer-PATP was used to immobilize on the GE though Au‒S bonds. It plays two important roles. Firstly, the multiple thiol groups of PATP provide tenacious attachment to GE surfaces due to the cooperative binding through multiple thiols bonds (Johnson and Levicky, 2003). Significantly, the remnant (unbound) thiols
moieties of PATP can provide reactive sites for modification with AuNPs to fix more antibodies. Secondly, the excellent conductivity can further enhance the sensitivity of immunosensors since the PATP acts as an effective mediator for electron transfer in redox (Luo and Do, 2004). As a result, the PATP and AuNPs could be utilized to build immunosensing platform. The immunosensing probes were fabricated by choosing excellent redox-active species. With two amine groups on the molecule, the OPD could be oxidized to be conjugated polymer with efficient redox-activity by one-step method which the HAuCl4 was used as the Oxidant. The remained amine groups could reduce the HAuCl4 to be AuNPs immobilized on the surface of AuNPs by covalent bond. For the VFc, it is not convenient to immobilize the biomolecule even if it has good redox-activity. But it could be copolymerized with the ATP to introduce the –SH and amine groups into the chain of the copolymer for the easy modification. By the chemical polymerization method, the HAuCl4 not only could oxidize the VFc and ATP to form copolymer, but also itself can be reduced to be AuNPs which attached on the copolymer by Au3S bond. Thus, The POPD/Au and poly(VFc-ATP)/Au nanocomposites integrate the advantages of simple preparation, easy modification, and excellent electrochemical signals. Subsequently, the immunosensing probes were successfully prepared based on immobilizing labeled anti-CEA and anti-AFP on POPD/Au and poly(VFc-ATP)/Au nanocomposites via AuNPs, respectively. With the sandwich-type assay format, the electrochemical signals were simultaneously obtained at the two different peak potentials. The peak currents were dependent on the concentration of the corresponding antigens, respectively. The DPV measurements were used to monitor the electrochemical behavior of the modification procedure after each step in 5 mM K3[Fe(CN)6]/K4[Fe(CN)6]-PBS (pH 6.98) in Fig. 2A. It was observed that the current of PATP modified electrode (curve b) was lower than that of a bare GE (curve a), which was attributed to the electron transfer ability of PATP is not as good as bare GE. In contrast, the adsorption of AuNPs led to an obvious increase of the peak current (curve c) owing to the formation of an excellent electron-transfer layer and the increase of the area of electrode. Subsequently, it was found that the current response further decreased after the electrode modified with anti-CEA and antiAFP (curve d). After the immunosensor was blocked with BSA (curve e) and incubated in a solution with 1 ng mL 1 CEA and AFP (curve f), the current responses were decreased gradually. This may originate from the insulating BSA, CEA and AFP protein layers on the electrode that retards the electron transfer. After that, the immunosensor was simultaneously incubated in the mixture of 1:1 diluted POPD/Au-anti-CEA and poly(VFc-ATP)/Au-anti-AFP in 0.01 M PB (pH 7.3) for 1 h, so that the two probes could be modified on the electrode. The two pairs of peaks at 0.43 V and 0.2 V were assigned to the redox-activity of PODP and poly (VFc-ATP), respectively, and monitored by the DPV in 0.1 M PBS (pH 6.98) as shown in Fig.2B. The peaks separation between two pairs of the peaks was about 630 mV (ΔEpc). Thus, a simultaneous detection of CEA and AFP was possible, according to the position of the corresponding peak. 3.3. Optimization of conditions for electrochemical detection
Scheme 1. The immunosensor.
fabrication
process
of
the
multiplexed
electrochemical
The pH value of detection solution is an important factor in the performance of immunosensor. It not only has a great influence on the activity of the antigens and antibodies, but also on the immunosensing matrix and redox-active species. As shown in Fig. S3. The current response increased rapidly with increasing pH value from 4.92 to 6.98 and then decreased as pH increase. That is because the activity of the immunoreaction between antibody and antigen could be seriously influenced by pH (Liu et al., 2014). The better the immunoreaction activity, the more redox-active probes
Z. Liu et al. / Biosensors and Bioelectronics 65 (2015) 307–313
311
Fig. 2. DPV (A) of the GE modified with (a) bare GE, (b) PATP, (c) PATP-AuNPs, (d) modified GE incubated with anti-CEA and anti-AFP, (e) blocked with 1% BSA, and (f) incubated with 1 ng mL 1 CEA and AFP after blocked with 1% BSA. All the experiments were performed in 5 mM K3[Fe(CN)6]/K4[Fe(CN)6]-PBS (pH 6.98); and DPV (B) the immunosensor incubated with excess PODP/Au-anti-CEA and poly(VFc-ATP)/Au-anti-AFP in PBS, pH 6.98.
could be modified on the electrode, resulting in the maximal current response. In general, the optimal reactivity of antibody and antigen was at neutral condition (Viswanathan et al., 2006; Chen et al., 2006; Li et al., 2009). Thus, pH 6.98 was optimal and corresponding PBS was used for the following measurements. 3.4. Evaluation of repeatability, cross-talk, cross-reactivity and selectivity As well known, repeatability is a key factor in the practical application for a successful immunosensor. In order to evaluate the repeatability of the immunoassay, two panel immunosensing experiments were carried out by the freshly prepared modified electrodes in five times. The relative standard deviation were 2.8% and 3.6% for 0.1 ng mL 1 CEA and AFP, 4.3% and 3.2% for 10 ng mL 1 CEA and AFP, respectively. The result indicated that the proposed immunosensor displayed good repeatability. To investigate the cross-reactivity between the analytes and noncognate antibodies, two different antibodies of CEA and AFP were immobilized on the electrode, and then immunoreacted with 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100 ng mL 1 of CEA and 1 ng mL 1 AFP (Fig. S4A), or 1 ng mL 1 CEA and 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100 ng mL 1 of AFP (Fig. S4B), respectively. Lastly it was incubated with the immunoprobes (POPD/Au–anti-CEA and poly(VFc-ATP)/ Au–anti-AFP) to carry out the sandwich-type immunoreactions. As expected, the results showed that the single detection of the corresponding analyte is good agreement with the simultaneous detection of two analytes (The DPV measurements are shown in Fig. S4.), indicating that the multiplex detection exhibited low interference with each other and the cross-reactivity between the
two analytes could be ignored. Additionally, no other substrate or mediator was required in the detection procedure, thus, no electrochemical cross-talk occurred. The results indicate that the multiplex detection exhibited low interference with each other and the cross-reactivity between the two analytes was negligible. In order to evaluate the selectivity of the immunosensor, the changes in the current responses of 1 ng mL 1 CEA and AFP induced by the 100 ng mL 1 interferences, such as UA, IgG, glucose, BSA, and AA were measured. As shown in Fig.S5, the interference was negligible, showing that the developed immunosensor with good selectivity. 3.5. Analytical performance of the multiplexed immunoassay Under optimal conditions, the sensitivity and dynamic range of the immunoassay were evaluated the multiplexed immunoassay were evaluated toward CEA and AFP. After incubation with various analyte levels and excess immunosensing probes, the DPV measurements of the modified electrodes were carried out in 0.1 M PBS (pH 6.98). As shown in Fig. 3A, the DPV peak currents of the multiplexed immunoassay increased as the CEA and AFP concentrations increased in the range of 0.01–100 ng mL 1 for both CEA (Fig.3B) and AFP (Fig.3C). The correlation coefficients were 0.9965 and 0.9986, respectively. The detection limits reached 0.006 ng mL 1 for CEA and 0.003 ng mL 1 for AFP at a signal-tonoise ratio of 3. Compared with other multiplex electrochemical immunosensors for the detection of CEA and AFP, this proposed immunosensor displayed a better performance than some earlier reported studies (as shown in Table 1).
Fig. 3. DPV responses (A) and calibration curves for different concentrations of (B) CEA and (C) AFP in PBS, pH 6.98.
312
Z. Liu et al. / Biosensors and Bioelectronics 65 (2015) 307–313
Table 1 Comparison of the analytical performance of some multiplex electrochemical immunosensors Linear range (ng mL 1)
Redox-active species
CGS-MB/CGS-PB FCA-MPS/HRP-MPS Hydroquinone Thi@HPtNP/Fc@HPtNP Fc/Thi AuNPs-PB/Thi@GS PODP/poly(VFc-ATP)
Detection limit (ng mL 1)
Refs.
CEA
AFP
CEA
AFP
0.5–80 0.5–45 25–150 0.5–50 0.01–50 0.6–80 0.01–100
0.5–50 1–90 10–200 0.3–45 0.01–50 0.6–80 0.01–100
0.05 0.2 1.2 0.08 0.01 0.12 0.006
0.1 0.5 1.0 0.05 0.01 0.08 0.003
Kong et al. (2013) Lin et al. (2011) Wilson (2005) Song et al. (2010) Lai et al. (2012) Feng et al. (2014) This work
Table 2 Assay results of clinical serum samples using the proposed and reference methods. Sample no.
1 2 3 4 5 6 7 8 9 10
Proposed method (ng mL 1)
ELISA (ng mL 1)
Relative error (%)
CEA
AFP
CEA
AFP
CEA
AFP
3.06 70.04 1.36 70.03 1.04 70.04 2.84 70.05 0.87 70.04 0.97 70.05 0.69 70.04 3.26 70.06 4.89 70.05 1.05 70.03
4.20 7 0.05 2.067 0.05 3.23 7 0.07 2.87 7 0.06 4.62 7 0.03 0.25 7 0.02 0.477 0.02 0.34 7 0.03 0.357 0.02 0.62 7 0.03
2.977 0.05 1.417 0.06 0.98 7 0.07 2.777 0.05 0.93 7 0.03 1.03 7 0.04 0.667 0.03 3.187 0.06 4.99 7 0.05 1.09 7 0.04
4.34 7 0.06 1.98 7 0.07 3.167 0.05 2.98 7 0.08 4.79 7 0.06 0.247 0.02 0.447 0.03 0.36 7 0.02 0.377 0.02 0.59 7 0.03
3.03 3.55 6.12 2.53 5.38 5.82 4.55 2.52 2.00 3.67
3.23 4.04 2.22 3.69 3.55 4.17 6.82 5.56 5.41 5.08
3.6. Application in analysis of serum samples In order to evaluate the effect of the present method to detect real samples, the immunoassay for clinical serum samples was investigated by analyzing five real samples in comparison with the enzyme-linked immunosorbent assay (ELISA) method. As shown in Table 2, the results obtained by proposed immunosensor are well consistent with the data determined by ELISA, showing that present method can be practically applied in clinical analysis for simultaneous determination of CEA and AFP.
4. Conclusion In this work, POPD/Au and poly(VFc-ATP)/Au) nanocomposites were synthesized by one-step method and used to fabricate a simple and sensitive electrochemical immunoassay for simultaneous detection of multiple biomarkers. As redox-active species, the POPD/Au and poly(VFc-ATP)/Au) nanocomposites integrated the advantages of excellent redox-activity, facile synthesis, easy modification, good biocompatibility and stability, which were successfully used as the immunosensing probes. The experimental results showed that the proposed immunosensor enabled simultaneous monitoring of CEA and AFP in a single run. In particular, the convenient operation and ultrahigh sensitivity of the proposed immunoassay method provided a promising potential in clinical applications.
Acknowledgments This research was financed by Grants from the National Natural Science Foundation of China (21273153), the Beijing Natural Science Foundation (2132008), the Project of Construction of
Innovative Teams and Teacher Career Development for Universities and Colleges Under Beijing Municipality (IDHT20140512), and the Research Base Construction Projects of Beijing Municipal Education Commission.
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.2014.10.012.
References Baker, C.O., Shedd, B., Tseng, R.J., Martinez-Morales, A.A., Ozkan, C.S., Ozkan, M., Yang, Y., Kaner, R.B., 2011. ACS Nano 5, 3469–3474. Barbero, C., Silber, J.J., Sereno, L., 1989. J. Electroanal. Chem. 236, 333–352. Chen, W., Lu, Z.S., Li, C.M., 2008. Anal. Chem. 80, 8485–8492. Chen, J., Tang, J.H., Yan, F., Ju, H.X., 2006. Biomaterials 27, 2313–2321. Cosnier, S., Lonescu, R.E., Holzinger, M., 2008. J. Mater. Chem. 18, 5129–5133. Cui, R., Huang, H., Yin, Z., Gao, D., Zhu, J.J., 2008. Biosens. Bioelectron. 23, 1666–1673. Devi, R., Relhan, S., Pundir, C.S., 2013. Sens. Actuators B 186, 17–26. Elyasi, M., Khalilzadeh, M.A., Karimi-Maleh, H., 2013. Food Chem. 141, 4311–4317. Ensafi, A.A., Karimi-Maleh, H., Mallakpour, S., Hatami, M., 2011. Sens. Actuators B 155, 464–472. Feng, D.X., Li, L.H., Han, X.W., Fang, X., Li, X.Z., Zhang, Y.Z., 2014. Sens. Actuators B 201, 360–368. Feng, L.Y., Chen, Y., Ren, J.S., Qu, X.G., 2011. Biomaterials 32, 2930–2937. Gao, Q., Han, J.M., Ma, Z.F., 2013. Biosens. Bioelectron. 49, 323–328. Hao, R., Xing, R.J., Xu, Z.C., Hou, Y.L., Gao, S., Sun, S.H., 2010. Adv. Mater. 22, 2729–2742. Harper, J.C., Polsky, R., Wheeler, D.R., Dirk, S.M., Brozik, S.M., 2007. Langmuir 23, 8285–8287. Ivanov, S., Tsakova, V., Bund, A., 2013. Electrochim. Acta 90, 157–165. Jeong, B.J., Akter, R., Han, O.H., Rhee, C.K., Rahman, M.A., 2013. Anal. Chem. 85, 1784–1791. Jia, X.L., Liu, Z.M., Liu, N., Ma, Z.F., 2014. Biosens. Bioelectron. 53, 160–166. Jia, W.Z., Su, L., Lei, Y., 2011. Biosens. Bioelectron. 30, 158–164. Johnson, P.A., Levicky, R., 2003. Langmuir 19, 10288–10294.
Z. Liu et al. / Biosensors and Bioelectronics 65 (2015) 307–313
Kong, F.Y., Li, X.R., Zhao, W.W., Xu, J.J., Chen, H.Y., 2012. Electrochem. Commun. 14, 59–62. Kong, F.Y., Xu, B.Y., Du, Y., Xu, J.J., Chen, H.Y., 2013. Chem. Commun. 49, 1052–1054. Karimi-Maleh, H., Biparva, P., Hatami, M., 2013. Biosens. Bioelectron. 48, 270–275. Lai, W.Q., Zhuang, J.Y., Tang, J., Chen, G.N., Tang, D.P., 2012. Microchim. Acta 178, 357–365. Li, Q.F., Zeng, L.X., Wang, J.C., Tang, D.P., Liu, B.Q., Chen, G.N., Wei, M.D., 2011. ACS Appl. Mater. Interfaces 3, 1366–1373. Li, X.H., Dai, L., Liu, Y., Chen, X.J., Yan, W., Jiang, L.P., Zhu, J.J., 2009. Adv. Funct. Mater. 19, 3120–3128. Lin, J.H., Wei, Z.J., Mao, C.M., 2011. Biosens. Bioelectron. 29, 40–45. Liu, N., Ma, Z.F., 2014. Biosens. Bioelectron. 51, 184–190. Liu, N., Liu, Z.M., Han, H.L., Ma, Z.F., 2014. J. Mater. Chem. B 2, 3292–3298. Liu, Z.M., Ma, Z.F., 2013. Biosens. Bioelectron. 46, 1–7. Luo, Y.C., Do, J.S., 2004. Biosens. Bioelectron. 20, 15–23. Mazeiko, V., Kausaite-Minkstimiene, A., Ramanaviciene, A., Balevicius, Z., Ramanavicius, A., 2013. Sens. Actuators B 189, 187–193. Moradi, R., Sebt, S.A., Karimi-Maleh, H., Sadeghi, R., Karimi, F., Bahari, A., Arabi, H., 2013. Phys.Chem. Chem. Phys. 15, 5888–5897. Pillalamarri, S.K., Blum, F.D., Tokuhiro, A.T., Bertino, M.F., 2005. Chem. Mater. 17, 5941–5944. Roginski, R.T., Shapley, J.R., Drickamer, H.G., 1988. J. Phys. Chem. 92, 4316–4319. Rosi, N.L., Mirkin, C.A., 2005. Chem. Rev. 105, 1547–1562.
313
Sun, X.B., Ma, Z.F., 2012. Biosens. Bioelectron. 35, 470–474. Song, Z.J., Yuan, R., Chai, Y.Q., Zhuo, Y., Jiang, W., Su, H.L., Che, X., Li, J.J., 2010. Chem. Commun. 46, 6750–6752. Tavana, T., Khalilzadeh, M.A., Karimi-Maleh, H., Ensafi, A.A., Beitollahi, H., Zareyee, D., 2012. J. Mol. Liq. 168, 69–74. Tzou, K., Gregory, R.V., 1992. Synth. Met. 47, 267–277. Viswanathan, S., Wu, L.C., Huang, M.R., Ho, J.A., 2006. Anal. Chem. 78, 1115–1121. Wang, S.P., Wu, Z.S., Qu, F.L., Zhang, S.B., Shen, G.L., Yu, R.Q., 2008. Biosens. Bioelectron. 24, 1020–1026. Wei, Q., Mao, K.X., Wu, D., Dsi, Y.X., Yang, J., Du, B., Yang, M.H., Li, H., 2010. Sens. Actuators B 149, 314–318. Wilson, M.S., 2005. Anal. Chem. 77, 1496–1502. Yan, W., Chen, X.J., Li, X.H., Feng, X.M., Zhu, J.J., 2008. J. Phys. Chem. B 112, 1275–1281. Yang, Z.H., Chai, Y.Q., Yuan, R., Zhuo, Y., Li, Y., Han, J., Liao, N., 2014. Sens. Actuators B 193, 461–466. Zang, S., Liu, Y.J., Lin, M.H., Kang, J.L., Sun, Y.M., Lei, H.T., 2013. Electrochim. Acta 90, 246–253. Zengin, H., Zhou, W., Jin, J., Czerw, R., Smith, J.D.W., Echegoyen, L., Carroll, D.L., Foulger, S.H., Ballato, J., 2002. Adv. Mater. 14, 1480–1483.
