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Multifunctional reduced graphene oxide trigged chemiluminescence resonance energy transfer: Novel signal amplification strategy for photoelectrochemical immunoassay of squamous cell carcinoma antigen
Biosensors and Bioelectronics 79 (2016) 55–62
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Multifunctional reduced graphene oxide trigged chemiluminescence resonance energy transfer: Novel signal amplification strategy for photoelectrochemical immunoassay of squamous cell carcinoma antigen Yan Zhang a, Guoqiang Sun a, Hongmei Yang a, Jinghua Yu a, Mei Yan a,n, Xianrang Song b,n a b
School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China Shandong Provincial Key Laboratory of Radiation Oncology, Shandong Cancer Hospital and Institute, Jinan 250117, China
art ic l e i nf o
a b s t r a c t
Article history: Received 24 September 2015 Received in revised form 28 November 2015 Accepted 5 December 2015 Available online 8 December 2015
Herein, a photoelectrochemical (PEC) immunoassay is constructed for squamous cell carcinoma antigen (SCCA) detection using zinc oxide nanoflower-bismuth sulfide (Bi2S3) composites as photoactive materials and reduced graphene oxide (rGO) as signal labels. Horseradish peroxidase is used to block sites against nonspecific binding, and then participated in luminol-based chemiluminescence (CL) system. The induced CL emission is acted as an inner light source to excite photoactive materials, simplifying the instrument. A novel signal amplification strategy is stem from rGO because of the rGO acts as an energy acceptor, while luminol serves as a donor to rGO, triggering the CL resonance energy transfer phenomenon between luminol and rGO. Thus, the efficient CL emission to photoactive materials decreases. Furthermore, the signal amplification caused by rGO labeled signal antibodies is related to photogenerated electron–hole pairs: perfect matching of energy levels between rGO and Bi2S3 makes rGO a sink to capture photogenerated electrons from Bi2S3; the increased steric hindrance hinders the electron donor to the surface of Bi2S3 for reaction with the photogenerated holes. On the basis of the novel signal amplification strategy, the proposed immunosensor exhibits excellent analytical performance for PEC detection of SCCA, ranging from 0.8 pg mL 1 to 80 ng mL 1 with a low detection limit of 0.21 pg mL 1. Meanwhile, the designed signal amplification strategy provides a general format for future development of PEC assays. & 2015 Elsevier B.V. All rights reserved.
Keywords: Chemiluminescence resonance energy transfer Reduced graphene oxide Zinc oxide nanoflower Bismuth sulfide Squamous cell carcinoma antigen
1. Introduction Squamous cell carcinoma antigen (SCCA), a glycoprotein with molecular mass of ∼45 kDa, was isolated from human cervical squamous carcinoma cells and served as a circulating marker for more advanced squamous cell tumors of the cervix, lung, and oropharynx (Bastide et al., 2010; Giannelli et al., 2007; Kato and Torigoe, 1977). The serum level of SCCA was increased in parallel to the growth of the tumor size or the recurrence of the disease (Hefler et al., 2005; Schedel et al., 2011). At present, chemiluminescence enzyme immunoassay, radio-immunoassay and enzymelinked immunosorbent assay were usually applied to detect SCCA in serum (Çataltepe et al., 2000; Chang et al., 2004; Zhang and Qi, 2011). However, several shortcomings of these methods limit its extensive application, such as poor reproducibility, low sensitivity, n
Corresponding authors. E-mail addresses: [email protected] (M. Yan), [email protected] (X. Song).
http://dx.doi.org/10.1016/j.bios.2015.12.008 0956-5663/& 2015 Elsevier B.V. All rights reserved.
radiation hazards, require expensive equipments and time-consuming. Therefore, there was an utmost requirement of low cost, precise, and high sensitivity immunoassay for SCCA detection. Photoelectrochemical (PEC) immunosensing was a recent yet vibrantly emergent and promising analytical method for rapid and high-throughput biological assays (Li et al., 2014, 2012; Yu et al., 2014). In the case of the PEC detection process, light was used to excite active species, and current was used as the detection signal (Zhao et al., 2012). Benefitting from two separate forms of signals for excitation and detection, the PEC immunosensor possessed the advantages of both high selectivity of immunoassay and sensitivity of PEC analysis (Wang et al., 2012). Meanwhile, the use of electronic readout made the instrument simpler, cheaper, and easier to miniaturize than that of optical methods (Wang et al., 2014). Over the years, PEC sensors have been employed to analyze various compounds (An et al., 2010; Bellani et al., 2015; Ma et al., 2014; Tang et al., 2014). In PEC immunoassay, physical light source and monochromator were usually needed to bring appropriate light emission (Huo
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et al., 2015), which made the instrument complicated and departed from the portable trend for biosensor. Chemiluminescent (CL) emission, a process in which excited molecules or atoms generated from chemical reactions released the excess of energy in light form, was a promising substitute for the physical light source. This was attributed to the fact that different CL systems or adjusting the reaction conditions of CL systems could bring light emission with various wavelengths (Abdolmohammad-Zadeh and Rahimpour, 2015; Chen et al., 2013; Xu et al., 2014). The oxidation of luminol with H2O2 was one of the most commonly known CL reactions (Iranifam and Kharameh, 2015; Khajvand et al., 2015; Li et al., 2011). Meanwhile, this CL system had been applied in a CL resonance energy transfer (CRET), in which the CRET involved a nonradiative dipole–dipole transfer of energy from a donor to an acceptor (Bi et al., 2015; Lee et al., 2012). CRET occurred by the oxidation of a luminescent substrate without an excitation source, thus, the signal-to-noise ratio and sensitivity of the detection could be improved in bioassay. From the above, CRET could be a promising signal amplification strategy in PEC immunoassay. In this work, zinc oxide nanoflower-bismuth sulfide composites (ZNF@Bi2S3) were used as photoactive materials because of combining fast electron transfer rate of ZNF and excellent PEC performance of Bi2S3 (Balachandran and Swaminathan, 2013; Manna et al., 2014; Zhang et al., 2014). Meanwhile, the presence of Bi2S3 nanoparticles avoided the use of Cd-contained quantum dots, which suffered from photocorrosion and cadmium contamination (Liang et al., 2012). After immobilizing of capture antibodies (Ab1), horseradish peroxidase (HRP) was dropped onto the electrode surface. The HRP could not only act as a substitute for bovine serum albumin (BSA) to block sites against nonspecific binding, but also participate in the luminol-HRP-H2O2 CL system to induce inner light source. Reduced graphene oxide labeled signal antibodies (rGO-Ab2) was introduced into the immunosensor via immunoreactions. Excellent analytical performance of the immunosensor was attributed to the excellent PEC performance of ZNF@Bi2S3 and the multiple function of rGO. Firstly, the rGO acted as an energy acceptor, while luminol served as a donor to rGO, triggering the CRET phenomenon between luminol and rGO. The reduction of CL emission to ZNF@Bi2S3 composites was controlled by rGO, which decreased the photocurrent intensity. Secondly, an additional electron transfer pathway from Bi2S3 to rGO decreased the photogenerated electrons to electrode. Thirdly, the rGO-Ab2 increased the steric hindrance and obstructed the diffusion of H2O2 to the surface of Bi2S3 for reaction with the photogenerated holes. On the basis of the novel signal amplification strategy, a sensitive PEC immunoassay for SCCA detection was proposed for the first time.
2. Experimental section 2.1. Preparation of chitosan functionalized ZNF@Bi2S3 composites The materials and apparatus were shown in supporting information. The ZNF was fabricated as follow: 0.0395 g of zinc acetate was dissolved into 15 mL of ultrapure water under stirring. After 15 min stirring, an aqueous solution of NaOH (6.9 mM, 0.6 mL) was added. The obtained solution was then transferred into Teflon-lined stainless steel autoclaves and maintained at 120 °C for 45 min. After the reaction was completed, the products were collected by centrifugation and washed with ethanol several times, and finally dried at 60 °C in air. The Bi2S3 nanoparticles were grown on surface of ZNF by a hydrothermal method. In a typical process, 0.21 mM bismuth nitrate pentahydrate and 0.16 mM sodium thiosulfate pentahydrate were added into a given amount (25 mL) of ultrapure water. After
adding 50 mg of as-synthesized ZNF, the mixture was transferred into Teflon-lined stainless steel autoclaves kept at 110 °C for 4 h. Finally, the obtained ZNF@Bi2S3 composites were purified by centrifugation, and further dried at 40 °C in a vacuum oven. 10 mg of ZNF@Bi2S3 composites were dissolved into 1.0 mL of 1% chitosan solution and stirred for 2 h. Subsequently, the precipitation was collected and washed with water several times, chitosan functionalized ZNF@Bi2S3 composites were obtained and then re-dissolved into 1.0 mL of water for further use. 2.2. Preparation of rGO-Ab2 Ab2 was immobilized on the surface of rGO with the aid of thionine. Typically, 0.1 mg of rGO (the synthesis process was shown in Supporting information) was dispersed in 1.0 mL of pH 7.4 phosphate buffer saline (PBS), followed by addition of 1.0 mL of thionine solution (1.0 mM). After stirring at room temperature for 12 h, the resultant mixture was centrifuged (10,000 rpm for 10 min) and washed with PBS (pH 7.4) for five times to remove the unbounded thionine. In this process, the amino of thionine could attach on to the rGO through π–π stacking (Liu et al., 2014). The obtained product was re-dispersed in 1.0 mL of PBS (pH 7.4). Then, 100 μL of glutaraldehyde (GA, 2.5 wt%) which could bridge the rGO/thionine and Ab2 with covalent bond force was added into the above solution and allowed to react for 1.0 h at room temperature. After repeating a centrifugation (10,000 rpm for 10 min) and washing procedure for five times, the mixture were re-dissolved into 1.0 mL of PBS (pH 7.4). Then, 100 μL of Ab2 (20 μg mL 1) was added and incubated for 3 h at room temperature, followed by centrifugation (10,000 rpm for 10 min) and washing with PBS (pH 7.4) for five times to remove free antibodies. Finally, 1.0 wt% BSA solution was added to block possible remaining active sites, the resulting rGO-Ab2 was washed with PBS for five times and redispersed in 1.0 mL of PBS (pH 7.4). 2.3. Construction of the immunosensor platform The fabrication process of the immunosensor on an indium tin oxide (ITO) device (the synthesis details of the ITO device was exhibited in supporting information) was shown in Scheme 1. For preparation of ZNF@Bi2S3 modified ITO electrode, the ITO electrode was first surface functioned with amino according to the literature (Aziz et al., 2008), and then modified with GA (2.5 wt%). Subsequently, 10 μL of chitosan functionalized ZnO@Bi2S3 composites was dropped on the electrode surface and incubated for 2 h. The modified electrode was rinsed with ultrapure water and dried at room temperature. Ab1 was conjugated the modified electrode with the aid of GA. After incubating with GA for 1 h, 5.0 μL of Ab1 (50 μg mL 1) was applied to the electrode and reacted at room temperature for 1 h. Then, the electrode was rinsed with PBS (pH 7.4) several times to eliminate excess antibodies. At last, the modified immunosensor was incubated with 10 μL of HRP solution (1.0 mg mL 1) at room temperature for 2 h to block the possible remaining active sites against non-specific absorption. The resulting electrode was stored at 4 °C when not in use. 2.4. PEC immunoassay procedure for SCCA In this study, SCCA was assayed with a sandwich-type immunoassay format using Ab1/ZNF@Bi2S3/ITO as immunosensing probe and rGO-Ab2 as molecular tags. Initially, 5.0 μL of sample solution containing different concentration of SCCA was dropped onto the working electrode and allowed to incubate for 40 min at room temperature, followed by washing with pH 7.4 PBS. Subsequently, 5.0 μL of rGO-Ab2 was added onto the electrode surface
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Scheme 1. The fabrication process of the (A) ITO device and (B) PEC immunosensor.
and incubated for 40 min. After washing with PBS, 40 μL of Tris– HCl buffer solution (pH 8.5, 0.1 M) containing 6.0 mM H2O2, 0.5 mM p-iodo-phenol and 0.5 mM luminol was added into the PEC cell. Finally, the PEC measurements were performed at an applied potential of 0 V.
3. Results and discussion 3.1. Characterization of the immunosensor platform The surface morphology of the etched ITO was an important factor affecting its sensing performance, and scanning electron microscopy (SEM) was utilized to characterize the morphologies and edge roughness of the etched ITO layer. As shown in Fig. S1, the etched electrode owned the good homogeneity, indicating that the surface etching was a promising technique for fabricating ITO device. The morphology of ZNF and ZNF@Bi2S3 composites were elucidated by SEM images. As shown in Fig. 1A, the products were formed as a flower-like ensemble that was composed of needlelike microstructures. Fig. 1B and C were high high-magnification SEM images of the as-synthesized ZNF. The total size of the products was 1 μm, while the length of the needles was 400 nm. The flower-like structures made the ZNF owned large surface area, which provided abundant sites for Bi2S3 loading. The SEM images of ZNF@Bi2S3 composites were exhibited in Fig. 1D–F. As shown in Fig. 1D, after growth of Bi2S3 on ZNF, no apparatus size change was observed. The high-magnification SEM images (Fig. 1E and F)
exhibited that the surface of the composites became rough, which was mainly attributed to the successfully deposition of Bi2S3. Fig. 2A was a transmission electron microscope (TEM) image of pure ZNF which composed with nanoneedles. The Fig. 2D manifested the TEM image of the Bi2S3 nanoparticles of ∼10 nm sizes. In addition, energy dispersive spectrometer (EDS) was also used to characterize the ZNF and ZNF@Bi2S3 composites. The EDS result of ZNF was shown in Fig. 2B, the sample consisted of Zn, O elements, and the atomic percentage of ZnO in ZNF was 36.63% while O was 63.37%. As expected, the elements of Bi, S, Zn, O were resolved for ZNF@Bi2S3 composites (Fig. 2E), and the atomic percentage were 1.12%, 0.69%, 31.34%, and 66.85%, respectively, indicating the successfully synthesis of the ZNF@Bi2S3 composites. The X-ray diffraction (XRD) patterns of pure ZNF and ZNF@Bi2S3 composites were shown in Fig. 2C. The diffraction peaks in the range of 20°o2θ o70° could be indexed as (100), (002), (101), (102), (110), (103), (200), (112), and (201) planes of wurtzite hexagonal phase ZnO, which were consistent with the value in the standard card (JCPDS 36-1451). The higher intensity of the (002) peak indicated the ZNF were grown preferably along c axis, and the exhibited sharp peaks indicated that the nanostructures possess large crystalline domains as well as a high degree of crystallinity. Meanwhile, No other peaks that corresponded to impurities were found in the spectrum. After deposition of Bi2S3, peaks corresponding to ZNF became weak and no evident diffraction peaks were observed for the Bi2S3 of the composite film of ZNF@Bi2S3. There may be two reasons caused for the absence of Bi2S3 peaks, the one was that only few Bi2S3 nanoparticles were attached to the ZNF, the other one was that the Bi2S3 were highly dispersed on ZNF surface.
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Fig. 1. SEM images of (A–C) ZNF and (D–F) ZNF@Bi2S3 composites under different magnification.
Fig. 2. (A) TEM image and (B) EDS of ZNF; (D) TEM image and (E) EDS of ZNF@Bi2S3 composites; (C) XRD patterns and (F) UV–vis absorption spectra of ZNF and ZNF@Bi2S3 composites.
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Fig. 3. (A) TEM image of rGO; (B) XRD patterns, (C) UV–vis absorption spectra and (D) Raman spectra of GO and rGO.
The light absorption properties of the as-synthesized products were characterized by the Ultraviolet–visible (UV–vis) absorption spectra. As shown in Fig. 2F, the bare ZNF only absorbed the UV light with wavelength shorter than 365 nm, which was attributed to its large energy gap ( 3.7 eV). Obviously, the ZNF@Bi2S3 composites greatly enhanced the UV light absorbance and extended the light utilization from UV to visible range. The decorated Bi2S3 were able to efficiently promote the visible light absorption of ZNF@Bi2S3 composites, contributing to the high absorption coefficient of Bi2S3 nanoparticles. Meanwhile, we compared PEC performance of ZNF, Bi2S3 and ZNF@Bi2S3 composites. As shown in Fig. S2, the electrode modified by ZNF@Bi2S3 composites showed largest photocurrent response, demonstrated that the sensitivity of ZNF@Bi2S3 composites was improved by introducing Bi2S3. 3.2. Characterization of the signal labels The TEM image (Fig. 3A) displayed a view of rGO clearly illustrating typical flake-like wrinkled shapes of graphene with irregular size. Many wrinkles and folds revealed that the two-dimensional structure of rGO sheet was flexible. The XRD patterns and UV–vis absorption spectra of GO and rGO were exhibited in Fig. 3B and C, respectively. The XRD pattern of GO (curve a) showed a strong and sharp diffraction peak at 2θ of 13.0°, which was in agreement with the lamellar structure of GO. After the chemical reduction of GO, a broad peak around 25° could be seen for rGO (curve b), which was due to the deep reduction of GO, indicating that most oxygen functional groups had been removed.
In the UV–vis absorption spectrum of GO (curve a), there were two absorption features: a peak at 230 nm due to the π-π* transition of C ¼C bond, and a band at a 299 nm corresponding to the n-p* transition of the C ¼O bond. Meanwhile, the typical band for GO with a maximum at 230 nm gradually red-shifted to around 269 nm after the hydrothermal reduction process (curve b) owing to n-π* transitions of aromatic C ¼C bonds, suggesting that the electronic conjugation within rGO was restored after reduction. The formation of rGO was also evidenced by the corresponding Raman spectra. It was proved that graphene exhibited two characteristic main peaks: the G mode at 1575 cm 1, arising from the first order scattering of the E2g phonons of the sp2 C atoms; the D mode at 1350 cm 1, arising from a breathing mode of the κpoint phonons of A1g symmetry. As shown in Fig. 3D, GO exhibited a D band at 1350 cm 1 and a G band at 1613 cm 1, while the corresponding bands of rGO were 1350 cm 1 and 1600 cm 1, respectively. The red-shifted of G band from 1616 to 1603 cm 1 was attributed to the high ability for recovery of the hexagonal network of carbon atom. Furthermore, the rGO showed relative higher intensity of D to G band than that of GO, confirmed that the rGO was formed after a hydrothermal process. 3.3. The feasible of the signal amplification strategy To confirm the effectiveness of rGO as an acceptor in CRET, we investigated CRET by adding SCCA and rGO-Ab2 in sequence to a reaction solution containing Ab1-HRP. As shown in Fig. 4A, the luminol-HRP-H2O2 CL system showed a high CL intensity (curve a).
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Fig. 4. (A) CL spectra of luminol-HRP-H2O2 system (a) before and after incubated with (b) 0.1 ng mL 1 SCCA, (c) 0.1 ng mL 1 SCCA þrGO-Ab2, (d) rGO-Ab2 only and (e) 1.0 ng mL 1 SCCA þ rGO-Ab2; (B) EIS of (a) bare ITO, ZNF@Bi2S3/ITO (b) before and after (c) incubated with Ab1, (d) blocked with HRP, (e) incubated with SCCA and (f) rGO-Ab2; (C) the photocurrent responses of Ab1 modified electrode (a) before and after (b) blocked with HRP, incubated with (c) 0.1 ng mL 1 SCCA, (d) 0.1 ng mL 1 SCCA þrGO-Ab2 and (e) 1.0 ng mL 1 SCCAþ rGO-Ab2; (D) logarithmic calibration curve for SCCA.
No apparent change of CL intensity (curve b) was observed after introducing of SCCA. Then, rGO-Ab2 was added to incubate with SCCA, and the CL intensity decreased significantly (curve c). If we added rGO-Ab2 to the solution directly without SCCA, the CL quenching was not occurred (curve d). This was attributed SCCA triggered rGO-Ab2 to undergo sandwich-type immunoreactions, serving as a bridge short enough for CRET to occur, indicated that the proximity of HRP to rGO was critical for efficient energy transfer to rGO from excited luminol. With the increasing of concentration of SCCA, more acceptors (rGO) was introduced into the system, thus, in turn, enabling higher energy transfer from the donor to the acceptor and more quenching of luminol CL (curve e). These observations confirmed that the CRET was feasible and could used to construct immunosensor. 3.4. Electrochemical impedance spectroscopy (EIS) of the immunosensor EIS was an effective method to monitor the changes of interfacial properties, allowing the understanding of chemical transformation and processes associated with the conductive electrode surface (Zhang et al., 2013). In EIS, the impedance spectrum included a semicircle portion at high frequencies corresponding to an electron transfer limited process and a linear portion at low frequencies represented a diffusion-limited electrochemical process. The semicircle diameter was equal to the electron-transfer resistance (Ret), which reflected the electron transfer kinetics of the redox probe at the electrode interface. Each reaction step of the modified electrode was analyzed by EIS in the frequency range
0.1–105 Hz, and in PBS (pH 7.4) containing [Fe(CN)6]3 /4 (5.0 mM) as probe. As shown in Fig. 4B, a relatively large semicircle diameter was observed at bare ITO (curve a). Subsequently, when ZNF@Bi2S3 composites were loaded on the electrode surface, the interfacial resistance increased (curve b) which suggested that the composites were successfully immobilized on the surface and the formed compact film hindered the access of the redox probe to the electrode surface. After the electrode was conjugated with Ab1, blocked with HRP, and subsequently incubated with SCCA, the Ret increased gradually as the experiment proceeds (curves c–e). This was attributed to the proteins with nonconductive property acted as an inert layer to obstruct electron transfer from the redox probe to the electrode surface, testifying the successful introduction of these substances into immunosensor. Finally, rGO-Ab2 was introduced into the immunosensor via immunoreactions and gave rise to the resistance (curve f), turning out the successfully immobilized of rGOx-Ab2. The increase of Ret verified the successful fabrication of the immunosensor. 3.5. PEC performance of the immunosensor The fabrication of the immunosensor was also be monitored by PEC experiments. The photocurrent responses of different electrodes were detected in mixed solution containing luminal and H2O2, and the results were shown in Fig. 4C. A CL emission, which was produced from the oxidation of luminol by H2O2 in the presence of HRP, was used as light source to excite ZNF@Bi2S3 composites due to the natural overlap between the absorption spectrum of ZNF@Bi2S3 and the CL spectrum of luminol-HRP-H2O2.
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Before blocked the immunosensor with HRP, almost no light source was produced, resulted in a negligible photocurrent (curve a). Then, HRP was introduced into the immunosensor to block sites against nonspecific binding, and then enhanced the CL emission intensity. A large photocurrent was observed (curve b), which was attributed to the ZNF@Bi2S3 composites owned good PEC performance and could be excited by CL emission, indicating the feasibility of the CL-excited PEC immunoassay. After incubating with SCCA, the photocurrent intensity decreased (curve c). This could be explained by the fact that the proteins act as an inert layer hindered the diffusion of H2O2 to the surface of Bi2S3 for efficient scavenging of photogenerated holes. In the final step, the rGO-Ab2 bioconjugates were immobilized on the developed immunosensor through specific antibody-antigen immunoreactions, the photocurrent decreased significantly (curve d). The decrement reasons of photocurrent response was concluded as follow: (i) the rGO acted as energy acceptor to compete the CL emission with ZNF@Bi2S3 composites, decreasing the effective of use light source; (ii) the rGO served as electrons sink to capture photogenerated electrons from Bi2S3 due to the perfect matching of energy levels between rGO and Bi2S3, decreasing the electrons amount to electrode; (iii) the introduced rGO-Ab2 hindered the transfer of electron donor to Bi2S3 surface. The detail charge transfer processes was shown in Scheme S1. Meanwhile, the photocurrent responses of the platform incubated with different signal labels were determined. As shown in Fig. S3, the platform incubated with rGO-Ab2 bioconjugates showed the lowest photocurrent response, confirmed that the signal amplification strategy induced by rGO was feasible. Furthermore, the photocurrent decreased with the increasing of the concentration of SCCA (curve e). With the increasing of concentration of SCCA, the amount of rGO-Ab2 connected to immunosensor increased, leading to a further decrement of photocurrent intensity. These results confirmed that the immunosensor was successfully constructed and could be applied to the sensitive determination of SCCA.
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3.7. Application in real samples In order to evaluate the analytical reliability and application potential of the designed immunosensing method, the recovery of the different concentrations of SCCA in serum was measured by standard addition methods. The spiked human serum samples were prepared through adding different amounts of SCCA into the human serum samples. The results were shown in Table S2. The recoveries of SCCA in human serum samples were obtained in the range of 95.2–104.2%. The results demonstrated the designed biosensor could be suitable to the clinical determination of SCCA levels in serum samples.
4. Conclusion In this work, SCCA was detected using a PEC method. ZNF@Bi2S2 composites were modified on an ITO device and served as photoactive materials. HRP was instead of BSA to block sites against non-specific binding, and then participated in the luminol-HRP-H2O2 CL system to provide inner light source, simplifying the instrument. A novel signal amplification strategy caused by rGO-Ab2 was adapted to enhance the analytical performance. Typically, rGO, as an energy acceptor, trigged the CRET phenomenon between luminol and rGO, decreasing the efficient CL emission to ZNF@Bi2S3 composites. Duo to perfect matching of energy levels between rGO and Bi2S3, the rGO acted as an electrons sink to capture photogenerated electrons from Bi2S3, decreasing the electrons amount to electrode surface. Finally, the introduced rGO-Ab2 increased the steric hindrance and hindered the electron donor to the surface of Bi2S3 for reaction with the photogenerated holes. Using this novel signal amplification strategy, the resulting immunosensor exhibited wide linear relation, low detection limit, high sensitivity and specificity, good reproducibility and well feasibility in real samples determination.
3.6. Analytical performance
Acknowledgments
In this work, SCCA detection was based a novel signal amplification strategy trigged by rGO. Under optimal conditions (details in Supporting information), after introduction of the rGO labeled Ab2, CRET, new photogenerated electrons transfer pathway and increased steric hindrance were all occurred to decrease the photocurrent response. Therefore, by tracking the photocurrent response related to the amount of SCCA, a sensitive immunoassay was accomplished. Fig. 4D depicted the photocurrent responses of the immunosensor to different concentrations of SCCA. With the increasing the concentration of SCCA, more rGO was loaded onto the electron surface, and then the photocurrent signal reduced regularly. The photocurrent was proportional to the logarithmic value of SCCA concentration, ranging from 0.8 pg mL 1 to 80 ng mL 1 with a correlation coefficient of 0.9989. The detection limit for SCCA concentration was estimated to be 0.21 pg mL 1 at a signal-to-noise ratio of 3. The detection performance was compared with other reported methods and the results were listed in Table S1. Compared with previous studies, the proposed immunosensor had a relative large linear range and low detection limit. The results demonstrated that the integration of the excellent PEC performance of ZNF@Bi2S3 composites and multifunction of rGO for signal enhancement would bring a new insightful horizon to PEC biosensors. Meanwhile, using this novel signal amplification strategy, the resulting immunosensor exhibited high sensitivity and specificity, good reproducibility and feasibility (details in Supporting information).
This work was financially supported by National High-tech R&D Program (863 Program) (SQ2015AAJY1562) and National Natural Science Foundation of China (51273084, 51473067, and 21475052).
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.2015.12.008.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Multifunctional reduced graphene oxide trigged chemiluminescence resonance energy transfer: Novel signal amplification strategy for photoelectrochemical immunoassay of squamous cell carcinoma antigen Yan Zhang a, Guoqiang Sun a, Hongmei Yang a, Jinghua Yu a, Mei Yan a,n, Xianrang Song b,n a b
School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China Shandong Provincial Key Laboratory of Radiation Oncology, Shandong Cancer Hospital and Institute, Jinan 250117, China
art ic l e i nf o
a b s t r a c t
Article history: Received 24 September 2015 Received in revised form 28 November 2015 Accepted 5 December 2015 Available online 8 December 2015
Herein, a photoelectrochemical (PEC) immunoassay is constructed for squamous cell carcinoma antigen (SCCA) detection using zinc oxide nanoflower-bismuth sulfide (Bi2S3) composites as photoactive materials and reduced graphene oxide (rGO) as signal labels. Horseradish peroxidase is used to block sites against nonspecific binding, and then participated in luminol-based chemiluminescence (CL) system. The induced CL emission is acted as an inner light source to excite photoactive materials, simplifying the instrument. A novel signal amplification strategy is stem from rGO because of the rGO acts as an energy acceptor, while luminol serves as a donor to rGO, triggering the CL resonance energy transfer phenomenon between luminol and rGO. Thus, the efficient CL emission to photoactive materials decreases. Furthermore, the signal amplification caused by rGO labeled signal antibodies is related to photogenerated electron–hole pairs: perfect matching of energy levels between rGO and Bi2S3 makes rGO a sink to capture photogenerated electrons from Bi2S3; the increased steric hindrance hinders the electron donor to the surface of Bi2S3 for reaction with the photogenerated holes. On the basis of the novel signal amplification strategy, the proposed immunosensor exhibits excellent analytical performance for PEC detection of SCCA, ranging from 0.8 pg mL 1 to 80 ng mL 1 with a low detection limit of 0.21 pg mL 1. Meanwhile, the designed signal amplification strategy provides a general format for future development of PEC assays. & 2015 Elsevier B.V. All rights reserved.
Keywords: Chemiluminescence resonance energy transfer Reduced graphene oxide Zinc oxide nanoflower Bismuth sulfide Squamous cell carcinoma antigen
1. Introduction Squamous cell carcinoma antigen (SCCA), a glycoprotein with molecular mass of ∼45 kDa, was isolated from human cervical squamous carcinoma cells and served as a circulating marker for more advanced squamous cell tumors of the cervix, lung, and oropharynx (Bastide et al., 2010; Giannelli et al., 2007; Kato and Torigoe, 1977). The serum level of SCCA was increased in parallel to the growth of the tumor size or the recurrence of the disease (Hefler et al., 2005; Schedel et al., 2011). At present, chemiluminescence enzyme immunoassay, radio-immunoassay and enzymelinked immunosorbent assay were usually applied to detect SCCA in serum (Çataltepe et al., 2000; Chang et al., 2004; Zhang and Qi, 2011). However, several shortcomings of these methods limit its extensive application, such as poor reproducibility, low sensitivity, n
Corresponding authors. E-mail addresses: [email protected] (M. Yan), [email protected] (X. Song).
http://dx.doi.org/10.1016/j.bios.2015.12.008 0956-5663/& 2015 Elsevier B.V. All rights reserved.
radiation hazards, require expensive equipments and time-consuming. Therefore, there was an utmost requirement of low cost, precise, and high sensitivity immunoassay for SCCA detection. Photoelectrochemical (PEC) immunosensing was a recent yet vibrantly emergent and promising analytical method for rapid and high-throughput biological assays (Li et al., 2014, 2012; Yu et al., 2014). In the case of the PEC detection process, light was used to excite active species, and current was used as the detection signal (Zhao et al., 2012). Benefitting from two separate forms of signals for excitation and detection, the PEC immunosensor possessed the advantages of both high selectivity of immunoassay and sensitivity of PEC analysis (Wang et al., 2012). Meanwhile, the use of electronic readout made the instrument simpler, cheaper, and easier to miniaturize than that of optical methods (Wang et al., 2014). Over the years, PEC sensors have been employed to analyze various compounds (An et al., 2010; Bellani et al., 2015; Ma et al., 2014; Tang et al., 2014). In PEC immunoassay, physical light source and monochromator were usually needed to bring appropriate light emission (Huo
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et al., 2015), which made the instrument complicated and departed from the portable trend for biosensor. Chemiluminescent (CL) emission, a process in which excited molecules or atoms generated from chemical reactions released the excess of energy in light form, was a promising substitute for the physical light source. This was attributed to the fact that different CL systems or adjusting the reaction conditions of CL systems could bring light emission with various wavelengths (Abdolmohammad-Zadeh and Rahimpour, 2015; Chen et al., 2013; Xu et al., 2014). The oxidation of luminol with H2O2 was one of the most commonly known CL reactions (Iranifam and Kharameh, 2015; Khajvand et al., 2015; Li et al., 2011). Meanwhile, this CL system had been applied in a CL resonance energy transfer (CRET), in which the CRET involved a nonradiative dipole–dipole transfer of energy from a donor to an acceptor (Bi et al., 2015; Lee et al., 2012). CRET occurred by the oxidation of a luminescent substrate without an excitation source, thus, the signal-to-noise ratio and sensitivity of the detection could be improved in bioassay. From the above, CRET could be a promising signal amplification strategy in PEC immunoassay. In this work, zinc oxide nanoflower-bismuth sulfide composites (ZNF@Bi2S3) were used as photoactive materials because of combining fast electron transfer rate of ZNF and excellent PEC performance of Bi2S3 (Balachandran and Swaminathan, 2013; Manna et al., 2014; Zhang et al., 2014). Meanwhile, the presence of Bi2S3 nanoparticles avoided the use of Cd-contained quantum dots, which suffered from photocorrosion and cadmium contamination (Liang et al., 2012). After immobilizing of capture antibodies (Ab1), horseradish peroxidase (HRP) was dropped onto the electrode surface. The HRP could not only act as a substitute for bovine serum albumin (BSA) to block sites against nonspecific binding, but also participate in the luminol-HRP-H2O2 CL system to induce inner light source. Reduced graphene oxide labeled signal antibodies (rGO-Ab2) was introduced into the immunosensor via immunoreactions. Excellent analytical performance of the immunosensor was attributed to the excellent PEC performance of ZNF@Bi2S3 and the multiple function of rGO. Firstly, the rGO acted as an energy acceptor, while luminol served as a donor to rGO, triggering the CRET phenomenon between luminol and rGO. The reduction of CL emission to ZNF@Bi2S3 composites was controlled by rGO, which decreased the photocurrent intensity. Secondly, an additional electron transfer pathway from Bi2S3 to rGO decreased the photogenerated electrons to electrode. Thirdly, the rGO-Ab2 increased the steric hindrance and obstructed the diffusion of H2O2 to the surface of Bi2S3 for reaction with the photogenerated holes. On the basis of the novel signal amplification strategy, a sensitive PEC immunoassay for SCCA detection was proposed for the first time.
2. Experimental section 2.1. Preparation of chitosan functionalized ZNF@Bi2S3 composites The materials and apparatus were shown in supporting information. The ZNF was fabricated as follow: 0.0395 g of zinc acetate was dissolved into 15 mL of ultrapure water under stirring. After 15 min stirring, an aqueous solution of NaOH (6.9 mM, 0.6 mL) was added. The obtained solution was then transferred into Teflon-lined stainless steel autoclaves and maintained at 120 °C for 45 min. After the reaction was completed, the products were collected by centrifugation and washed with ethanol several times, and finally dried at 60 °C in air. The Bi2S3 nanoparticles were grown on surface of ZNF by a hydrothermal method. In a typical process, 0.21 mM bismuth nitrate pentahydrate and 0.16 mM sodium thiosulfate pentahydrate were added into a given amount (25 mL) of ultrapure water. After
adding 50 mg of as-synthesized ZNF, the mixture was transferred into Teflon-lined stainless steel autoclaves kept at 110 °C for 4 h. Finally, the obtained ZNF@Bi2S3 composites were purified by centrifugation, and further dried at 40 °C in a vacuum oven. 10 mg of ZNF@Bi2S3 composites were dissolved into 1.0 mL of 1% chitosan solution and stirred for 2 h. Subsequently, the precipitation was collected and washed with water several times, chitosan functionalized ZNF@Bi2S3 composites were obtained and then re-dissolved into 1.0 mL of water for further use. 2.2. Preparation of rGO-Ab2 Ab2 was immobilized on the surface of rGO with the aid of thionine. Typically, 0.1 mg of rGO (the synthesis process was shown in Supporting information) was dispersed in 1.0 mL of pH 7.4 phosphate buffer saline (PBS), followed by addition of 1.0 mL of thionine solution (1.0 mM). After stirring at room temperature for 12 h, the resultant mixture was centrifuged (10,000 rpm for 10 min) and washed with PBS (pH 7.4) for five times to remove the unbounded thionine. In this process, the amino of thionine could attach on to the rGO through π–π stacking (Liu et al., 2014). The obtained product was re-dispersed in 1.0 mL of PBS (pH 7.4). Then, 100 μL of glutaraldehyde (GA, 2.5 wt%) which could bridge the rGO/thionine and Ab2 with covalent bond force was added into the above solution and allowed to react for 1.0 h at room temperature. After repeating a centrifugation (10,000 rpm for 10 min) and washing procedure for five times, the mixture were re-dissolved into 1.0 mL of PBS (pH 7.4). Then, 100 μL of Ab2 (20 μg mL 1) was added and incubated for 3 h at room temperature, followed by centrifugation (10,000 rpm for 10 min) and washing with PBS (pH 7.4) for five times to remove free antibodies. Finally, 1.0 wt% BSA solution was added to block possible remaining active sites, the resulting rGO-Ab2 was washed with PBS for five times and redispersed in 1.0 mL of PBS (pH 7.4). 2.3. Construction of the immunosensor platform The fabrication process of the immunosensor on an indium tin oxide (ITO) device (the synthesis details of the ITO device was exhibited in supporting information) was shown in Scheme 1. For preparation of ZNF@Bi2S3 modified ITO electrode, the ITO electrode was first surface functioned with amino according to the literature (Aziz et al., 2008), and then modified with GA (2.5 wt%). Subsequently, 10 μL of chitosan functionalized ZnO@Bi2S3 composites was dropped on the electrode surface and incubated for 2 h. The modified electrode was rinsed with ultrapure water and dried at room temperature. Ab1 was conjugated the modified electrode with the aid of GA. After incubating with GA for 1 h, 5.0 μL of Ab1 (50 μg mL 1) was applied to the electrode and reacted at room temperature for 1 h. Then, the electrode was rinsed with PBS (pH 7.4) several times to eliminate excess antibodies. At last, the modified immunosensor was incubated with 10 μL of HRP solution (1.0 mg mL 1) at room temperature for 2 h to block the possible remaining active sites against non-specific absorption. The resulting electrode was stored at 4 °C when not in use. 2.4. PEC immunoassay procedure for SCCA In this study, SCCA was assayed with a sandwich-type immunoassay format using Ab1/ZNF@Bi2S3/ITO as immunosensing probe and rGO-Ab2 as molecular tags. Initially, 5.0 μL of sample solution containing different concentration of SCCA was dropped onto the working electrode and allowed to incubate for 40 min at room temperature, followed by washing with pH 7.4 PBS. Subsequently, 5.0 μL of rGO-Ab2 was added onto the electrode surface
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Scheme 1. The fabrication process of the (A) ITO device and (B) PEC immunosensor.
and incubated for 40 min. After washing with PBS, 40 μL of Tris– HCl buffer solution (pH 8.5, 0.1 M) containing 6.0 mM H2O2, 0.5 mM p-iodo-phenol and 0.5 mM luminol was added into the PEC cell. Finally, the PEC measurements were performed at an applied potential of 0 V.
3. Results and discussion 3.1. Characterization of the immunosensor platform The surface morphology of the etched ITO was an important factor affecting its sensing performance, and scanning electron microscopy (SEM) was utilized to characterize the morphologies and edge roughness of the etched ITO layer. As shown in Fig. S1, the etched electrode owned the good homogeneity, indicating that the surface etching was a promising technique for fabricating ITO device. The morphology of ZNF and ZNF@Bi2S3 composites were elucidated by SEM images. As shown in Fig. 1A, the products were formed as a flower-like ensemble that was composed of needlelike microstructures. Fig. 1B and C were high high-magnification SEM images of the as-synthesized ZNF. The total size of the products was 1 μm, while the length of the needles was 400 nm. The flower-like structures made the ZNF owned large surface area, which provided abundant sites for Bi2S3 loading. The SEM images of ZNF@Bi2S3 composites were exhibited in Fig. 1D–F. As shown in Fig. 1D, after growth of Bi2S3 on ZNF, no apparatus size change was observed. The high-magnification SEM images (Fig. 1E and F)
exhibited that the surface of the composites became rough, which was mainly attributed to the successfully deposition of Bi2S3. Fig. 2A was a transmission electron microscope (TEM) image of pure ZNF which composed with nanoneedles. The Fig. 2D manifested the TEM image of the Bi2S3 nanoparticles of ∼10 nm sizes. In addition, energy dispersive spectrometer (EDS) was also used to characterize the ZNF and ZNF@Bi2S3 composites. The EDS result of ZNF was shown in Fig. 2B, the sample consisted of Zn, O elements, and the atomic percentage of ZnO in ZNF was 36.63% while O was 63.37%. As expected, the elements of Bi, S, Zn, O were resolved for ZNF@Bi2S3 composites (Fig. 2E), and the atomic percentage were 1.12%, 0.69%, 31.34%, and 66.85%, respectively, indicating the successfully synthesis of the ZNF@Bi2S3 composites. The X-ray diffraction (XRD) patterns of pure ZNF and ZNF@Bi2S3 composites were shown in Fig. 2C. The diffraction peaks in the range of 20°o2θ o70° could be indexed as (100), (002), (101), (102), (110), (103), (200), (112), and (201) planes of wurtzite hexagonal phase ZnO, which were consistent with the value in the standard card (JCPDS 36-1451). The higher intensity of the (002) peak indicated the ZNF were grown preferably along c axis, and the exhibited sharp peaks indicated that the nanostructures possess large crystalline domains as well as a high degree of crystallinity. Meanwhile, No other peaks that corresponded to impurities were found in the spectrum. After deposition of Bi2S3, peaks corresponding to ZNF became weak and no evident diffraction peaks were observed for the Bi2S3 of the composite film of ZNF@Bi2S3. There may be two reasons caused for the absence of Bi2S3 peaks, the one was that only few Bi2S3 nanoparticles were attached to the ZNF, the other one was that the Bi2S3 were highly dispersed on ZNF surface.
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Fig. 1. SEM images of (A–C) ZNF and (D–F) ZNF@Bi2S3 composites under different magnification.
Fig. 2. (A) TEM image and (B) EDS of ZNF; (D) TEM image and (E) EDS of ZNF@Bi2S3 composites; (C) XRD patterns and (F) UV–vis absorption spectra of ZNF and ZNF@Bi2S3 composites.
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Fig. 3. (A) TEM image of rGO; (B) XRD patterns, (C) UV–vis absorption spectra and (D) Raman spectra of GO and rGO.
The light absorption properties of the as-synthesized products were characterized by the Ultraviolet–visible (UV–vis) absorption spectra. As shown in Fig. 2F, the bare ZNF only absorbed the UV light with wavelength shorter than 365 nm, which was attributed to its large energy gap ( 3.7 eV). Obviously, the ZNF@Bi2S3 composites greatly enhanced the UV light absorbance and extended the light utilization from UV to visible range. The decorated Bi2S3 were able to efficiently promote the visible light absorption of ZNF@Bi2S3 composites, contributing to the high absorption coefficient of Bi2S3 nanoparticles. Meanwhile, we compared PEC performance of ZNF, Bi2S3 and ZNF@Bi2S3 composites. As shown in Fig. S2, the electrode modified by ZNF@Bi2S3 composites showed largest photocurrent response, demonstrated that the sensitivity of ZNF@Bi2S3 composites was improved by introducing Bi2S3. 3.2. Characterization of the signal labels The TEM image (Fig. 3A) displayed a view of rGO clearly illustrating typical flake-like wrinkled shapes of graphene with irregular size. Many wrinkles and folds revealed that the two-dimensional structure of rGO sheet was flexible. The XRD patterns and UV–vis absorption spectra of GO and rGO were exhibited in Fig. 3B and C, respectively. The XRD pattern of GO (curve a) showed a strong and sharp diffraction peak at 2θ of 13.0°, which was in agreement with the lamellar structure of GO. After the chemical reduction of GO, a broad peak around 25° could be seen for rGO (curve b), which was due to the deep reduction of GO, indicating that most oxygen functional groups had been removed.
In the UV–vis absorption spectrum of GO (curve a), there were two absorption features: a peak at 230 nm due to the π-π* transition of C ¼C bond, and a band at a 299 nm corresponding to the n-p* transition of the C ¼O bond. Meanwhile, the typical band for GO with a maximum at 230 nm gradually red-shifted to around 269 nm after the hydrothermal reduction process (curve b) owing to n-π* transitions of aromatic C ¼C bonds, suggesting that the electronic conjugation within rGO was restored after reduction. The formation of rGO was also evidenced by the corresponding Raman spectra. It was proved that graphene exhibited two characteristic main peaks: the G mode at 1575 cm 1, arising from the first order scattering of the E2g phonons of the sp2 C atoms; the D mode at 1350 cm 1, arising from a breathing mode of the κpoint phonons of A1g symmetry. As shown in Fig. 3D, GO exhibited a D band at 1350 cm 1 and a G band at 1613 cm 1, while the corresponding bands of rGO were 1350 cm 1 and 1600 cm 1, respectively. The red-shifted of G band from 1616 to 1603 cm 1 was attributed to the high ability for recovery of the hexagonal network of carbon atom. Furthermore, the rGO showed relative higher intensity of D to G band than that of GO, confirmed that the rGO was formed after a hydrothermal process. 3.3. The feasible of the signal amplification strategy To confirm the effectiveness of rGO as an acceptor in CRET, we investigated CRET by adding SCCA and rGO-Ab2 in sequence to a reaction solution containing Ab1-HRP. As shown in Fig. 4A, the luminol-HRP-H2O2 CL system showed a high CL intensity (curve a).
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Fig. 4. (A) CL spectra of luminol-HRP-H2O2 system (a) before and after incubated with (b) 0.1 ng mL 1 SCCA, (c) 0.1 ng mL 1 SCCA þrGO-Ab2, (d) rGO-Ab2 only and (e) 1.0 ng mL 1 SCCA þ rGO-Ab2; (B) EIS of (a) bare ITO, ZNF@Bi2S3/ITO (b) before and after (c) incubated with Ab1, (d) blocked with HRP, (e) incubated with SCCA and (f) rGO-Ab2; (C) the photocurrent responses of Ab1 modified electrode (a) before and after (b) blocked with HRP, incubated with (c) 0.1 ng mL 1 SCCA, (d) 0.1 ng mL 1 SCCA þrGO-Ab2 and (e) 1.0 ng mL 1 SCCAþ rGO-Ab2; (D) logarithmic calibration curve for SCCA.
No apparent change of CL intensity (curve b) was observed after introducing of SCCA. Then, rGO-Ab2 was added to incubate with SCCA, and the CL intensity decreased significantly (curve c). If we added rGO-Ab2 to the solution directly without SCCA, the CL quenching was not occurred (curve d). This was attributed SCCA triggered rGO-Ab2 to undergo sandwich-type immunoreactions, serving as a bridge short enough for CRET to occur, indicated that the proximity of HRP to rGO was critical for efficient energy transfer to rGO from excited luminol. With the increasing of concentration of SCCA, more acceptors (rGO) was introduced into the system, thus, in turn, enabling higher energy transfer from the donor to the acceptor and more quenching of luminol CL (curve e). These observations confirmed that the CRET was feasible and could used to construct immunosensor. 3.4. Electrochemical impedance spectroscopy (EIS) of the immunosensor EIS was an effective method to monitor the changes of interfacial properties, allowing the understanding of chemical transformation and processes associated with the conductive electrode surface (Zhang et al., 2013). In EIS, the impedance spectrum included a semicircle portion at high frequencies corresponding to an electron transfer limited process and a linear portion at low frequencies represented a diffusion-limited electrochemical process. The semicircle diameter was equal to the electron-transfer resistance (Ret), which reflected the electron transfer kinetics of the redox probe at the electrode interface. Each reaction step of the modified electrode was analyzed by EIS in the frequency range
0.1–105 Hz, and in PBS (pH 7.4) containing [Fe(CN)6]3 /4 (5.0 mM) as probe. As shown in Fig. 4B, a relatively large semicircle diameter was observed at bare ITO (curve a). Subsequently, when ZNF@Bi2S3 composites were loaded on the electrode surface, the interfacial resistance increased (curve b) which suggested that the composites were successfully immobilized on the surface and the formed compact film hindered the access of the redox probe to the electrode surface. After the electrode was conjugated with Ab1, blocked with HRP, and subsequently incubated with SCCA, the Ret increased gradually as the experiment proceeds (curves c–e). This was attributed to the proteins with nonconductive property acted as an inert layer to obstruct electron transfer from the redox probe to the electrode surface, testifying the successful introduction of these substances into immunosensor. Finally, rGO-Ab2 was introduced into the immunosensor via immunoreactions and gave rise to the resistance (curve f), turning out the successfully immobilized of rGOx-Ab2. The increase of Ret verified the successful fabrication of the immunosensor. 3.5. PEC performance of the immunosensor The fabrication of the immunosensor was also be monitored by PEC experiments. The photocurrent responses of different electrodes were detected in mixed solution containing luminal and H2O2, and the results were shown in Fig. 4C. A CL emission, which was produced from the oxidation of luminol by H2O2 in the presence of HRP, was used as light source to excite ZNF@Bi2S3 composites due to the natural overlap between the absorption spectrum of ZNF@Bi2S3 and the CL spectrum of luminol-HRP-H2O2.
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Before blocked the immunosensor with HRP, almost no light source was produced, resulted in a negligible photocurrent (curve a). Then, HRP was introduced into the immunosensor to block sites against nonspecific binding, and then enhanced the CL emission intensity. A large photocurrent was observed (curve b), which was attributed to the ZNF@Bi2S3 composites owned good PEC performance and could be excited by CL emission, indicating the feasibility of the CL-excited PEC immunoassay. After incubating with SCCA, the photocurrent intensity decreased (curve c). This could be explained by the fact that the proteins act as an inert layer hindered the diffusion of H2O2 to the surface of Bi2S3 for efficient scavenging of photogenerated holes. In the final step, the rGO-Ab2 bioconjugates were immobilized on the developed immunosensor through specific antibody-antigen immunoreactions, the photocurrent decreased significantly (curve d). The decrement reasons of photocurrent response was concluded as follow: (i) the rGO acted as energy acceptor to compete the CL emission with ZNF@Bi2S3 composites, decreasing the effective of use light source; (ii) the rGO served as electrons sink to capture photogenerated electrons from Bi2S3 due to the perfect matching of energy levels between rGO and Bi2S3, decreasing the electrons amount to electrode; (iii) the introduced rGO-Ab2 hindered the transfer of electron donor to Bi2S3 surface. The detail charge transfer processes was shown in Scheme S1. Meanwhile, the photocurrent responses of the platform incubated with different signal labels were determined. As shown in Fig. S3, the platform incubated with rGO-Ab2 bioconjugates showed the lowest photocurrent response, confirmed that the signal amplification strategy induced by rGO was feasible. Furthermore, the photocurrent decreased with the increasing of the concentration of SCCA (curve e). With the increasing of concentration of SCCA, the amount of rGO-Ab2 connected to immunosensor increased, leading to a further decrement of photocurrent intensity. These results confirmed that the immunosensor was successfully constructed and could be applied to the sensitive determination of SCCA.
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3.7. Application in real samples In order to evaluate the analytical reliability and application potential of the designed immunosensing method, the recovery of the different concentrations of SCCA in serum was measured by standard addition methods. The spiked human serum samples were prepared through adding different amounts of SCCA into the human serum samples. The results were shown in Table S2. The recoveries of SCCA in human serum samples were obtained in the range of 95.2–104.2%. The results demonstrated the designed biosensor could be suitable to the clinical determination of SCCA levels in serum samples.
4. Conclusion In this work, SCCA was detected using a PEC method. ZNF@Bi2S2 composites were modified on an ITO device and served as photoactive materials. HRP was instead of BSA to block sites against non-specific binding, and then participated in the luminol-HRP-H2O2 CL system to provide inner light source, simplifying the instrument. A novel signal amplification strategy caused by rGO-Ab2 was adapted to enhance the analytical performance. Typically, rGO, as an energy acceptor, trigged the CRET phenomenon between luminol and rGO, decreasing the efficient CL emission to ZNF@Bi2S3 composites. Duo to perfect matching of energy levels between rGO and Bi2S3, the rGO acted as an electrons sink to capture photogenerated electrons from Bi2S3, decreasing the electrons amount to electrode surface. Finally, the introduced rGO-Ab2 increased the steric hindrance and hindered the electron donor to the surface of Bi2S3 for reaction with the photogenerated holes. Using this novel signal amplification strategy, the resulting immunosensor exhibited wide linear relation, low detection limit, high sensitivity and specificity, good reproducibility and well feasibility in real samples determination.
3.6. Analytical performance
Acknowledgments
In this work, SCCA detection was based a novel signal amplification strategy trigged by rGO. Under optimal conditions (details in Supporting information), after introduction of the rGO labeled Ab2, CRET, new photogenerated electrons transfer pathway and increased steric hindrance were all occurred to decrease the photocurrent response. Therefore, by tracking the photocurrent response related to the amount of SCCA, a sensitive immunoassay was accomplished. Fig. 4D depicted the photocurrent responses of the immunosensor to different concentrations of SCCA. With the increasing the concentration of SCCA, more rGO was loaded onto the electron surface, and then the photocurrent signal reduced regularly. The photocurrent was proportional to the logarithmic value of SCCA concentration, ranging from 0.8 pg mL 1 to 80 ng mL 1 with a correlation coefficient of 0.9989. The detection limit for SCCA concentration was estimated to be 0.21 pg mL 1 at a signal-to-noise ratio of 3. The detection performance was compared with other reported methods and the results were listed in Table S1. Compared with previous studies, the proposed immunosensor had a relative large linear range and low detection limit. The results demonstrated that the integration of the excellent PEC performance of ZNF@Bi2S3 composites and multifunction of rGO for signal enhancement would bring a new insightful horizon to PEC biosensors. Meanwhile, using this novel signal amplification strategy, the resulting immunosensor exhibited high sensitivity and specificity, good reproducibility and feasibility (details in Supporting information).
This work was financially supported by National High-tech R&D Program (863 Program) (SQ2015AAJY1562) and National Natural Science Foundation of China (51273084, 51473067, and 21475052).
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.2015.12.008.
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