- Email: [email protected]
Sandwich-like electrochemiluminescence aptasensor based on dual quenching effect from hemin-graphene nanosheet and enzymatic biocatalytic precipitation for sensitive detection of carcinoembryonic antigen
Journal of Electroanalytical Chemistry 787 (2017) 88–94
Contents lists available at ScienceDirect
Journal of Electroanalytical Chemistry journal homepage: www.elsevier.com/locate/jelechem
Sandwich-like electrochemiluminescence aptasensor based on dual quenching effect from hemin-graphene nanosheet and enzymatic biocatalytic precipitation for sensitive detection of carcinoembryonic antigen Jiu-Jun Yang a,b, Jun-Tao Cao a,b,⁎, Yu-Ling Wang a,b, Hui Wang a,b, Yan-Ming Liu a,b,⁎, Shu-Hui Ma c a b c
College of Chemistry and Chemical Engineering, Xinyang Normal University, Xinyang 464000, P.R. China Institute for Conservation and Utilization of Agro-bioresources in Dabie Mountains, Xinyang Normal University, Xinyang 464000, P.R. China Xinyang Central Hospital, Xinyang 464000, P.R. China
a r t i c l e
i n f o
Article history: Received 10 December 2016 Received in revised form 20 January 2017 Accepted 21 January 2017 Available online 23 January 2017 Keywords: Dual-quenching effect Hemin-rGO nanosheet Enzymatic biocatalytic precipitation Flower-like spherical Au-CdS Electrochemiluminescence Carcinoembryonic antigen
a b s t r a c t A new and simple sandwich-like electrochemiluminescence (ECL) aptasensor for carcinoembryonic antigen (CEA) assay was fabricated based on the dual quenching effect from hemin-graphene (H-rGO) nanosheet and enzymatic biocatalytic precipitation (BCP) on the Au-CdS nanocomposites-based ECL system. In this aptasensor platform, flower-like spherical Au-CdS nanocomposites were used as ECL luminophores and exhibit a strong ECL signal. The rGO nanosheet was used as a supporter to immobilize hemin molecules via π-π stacking interactions. Due to the steric hindrance and quenching effect of rGO, the ECL intensity decreased by the construction of the sandwich “CEA aptamer I (NH2-DNA)-CEA-aptamer II” (H-rGO-aptamer II) mode. In the process of BCP, the ECL intensity further decreased because the hemin with intrinsic peroxidase-like catalytic activity could oxidize the 4-chloro-1-naphthol (4-CN) to produce an insoluble precipitation on the sensor. Using this dual quenching strategy, the prepared aptasensor exhibits a linear range from 0.8 pg/mL to 4 ng/mL and a detection limit of 0.28 pg/mL. This ECL aptasensor has simple design and undemanding in operation and was utilized to determine the content of CEA in complex samples with recoveries of 95.0% to 115.8%. Moreover, no any chemical modification of aptamer was required, suggesting that the proposed ECL aptasensor could be applied for the detection of diverse proteins just by altering the aptamer sequence. © 2017 Elsevier B.V. All rights reserved.
1. Introduction The increasing demand of determination of disease-related proteins, especially cancer biomarkers, has received more and more attention in many fields. Carcinoembryonic antigen (CEA), a type of glycoprotein generated by tumor cells, was widely used as a tumor marker [1]. The specific and sensitive determination of CEA has great significance for clinical diagnosis and treatment assessment of cancers. Thus, many analytical methods for CEA detection have been studied, such as amperometric assay [2], fluorescence analysis [3], electrochemical method [4], photoelectrochemical assay [5], enzyme-linked immunosorbent assays [6] and capillary electrophoresis [7]. Among these methods, antibodies were mainly acted as recognition elements. However, the limits of antibodies are easy deactivation and instability. Aptamer, a kind of new recognition element, shows high bind affinity to its target and possesses numerous remarkable advantages such as design flexibility, easy ⁎ Corresponding authors. E-mail addresses: [email protected] (J.-T. Cao), [email protected] (Y.-M. Liu).
http://dx.doi.org/10.1016/j.jelechem.2017.01.044 1572-6657/© 2017 Elsevier B.V. All rights reserved.
synthesis and specificity [8]. So far, aptameric-based protein analytical systems have been greatly developed. For instance, in Guo's group [9], two different kinds of aptamers (with amidogen and thiol group) specific to CEA were modified on the surface of Ru@SiO2 and Au NPs based on localized surface plasmon resonance for CEA detection. Wu et al. [10] constructed an aptasensor for CEA detection based on fluorescence resonance energy transfer, an amino group modified CEA aptamer was covalently tagged on the PAA-UCPs which was employed as the energy donor. Noticed that the aptamers for protein detection were all chemically modified, which not only limited the association with the number of target binding sites, but also increased the operation and expenses. In addition, many literatures reported that graphene sheet can be served as a supporter to adsorb ss-DNA due to its high surface area and π-π conjunction [11]. Therefore, attaching aptamer onto the graphene sheet as a probe may be an alternative for constructing a sensor with good property. Electrochemiluminescence (ECL) approach is competitive with conventional assays because of its high sensitivity, rapid response and low background [12,13]. In recent years, various semiconductor
J.-J. Yang et al. / Journal of Electroanalytical Chemistry 787 (2017) 88–94
nanomaterials with ECL activity have been growing [14]. CdS nanomaterials are the most promising and effective materials, owing to their excellent luminescent properties in the presence of coreactant. According to reports, Au nanoparticles exhibited a strong surface plasmon resonance effect on the CdS because Au can increase the light absorption and enhance the charge separation [15–17]. Wang et al. [15] reported an obvious enhancement of photocatalytic activity in the CdS-Au-CdS nanorod arrays, which was relative to that of pure CdS because surface plasmon resonance effect of Au segments increased the effective optical length inside the CdS. Therefore, depositing AuNPs on the spherical CdS is also expected to obtain enhanced intensity compared with the pure CdS. Hemin, the active center of heme-protein, has the peroxidase-like activity similar to the peroxidase enzyme. In general, hemin could intercalate into G4 structure to form G4H DNAzyme with high peroxidaselike catalytic activity and there are no disadvantages of the natural enzymes such as instability, long-time consumption. In our previous work [18], hemin-DNAzyme with high electrocatalytic ability was used to quench ECL signal of MoS2-CdS nanocomposites for sensitive detection of IgE. Nevertheless, it is difficult for hemin to maintain high catalytic activity in aqueous solution due to its low solubility and easy aggregation [19]. In order to solve this problem, various nanomaterials have been used as nanocarriers to load hemin, such as ZnO [20], metal-Organic frameworks [21], supramolecular hydrogels [22]. Graphene [23], with large surface area and good biocompatibility, has been also served as a valuable candidate for immobilization hemin. In addition, graphene possesses a rich surface chemistry and has the potential to promote the catalytic activity and stability of the supported molecular systems by cation-π interactions or π-π stacking. Duan's group [24] reported that hemin attached on the graphene still retained the catalytic-active monomer form as in natural enzymes. So far, H-rGO with peroxidase activity has been widely applied in many aspects, such as distinguishing between ss- and ds-DNA [25], electrochemical and ECL sensor [26,27]. Tao and co-workers [28] described a sensing strategy by employing ss-DNA probe and H-rGO sheets to detect a wide range of targets including metal ions, small molecules and DNA. A label-free colorimetric method for PDGF-BB and thrombin assay on the basis of HrGO-DNA composite was reported by Zhang's group [29]. However, as far as we know, little attention has been paid to the applications of the above-mentioned rGO-based hybrid peroxidase mimetics in ECL aptasensor. Except for the nanomaterial-based labeling strategy, employing enzymatic reaction to form insoluble product on the sensing interface, e.g. enzymatic biocatalytic precipitation (BCP), is an important concern for obtaining low limits of detection. Tang et al. [30] utilized the formed hemin-based DNAzyme concatamers toward catalytic precipitation of 4-CN for recognizing Cu2+. Our group [31] prepared a competitive ECL aptasensor by introducing BCP technique for the sensitive detection of IgE. Herein, we fabricated a new and simple sandwich-like ECL aptasensor for sensitive CEA assay by coupling with H-rGO-aptamer II composite as probe. The probe was used as both quencher and catalyst of BCP. The monodisperse CdS nanoparticles were synthetized via a hydrothermal method and used as a support to load high amounts of AuNPs to prepare flower-like spherical Au-CdS nanocomposite. HrGO-aptamer II composite could use as an excellent platform for assembling ss-DNA containing a 24-base tail and a 19-base CEA aptamer sequence. In the presence of CEA, aptamers specifically combined with CEA to form a sandwich structure with the 24-base fragment still adsorbed on rGO. The ECL intensity was quenched due to the steric hindrance and quenching effect of rGO. Simultaneously, in view of the intrinsic peroxidase-like activity of hemin catalyzed to produce insoluble product on the electrode after incubated with 4-CN, further quenched the ECL signal. Under the optimum conditions, the aptasensor for CEA assay was well established and exhibits good performance to determine CEA in human serum samples.
89
2. Experimental 2.1. Materials and reagents Carcinoembryonic antigen (CEA) aptamers 5′-NH2-(CH2)6-TTT TAT ACC AGC TTA TTC AAT T-3′ (aptamer I, NH2-DNA) and 5′-CCC ATA GGG AAG TGG GGG ATG TGT GTG TGT GTG TGT GTG TGT G (aptamer II) were synthesized by Sangon biotech Co. Ltd. (Shanghai, China). CEA was purchased from Zhengzhou Immuno Biotech Co., Ltd. (Zhengzhou, China). Hemin was from Sigma-Aldrich Chemical Co. (St. Louis, MO). Cadmium acetate [Cd(CH3COO)2·4H2O] and thiourea were got from Sinopharm Group Chemical Reagent Co., Ltd. (Shanghai, China). Sodium L-glutamate monohydrate was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). HAuCl4·3H2O and glutaraldehyde (GA) were acquired from Alfa Aesar (Tianjin, China) and Tianjin Yongda Chemical Reagent Development Center (Tianjin, China), respectively. Human IgG (hIgG), human serum albumin (HSA), and bovine serum albumin (BSA) were purchased from Shanghai Solarbio Bioscience & Technology Co., Ltd. (see bio Biotechnology). 4-chloro-1-naphthol (4-CN) was from Shanghai Ziyi Reagent Company (Shanghai, China). Ethylene glycol, Ammonia (28%) and hydrazine hydrate (35%) were from Aladdin industrial corporation (Shanghai, China). Phosphate-buffered saline (PBS) solutions with various pHs were prepared by mixing different volumes of NaH2PO4 and K2HPO4 containing 0.1 M KCl as the supporting electrolyte. Ultrapure water (Kangning Water Treatment Solution Provider, Chengdu, China) was used throughout the experiments. 2.2. Apparatus The ECL emissions were recorded using a BPCL ultra-weak luminescence analyzer (Institute of Biophysics, Chinese Academy of Science, Beijing, China) with a CR 120 type photomultiplier tube (Binsong Photonics, Beijing, China). All ECL experiments were performed with a conventional three-electrode system comprised of a Pt wire as the counter electrode, an Ag/AgCl electrode as reference electrode and bare or modified glass carbon electrodes (GCE, φ = 3 mm) as the working electrodes. Electrochemical impedance spectroscopy (EIS) was performed using an RST5200F electrochemical workstation (Zhengzhou Shiruisi Technology Co., Ltd., Zhengzhou, China) in the solution of 5 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) containing 0.1 M KCl. Scanning electron microscopy (SEM) images and transmission electron microscope (TEM) images were acquired using a S-4800 (Hitachi, Tokyo, Japan) and Tecnai G2 F20 TEM (FEI Co., Hillsboro, Oregon, USA), respectively. X-ray photoelectron spectra (XPS) analysis was performed on a KAlpha X-ray photoelectron spectrometer (Thermo Fisher Scientific Co., USA). UV–visible detection was carried out using an UV mini-1240 UV–vis spectrophotometer (Shimadzu Corp., Kyoto, Japan). 2.3. Synthesis of Au-CdS nanocomposites 2.3.1. Synthesis of spherical CdS nanoparticles The monodisperse CdS nanoparticles were synthesized following a typical procedure [32]. Firstly, 2.4 mmol Cd(CH3COO)2 was dissolved in 60 mL water and sonicated for a few minutes. Then 24 mmol thiourea was added in the above solution after vigorous stirring for 30 min, the mixture was transferred to a Tefion-lined autoclave (100 mL) and heated to 200 °C for 5 h. After naturally cooled, the precipitate was washed with water and ethanol for three times, respectively, and dried at 60 °C. 2.3.2. Synthesis of Au-CdS nanocomposites Au-CdS nanocomposites were prepared as previously described [33]. Briefly, 0.55 mL HAuCl4 aqueous solution was added into 50 mL of ethylene glycol, followed by the dissolution of 55 mg glutamate into the above solution with vigorous stirring. The pH was then adjusted to 11 by NaOH and 53 mg of CdS nanomaterial was subsequently slowly
90
J.-J. Yang et al. / Journal of Electroanalytical Chemistry 787 (2017) 88–94
added. The reaction solution was stirred at room temperature for 24 h. Finally, the solution was centrifuged at 9000 rpm for 15 min to collect the precipitate, which was then washed separately with water and ethanol for several times, and dried at 60 °C. 2.4. Preparation of hemin functionalized graphene nanosheet (H-rGO) Graphene oxide (GO) was prepared using modified Hummer's method [34]. Graphene–hemin composites were synthesized according to Guo's method [25]. Briefly, 10 mg of GO was added into 20 mL of water and sonicated for 1 h to obtain a homogeneous dispersion. The resulting dispersion was mixed with 20 mL of 0.5 mg/mL hemin solution in a flask and stirred for 30 min. Then 200 μL of ammonia solution and 30 μL of hydrazine solution were added. After being vigorously stirred for 30 min, the above solution was put in an oil bath at 60 °C for another 4 h. The stable black dispersion was obtained. 2.5. Synthesis of H-rGO-aptamer II composites According to the literature [28,29], a certain concentration of H-rGO aqueous solution and aptamer (4 μM) were mixed in Tris-EDTA buffer (10 mM Tris-HCl, 10 mM EDTA, pH 8.0) by sonicating for 10 min and incubated at room temperature for 12 h. After that, 100 μL 0.005 M NaCl solutions were added. Finally, the solution was centrifuged at 12,000 rpm for 20 min and the precipitated H-rGO-aptamer conjugates were redispersed in Tris-EDTA buffer and stored in 4 °C. 2.6. Fabrication of ECL aptasensor The construction process of the ECL aptasensor was displayed in Scheme 1. At first, the Au-CdS composites were mixed with 0.1 mg/mL chitosan (CS) solution by sonication and the Au-CdS/CS composites film was obtained by dropping 6 μL of the obtained composites solution onto a polished and cleaned GCE, drying in air. Subsequently, 6 μL of 5% (v/v) GA in PBS was covered on the electrode and remained at room temperature for 1 h. Then the electrode was rinsed with water thoroughly to remove physically adsorbed GA. The amino-modified DNA were introduced onto the GCE/Au-CdS/CS/GA-activated electrode by dropping 6 μL of NH2-DNA on the GCE and incubated at 4 °C overnight. After the nonspecific bind sites were thoroughly washed
and blocked by 1% BSA, the prepared electrodes were immersed into a series of CEA solution at different concentrations and incubated at 37 °C for 1 h. Following by washed with water, the above-prepared HrGO-aptamer II solution was coated onto the surface of CEA/BSA/NH2DNA/Au-CdS/CS/GCE and incubated at 37 °C for 45 min, and again washed thoroughly with water to remove the unbound probes. Finally, the above electrodes were further reacted with BCP solution consisting of 1 mM 4-CN and 0.15 mM H2O2 at 25 °C for 15 min. After thoroughly rinsed with water, the electrodes were prepared for ECL detection. The ECL responses of the electrodes were recorded in 0.1 M PBS (pH 7.4) containing 0.1 M K2S2O8 as a coreactant. The voltage of the PMT was set at 800 V. ECL signals related to the CEA concentrations could be measured. 3. Results and discussion 3.1. Characterization of CdS nanoparticles, Au-CdS nanocomposites and hemin-rGO sheets The morphologies of CdS nanoparticles and Au-CdS nanocomposites were characterized by SEM and TEM. As shown in Fig. 1A, the monodisperse CdS nanoparticles are flower-like spherical structure in shape and have a uniform morphology with a size of about 200 nm. TEM result also revealed relatively uniform CdS structure (Fig. 1C). When HAuCl4 was introduced and reduced, some highlights could be observed on the surface of flower-like spherical CdS, the corresponding SEM micrograph of Au-CdS nanocomposites were shown in Fig. 1B, indicating the AuNPs were successfully reduced on the surface of the CdS. The inset of the Fig. 1C showed corresponding high-magnification TEM image, it could be seen that AuNPs were uniformly deposited on the surface of the CdS. GO and rGO-hemin were characterized by UV–vis absorption spectra (Fig. 2A). The GO dispersion (curve a) exhibited a maximum absorption peak at 227 nm owing to the π-π* transition of aromatic C _C bonds and a shoulder at ca. 290–300 nm which corresponds to the nπ* transition of the C _O bond [35]. The spectrum of hemin solution (curve b) displayed an absorption peak at 390 nm attributed to the Soret band. After reduced, two characteristic absorption peaks of GH at 262 nm and 416 nm were obviously observed. An absorption peak at 262 nm was corresponding to the reduced graphene oxide which was 35 nm red shifted compared to GO, while a second peak at
Scheme 1. H-rGO-aptamer composite synthesis process (A); schematic illustration of the ECL biosensor for detection of PSA (B).
J.-J. Yang et al. / Journal of Electroanalytical Chemistry 787 (2017) 88–94
91
Fig. 1. SEM images of the as-prepared CdS (A) and Au-CdS (B), the insets of A and B correspond to the single CdS and Au-CdS, respectively; TEM image of CdS (C), the inset of the C corresponds to the TEM image of AuNPs uniformly deposited on the surface of the CdS.
416 nm was attributed to the Soret band of hemin with bathochromic shift of 26 nm. These findings indicate the existence of the π-π interactions between the GN and the adsorbed hemin molecules, which are in agreement with the previous report about the interactions of cationic porphyrin derivative with chemical converted graphene [36]. To conclude, hemin molecules could be attached onto graphene via π-π interaction. Attaching hemin on rGO nanosheet was further verified by X-ray photoelectron spectroscopy (XPS). As seen from Fig. 2B, the fully scanned spectra demonstrated the existence of iron, nitrogen and carbon elements in hemin-rGO nanosheet. The characteristic peaks of Fe 2P (2P3/2 710.88 ev, 2P1/2 724.48 ev), N 1S (399.2 ev), C 1S (283.98 ev), O 1S (531.58 ev) were respectively derived from hemin and rGO, clearly confirming the successfully synthesis of hemin-rGO nanosheet. 3.2. Electrochemical and electrochemiluminescence characterization of the fabricated aptasensor In order to characterize the modified process of the sensing interface, EIS was performed to investigate the modification of the electrode. As shown in Fig. 3A, the bare GCE exhibited a small semicircle (curve a). After the CS-Au-CdS covered on the electrode (curve b), an increased semicircle was obtained due to the CS decreased the electron-transfer efficiency. Moreover, with the sequential assembly of negative NH2DNA (curve c), inert BSA (curve d) and CEA (curve e), the resistance
increased steadily. The reason was that the formation of DNA or protein layer hindered the electron transfer. Similarly, continuous increases of Ret values were obtained after binding with H-rGO-aptamer II, reflecting the signal probe being successfully captured. Finally, the semicircle diameter increased greatly after incubated with 4-CN solution (curve g) as a result of yielding the insulating product prevented the diffusion of redox probe to the electrode surface. In addition, the ECL measurement was used to further monitor the stepwise assembly of the aptasensor. As shown in Fig. 3B, it can be seen that no ECL signal was found at the bare GCE (curve a). A strong ECL signal was obtained after assembling the Au-CdS-CS composites on the bare electrode (curve b). Two sensors, with Au (Au-CdS/GCE) and without Au (CdS/GCE), were constructed and tested under the same experimental conditions. The results were presented in insert figure in Fig. 3B. The results show that the ECL intensity of the former was higher than the latter as a result of Au nanoparticles on the CdS material exhibiting SPR effect. When the resultant electrode was immobilized of NH2-DNA, BSA, and CEA, successively, obvious gradual decreases in ECL intensity were observed (curves c, d, and e) due to the nonelectroactive property of DNA and protein and hindering the electron transfer. The ECL signal yet further declined after conjugation of H-rGO-aptamer II (curve f), which was attributed to the steric hindrance and quenching effect of rGO. After the successive incubation with 4-CN solution, the ECL intensity still decreased (curve g). The phenomena can be explained by the insoluble substance forming a barrier for electron transfer and
Fig. 2. (A) UV–vis absorption spectra of GO suspension (a), hemin solution (b), and hemin-graphene suspension (c); (B) XPS analyses for the full region of XPS for GH sheets, (C) the Fe 2P region, N 1S region, C 1S region, and O 1S region.
92
J.-J. Yang et al. / Journal of Electroanalytical Chemistry 787 (2017) 88–94
Fig. 3. The curves of EIS (A) and ECL (B) characterization of electrodes at different modify stages: bare GCE (a); Au-CdS-CS/GCE (b); NH2-DNA/Au-CdS-CS/GCE (c); BSA/NH2-DNA/Au-CdSCS/GCE (d), CEA/BSA/NH2-DNA/Au-CdS-CS/GCE (e); H-rGO-aptamer II/CEA/BSA/NH2-DNA/Au-CdS-CS/GCE (f); BCP/H-rGO-aptamer II/CEA/BSA/NH2-DNA/Au-CdS-CS/GCE (g). Inset in B: ECL curves in the presence (curve b′) and absence (curve a′) of AuNPs. EIS was measured in 0.1 M KCl solution containing 5.0 mM [Fe(CN)6]3−/4− at a scan rate of 100 mV/s. ECL detection buffer: 0.1 M PBS solution (pH 7.4) containing 0.1 M K2S2O8. Scan rate, 100 mV/s.
hindering the diffusion ECL coreactant toward the electrode surface. Therefore, both EIS and ECL characterization suggested the successful preparation of the aptasensor. 3.3. Optimization of experimental conditions In order to achieve sensitive detection of target protein, the optimal conditions including the pH and incubation time was studied. The effect of the pH was investigated in the range of 6.5–8.5. With the increase of pH from 6.5 to 7.4, the ECL intensity gradually decreased. However, when the pH increased beyond 7.4, the ECL response increased. Therefore, pH 7.4 was selected. Meanwhile, the incubation time influenced the ECL intensity directly. The ECL response decreased with the increment of the incubation time and reached a plateau at 45 min. Longer incubation time did not improve the response. Consequently, 45 min was chosen as the incubation time for determination of CEA. 3.4. Comparison of ECL responses with different labeled signal probes In order to investigate the ECL quenching ability of signal probe, two probes, GO-aptamer II and H-rGO-aptamer II/BCP, were prepared under the same conditions. The changed ECL values (ΔI) of the aptasensors with different probes were displayed in Fig. 4. The ΔI of the aptasensor incubated with H-rGO-aptamer II/BCP probe was 11,664 a.u. (Fig. 4B) compared with background values (the ECL responses of the CEA/BSA/ NH2-DNA/Au-CdS-CS), while that decreased to 5454 a.u after incubated
with GO-aptamer II (ΔI represents the degree of ECL intensity change, ΔI = (I − I0), where I and I0 are the ECL intensity in the presence and absence of CEA, respectively) (Fig. 4A). The reason was that the hemin attached on the graphene sheets still retained the catalytic-active monomer form, which can catalyze the reaction of peroxidase substrate in the presence of H2O2. After incubated with 4-CN solution, in view of hemin can oxidize the 4-CN by H2O2 to produce an insulating barrier on the electrode surface, thereby effectively inhibiting the reaction between coreactant K2S2O8 and Au-CdS for the consequent high ECL quenching efficiency.
3.5. Analytical performance of the ECL aptasensor In order to ensure that the proposed strategy can be quantitatively used to detected CEA, a series of CEA concentrations (4 ng/mL, 0.8 ng/mL, 0.4 ng/mL, 0.08 ng/mL, 0.04 ng/mL, 0.01 ng/mL, 0.008 ng/mL, 0.004 ng/mL, 0.001 ng/mL, 0.0008 ng/mL) was investigated. The plot of ECL change vs the logarithmic value of CEA concentration showed a linear relationship in the range of 0.8 pg/mL to 4 ng/mL with a correlation coefficient of 0.995. The linear equation was I = 12,127.61 − 2472.38 lg c (Fig. 5A). Meanwhile, the limit of detection (LOD, S/N = 3) for CEA concentration was calculated to be 0.28 pg/mL. Furthermore, the analytical performance of the proposed method was compared with other reported CEA detection methods (listed in Table 1). As shown, the constructed ECL aptasenor in this
Fig. 4. ECL responses of aptasensor by using different labeled probes: GO-aptamer II probe (A); H-rGO-aptamer II/BCP probe (B) in 0.1 M PBS (pH 7.4) containing 0.1 M K2S2O8.
J.-J. Yang et al. / Journal of Electroanalytical Chemistry 787 (2017) 88–94
93
Fig. 5. (A) The calibration curve between the logarithm of CEA concentrations and ECL intensity. (B) The specificity of the ECL aptasensor toward different targets. CIgG: 10 mg/mL, CHSA: 40 mg/mL, CCEA: 0.8 ng/mL. (C) Continuous cyclic potential scans of the proposed aptasensor. (D) Repeatability of the aptasensor with five electrodes. Dection solution: 0.1 mol/L PBS (pH 7.4) containing 0.1 mol/L K2S2O8 at a scanning rate of 100 mV/s (error bar = SD, n = 3).
work represented a relatively wide linear range with a low detection limit. 3.6. Specificity, stability and repeatability of ECL response To study the specificity of the proposed method, the non-target protein HSA, and IgG were chosen as the possible interfering substances. As seen from Fig. 5B, the ECL intensity of the mixture containing HSA, IgG and CEA was almost the same as the value obtained from CEA only. When the aptasensor was incubated with interfering substance, there was no obvious effect on the ECL intensity compared with the blank sample. These results indicate that the aptasensor possesses good specificity for CEA. The stability was also monitored by continuous cyclic potential scan for 18 circles with RSD of 2.4% (Fig. 5C). Moreover, the repeatability of the method was evaluated by analyzing five different sensors in the same conditions. The results were shown in Fig. 5D, the RSD was 3.2%. 3.7. Application in complex biological samples To further evaluate the application potential of the aptasensor in the biological samples, nine human serum including three healthy persons (No. 1–3) and six cancer patients (No. 4–9) were measured by the ECL aptasensor and referenced method of ROCHE ELISA ECL analyzer used by Xinyang Central Hospital, respectively. Three of the serum samples (No. 1–3) were not diluted during the experiments and the rest of the Table 1 Analytical performance of various methods for CEA detection. Methods
Linear range (ng/mL)
Detection limits (pg/mL)
Reference
Electrochemistry Impedimetry Fluorescence PEC CE-CL ECL ECL
0.1–750.0 0.001–20 0.02–0.2 0.0005–100 0.05–20 0.05–20 0.0008–4
90 0.42 1 0.1 34 31 0.28
[37] [38] [40] [39] [7] [41] This work
serum samples (No. 4–9) were diluted because the content of CEA was beyond the linear range. The analytical results were listed in Table 2. By comparison, the results of CEA are in acceptable agreement with the reference values. The relative errors were within 12.2%. The linear regression equation of the two methods is Y = 0.104 + 0.959 X (X, the reference method; Y, the proposed method) with a correlation coefficient of 0.993. Whereafter, the recovery experiments were performed by the standard addition method. Three different concentrations of CEA, 0.04 ng/mL, 0.4 ng/mL and 4 ng/mL, were spiked into the serum samples, respectively. The recoveries ranged from 95.0% to 115.8% with the RSDs varied from 4.5% to 9.1%. These results demonstrated that the matrix effect has no obvious influence on the response to CEA and the provided aptasensor held the reliability to determine CEA in biological sample. 4. Conclusions In summary, this study developed a dual-quenching sandwich-like ECL sensing system by taking advantage of H-rGO-aptamer II composite specifically identified the target and the attractively catalytic property of H-rGO to BCP. The flower-like spherical Au-CdS nanocomposites as ECL
Table 2 Assay results of CEA in human serum samples using the proposed method and reference method. Sample no.
Reference method (ng/mL)
Proposed method (ng/mL) ± SD
Relative errors (%)
1 2 3 4 5 6 7 8 9
1.27 0.94 2.78 8.06 6.74 9.34 4.50 7.67 11.21
1.35 ± 0.05 1.01 ± 0.13 2.56 ± 0.07 7.87 ± 0.22 7.15 ± 0.24 8.99 ± 0.07 3.95 ± 0.08 7.07 ± 0.14 10.53 ± 0.17
6.3 7.5 −7.9 −2.3 6.1 −3.7 −12.2 −7.8 −6.1
94
J.-J. Yang et al. / Journal of Electroanalytical Chemistry 787 (2017) 88–94
luminophores show excellent ECL performance. The quenching efficiency of H-rGO and the subsequent formation of insoluble product induced by hemin with intrinsic peroxidase-like catalytic activity achieved sensitive detection of CEA. In addition, the aptamer adsorbed on the surface of H-rGO sheets was realized just by π-π stacking interaction, any complicated or chemical modification was unnecessary, avoiding limitation the number of target binding sites, and also providing the universality for the design by virtue of altering the sequence of aptamer. Simultaneously, this developed method could be successfully applied to the determination of CEA in complex biosamples. This ECL sensing strategy promises a great potential of becoming a useful tool for quantitative detection of diverse proteins in biochemical analysis. Acknowledgements This work was supported by the National Natural Science Foundation of China (21675136, 21375114, and 21405129), Plan for Scientific Innovation Talent of Henan Province (2017JR0016), and Nan Hu Young Scholar Supporting Program of XYNU. References [1] M.D. Greg, L. Perkins, M.D. Evan, D. Slater, M.D. Georganne, K. Sanders, M.D. John, G. Prichard, Am. Fam. Physician 68 (2003) 1075–1081. [2] N. Laboria, A. Fragoso, W. Kemmner, D. Latta, O. Nilsson, M.L. Botero, K. Drese, C.K. O'Sullivan, Anal. Chem. 82 (2010) 1712–1719. [3] Q.L. Yu, X.F. Zhan, K.P. Liu, H. Lv, Y.X. Duan, Anal. Chem. 85 (2013) 4578–4585. [4] D.J. Lin, J. Wu, H.X. Ju, F. Yan, Biosens. Bioelectron. 52 (2014) 153–158. [5] G.M. Wen, X.J. Yang, X.L. Xi, J. Electroanal. Chem. 757 (2015) 192–197. [6] R.Y. Dian, P. Tang, Y.Q. Chai, Anal. Chem. 80 (2008) 1582–1588. [7] J. Jiang, S.L. Zhao, Y. Huang, G.X. Qin, F.G. Ye, J. Chromatogr. A 1282 (2013) 161–166. [8] S.P. Song, L.H. Wang, J. Li, J. Zhao, C.H. Fan, Anal. Chem. 27 (2008) 108–117. [9] D.F. Wang, Y.Y. Li, Z.Y. Lin, B. Qiu, L.H. Guo, Anal. Chem. 87 (2015) 5966–5972. [10] Z.J. Wu, H. Li, Z.H. Liu, Sensors Actuators B Chem. 206 (2015) 531–537. [11] W. Lv, M. Guo, M.H. Liang, F.M. Jin, L. Cui, L.J. Zhi, Q.H. Yang, J. Mater. Chem. 20 (2010) 6668–6673. [12] A.J. Stewart, J. Hendry, L. Dennany, Anal. Chem. 87 (2015) 11847–11853. [13] Q.M. Feng, Y.Z. Shen, M.X. Li, Z.L. Zhang, W. Zhao, J.J. Xu, H.Y. Chen, Anal. Chem. 88 (2016) 937–944. [14] P. Wu, X. Hou, J.J. Xu, H.Y. Chen, Chem. Rev. 114 (2014) 11027–11059. [15] X.T. Wang, C.H. Liow, D.P. Qi, B.W. Zhu, W.R. Leow, H. Wang, C. Xue, X.D. Chen, S.Z. Li, Adv. Mater. 26 (2014) 3506–3512.
[16] E. Khon, A. Mereshchenko, A.N. Tarnovsky, K. Acharya, A. Klinkova, N.N. HewaKasakarage, I. Nemitz, M. Zamkov, Nano Lett. 11 (2011) 1792–1799. [17] M. Zayats, A.B. Kharitonov, S.P. Pogorelova, O. Lioubashevski, E. Katz, I. Willner, J. Am. Chem. Soc. 125 (2003) 16006–16014. [18] G.F. Shi, J.T. Cao, J.J. Zhang, Y.M. Liu, Y.H. Chen, S.W. Ren, Sensors Actuators B Chem. 220 (2015) 340–346. [19] R. Qu, L.L. Shen, Z.H. Chai, C. Jing, Y.F. Zhang, Y.L. An, L.Q. Shi, ACS Appl. Mater. Interfaces 6 (2014) 19207–19216. [20] Z.H. Yang, Y. Zhuo, R. Yuan, Y.Q. Chai, Biosens. Bioelectron. 78 (2016) 321–327. [21] F.Q. Luo, Y.L. Lin, L.Y. Zheng, X.M. Lin, Y.W. Chi, ACS Appl. Mater. Interfaces 7 (2015) 11322–11329. [22] Q.G. Wang, Z.M. Yang, X.Q. Zhang, X.D. Xiao, C.K. Chang, B. Xu, Angew. Chem. Int. Ed. 46 (2007) 4285–4289. [23] Y.J. Li, X.Q. Huang, Y. Huang, Y.J. Li, Y.X. Xu, Y. Wang, E.B. Zhu, X.F. Duan, Sci. Rep. 3 (2013) 1787. [24] T. Xue, S. Jiang, Y.Q. Qu, Q. Su, R. Cheng, S. Dubin, C.Y. Chiu, R. Kaner, Y. Huang, X.F. Duan, Angew. Chem. Int. Ed. 51 (2012) 3822–3825. [25] Y.J. Guo, L. Deng, J. Li, S.J. Guo, E.K. Wang, S.J. Dong, ACS Nano 5 (2011) 1282–1290. [26] Y.L. Zhou, M. Wang, Z.N. Xu, C.L. Ni, H.S. Yin, S.Y. Ai, Biosens. Bioelectron. 54 (2014) 244–250. [27] Z.T. Yang, J. Qian, X.W. Yang, D. Jiang, X.J. Du, K. Wang, H.P. Mao, K. Wang, Biosens. Bioelectron. 65 (2015) 39–46. [28] Y. Tao, Y.H. Lin, J.S. Ren, X.G. Qu, Biomaterials 34 (2013) 4810–4817. [29] B. Lin, Q.Q. Sun, K. Liu, D.Q. Lu, Y. Fu, Z.A. Xu, W. Zhang, Langmuir 30 (2014) 2144–2151. [30] M.D. Xu, Z.Q. Gao, Q.H. Wein, G.N. Chen, D.P. Tang, Biosens. Bioelectron. 74 (2015) 1–7. [31] Y.M. Liu, J.J. Yang, J.T. Cao, J.J. Zhang, Y.H. Chen, S.W. Ren, Sensors Actuators B Chem. 232 (2016) 538–544. [32] S.C. Han, L.F. Hu, N. Gao, A.A. Al-Ghamdi, X.S. Fang, Adv. Funct. Mater. 24 (2014) 3725–3733. [33] Y. Jiao, X.M. Ma, F.F. Wang, K. Wang, P.P. Zhang, Z.Z. Wang, L. Yang, Micro Nano Lett. 7 (2012) 631–633. [34] W.S. Hummers, R.E. Offeman, J. Am. Chem. Soc. 80 (1958) 1339. [35] L. Dan, M.B. Müller, S. Gilje, R.B. Kaner, G.G. Wallace, Nat. Nanotechnol. 3 (2008) 101–105. [36] Y.X. Xu, L. Zhao, H. Bai, W.J. Hong, C. Li, G.Q. Shi, J. Am. Chem. Soc. 131 (2009) 13490–13497. [37] J.Y. Liu, J. Wang, T.S. Wang, D. Li, F.N. Xi, J. Wang, E.K. Wang, Biosens. Bioelectron. 15 (2015) 281–286. [38] L. Hou, X.P. Wu, G.N. Chen, H.H. Yang, M.H. Lu, D.P. Tang, Biosens. Bioelectron. 68 (2015) 487–493. [39] G.C. Fan, H. Zhu, D. Du, J.R. Zhang, J.J. Zhu, Y.H. Lin, Anal. Chem. 88 (2016) 3392–3399. [40] J. Zhu, J.F. Wang, J.J. Li, J.W. Zhao, Sensors Actuators B Chem. 233 (2016) 214–222. [41] Y.L. Wang, J.T. Cao, Y.H. Chen, Y.M. Liu, Anal. Methods 8 (2016) 5242–5247.
Contents lists available at ScienceDirect
Journal of Electroanalytical Chemistry journal homepage: www.elsevier.com/locate/jelechem
Sandwich-like electrochemiluminescence aptasensor based on dual quenching effect from hemin-graphene nanosheet and enzymatic biocatalytic precipitation for sensitive detection of carcinoembryonic antigen Jiu-Jun Yang a,b, Jun-Tao Cao a,b,⁎, Yu-Ling Wang a,b, Hui Wang a,b, Yan-Ming Liu a,b,⁎, Shu-Hui Ma c a b c
College of Chemistry and Chemical Engineering, Xinyang Normal University, Xinyang 464000, P.R. China Institute for Conservation and Utilization of Agro-bioresources in Dabie Mountains, Xinyang Normal University, Xinyang 464000, P.R. China Xinyang Central Hospital, Xinyang 464000, P.R. China
a r t i c l e
i n f o
Article history: Received 10 December 2016 Received in revised form 20 January 2017 Accepted 21 January 2017 Available online 23 January 2017 Keywords: Dual-quenching effect Hemin-rGO nanosheet Enzymatic biocatalytic precipitation Flower-like spherical Au-CdS Electrochemiluminescence Carcinoembryonic antigen
a b s t r a c t A new and simple sandwich-like electrochemiluminescence (ECL) aptasensor for carcinoembryonic antigen (CEA) assay was fabricated based on the dual quenching effect from hemin-graphene (H-rGO) nanosheet and enzymatic biocatalytic precipitation (BCP) on the Au-CdS nanocomposites-based ECL system. In this aptasensor platform, flower-like spherical Au-CdS nanocomposites were used as ECL luminophores and exhibit a strong ECL signal. The rGO nanosheet was used as a supporter to immobilize hemin molecules via π-π stacking interactions. Due to the steric hindrance and quenching effect of rGO, the ECL intensity decreased by the construction of the sandwich “CEA aptamer I (NH2-DNA)-CEA-aptamer II” (H-rGO-aptamer II) mode. In the process of BCP, the ECL intensity further decreased because the hemin with intrinsic peroxidase-like catalytic activity could oxidize the 4-chloro-1-naphthol (4-CN) to produce an insoluble precipitation on the sensor. Using this dual quenching strategy, the prepared aptasensor exhibits a linear range from 0.8 pg/mL to 4 ng/mL and a detection limit of 0.28 pg/mL. This ECL aptasensor has simple design and undemanding in operation and was utilized to determine the content of CEA in complex samples with recoveries of 95.0% to 115.8%. Moreover, no any chemical modification of aptamer was required, suggesting that the proposed ECL aptasensor could be applied for the detection of diverse proteins just by altering the aptamer sequence. © 2017 Elsevier B.V. All rights reserved.
1. Introduction The increasing demand of determination of disease-related proteins, especially cancer biomarkers, has received more and more attention in many fields. Carcinoembryonic antigen (CEA), a type of glycoprotein generated by tumor cells, was widely used as a tumor marker [1]. The specific and sensitive determination of CEA has great significance for clinical diagnosis and treatment assessment of cancers. Thus, many analytical methods for CEA detection have been studied, such as amperometric assay [2], fluorescence analysis [3], electrochemical method [4], photoelectrochemical assay [5], enzyme-linked immunosorbent assays [6] and capillary electrophoresis [7]. Among these methods, antibodies were mainly acted as recognition elements. However, the limits of antibodies are easy deactivation and instability. Aptamer, a kind of new recognition element, shows high bind affinity to its target and possesses numerous remarkable advantages such as design flexibility, easy ⁎ Corresponding authors. E-mail addresses: [email protected] (J.-T. Cao), [email protected] (Y.-M. Liu).
http://dx.doi.org/10.1016/j.jelechem.2017.01.044 1572-6657/© 2017 Elsevier B.V. All rights reserved.
synthesis and specificity [8]. So far, aptameric-based protein analytical systems have been greatly developed. For instance, in Guo's group [9], two different kinds of aptamers (with amidogen and thiol group) specific to CEA were modified on the surface of Ru@SiO2 and Au NPs based on localized surface plasmon resonance for CEA detection. Wu et al. [10] constructed an aptasensor for CEA detection based on fluorescence resonance energy transfer, an amino group modified CEA aptamer was covalently tagged on the PAA-UCPs which was employed as the energy donor. Noticed that the aptamers for protein detection were all chemically modified, which not only limited the association with the number of target binding sites, but also increased the operation and expenses. In addition, many literatures reported that graphene sheet can be served as a supporter to adsorb ss-DNA due to its high surface area and π-π conjunction [11]. Therefore, attaching aptamer onto the graphene sheet as a probe may be an alternative for constructing a sensor with good property. Electrochemiluminescence (ECL) approach is competitive with conventional assays because of its high sensitivity, rapid response and low background [12,13]. In recent years, various semiconductor
J.-J. Yang et al. / Journal of Electroanalytical Chemistry 787 (2017) 88–94
nanomaterials with ECL activity have been growing [14]. CdS nanomaterials are the most promising and effective materials, owing to their excellent luminescent properties in the presence of coreactant. According to reports, Au nanoparticles exhibited a strong surface plasmon resonance effect on the CdS because Au can increase the light absorption and enhance the charge separation [15–17]. Wang et al. [15] reported an obvious enhancement of photocatalytic activity in the CdS-Au-CdS nanorod arrays, which was relative to that of pure CdS because surface plasmon resonance effect of Au segments increased the effective optical length inside the CdS. Therefore, depositing AuNPs on the spherical CdS is also expected to obtain enhanced intensity compared with the pure CdS. Hemin, the active center of heme-protein, has the peroxidase-like activity similar to the peroxidase enzyme. In general, hemin could intercalate into G4 structure to form G4H DNAzyme with high peroxidaselike catalytic activity and there are no disadvantages of the natural enzymes such as instability, long-time consumption. In our previous work [18], hemin-DNAzyme with high electrocatalytic ability was used to quench ECL signal of MoS2-CdS nanocomposites for sensitive detection of IgE. Nevertheless, it is difficult for hemin to maintain high catalytic activity in aqueous solution due to its low solubility and easy aggregation [19]. In order to solve this problem, various nanomaterials have been used as nanocarriers to load hemin, such as ZnO [20], metal-Organic frameworks [21], supramolecular hydrogels [22]. Graphene [23], with large surface area and good biocompatibility, has been also served as a valuable candidate for immobilization hemin. In addition, graphene possesses a rich surface chemistry and has the potential to promote the catalytic activity and stability of the supported molecular systems by cation-π interactions or π-π stacking. Duan's group [24] reported that hemin attached on the graphene still retained the catalytic-active monomer form as in natural enzymes. So far, H-rGO with peroxidase activity has been widely applied in many aspects, such as distinguishing between ss- and ds-DNA [25], electrochemical and ECL sensor [26,27]. Tao and co-workers [28] described a sensing strategy by employing ss-DNA probe and H-rGO sheets to detect a wide range of targets including metal ions, small molecules and DNA. A label-free colorimetric method for PDGF-BB and thrombin assay on the basis of HrGO-DNA composite was reported by Zhang's group [29]. However, as far as we know, little attention has been paid to the applications of the above-mentioned rGO-based hybrid peroxidase mimetics in ECL aptasensor. Except for the nanomaterial-based labeling strategy, employing enzymatic reaction to form insoluble product on the sensing interface, e.g. enzymatic biocatalytic precipitation (BCP), is an important concern for obtaining low limits of detection. Tang et al. [30] utilized the formed hemin-based DNAzyme concatamers toward catalytic precipitation of 4-CN for recognizing Cu2+. Our group [31] prepared a competitive ECL aptasensor by introducing BCP technique for the sensitive detection of IgE. Herein, we fabricated a new and simple sandwich-like ECL aptasensor for sensitive CEA assay by coupling with H-rGO-aptamer II composite as probe. The probe was used as both quencher and catalyst of BCP. The monodisperse CdS nanoparticles were synthetized via a hydrothermal method and used as a support to load high amounts of AuNPs to prepare flower-like spherical Au-CdS nanocomposite. HrGO-aptamer II composite could use as an excellent platform for assembling ss-DNA containing a 24-base tail and a 19-base CEA aptamer sequence. In the presence of CEA, aptamers specifically combined with CEA to form a sandwich structure with the 24-base fragment still adsorbed on rGO. The ECL intensity was quenched due to the steric hindrance and quenching effect of rGO. Simultaneously, in view of the intrinsic peroxidase-like activity of hemin catalyzed to produce insoluble product on the electrode after incubated with 4-CN, further quenched the ECL signal. Under the optimum conditions, the aptasensor for CEA assay was well established and exhibits good performance to determine CEA in human serum samples.
89
2. Experimental 2.1. Materials and reagents Carcinoembryonic antigen (CEA) aptamers 5′-NH2-(CH2)6-TTT TAT ACC AGC TTA TTC AAT T-3′ (aptamer I, NH2-DNA) and 5′-CCC ATA GGG AAG TGG GGG ATG TGT GTG TGT GTG TGT GTG TGT G (aptamer II) were synthesized by Sangon biotech Co. Ltd. (Shanghai, China). CEA was purchased from Zhengzhou Immuno Biotech Co., Ltd. (Zhengzhou, China). Hemin was from Sigma-Aldrich Chemical Co. (St. Louis, MO). Cadmium acetate [Cd(CH3COO)2·4H2O] and thiourea were got from Sinopharm Group Chemical Reagent Co., Ltd. (Shanghai, China). Sodium L-glutamate monohydrate was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). HAuCl4·3H2O and glutaraldehyde (GA) were acquired from Alfa Aesar (Tianjin, China) and Tianjin Yongda Chemical Reagent Development Center (Tianjin, China), respectively. Human IgG (hIgG), human serum albumin (HSA), and bovine serum albumin (BSA) were purchased from Shanghai Solarbio Bioscience & Technology Co., Ltd. (see bio Biotechnology). 4-chloro-1-naphthol (4-CN) was from Shanghai Ziyi Reagent Company (Shanghai, China). Ethylene glycol, Ammonia (28%) and hydrazine hydrate (35%) were from Aladdin industrial corporation (Shanghai, China). Phosphate-buffered saline (PBS) solutions with various pHs were prepared by mixing different volumes of NaH2PO4 and K2HPO4 containing 0.1 M KCl as the supporting electrolyte. Ultrapure water (Kangning Water Treatment Solution Provider, Chengdu, China) was used throughout the experiments. 2.2. Apparatus The ECL emissions were recorded using a BPCL ultra-weak luminescence analyzer (Institute of Biophysics, Chinese Academy of Science, Beijing, China) with a CR 120 type photomultiplier tube (Binsong Photonics, Beijing, China). All ECL experiments were performed with a conventional three-electrode system comprised of a Pt wire as the counter electrode, an Ag/AgCl electrode as reference electrode and bare or modified glass carbon electrodes (GCE, φ = 3 mm) as the working electrodes. Electrochemical impedance spectroscopy (EIS) was performed using an RST5200F electrochemical workstation (Zhengzhou Shiruisi Technology Co., Ltd., Zhengzhou, China) in the solution of 5 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) containing 0.1 M KCl. Scanning electron microscopy (SEM) images and transmission electron microscope (TEM) images were acquired using a S-4800 (Hitachi, Tokyo, Japan) and Tecnai G2 F20 TEM (FEI Co., Hillsboro, Oregon, USA), respectively. X-ray photoelectron spectra (XPS) analysis was performed on a KAlpha X-ray photoelectron spectrometer (Thermo Fisher Scientific Co., USA). UV–visible detection was carried out using an UV mini-1240 UV–vis spectrophotometer (Shimadzu Corp., Kyoto, Japan). 2.3. Synthesis of Au-CdS nanocomposites 2.3.1. Synthesis of spherical CdS nanoparticles The monodisperse CdS nanoparticles were synthesized following a typical procedure [32]. Firstly, 2.4 mmol Cd(CH3COO)2 was dissolved in 60 mL water and sonicated for a few minutes. Then 24 mmol thiourea was added in the above solution after vigorous stirring for 30 min, the mixture was transferred to a Tefion-lined autoclave (100 mL) and heated to 200 °C for 5 h. After naturally cooled, the precipitate was washed with water and ethanol for three times, respectively, and dried at 60 °C. 2.3.2. Synthesis of Au-CdS nanocomposites Au-CdS nanocomposites were prepared as previously described [33]. Briefly, 0.55 mL HAuCl4 aqueous solution was added into 50 mL of ethylene glycol, followed by the dissolution of 55 mg glutamate into the above solution with vigorous stirring. The pH was then adjusted to 11 by NaOH and 53 mg of CdS nanomaterial was subsequently slowly
90
J.-J. Yang et al. / Journal of Electroanalytical Chemistry 787 (2017) 88–94
added. The reaction solution was stirred at room temperature for 24 h. Finally, the solution was centrifuged at 9000 rpm for 15 min to collect the precipitate, which was then washed separately with water and ethanol for several times, and dried at 60 °C. 2.4. Preparation of hemin functionalized graphene nanosheet (H-rGO) Graphene oxide (GO) was prepared using modified Hummer's method [34]. Graphene–hemin composites were synthesized according to Guo's method [25]. Briefly, 10 mg of GO was added into 20 mL of water and sonicated for 1 h to obtain a homogeneous dispersion. The resulting dispersion was mixed with 20 mL of 0.5 mg/mL hemin solution in a flask and stirred for 30 min. Then 200 μL of ammonia solution and 30 μL of hydrazine solution were added. After being vigorously stirred for 30 min, the above solution was put in an oil bath at 60 °C for another 4 h. The stable black dispersion was obtained. 2.5. Synthesis of H-rGO-aptamer II composites According to the literature [28,29], a certain concentration of H-rGO aqueous solution and aptamer (4 μM) were mixed in Tris-EDTA buffer (10 mM Tris-HCl, 10 mM EDTA, pH 8.0) by sonicating for 10 min and incubated at room temperature for 12 h. After that, 100 μL 0.005 M NaCl solutions were added. Finally, the solution was centrifuged at 12,000 rpm for 20 min and the precipitated H-rGO-aptamer conjugates were redispersed in Tris-EDTA buffer and stored in 4 °C. 2.6. Fabrication of ECL aptasensor The construction process of the ECL aptasensor was displayed in Scheme 1. At first, the Au-CdS composites were mixed with 0.1 mg/mL chitosan (CS) solution by sonication and the Au-CdS/CS composites film was obtained by dropping 6 μL of the obtained composites solution onto a polished and cleaned GCE, drying in air. Subsequently, 6 μL of 5% (v/v) GA in PBS was covered on the electrode and remained at room temperature for 1 h. Then the electrode was rinsed with water thoroughly to remove physically adsorbed GA. The amino-modified DNA were introduced onto the GCE/Au-CdS/CS/GA-activated electrode by dropping 6 μL of NH2-DNA on the GCE and incubated at 4 °C overnight. After the nonspecific bind sites were thoroughly washed
and blocked by 1% BSA, the prepared electrodes were immersed into a series of CEA solution at different concentrations and incubated at 37 °C for 1 h. Following by washed with water, the above-prepared HrGO-aptamer II solution was coated onto the surface of CEA/BSA/NH2DNA/Au-CdS/CS/GCE and incubated at 37 °C for 45 min, and again washed thoroughly with water to remove the unbound probes. Finally, the above electrodes were further reacted with BCP solution consisting of 1 mM 4-CN and 0.15 mM H2O2 at 25 °C for 15 min. After thoroughly rinsed with water, the electrodes were prepared for ECL detection. The ECL responses of the electrodes were recorded in 0.1 M PBS (pH 7.4) containing 0.1 M K2S2O8 as a coreactant. The voltage of the PMT was set at 800 V. ECL signals related to the CEA concentrations could be measured. 3. Results and discussion 3.1. Characterization of CdS nanoparticles, Au-CdS nanocomposites and hemin-rGO sheets The morphologies of CdS nanoparticles and Au-CdS nanocomposites were characterized by SEM and TEM. As shown in Fig. 1A, the monodisperse CdS nanoparticles are flower-like spherical structure in shape and have a uniform morphology with a size of about 200 nm. TEM result also revealed relatively uniform CdS structure (Fig. 1C). When HAuCl4 was introduced and reduced, some highlights could be observed on the surface of flower-like spherical CdS, the corresponding SEM micrograph of Au-CdS nanocomposites were shown in Fig. 1B, indicating the AuNPs were successfully reduced on the surface of the CdS. The inset of the Fig. 1C showed corresponding high-magnification TEM image, it could be seen that AuNPs were uniformly deposited on the surface of the CdS. GO and rGO-hemin were characterized by UV–vis absorption spectra (Fig. 2A). The GO dispersion (curve a) exhibited a maximum absorption peak at 227 nm owing to the π-π* transition of aromatic C _C bonds and a shoulder at ca. 290–300 nm which corresponds to the nπ* transition of the C _O bond [35]. The spectrum of hemin solution (curve b) displayed an absorption peak at 390 nm attributed to the Soret band. After reduced, two characteristic absorption peaks of GH at 262 nm and 416 nm were obviously observed. An absorption peak at 262 nm was corresponding to the reduced graphene oxide which was 35 nm red shifted compared to GO, while a second peak at
Scheme 1. H-rGO-aptamer composite synthesis process (A); schematic illustration of the ECL biosensor for detection of PSA (B).
J.-J. Yang et al. / Journal of Electroanalytical Chemistry 787 (2017) 88–94
91
Fig. 1. SEM images of the as-prepared CdS (A) and Au-CdS (B), the insets of A and B correspond to the single CdS and Au-CdS, respectively; TEM image of CdS (C), the inset of the C corresponds to the TEM image of AuNPs uniformly deposited on the surface of the CdS.
416 nm was attributed to the Soret band of hemin with bathochromic shift of 26 nm. These findings indicate the existence of the π-π interactions between the GN and the adsorbed hemin molecules, which are in agreement with the previous report about the interactions of cationic porphyrin derivative with chemical converted graphene [36]. To conclude, hemin molecules could be attached onto graphene via π-π interaction. Attaching hemin on rGO nanosheet was further verified by X-ray photoelectron spectroscopy (XPS). As seen from Fig. 2B, the fully scanned spectra demonstrated the existence of iron, nitrogen and carbon elements in hemin-rGO nanosheet. The characteristic peaks of Fe 2P (2P3/2 710.88 ev, 2P1/2 724.48 ev), N 1S (399.2 ev), C 1S (283.98 ev), O 1S (531.58 ev) were respectively derived from hemin and rGO, clearly confirming the successfully synthesis of hemin-rGO nanosheet. 3.2. Electrochemical and electrochemiluminescence characterization of the fabricated aptasensor In order to characterize the modified process of the sensing interface, EIS was performed to investigate the modification of the electrode. As shown in Fig. 3A, the bare GCE exhibited a small semicircle (curve a). After the CS-Au-CdS covered on the electrode (curve b), an increased semicircle was obtained due to the CS decreased the electron-transfer efficiency. Moreover, with the sequential assembly of negative NH2DNA (curve c), inert BSA (curve d) and CEA (curve e), the resistance
increased steadily. The reason was that the formation of DNA or protein layer hindered the electron transfer. Similarly, continuous increases of Ret values were obtained after binding with H-rGO-aptamer II, reflecting the signal probe being successfully captured. Finally, the semicircle diameter increased greatly after incubated with 4-CN solution (curve g) as a result of yielding the insulating product prevented the diffusion of redox probe to the electrode surface. In addition, the ECL measurement was used to further monitor the stepwise assembly of the aptasensor. As shown in Fig. 3B, it can be seen that no ECL signal was found at the bare GCE (curve a). A strong ECL signal was obtained after assembling the Au-CdS-CS composites on the bare electrode (curve b). Two sensors, with Au (Au-CdS/GCE) and without Au (CdS/GCE), were constructed and tested under the same experimental conditions. The results were presented in insert figure in Fig. 3B. The results show that the ECL intensity of the former was higher than the latter as a result of Au nanoparticles on the CdS material exhibiting SPR effect. When the resultant electrode was immobilized of NH2-DNA, BSA, and CEA, successively, obvious gradual decreases in ECL intensity were observed (curves c, d, and e) due to the nonelectroactive property of DNA and protein and hindering the electron transfer. The ECL signal yet further declined after conjugation of H-rGO-aptamer II (curve f), which was attributed to the steric hindrance and quenching effect of rGO. After the successive incubation with 4-CN solution, the ECL intensity still decreased (curve g). The phenomena can be explained by the insoluble substance forming a barrier for electron transfer and
Fig. 2. (A) UV–vis absorption spectra of GO suspension (a), hemin solution (b), and hemin-graphene suspension (c); (B) XPS analyses for the full region of XPS for GH sheets, (C) the Fe 2P region, N 1S region, C 1S region, and O 1S region.
92
J.-J. Yang et al. / Journal of Electroanalytical Chemistry 787 (2017) 88–94
Fig. 3. The curves of EIS (A) and ECL (B) characterization of electrodes at different modify stages: bare GCE (a); Au-CdS-CS/GCE (b); NH2-DNA/Au-CdS-CS/GCE (c); BSA/NH2-DNA/Au-CdSCS/GCE (d), CEA/BSA/NH2-DNA/Au-CdS-CS/GCE (e); H-rGO-aptamer II/CEA/BSA/NH2-DNA/Au-CdS-CS/GCE (f); BCP/H-rGO-aptamer II/CEA/BSA/NH2-DNA/Au-CdS-CS/GCE (g). Inset in B: ECL curves in the presence (curve b′) and absence (curve a′) of AuNPs. EIS was measured in 0.1 M KCl solution containing 5.0 mM [Fe(CN)6]3−/4− at a scan rate of 100 mV/s. ECL detection buffer: 0.1 M PBS solution (pH 7.4) containing 0.1 M K2S2O8. Scan rate, 100 mV/s.
hindering the diffusion ECL coreactant toward the electrode surface. Therefore, both EIS and ECL characterization suggested the successful preparation of the aptasensor. 3.3. Optimization of experimental conditions In order to achieve sensitive detection of target protein, the optimal conditions including the pH and incubation time was studied. The effect of the pH was investigated in the range of 6.5–8.5. With the increase of pH from 6.5 to 7.4, the ECL intensity gradually decreased. However, when the pH increased beyond 7.4, the ECL response increased. Therefore, pH 7.4 was selected. Meanwhile, the incubation time influenced the ECL intensity directly. The ECL response decreased with the increment of the incubation time and reached a plateau at 45 min. Longer incubation time did not improve the response. Consequently, 45 min was chosen as the incubation time for determination of CEA. 3.4. Comparison of ECL responses with different labeled signal probes In order to investigate the ECL quenching ability of signal probe, two probes, GO-aptamer II and H-rGO-aptamer II/BCP, were prepared under the same conditions. The changed ECL values (ΔI) of the aptasensors with different probes were displayed in Fig. 4. The ΔI of the aptasensor incubated with H-rGO-aptamer II/BCP probe was 11,664 a.u. (Fig. 4B) compared with background values (the ECL responses of the CEA/BSA/ NH2-DNA/Au-CdS-CS), while that decreased to 5454 a.u after incubated
with GO-aptamer II (ΔI represents the degree of ECL intensity change, ΔI = (I − I0), where I and I0 are the ECL intensity in the presence and absence of CEA, respectively) (Fig. 4A). The reason was that the hemin attached on the graphene sheets still retained the catalytic-active monomer form, which can catalyze the reaction of peroxidase substrate in the presence of H2O2. After incubated with 4-CN solution, in view of hemin can oxidize the 4-CN by H2O2 to produce an insulating barrier on the electrode surface, thereby effectively inhibiting the reaction between coreactant K2S2O8 and Au-CdS for the consequent high ECL quenching efficiency.
3.5. Analytical performance of the ECL aptasensor In order to ensure that the proposed strategy can be quantitatively used to detected CEA, a series of CEA concentrations (4 ng/mL, 0.8 ng/mL, 0.4 ng/mL, 0.08 ng/mL, 0.04 ng/mL, 0.01 ng/mL, 0.008 ng/mL, 0.004 ng/mL, 0.001 ng/mL, 0.0008 ng/mL) was investigated. The plot of ECL change vs the logarithmic value of CEA concentration showed a linear relationship in the range of 0.8 pg/mL to 4 ng/mL with a correlation coefficient of 0.995. The linear equation was I = 12,127.61 − 2472.38 lg c (Fig. 5A). Meanwhile, the limit of detection (LOD, S/N = 3) for CEA concentration was calculated to be 0.28 pg/mL. Furthermore, the analytical performance of the proposed method was compared with other reported CEA detection methods (listed in Table 1). As shown, the constructed ECL aptasenor in this
Fig. 4. ECL responses of aptasensor by using different labeled probes: GO-aptamer II probe (A); H-rGO-aptamer II/BCP probe (B) in 0.1 M PBS (pH 7.4) containing 0.1 M K2S2O8.
J.-J. Yang et al. / Journal of Electroanalytical Chemistry 787 (2017) 88–94
93
Fig. 5. (A) The calibration curve between the logarithm of CEA concentrations and ECL intensity. (B) The specificity of the ECL aptasensor toward different targets. CIgG: 10 mg/mL, CHSA: 40 mg/mL, CCEA: 0.8 ng/mL. (C) Continuous cyclic potential scans of the proposed aptasensor. (D) Repeatability of the aptasensor with five electrodes. Dection solution: 0.1 mol/L PBS (pH 7.4) containing 0.1 mol/L K2S2O8 at a scanning rate of 100 mV/s (error bar = SD, n = 3).
work represented a relatively wide linear range with a low detection limit. 3.6. Specificity, stability and repeatability of ECL response To study the specificity of the proposed method, the non-target protein HSA, and IgG were chosen as the possible interfering substances. As seen from Fig. 5B, the ECL intensity of the mixture containing HSA, IgG and CEA was almost the same as the value obtained from CEA only. When the aptasensor was incubated with interfering substance, there was no obvious effect on the ECL intensity compared with the blank sample. These results indicate that the aptasensor possesses good specificity for CEA. The stability was also monitored by continuous cyclic potential scan for 18 circles with RSD of 2.4% (Fig. 5C). Moreover, the repeatability of the method was evaluated by analyzing five different sensors in the same conditions. The results were shown in Fig. 5D, the RSD was 3.2%. 3.7. Application in complex biological samples To further evaluate the application potential of the aptasensor in the biological samples, nine human serum including three healthy persons (No. 1–3) and six cancer patients (No. 4–9) were measured by the ECL aptasensor and referenced method of ROCHE ELISA ECL analyzer used by Xinyang Central Hospital, respectively. Three of the serum samples (No. 1–3) were not diluted during the experiments and the rest of the Table 1 Analytical performance of various methods for CEA detection. Methods
Linear range (ng/mL)
Detection limits (pg/mL)
Reference
Electrochemistry Impedimetry Fluorescence PEC CE-CL ECL ECL
0.1–750.0 0.001–20 0.02–0.2 0.0005–100 0.05–20 0.05–20 0.0008–4
90 0.42 1 0.1 34 31 0.28
[37] [38] [40] [39] [7] [41] This work
serum samples (No. 4–9) were diluted because the content of CEA was beyond the linear range. The analytical results were listed in Table 2. By comparison, the results of CEA are in acceptable agreement with the reference values. The relative errors were within 12.2%. The linear regression equation of the two methods is Y = 0.104 + 0.959 X (X, the reference method; Y, the proposed method) with a correlation coefficient of 0.993. Whereafter, the recovery experiments were performed by the standard addition method. Three different concentrations of CEA, 0.04 ng/mL, 0.4 ng/mL and 4 ng/mL, were spiked into the serum samples, respectively. The recoveries ranged from 95.0% to 115.8% with the RSDs varied from 4.5% to 9.1%. These results demonstrated that the matrix effect has no obvious influence on the response to CEA and the provided aptasensor held the reliability to determine CEA in biological sample. 4. Conclusions In summary, this study developed a dual-quenching sandwich-like ECL sensing system by taking advantage of H-rGO-aptamer II composite specifically identified the target and the attractively catalytic property of H-rGO to BCP. The flower-like spherical Au-CdS nanocomposites as ECL
Table 2 Assay results of CEA in human serum samples using the proposed method and reference method. Sample no.
Reference method (ng/mL)
Proposed method (ng/mL) ± SD
Relative errors (%)
1 2 3 4 5 6 7 8 9
1.27 0.94 2.78 8.06 6.74 9.34 4.50 7.67 11.21
1.35 ± 0.05 1.01 ± 0.13 2.56 ± 0.07 7.87 ± 0.22 7.15 ± 0.24 8.99 ± 0.07 3.95 ± 0.08 7.07 ± 0.14 10.53 ± 0.17
6.3 7.5 −7.9 −2.3 6.1 −3.7 −12.2 −7.8 −6.1
94
J.-J. Yang et al. / Journal of Electroanalytical Chemistry 787 (2017) 88–94
luminophores show excellent ECL performance. The quenching efficiency of H-rGO and the subsequent formation of insoluble product induced by hemin with intrinsic peroxidase-like catalytic activity achieved sensitive detection of CEA. In addition, the aptamer adsorbed on the surface of H-rGO sheets was realized just by π-π stacking interaction, any complicated or chemical modification was unnecessary, avoiding limitation the number of target binding sites, and also providing the universality for the design by virtue of altering the sequence of aptamer. Simultaneously, this developed method could be successfully applied to the determination of CEA in complex biosamples. This ECL sensing strategy promises a great potential of becoming a useful tool for quantitative detection of diverse proteins in biochemical analysis. Acknowledgements This work was supported by the National Natural Science Foundation of China (21675136, 21375114, and 21405129), Plan for Scientific Innovation Talent of Henan Province (2017JR0016), and Nan Hu Young Scholar Supporting Program of XYNU. References [1] M.D. Greg, L. Perkins, M.D. Evan, D. Slater, M.D. Georganne, K. Sanders, M.D. John, G. Prichard, Am. Fam. Physician 68 (2003) 1075–1081. [2] N. Laboria, A. Fragoso, W. Kemmner, D. Latta, O. Nilsson, M.L. Botero, K. Drese, C.K. O'Sullivan, Anal. Chem. 82 (2010) 1712–1719. [3] Q.L. Yu, X.F. Zhan, K.P. Liu, H. Lv, Y.X. Duan, Anal. Chem. 85 (2013) 4578–4585. [4] D.J. Lin, J. Wu, H.X. Ju, F. Yan, Biosens. Bioelectron. 52 (2014) 153–158. [5] G.M. Wen, X.J. Yang, X.L. Xi, J. Electroanal. Chem. 757 (2015) 192–197. [6] R.Y. Dian, P. Tang, Y.Q. Chai, Anal. Chem. 80 (2008) 1582–1588. [7] J. Jiang, S.L. Zhao, Y. Huang, G.X. Qin, F.G. Ye, J. Chromatogr. A 1282 (2013) 161–166. [8] S.P. Song, L.H. Wang, J. Li, J. Zhao, C.H. Fan, Anal. Chem. 27 (2008) 108–117. [9] D.F. Wang, Y.Y. Li, Z.Y. Lin, B. Qiu, L.H. Guo, Anal. Chem. 87 (2015) 5966–5972. [10] Z.J. Wu, H. Li, Z.H. Liu, Sensors Actuators B Chem. 206 (2015) 531–537. [11] W. Lv, M. Guo, M.H. Liang, F.M. Jin, L. Cui, L.J. Zhi, Q.H. Yang, J. Mater. Chem. 20 (2010) 6668–6673. [12] A.J. Stewart, J. Hendry, L. Dennany, Anal. Chem. 87 (2015) 11847–11853. [13] Q.M. Feng, Y.Z. Shen, M.X. Li, Z.L. Zhang, W. Zhao, J.J. Xu, H.Y. Chen, Anal. Chem. 88 (2016) 937–944. [14] P. Wu, X. Hou, J.J. Xu, H.Y. Chen, Chem. Rev. 114 (2014) 11027–11059. [15] X.T. Wang, C.H. Liow, D.P. Qi, B.W. Zhu, W.R. Leow, H. Wang, C. Xue, X.D. Chen, S.Z. Li, Adv. Mater. 26 (2014) 3506–3512.
[16] E. Khon, A. Mereshchenko, A.N. Tarnovsky, K. Acharya, A. Klinkova, N.N. HewaKasakarage, I. Nemitz, M. Zamkov, Nano Lett. 11 (2011) 1792–1799. [17] M. Zayats, A.B. Kharitonov, S.P. Pogorelova, O. Lioubashevski, E. Katz, I. Willner, J. Am. Chem. Soc. 125 (2003) 16006–16014. [18] G.F. Shi, J.T. Cao, J.J. Zhang, Y.M. Liu, Y.H. Chen, S.W. Ren, Sensors Actuators B Chem. 220 (2015) 340–346. [19] R. Qu, L.L. Shen, Z.H. Chai, C. Jing, Y.F. Zhang, Y.L. An, L.Q. Shi, ACS Appl. Mater. Interfaces 6 (2014) 19207–19216. [20] Z.H. Yang, Y. Zhuo, R. Yuan, Y.Q. Chai, Biosens. Bioelectron. 78 (2016) 321–327. [21] F.Q. Luo, Y.L. Lin, L.Y. Zheng, X.M. Lin, Y.W. Chi, ACS Appl. Mater. Interfaces 7 (2015) 11322–11329. [22] Q.G. Wang, Z.M. Yang, X.Q. Zhang, X.D. Xiao, C.K. Chang, B. Xu, Angew. Chem. Int. Ed. 46 (2007) 4285–4289. [23] Y.J. Li, X.Q. Huang, Y. Huang, Y.J. Li, Y.X. Xu, Y. Wang, E.B. Zhu, X.F. Duan, Sci. Rep. 3 (2013) 1787. [24] T. Xue, S. Jiang, Y.Q. Qu, Q. Su, R. Cheng, S. Dubin, C.Y. Chiu, R. Kaner, Y. Huang, X.F. Duan, Angew. Chem. Int. Ed. 51 (2012) 3822–3825. [25] Y.J. Guo, L. Deng, J. Li, S.J. Guo, E.K. Wang, S.J. Dong, ACS Nano 5 (2011) 1282–1290. [26] Y.L. Zhou, M. Wang, Z.N. Xu, C.L. Ni, H.S. Yin, S.Y. Ai, Biosens. Bioelectron. 54 (2014) 244–250. [27] Z.T. Yang, J. Qian, X.W. Yang, D. Jiang, X.J. Du, K. Wang, H.P. Mao, K. Wang, Biosens. Bioelectron. 65 (2015) 39–46. [28] Y. Tao, Y.H. Lin, J.S. Ren, X.G. Qu, Biomaterials 34 (2013) 4810–4817. [29] B. Lin, Q.Q. Sun, K. Liu, D.Q. Lu, Y. Fu, Z.A. Xu, W. Zhang, Langmuir 30 (2014) 2144–2151. [30] M.D. Xu, Z.Q. Gao, Q.H. Wein, G.N. Chen, D.P. Tang, Biosens. Bioelectron. 74 (2015) 1–7. [31] Y.M. Liu, J.J. Yang, J.T. Cao, J.J. Zhang, Y.H. Chen, S.W. Ren, Sensors Actuators B Chem. 232 (2016) 538–544. [32] S.C. Han, L.F. Hu, N. Gao, A.A. Al-Ghamdi, X.S. Fang, Adv. Funct. Mater. 24 (2014) 3725–3733. [33] Y. Jiao, X.M. Ma, F.F. Wang, K. Wang, P.P. Zhang, Z.Z. Wang, L. Yang, Micro Nano Lett. 7 (2012) 631–633. [34] W.S. Hummers, R.E. Offeman, J. Am. Chem. Soc. 80 (1958) 1339. [35] L. Dan, M.B. Müller, S. Gilje, R.B. Kaner, G.G. Wallace, Nat. Nanotechnol. 3 (2008) 101–105. [36] Y.X. Xu, L. Zhao, H. Bai, W.J. Hong, C. Li, G.Q. Shi, J. Am. Chem. Soc. 131 (2009) 13490–13497. [37] J.Y. Liu, J. Wang, T.S. Wang, D. Li, F.N. Xi, J. Wang, E.K. Wang, Biosens. Bioelectron. 15 (2015) 281–286. [38] L. Hou, X.P. Wu, G.N. Chen, H.H. Yang, M.H. Lu, D.P. Tang, Biosens. Bioelectron. 68 (2015) 487–493. [39] G.C. Fan, H. Zhu, D. Du, J.R. Zhang, J.J. Zhu, Y.H. Lin, Anal. Chem. 88 (2016) 3392–3399. [40] J. Zhu, J.F. Wang, J.J. Li, J.W. Zhao, Sensors Actuators B Chem. 233 (2016) 214–222. [41] Y.L. Wang, J.T. Cao, Y.H. Chen, Y.M. Liu, Anal. Methods 8 (2016) 5242–5247.