Article pubs.acs.org/ac
Surface-Enhanced Electrochemiluminescence of Ru@SiO2 for Ultrasensitive Detection of Carcinoembryonic Antigen Daifang Wang, Yanyan Li, Zhenyu Lin, Bin Qiu, and Longhua Guo* Institute of Nanomedicine and Nanobiosensing, Ministry of Education Key Laboratory of Analysis and Detection Technology for Food Safety, College of Chemistry, Fuzhou University, Fuzhou, 350116, China S Supporting Information *
ABSTRACT: Carcinoembryonic antigen (CEA) is recognized as a disease biomarker to reflect the existence of various cancers and tumors in the human body. Sensitive detection of CEA in body fluid is valuable for clinical diagnosis and treatment assessment of cancers. Herein, we present a new approach for ultrasensitive determination of CEA in human serum based on localized surface plasmon resonance (LSPR) enhanced electrochemiluminescence (ECL) of Ru(bpy)32+. In this surface-enhanced ECL (SEECL) sensing scheme, Ru(bpy)32+-doped SiO2 nanoparticles (Ru@SiO2) act as ECL luminophores, and AuNPs are used as LSPR source to enhance the ECL signal. Two different kinds of aptamers specific to CEA are modified on the surface of Ru@SiO2 and AuNPs, respectively. In the presence of CEA, a multilayer of Ru@SiO2− AuNPs nanoarchitectures would be formed. Our investigation reveals that the ECL signal of Ru@SiO2 can be effectively enhanced by AuNPs. One layer of Ru@SiO2−AuNPs nanoarchitectures would generate about 3-fold ECL enhancement compared with the ECL of the nanoarchitectures without the presence of AuNPs. As much as 30-fold ECL enhancement could be obtained by a multilayer of Ru@SiO2−AuNPs nanoarchitectures. Under the optimal conditions, a detection limit of 1.52 × 10−6 ng/mL of CEA in human serum was achieved. To the best of our knowledge, CEA assays with such a low LOD have never been reported for an ECL sensor. of ∼104−109 can be obtained.20−23 Most of the approaches for sensitive detection of CEA by SES were focused on surfaceenhanced Raman scattering (SERS)24,25 and surface-enhanced fluorescence (SEF).26 Electrochemiluminescence (ECL) is regarded as a simple, sensitive, and powerful analytical technique triggered by an electrochemical reaction.27−30 Especially, with the superiority of Ru(bpy)32+ and its derivatives in electrochemical reaction,31,32 the ECL systems have been applied extensively in the areas of biological and chemical sensing in the past 2 decades.33−35 In recent years, with the rapid development of nanotechnology, the utility of different nanotechnologies for enhancing the ECL responses has garnered special attention within the research community. For example, it is reported that the ECL behavior of semiconductor nanocrystals could be greatly affected by noble metal nanoparticles.36−41 Mostly recently, we have systematically investigated the ECL enhancement by LSPR of gold nanoparticles (AuNPs), and we termed this phenomenon surfaceenhanced ECL (SEECL).42 Our investigation revealed that the LSPR of AuNPs could increase both the excitation rate and the emission factor of luminophores. Thus, the ECL intensity was
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ancer is one of the major causes of death for human beings. The clinic diagnosis and cure of cancer at an early stage is effective to reduce the death rate of patients. Carcinoembryonic antigen (CEA) is a type of glycoprotein generated by tumor cells in the human body. It is one of the common tumor markers for clinic diagnosis and treatment of cancer.1 Many reports2,3 have declared that higher levels of CEA existed in the serum of patients with colon cancer and other carcinomas than in healthy individuals. Thus, sensitive detection of CEA has attracted broad interest of scientists, and a series of analysis techniques has been developed, including those based on fluorescence immunoassay (FIA), 4−6 enzyme-linked immunosorbent assays (ELISA),7,8 electrochemical immunoassays,9−11 and other immunoassays.12,13 Up to now, multiple strategies have been developed to promote the sensitivity of CEA detection. For example, nanoparticle-amplified capillary electrophoresis,14 a dual amplification system combining with an electrochemical-redox cycling and coulometric signal transduction,15 various signal-gathering methods based on the surface effect of nanomaterials,16,17 and signal amplification technology based on surface plasmon resonance (SPR) of noble metal nanostructures18,19 have been previous reported with high sensitivity for CEA determination. Among all these signal amplification technologies, surfaceenhanced spectroscopy (SES) based on SPR of noble metal nanostructures is attractive because a signal amplification factor © XXXX American Chemical Society
Received: December 18, 2014 Accepted: May 26, 2015
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DOI: 10.1021/acs.analchem.5b01038 Anal. Chem. XXXX, XXX, XXX−XXX
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rpm for 10 min and the precipitate was redispersed in H2O. A 10 μL portion of 0.1 M pH 7.4 Tris-HCl solution containing 100 μM aptamer 1 (Apt1) with a thiol group was added to the assynthesized AuNP solution, and the reaction was maintained in a dark place for 12 h. Then 100 μL of 1.0 M NaCl was injected into the reaction solution and the reaction continued for 12 h. Finally, the AuNR−Apt1 solution was centrifuged at 5000 rpm for 10 min and the precipitate was redispersed in H2O for further characterization. Preparation of Ru@SiO2−Apt2. A 100 mL portion of ethanol, 4.0 mL of H2O, and 3.2 mL of NH3·H2O (25%) were add into a 200 mL flask in turn, and 3.0 mL of TEOS was injected into the mixture solution with stirring, followed by the addition of 10 μL of 10.0 mM Ru(bpy)3Cl2 solution. The mixture solution was kept stirring for at least 24 h at room temperature to obtain Ru(bpy)3Cl2−SiO2 composite nanoparticles (Ru@SiO2). Next, the solution of composite nanoparticles was centrifuged at 8500 rpm for 10 min and the precipitate was redispersed in ethanol. A 10 μL aliquot of vinyl trimethoxysilane (VTES) and 3.2 mL of NH3·H2O (25%) were added to the above ethanol solution of composite particles under continuous stirring, and the mixture was allowed to react overnight. Next, the particles were eventually washed as described above and redispersed in the solution mixture of 0.1 mg/mL KMnO4 and 4.2 mg/mL NaIO4 for formation of carboxy functions on the surface of
[email protected] The Ru@SiO2 was centrifuged at 8500 rpm for 10 min, and the precipitate was redispersed in 0.1 M pH 7.4 Tris-HCl−NaCl solution, followed by the addition of 10 μL of 0.1 M pH 7.4 TrisHCl solution containing 100 μM aptamer 2 (Apt2) with amidogen. Finally, Ru@SiO2−Apt2 was centrifuged and redispersed in 0.1 M pH 7.4 Tris-HCl solution. Ru@SiO2− Apt1 was prepared by the same process. Fabrication of SEECL Sensors. Gold electrodes were polished to a mirror finish with 0.5 and 0.05 μm alumina aqueous slurry in turn. The bare gold electrodes were then immersed in 50% (v/v) HNO3 for 3 min under sonication; next, the electrodes were taken out and washed with doubly distilled water and then soaked in 50% (v/v) ethanol for 3 min under sonication. The electrodes were then immersed into doubly distilled water for another 3 min under sonication. The electrodes were electrochemically scanned with cyclic voltammetry (CV) in a potential range from −0.4 to +1.6 V in 0.5 M H2SO4 solution 10 times until steady CV curves were obtained. Next, the gold electrode was immersed in 0.1 M pH 7.4 TrisHCl−NaCl solution containing 0.1 μM thiol-functionalized Apt1 and 0.1 μM thiol-functionalized Apt2 for 5 h and 0.1 mM βmercaptoethanol (β-ME) for 30 min, respectively. This procedure would generate a dense aptamer monolayer at the surface of the gold electrode (GE/Apt). After careful washing with water, the electrode was dipped in CEA aqueous solution for 2 h to obtain the CEA-modified gold electrode (GE/Apt/CEA). Next, 10 μL of a solution of 4 μL of AuNP−Apt1 solution, 2 μL of CEA solution, and 4 μL of Ru@SiO2−Apt2 solution was dropped on the surface of the modified electrode, and the electrode was incubated at room temperature for 12 h to obtain the DNA-probe-modified gold electrode (GE/Apt/CEA/Ru@ SiO2−AuNP). The modified electrode was washed carefully with 0.1 M pH 7.4 Tris-HCl−NaCl solution to remove the unbound nanoparticles and stored at 4 °C. ECL Measurements. A 5.0 mL cylindroid glass cell was used as an ECL cell, which was successively washed with 0.5 M HNO3 and doubly distilled water and dried before use. Then the biosensor was electrochemically cleaned in 0.1 M pH 7.4 PBS by
enhanced greatly. Thereafter, we developed a method for the ultrasensitive detection of Hg2+ in drinking water based on this SEECL mechamism.43 In this paper, we described a new approach for ultrasensitive determination of CEA based on SEECL. Ru(bpy)32+-doped SiO2 nanoparticles (Ru@SiO2) were used as ECL luminophores, while AuNPs were used as surface-enhanced sources for ECL signal amplification. Ru(bpy)32+ is selected as the luminophore rather than any other ECL luminophore due to its superior properties, such as high sensitivity and good stability under moderate conditions in aqueous solution.44−46 Two different aptamers targeting different binding sites of CEA were modified on Ru@SiO2 and AuNPs, respectively. In the presence of CEA, the aptamer-modified nanoparticles (e.g., Ru@SiO2 and AuNPs) would aggregate on the surface of the working electrode. Then, ECL emission of this nanoparticle-modified electrode was generated by electrochemical reaction of Ru@SiO2 and enhanced by LSPR of AuNPs. The amount of nanoparticles linked on the electrode surface is proportional to the concentration of CEA. In the absence of CEA, no Ru@SiO2 and AuNPs were immobilized on the surface of the electrode; thus, no ECL signal can be detected. Therefore, this senor can be utilized for sensitive determination of CEA.
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EXPERIMENTAL SECTION Apparatus and Reagents. The ECL measurements were recorded by a model BPCL-1-TIC ultraweak luminescence analyzer at room temperature, and the voltage of the photomultiplier tube was set at 1000 V. The electrochemical measurements were detected with a CHI 660D electrochemical workstation (Shanghai CHI Instruments Co.). All experiments were carried out with a conventional three-electrode system. The reference electrode was an Ag/AgCl electrode, the working electrode was a modified gold electrode (GE, φ = 2 mm), and a Pt wire served as the counter electrode. The UV−vis absorption spectra were obtained on a Shimadzu UV-3600 UV−vis-NIR photospectrometer (PerkinElmer). CEA was obtained from Biocell Biotechnology Co., Ltd. (Zhengzhou, China). All aptamers for CEA were acquired from Sangon Biotech Co., Ltd. (Shanghai, China). The aptamers squences are obtained from a previous report (aptamer 1, 3′TTA ACT TAT TCG ACC TAT-5′; aptamer 2, 3′-CCC ATA GGG AAG TGG GGG A-5′).47 Herein, the aptamers with a thiol on the 3′-end were used to modify the surface of the gold electrode and AuNPs, and the aptamers with an amino on the 3′end were immobilized on the surface of Ru@SiO2. Tris(2,2′bipyridyl)dichlororuthenium(II) hexahydrate [Ru(bpy)3Cl2· 6H2O] was purchased from Sigma-Aldrich. Hydrogen tetrachloroaurate tetrahydrate (HAuCl4·4H2O) and sodium citrate was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Tri-n-propylamine (TPrA), tris(hydroxymethyl)aminomethane (Tris), and tetraethyl orthosilicate (TEOS) were purchased from Aladdin (Shanghai, China), and other reactants were obtained from Fuchen Chemical Reagents Ltd. Co. (Tianjin, China). All reagents were of analytical grade and used as received. Millipore ultrapure water (resistivity >18.2 MΩ cm) was used throughout the experiment. Preparation of AuNP−Apt1. Gold nanoparticles were synthesized by an approach based on the chemical reduction method.48 Briefly, 3 mL of 1% sodium citrate aqueous solution was injected into 50 mL of boiling 0.15 mM HAuCl4 aqueous solution under vigorous stirring and kept as such for 30 min to obtain AuNPs. Next, the AuNP solution was centrifuged at 8500 B
DOI: 10.1021/acs.analchem.5b01038 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry Scheme 1. Schematic Diagram of SEECL Biosensor for CEA Detection
Figure 1. (A) UV−vis spectra of different as-synthesized AuNP: AuNP−Apt2 (red) and bare AuNP (black). (B) TEM images of AuNP samples. (C) UV−vis spectra of different as-synthesized Ru@SiO2: Ru@SiO2−Apt2 (green), Ru@SiO2 (red), and bare SiO2 nanoparticles (black). (D) TEM images of Ru@SiO2 samples.
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Analytical Chemistry scanning from −0.5 to +1.5 V via cyclic voltammetry. ECL measurements of biosensors were obtained in 0.1 M pH 7.4 PBS containing 1 mM TPrA with a scanning rate of 0.1 V/s. CEA Detection. A series of CEA standard solutions with different concentrations was prepared to investigate the relationship between the CEA concentration and the ECL intensity of the biosensor. Then, the relational expression was established. A human serum sample was obtained from the First Affiliated Hospital of Fujian Medical University, and it was diluted 1000 times with 0.1 M pH 7.4 PBS before further experiments. The diluted human serum sample was used for fabrication of biosensor instead of CEA standard samples.
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RESULTS AND DISCUSSION Principle of the SEECL Biosensor. A schematic diagram of the proposed SEECL biosensor for CEA is shown in Scheme 1. Apt1 and Apt2 were first immobilized on the surface of the gold electrode via Au−S interaction. It was reported that Apt1 and Apt2 bound to different sites of CEA.47 Thus, Apt2-modified Ru@SiO2 could bind to the CEA immobilized on the electrode surface by Apt1. Similarly, the Apt1-modified AuNPs could also be immobilized on the surface of electrode via the formation of double-aptamer sandwich structures by interaction with Apt2immobilized CEA. Then ECL emission would generate surface plasmon at the surface of AuNP as well. As discussed in our previous works,43 this kind of strong electromagnetic oscillation could effectively improve the ECL efficiency of Ru(bpy)32+; hence, the ECL intensity could be greatly enhanced. It should be noted that in the case of an excess of Ru@SiO2 and AuNPs being present, the number of nanoparticles immobilized on the surface of the electrode is proportional to the CEA concentration of the solution. Thus, the proposed sensing strategy could be used for quantitative determination of CEA. Characterization of Nanoparticle-Modified Electrodes. Transmission electron microscopy was used to measure the morphology of the as-synthetic nanoparticles. As shown in parts B and D of Figure 1, AuNP and Ru@SiO2 show uniform spherical structures with diameters of ∼45 and ∼55 nm, respectively. The immobilization of AuNPs and Ru@SiO2 on the electrode surface was monitored by UV−vis spectroscopy, and the results are shown in Figure 1A,C. AuNPs has a LSPR absorption peak located at ∼520 nm, while the peak red-shifted to 526 nm after Apt1 modification. This kind of peak shift is derived from the refractive index change around the AuNP surface.50−52 Comparing with the UV−vis spectroscopy of SiO2 nanoparticles, two additional absorption peaks are observed for the UV−vis spectroscopy of Ru@SiO2 (see Figure 1C): the absorption peak located at ∼460 nm is assigned to the metal-to-ligand charge transfer (MLCT) of Ru(bpy)32+, and the other absorption peak located at ∼285 nm is the result from an intraligand (π → π*) transition.53 Another slight peak at ∼260 nm appeared on the UV−vis curve after the modification of Ru@SiO2 by Apt2. This absorption peak corresponds to the absorbance of oligonucleotide aptamers. Figure 2 depicts the distinct electrochemical impedance spectroscopy (EIS) of different modified electrodes. The electron transfer resistance (Rct) increased greatly after modification with the aptamer and CEA. This kind of electrochemical response variations derived from the low conductivity of aptamer and CEA, which hindered the electron transfer of Fe(CN)63− on the surface of the electrode. Although the bare AuNPs have good ability to transfer electrons, the
Figure 2. Nyquist plots of different modified electrodes. The Nyquist plots were recorded in PBS solution (0.1 M, pH 7.0) containing 5.0 mM K3Fe(CN)6, with the biasing potential 0.21 V and 5 mV alternative voltage in the frequency range of 1−100 000 Hz.
surface of AuNPs is electronegative when AuNPs bond with aptamers. Then, the electronegative nanoparticles immobilized on the surface of the electrode would reject the approaching of Fe(CN)63−; hence, a higher Rct is observed. Similarly, modification of the electronegative Ru@SiO2 would bring the same results as for AuNPs. In addition, as shown in Figure S1 (Supporting Information), the CV responses of electrodes gradually changed from quasireversible to irreversible during the modification process of nanoparticles. The different electrochemical responses indicate the successful modification of different nanoparticles on the electrodes. The distributions of nanoparticles modified on electrode surfaces are demonstrated by scanning electron microscopy (SEM, Figure 3). It should be noted that only Ru@SiO2 was used to prepare the modified electrodes for Figure 3a,c, while both Ru@SiO2 and AuNPs were immobilized on the electrode surface for Figure 3b,d. Before the CEA-induced amplification, the nanoparticles were single-layer distributed on the electrode surfaces (see Figure 3a,b). After repeatedly adding the nanoparticles and CEA to the surface of electrodes during the amplification process, more and more nanoparticles bonded on the electrode to form a multilayer structure via the interaction between aptamers and CEA. As shown in Figure 3c,d, compared with the single-layer modification, the coverage of nanoparticles increased significantly after the amplification process. It is reported that AuNPs are brighter than Ru@SiO2 in the SEM image due to its strong emission of secondary electrons and superior electron density.54 It can be seen from Figure 3b,d that the bright and dark dots mixed evenly to build the nanoarchitectures. Thus, we concluded that the AuNP and Ru@SiO2 were evenly distributed on the surface of the electrode. ECL Enhancement. In the proposed sensing scheme, there are two factors that lead to the ECL enhancement: targets induced formation of multilayer Ru@SiO2 and SEECL by AuNPs. In order to show the contribution of these factors, we prepared four different modified electrodes and detected the ECL response of them in the same PBS solution. The results of ECL responses from the four different electrodes are shown in Figure 4. As can be seen from Figure 4, only a weak ECL signal D
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Figure 3. SEM images of different modified electrodes: (a) single-layer GE/Apt/CEA/s-Ru@SiO2, (b) single-layer GE/Apt/CEA/s-Ru@SiO2−AuNP, (c) multilayer GE/Apt/CEA/Ru@SiO2, and (d) multilayer GE/Apt/CEA/Ru@SiO2−AuNP.
Figure 4. ECL intensity of four different electrodes: GE/Apt/CEA/s-Ru@SiO2 (black, electrode a), GE/Apt/CEA/s-Ru@SiO2−AuNP (red, electrode b), GE/Apt/CEA/Ru@SiO2 (green, electrode c), and GE/Apt/CEA/Ru@SiO2−AuNP (violet, electrode d) in the same 0.1 M pH 7.4 PBS solution containing with 1.0 mM TPrA. The scanning rate is 100 mV/s, and the concentration of CEA is 10.0 ng/mL.
electrode c in the inset scheme of Figure 4). This phenomenon was attributed to SEECL in the presence of AuNPs, which has been investigated in detail in our previous works.42,43 After the formation of multiple-layer Ru@SiO2−AuNPs composites on the surface of the electrode (GE/Apt/CEA/ Ru@SiO2−AuNP, see electrode d in the inset scheme of Figure 4), significant ECL enhancement was observed. The ECL intensity after amplification was more than 30 times higher than that of GE/Apt/CEA/Ru@SiO2. With the nanoparticles amplification process, more AuNPs and Ru@SiO2 was
occurred after the electrode was modified with a single layer of Ru@SiO2 (GE/Apt/CEA/s-Ru@SiO2; see electrode a in the inset scheme of Figure 4). However, significant ECL enhancement was observed after the addition of AuNPs to form the single-layer AuNP−Ru@SiO2 composite (GE/Apt/CEA/sRu@SiO2−AuNP; see electrode b in the inset scheme of Figure 4). It is worth noting that the ECL intensity of the electrode modified with a single layer of AuNP−Ru@SiO2 composite was even higher than that of the electrode modified with multiple layers of Ru@SiO2 composite (GE/Apt/CEA/Ru@SiO2; see E
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Figure 5. (A) Concentration-dependent ECL behaviors of the biosensor. CEA from top to bottom is 5.00, 0.500, 5.00 × 10−2, 5.00 × 10−3, 5.00 × 10−4, 5.00 × 10−5, and 5.00 × 10−6 ng/mL, respectively. (B) The calibration curve for quantification of CEA. The ECL buffer containing 0.1 M pH 7.4 PBS and 1.0 mM TPrA. The scanning rate is 100 mV/s for all measurements.
sensitivity and dynamic range) of the proposed approach is superior to that of most methods developed for CEA detection (Table 1).
immobilized on the surface of electrode; thus, this kind of strong ECL enhancement was due to the combination of both SEECL and the amplification process. Selectivity, Stability, and Reproducibility of the Sensor. In order to show the feasibility of the proposed sensor for real sample assays, we needed to investigate the specificity of the sensor for CEA. We tested the responses of the sensor for other proteins such as bovine serum albumin (BSA), lysozyme, trypsin, and thrombin. For the selectivity test, the concentration of the CEA was 2.00 ng/mL, while the concentration for the others proteins was 50.0 ng/mL (see Figure S2 in the Supporting Information). The results revealed that only slight ECL responses were observed after modification with different proteins. It can be clearly seen that the ECL response of CEA was much higher than that of other types of proteins, even at a concentration 25 times less than for the other proteins. These results indicated that this type of biosensor had good selectivity for the determination of CEA. The stability and reproducibility of the biosensor are vitally important for analytical application. The stability of the sensor can be evaluated by repeatedly testing the ECL signal from the same modified electrode, while the reproducibility can be evaluated by testing the ECL responses of different modified electrodes collected from different batches. The RSD of ECL responses was 2.67% (n = 10) for the same modified electrode (see Figure S3A, Supporting Information) and 2.74% for six different modified electrodes collected from six different batches (see Figure S3B, Supporting Information). The good stability and reproducibility of the sensor indicated the potential for utilizing the method for ECL signal enhancement. Determination of CEA. We investigated the concentrationdependent response of the sensor by testing a series of standard solutions containing different CEA concentrations varied from 5.00 to 5.00 × 10−6 ng/mL, and the results are shown in Figure 5A. The limit of detection (LOD) is 1.52 × 10−6 ng/mL on the basis of a signal-to-noise ratio of 3. A linear response is observed from 5.00 × 10−2 to 5.00 × 10−6 ng/mL (Figure 5B). The linear relation equation is ΔI = 684 + 117 × log CCEA with a linear relation coefficient of 0.991. The analytical figure of merits (e.g.,
Table 1. Comparison of Different Approaches for CEA Detection in Terms of LOD and Dynamic Range ref
LOD of CEA (ng/mL)
dynamic range (ng/mL)
Lin et al.4 Yuan et al.5 Hou et al.6 Zhou et al.8 Limbut et al.10 Zhang et al.11 Thomson et al.12 Pino et al.13 this work
0.1 0.07 0.5 0.025 0.01 1.1 2.5 0.01 1.52 × 10−6
0.1−10 0.07−100 1−1000 0−40 0.01−10 2.5−40 2.5−320 0−50 (5.00 × 10−6)−0.0500
Finally, we demonstrated the use of the proposed sensor for CEA detection in human serum. A human serum sample obtained from the hospital with an original CEA concentration of 5.81 ng/mL was utilized to test the feasibility of the sensor for real sample assays. Since the CEA concentration of the sample was much higher than the upper limit of the linear range of the sensor, the sample was diluted with buffer 1000 times before assay. The average measured concentration of CEA is 5.86 ng/ mL by the biosensor in this work (see Table S1, Supporting Information), which is consistent with the results obtained from the hospital. In addition, the standard addition method was used to test the reliability of the sensor for determination of CEA in human serum. The results showed that the recoveries of CEA are between 96.0% and 122% (Table S1, Supporting Information).
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CONCLUSION In summary, we demonstrated a biosensor for ultrasensitive detection of CEA based on SEECL of Ru(bpy)32+. In the presence of CEA, Ru@SiO2 and AuNPs formed into reticular nanoarchitectures. These reticular nanoarchitectures enhanced the ECL intensity as much as 30 times compared with a single F
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layer of Ru@SiO2. Our investigation revealed that SEECL of AuNPs was the main cause of this ECL enhancement. The proposed SEECL aptamer biosensor was demonstrated to be effective for selective detection of CEA, and the LOD was 1.52 × 10−6 ng/mL under optimal conditions (S/N = 3). In view of the large amount of aptamers developed for all kinds of targets, the proposed method is potentially applicable for sensitive determination of these analytes.
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ASSOCIATED CONTENT
S Supporting Information *
The electrochemical responses of different modified electrodes; the selectivity, stability and reproducibility of the biosensor; and the recoveries of CEA in human serum samples. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b01038.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Fax: (+)86-591-22866135. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support of the National Natural Science Foundation of China (21277025, 21205017, 21375021), the Foundation of Fujian Educational Committee (JA12039, JA13024), and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry.
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DOI: 10.1021/acs.analchem.5b01038 Anal. Chem. XXXX, XXX, XXX−XXX
