Short Communication
A dual-signaling electrochemical ratiometric method for sensitive detection of carcinoembryonic antigen based on Au-Cu2S-CuS/graphene and Au-CeO2 supported toluidine blue complex

https://doi.org/10.1016/j.snb.2017.10.136Get rights and content

Highlights

  • Cu2S-CuS and Au-CeO2 supported toluidine blue complexe were introduced into the electrochemical ratiometric assay.

  • A novel two dual-signal based dual-potential electrochemical system was constructed.

  • Carboxymethyl chitosan doped ionic liquids prevents the leak of toluidine blue and facilitates the electron transfer.

  • This strategy revealed a great attraction for designing electrochemical ratiometry for biological small molecule detection.

Abstract

Developing electrochemical biosensor, particularly for high sensitive recognizing and detecting tumor marker at low level under benign conditions, is highly desired, but still remains a severe challenge. In this communication, we report a dual-signaling ratiometric method for sensitive detection of carcinoembryonic antigen (CEA) based on gold nanoparticles (Au NPs) functionalized Cu2S-CuS/graphene composite (Au-Cu2S-CuS/graphene) as the matrix material and carboxyl-Au nanoparticles (Au-COOH) functionalized mesoporous CeO2 nanoparticles (Au-CeO2) supported toluidine blue (TB) complex as signal label. The oxidation peak current of Cu2S-CuS decreases with the increase of CEA concentration, while the oxidation peak current of TB increases. The change of dual signals “ΔI = ΔITB + ICu2S-CuS|” (ΔITB and |ΔICu2S-CuS| are the change values of the oxidation peak currents of TB and Cu2S-CuS, respectively) is used as the response signal for quantitative determination of CEA. Notably, the linear range is 0.001  100 ng/mL with a detection limit of 0.78 pg/mL (S/N = 3). The ratiometric electrochemical system provides an extremely simple strategy for detecting tumor markers in a reliable, facile, sensitive, and specific manner, which has a promising application for quantitative detection of other tumor markers in clinical diagnosis.

Graphical abstract

The oxidation peak current of Cu2S-CuS decreases with the increase of CEA concentration, while the oxidation peak current of TB increases. The change of dual signals “ΔI = ΔITB + ICu2S-CuS|” (ΔITB and |ΔICu2S-CuS| are the change values of the oxidation peak currents of TB and Cu2S-CuS, respectively) is used as the response signal for quantitative determination of CEA. Notably, the linear range is 0.001  100 ng/mL with a detection limit of 0.78 pg/mL (S/N = 3). The ratiometric electrochemical system provides an extremely simple strategy for detecting tumor markers in a reliable, facile, sensitive, and specific manner, which has a promising application for quantitative detection of other tumor markers in clinical diagnosis.

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Introduction

To evaluate tumor marker level is a of valuable characteristic in diagnosis, prognosis, staging and monitoring the growth of the tumor [1], [2]. Carcinoembryonic antigen (CEA), as a broad-spectrum tumor marker, can reflect the existence of multiple neoplastic diseases, like colorectal cancer, breast cancer, and lung cancer, etc [3], [4], [5]. Currently, various analytical techniques such as enzyme-linked immunosorbent assay (ELISA), electrochemistry, electrochemiluminescence (ECL), photoelectrochemistry, imaging, fluoroimmunoassay and colorimetry [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16] have been utilized for disease studies and tumor clinical diagnostics [17], [18], [19], [20]. Among these promising analytical approaches, electrochemical method is particularly attractive for early identification and detection at low levels toward tumor markers because of its advantages of fast and direct detection, simplicity, high sensitivity, good selectivity, real-time monitoring and low cost [21], [22], [23]. As well known, signal amplification is considered to be of great importance for the development of electrochemical biosensors. Various electrochemical biosensors have been reported for the detection of tumor markers based on multi-enzymatic reactions [24], [25], synergistic effect of nano-materials signal amplification strategy [26] or Exo III-assisted signal amplification strategy [27], [28]. However, most of the conventional electrochemical biosensing detection have involved only one signal, either the “signal-on” or “signal-off”.

Recently, a variety of the ratiometric electrochemical assays that combine “signal-on” with “signal-off” strategies have been developed for quantitative detection of other tumor markers [23]. It is demonstrated that the strategy fabricates the ratiometric electrochemical sensor to reduce environmental influence and background noise and provide a more accurate signal, wide linear range and low limit of detection [29], [30], [31], but such ratiometric electrochemical sensors still need complex construction process and high cost. In addition, screen-printed carbon electrode (SPCE), as a biomedical analytical method, has easily realized commercialization because of its advantages of simple instrumentation, convenient miniaturization and low cost [32]. To achieve high-efficiency detection of CEA, it is thus highly attractive to develop a dual-signals ratiometric electrochemical immunosensor. This work uses gold nanoparticles (Au NPs) functionalized Cu2S-CuS/graphene composite (Au-Cu2S-CuS/graphene) as the matrix material and Au-CeO2 supported TB complex as signal label to design a novel dual-signals ratiometric electrochemical immunosensing strategy.

Graphene oxide sheets (GO), a typical carbon material, has been applied in the field of biosensor as an ideal platform for growing and supporting Cu2S-CuS particles because of its advantages of large surface area-to-volume ratio, good electron transfer property, good water dispersibility and low cytotoxicity [33], [34], [35], [36], [37]. Additional incorporation of GO into the Cu2S-CuS particles leads to effectively prevent from agglomerating, but also makes them smaller. Au-Cu2S-CuS/graphene not only immobilizes abundant primary anti-CEA (Ab1), but also further promotes electron transfer property. Mesoporous CeO2 nanoparticles have been applied as a platform for supporting detection anti-CEA antibody (Ab2) and the electron transfer mediator TB because of its large surface area, good adsorption property, uniform size distribution, well-defined pore topology and good water dispersibility [38]. Carboxyl-Au nanoparticles (Au-COOH) decorated mesoporous CeO2 nanoparticles (Au-CeO2) can increase electron transfer property and binding force between CeO2 and Ab2. TB, a widely used electron transfer mediator, is loaded on Au-CeO2 to produce electrochemical signal. In addition, carboxymethyl chitosan doped ionic liquids (CMC/ILs) increases adsorption capacity towards TB because CMC contains active −OH, −COOH and −NH- in the molecule, which can offer enough adsorption groups to prevent TB from leaking. On the other side, CMC/ILs effectively facilitates the electron transfer property because ILs is composed of an organic cation and an inorganic anion of salt which can keep liquid at ambient conditions [39], [40]. This method has eliminated the possible negative effects of enzyme and reduced the cost. It also has readily improved analytical performance and clinical reliability, thus improving the accuracy of the results. The simplicity in operation together with the excellent analytical performance of the immunosensor should make it useful and powerful for applications in biochemical investigations and easy realize commercialization.

Section snippets

Labeling of detection antibody

Scheme 1A shows the preparation procedure of the TB/Au-CeO2/CMC/ILs-Ab2. The Au-CeO2 solution (1 mL, 1.8 mg/mL) was ultrasoniced treatment for 30 min, and detection antibody dispersion (1 mL, 10 μg/mL) was added and incubated for 12 h at 4 °C. Then toluidine blue (TB, 2 mg) and EDC/NHS (10 mM/2 mM) were added and incubated for 12 h at 4 °C. After centrifugation, 1-butyl-pyridine tetrafluoroborate, as an ionic liquid (ILs, 8 mg), was mixed with CMC (10 mg) and incubated for 1 h at 4 °C. Following the

Characterization of materials

The structure and morphology of Cu2S-CuS and the obtained Cu2S-CuS/graphene composite were studied by XRD and SEM. As confirmed by XRD results in Fig. 1A, a mixture of Cu2S and CuS was obtained, with Cu2S being the major product. In XRD pattern all the peaks can be indexed to Cu2S (JCPDS Card No.33-0490) and CuS (JCPDS Card No.65-3561), respectively. As shown in Fig. 1B, the pristine Cu2S-CuS particles display irregular particles, the most of which ranges from 45 to 60 nm in size. It is also

Conslusions

The ratiometric immunosensor was successfully achieved by the combination of the signal of Cu2S-CuS and the signal of TB to form the integrated dual signal-tagged CEA. Under the optimal experimental conditions, the change of dual signals “ΔI = ΔITB + ICu2S-CuS|” is used as the response signal for quantitative determination of CEA, the linear range is 0.001  100 ng/mL with a detection limit of 0.78 pg/mL (S/N = 3). The electrochemical strategy was analyzed for detection of the CEA by real sample

Acknowledgments

This work was supported by the National Key Scientific Instrument and Equipment Development Project of China (No. 21627809), the National Natural Science Foundation of China (Nos. 21775053, 21601064, 21575050, 21375047), the Natural Science Foundation of Shandong Province (No.ZR2017MB027), the China Postdoctoral Science Foundation (2017M612170), the Special Foundation for Taishan Scholar Professorship of Shandong Province (No. ts20130937) and the Foundation of Key Laboratory of Sensor Analysis

Yicheng Wei studies in school of chemistry and chemical engineering, University of Jinan as postgraduate student. His current research interest focuses on analytical chemistry.

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  • Cited by (0)

    Yicheng Wei studies in school of chemistry and chemical engineering, University of Jinan as postgraduate student. His current research interest focuses on analytical chemistry.

    Hongmin Ma received both his B.S. and M.S. degree in Applied Chemistry from University of Jinan in 2005 and 2008 respectively. And he has received his Ph.D. degree in Colloid and Interface Chemistry at Shandong University, investigating self-assembly at all scales at surfaces in 2011. Now, he is an associate professor at University of Jinan, interested in the assembly of nano-composites and the construction of ordered porous films as well as their analytical applications.

    Xiang Ren studies in University of Jinan as doctoral candidate. His research is focused on the immunoassay and energy environmental chemistry. He has published over 20 research papers.

    Caifeng Ding joined College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology.

    Huan Wang completed his master degree studies in University of Jinan in 2013. He joined University of Jinan. His research interests are in the area of biosensor and chemical sensor.

    Xu Sun joined University of Jinan, as a tutor working on the synthesis and performance of advanced functional materials.

    Bin Du a professor and DSc, doctoral supervisor, has been engaged in analytic chemistry and environmental science research. He acquired the degree of Doctor of Science from Chinese Academy of Sciences. He has been investigating deeply the composition, property and application of micro emulsion.

    Yong Zhang received his BSc degree in applied chemistry from University of Jinan, his MSc degree in applied chemistry from Nanjing University of Technology and PhD in school of materials science and engineering form Beijing Institute of Technology. He is now working as an associate professor in University of Jinan. He has coauthored over 60 peer-reviewed publications. To host three of national and provincial scientific research projects. His current research interests include preparing functional nanomaterials and developing new biosensors and bioelectronics devices. His other research activities include synthesizing functional nanomaterials for energy and environmental applications.

    Qin Wei a professor and DSc, has devoted herself to analytical teaching and scientific research. Her main research interests are the determination of protein and nucleic acid by photometry and the electrochemical immunosensor preparation. She has published over one hundred articles on analysis, immunosensor and applied successfully for many research projects, such as Analytical Chemistry, ACS Applied Materials & Interfaces, Biomaterials, Advanced Functional Materials, Biosensors and Bioelectronics and Sensors and Actuators B: Chemical, etc.

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