Elsevier

Biosensors and Bioelectronics

Volume 135, 15 June 2019, Pages 22-29
Biosensors and Bioelectronics

Bimetallic cerium and ferric oxides nanoparticles embedded within mesoporous carbon matrix: Electrochemical immunosensor for sensitive detection of carbohydrate antigen 19-9

https://doi.org/10.1016/j.bios.2019.04.018Get rights and content

Highlights

  • Bimetallic CeFe-MOF derived mesoporous carbon matrix embedded with CeO2 and FeOx nanoparticles.

  • The nanocomposite by calcination at 500 °C with abundant sites for binding antibody molecules.

  • The electrochemical immunosensor for ultra-sensitive detection of carbohydrate antigen 19–9.

  • High selectivity, stability, reproducibility and applicability of the proposed immunosensor.

Abstract

A label-free electrochemical immunosensor was successfully developed for sensitively detecting carbohydrate antigen 19–9 (CA19-9) as a cancer marker. To achieve this, a series of bimetallic cerium and ferric oxide nanoparticles embedded within the mesoporous carbon matrix (represented by CeO2/FeOx@mC) was obtained from the bimetallic CeFe-based metal organic framework (CeFe-MOF) by calcination at different high temperatures. The formed CeO2 or FeOx nanoparticles were uniformly distributed within the highly graphitized mesoporous carbon matrix at the calcination temperature of 500 °C (represented by CeO2/FeOx@mC500). However, the obtained nanoparticles were aggregated into large size when calcined at the temperatures of 700 and 900 °C. The CA 19–9 antibody can be anchored to the CeO2/FeOx@mC network through chemical absorption between carboxylic groups of antibody and CeO2 or FeOx by ester-like bridging. The CeO2/FeOx@mC500-based immunosensor displayed superior sensing performance to the pristine CeFe-MOF, CeO2/FeOx@mC700- and CeO2/FeOx@mC900-based ones. Electrochemical impedance spectroscopy results showed that the developed immunosensor exhibited an extremely low detection limit of 10 μU·mL−1 (S/N = 3) within a wide range from 0.1 mU·mL−1 to 10 U·mL−1 toward CA 19–9. It also illustrated excellent specificity, good reproducibility and stability, and acceptable application analysis in the human serum solution which was diluted 100-fold with 0.01 M PBS solution (pH 7.4) and spiked with different amounts of CA19-9. Consequently, the proposed electrochemical immunosensor is capable enough of determining CA 19–9 in clinical diagnostics.

Introduction

Early diagnosis for cancers is extremely important in the treatment efficacy and quality of life of cancer patients. In china, pancreatic cancer is ninth most common malignant tumor (Li et al., 2007). Owning to the high degree of malignancy of the disease, the 5-year survival rate of pancreatic cancer patients is less than 5% (Hidalgo, 2010). Biomarkers secreted from tumor tissue, which can found in blood, tissue, and body fluids, are important diagnostic basis for clinic cancer. Carbohydrate antigen 19–9 (CA19-9), a kind a of carbohydrate antigen, is a glycoprotein highly associated with malignant tumors and shows great promise for malignant tumor detection, such as pancreatic cancer, colorectal cancer, liver cancer, gastric cancer, and ovarian cancer (Yang et al., 2015). The CA19-9 levels of normal healthy people are significantly lower than 37 U·mL−1 (Imaoka et al., 2016). Therefore, a slight elevation of CA19-9 level in blood means the possibility of the pancreatic cancer incidence and development (Humphris et al., 2012). Ultrasensitive detection of CA19-9 plays key roles in the early diagnosis, treatment, and prognosis of pancreatic cancer (Ludwig and Weinstein, 2005). At moment, several immunoassay approaches have been employed for detecting CA19-9 including electrochemical immunoassay (Zhu et al., 2016), chemiluminescent immunoassay (Lin et al., 2004), enzyme-linked immunosorbent assay (Parker et al., 1992) and radioimmunoassay (Ching and Rhodes, 1989). Despite of these efforts, these methods typically require labeling of the antibodies or antigens, leading to the assay process more complex, time consuming and expensive. Among different immunoassay methods, electrochemical label-free immunosensors have attracted increasing attention since they feature easy control, simple apparatus, and low-cost (Santharaman et al., 2016). Furthermore, aiming at designing and constructing highly sensitive electrochemical immunosensors, efficiently anchoring antibodies and producing amplified signals are the crucial steps (Sánchez et al., 2008). The sensitivity of the electrochemical immunosensors for the early detection of trace antigen depends on the adsorbing antibodies over the bioplatform, as well as the orientation of immobilized antibodies on the electrode surface (López-Alarcón and Denicola, 2013). Therefore, it is highly attractive to develop a novel nanomaterial-based bioplatform, which possesses good electrochemical and strong adsorption ability for antibodies.

Metal-organic frameworks (MOFs), known as coordination polymers or coordination networks, are a class of hybrid materials that consist of inorganic connectors and organic linker molecules (Zhou and Kitagawa, 2014). Owning to the specific features, including well-defined pore structure, large surface area, and specific functional sites that endows them with strong-bioaffinity toward biomolecules, have extended the application region of MOFs in various sensing fields (Li et al., 2015). The combined effects of the dynamics of analyte transport (within the MOF) and high loading capacity (due to a high volume-to surface-area ratio) can make contributions to the enhanced sensitivity of MOFs (Wang, 2017). For instance, Al-MOF (Liu et al., 2017a), Fe-MOF (Tang et al., 2018), Zr-MOF (Liu et al., 2017b), Cd-MOF (Kumar et al., 2016), Co-MOF (Xu et al., 2018a), Cu-MOF (Bhardwaj et al., 2017), and Zn-MOF (Wang et al., 2018a) have been employed as the scaffolds for immobilizing antibodies or other probes to detect various analytes. Additionally, recruiting the MOFs as precursors, recent work has presented a myriad of MOF-derived porous or hollow metal oxide nanostructures by thermal treatment (Zhang et al., 2017a), which has already found their applications in different fields (Mai et al., 2017). Regarding the porous structure and tailorable composition, the possibility of MOF-templated metal oxides as an alternative to extract biomolecules instead of MOFs have been evaluated. In the past decade, various metal oxides, such as CuO, ZnO, NiO, Co3O4, and Fe2O3, were synthesized by calcining their corresponding MOFs (Zou and Li, 2018). Because of their large specific surface area, superior electrochemical performance, and excellent biocompatibility, the derivatives from MOFs have been paid more attention to be employed as the substrate to construct electrochemical biosensors for trace detection of different analytes (Dang et al., 2017). Nevertheless, most of metal oxides derived from MOFs were exploited as the non-enzyme electrocatalysts for detecting small biomolecules (Zhang et al., 2016a). Rare reports have been observed about their applications in immunosensors or immunosensor fields for detecting the cancer markers. For instance, Fe3O4@mC nanocomposite derived from Fe-MOF was applied as the scaffold for immunosensor for sensitively detecting oxytetracycline, giving a low limit of detection (LOD) of 0.027 pg mL−1 (Song et al., 2017). NiOxFeOy@mC derived from the hollow NiFe Prussian blue analogue was used as the scaffold for sensitively detecting adenosine triphosphate, showing a LOD of 0.98 fg mL−1 (Wang et al., 2018b).

Bimetallic nanoparticles composed of two different metal elements have also received wide spread attention especially in the field of electrochemical immunoassays. They often show improved sensing performance than their monometallic counterparts due to the synergistic effect and the electronic effect (Yang et al., 2017a). Among different MOFs, a mixed valence state of Ce-MOF and Fe-MOF possessed an excellent mimics of catalytic activity, hence showing a sensitive electrochemical approach for the detection of small biomolecules (Peng et al., 2018). Especially, CeO2 as a typical rare earth oxide has attracted extensive attention due to the special 4f electronic structure, along with the excellent electrochemical performance, chemical inertness, non-toxicity, and negligible swelling (Kašpar et al., 1999). A wide scope to explore the electrochemical properties of the CeO2-based nanocomposite for biosensor application and biomarker detection has been found. CeO2-reduced graphene oxide nanocomposite was explored to be as the immunosensor for detecting the cancer marker, Cyfra-21-1 (Pachauri et al., 2018). CuMn-CeO2 was developed as redox probe, signal amplifier and matrix for sensitive detection of procalcitonin since CeO2 possesses special surface mixed-valence properties (Yang et al., 2017a). Au nanoparticles were used to combine anti-PSA1 (Ab1) to CeO2 via the Au-NH2 covalent bond and meanwhile to enhance the sensitivity of the immunosensor (Zhao et al., 2016). GO/MWCNTs-COOH/Au@CeO2 was served as the sensing platform for detecting carcinoembryonic antigen, giving a LOD of 0.02 ng mL−1 (Pang et al., 2015). Fe2O3@C composite was synthesized by annealing of Fe-MOFs. The Fe2O3@C-based biosensor exhibited wide linear range and the low LODs for the determination of H2O2 and paraoxon (Wei et al., 2018). Antibody against CA19-9 was covalently immobilized on the magnetized carbon nanotubes (Fe3O4-MCNTs) and was employed to capture CA19-9 in blood with LOD of 30 U·mL−1 (Huang et al., 2017). At moment, however, no report was observed for the application of CeO2/FeOx hybrid as the immunosensor for detecting CA19-9.

In our previous work, cerium and cupric oxide nanoparticles embedded within the mesoporous carbon matrix were obtained by the calcination of bimetallic CeCu-MOF in N2 atmosphere (CeO2/CuOx@mC) (Wang et al., 2019). The CeO2/CuOx@mC-based aptasensor was fabricated for the tobramycin detection, giving an extremely low LOD of 2.0 fg mL−1 within a broad linear range from 0.01 pg mL−1 to 10 ng mg L−1. However, the optimized bioplatform of aptasensor, CeO2/CuOx@mC calcined at 900 °C, exhibited the favorable aggregation behavior, showing the uneven surface morphology. As such, it would be very desirable to develop a novel bimetallic oxides composed of cerium for the biosensor fabrication at a relatively low calcination temperature. Considering the advantages of CeO2 and FeOx in the sensing fields, we designed and prepared a novel bimetallic CeFe-MOF, following by pyrolyzing it to form a series of structure-controlled mixed CeFe oxides under different high temperatures (500, 700, and 900 °C), which was embedded within the mesoporous carbon matrix (represented by CeO2/FeOx@mC) for the first time. Subsequently, the series of CeO2/FeOx@mC were employed as new scaffolds for binding the CA19-9 antibody to sensitively detect CA19-9 (Scheme 1). As compared with the routine immunosensors for detecting CA19-9, the constructed CeO2/FeOx@mC-based electrochemical immunosensor displayed the superior sensing performances. It is mainly attributed to the following reasons: (i) antibody molecules can be adsorbed on CeO2 through chemical absorption between carboxylic groups of antibody and CeO2 by ester-like bridging (Yang et al., 2017b); (ii) Fe2O3 can improve electrochemical properties of the transducers and strengthen the conjugation with biological compounds (Wei et al., 2018); (iii) the formed graphitized carbon layer with porous structure and high specific surface area was also implemented as the matrix to provide interface with abundant sites for antibody immobilization (Wang and Dai, 2015). After blocking with BSA and incubation of target CA19-9, the electrochemical response signal variation can be detected owing to the antigen-antibody reaction (Sha et al., 2015). Therefore, it would be extremely expectant to construct a novel electrochemical immunosensor based on CeO2/FeOx@mC for early detecting cancer markers and further find the possible applications in early cancer diagnosis.

Section snippets

Experimental section

The detailed description of reagents and materials, preparation of solutions, characterizations, and pretreatment of the bare Au electrode (AE) were supplied in the part of “S1. Experimental section” in the Supporting Information (SI).

Crystal and chemical structure of the CeFe-MOF and the series of CeO2/FeOx@mC composites

The crystal and chemical structure of the CeFe-MOF and the series of CeO2/FeOx@mC composites were characterized by powder X-ray diffraction measurements (PXRD), Raman spectra, Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS) (Fig. S4) and analyzed in detail in the part of S3 (See the Supporting Information). Briefly, the XRD pattern of bimetallic CeFe-MOF shows main diffraction peaks of the Ce-MOF, indicating the preferential orientation of Ce-MOF. In

Conclusions

In summary, a label-free electrochemical immunosensor based on the bimetallic CeFe-MOF-derived CeO2/FeOx@mC nanocomposite for the ultrasensitive detection of CA19-9 was developed. The calcination temperature used in the CeO2/FeOx@mC nanocomposite preparation has played an important role in their nanostructures. Among different nanocomposites, the CeO2/FeOx@mC500 displays highly graphitized mesoporous carbon structure, rich chemical functional groups, and homogeneous distribution of

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We are grateful to the support from the National Natural Science Foundation of China (Nos. U1604127 and U1704256), Scientific and Technological Project of Henan Province (Nos.192102310261 and 192102310460), and Young Backbone Teacher Training Program in Universities of Henan Province (No. 2018GGJS089).

References (61)

  • S.K. Bhardwaj et al.

    Sensor. Actuator. B Chem.

    (2017)
  • J.L. Humphris et al.

    Ann. Oncol.

    (2012)
  • H. Imaoka et al.

    Pancreatology

    (2016)
  • J. Kašpar et al.

    Catal. Today

    (1999)
  • P. Kumar et al.

    Microchem. J.

    (2016)
  • W. Li et al.

    Biosens. Bioelectron.

    (2014)
  • J. Lin et al.

    J. Immunol. Methods

    (2004)
  • C. Liu et al.

    Biosens. Bioelectron.

    (2017)
  • C. López-Alarcón et al.

    Anal. Chim. Acta

    (2013)
  • P. Santharaman et al.

    Sensor. Actuator. B Chem.

    (2016)
  • Y. Sha et al.

    Biosens. Bioelectron.

    (2015)
  • E. Sheikhzadeh et al.

    Biosens. Bioelectron.

    (2016)
  • M. Shi et al.

    Talanta

    (2014)
  • Z. Tang et al.

    Biosens. Bioelectron.

    (2018)
  • H. Wang

    Coord. Chem. Rev.

    (2017)
  • M. Wang et al.

    Biosens. Bioelectron.

    (2018)
  • S. Wang et al.

    Anal. Chim. Acta

    (2019)
  • W. Wei et al.

    Sensor. Actuator. B Chem.

    (2018)
  • X. Weng et al.

    Sensor. Actuator. B Chem.

    (2017)
  • W. Xu et al.

    Biosens. Bioelectron.

    (2018)
  • F. Yang et al.

    Biosens. Bioelectron.

    (2015)
  • L. Yang et al.

    Biosens. Bioelectron.

    (2017)
  • H. Yu et al.

    Biosens. Bioelectron.

    (2018)
  • A. Zhang et al.

    Biosens. Bioelectron.

    (2016)
  • E. Zhang et al.

    Biosens. Bioelectron.

    (2016)
  • F. Zhang et al.

    Surf. Sci.

    (2004)
  • T. Zhang et al.

    Chem. Eng. J.

    (2018)
  • Z. Zhang et al.

    Biosens. Bioelectron.

    (2017)
  • H. Zhu et al.

    Biosens. Bioelectron.

    (2016)
  • A.N. Alarfaj et al.

    Int. J. Mol. Sci.

    (2018)
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