Elsevier

Biosensors and Bioelectronics

Volume 102, 15 April 2018, Pages 189-195
Biosensors and Bioelectronics

Ultrasensitive electrochemical immunosensor for quantitative detection of HBeAg using Au@Pd/MoS2@MWCNTs nanocomposite as enzyme-mimetic labels

https://doi.org/10.1016/j.bios.2017.11.032Get rights and content

Highlights

  • Using the p-GO@Au as sensing platform increased the loading of primary antibodies.

  • Dandelion-like Au@Pd nanoparticles were used in immunosensor.

  • Au@Pd/MoS2@MWCNTs has better electrocatalytic activity towards reduction of H2O2.

  • The sandwich-type immunosensor showed desired sensitivity and stability for HBeAg detection.

Abstract

A sensitive sandwich-type electrochemical immunosensor for the detection of hepatitis B e antigen (HBeAg) was successfully developed based on the gold@palladium nanoparticles (Au@Pd NPs) loaded by molybdenum disulfide functionalized multiwalled carbon nanotubes (Au@Pd/MoS2@MWCNTs). The resultant nanocomposites not only possessed high specific surface area and good biocompatibility, but also exhibited excellent electro-catalytical property. Au NPs functionalized porous graphene oxide (p-GO@Au) were used as sensing platforms and primary antibodies carriers, which can accelerate the electron transfer and improve the load capacity of primary antibodies (Ab1), improving the sensitivity of the immunosensor. Under optimal conditions, the designed immunosensor could detect target HBeAg concentration in the range from 0.1pg/mL to 500 pg/mL, with a low detection limit of 26 fg/mL (S/N = 3) for HBeAg. Additionally, the designed immunosensor showed excellent specificity, good reproducibility and acceptable stability. The satisfactory results in analysis of human serum samples indicated that it had potential application in clinical monitoring of tumor markers.

Introduction

As one of the most severe public health problems, the hepatitis B virus (HBV) infection can trigger cirrhosis and hepatocellular carcinoma (HCC), the third leading fearful cause of cancer death (Ledesma et al., 2011, da Costa Ferreira et al., 2017). Hepatitis B e antigen (HBeAg) has been identified as the reliable tumor marker for diagnosis and prognosis of disease caused by HBV infection (Magnius and Espmark, 1972, Ott et al., 2012). The HBeAg could increase immune tolerance during neonatal transmission and hinder immune response against the related core protein (Milich and Liang, 2003, Milich et al., 1990). The previous research has shown that relative risks for developing HCC of HBeAg positivity was about 50% higher than HBeAg negativity (Ganem and Prince, 2004). Thus, accurate detection of HBeAg is significantly crucial to the HCC diagnosis.

With the rapid development of technology, many methods have been applied to the biomarker detection. However, there were few research methods for HBeAg detection. Recently, the sandwich-type electrochemical immunosensor has attracted burgeoning attention, due to its unique advantages of rapid response, good sensitivity, convenient operation and excellent specificity (Teymourian et al., 2013). As a diagnostic tool, it is a great promising approach to detect HBeAg.

For sandwich-type electrochemical immunosensors, a crucial strategy of detection is to achieve signal amplification by coupling different labels of secondary antibody (Ab2) (Li et al., 2017a). In this work, gold@palladium nanoparticles loaded by molybdenum disulfide functionalized multiwalled carbon nanotubes (Au@Pd/MoS2@MWCNTs) were proposed to label secondary antibodies (Ab2), improving the sensitivity of immunosensor. Due to excellent stability, superior catalytic efficiency and ease of mass production, enzyme mimics such as metal complexes, polymers and supramolecules have drawn widespread attention in bionanotechnology, such as biosensing, bioimaging, tissue engineering and therapeutics (Wang et al., 2016, Wei and Wang, 2013). Metal complexes as the representative have widely used in fabrication of immunosensors, because they could increase the sensing surface area and amplify the signal. MoS2, as two-dimensional layered group-VI transition metal dichalcogenides, has attracted increasing interest in catalysis, owing to the unique structure and properties such as semiconducting property, direct band gap and conductance. (Cao et al., 2012, Splendiani et al., 2010, Zhang et al., 2015). And the free energy of atomic hydrogen bonding to the sulfur edge of the MoS2 structure is approach to that of Pt, illustrating that MoS2 has a promising potential to substitute Pt-group catalysts (Ren et al., 2015). Zhu et al. (Zhu et al., 2016) integrated PtW nanocrystals and MoS2 nanosheets in conjunction with each other to detect H2O2, obtaining a high sensitivity. And Yoon's group (Yoon et al., 2017) synthesized MoS2 nanoparticle hybrid graphene oxide to catalyze H2O2, achieving satisfying results. Molybdenum disulfide functionalized MWCNTs synthesized in this work can control the size of MoS2 held together by van der Waals interactions and accelerate the electron transfer to improve the conductivity (Radisavljevic et al., 2011). Meanwhile, as the result of the enhanced physical, and chemical catalytic properties, bimetallic composite nanostructures, such as core-shell nanoparticles and nano-dendrites, enhanced catalytic activity and tolerability for oxygen reduction over the corresponding monometallic counterparts (Shi et al., 2013). And they can also accelerate reaction kinetics (Henning et al., 2013). Most importantly, bimetallic catalysts often displayed superior catalytic performance compared to their single metal counterparts, owing to synergistic effects between two metals (Enache et al., 2006). And a novel Au@Pd nanoparticles (Au@Pd NPs) synthesized was firstly used to further improve the sensitivity. Large numbers of catalytically active sites formed by the dendrite Au@Pd NPs efficiently facilitated oxygen reduction reaction (ORR) (Fujita et al., 2012, Huang et al., 2015). Meanwhile, Au@Pd NPs had wonderful biocompatibility with biomolecules, leading to load a larger number of Ab2 via the affinity interaction between Pd NPs and amine group of Ab2.

Hence, a novel and ultrasensitive sandwich-type electrochemical immunosensor was designed for quantitative HBeAg detection, using p-GO@Au as the sensing platform and Au@Pd/MoS2@MWCNTs as signal labels. The proposed immunosensor showed admirable linear range, low detection limit, excellent specificity, good reproducibility and acceptable stability.

Section snippets

Reagents and apparatus

The HBe antibody and antigen were supplied from Shanghai Linc-Bio Science Co., Ltd (Shanghai, China). Bovine serum albumin (BSA, 96–99%) was purchased from Sigma reagent Co., Ltd. (St. Louis, MO, USA). Multi-walled carbon nanotubes (MWCNTs) were purchased from Alfa Aesar Co., Ltd. (Shanghai, China). HAuCl4·4H2O was obtained from Sigma-Aldrich Co., Ltd. (Beijing, China). Ammonium thiomolybdate [(NH4)2MoS4] was obtained from J&K Scientific Ltd. (Beijing, China). cetyltrimethylammonium bromide

Characterization of the structure and morphology

Fig. 2A showed the SEM image of the p-GO@Au composites. Clearly, plenty of nanoparticles distributed on the surface and inside of GO porous composites evenly, with a mean diameter of 25 nm. And it was clear to see that obviously porous structures (inset in Fig. 2A) which further gave a high specific surface area for antibody attachment and large amounts of defects/functional groups for the growth of Au NPs (Li et al., 2017a, Li et al., 2017b, Li et al., 2017). More importantly, the internal

Conclusions

In this research, a novel immunosensor was proposed for ultrasensitive determination of HBeAg by loading Au@Pd nanoparticles on MoS2@MWCNTs as label to amplify the current signal. Using p-GO@Au as platform can enlarge the captured biomolecules and accelerate the electron transfer. Overall, the developed immunosensor possessed excellent specificity, good reproducibility and acceptable stability. The prepared immunosensor which exhibited excellent properties would provide a potential application

Acknowledgments

This study was financially supported by the National Natural Science Foundation of China (Nos. 21575079 and 21575050), and Natural Key Science Foundation of Shandong Province (ZR2013FB001). All of the authors express their deep thanks.

References (39)

  • V. Datsyuk et al.

    Carbon

    (2008)
  • J. Feng et al.

    Biosens. Bioelectron.

    (2017)
  • M. Grilc et al.

    Appl. Catal. B: Environ.

    (2015)
  • J. Han et al.

    Anal. Chim. Acta

    (2013)
  • K.-J. Huang et al.

    Energy

    (2014)
  • B. Kavosi et al.

    Biosens. Bioelectron.

    (2015)
  • N. Larouche et al.

    Carbon

    (2010)
  • M.M.G.L. Ledesma et al.

    Int. J. Infect. Dis.

    (2011)
  • X. Li et al.

    Biosens. Bioelectron.

    (2017)
  • Y. Li et al.

    Biosens. Bioelectron.

    (2017)
  • M. Li et al.

    Biosens. Bioelectron.

    (2017)
  • D. Milich et al.

    Hepatology

    (2003)
  • H. Teymourian et al.

    Biosens. Bioelectron.

    (2013)
  • Y. Wang et al.

    Biosens. Bioelectron.

    (2014)
  • M. Yan et al.

    Anal. Chim. Acta

    (2013)
  • J. Yoon et al.

    Biosens. Bioelectron.

    (2017)
  • L. Zhu et al.

    Biosens. Bioelectron.

    (2016)
  • T. Cao et al.

    Nat. Commun.

    (2012)
  • S. da Costa Ferreira et al.

    Hum. Immunol.

    (2017)
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