Elsevier

Journal of Electroanalytical Chemistry

Volume 781, 15 November 2016, Pages 410-417
Journal of Electroanalytical Chemistry

Novel graphene/Au-CdS:Eu composite-based electrochemiluminescence immunosensor for cancer biomarker detection by coupling resonance energy transfer and enzyme catalytic reaction

https://doi.org/10.1016/j.jelechem.2016.06.045Get rights and content

Highlights

  • A ternary composite graphene/Au-CdS:Eu QDs is presented for ECL luminescence.

  • The ECL immunosensor relies on dual-quenching strategy.

  • Sensitive detection of AFP is achieved with good selectivity and stability.

Abstract

Recent studies show that ECL resonance energy transfer (ECL-RET) coupled with H2O2-related enzymatic reactions is an efficient strategy for designing excellent ECL biosensors with high sensitivity. Here we find that a ternary composite named graphene/Au-CdS:Eu QDs demonstrated ECL-RET effect with Au nanorods (Au NRs) and horseradish peroxidase (HRP) on the Au NRs could catalyze hydrogen peroxide reduction in the presence of hydroquinone (HQ). Therefore, we developed an efficient dual-quenching ECL immunosensor based on the novel ternary composite with high ECL intensity coupled with H2O2-related enzymatic reactions and ECL-RET strategy from Au NRs. This ultrasensitive ECL sensing strategy combined the advantages of the excellent ECL sensing properties of graphene/Au-CdS:Eu QDs, and dual-quenching effect and achieve sensitive detection of alpha fetoprotein (AFP).

Introduction

In diseases diagnostics and therapeutic evaluation, cancer biomarkers as a kind of disease-related proteins still play an important role for its sensitive response of disease severity [1], [2], [3]. During the diagnostics very high sensitivity is required for early discovery and accurate treatment of malignant tumor. Therefore many signal amplification strategies coupling various photo-electrochemical detecting technologies were studied to achieve extremely low detection limit of cancer biomarker. Among these technologies, multienzyme probes which possess large amounts of enzymes on nanocarriers are usually employed to enhance the enzymatically catalytic signal [4], [5], [6], [7], [8], [9], [10], [11]. Combination of multienzyme probes and the electrochemiluminescence (ECL) technique showed powerful properties due to the peculiar advantages of ECL such as low background and high sensitivity [12], [13], [14]. Since 2002 in which the first ECL work of silicon quantum dots (QDs) was reported, QDs-based ECL sensing technology has been developed at full speed during the past decade [15], [16], [17], [18], [19], [20].

Although QDs hold many excellent properties, they have lower ECL signal compared with conventional luminescent reagents, such as luminol or Ru(bpy)32 +, which greatly limits its application in biomolecules analysis. Recent reports showed that metal ion doped QDs could obviously improve the luminescence efficiency by perturbing the host energy level or providing a new electron energy level [21], [22], [23]. Xu's group has reported a series of work. For example, ECL behavior of Mn2 + ions doped ZnS QDs was first studied [21] and two ECL emission peaks at different potentials during a negative potential scan were found after the reaction of the doped ZnS QDs and H2O2. The peak at − 2.38 V is ECL emission of main body ZnS QDs and the other peak at − 1.5 V was due to the launching of surface states associated with Mn2 + ions. This discovery provided a new approach for construction of QDs-based biosensor with improved ECL performance. Also they successfully synthetize Eu3 + ions doped CdS QDs which could enhance the ECL intensity by creating a new surface state. And this study may greatly broaden QDs' application for highly sensitive biosensing [22]. Just recently, many new functional materials including nanogold, carbon nanotubes (CNTs), graphene oxide, dendrimers, etc. were employed for fabrication of QDs family composites to improve the biocompatibility of the materials and the ECL intensity [24], [25], [26], [27], [28], [29], [30], [31]. For example, Jie et al. developed a kind of CdSe QDs film which was combined of carbon nanotubes and poly(diallyldimethylammonium chloride) (PDDA), and the decoration was found to greatly improve the ECL ability of CdSe QDs and could receive sensitive detection of human immunoglobulin (IgG) [25]. Moreover, nitrogen-doped CNTs and CdSe QDs complexes were studied by Ju's group [26], and it showed five times higher ECL cathode luminescence than that of pure CdSe QDs and three times higher than that of the CdSe QDs-CNTs composites when modified on the electrode with hydrogen peroxide as a coreactant. Besides this, Zhu's group presented a preparation strategy of graphene-CdSe composites [28]. And this graphene-CdSe composite film showed higher ECL intensity, better electronic conductivity and stability compared with pure CdSe material. These studies demonstrated that the fabrication of doped ECL biosensors holds great promise for disease-related biomolecules detection.

During the recent studies, ECL resonance energy transfer (ECL-RET) was found to be a promising strategy for designing excellent biosensors with high sensitivity. The key point of this ECL-RET biosensing design is to obtain a suitable ECL donor and corresponding acceptor, and the donor's ECL spectrum is required to be overlapped with the acceptor's absorption spectrum. And we recently find that a ternary composite named graphene/Au-CdS:Eu QDs and Au nanorods (Au NRs) just meet this requirement, moreover it showed many other advantages in construction of biosensor including its high sensitivity and good stability due to high electron transfer efficiency and excellent conductivity of RGO/Au. Therefore, we developed a novel ECL-ET based immunosensor for the detection of tumor markers, using graphene/Au-CdS:Eu QDs and Au nanorods (Au NRs) as the donor and acceptor, respectively. QDs could produce an ECL emission through the reaction with coreactant H2O2, however, in the presence of substrate hydroquinone (HQ) which could consume H2O2, the ECL emission would be decreased. And HRP modified on the electrode via the sandwich structure could catalyzed the reaction process of HQ and H2O2, resulting the ECL quenching. Moreover, Au NRs could also quench ECL signal through ECL-RET due to the spectrum overlap between the Au-CdS:Eu QDs' ECL spectrum and Au NRs' absorption spectrum.

Based on Au NRs assistant-ECL-RET and enzymatic cycling catalysis, the proposed ECL immunosensing system showed a high sensitivity and selectivity which present excellent prospect for clinical protein detection. Scheme 1 illustrates the schematic representation of graphene/Au-CdS:Eu QDs modified ECL biosensor based on enzymatic cycling signal amplification assistant-ECL-RET system.

Section snippets

Reagents and apparatus

Thioglycolic acid (TGA), poly(diallyldimethylammonium chloride) (PDDA), N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), horseradish peroxidase (HRP), Cd(NO3)2·4H2O, CTAB, Eu(NO3)3, Na2S·9H2O, alpha fetoprotein (AFP) and anti-AFP were obtained from Aladdin. Poly(sodium 4-styrenesulfonate) (PSS) and Bovine serum albumin (BSA), hydroquinone (HQ) were purchased from Sigma-Aldrich. Human serum samples containing AFP were kindly provided by Linyi Cancer

Characterization of the materials

As shown in Scheme 1A, two steps were needed in the synthesis of reduced graphene oxide (RGO)/Au-CdS:Eu QDs. First, Au NPs-modified RGO was prepared by reduction of HAuCl4 on the surface of the RGO. And then, RGO/Au-CdS:Eu composite formed via electrostatic interactions between negatively charged thioglycolic acid (TGA) which was in advance modified on CdS:Eu QDs and positively charged poly(diallyldimethylammonium chloride) (PDDA) functionalized RGO/Au.

TEM images of above materials including

Conclusion

In summary, we presented a dual-quenching ECL immunosensor based on the novel ternary composite RGO/Au-CdS:Eu QDs with high ECL intensity coupled with H2O2-related enzymatic reactions and ECL-RET strategy from Au NRs. HRP molecules on Au NRs/Ab2 composite could catalyze the redox reaction between substrate hydroquinone and H2O2. Then the consumption of ECL co-reactant H2O2 thus resulted in a quenching effect of ECL signal. Moreover, the ECL-RET strategy between the Au NRs and the CdS:Eu QDs

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant Nos. 21405072, 21505065, 21275086 and 21535002), the “Innovation Team Development Plan” of the Ministry of Education Rolling Support (IRT_15R31), the Project of Shandong Province Higher Educational Science and Technology Program (J14LC14 and J14LC15), the Shandong Provincial Natural Science Foundation (ZR2014BL022, ZR2014BL023 and ZR2014BL026), Special Funds for the Construction of Taishan Scholars (Grant No.

References (40)

  • R. de la Rica et al.

    Plasmonic ELISA for the ultrasensitive detection of disease biomarkers with the naked eye

    Nat. Nanotechnol.

    (2012)
  • S.I. Stoeva et al.

    Multiplexed detection of protein cancer markers with biobarcoded nanoparticle probes

    J. Am. Chem. Soc.

    (2006)
  • S.-J. Xu et al.

    Positive potential operation of a cathodic electrogenerated chemiluminescence immunosensor based on luminol and graphene for cancer biomarker detection

    Anal. Chem.

    (2011)
  • Z.-Q. Gao et al.

    Enhanced colorimetric immunoassay accompanying with enzyme cascade amplification strategy for ultrasensitive detection of low-abundance protein

    Sci. Rep.

    (2014)
  • W.-W. Zhao et al.

    In situ enzymatic ascorbic acid production as electron donor for CdS quantum dots equipped TiO2 nanotubes: a general and efficient approach for new photoelectrochemical immunoassay

    Anal. Chem.

    (2012)
  • D.-J. Lin et al.

    Ultrasensitive immunoassay of protein biomarker based on electrochemiluminescent quenching of quantum dots by hemin bio-bar-coded nanoparticle tags

    Anal. Chem.

    (2011)
  • W.-W. Zhao et al.

    Immunogold labeling-induced synergy effect for amplified photoelectrochemical immunoassay of prostate-specifc antigen

    Chem. Commun.

    (2012)
  • Y.-J. Li et al.

    Dual-signal amplication strategy for ultrasensitive photoelectrochemical immunosensing of α-fetoprotein

    Anal. Chem.

    (2012)
  • M.M. Richter

    Electrochemiluminescence (ECL)

    Chem. Rev.

    (2004)
  • W.-J. Miao

    Electrogenerated chemiluminescence and its biorelated applications

    Chem. Rev.

    (2008)
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