Decoration of reduced graphene oxide with rhodium nanoparticles for the design of a sensitive electrochemical enzyme biosensor for 17β-estradiol

https://doi.org/10.1016/j.bios.2016.07.018Get rights and content

Highlights

  • One pot synthesis of novel graphene oxide/Rh nanoparticles 2D composite material.

  • Highly sensitive laccase-based biosensor for 17β-estradiol.

  • Accurate determination of the hormone in urine samples.

Abstract

A novel nanocomposite material consisting of reduced graphene oxide/Rh nanoparticles was prepared by a one-pot reaction process. The strategy involved the simultaneous reduction of RhCl3 and graphene oxide with NaBH4 and the in situ deposition of the metal nanoparticles on the 2D carbon nanomaterial planar sheets. Glassy carbon electrode coated with this nanocomposite was employed as nanostructured support for the cross-linking of the enzyme laccase with glutaraldehyde to construct a voltammperometric biosensor for 17β-estradiol in the 0.9–11 pM range. The biosensor showed excellent analytical performance with high sensitivity of 25.7 A µM−1 cm−1, a very low detection limit of 0.54 pM and high selectivity. The biosensor was applied to the rapid and successful determination of the hormone in spiked synthetic and real human urine samples.

Introduction

Nanomaterials engineering has greatly impacted bioelectroanalytical chemistry by providing a large variety of novel and advanced nanosized materials with well-defined morphology and physicochemical properties (Walcarius et al., 2013). These nanomaterials have been successfully employed as transducers for the design of novel, sensitive and reliable biosensor devices. Tailor made nanomaterials have been also widely employed as signalling and amplification tools for affinity-based electrochemical biosensors (Lei and Ju, 2012, Yáñez-Sedeño et al., 2015).

Among these, two dimensional nanomaterials have recently attracted great attention for designing electrochemical biosensors due to the unique thickness dependent physical and chemical properties of these materials (Kannan et al., 2015). In this sense, several 2D layered inorganic nanomaterials such as MoS2, WS2, CuS and SnS2 have been explored as transduction elements for bioelectroanalytical applications (Sarkar et al., 2014, Wang et al., 2014, Vasilescu et al., 2016, Yuan et al., 2014, Huang et al., 2015a, Huang et al., 2015b, Yang et al., 2011). Mesoporous silica thin films have been also recently employed as two dimensional scaffolds for the assembly of label free electrochemical biosensors (Fernández et al., 2014, Saadaoui et al., 2015). However, graphene-based nanomaterials are, by far, the 2D nanomaterials more widely used for biosensing purposes (Araque et al., 2014a, Araque et al., 2013, Borisova et al., 2015, Kailashiya et al., 2015, Khatayevich et al., 2014, Lian et al., 2015). These 2D carbon nanomaterials have emerged as useful tools for bioelectroanalysis due to their unique electroconductive properties, large surface-to-volume ratio, flexibility, high mechanical and thermal stability, remarkable biocompatibility and low cost (Allen et al., 2009).

Despite these outstanding properties, high hydrophobicity and lack of chemical functional groups to be employed as linking points for the covalent immobilization of bioreceptors has limited the use of graphene as scaffold for the assembly of stable biosensing interfaces (Liu et al., 2012). These problems can be partially overcome by using graphene oxide (GO), a water soluble derivative with carboxyl groups at the sheet edges, and hydroxyl, epoxide and carbonyl groups on the graphene basal planes (Allen et al., 2009; Rourke et al., 2011). These oxygen functional groups confer hydrophilicity to the 2D nanomaterial, and can be easily employed as anchoring point for the further surface immobilization of biomolecules. Bioelectroanalytical potential of GO can be boosted by proper covalent and non-covalent decoration with metal nanostructures, yielding advanced nanohybrids and nanocomposites with improved electroconductive and catalytic properties (Araque et al., 2014b, Boujakhrout et al., 2015, Mei et al., 2015, Zhao et al., 2015).

In this work we describe the facile preparation of a novel GO nanocomposite by in situ non-covalent decoration of the 2D carbon nanomaterial with rhodium nanoparticles. The resulting nanocomposite was employed as coating material for glassy carbon electrode to construct an electrochemical laccase enzyme biosensor for the determination of 17β-estradiol. The steroid 17β-estradiol is a natural endocrine disrupting compound excreted by humans and animals, which has been classified as emerging contaminant due to its harmful effects on endocrine function of animals, humans and aquatic organisms (Ying et al., 2002). In fact, it has been previously reported that high amounts of 17β-estradiol may interfere with the normal physiological processes causing sexual abnormalities, a declined male birth rate, and even cancer development (Storgaard et al., 2006, De Assis et al., 2013). That is why the development of simple, sensitive, reliable and cost-effective analytical methods and devices for 17β-estradiol receives considerable attention. In this work we selected laccase as catalytic bioreceptor for the construction of the electrochemical biosensor due to the ability of this enzyme to recognize 17β-estradiol as substrate (Suzuki et al., 2003, Riva, 2006, Torres-Duarte et al., 2012).

Section snippets

Materials

Single layer graphene oxide (GO), prepared according to Hummers method (Hummers and Offeman, 1958) was provided by Orion High Technologies (Spain). Laccase from Trametes versicolor (Lac, EC 1.10.3.2, 10 U/mg), 17β-estradiol, thionine, RhCl3, poly(ethylene glycol) diglycidyl ether, NaBH4 and glutaraldehyde were purchased from Sigma-Aldrich Co. (USA). All other chemicals were analytical grade.

Electrochemical measurements

A dual-channel Inbea potentiostat was used for amperometric measurements (Inbea Biosensores, Spain).

Preparation and characterization of the transducer nanomaterial

Scheme 1 illustrates the strategy employed to prepare the rGO-RhNP nanocomposite and to assemble the laccase amperometric biosensor for 17β-estradiol. First, the nanocomposite material was prepared through a one-pot scheme by reducing Rh3+ ions with NaBH4 in the presence of graphene oxide and sodium citrate. Through this process, citrate-capped Rh nanoparticles were rapidly formed and, at the same time, the epoxy and carbonyl residues on the GO planar sheets were transformed to hydroxyl groups.

Conclusions

A novel nanocomposite material was prepared by in situ reduction and decoration of graphene oxide with rhodium nanoparticles. This nanomaterial was employed to modify GCE and then used as scaffold to immobilize the enzyme laccase on the electrode surface through a glutaraldehyde-mediated cross-linking reaction. This enzyme electrode was employed to construct a voltammperometric biosensor for 17β-estradiol which exhibited high sensitivity, selectivity and reproducibility, and was able to detect

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

Financial support from the Spanish Ministry of Economy and Competitiveness CTQ2014-58989-P, CTQ2011-24355, CTQ2015-71936-REDT, CTQ2012-34238 and Comunidad de Madrid S2013/MIT-3029, Programme NANOAVANSENS is gratefully acknowledged.

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