Elsevier

Biosensors and Bioelectronics

Volume 62, 15 December 2014, Pages 357-364
Biosensors and Bioelectronics

Enzyme-integrated cholesterol biosensing scaffold based on in situ synthesized reduced graphene oxide and dendritic Pd nanostructure

https://doi.org/10.1016/j.bios.2014.06.063Get rights and content

Highlights

  • New method for the synthesis of rGO-Pd nanoparticle functional hybrid is developed.

  • Amperometric sensing of hydrogen peroxide with detection limit of 0.2 nM is achieved.

  • Biosensing of total cholesterol is demonstrated at 0.25 V without any interference.

  • Total cholesterol in human serum is quantified.

Abstract

A new approach for the one-pot synthesis of reduced graphene oxide–dendritic Pd nanoparticle (rGO–nPd) hybrid material and the development of biosensing scaffold for the amperometric sensing of H2O2 and total cholesterol in human serum are described. In situ reduction of both graphene oxide (GO) and PdCl42− in acidic solution was achieved with Zn to obtain rGO–nPd hybrid material. The oxygen containing functionalities of GO were reduced by the liberated atomic hydrogen in 20 min. The formal potential of Zn/Zn2+ and PdCl42−/Pd couples favor the facile reduction of PdCl42−. The in situ produced Pd nanoparticles behave like hydrogen sponge and increase the local concentration of atomic hydrogen. Biosensing platforms for H2O2 and cholesterol in human serum and butter sample were developed using the hybrid material. Amperometric sensing of H2O2 at 0.2 nM level without any redox mediator or enzymes in neutral pH is demonstrated. The cholesterol biosensing platform was developed by integrating cholesterol oxidase and cholesterol esterase with the hybrid material. The biosensor is highly sensitive (5.12±0.05 μA/μM cm2) and stable with a fast response time of 4 s; it could detect cholesterol ester as low as 0.05 µM (S/N=3). The biosensor is successfully used for the analysis of total cholesterol in human serum and butter.

Introduction

The design and development of amperometric sensing and biosensing platforms for the quantification of bioanalytes such as H2O2, one of the reactive oxygen metabolites, in cellular environment and cholesterol, uric acid, etc. in biological fluids is of great interest in biology and biomedicine. H2O2 has dual role in the physiological system and it has been considered as a “necessary evil” (Rhee, 2006, Dröge and Schipper, 2007, Sevanian and Hochstein, 1985). It involves in the normal function of cell signaling and it is a killing agent released from immune cell. However, its abnormal generation causes the oxidative stress and is associated with ageing, cancer and Parkinson's and Alzheimer's diseases (Li et al., 2011). The precise quantification of H2O2 in cellular environment is of broad interest for the diagnosis and monitoring of the diseases. On the other hand, cholesterol and its fatty acids esters are the primary constituents of all mammalian cells, and is also precursor for the biosynthesis of steroid hormones, vitamin D and bile acids (Myant, 1981). The undesired accumulation of cholesterol in serum leads to serious life threatening diseases like coronary heart diseases, cerebral thrombosis, myocardial infarction, hypertension and atherosclerosis. The normal concentration of total cholesterol in serum for a healthy person should be less than 200 mg/dL (<5.17 mM). The borderline high value of total cholesterol is defined as 200–239 mg/dL (5.17–6.18 mM) and the high value is above 240 mg/dL (≥6.21 mM) (Motonaka and Faulkner, 1993). The colorimetric methods have been traditionally used for the sensing of these bioanalytes. The poor sensitivity, selectivity and reliability of these conventional methods demand for alternative procedures or sensing devices for the quantification of the analytes without compromising the sensitivity and selectivity. The electrochemical methods are of great interest and received significant attention in the biosensor and bioelectronic industries. The transducers based on metal nanoparticles are known to have enhanced sensitivity, selectivity and fast response time towards H2O2 and other metabolites (Safavi et al., 2007, Karam and Halaoui, 2008, Hrapovic et al., 2004, Yang et al., 2006, Lu et al., 2008). Among these, Pt and Pt-based alloys have been widely used in the past and good sensitivity and low detection limit have been achieved (Hrapovic et al., 2004, Yang et al., 2006, Lu et al., 2008). One of the major concerns with the Pt-based catalysts is that they require high detection potential (>0.4 V vs Ag/AgCl), which actually invites interference due to coexisting other easily oxidizable analytes. As the biological fluids such as serum contains high concentration of other analytes, protein, etc., it is desirable to develop a sensing platform which can be operated at low potential for the measurement of H2O2, cholesterol, etc. The nanostructured Pd with suitable shape and morphology can be an ideal choice due to its high catalytic activity, high abundance and relatively cheaper than Pt.

Synthesis of new multifunctional materials is of great interest in the development of biosensors and bioelectronic devices (Pumera, 2011, Wang et al., 2013, Lightcap and Kamat, 2013). The functional materials required for the development of such devices should have high electronic conductivity, mechanical strength, flexibility and good biocompatibility. The organic–inorganic hybrid materials derived from one atom thick graphene or reduced graphene oxide (rGO) and metal/metal oxide nanostructures are very promising for biosensing and bioelectronic applications, as they can provide ideal environment for the redox proteins and enzymes (1, Wang et al., 2013, Zan et al., 2013, Cui et al., 2013). The exceptional electronic conductivity, mechanical strength, biocompatibility, large accessible surface area and flexibility of rGO make it an excellent candidate in the development of biosensors (Dey et al., 2013; Wang et al., 2013; Zan et al., 2013; Cui et al., 2013). The hybrid material of rGO and transition metal nanostructures is anticipated to show outstanding biosensing performance in terms of sensitivity, low detection limit, fast response time, etc. The metal nanostructures supported on the electronically conductive rGO sheets can efficiently catalyze the electrode reaction and hence high sensitivity and fast response time can be achieved (Zhu et al., 2010). The enhanced performance of the biosensors based on metal nanoparticles decorated rGO sheets has been recently documented in the literature (Tian et al., 2013, Artiles et al., 2011, Gutés et al., 2012).

Traditionally, the rGO–metal nanoparticle hybrid materials are synthesized by two-step approach involving the reduction of GO to rGO and metal precursors to corresponding zero valent metals followed by the mixing of both rGO and metal nanoparticles under optimized condition (Fang et al., 2010). One of the major disadvantages in the two-step approach is the re-stacking of the exfoliated individual rGO sheets. The hybrid materials have also been obtained by the reduction of metal precursors in the presence of rGO (Guo et al., 2010). Another interesting approach for the synthesis of rGO–metal nanoparticle is the in situ reduction of both GO and metal precursors in one-pot. The reducing agents capable of reducing both GO and metal precursors are critically required in this approach. The advantages of this approach is that the (i) unwanted restacking of rGO sheets can be avoided, (ii) uniform distribution of nanoparticles on the rGO sheets can be achieved and (iii) GO/rGO can function as a template for the growth of metal nanoparticles. The nature of reducing agent and the reaction conditions are known to play key role in controlling the reduction of oxygen functionalities of GO and regulating shape and morphology of the metal nanoparticles. The controlled reduction can yield anisotropic metal nanostructures. Various chemical methods with different reducing agents such as hydrazine, sodium borohydride, ascorbic acid, metals, etc. have been used in the past for the reduction of GO (Guo et al., 2010, Yang et al., 2011, Jin et al., 2010, He et al., 2011, Barman and Nanda, 2013, Kamat,, 2010). Most of these methods are time consuming and involve toxic reducing agents and non-eco-friendly solvents or hydrothermal conditions. The harsh reaction condition is known to introduce unwanted defects in the rGO sheets and severely alter the properties. Development of room temperature eco-friendly methods for the synthesis of rGO–metal nanoparticle hybrid materials for biosensing application is desired in order to retain the integrity of the individual components.

Herein we describe a new eco-friendly method for the synthesis of catalytically active hybrid material based on rGO and Pd nanoparticles (nPd) and the development of sensitive amperometric sensing platform for the low potential detection of H2O2 and cholesterol ester. The rGO–nPd hybrid material is able to sense H2O2 at the potential of ∼0.25 V, which is significantly lower than the traditional Pt-based catalyst. The cholesterol biosensing platform was developed by integrating the enzymes with the hybrid material.

Section snippets

Reagents and materials

Graphite powder (<45 μm, >99.99%), H2O2 (30%), K2PdCl4, Nafion® (5 wt % solution in lower aliphatic alcohol containing 15–20% water), cholesterol, cholesteryl stearate, uric acid (UA), ascorbic acid (AA), ethanol (99.9%), cholesterol oxidase (ChOx) (from Streptomyces sp., 54 units/mg), and cholesterol esterase (ChEt) (from Pseudomonas fluorescens, 13.16 units/mg) were obtained from Sigma-Aldrich and used as received. All other chemicals used in this investigation were of analytical grade (99.9%).

Synthesis of rGO–nPd hybrid materials

The hybrid material was synthesized by one-pot approach at room temperature using Zn and mineral acid. Scheme 1 depicts the plausible mechanistic pathway for the reduction of GO and PdCl42− by metallic Zn in acid medium. Zn is capable of effectively reducing the oxygen containing functionalities of GO [Dey et al., 2012] at room temperature in 2 h (Fig. S1) in the absence of PdCl42−. However, in the presence of PdCl42−, the reaction is highly facile and complete reduction of GO was achieved in 20 

Conclusions

We have demonstrated a new protocol for the synthesis of hybrid material based on Pd nanoparticles and rGO. Facile reduction of GO was achieved using Zn and Pd precursor. The in situ generated nPd essentially increase the local concentration of nascent hydrogen. The in situ generated atomic hydrogen and hydrogen intercalated nPd efficiently reduces GO to rGO. The as-synthesized rGO–nPd material has high electrocatalytic activity towards oxidation of H2O2 at the potential of 0.25 V, which is

Acknowledgments

R. S. Dey is recipient of UGC research fellowship. This work was financially supported by Department of Science and Technology and Council of Scientific and Industrial Research, New Delhi. Authors are thankful to B. C. Roy Technology Hospital, IIT Kharagpur for human serum samples and the clinical laboratory analysis of cholesterol. We acknowledge DST-FIST for X-ray diffractometer facility.

References (46)

  • M.S. Artiles et al.

    Adv. Drug Deliv. Rev

    (2011)
  • S. Chakraborty et al.

    Biosens. Bioelectron.

    (2009)
  • A. Gutés et al.

    Biosens. Bioelectron.

    (2012)
  • K.H. Lee et al.

    J. Colloid Interface Sci.

    (2013)
  • C. Nethravathi et al.

    Carbon

    (2008)
  • M. Pumera

    Mater. Today

    (2011)
  • D.A.J. Rand et al.

    J. Electroanal. Chem.

    (1971)
  • A. Safavi et al.

    Electrochem. Commun.

    (2007)
  • S. Singh et al.

    Anal. Chim. Acta

    (2004)
  • S. Stankovich et al.

    Carbon

    (2007)
  • J. Tian et al.

    Biosens. Bioelectron.

    (2013)
  • M. Yang et al.

    Biosens. Bioelectron.

    (2006)
  • T. Yao et al.

    Biosens. Bioelectron.

    (1998)
  • X. Zan et al.

    Biosens. Bioelectron

    (2013)
  • B.K. Barman et al.

    Chem. Commun.

    (2013)
  • I.F. Cheng et al.

    Environ. Sci. Technol.

    (1997)
  • S. Cui et al.

    J. Phys. Chem. Lett.

    (2013)
  • R.S. Dey et al.

    Chem. Commun.

    (2012)
  • R.S. Dey et al.

    RSC Adv.

    (2013)
  • W. Dröge et al.

    Aging Cell

    (2007)
  • Y. Fang et al.

    Langmuir

    (2010)
  • M. Guo et al.

    Electroanalysis

    (2004)
  • S. Guo et al.

    ACS Nano

    (2010)
  • Cited by (0)

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