Elsevier

Enzyme and Microbial Technology

Volume 57, 10 April 2014, Pages 69-77
Enzyme and Microbial Technology

Construction of glutamate biosensor based on covalent immobilization of glutmate oxidase on polypyrrole nanoparticles/polyaniline modified gold electrode

https://doi.org/10.1016/j.enzmictec.2014.02.001Get rights and content

Highlights

  • Constructed a PPyNPs/PANI/AuE hybrid film.

  • Fabricated an improved amperometric glutamate biosensor based on this film.

  • Biosensor had a detection limit of 0.1 nM and linear range 0.02–400 μM.

  • Employed for glutamate determination in different food stuffs.

  • It also showed improved storage stability and reusability.

Abstract

A method is described for construction of a highly sensitive electrochemical biosensor for detection of glutamate. The biosensor is based on covalent immobilization of glutamate oxidase (GluOx) onto polypyrrole nanoparticles and polyaniline composite film (PPyNPs/PANI) electrodeposited onto Au electrode. The enzyme electrode was characterized by cyclic voltammetry (CV), scanning electron microscopy (SEM), X-ray diffraction (XRD), transmission electron microscopy (TEM), Fourier transform infra-red spectroscopy (FTIR) and electrochemical impedance spectroscopy (EIS). The biosensor showed optimum response within 3 s at pH 7.5 (0.1 M sodium phosphate) and 35 °C, when operated at 50 mV s−1. It exhibited excellent sensitivity (detection limit as 0.1 nM), fast response time and wider linear range (from 0.02 to 400 μM). Analytical recovery of added glutamate (5 mM and 10 mM) was 95.56 and 97%, while within batch and between batch coefficients of variation were 3.2% and 3.35% respectively. The enzyme electrode was used 100 times over a period of 60 days, when stored at 4 °C. The biosensor measured glutamate level in food stuff, which correlated well with a standard colorimetric method (r = 0.99).

Introduction

Glutamate (Glu) is one of the 22 amino acids, which are used to synthesize proteins and takes part in typical metabolic functions like energy production and ammonia detoxification. Glutamate is probably best known as “monosodium glutamate (monosodium salt)” or “MSG” which is employed as a flavor or taste enhancer in food. It is usually available together with other food additives and spices in most large food stores, to give a taste known as Umami. The excessive intake of it simulates glutamate receptors in CNS of vertebrates & thereby release glutamate from neurons which lead to neuronal degeneration & cell death beside several neurological disorders including stroke, epilepsy, Alzheimer's diseases & Parkinson's disease as well as learning & memory power [1], [2], [3], [4], [5], [6]. It has also been linked to Chinese Restaurant Syndrome (CRS) [7], being a common ingredient of Chinese food [8]. The term “Chinese restaurant syndrome” is a sudden fall in blood pressure with subsequent fainting after ingestion of very spicy food which is rich in MSG. Hence, there is need to determine its presence in variety of foods. India's Prevention of Food Adulteration Act has set an upper limit of MSG in food, which is 1% [9]. Different techniques have been developed to determine Glu, e.g. potentiometric titration [10], chromatographic [11], [12], [13], [14], [15], spectrophotometric [16], [17], [18] and fluorimetric [19], [20], [21], [22], but these methods require time consuming sample preparation, costly equipment and skilled persons to operate. Biosensors overcome these drawbacks, as these are simple, sensitive, rapid and specific. Recently biosensors have been improved using combination of nanomaterials and conducting polymers.

Polypyrrole, obtained by polymerization of pyrrole is a conducting polymer. The film of polypyrrole is of yellow in color but get darken in air due to oxidation. Although polypyrrole is an insulator, its oxidized derivatives are good electrical conductor with a conductivity range of 2–100 S cm−1 [23]. Nanoparticles of polypyrrole have shown the higher conductivity when doped with short alkyl chain than long alkyl chain [24] due to large surface area for reactions and highly porous in sol form [25], [26]. PPyNPs sandwiched with core shell Fe3O4 nanoparticles have been recently used to improve the analytic performance of potentiometric glucose biosensor [27]. Similarly polyanilne (PANI) is also a polymer of aniline, which has been used in biosensor architecture as transducer, due to its electronic & biomolecular properties [28], [29]. The present work describes a unique approach of immobilizing glutamate oxidase onto PPyNPs/PANI, modified Au electrode and its application in construction of an amperometric biosensor for the determination of l-glutamte. PPyNPs/PANI, based glutamate biosensor is expected to provide high sensitivity, high biocompatibility, high charge transfer rate and good stability.

Section snippets

Materials

Glutamate oxidase (GluOx) from Sigma–Aldrich, St. Louis, USA, potassium ferrocynide (K2Fe2CN2) & potassium ferricynide (K2(FeCN)6·3H2O), aniline, sodium dodecylsulfate (SDS) ((NH4)2S2O8), APS and polypyrrole, and potassium chloride (KCl), from SISCO Research Lab., Mumbai, India, Glutamic acid & Glutraldehyde from LOBA cheme. PVT. LTD. Mumbai, were used. Double distilled water (DW) was used throughout the experimental studies.

Apparatus used

Potentiostat/Galvanostat (Autolab, model: AUT83785, manufactured by

Characterization of PPyNPs

The characterization of polypyrrole nanoparticles was carried out by recording its UV and visible spectra, X-ray diffraction (XRD) pattern, transmission electron micrograph (TEM) and Fourier transform infra red spectroscopy (FTIR). The UV and visible spectra exhibited strong absorbance peak at 440 nm, confirming the synthesis of PPyNPs (Fig. 3A). The XRD patterns of PPyNPs clearly showed its characteristics peak at (Fig. 3B). No characteristic peaks of impurities were observed, revealing the

Conclusion

An improved amperometric bilirubin biosensor was constructed by immobilizing covalently a glutamate oxidase onto PPyNPs/PANI electrodeposited onto Au electrode which exhibited relatively rapid response (3 s), broad linear range (0.02–400 μM), low detection limit (0.1 nM), good reproducibility and long stability (60 days at 4 °C).

Acknowledgements

Author (Bhawna Batra) is thankful to the Council of Scientific and Industrial Research (CSIR), India, for the award of Junior Research Fellowship during this study.

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