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20 A novel nanobiocomposite for immobilization of xanthine oxidase (XO) was developed by 21 incorporating functionalized MWCNT in nanogold doped poly(o-phenylenediamine)(PPD) (Au-22 PPD) film on glassy carbon electrode(GCE) for selective and sensitive detection of xanthine in 23 real samples e.g. blood, urine, fish. Stable colloid of o-phenylenediamine(OPD) and HAuCl 4 in 24 acidic environment was electropolymerized on working electrode(GCE) to form ultrathin film of 25 AuNP-PPD which possessed permselectivity and no interference against electroactive species 26 such as ascorbic acid and uric acid. Spectrophotometric and microscopic analysis confirmed the 27 doping behaviour of AuNP. Electrodeposition of carboxylated MWCNT onto the Au-PPD film 28 increased conductivity, sensitivity and also facilitated a microenvironment to entrap XO enzyme 29 by covalent bonding enhancing storage stability. The conductive nature of the electrode after 30 every step of modification was investigated by electrochemical impendence spectroscopy. High 31 I max /K map value was achieved by XO/fMWCNT/Au-PPD modified electrode. Oxidation of 32 xanthine on this modified electrode was diffusion-controlled involving two-electron in the rate-33 determining step with a transfer coefficient (α) of about 0.596. Differential pulse voltammetric 34 study of XO/fMWCNT/Au-PPD/GCE exhibited good analytical characteristics e.g. low 35 detection limit (12nM) (S/N=3), a wide linear range of 0.01-300 µM (R 2 =0.994), good sensitivity 36 (14.03 µA µM −1 cm −2 ), fast response (6s) at anodic potential of +0.625V vs. Ag/AgCl (pH 7.0). It 37 retained 91% of its initial activity even after 210 times of use over a period of 4 months when 38 stored at 4°C. The applicability of the xanthine biosensor was tested by performing 39 reproducibility, repeatability and interference study on real samples.40

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