An interference-free new xanthine biosensor based on immobilized enzyme-nanogold conjugate on carbon nanotube doped poly(3,4-Ethylenedioxythiophene) composite film

https://doi.org/10.1016/j.ijbiomac.2021.12.094Get rights and content

Highlights

  • One-step rapid electrodeposition of fMWCNT doped PEDOT:NO3.

  • Purified xanthine oxidoreductase (XOR) from a cloned gene of Pseudomonas aerogenosa strain CEBP1.

  • Better substrate affinity and storage stability of nanogold conjugated XOR.

  • Biocompatibility of enzyme immobilization ensured by polymeric nanocomposite.

  • High sensitivity, nanomolar detection limits, and excellent stability of xanthine biosensor.

Abstract

A new design of biosensor based on polymeric nano(bio)composite has been proposed for the selective detection of xanthine to be used in the clinical analysis as well as food quality control. The xanthine oxidoreductase (XOR) gene of Pseudomonas aerogenosa strain CEBP1 was cloned to obtain purified enzyme through affinity chromatography. fMWCNT doped PEDOT was electrodeposited on the working electrode to enhance the sensitivity and selectivity of the biosensor. Bio-synthesized gold nanoparticles conjugated XOR (Au-XOR) was covalently immobilized on the polymeric nanocomposite. The enzymatic activity was enhanced 1.12 times with increased substrate affinity. The surface morphology and structural properties of the polymeric layer were investigated using SEM, FESEM, TEM. Electrochemical characteristics were performed by cyclic voltammetry, differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy. Xanthine was oxidized (pH 7.0) on the uniquely designed polymeric nano(bio)composite modified electrode at a lower anodic potential of + 0.446 V vs. Ag/AgCl (3 M NaCl) at optimized DPV conditions. The simple, newly designed Au-XOR/fMWCNT-PEDOT/GCE exhibited interference-free reproducibility and stability (∼4 months) with excellent sensitivity of 16.075 µA.µM−1.cm−2 for the quantification of xanthine in biological samples such as blood, tissue, urine. The applicability of the biosensor was validated by comparing the sensing results for the real biological fluidic solutions with HPLC data (RE = 0.5–3.1%).

Introduction

Xanthine (XN) is an important bioanalyte for biomedical and food processing industries [1]. XN is an evaluating index for monitoring the freshness of fish, meat, and other protein-derived products as it starts to accumulate right after the animal is slaughtered due to microbial degradation of ATP following the pathway ATP → ADP → AMP → IMP → HxP → HX → XN [2], [3], [4], [5]. XN is the first indicator of an abnormal purine profile, as it is the metabolic precursor of uric acid. The generated reactive oxygen species (ROS) in this process are also involved in numerous vital biological downstream processes. Thus abnormalities in the purine degradation pathway may cause various clinical disorders, including perinatal asphyxia, adult respiratory distress syndrome, cerebral ischemia, tumor, hyperthermia, and pre-eclampsia [6], [7], [8]. Extreme abnormal levels of XN in body fluids are symptoms of several diseases, such as renal failure, hyperuricemia, gout, xanthinuria, leukemia, and pneumonia [5], [7].

Xanthine oxidoreductase (XOR) is a prime enzyme that oxidizes hypoxanthine to xanthine and xanthine to uric acid [3], [8], [9]. XOR is a homodimeric cytosolic molybdenum protein with a molecular mass of around 290 kDa [8], [9]. Each subunit acts independently during the oxidation process, having two distinct iron-sulfur ([2Fe-2S]) centers, one molybdopterin (Mo) cofactor, and one flavin adenine dinucleotides (FAD) [8], [9], [10]. Mo(VI) transforms to its reduced form Mo(IV) during the oxidative hydroxylation process. Two generated electrons pass successively through [2Fe-2S] centers to FAD, and finally to molecular oxygen (O2) [7], [8], [9], [10]. Reactive oxygen species (ROS) such as superoxide radical (O2•−) and hydrogen peroxide (H2O2) are produced at the end of the oxidative reaction [8].

It was noted from our previous study that Pseudomonas aerogenosa strain CEBP1 exhibited the highest XOR activity in comparison to other isolated strains when it was incubated in selective xanthine media (SXM) at optimized conditions [11]. In the present research work, the XOR gene of P. sp. CEBP1 was expressed in E.coli BL21 and recombinant XOR enzyme was purified for sensor fabrication. Since XOR is an intracellular enzyme, after bacterial cell disruption for extraction of XOR, the cell-free extracellular extract (CFE) was used for biosynthesis of gold nanoparticles (Au NP). This study aimed to utilize the AuNP for enhanced activity of XN oxidizing enzyme, and eventually further enhancement of the stability of the biosensing platform. This Biosynthesised AuNP was conjugated with purified XOR enzyme (Au-XOR) and the interaction of AuNP with XOR was studied by various spectrophotometric techniques such as UV–vis, fluorescence, circular dichroism (CD). The proposed technique can be applied for the economical production of purified XOR enzyme to be used in biomedical applications.

Green synthesized nanoparticles such as gold (Au), silver (Ag), platinum (Pt) have immense importance in modern science [12], [13], [14]. The non-toxic and cost-effective biosynthesized nanoparticles could be isolated from various microbes and, plant sources [12], [15], [16]. Gold NP has been widely used in biomedical applications due to its antibacterial, anticancer, imaging, and conducting nature. AuNP provides a suitable microenvironment to retain the biological activity of proteins upon adsorption and enhance storage stability [17], [18]. It has an attractive role in the fabrication of biosensing matrix because of its unique properties such as large effective surface area, excellent electron transfer, fast mass transport rate, and good biocompatibility [2], [17], [19], [20], [21]. In the last two decades, different materials such as metal nanomaterials (Au, Ag, Pt, Cu, ZnO, Fe3O4), carbon (e.g. nanotube, graphene, nanofiber, nanohorn), self-assembled monolayers, conducting and nonconducting polymers have widely been incorporated to enhance the electron transfer rate of enzyme electrodes, that further helps to increase the sensitivity of biosensor [2], [17], [19], [20], [21], [22], [23], [24]. Carbon nanotubes and conducting polymers both offer excellent biocompatibility as well as catalytic activities [2], [4], [7], [17], [19], [22], [24], [25], [26]. In this work, we have synthesized a uniform thin film of functionalized multiwalled carbon nanotube (fMWCNTs) doped poly(3,4-ethylenedioxythiophene) (PEDOT) through a rapid one-step electropolymerization process for immobilization of nanogold conjugated purified XOR (Au-XOR) to fabricate an enzyme-based biosensing matrix.

Section snippets

Chemicals

3,4 ethylenedioxythiophene (EDOT), auric chloride (HAuCl4·3H2O), and multiwalled carbon nanotube (MWCNT) were supplied by Sigma (US). Potassium nitrate, potassium dihydrogen phosphate (KH2PO4) and dipotassium hydrogen phosphate (K2HPO4), sodium chloride (NaCl), peptone, beef extract, yeast extract, agar-agar were purchased from E-merck (Mumbai, India). Xanthine (XN), N-hydroxy succinamide (NHS), and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDC) have been procured from

Cloning, expression, and purification of XOR enzyme

About ten colonies of the appropriate clone for the XOR gene were selected and transformed into the E.coli host (BL21 DE3 Rosetta). Fig. 1a displays the schematic design of pET22b plasmid that was present in the appropriate clone. The plasmid contained T7 promoter region responsible for XOR enzyme synthesis coding DNA sequence followed by His tag for better purification with two restriction endonuclease (NdeI and Xho I) cutting sites. The Rosetta cell having successfully transformed recombinant

Conclusions

In conclusion, it can be stated that BSGNP conjugated recombinant XOR enhanced enzymatic activity and storage stability compared to free XOR. BSGNP facilitated rapid electron transfer through the polymeric matrix of the modified electrode. The modified electrode of Au-XOR/fMWCNT-PEDOT/GCE exhibited low detection limits (54.95 nM), high sensitivity (16.075 µA.µM−1.cm−2), and rapid response (4 s) at low oxidation potential (0.446 V) for the detection of xanthine in real samples. It also showed

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This study was funded by Department of Biotechnology, Government of India [No. BT/PR13282/NNT/28/802/2015]. The authors are thankful to Centre for research in Nanoscience and Nanotechnology (CRNN, CU) for SEM, FESEM. TEM facilities.

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