Modelling of growth kinetics of isolated Pseudomonas sp. and optimisation of parameters for enhancement of xanthine oxidoreductase production by statistical design of experiments.

This report presents the substrate inhibitory effect of xanthine (XN) on microbial growth and optimisation of effective parameters to achieve high enzyme activity of xanthine oxidoreductase (XOR) through statistical design. Three efficient isolated strains (Pseudomonas aeruginosa CEBP1 and CEBP2, Pseudomonas sp. CEB1G) were screened for growth kinetic studies. Substrate inhibitory models (eg. Aiba, Edward) could explain the growth kinetics of CEBP1, CEBP2 and CEB1G very well with various initial [XN] (S0), e.g., 0.1-35 g L-1. Highest XOR activity was obtained at stationary phase when biomass yield was high. Highest catalytic efficiency (kcat/KM) of XOR was obtained by CEBP1 at optimum specific growth rate of 0.082 h-1 and biomass yield of 0.196 g g-1 at S0 = 5 g L-1. The effects of S0, pH and temperature were studied by Box-Behnken experimental design to evaluate the interactive effects of the significant variables influencing XOR production by CEBP1. ANOVA with high correlation coefficient (R2 > 0.99) and lower 'Prob > F'value (< 0.05) validated the second order polynomial model for the enzyme production. The highest XOR activity of 31.2 KU min-1 mg-1 was achieved by CEBP1 under optimised conditions (35 °C; S0=5 g L-1; pH = 7.0) as compared to any report in literature. A sevenfold substrate affinity of the enzyme was observed after purification.

Impedance nanobiosensor based on enzyme-conjugated biosynthesized gold nanoparticles for the detection of Gram-positive bacteria.

In this report, gold nanoparticles (GNPS) were synthesized using cell-free extracts of seven different isolates, namely, Pseudomonas aerogenosa CEBP2, Pseudomonas sp. CEBP1, Pseudomonas pseudoalcaligenes CEB1G, Acinetobactor baumani CEBS1, Cuprividus sp. CEB3, Micrococcus luteus CUB12, and Pandoraea sp. CUB2S. The spectroscopic (UV-vis, FTIR, DLS, XRD, EDS) and microscopic (FESEM, TEM) results confirm the reduction of Au3+ to Au0 in the presence of biomolecules having reducing as well as self-stabilizing activity. In this green synthesis approach, the average particle size of biosynthesized GNPS might vary (4-60 nm) depending on the bacterial species, pH of the media, incubation time, and temperature. In this study, GSH-modified BSGNPs (Au-GSH) have shown antimicrobial activity with better stability against Gram-positive bacteria. After conjugation of lysozyme with Au-GSH (lyso@Au-GSH), the zone of inhibition was enhanced from 12 to 23 mm (Au-GSH). The TEM study shows the spherical GNP (16.65 ± 2.84) turns into a flower-shaped GNP (22.22 ± 3.12) after conjugation with lysozyme due to the formation of the protein corona. Furthermore, the nanobioconjugate (lyso@Au-GSH) was immobilized with Nafion on a glassy carbon electrode to fabricate a label-free impedance biosensor that is highly sensitive to monitor changes in the transducer surface due to biomolecular interactions. The uniquely designed biosensor could selectively detect Gram-positive bacteria in the linear range of 3.0 × 101-3 × 1010 cfu mL-1 with RE <5%. The proposed simplest biosensor exhibited good reproducibility (RSD = 3.1%) and excellent correlation (R2 = 0.999) with the standard plate count method, making it suitable for monitoring Gram-positive bacterial contamination in biofluids, food, and environmental samples.

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

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 ofPseudomonas aerogenosastrain CEBP1 wascloned to obtainpurifiedenzyme through affinity chromatography. fMWCNTdoped PEDOTwas electrodeposited on the working electrodeto 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-2for the quantification of xanthine in biological samples such as blood, tissue, urine. The applicability of thebiosensor was validatedby comparing the sensing results for the real biological fluidic solutions with HPLC data (RE = 0.5-3.1%).

A nonenzymatic reduced graphene oxide-based nanosensor for parathion.

Organophosphate-based pesticides (e.g., parathion (PT)) have toxic effects on human health through their residues. Therefore, cost-effective and rapid detection strategies need to be developed to ensure the consuming food is free of any organophosphate-residue. This work proposed the fabrication of a robust, nonenzymatic electrochemical-sensing electrode modified with electrochemically reduced graphene oxide (ERGO) to detect PT residues in environmental samples (e.g., soil, water) as well as in vegetables and cereals. The ERGO sensor shows a significantly affected electrocatalytic reduction peak at -0.58 V (vs Ag/AgCl) for rapid quantification of PT due to the amplified electroactive surface area of the modified electrode. At optimized experimental conditions, square-wave voltammetric analysis exhibits higher sensitivity (50.5 µA·µM-1·cm-2), excellent selectivity, excellent stability (≈180 days), good reproducibility, and repeatability for interference-free detection of PT residues in actual samples. This electrochemical nanosensor is suitable for point-of-care detection of PT in a wide dynamic range of 3 × 10-11-11 × 10-6 M with a lower detection limit of 10.9 pM. The performance of the nanosensor was validated by adding PT to natural samples and comparing the data via absorption spectroscopy. PT detection results encourage the design of easy-to-use nanosensor-based analytical tools for rapidly monitoring other environmental samples.

Interference-free electrocatalysis of p-chloro meta xylenol (PCMX) on uniquely designed optimized polymeric nanohybrid of P(EDOT-co-OPD) and fMWCNT modified glassy carbon electrode.

p-Chloro-meta-Xylenol (PCMX) is an environmentally hazardous phenolic compound having biocidal and antiseptic activity. Very few research publications addressed monitoring this contaminant. This paper presents a rapid sensing system to quantify it in waste water samples. The electrochemical activity of PCMX was exploited through a unique polymeric nanocomposite modified transducer for its quantification. Poly[(3,4-Ethylenedioxythiophene)-co-(o-phenylenediamine)] [P(EDOT-co-OPD)] was deposited through one-step electropolymerization technique on the glassy carbon electrode (GCE) modified by functionalized multi-wall carbon nanotubes (fMWCNTs). An optimized combination of these constituents was evaluated using response surface methodology (RSM) based Box-Behnken experimental design. This maximized the response for PCMX using differential pulse voltammetry (DPV). The sensing matrix was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The structural and morphological study of the modified film was conducted by Fourier transform-infrared spectroscopy (FT-IR), Raman spectroscopy, scanning electron microscopy (SEM), and field emission scanning electron microscope (FESEM). The anodic peak current could be read from a wide range of 0.5-225 µM calibration curve with a detection limit of 0.2545 µmol L-1. Interestingly this work did not use any biomaterial in the modification but achieved interference-free response with excellent selectivity, sensitivity (0.4668 µA µM-1 cm-2), reproducibility (RSD = 2.2%), and repeatability. The sensing platform showed good stability (85.7%) of 3 months even after 150 times repetitive use. Its applicability for real samples was established by good correlation with standard methods.

A simple electrochemical approach to fabricate functionalized MWCNT-nanogold decorated PEDOT nanohybrid for simultaneous quantification of uric acid, xanthine and hypoxanthine.

Medical diagnostics and detection of food spoilage require estimation of hypoxanthine (HX), xanthine (XN), and uric acid (UA). A selective sensing platform has been proposed for simultaneous detection of all these species. Functionalized multi-walled carbon nanotube (fMWCNT) stabilized nanogold decorated PEDOT:TOS polymeric nanocomposite (Au-PEDOT-fMWCNT) was synthesized through rapid one-step electropolymerization to enhance conductivity and active surface area by several folds. Electrochemical activities of the proposed sensing platform were analyzed by cyclic voltammetry (CV) and differential pulse voltammetry (DPV), electrochemical impedance spectroscopy (EIS). Analyses through SEM, FESEM and TEM were performed to explore the surface morphology and elemental analysis of the polymeric nanohybrid was investigated by XPS, Raman, FTIR, XRD spectroscopy. Electro-catalysis of UA, XN and HX occurred at low oxidation potentials i.e. 0.082, 0.463 and 0.808 V, respectively in the optimized conditions. The uniquely designed simple, interference free Au-PEDOT-fMWCNT/GCE sensor exhibited high selectivity, good reproducibility, reusability (∼180 times) and stability (∼3 month) with excellent sensitivity of 1.73, 14.31 and 3.82 µA µM-1 cm-2 for UA, XN and HX, respectively. The sensor exhibited linear ranges of detection as 0.1-800, 0.05-175 and 0.1-150 µM with detection limits of 199.3, 24.1 and 90.5 nM for quantification of UA, XN and HX respectively. The performance of the proposed sensor was validated by addition of UA, XN and HX in human serum, urine and fish samples by comparing to those using HPLC. The results indicated good applicability of the proposed sensor for simultaneous detection of UA, XN, HX in real biological fluids.