Optimised two-dimensional electrophoresis procedures for the protein characterisation of structural tissues

The protein analysis of structural tissues is typically highly problematic. Amniotic membrane displays unique wound healing and anti-scarring properties; however, little is known concerning its active protein content. The structural nature of amniotic membrane necessitated development and extensive optimisation of the entire two-dimensional (2-D) workflow. Proteins were extracted using powerful solubilisation buffers and analysis carried out using 2-D electrophoresis followed by mass spectrometry (MS) identification. Preservation and processing resulted in prefractionation of soluble from structural and membrane-associated proteins. Enhanced protein solubility was achieved by cysteine blocking using both N,N-dimethylacrylamide (DMA) alkylation and bis(2-hydroxyethyl) disulphide (HED); an alternative procedure for the effective application of HED is demonstrated. The benefits of precipitation and cup-loading versus in-gel rehydration were also assessed, with procedures for the employment of HED with the latter described. Following optimisation, a representative sample 21 proteins were identified from amniotic membrane using MS verify procedures were MS-compatible. Our results demonstrate that techniques for the reproducible separation of proteins from a proteinaceous structural tissue have been optimised. Briefly, proteins are extracted using a thiourea/urea extraction buffer containing carrier ampholytes, dithiothreitol (DTT), and 3-(cyclohexylamino)-1-propanesulfonic acid (CHAPS). After DMA alkylation, proteins were precipitated (using the 2-D clean-up kit from Amersham Biosciences) and resolubilised in extraction buffer containing a lower concentration of DTT. Samples were either cup-loaded onto rehydrated HED-containing strips or rebuffered into HED-containing buffer followed by in-gel rehydration.     

Membrane phase behavior of Escherichia coli during desiccation, rehydration, and growth recovery

The membrane lipid bilayer is one of the primary cellular components affected by variations in hydration level, which cause changes in lipid packing that may have detrimental effects on cell viability. In this study, Fourier transform infrared (FTIR) spectroscopy was used to quantify changes in the membrane phase behavior, as identified by membrane phase transition temperature (T_m), of Escherichia coli during desiccation and rehydration. Extensive cell desiccation (1 week at 20%-40% RH) resulted in an increase in T_m from 8.4 ± 1.7 °C (in undried control samples) to 16.5 ± 1.3 °C. Fatty acid methyl ester analysis (FAME) on desiccated samples showed an increase in the percent composition of saturated fatty acids (FAs) and a decrease in unsaturated FAs in comparison to undried control samples. However, rehydration of E. coli resulted in a gradual regression in T_m, which began approximately 1 day after initial rehydration and plateaued at 12.5 ± 1.8 °C after approximately 2 days of rehydration. FAME analysis during progressive rehydration revealed an increase in the membrane percent composition of unsaturated FAs and a decrease in saturated FAs. Cell recovery analysis during rehydration supported the previous findings that showed that E. coli enter a viable but non-culturable (VBNC) state during desiccation and recover following prolonged rehydration. In addition, we found that the delay period of approximately 1 day of rehydration prior to membrane reconfiguration (i.e. decrease in T_m and increase in membrane percent composition of unsaturated FAs) also preceded cell recovery. These results suggest that changes in membrane structure and state related to greater membrane fluidity may be associated with cell proliferation capabilities.     

Factor affecting the endogenous β-glucuronidase activity in rapeseed haploid cells: how to avoid interference with the Gus transgene in transformation studies

The gus gene is one of the most frequently used reporter genes in transgenic plants. However, this gene can only be used if the selected plant species does not show endogenous GUS activity. Rapeseed (Brassica napus) microspores and microspore-derived embryos (MDEs) were found to exhibit high activity of endogenous β-glucuronidase which interferes with the expression of bacterial β-glucuronidase that was transferred into these tissues by biolistic transformation. In order to eliminate this background activity from rapeseed MDEs, different pHs of the assay buffer (5.8, 7 and 8) with or without methanol in the reaction buffer and incubation of these tissues at different temperatures (24 °C, 38 °C and 55 °C) were investigated. To avoid this problem in microspores, two incubation temperatures (38 °C and 55 °C) at different periods after GUS assay (4, 24 and 48 h) and in the presence of 1 mM potassium ferricyanide and 1 mM potassium ferrocyanide were tested. The endogenous GUS activity was significantly decreased in transformed and untransformed MDEs, when the phosphate buffer was adjusted to pH 8 and 28% methanol in the reaction solution was used. In rapeseed microspores, use of 1 mM potassium ferricyanide and 1 mM potassium ferrocyanide in the reaction buffer enhanced the expression rate of gus transgene rather than endogenous GUS activity where the high levels of gus transgene expression was observed 4 h after histochemical GUS assay. Incubation of rapeseed microspores and MDEs at 55 °C completely eliminated the endogenous GUS activity. In this study, we also examined changes in endogenous GUS activity in rapeseed MDEs at several stages including the globular, heart, torpedo and cotyledonary stages. The level of endogenous GUS activity was increased 4.33 folds in heart embryos, 6.54 folds in torpedo embryos and 8.5 folds in cotyledonary embryos. Furthermore, the level of GUS activity increased 1.72 folds in MDEs of B. napus in 12-h treatment with 2 μM gibberellic acid.     

An improved bioprocess for synthesis of acetohydroxamic acid using DTT (dithiothreitol) treated resting cells of Bacillus sp. APB-6

Acyltransferase activity of amidase from Bacillus sp. APB-6 was enhanced (24 U) by multiple feedings of N-methylacetamide (70 mM) into the production medium. Hyperinduced whole resting cells of Bacillus sp. APB-6 corresponding to 4 g/L (dry cell weight), when treated with 10 mM DTT (dithiothreitol) resulted in 93% molar conversion of acetamide (300 mM) to acetohydroxamic acid in presence of hydroxylamine-HCl (800 mM) after 30 min at 45 °C in a 1 L reaction mixture. After lyophilization, a 62 g powder containing 34% (wt wt⁻¹) acetohydroxamic acid was recovered. This is the first report where DTT has been used to enhance acyltransfer reaction and such high molar conversion (%) of amide to hydroxamates was recorded at 1 L scale.     

A quantitative investigation into the losses of proteins at different stages of a two-dimensional gel electrophoresis procedure

We report the results of a systematic investigation to quantify the losses of protein during a well-established two-dimensional polyacrylamide gel electrophoresis (2-DE) procedure. Radioactively labelled proteins ([(14)C]bovine serum albumin and a homogenate prepared from the liver of a rat that had been injected with [(35)S]methionine) were used, and recovery was quantified by digesting pieces of gel in H(2)O(2) and subjecting the digests to liquid scintillation counting. When samples were loaded onto the first dimension immobilised pH gradient strips by in-gel rehydration, recovery of protein from the strips was 44-80% of the amount of protein loaded, depending on the amount of protein in the sample. Most of the unrecovered protein appeared to have adhered to the reswelling tray. Losses during isoelectric focusing (IEF) were much smaller (7-14%), although approximately 2% of the protein appeared to migrate from sample strips to adjacent blank strips in the focussing apparatus. A further 17-24% of the proteins were lost into the buffers during equilibration prior to running in the second dimension. Losses during the second dimension run and subsequent staining with SYPRO Ruby amounted to less than 10%. The overall loss during 2-DE was reduced by approximately 25% when proteins were loaded onto the IEF strips using sample cups instead of by in-gel rehydration. These extensive and variable losses during the 2-DE procedure mean that spot intensities on 2-DE gels cannot be used to derive reliable, quantitative information on the amounts of proteins present in the original sample.     

Rehydration temperature is critical for metabolic competence and for membrane integrity in active dry yeast (ADY)

Respiration and fermentation were lower in active dry yeast (ADY) rehydrated at 0°C than in ADY rehydrated at 40°C. In agreement with other reports, it was found that membrane permeability increased during rehydration. In addition, ADY rehydrated at 0° did not reseal, even after hours of incubation at 40°C. Using ³²P-nuclear magnetic resonance it was found that the cellular concentration of sugar phosphates, phosphate, pyrophosphate, NADH and ATP were lower in ADY rehydrated at 0°C. In addition, the phospholipid peak had a higher height to broadness ratio at 0°C than at 40°C, suggesting that membranes in the 0° sample were more disordered. The lower fermentation rate in ADY rehydrated at 0° could not be due solely to membrane permeation since addition of cofactors that leaked from these cells did not reactivate fermentation. In cell free extracts or in toluenized cells it was observed that some activities were modified after rehydration at 0°C. In the 40°C sample a lower activity of pyruvate decarboxylase and higher fructose-1,6-bisphosphatase and ATPase activities were detected. As a result, higher levels of ADP and pyruvate were found in the cell. Higher ADP levels could contribute to the higher fermentation rate of the cells rehydrated at 40°C. Enzyme modification might explain the low viability of ADY observed by a plating method, even in cells that were impermeable to a vital dye.Abbreviations ADY Active dry yeast - MES 2(N-morpholino)-ethanesulfonic acid     

Debilitation in conidia of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae and implication with respect to viability determinations and mycopesticide quality assessments

Germination of Beauveria bassiana (Bb) and Metarhizium anisopliae (Ma) conidia determined from a fast-rehydration (FR) protocol were compared to those obtained when dry conidia were subjected to slow rehydration (SR) by holding under high humidity conditions prior to aqueous suspension. Differences in viability estimates obtained using the FR vs. SR protocols increased markedly after conidia were exposed to various stress factors in storage (high a_w, temperature, and O₂ concentrations), with the SR protocol producing higher estimates of viability in all cases. After Bb conidia were stored under moist conditions for 21 days at 25 °C, the SR estimate of viability was >21% greater than the FR estimate. In jars flushed with different O₂ concentrations and stored at 50 °C for 34 days, proportional differences between protocols varied, depending on water activity, from 18-44% in jars flushed with 0% O₂ (100% N₂) to as high as 63-93% when treated with 21-22% O₂. For conidia stored over a broad range of moderate to high temperatures in the absence of O₂, SR-FR differences were ?9% at 25-40 °C but 30% at 50 °C. Germination of stressed Bb and Ma conidia increased substantially when incubation time on the germination substrate was increased from 24 to 72 h, whereas germination of non-stressed conidia showed little change. Conidia debilitated by stress were characterized by hypersensitivity to lethal imbibitional damage (damage that is mitigated by slow rehydration) and slow germination. Viability protocols that may provide more reliable assessments of overall mycopesticide quality are discussed.     

The hydrolytic route to Co-porphyrin-doped SnO2 gas-sensing materials: Chemical study of Co-porphyrin versus Sn(IV) oxide interactions

SnO₂ and SnO₂ + Co-porphyrin solids were prepared from SnCl₄ in propanol and hydrolyzed to sol. Thermal behavior of samples obtained at 110 °C was studied in the 20-600 °C interval by thermal analysis coupled with mass spectrometry for identification of released species. The original samples maintain residual Sn-OR, Sn-OH and Sn-Cl groups up to 350 °C. The sample doped with 1% Co-porphyrin differs for a significant presence of residual Sn-Cl species, accounting for SnCl₄ release in the 300-340 °C range.¹¹⁹Sn solid state NMR analysis reveals disordered SnO₂ species in the sample heated at 250 °C and non-uniform SnO₆ units in the SnO₂ + Co-porphyrin sample at 110 °C, due to persistence of Sn-OR and Sn-OH groups. This complexity is lost at 250 °C. X-ray diffraction analysis confirms all these data. The sensing efficiency of these materials versus alcohols is ascribed to the presence of an open, incomplete SnO₂ structure, which is more pronounced in the Co-porphyrin-doped sample.     

Inherent toxicity of organ preservation solutions to cultured hepatocytes

Organ preservation solutions have been designed to protect grafts against the injury inflicted by cold ischemia. However, toxicity of University of Wisconsin (UW) solution during rewarming has been reported. Therefore, we here assessed the toxicity of UW, histidine-tryptophan-ketoglutarate (HTK), Euro-Collins, histidine-lactobionate (HL), sodium-lactobionate-sucrose and Celsior solutions in cultured hepatocytes under hypothermic (4 °C), intermediate (21 °C) and physiological (37 °C) conditions. Marked toxicity of UW, HTK, HL and Euro-Collins solutions was observed at both 37 and 21 °C. With the exception of UW solution, these solutions also increased cell injury during cold incubation (LDH release after 18 h at 4 °C: HTK 76 ± 2%, Euro-Collins 78 ± 17%, HL 81 ± 15%; control: Krebs-Henseleit buffer 20 ± 6%). Testing of individual components using modified Krebs-Henseleit buffers suggested that histidine and phosphate are responsible for (part of) this toxicity. These potential toxicities should be taken into account in the development of future preservation solutions.     

The biological activity of Humanin analogs correlates with structure stabilities in solution

A single mutation has resulted in large differences in neuroprotective activity of a 24 amino acid Humanin (HN). A mutation of Ser7Ala (S7A-HN) resulted in loss of activity, while a mutation of Ser14Gly (S14G-HN) resulted in about 1000-fold increase. The mechanism of the effects conferred by these mutations have been totally unclear, although our recent structure analysis suggested a possibility of the effect of mutation on the structure stability. Here, we have studied the effects of buffer and temperature on the structure of these three HN peptides. These peptides showed a similar disordered structure at 10 °C in 10 mM phosphate, pH 6.0. They were also similar in phosphate-buffered saline (PBS) as long as the temperature was kept low at 10 °C. However, a large difference was observed in both phosphate buffer and PBS between the peptides, when the temperature was raised to a physiological temperature of 37 °C. While S14G-HN showed small changes in both solutions at 37 °C, the less active HN and inactive S7A-HN showed much larger changes under the identical conditions. In addition, it appeared that structure change at 37 °C was faster for S7A-HN than HN. These results show that the structure stability at 37 °C increases in the order of S7A-HN, HN and S14G-HN, in correlation with their neuroprotective activities.     

Microwave-assisted digestion combined with silica-based spin column for DNA isolation from human bones

A protocol for the extraction of DNA from ancient skeletal material was developed. Bone specimen samples (powder or slice), buffer, pretreatment, and extraction methodologies were compared to investigate the best conditions yielding the highest concentration of DNA. The degree of extract contamination by polymerase chain reaction (PCR) inhibitors was compared as well. Pretreatment was carried out using agitation in an incubator shaker and microwave digestion. Subsequently, DNA from bones was isolated by the classical organic phenol–chloroform extraction and silica-based spin columns. Decalcification buffer for total demineralization was required as well as lysis buffer for cell lysis to obtain DNA, whereas microwave-assisted digestion proved to be very rapid, with an incubation time of 2 min instead of 24 h at an incubator shaker without using lysis buffer. The correction of isolated DNA was detected using real-time PCR with melt curve analysis, which was 82.8 ± 0.2 °C for highly repetitive α-satellite gene region specific for human chromosome 17 (locus D17Z1). Consequently, microwave-based DNA digestion followed by silica column yielded a high-purity DNA with a concentration of 19.40 ng/μl and proved to be a superior alternative to the phenol–chloroform method, presenting an environmentally friendly and efficient technique for DNA extraction.     

Detection of microRNAs in dried serum blots

We observed the preservation of microRNAs in unrefrigerated dried serum blots. Preservation was not adversely affected by drying or storing at 37, 45, or 60 °C instead of room temperature, but it was harmed when blots were dried incompletely before storage. Preservation of microRNAs in serum was not diminished if, instead of being kept frozen at −80 °C, it was stored as dried blots at room temperature for 5 months or at 37 °C for 4 weeks. Thus, dried blots can be a convenient and safer way to save, transport, and store serum for microRNA assays.     

Development and validation of a liquid chromatography/mass spectrometry method for pharmacokinetic studies of OZ78, a fasciocidal drug candidate

Fascioliasis is a zoonotic disease of considerable public health and great veterinary significance and new drugs are needed. OZ78 is a promising fasciocidal drug candidate. In order to support the development of OZ78, including pharmacokinetic (PK) studies an accurate, precise, and selective liquid chromatography/mass spectrometry (LC/MS) method for OZ78 was developed for sheep plasma and validated in accordance with the US Food and Drug Administration Guidance on Bioanalytical Method Validation. Protein precipitation was used for sample clean up. Separation was performed through a Phenomenex C8(2) analytical column (50.0 mm × 2.0 mm, 5 μm) with a mobile phase of acetonitrile (buffer B) and 5 mM ammonium formate (buffer A) at a flow-rate of 0.3 mL/min and a gradient from 20% to 95% acetonitrile. The mass spectrometer was operated under selected ion monitoring, and orifice voltage set to −4.1 kV and ion spray temperature to 400 °C. Nitrogen was used as a nebulizer, curtain, and collision gas. OZ78 was monitored at 321.4 m/z (deprotonated parent compound, M-). The validated linear dynamic range was between 156.25 ng/mL and 5 μg/mL and the achieved correlation coefficient (r²) was greater than 0.99. The validation results demonstrated that the developed LC/MS method is precise, accurate, and selective for the determination of OZ78 in sheep plasma. The method was successfully applied to the evaluation of the PK profile of OZ78 in sheep.     

Dissociation and reduction of covalent β-lactoglobulin–quinone adducts by dithiothreitol,tris(2-carboxyethyl)phosphine,or sodium sulfite

Covalent protein–phenol adducts, generated by reaction of protein nucleophiles with quinones, have recently attracted increased attention because the interactions change the functionality and physicochemical properties of proteins in biological and food systems. The formation of such covalent adducts between β-lactoglobulin (β-LG) and the quinone of 4-methylcatechol, 4-methylbenzoquinone (4MBQ), and subsequent reduction by dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or sodium sulfite was investigated by mass spectrometry. The results showed that 19.0 ± 8.8% of β-LG reacted with 4MBQ when present in equimolar ratio at 20 °C (pH 8.0) to yield the protein–phenol adduct (β-LG-Q). Following treatment with sulfite, DTT, or TCEP, 75, 68, or 36%, respectively, of the formed β-LG-Q adduct dissociated. Different reaction mechanisms were proposed for the reduction of β-LG and β-LG-Q by each of the reducing agents. These results show that on reductive sample preparation for analysis of protein samples, not only are protein polymers formed through oxidative disulfide bonds reduced into the individual protein constituents but also a large part of any protein–phenol adducts present will dissociate and, thus, give a false picture of the level of protein–protein interactions that have occurred in the sample.     

Numerical simulation of the effect of superparamagnetic nanoparticles on microwave rewarming of cryopreserved tissues

In this study, the microwave rewarming process of cryopreserved samples with embedded superparamagnetic (SPM) nanoparticles was numerically simulated. The Finite Element Method (FEM) was used to calculate the coupling of the electromagnetic field and the temperature field in a microwave rewarming system composed of a cylindrical resonant cavity, an antenna source, and a frozen sample phantom with temperature-dependent properties. The heat generated by the sample and the nanoparticles inside the electromagnetic field of the microwave cavity was calculated. The dielectric properties of the biological tissues were approximated using the Debye model, which is applicable at different temperatures. The numerical results showed that, during the rewarming process of the sample phantom without nanoparticles, the rewarming rate was 29.45 °C/min and the maximum temperature gradient in the sample was 3.58 °C/mm. If nanoparticles were embedded in the sample, and the cavity power was unchanged, the rewarming rate was 47.76 °C/min and the maximum temperature gradient in the sample was 1.64 °C/mm. In the presence of SPM nanoparticles, the rewarming rate and the maximum temperature gradient were able to reach 20.73 °C/min and 0.68 °C/mm at the end of the rewarming under the optimized cavity power setting, respectively. The ability to change these temperature behaviors may prevent devitrification and would greatly diminish thermal stress during the rewarming process. The results indicate that the rewarming rate and the uniformity of temperature distribution are increased by nanoparticles. This could be because nanoparticles generated heat in the sample homogeneously and the time-dependent parameters of the sample improved after nanoparticles were homogeneously embedded within it. We were thus able to estimate the positive effect of SPM nanoparticles on microwave rewarming of cryopreserved samples.     

Staphylococcal enterotoxin A: Partial unfolding caused by high pressure or denaturing agents enhances superantigenicity

The effect of transient exposure of Staphylococcus aureus enterotoxin A (SEA) to high pressure and/or denaturing agents was examined by assessing the toxin superantigenicity and immunoreactivity, and by monitoring pressure-induced changes in fluorescence emission spectra. Pressurization of SEA at 600 MPa and 45 °C in Tris–HCl buffer (20 mM, pH 7.4) resulted in a marked increase in both T-cell proliferation (superantigenicity) and immunoreactivity. In opposite, pressurization at 20 °C did not change significantly SEA superantigenicity and immunoreactivity, indicating some toxin baro-resistance. Exposure of SEA to 8 M urea at atmospheric pressure or at 600 MPa and 20 °C, also led to a marked increase of superantigenicity (but not of immunoreactivity). In contrast, exposure of SEA to sodium-dodecylsulfate (30 mM) led to an increase of immunoreactivity with some effect on superantigenicity after pressurization at 45 °C only. High pressure up to 600 MPa induced spectral changes which at 20 °C were fully reversible upon decompression. At 45 °C, however, a sharp break of the centre of spectral mass mainly due to tryptophan residues was observed at 300 MPa, and irreversible spectral changes mainly related to tyrosine residues subsisted after pressure release, indicating a marked protein conformational transition. Urea 8 M further increased SEA structural changes at 600 MPa and 20 °C. These results indicate that SEA, under a combination of high pressure and mild temperature, as well as in the presence of urea, partly unfolds to a structure of strongly increased T-cell proliferative ability.     

Amphiphilic and thermosensitive copolymers based on pullulan and Jeffamine: Synthesis, characterization and physicochemical properties

The synthesis of thermosensitive copolymers based on pullulan and polyether amine was performed in water using a water-soluble carbodiimide and N-hydroxysuccinimide as activators. Jeffamine^® M2005 was chosen as a polyether to impart thermosensitive character to the copolymer. Pullulan was modified into carboxymethylpullulan, to bring carboxylate groups to the polysaccharide so as to further the grafting reaction. The copolymers were characterized by FT-IR, ¹H NMR spectroscopy and molecular weights measurements (by SEC coupled with MALS/DRI/Viscometer lines). The thermosensitive behaviour of CMP-g-M2005 copolymers was studied by fluorescence spectroscopy of pyrene, by rheometry and microDSC measurements. The sol-gel transition temperature was found dependent on the solvent, the grafting degree of M2005 and the concentration of the copolymer. For example it was 35 °C in water, 28 °C in acid buffer (0.1 M, pH 5.4) and 26 °C in saline phosphate buffer (0.15 M, pH 7.4) for a grafting degree of 0.20 at a concentration of 5 wt%.     

Effect of storage temperature on survival and infectivity of Steinernema rarum (OLI strain) (Rhabditida: Steinernematidae)

Nematode strains of the entomopathogenic family Steinernematidae differ in their ability to infect insects at different temperatures. Survival and infectivity of infective juveniles (IJs) of Steinernema rarum (OLI) were studied after their storage at 23 ± 2 °C and at 5 ± 1 °C. Survival at 23 ± 2 °C was always above 95%. At 5 ± 1 °C, survival decreased at week 5, but infectivity did the same after week 2. Unlike other steinernematids, both infectivity and survival of IJs would be higher for S. rarum (OLI) when stored at 23 ± 2 °C.     

Gelation of high-methoxy pectin by enzymic de-esterification in the presence of calcium ions: a preliminary evaluation

Cohesive gels have been obtained by de-esterification of 1.0 wt % high-methoxy citrus pectin (degree of esterification ≈ 68%) in the presence of Ca²⁺ cations, using a commercial preparation (NovoShape) of fungal methyl esterase cloned from Aspergillus aculeatus. A convenient rate of network formation (gelation within ∼30 min) was achieved at an enzyme concentration of 0.2 PEU/g pectin. At a Ca²⁺-concentration of 40 mM and incubation temperature of 20 °C, severe syneresis (>7% of sample mass) was observed, but release of fluid decreased with decreasing concentration of Ca²⁺ and increasing temperature of incubation, becoming undetectable for 10 mM Ca²⁺ at 30 °C. Under these conditions, progressive development of solid-like character (storage modulus, G′) was observed during 160 min of enzymic de-esterification, and the mechanical spectrum recorded at the end of the incubation period had the form typical of a biopolymer gel. On subsequent heating to 70 °C, dissociation of the gel network (sigmoidal reduction in G′ and G″) was observed. At or above the midpoint temperature of this melting process (∼50 °C), there was no indication of gel formation on enzymic de-esterification (at 50 or 60 °C). At lower temperatures (20, 30 and 40 °C), the rate of gelation (assessed visually) showed no systematic increase as the incubation temperature was increased towards the temperature-optimum of the enzyme (∼50 °C). This unexpected behaviour is attributed to competition between faster de-esterification and slower formation of Ca²⁺-induced ‘egg-box’ junctions.     

Fabrication of polyphenol biosensor based on laccase immobilized on copper nanoparticles/chitosan/multiwalled carbon nanotubes/polyaniline-modified gold electrode

A high-performance amperometric polyphenol biosensor was developed, based on covalent immobilization of Ganoderma sp. laccase onto copper nanoparticles (CuNP's)/chitosan (CHIT)/carboxylated multiwalled carbon nanotube (cMWCNT)/polyaniline (PANI)-modified gold (Au) electrode. The CuNP's and cMWCNT had a synergistic electrocatalytic effect in the matrix of CHIT. The biosensor showed optimum response at pH 6.0 (0.1 M acetate buffer) and 35 °C, when operated at 50 mV s⁻¹. The biosensor exhibited excellent sensitivity (the detection limit was down to 0.156 μM for guaiacol), fast response time (less than 4 s) and wide linear range (from 1 to 500 μM). Analytical recovery of added guaiacol was 96.40-98.46%. Within batch and between batch coefficients of variation were <2.6% and <5.3%, respectively. The enzyme electrode was used 300 times over a period of 7 months, when stored at 4 °C.