Disodium Phenyl Phosphate Research Articles

Self-Assembled Integrated Nanozyme Cascade Biosensor with Dual Catalytic Activity for Portable Urease Analysis.

In this work, a novel nanozyme (Cu@Zr) with all-in-one dual enzyme and fluorescence properties is designed by simple self-assembly. A nanozyme cascade sensor with disodium phenyl phosphate (PPDS) as substrate was first established by exploiting the dual enzymatic activities of phosphatase and laccase. Specifically, phosphatase cleaves the P-O bond of PPDS to produce colorless phenol, which is then oxidized by laccase and complexed with the chromogenic agent 4-aminoantipyrine (4-AP) to produce red quinoneimine (QI). Strikingly, the NH3 produced by the urease hydrolysis of urea can interact with Cu@Zr, accelerating the electron transfer rate and ultimately leading to a significantly improved performance of the cascade reaction. Moreover, the fluorescence at 440 nm of Cu@Zr is further quenched by the inner filter effect (IFE) of QI. Thus, the colorimetric and fluorescence dual-mode strategy for sensitive urease analysis with LODs of 3.56 and 1.83 U/L was established by the proposed cascade sensor. Notably, a portable swab loaded with Cu@Zr was also prepared for in situ urease detection with the aid of a smartphone RGB readout. It also provides a potentially viable analytical avenue for environmental and biological analysis.

Dynamics of biochemical parameters of blood of cows during the dry period for stall keeping

The period of stable keeping of cows is one of the most difficult periods for the body, as it is the most saturated with stressful influences, and precisely due to the lack of solar insolation and exercise, the action of potentially pathogenic microflora and increased gassing of livestock premises, shortage vitamins, micro- and macroelements in feed. Deep calving animals have metabolic disorders without clinical signs. Such a form pathologies can be detected only through biochemical research.The content of total protein was determined by biuret`s method, albumins ‒ by reaction with bromocresol green, activity alanine and aspartate aminotransferase ‒ by the Reitman-Frenkel method, activity alkaline phosphatase ‒ with disodium phenyl phosphate, acid capacity ‒ by Bolshakov-Belyaev technique, carotene content ‒ by photometric method, content of total calcium ‒ by the complexometric method, the content of carotene ‒ photometric method. The purpose of our work was to study the dynamics of protein and mineral meabolism in cows during the dry period for stall keeping. The object of the study was cows (15 heads) of the Holstein breed with a milk yield of 5‒6 thousand kg per lactation for 8‒9 months of pregnancy. It was found that the total protein of the cows organism decreased in the last months of pregnancy during the dry period from 87.25 ± 1.20 to 81.60 ± 1.20 g/l (Р < 0.05), including the globulin fraction from 51.96 ± 2.08 to 43.75 ± 2.07 g/l (Р < 0.05) and other indicators of protein metabolism changed. The g-globulin fraction significantly decreased from 28.53 g/l to 22.97 g/l (Р < 0.01). In our opinion, this is due to a decrease in the activity of the mother's body defenses to prevent the development of a reaction to the fetal tissue, which is a foreign organism. The activity of aspartate aminotransferase decreased by 8.5 months of pregnancy to 150.26 ± 13.59 nM/s·L (Р < 0.01), and then tended to increase to 187.19 ± 9.21 nM/s·L (Р < 0.05). Such a change in the activity of this enzyme in late pregnancy is associated with an increase in the processes of self-renewal of proteins due to transamination, which can be used more intensively by the fetus. The calcium content decreased by 1.14 times during the dry period. This fact, in our opinion, is associated with the intensive development and formation of the fetal skeleton. This is also evidenced by a significant increase in the activity of alkaline phosphatase (1.29 times, Р < 0.01). Changes in total calcium and inorganic phosphorus were strongly correlated (r = 1.0). The results obtained allow us to point out the causes of the identified deviations and offer recommendations for their elimination and correction of animal feeding.

MicroRNA-136-5p from bone marrow mesenchymal stem cell-derived exosomes facilitates fracture healing by targeting LRP4 to activate the Wnt/β-catenin pathway.

AimsExosomes derived from bone marrow mesenchymal stem cells (BMSCs) have been reported to be a promising cellular therapeutic approach for various human diseases. The current study aimed to investigate the mechanism of BMSC-derived exosomes carrying microRNA (miR)-136-5p in fracture healing.MethodsA mouse fracture model was initially established by surgical means. Exosomes were isolated from BMSCs from mice. The endocytosis of the mouse osteoblast MC3T3-E1 cell line was analyzed. CCK-8 and disodium phenyl phosphate microplate methods were employed to detect cell proliferation and alkaline phosphatase (ALP) activity, respectively. The binding of miR-136-5p to low-density lipoprotein receptor related protein 4 (LRP4) was analyzed by dual luciferase reporter gene assay. HE staining, tartrate-resistant acid phosphatase (TRAP) staining, and immunohistochemistry were performed to evaluate the healing of the bone tissue ends, the positive number of osteoclasts, and the positive expression of β-catenin protein, respectively.ResultsmiR-136-5p promoted fracture healing and osteoblast proliferation and differentiation. BMSC-derived exosomes exhibited an enriched miR-136-5p level, and were internalized by MC3T3-E1 cells. LRP4 was identified as a downstream target gene of miR-136-5p. Moreover, miR-136-5p or exosomes isolated from BMSCs (BMSC-Exos) containing miR-136-5p activated the Wnt/β-catenin pathway through the inhibition of LRP4 expression. Furthermore, BMSC-derived exosomes carrying miR-136-5p promoted osteoblast proliferation and differentiation, thereby promoting fracture healing.ConclusionBMSC-derived exosomes carrying miR-136-5p inhibited LRP4 and activated the Wnt/β-catenin pathway, thus facilitating fracture healing.Cite this article: Bone Joint Res 2021;10(12):744–758.

Inhibitory mechanism of icariin against oxidative stress-induced calcification in aortic vascular smooth muscle cells

This study aimed to observe the inhibitory effect of icariin against oxidative stress-induced calcification in aortic vascular smooth muscle cells(VSMCs) and elucidate the molecular mechanism of icariin in inhibiting endoplasmic reticulum stress(ERS)-mediated atherosclerotic calcification, so as to provide new ideas for exploring the anti-atherosclerotic mechanism of Epimedii Folium. The VSMCs in rat thoracic aorta were subjected to adherent culture and then treated with the complete calcification DMEM containing high glucose and hydrogen peroxide(H_2O_2) for three weeks. The resulting calcified VSMCs were divided into different treatment groups. Icariin was added one week after calcification induction for protecting the VSMCs, whose viability was then detected using cell counting kit-8(CCK-8). Alizarin red-S staining was conducted to observe the calcification degree. The activity of alkaline phosphatase(ALP) in VSMCs was measured using the disodium phenyl phosphate substrate and the calcium content was measured by arsenazo Ⅲ method. The mRNA expression levels of ossification-related factors including osteocalcin(OC), osteopontin(OPN), Runt-related transcription factor 2(Runx2), and type Ⅰ collagen(Col Ⅰa) were detected by real-time PCR. Western blot was carried out to determine the protein expression levels of α-smooth muscle actin(α-SMA), Runx2, activating transcription factor 4(ATF4), and eukaryotic translation initiation factor(eIF)-2α. The results showed that H_2O_2 significantly induced the calcification of VSMCs, increased the ALP activity and calcium content in VSMCs, promoted OC, OPN, Runx2, and Col Ⅰa mRNA expression and Runx2 protein expression, and reduced α-SMA protein expression. The ATF4 protein expression and eIF2α phosphorylation were also elevated significantly. Icariin reversed the calcification of VSMCs induced by H_2O_2, inhibited ALP activity and calcium content in VSMCs, down-regulated the mRNA expression levels of OC, OPN, Runx2 and Col Ⅰa and Runx2 protein expression, and relatively up-regulated the expression of α-SMA. The expression of ATF4 and phosphorylation of eIF2α also declined significantly. All these have demonstrated that icariin inhibited VSMCs calcification by down-regulating the ossification-related factors and lowering ALP activity and calcium content in VSMCs. Besides, the down-regulation of Runx2 expression and the inhibition of ATF4 and eIF2α-mediated cellular calcification pathway in ERS might also be involved in such calcification-suppressing process.

Enzymatic electrochemical biosensor for glyphosate detection based on acid phosphatase inhibition.

A novel enzymatic electrochemical biosensor was fabricated for the indirect detection of glyphosate-based acid phosphatase inhibition. The biosensor was constructed on a screen-printed carbon electrode modified with silver nanoparticles, decorated with electrochemically reduced graphene oxide, and chemically immobilized with acid phosphatase via glutaraldehyde cross-linking. We measured the oxidation current by chronoamperometry. The current arose from the enzymatic reaction of acid phosphatase and the enzyme-substrate disodium phenyl phosphate. The biosensing response is a decrease in signal resulting from inhibition of acid phosphatase in the presence of glyphosate inhibitor. The inhibition of acid phosphatase by glyphosate was investigated as a reversible competitive-type reaction based on the Lineweaver-Burk equation. Computational docking confirmed that glyphosate was the inhibitor bound in the substrate-binding pocket of acid phosphatase and that it was able to inhibit the enzyme efficiently. Additionally, the established method was applied to the selective analysis of glyphosate in actual samples with satisfactory results following a standard method.

Isolation and Activity Determination of Enzyme Phosphatase Secreted by Aspergillus niger

The use of enzymes on industrial scale saves a lot of energy and avoids pollution, thus holding a promise for green and economically sustainable alternative strategies in industrial transformations. Generally, the fungi Aspergillus niger secretes enzymes which can be used in different industries. Thus, coming up with these enzymes in large amounts will definitely result in reduced costs encountered in importing them for industrial use. This study focussed on isolation and activity determination of an enzyme phosphatase secreted by Aspergillus niger. This enzyme can be of great importance in molecular biology industries, particularly for recombinant DNA technology. For this study, pure cultures of Aspergillus niger were used. Aspergillus niger was resuscitated on potato dextrose agar and then subcultured in Adam’s medium, a medium specific for the production of phosphatase. Cells were centrifuged and the filtrate was collected whilst the residue was discarded. The filtrate was expected to contain the crude enzyme phosphatase since Aspergillus niger secretes the extracellular enzyme into the medium. Disodium phenyl phosphate was used as a substrate for the determination of the phosphatase activity. The enzyme activity was determined spectrophotometrically by reading absorbance of phenol formed in the presence of enzyme and the substrate. The concentration of phenol liberated was then used to calculate the enzyme activity expressed in King Armstrong Units (KAU). Further work on enzyme activity determination was done by varying enzyme and substrate concentrations. Results showed that the isolated alkaline phosphatase had activity of 4.0 KAU and 4.5 KAU at 25 ºC and 37 ºC respectively. Acidic phosphatase had activity of 5 KAU and 7 KAU at 25 ºC and 37 ºC respectively. Rate of activity increased upon increasing enzyme concentration and substrate. Thus, Aspergillus niger produces the enzyme phosphatase, however, there is need to induce the production of these enzymes for industrial use.

Improving the flame-retardant efficiency of layered double hydroxide with disodium phenylphosphate for epoxy resin

CuAl layered double hydroxide (CuAl-LDH) was synthesized by co-precipitation. Sodium phenyl phosphate (SPP) and sodium dodecyl sulfate (SDS) are used to modify CuAl-LDH for preparing CuAl-(SPP)LDH and CuAl-(SDS)LDH, which were incorporated into epoxy resin (EP) to obtain EP/CuAl-(SPP)LDH and EP/CuAl-(SPP)LDH nanocomposites. The results indicate that SPP and SDS are intercalated into the interlayers of CuAl-LDH, and CuAl-(SPP)LDH has larger layer spacing than CuAl-(SDS)LDH. The thermal stability and flame-retardant performances of EP/CuAl-(SPP)LDH nanocomposites were better than those of EP/CuAl-(SDS)LDH composites. Compared with those of EP/4CuAl-(SDS)LDH nanocomposites, the peak heat release rate (PHRR) of EP/4CuAl-(SPP)LDH nanocomposites is reduced 25.8% and 55.6%, and peak smoke production rate (PSPR) value of EP/4CuAl-(SPP)LDH nanocomposites is reduced 27.6% and 46.2%, value of EP/4CuAl-(SPP)LDH nanocomposites is reduced 27.6% and 46.2%, respectively. The improved flame retardancy and smoke suppression performances of EP/CuAl-(SPP)LDH nanocomposites were attributed to the combination of copper compounds and SPP, promoting the formation of swollen, continuous and compact char layers on the surface of EP nanocomposites during combustion, eventually restraining the decomposition of EP nanocomposites.

An alkaline phosphatase from Bacillus amyloliquefaciens YP6 of new application in biodegradation of five broad-spectrum organophosphorus pesticides

In recent decades, biodegradation has been considered a promising and eco-friendly way to eliminate organophosphorus pesticides (OPs) from the environment. To enrich current biodegrading-enzyme resources, an alkaline phosphatase (AP3) from Bacillus amyloliquefaciens YP6 was characterized and utilized to test the potential for new applications in the biodegradation of five broad-spectrum OPs. Characterization of AP3 demonstrated that activity was optimal at 40 °C and pH 10.3. The activity of AP3 was enhanced by Mg2+, Ca2+, and Cu2+, and strongly inhibited by Mn2+, EDTA, and L-Cys. Compared to disodium phenyl phosphate, p-nitrophenyl phosphate (pNPP) was more suitable to AP3, and the Vm, Km, kcat, kcat/Km values of AP3 for pNPP were 4,033 U mg−1, 12.2 mmol L−1, 3.3 × 106 s−1, and 2.7 × 108 s−1mol−1L, respectively. Degradation of the five OPs, which included chlorpyrifos, dichlorvos, dipterex, phoxim, and triazophos, was 18.7%, 53.0%, 5.5%, 68.3%, and 96.3%, respectively, after treatment with AP3 for 1 h. After treatment of the OP for 8 h, AP3 activities remained more than 80%, with the exception of phoxim. It can be postulated that AP3 may have a broad OP-degradation ability and could possibly provide excellent potential for biodegradation and bioremediation in polluted ecosystems.

Determination of staphylococcus aureus mycoprotein by using ELISA based on oscillating chemical kinetic detection

Oscillating chemical detection has the advantages of high sensitivity and fast and simple operation, but it is also very vulnerable to many additional species and has low selectivity, poor qualitative analysis and poor detection of macromolecules. This article establishes a Belousov–Zhabotinskii oscillating chemical detection system with good stability and repeatability, which involves ([CuL](ClO4)2) as a catalyst and C3H6O and C6H12O6 as a mixed substrate, as well as an enzyme-linked immunosorbent assay (ELISA) detection system, with disodium phenyl phosphate dihydrate (DPPD) as the key substrate and alkaline phosphatase (ALP) as a catalyst. This article introduces the oscillating chemical detection system into the enzyme-linked immune detection system and establishes the detection method, which combines the advantages of chemical oscillation with those of ELISA, including good selectivity and high specificity, to test the Staphylococcus aureus (S. aureus) protein. There is a good linear proportionality between the concentration of the S. aureus protein and the oscillating amplitude change ΔA, and the detection sensitivity of the method is much higher than that of normal spectrophotometry. The sensitivity of biotin-streptomycin ELISA with a detection limit (3σ) of 1.56 × 10−11 g/mL is better than that of double-antibody sandwich ELISA with a detection limit (3σ) of 1.06 × 10−9 g/mL. Therefore, this method can be applied to the detection of the S. aureus protein antigen.

A novel and sensitive chemiluminescence immunoassay based on AuNCs@pepsin@luminol for simultaneous detection of tetrabromobisphenol A bis(2-hydroxyethyl) ether and tetrabromobisphenol A mono(hydroxyethyl) ether

A novel chemiluminescence immunoassay based on luminol-modified gold nanoclusters (AuNCs@Peps@luminol) was developed for simultaneous detection of tetrabromobisphenol A bis(2-hydroxyethyl) ether (TBBPA-DHEE) and tetrabromobisphenol A mono(hydroxyethyl) ether (TBBPA-MHEE), an important derivative and byproduct of tetrabromobisphenol A (TBBPA), respectively. In the system, alkaline phosphatase (ALP) was labeled on the second antibody (Ab2) for signal amplification. When ALP-Ab2 was captured by antigen-primary antibody (Ab1) complex, disodium phenyl phosphate (PPNa) generated massive phenol under the catalysis of ALP, markedly inhibiting the chemiluminescence intensity of AuNCs@Peps@luminol. Under the optimized conditions, the calculated detection of limit (LOD, 90% inhibition) was 0.078 μg/L for TBBPA-DHEE with a linear range of 0.23–9.32 μg/L, which was lower 9 times than that of conventional ELISA with the same antibody. In addition, our method showed satisfactory accuracy and precision (recoveries, 88.00–113.4%; CV, 2.75–8.14%), it can be applied to systematically investigate the concentration of the trace TBBPA-DHEE and TBBPA-MHEE in environmental and food samples.

A Graphene Quantum Dots-Enzyme Hybrid System for the Fluorescence Assay of Alkaline Phosphatase Activity and Inhibitor Screening.

A graphene quantum dots (GQDs) and horse radish peroxidase (HRP) hybrid system was designed for the sensing of alkaline phosphatase (ALP) activity and inhibitor screening. We found that the photoluminescence (PL) intensity of GQDs could be quenched efficiently in the presence of phenol, H2O2 and HRP. Moreover, ALP could hydrolyze disodium phenyl phosphate (DPP) to produce phenol, and also could result in the photoluminescence quenching of GQDs. The decrease in the PL intensity was linear to the activity of ALP in the concentration range of 0.02 - 0.8 U/L, with a detection limit of 0.008 U/L. The proposed GQDs/HRP hybrid system was successfully applied to ALP determination in human serum samples. The inhibition study was further analyzed, and Na3VO4 (as an ALP inhibitor) showed a clear inhibition effect. The results suggest that the GQDs/HRP hybrid system has good potential applications for the assay of ALP activity and inhibitors screening in related biochemical fields.

Response of soil enzymes and microbial communities to root extracts of the alien Alternanthera philoxeroides

ABSTRACTAlternanthera philoxeroides is an alien invasive species, which can cause substantial damage to the local ecosystem by secreting allelochemicals. The objective of the present study was to appraise the effects of root extracts of the invasive plant Alternanthera philoxeroides on soil enzyme and native microbial community. Urease activity was measured using the phenol sodium hypochlorite colorimetric method, sucrase activity was determined by 3, 5-dinitrosalicylic acid colorimetric method, catalase activity was determined using the KMnO4 titration method and alkaline phosphatase activity was determined by the disodium phenyl phosphate colorimetric method. The soil microbial community was investigated by pyrosequencing of the 16S rRNA gene. Results showed that the alkaline phosphatase, sucrase and urease activities were all significantly lower than that of the control treatment. The structure of the soil microbial community in the treatments with root extracts addition clearly differed from that of the control treatment. The result showed that the relative abundance of microorganism decreased including Burkhholderia, Gp6, Gp1 and Gp4 in the applied treatments at genus level as compared to control treatment. Hence, we inferred that A. philoxeroides could inhibit the growth of native plant species through toxic effects on soil enzyme activities and the microbial community.

Determination of Alkaline Phosphatase in Activated Sludge

Experiment disodium phenyl phosphate as a reaction basic, stromal hydrolysis produce phenol, the amount of phenol indirectly indicates the activity of Alkaline Phosphatase.Experiment by absorption wavelength, activated sludge volume, disodium phenyl phosphate, aluminum sulfate, reagent, temperature and other factors affecting the determination of Alkaline Phosphatase were studied.The experiments optimal reaction conditions initially identified. At 37°C, when activated sludge volume of 5mL, 15mL disodium phenyl phosphate absorbance measured at the maximum value, highest levels of Alkaline Phosphatase. Results show that, 0.25mL reagent optimum dosage for the trial, the best absorption wavelength through fitting the peak-valley is 590nm.

Influence of pH value of mobile phase on phosphopeptide enrichment selectivity under hydrophilic interaction liquid chromatography mode by using Click OEG-CD matrix

The digest of the standard protein alpha-casein was used as the objective to investigate the influence of pH value of mobile phase on phosphopeptide enrichment selectivity by using Click OEG-CD matrix. Disodium phenyl phosphate was primarily selected as the model sample. The results indicated that phosphate group was difficultly to be ionized when the pH value of mobile phase was below its pKa value. The retention of disodium phenyl phosphate on the Click OEG-CD matrix was not based on ion exchange interaction, leading to the weak retention. The influence of pH value (2, 4 and 6) of mobile phase under hydrophilic interaction liquid chromatography (HILIC) mode on phosphopeptide enrichment selectivity was investigated taking the digest of alpha-casein as a sample. Phosphopeptides could not be enriched on the matrix with the pH value of 2; phosphopeptides could be enriched on the matrix and the elution window was narrow with the pH value of 4; phosphopeptides could be enriched on the matrix while the elution window was broad with the pH value of 6. According to the investigation, Click OEG-CD matrix could be better applied in phosphopeptide enrichment.

Oxidatively modified low density lipoprotein promote proliferation and osteodifferentiation of bone marrow mesenchymal stem cells that co-cultured with vascular smooth muscle cells by osteogenic inductor

ObjectiveTo investigate the differentiation action of BMSCs into osteocytes by ox-LDL and osteogenic inductor in indirect co-culture system.MethodsThe proliferation of BMSCs was determined by MTT assay. Expression of OPN mRNA...

Modified phosphate pigments as high performance reinforcing materials for rubber vulcanizates

This study is focusing on the synthesis of novel modified micronized phosphate pigments as reinforcing materials for the vulcanizates of styrene-butadiene rubber (SBR), natural rubber (NR) and their blends. The metal phenyl phosphate pigments were prepared via co-precipitation process from the reaction of equimolar ratios of the disodium phenyl phosphate solution and the water soluble salts of the investigated metals. The white prepared phosphate pigments were introduced in the rubber formulations to replace carbon black the highly common and commercial reinforcing material in rubber vulcanizates. The rheometric characteristics, physico-mechanical properties in addition to the accelerated aging properties of the rubber vulcanizates were investigated, discussed and interpreted in the light of previous studies. The results showed that, phenyl phosphate pigments exercised a great effect on the rheological characteristics (scorch time, cure time…etc.), and achieved high performance and pronounced mechanical properties. The stress and strain at yield and at rupture of the loaded rubber with modified phosphates are better than that loaded with carbon black and Hisil e.g. tensile strength data were (20.0–23.4), 18.01 MPa and 15.05 for rubber blend vulcanizates loaded with 30 phr of modified phosphate pigments, carbon black and Hisil, respectively. These results open a new direction for various applications of non-black rubber vulcanizates.

Ultrasensitive electrochemical immunoassay based on counting single magnetic nanobead by a combination of nanobead amplification and enzyme amplification

An ultrasensitive electrochemical immunoassay method based on counting single magnetic nanobead (MNB) with combined amplification of nanobead and enzyme. Carcinoembryonic antigen (CEA) and MNB were initially immobilized on substrate at 1:1 molar ratio through sandwich immunoreactions. The MNBs were then labeled with alkaline phosphatase (AP) and continuously introduced to capillary with disodium phenyl phosphate (DPP). APs convert DPPs into a phenol zone around each moving MNB, i.e., one CEA. The phenol zones can be electrochemically detected as peaks and counted for CEA quantification. The detection limit for CEA is 5.0 × 10 −17 mol/L.

Ultrasensitive Electrochemical DNA Assay Based on Counting of Single Magnetic Nanobeads by a Combination of DNA Amplification and Enzyme Amplification

An ultrasensitive electrochemical method for determination of DNA is developed based on counting of single magnetic nanobeads (MNBs) corresponding to single DNA sequences combined with a double amplification (DNA amplification and enzyme amplification). In this method, target DNA (t-DNA) is captured on a streptavidin-coated substrate via biotinylated capture DNA. Then, MNBs functionalized with first-probe DNAs (p1-DNA-MNBs) are conjugated to t-DNA sequences with a ratio of 1:1. Subsequently, the p1-DNA-MNBs are released from the substrate via dehybridization. The released p1-DNA-MNBs are labeled with alkaline phosphatase (AP) using biotinylated second-probe DNAs (p2-DNAs) and streptavidin-AP conjugates. The resultant AP-p2-DNA-p1-DNA-MNBs with enzyme substrate disodium phenyl phosphate (DPP) are continuously introduced through a capillary as the microsampler and microreactor at 40 degrees C. AP on the AP-p2-DNA-p1-DNA-MNBs converts a huge number of DPP into its product phenol, and phenol zones are produced around each moving AP-p2-DNA-p1-DNA-MNB. The phenol zones are continuously delivered to the capillary outlet and detected by a carbon fiber disk bundle electrode at 1.05 V. An elution curve with peaks is obtained. Each peak is corresponding to a phenol zone relative to single t-DNA sequence. The peaks on the elution curve are counted for quantification. The number of the peaks is proportional to the concentration of t-DNA in a range of 5.0 x 10(-16) to 1.0 x 10(-13) mol/L.

Immobilization of Enzymes through One-Pot Chemical Preoxidation and Electropolymerization of Dithiols in Enzyme-Containing Aqueous Suspensions To Develop Biosensors with Improved Performance

A protocol of one-pot chemical preoxidation and electropolymerization of monomers (CPEM) in enzyme-containing aqueous suspensions (or solutions) was proposed as a universal strategy for high-activity and high-load immobilization of enzymes to construct amperometric biosensors, which was proven to be effective for the monomer of 1,4-benzenedithiol (BDT), 1,6-hexanedithiol, o-phenylenediamine, o-aminophenol or pyrrole, the preoxidant of K3Fe(CN)6 or p-benzoquinone, and the enzyme of glucose oxidase (GOx) or alkaline phosphatase (AP) to develop GOx-based glucose biosensors or AP-based disodium phenyl phosphate biosensors. As a case examined in detail, a well-dispersed aqueous suspension of the poorly soluble BDT was obtained through its dispersion assisted by ultrasonication and coexisting GOx, which was then subject to chemical preoxidation through adding K3Fe(CN)6, yielding many composites of insoluble BDT oligomers with lots of high-activity enzyme molecules entrapped. Some insoluble composites were then electrochemically codeposited with poly(1,4-benzenedithiol) on an Au electrode, yielding an enzyme film with high-load and high-activity enzyme immobilized. The glucose biosensor prepared here from the CPEM protocol showed much better performance than that from the preoxidant-free conventional electropolymerization (CEP) protocol, with a detection sensitivity increase by a factor of 32 in this case. The GOx-based and AP-based first-generation biosensors developed from the present CPEM protocol all exhibited notably improved performance compared with the analogues from the preoxidant-free CEP protocol. The electrochemical quartz crystal microbalance (EQCM) technique was used to investigate various electrode modification processes. The values of quantity and enzymatic specific activity (ESA) of the immobilized enzymes were evaluated through the EQCM and the conventional UV-vis spectrophotometric method, given that the CPEM protocol notably improved the quantity and the ESA of immobilized enzymes as compared with the preoxidant-free CEP protocol. The proposed CPEM protocol may be interesting in a number of fields, including biosensing, biocatalysis, biofuel cells, bioaffinity chromatography, and biomaterials, and the successful electropolymerization of dithiols in aqueous suspensions (two-phase electropolymerization) may open a new avenue for many monomers that are poorly soluble in neutral aqueous solutions to in situ immobilize biomolecules for bioapplications.

Regulation of extracellular acid phosphatase biosynthesis by culture conditions in entomopathogenic fungusMetarhizium anisopliae strain CQMa102

Metarhizium anisopliae is an imperfect entomopathogenic fungus. Once invading into its host,M. anisopliae needs to absorb basic nutrients such as phosphorus from the host haemolymph. A large number of phosphorylated compounds in haemolymph cannot be directly utilised by the fungal cell and must be hydrolysed into available form by phosphatase before ingested. Aims of this paper were to investigate optimum fermentation conditions for production of acid phosphatase and phosphatase isoenzymes byMetarhizium anisopliae. The optimum fermentation conditions were: glucose, 20 g/l; (NH4)2SO4, 2 g/l; casein, 4 g/l; MgSO4, 0.5 g; KCl, 0.5 g; microelement salt solution, 10 ml; inoculum size, 1×107 spores per 100 ml medium; initial medium pH, 6.0. Under these conditions, the highest total acid phosphatase activity was 3.05 U/ml in 4 days at 27 °C and 160 rpm. Synthesis of the acid phosphatase was repressed by 0.01% inorganic phosphate in culture medium. The spectrum of isoenzymes produced byM. anisopliae varied depending on the phosphorus source employed in the culture. A specific isoform with pI 9.45 was induced by casein, and another isoform of pI 8.21 was induced by phytic acid and disodium phenyl phosphate.