Zhi Shan

BSA-stabilized Au clusters as peroxidase mimetics for use in xanthine detection

In this paper, we demonstrated that bovine serum albumin (BSA) stabilized Au clusters exhibited highly intrinsic peroxidase-like activity. Unlike nature enzymes, the BSA-Au clusters have strong robustness and can be used over a wide range of pH and temperature. Because of ultra-small size, good stability and high biocompatibility in water solution compare with other kinds of nanoparticles as peroxidase mimetics, such as Fe(3)O(4), FeS or graphene oxide, it is more competent for bioanalysis. Furthermore, we make use of the novel properties of BSA-Au clusters as peroxidase mimetics to detect H(2)O(2). The as-prepared BSA-Au clusters were used to catalyze the oxidation of a peroxidase substrate 3,3,5,5-tetramethylbenzidine (TMB) by H(2)O(2) to the oxidized colored product, and which provides a colorimetric detection of H(2)O(2). As low as 2.0 × 10(-8)M H(2)O(2) could be detected with a linear range from 5.0 × 10(-7) to 2.0 × 10(-5)M via this method. More importantly, a sensitive and selective method for xanthine detection was developed using xanthine oxidase (XOD) and the as-prepared BSA-Au clusters. The detection limit of this assay for xanthine was 5 × 10(-7)M and the proposed method was successfully applied for the determination of xanthine in urine and human serum sample.

PCR-ready human DNA extraction from urine samples using magnetic nanoparticles

Urine-derived human genomic DNA (gDNA) has wide application in a variety of disciplines including clinical medicine, sports, and forensic science. We describe a novel method for gDNA extraction from urine samples using carboxylated magnetic nanoparticles (CMNPs) as solid-phase adsorbents. Sedimentation associated with freezing of urine samples significantly reduces cell capture by CMNPs. However, the addition of 10 mM EDTA and subsequent pH modification (pH 6.0-7.1) can re-dissolve urine sediments. Purified gDNA ranged from around 0.1 kb to more than 23 kb. PCR using specific primers targeting K-ras, GAPDH, CYP3A4 and GDF5 amplified 100% of varying sized gene fragments, verifying the high quality of the isolated DNA. Successful PCR amplifications using DNA isolated from urine samples as small as 50 μl were demonstrated. Enrichment of urine cells and subsequent adsorption of DNA can be achieved with the same CMNPs, greatly simplifying extraction procedures. The CMNP gDNA extraction technique proved to be simple, rapid, sensitive and environmentally friendly, with application for routine laboratory use and potentially within automated urine extraction platforms.

Improving Phytase Enzyme Activity in a Recombinant phyA Mutant Phytase from Aspergillus niger N25 by Error-Prone PCR

The mutant acid phytase (phyAm) gene was modified by random mutagenesis to improve enzymatic activity by using an error-prone PCR (ep-PCR) strategy. The mutated gene was linearized and inserted into plasmid vector pPIC9K and transformed by electroporation into Pichia pastoris GS115. A single transformant, PP-NPep-6A, showing the strongest phytase activity from among the 5,500 transformants, was selected for detailed analyses. Southern blot analysis of the mutant yeast transformant showed that phyAep gene was integrated into the chromosome genome through single crossover with one copy of phyA. The kinetic parameters indicated that the mutant one showed 61% higher specific activity and 53% lower km value than that of PP-NPm-8 (P < 0.05). In addition, the overall catalytic efficiency (kcat/km) of the mutant one was 84% higher (P < 0.05) than that of PP-NPm-8. Nine bases were altered in the mutant sequences, which resulted in three amino acid changes, namely, Glu156Gly, Thr236Ala, and Gln396Arg. The structural predictions indicated that the mutations generated by ep-PCR somehow reorganized or remodeled the active site, which could lead to increasing catalytic efficiency.

Promoting DNA loading on magnetic nanoparticles using a DNA condensation strategy.

Maximizing DNA loading on magnetic nanoparticles (MNPs) is crucial for their successful utilization in gene transfer, DNA isolation, and bio-analytical applications. This enhancement is typically achieved by altering particle size and surfaces as well as charge density and ionic strength. We demonstrate a novel route for promoting DNA loading on amino-modified silica-coated magnetic nanoparticles (ASMNPs) by prior condensation of elongated DNA to a compact globule before adsorption. The enhanced DNA-loading capacity, as demonstrated by a reduction in the number of ASMNPs needed to achieve complexation, was presumably due to the elimination of DNA wrapping around nanoparticles and substantially reduced electrostatic interactions of DNA with nanoparticles because the compacted DNA globule conformation decreases its exposed surface charge. The maximum loading capacity of ASMNPs for condensed DNA was 4.4 times greater than that for elongated coiled DNA, achieving the highest ever reported value of 385 μg mg(-1). Practical applications for plasmid DNA isolation from cleared lysate confirmed the reliability of the proposed method.

Application of magnetic hydroxyapatite nanoparticles for solid phase extraction of plasmid DNA.

We developed a facile method for plasmid DNA (pDNA) extraction from crude Escherichia coli lysate using magnetic hydroxyapatite nanoparticles (MHapNPs) in the presence of polyethylene glycol (PEG)/NaCl. DNA condensation induced by PEG/NaCl is a prerequisite for achieving pronounced DNA recovery. The quality and quantity of MHapNP-purified pDNA under optimal binding buffer conditions (0.5 volume of 20% PEG 8000/2M NaCl) were comparable to those obtained using organic solvents or commercial kits. This MHapNP technique is rapid, simple, cost-effective, and environmentally friendly and has the potential to extract DNA from other cell lysates.

Temperature-dependent selective purification of plasmid DNA using magnetic nanoparticles in an RNase-free process

Carboxyl group-functionalized magnetic nanoparticles were used to develop an RNase-free method for plasmid DNA (pDNA) purification directly from RNA-containing crude Escherichia coli lysates. This method takes advantage of differing adsorption behaviors of pDNA and RNA onto magnetic nanoparticle surfaces at different temperatures. Pure pDNA can be isolated between 70 and 80°C without sacrificing DNA quality and quantity, as evidenced by comparison with that obtained using organic solvents or commercial kits. This RNase-free method is rapid, simple, cost-effective, and environmentally friendly, and it can be easily scaled up for the production of pharmacological-grade pDNA.

Chemisorption Mechanism of DNA on Mg/Fe Layered Double Hydroxide Nanoparticles: Insights into Engineering Effective SiRNA Delivery Systems

Layered double hydroxide nanoparticles (LDH NPs) have attracted interest as an effective gene delivery vehicle in biomedicine. Recent advances in clinic trials have demonstrated the efficacy of Mg/Fe LDHs for hyperphosphatemia treatment, but their feasibility for gene delivery has not been systematically evaluated. As a starting point, we aimed to study the interaction between oligo-DNA and Mg/Fe LDH NPs. Our investigation revealed the chemisorption mechanism of DNA on Mg/Fe LDH surfaces, wherein the phosphate backbone of the DNA polymer coordinates with the metal cations of the LDH lattice via the ligand-exchange process. This mechanistic insight may facilitate future gene delivery applications using Mg/Fe LDH NPs.

Research on homology modeling, molecular docking of the cellulase and highly expression of the key enzyme (Bgl) in Pichia pastoris

Cellulose is the most abundant and renewable biological resource on earth. As nonrenewable resources are becoming scarce, cellulose is expected to become a major raw material for food, energy, fuel and other products. 1,4-β-glucosidase (Bgl), as a kind of cellulose, can be degraded cellulose into industrial available glucose. In this study, we constructed mutants of Bgl with enhanced activity based on homology modeling, molecular docking, and the site-directed mutagenesis of target residues to modify spatial positions, steric hindrances, or hydrophilicity/hydrophobicity. On the basis of the high-activity mutations were got (N347S and G235 M) by using site-directed mutagenesis and screening methods and introduced in the Pichia pastoris expression system, the enzymatic properties of mutant enzymes were analysed. Assays of the activity of the purified Bgl revealed that the two mutants exhibited increased activity. The pPICZαA-G235 M and pPICZαA-N347S mutants exhibited a >33.4% and 44.8% increase in specific activity respectively, with similar pH, temperature and metal ion requirements, compared to wild-type Bgl. These findings would be good foundation for improving production properties of Bgl in the future.

Applications of Immobilized Metal Affinity Magnetic Nanoparticles for Protein Separation/Purification

Immobilized metal ion affinity chromatography(IMAC) has been widely applied to the separation and purification of protein.And when replacing the solid matrix part of the IMAC with magnetic nanoparticles(MNPs),it makes immobilized metal affinity magnetic nanoparticles(IMAN).The introduction of MNPs makes IMAN having more extensive application value than IMAC.IMAN has been widely used in the separation and purification of protein in recent years.This review first addresses the composition of IMAN,then summarizes its principle,application and affecting factors in the separation and purification of protein.Finally,the trends and future perspectives in this research area are outlined.