Modulation of DNA Polymerases with Gold Nanoparticles and their Applications in Hot‐Start PCR

A new gold‐nanoparticle (AuNP)‐based strategy to dynamically modulate the activity of DNA polymerases and realize a hot‐start (HS)‐like effect in the polymerase chain reaction (PCR) is reported, which effectively prevents unwanted nonspecific amplification and improves the performance of PCRs. A high‐fidelity Pfu DNA polymerase is employed as the model system. Interestingly, AuNPs inactivate the polymerase activity of Pfu at low temperature, thus resembling an antibody‐based HS PCR. This inhibition effect of AuNPs is demonstrated for the preamplification polymerization activity of the PCR, which largely suppresses nonspecific amplification at temperatures between 30 and 60 °C and leads to highly specific and sensitive PCR amplification with Pfu. Significantly, the fidelity of Pfu is not sacrificed in the presence of AuNPs. Therefore, this AuNP‐based HS strategy provides a straightforward and potentially versatile approach to realize high‐performance PCR amplification.     

Water chemistry and processing effects on the corrosion degradation of copper tubing in cooling water systems

Specimens of copper tubing from an industrial scale chiller were subjected to a complete corrosion failure analysis. Nondestructive inspection of the tubing indicated substantial corrosion damage and provided the impetus for the corrosion analysis. By application of the typical methods encountered in metallurgical failure investigations, as well as additional chemical analysis techniques, the most probable cause of failure was identified to be a change in the water chemistry during the service life of the tubes. Additional items that may have contributed to the failure include the geometric design of the tube and the post-manufacture cleaning process prior to service. Recommendations were made regarding water treatment and the requirements for any replacement tubes. In addition to discussion of the specific failure(s) investigated, notes regarding the general procedures for corrosion failure analysis are also presented.     

Rapid,cost-effective DNA quantification via a visually-detectable aggregation of superparamagnetic silica-magnetite nanoparticles

DNA and silica-coated magnetic particles entangle and form visible aggregates under chaotropic conditions with a rotating magnetic field, in a manner that enables quantification of DNA by image analysis. As a means of exploring the mechanism of this DNA quantitation assay, nanoscale SiO2-coated Fe304 （Fe3O4@SiO2） particles are synthesized via a solvothermal method. Characterization of the particles defines them to be -200 nm in diameter with a large surface area （141.89 m2/g）, possessing superparamagnetic properties and exhibiting high saturation magnetization （38 emu/g）. The synthesized Fe3O4@SiO2 nanoparticles are exploited in the DNA quantification assay and, as predicted, the nanoparticles provide better sensitivity than commercial microscale Dynabeads for quantifying DNA, with a detection limit of 4 kilobase-pair fragments of human DNA. Their utility is proven using nanoparticle DNA quantification to guide efficient polymerase chain reaction （PCR） amplification of short tandem repeat loci for human identification.     

重组质粒pcDNKCpG在动物体内分布、残留及水平传播检测

通过肌肉注射的途径将重组质粒pcDNKCpG导入到鸡和猪体内,提取不同时间段的动物组织DNA,特异性的PCR扩增结果提示重组质粒pcDNKCpG主要分布在鸡和猪的血液和注射部位肌肉中,残留时间分别不超过16天和8天;凝胶电泳回收的组织DNA经过酶切、电泳和转膜后的Southern检测表明,重组质粒pcDNKCpG没有整合到鸡和猪的任何组织DNA中去;地高辛标记的CpG核酸探针的Southern检测结果显示重组质粒pcDNKCpG没有整合入宿主的基因组中;特异性的PCR扩增结果证实重组质粒pcDNKCpG没有转移到其他的环境微生物中去.因此,重组质粒pcDNKCpG具有良好的生物安全性.     

Accumulation of amplified target DNAs using thiol/biotin labeling, S1 nuclease, and ferrocene-streptavidin-magnetic system and a direct detection of specific DNA signals with screen printed gold electrode

Combinations of PCR-based amplification platform using 5′ thiolated and biotinylated specific primers, S1 nuclease-PCR products treatment, ferrocene-streptavidin (Fc-Stv)-magnetic binding for DNA accumulation, and screen printed gold electrode for the DNA allocation, were applied to Hoechst 33258-induced DNA aggregation and signals induction system for direct signals detection and DNA quantification in food samples. Thiolated and biotinylated at each 5′ terminus enabled DNA purification through S1 nuclease treatment for primers and non-specific DNA elimination and enabled DNA trapping with a ferrocene-streptavidin-magnetic system. This facilitated the accumulation of target DNAs at higher concentration, resulting in enhanced signals. After allocation of DNA on the surface of gold electrode via thiol binding, intensity of DNA signals through these treatments could be measured directly after being induced by Hoechst 33258. Wider amplitude changes in anodic current peaks between negative and positive samples (increasing from 3.70 to 10.10 μA) compared with those applied with no treatment combinations (decreasing from 3.92 to 1.23 μA) were observed. This enhancement of the signals allowed a greater efficiency of DNA quantification. When this combination was used for GMOs content estimation in reference samples, results revealed an improved accuracy from 66% to 96%. The combined biosensor system, although more costly than the standard Hoechst 33258/carbon electrode system, provided an alternative choice for DNA quantification, offering labor-free immobilization of probe onto electrode surface, easy test administration, and efficient semi-quantitative test without expensive instruments.     

Electrochemical, atomic force microscopy and infrared reflection absorption spectroscopy studies of pre-formed mussel adhesive protein films on carbon steel for corrosion protection

Electrochemical measurements, in situ and ex situ atomic force microscopy (AFM) experiments and infrared reflection absorption spectroscopy (IRAS) analysis were performed to investigate the formation and stability as well as corrosion protection properties of mussel adhesive protein (Mefp-1) films on carbon steel, and the influence of cross-linking by NaIO₄ oxidation. The in situ AFM measurements show flake-like adsorbed protein aggregates in the film formed at pH 9. The ex situ AFM images indicate multilayer-like films and that the film becomes more compact and stable in NaCl solution after the cross-linking. The IRAS results reveal the absorption bands of Mefp-1 on carbon steel before and after NaIO₄ induced oxidation of the pre-adsorbed protein. Within a short exposure time, a certain corrosion protection effect was noted for the pre-formed Mefp-1 film in 0.1 M NaCl solution. Cross-linking the pre-adsorbed film by NaIO₄ oxidation significantly enhanced the protection efficiency by up to 80%.     

Advances in piezoelectric thin films for acoustic biosensors,acoustofluidics and lab-on-chip applications

Recently, piezoelectric thin films including zinc oxide (ZnO) and aluminium nitride (AlN) have found a broad range of lab-on-chip applications such as biosensing, particle/cell concentrating, sorting/patterning, pumping, mixing, nebulisation and jetting. Integrated acoustic wave sensing/microfluidic devices have been fabricated by depositing these piezoelectric films onto a number of substrates such as silicon, ceramics, diamond, quartz, glass, and more recently also polymer, metallic foils and bendable glass/silicon for making flexible devices. Such thin film acoustic wave devices have great potential for implementing integrated, disposable, or bendable/flexible lab-on-a-chip devices into various sensing and actuating applications. This paper discusses the recent development in engineering high performance piezoelectric thin films, and highlights the critical issues such as film deposition, MEMS processing techniques, control of deposition/processing parametres, film texture, doping, dispersion effects, film stress, multilayer design, electrode materials/designs and substrate selections. Finally, advances in using thin film devices for lab-on-chip applications are summarised and future development trends are identified.