[Validation of GlobalFiler® PCR Amplification Kit and the STR Polymorphism].

OBJECTIVE: To test the technical parameters of GlobalFiler® PCR Amplification Kit for its application to forensic application value and to investigate the genetic polymorphisms. METHODS: The validation was conducted in sensitivity, mixed samples, species specificity, adaptability, survivability, consistency, peak height balance and stability. The amplification and detection of the genomic DNA from 373 unrelated individuals from Beijing Han nationality were extracted by automation workstation. RESULTS: Global-Filer® PCR Amplification Kit was adaptive to some mixed, degraded and inhibited samples. The power of sensitivity and adaptability and peak height balance showed well. The distributions of genotype frequencies for 21 STR loci in the population were all in accordance with Hardy-Weinberg equilibrium (P > 0.05). The PIC value of the 21 STR loci was among 0.536 to 0.940; the H value was among 0.558 to 0.933; the DP value was among 0.783 to 0.992; the PE value was among 0.243 to 0.874. CONCLUSION: GlobalFiler® PCR Amplification Kit is suitable for criminal cases and DNA database in forensic practice. And 21 STR loci in Beijing Han nationality have high polymorphism, which have application value in forensic practice and population genetics.

Evaluation of parameters in mixed male DNA profiles for the Identifiler® multiplex system.

The analysis of complex DNA mixtures is challenging for forensic DNA testing. Accurate and sensitive methods for profiling these samples are urgently required. In this study, we developed 11 groups of mixed male DNA samples (n=297) with scientific validation of D­value [>95% of D­values ≤0.1 with average peak height (APH) of the active alleles ≤2,500 rfu]. A strong linear correlation was detected between the peak height (PH) and peak area (PA) in the curve fit using the least squares method (P<2e-16). The Kruskal-Wallis rank-sum test revealed significant differences in the heterozygote balance ratio (H(b)) at 16 short tandem repeat (STR) loci (P=0.0063) and 9 mixed gradients (P=0.02257). Locally weighted regression fitting of APH and Hb (inflection point at APH = 1,250 rfu) showed 92.74% of H(b) >0.6 with the APH ≥1,250. The variation of H(b) distribution in the different STR loci suggested the different forensic efficiencies of these loci. Allelic drop-out (ADO) correlated with the APH and mixed gradient. All ADOs had an APH of <1,000 rfu, and the number of ADO increased when the APH of mixed DNA profiles gradually decreased. These results strongly suggest that calibration parameters should be introduced to correct the deviation in the APH at each STR locus during the analysis of mixed DNA samples.

Choline acetate enhanced the catalytic performance of Candida rogusa lipase in AOT reverse micelles.

Choline acetate is an ionic liquid composed of a kosmotropic anion and a chaotropic cation. According to Hofmeister series, a kosmotropic anion and/or a chaotropic cation could stabilize an enzyme, thereby facilitating the retention of the catalytic activity of the enzyme. In this work, we first report the influence of choline acetate on the activity and stability of lipase in AOT/water/isooctane reverse micelles. The indicator reaction is the lipase-catalyzed hydrolysis of 4-nitrophenyl butyrate. The results show that a low level of choline acetate does not affect the microstructure of the AOT reverse micelles, but the ionic liquid can improve the catalytic efficiency of lipase. Fluorescence spectra show that a high level of choline acetate has an impact on the conformation of lipase, so the activation is mainly due to the influence of choline acetate on the nucleophilicity of water. Infrared spectra demonstrate that choline acetate can form stronger hydrogen bonds with water surrounding lipase, and therefore enhance the nucleophilicity of the water, which makes it easier to attack the acyl enzyme intermediate, thereby increasing the activity of the lipase-catalyzed hydrolysis of the ester. A study on the stability of lipase in AOT reverse micelles indicates that the ionic liquid is able to maintain the activity of lipase to a certain extent. The effect of choline acetate is consistent with that predicted based on Hofmeister series.

Nitrile group as infrared probe for the characterization of the conformation of bovine serum albumin solubilized in reverse micelles.

Infrared spectroscopy is a powerful technique for structure characterization. For a protein hosted in a reversed micellar medium, the spectral features of the protein are always interfered by the IR absorption bands of the medium in addition to the congestion in their IR spectra. Fortunately, there is a transparent window in the 2500-2200 cm(-1) region. Incorporation of a vibrational probe with IR absorption frequencies in this region into proteins represents a promising strategy for the study of the conformation of a protein in a reverse micelle. In the present work, we incorporated 4-cyanobenzyl group (CN) into bovine serum albumin (BSA) via cysteine alkylation reactions under mild conditions. Circular dichroism spectroscopy showed that the CN modified BSA (CNBSA) could retain its conformation. When CNBSA was hosted in AOT reverse micelle, it was found that the nitrile group on BSA was sensitive to the conformational change of BSA induced by urea as an additive in the reverse micelle. The peak splitting of nitrile group was also observed when the size of AOT reverse micelle and the concentration of an electrolyte were varied. Obviously, the shift of the IR absorption peak and/or peak splitting of nitrile group on BSA are correlated with the change of BSA conformation in AOT reverse micelle. So we conclude that the nitrile infrared probe can be used to study protein conformation in a reverse micelle.

The catalytic efficiency of lipase in a novel water-in-[Bmim][PF6] microemulsion stabilized by both AOT and Triton X-100.

In the water-in-[Bmim][PF(6)] microemulsion stabilized by both AOT and Triton X-100, the lipase-catalyzed hydrolysis of 4-nitrophenyl butyrate (p-NPB) was investigated to evaluate the catalytic efficiency of lipase in this novel microemulsion. The structural parameters of the microemulsion and the conditions of the enzymatic reaction affect the catalytic activity of lipase, especially the concentration of Tris-HCl buffer. Under optimum conditions, the catalytic activity of lipase in the present microemulsion is much higher than that in H(2)O saturated [Bmim][PF(6)]. When the partitioning of the substrate in the microemulsion is taken into account, the catalytic efficiency of lipase in this novel microemulsion is 14.3 times that in H(2)O saturated [Bmim][PF(6)] due to the significant decrease of the Michaelis constant in the microemulsion. Due to the large interface, high water activity, and probably the activating effect of the imidazolium cation in the water pool, the present microemulsion is demonstrated to be a promising medium for the lipase-catalyzed hydrolytic reaction. To demonstrate an important biocatalytic application in the IL-based microemulsion, the lipase-catalyzed synthesis of the flavoring agent benzyl acetate via transesterification of vinyl acetate with benzyl alcohol was also studied in the medium. Due to the high dispersion of lipase, large interface and removal of the byproduct, a maximum yield of 94% was obtained, indicating that the novel microemulsion is really important and useful.

A novel water-in-ionic liquid microemulsion and its interfacial effect on the activity of laccase.

It is of great significance to develop an appropriate water-in-ionic liquid (W/IL) microemulsion suitable for the expression of the catalytic activity of a given enzyme. In this paper, the phase diagram of a new AOT/Triton X-100/H(2)O/[Bmim][PF(6)] pseudo ternary system is presented. With the aid of nonionic surfactant Triton X-100, AOT could be dissolved in hydrophobic ionic liquid [Bmim][PF(6)], forming a large single phase microemulsion region. The water-in-[Bmim][PF(6)] (W/IL) microemulsion domain was identified electrochemically by using K(3)Fe(CN)(6) as a probe. The existence of W/IL microemulsions was demonstrated spectrophotometrically by using CoCl(2) as a probe. New evidences from the FTIR spectroscopic study, which was first introduced to the W/IL microemulsion by substituting D(2)O for H(2)O to eliminate the spectral interference, demonstrated that there existed bulk water at larger ω(0) values (ω(0) was defined as the molar ratio of water to the total surfactant) in the W/IL microemulsion, which had remained unclear before. In addition to the inorganic salts, biomacromolecule laccase could be solubilized in the W/IL microemulsion. The laccase hosted in the microemulsion exhibited a catalytic activity and the activity could be regulated by the composition of the interfacial membrane.

Enzyme-modified nanoporous gold-based electrochemical biosensors.

On the basis of the unique physical and chemical properties of nanoporous gold (NPG), which was obtained simply by dealloying Ag from Au/Ag alloy, an attempt was made in the present study to develop NPG-based electrochemical biosensors. The NPG-modified glassy carbon electrode (NPG/GCE) exhibited high-electrocatalytic activity toward the oxidation of nicotinamide adenine dinucleotide (NADH) and hydrogen peroxide (H(2)O(2)), which resulted in a remarkable decrease in the overpotential of NADH and H(2)O(2) electro-oxidation when compared with the gold sheet electrode. The high density of edge-plane-like defective sites and large specific surface area of NPG should be responsible for the electrocatalytic behavior. Such electrocatalytic behavior of the NPG/GCE permitted effective low-potential amperometric biosensing of ethanol or glucose via the incorporation of alcohol dehydrogenase (ADH) or glucose oxidase (GOD) within the three-dimensional matrix of NPG. The ADH- and GOD-modified NPG-based biosensors showed good analytical performance for biosensing ethanol and glucose due to the clean, reproducible and uniformly distributed microstructure of NPG. The stabilization effect of NPG on the incorporated enzymes also made the constructed biosensors very stable. After 1 month storage at 4 degrees C, the ADH- and GOD-based biosensors lost only 5.0% and 4.2% of the original current response. All these indicated that NPG was a promising electrode material for biosensors construction.