Development and validation of a new multiplex Y-STR panel designed to increase the power of discrimination

In an attempt to increase the discrimination capacity (DC) and reduce the adventitious match probability, a 6-dye multiplex Y-chromosomal short tandem repeat (Y-STR) panel named Y34plex was constructed that combined 25 Y-chromosomal markers (DYS456, DYS627, DYS390, DYS570, DYS635, DYS385a/b, DYS448, DYS437, DYS533, DYS449, DYS481, DYS392, DYS391, DYS389I, DYS460, YGATAH4, DYS438, DYS389II, DYS19, DYS458, DYF387S1a/b, DYS439, DYS393, DYS576, and DYS518) in widely used commercial kits, with nine highly polymorphic Y-STR loci (DYS557, DYS527a/b, DYS593, DYS444, DYS596, DYS643, DYS447, DYS549, and DYS645). The Y34plex is a promising type system to distinguish both unrelated and related male individuals due to the incorporation of rapidly mutated Y-STR loci. A validation study of the Y34plex was performed and followed the guidelines of the Scientific Working Group on DNA analysis methods. Results show that full Y-STR profiles were obtained from male/female DNA mixtures with 125 pg of male DNA in the presence of 50 ng of female DNA. The ability to tolerate polymerase chain reaction inhibitors commonly contained in forensic casework samples demonstrated the applicability and robustness of the Y34plex. Compared with the Yfiler Plus kit, the novel panel showed an increased power of discrimination in Chinese Wuxi Han population (n = 434). The overall haplotype diversity of the Y34plex was 0.999606, whereas DC value was 0.956221, which is suitable for use on forensic paternal investigation.     

Development of a novel forensic STR multiplex for ancestry analysis and extended identity testing

There is growing interest in developing additional DNA typing techniques to provide better investigative leads in forensic analysis. These include inference of genetic ancestry and prediction of common physical characteristics of DNA donors. To date, forensic ancestry analysis has centered on population‐divergent SNPs but these binary loci cannot reliably detect DNA mixtures, common in forensic samples. Furthermore, STR genotypes, forming the principal DNA profiling system, are not routinely combined with forensic SNPs to strengthen frequency data available for ancestry inference. We report development of a 12‐STR multiplex composed of ancestry informative marker STRs (AIM‐STRs) selected from 434 tetranucleotide repeat loci. We adapted our online Bayesian classifier for AIM‐SNPs: Snipper, to handle multiallele STR data using frequency‐based training sets. We assessed the ability of the 12‐plex AIM‐STRs to differentiate CEPH Human Genome Diversity Panel populations, plus their informativeness combined with established forensic STRs and AIM‐SNPs. We found combining STRs and SNPs improves the success rate of ancestry assignments while providing a reliable mixture detection system lacking from SNP analysis alone. As the 12 STRs generally show a broad range of alleles in all populations, they provide highly informative supplementary STRs for extended relationship testing and identification of missing persons with incomplete reference pedigrees. Lastly, mixed marker approaches (combining STRs with binary loci) for simple ancestry inference tests beyond forensic analysis bring advantages and we discuss the genotyping options available.     

A novel hybrid mode of sample injection to enhance CZE sensitivity for simultaneous determination of a pyridine-triphenylborane anti-fouling agent and its degradation products

We developed a novel hybrid sample injection mode (HSIM) that presents the combination of electrokinetic injection and vacuum injection to enhance detection sensitivity in CZE. Samples were introduced using both vacuum and electrokinetic injections simultaneously, with a water plug injected into the capillary prior to sample introduction (i.e. similarly to field-amplified sample injection, FASI). Using a sample mixture containing an anti-fouling agent applied to ship hulls, pyridine-triphenylborane and its degradation products (diphenylborinic acid, phenylboronic acid, and phenol) dissolved in ACN, the length of water plug, time, and voltage for sample introduction were optimized. The signal intensity (peak height) was found to be up to a 30-fold increased using HSIM by applying 4 kV for 4 s at the inlet end of the capillary as the cathode with supplementary vacuum in comparison with only vacuum injection for 4 s. The LODs (at a S/N of 3) for pyridine-triphenylborane, diphenylborinic acid, phenylboronic acid, and phenol were 0.88, 1.0, 21, and 23 μg/L, respectively. At the level of 0.04 mg/L, the RSDs (n=4, intra-day) for the above analytes were in the ranges of 1.9-11, 4.3-9.2, and 0.34-0.66% for peak area, peak height, and migration time, respectively. The HSIM is a simple and promising procedure useful for enhancing the sensitivity for both low-and high-mobility ions in CZE.     

On-line sample stacking and short-end injection CE for the determination of fluoxetine and norfluoxetine in plasma: Method development and validation using experimental designs

A short-end injection CE method combining field-amplified sample stacking (FASS) is presented for the analysis of fluoxetine (FL) and norfluoxetine in plasma. In this study, FASS enhanced the sensitivity about 1100-fold, while short-end injection reduced the analysis time to less than 4 min. Parameters involved in the separations were investigated using a central composite design (CCD) and response surface methodology to optimize the separation conditions in a total of only 32 runs. Samples injected into the capillary for 99.9 s at a voltage of -5 kV were stacked in a water plug (0.5 psi, 9 s). Baseline resolution of FL and its major metabolite was achieved using a BGE formulation consisting of phosphate-triethanolamine at low pH, and a separation voltage of -10 kV. Five percent methanol was added as organic modifier to enhance selectivity and resolution. The linear range was between 10 and 500 ng/mL (r >0.9946), covering the expected plasma therapeutic ranges. The LOD in plasma were 4 ng/mL (S/N = 3), a value comparable to that obtained using LC-MS, showing the success of the on-line stacking technique. Our method was also successfully validated in quantification and pharmacokinetic studies with three volunteer plasma samples and could be applied to pharmacogenetic studies.     

Development and validation of a custom panel including 183 Y-SNPs for Chinese Y-chromosomal haplogroups dissection using a MALDI-TOF MS system

Y-chromosome SNP haplogroups exhibit geographic structuring in many populations around the world. Therefore, Y-chromosome haplogroups have been widely used to infer paternal biogeographical ancestry and high-resolution paternal lineage classification. In the present study, we designed a customized panel containing 183 Y-SNPs based on previous studies and evaluated the genotyping performance and repeatability, concordance, sensitivity, and ability of analysing case-type samples using a MALDI-TOF MS platform. The average call rate for duplicate typing of any one SNP in the panel was 97.0%. In the concordance and accuracy study, the results of haplogroup designation obtained from MALDI-TOF MS platform were fully consistent with those obtained from the next-generation sequencing (NGS) platform. The optimal amount of template DNA in the PCR seemed to be 10 ng. However, if less DNA (≥156.25 pg) was available, it was still possible to obtain meaningful haplogroup information. For the application part, this panel could be applied for the detection of blood, semen, and buccal swabs samples. Particularly, blood stain on FTA card samples could be dissected by direct PCR amplification on the MALDI-TOF MS platform. Besides, 371 unrelated male individuals from four Chinese ethnic groups (Han, Hui, Mongolian, and Kazak) were detected using this panel. Total 78 terminal haplogroups were found and the haplogroup diversity was 0.933576. The results demonstrate that this panel could be an accurate, fast, and cost-effective method for database construction where the amount of sample material is less of a concern and when the cost of the assay is taken into consideration.     

Meta-analysis of gene expression for development and validation of a diagnostic biomarker panel for Oral Squamous Cell Carcinoma

We use a newly developed feature extraction and classification method to analyze previously published gene expression data sets in Oral Squamous Cell Carcinoma and in healthy oral mucosa in order to find a gene set sufficient for diagnoses. The feature selection technology is based on the relative dichotomy power concept published by us earlier. The resulting biomarker panel has 100% sensitivity and 95% specificity, is enriched in genes associated with oncogenesis and invasive tumor growth, and, unlike marker panels devised in earlier studies, shows concordance with previously published marker genes.     

Optimization and validation of a method for the direct determination of catechin and epicatechin in red wines by HPLC/fluorescence

The present paper describes a direct procedure for the determination of catechin and epicatechin concentrations in red wines employing reverse-phase high performance liquid chromatography (RP HPLC) and detection by fluorescence. The method was performed using a sample volume of 10 µL without dilution. The separation process employed a Chromolith performance RP-18e (100 mm × 4.6 mm) column, and the mobile phase was composed of solvent A: methanol-acetic acid-water (90:8:2) and solvent B: water-acetic acid-methanol (10:2:88) at a flow rate of 1.0 mL min^− 1. Linearity was observed in the range of 1 to 30 mg L^− 1, with limits of detection and quantification of 0.27 and 0.89 mg L^− 1, respectively, for catechin and 0.33 and 1.01 mg L^− 1, respectively, for epicatechin. The precisions estimated by the relative standard deviation were 3.34 and 1.09% for catechin concentrations of 0.5 and 20 mg L^− 1 respectively and 2.82 and 0.49% for epicatechin concentrations of 0.5 and 20 mg L^− 1, respectively. The evaluation of the accuracy was done using an addition/recovery assay. Four wine samples were used, and the recoveries varied from 105 to 108% for catechin and from 97 to 119% for epicatechin. The method was applied to the analysis of red wine samples collected from the São Francisco region, Bahia State, Brazil. Nine samples were analyzed, and the catechin and epicatechin concentrations varied from 7.51 to 73.20 and from 5.08 to 43.32 mg L^− 1, respectively. The concentrations found agree with data reported in the literature.     

Development a multiplex panel of AISNPs,multi-allelic InDels,microhaplotypes, and Y-SNP/InDel loci for multiple forensic purposes via the NGS

Recently, next generation sequencing has shown the promising application value in forensic research. In this study, we constructed a multiplex amplification system of different molecular genetic markers based on the previous selected ancestry informative single nucleotide polymorphisms (SNPs), multi-allelic insertion/deletion (InDel) polymorphisms, microhaplotypes, and Y-chromosomal SNP/InDel loci, and evaluated forensic efficiencies of the system in Chinese Shaanxi Han, Chinese Hui, and Chinese Mongolian groups via the next generation sequencing platform. Ancestry information analyses of Shaanxi Han, Hui, and Mongolian groups revealed that most Mongolian individuals could be differentiated from Shaanxi Hans and Huis based on the selected ancestry informative SNPs. Multi-allelic InDels and microhaplotypes showed the multiple allele variations and possessed relatively high genetic polymorphisms in these three groups, indicating these loci could provide higher forensic efficiencies for individual identification and paternity testing. Based on Y-chromosomal SNPs, different haplogroup distributions were observed among Shaanxi Han, Hui, and Mongolian groups. In conclusion, the self-developed system could be used to simultaneously carry out the individual identification, paternity analysis, mixture deconvolution, forensic ancestry information analysis, and Y-chromosomal haplogroup inference, which could provide more valuable investigative clues in forensic practices.     

Design of a novel mitochondria targetable turn-on fluorescence probe for hydrogen peroxide and its two-photon bioimaging applications

Considering that hydrogen peroxide (H₂O₂) plays significant roles in oxidative stress, the cellular signal transduction and essential biological process regulation, the detection and imaging of H₂O₂ in living systems undertakes critical responsibility. Herein, we have developed a novel two-photon fluorescence turn on probe, named as Pyp-B for mitochondria H₂O₂ detection in living systems. Selectivity studies show that probe Pyp-B exhibit highly sensitive response toward H₂O₂ than other reactive oxygen species (ROS) and reactive nitrogen species (RNS) as well as biologically relevant species. The fluorescence colocalization studies demonstrate that the probe can localize in the mitochondria solely. Furthermore, as a bio-compatibility molecule, the highly selective and sensitive of fluorescence probe Pyp-B have been confirmed by its cell imaging application of H₂O₂ in living A549 cells and zebrafishes under the physiological conditions.     

A novel redox switch for fluorescence: drastic UV-vis and fluorescence spectral changes upon electrolysis of a hexaphenylethane derivative of 10,10′-dimethylbiacridan

The newly prepared title donor 1 has a very long CC bond [1.635(2) Å], which was readily cleaved upon oxidation to give dication 2²⁺ containing two 10-methylacridinium chromophores. Colorless donor 1 (E^ox +0.18 V) is non-fluorescent whereas orange dication 2²⁺ (E^red −0.27 V) emits strong green fluorescence, so that, this pair can be considered as a novel redox switch for fluorescence with high electrochemical bistability.     

Design of a novel mitochondria targetable turn-on fluorescence probe for hydrogen peroxide and its two-photon bioimaging applications

Considering that hydrogen peroxide (H₂O₂) plays significant roles in oxidative stress, the cellular signal transduction and essential biological process regulation, the detection and imaging of H₂O₂ in living systems undertakes critical responsibility. Herein, we have developed a novel two-photon fluorescence turn on probe, named as Pyp-B for mitochondria H₂O₂ detection in living systems. Selectivity studies show that probe Pyp-B exhibit highly sensitive response toward H₂O₂ than other reactive oxygen species (ROS) and reactive nitrogen species (RNS) as well as biologically relevant species. The fluorescence colocalization studies demonstrate that the probe can localize in the mitochondria solely. Furthermore, as a bio-compatibility molecule, the highly selective and sensitive of fluorescence probe Pyp-B have been confirmed by its cell imaging application of H₂O₂ in living A549 cells and zebrafishes under the physiological conditions.     

Development and validation of a sensitive quantification method for hematoporphyrin monomethyl ether in plasma using high-performance liquid chromatography with fluorescence detection

A rapid, sensitive, precise and specific method for determination of hematoporphyrin monomethyl ether (HMME), a novel photodynamic therapy (PDT) drug, was developed and validated using high-performance liquid chromatography (HPLC) with fluorescence detection. HMME was isolated from the plasma by a single-step liquid-liquid extraction with ethyl acetate. The analyte and internal standard fluorescein were baseline separated on a Diamonsil C(18) analytical column (4.6 x 150 mm, 5 microm) and analyzed using a fluorescence detector with the excitation and emission wavelengths set at 395 and 613 nm, respectively. The method was linear in the concentration range 0.025-5 microg/mL with a lower limit of quantitation (LLOQ) of 10 ng/mL. The inter- and intra-day accuracies and precisions were all within 10% and the mean recoveries of HMME and fluorescein were 95 +/- 3.7 and 90 +/- 2.3%, respectively. The analyte was stable during all sample storage, preparation and analysis periods. This method was successfully applied to a pharmacokinetic study after a single-dose intravenous administration of HMME (5 mg/kg) to beagle dogs. This method was reproducible and sensitive enough for the pharmacokinetic study of HMME. Based on the results of the pharmacokinetic study, we suggest that a rather long light-avoiding time is essential for patients under HMME therapy.     

High‐performance liquid chromatographic determination of anti‐ hypertensive drugs on dried blood spots using a fluorescence detector – method development and validation

A selective and sensitive high‐performance liquid chromatography method with fluorescence detection for simultaneous determination of irbesartan, losartan and valsartan on dried blood spots (DBS) has been developed and validated. It involves solvent extraction of a punch of DBS followed by reversed‐phase liquid chromatography on a Lichrospher^® 100 RP‐18e column. Fluorescence detection was performed at 259 and 385 nm as excitation and emission wavelengths, respectively. The detection limits of irbesartan, losartan and valsartan were 1.8, 3.6 and 1.8 ng/mL respectively. The mean recoveries of irbesartan, losartan and valsartan were 98.68, 98.42 and 97.81%, respectively. The mean inter‐day and intra‐day precisions of irbesartan, losartan and valsartan were 2.07 and 1.34%, 1.42 and 1.48%, and 3.20 and 2.15% respectively. The proposed method was simple and rapid. Design of experiments was used to evaluate the robustness of the method. Copyright © 2011 John Wiley & Sons, Ltd.     

基于三苯胺-罗丹明染料荧光探针对pH和Cu^2+的选择性识别

基于罗丹明B酰肼和三苯胺染料合成了pH和Cu^2+双识别分子探针(RTPA),并利用核磁氢谱、核磁碳谱和高分辨质谱表征了RTPA的分子结构。通过紫外-可见光谱法和荧光光谱法研究了该荧光分子探针在极性不同的有机溶剂中的光谱性能以及在THF/H2O(V:V=1:1)溶液中对Cu^2+和质子的选择性识别性能。结果表明:随着溶剂的极性增大,化合物RTPA的最大发射波长发生红移,从419 nm红移到500 nm。该探针在水溶液中可以实现高选择性识别Cu^2+离子,不受其他常见金属离子的竞争性干扰。通过Job’s plot曲线证明了探针RTPA识别Cu^2+的化学计量比是1:1,Cu^2+的检出限为0.635μmol/L。当pH在1.87~4.48区间内,探针的荧光强度与pH呈良好的线性关系,线性相关系数为0.9957,其pKa为3.13。探针RTPA也可用于极酸性条件下(pH<4)质子浓度的定量检测。     

Design and application of a novel integrated electrochemical hydride generation cell for the determination of arsenic in seaweeds by atomic fluorescence spectrometry

An integrated electrochemical hydride generation cell, mainly composed of three components (a gas liquid separator, a graphite tube cathode and a reticulate Pt wire anode), was laboratory constructed and employed for the detection of arsenic by coupling to atomic fluorescence spectrometry. This integrated cell was free of ion-exchange membrane and individual anolyte, with the virtues of low-cost, easy assembly and environmental-friendly. Using flow injection mode, the sample throughput could come to 120 h⁻¹ attributed to the small dimension of the cathode chamber. The operating conditions for the electrochemical hydride generation of arsenic were investigated in detail and the potential interferences from oxygen or various ions were also evaluated. Under the optimized conditions, no obvious oxygen quenching effects were observed. The limit of detection of As (III) for the sample blank solution was 0.2 ng mL⁻¹ (3σ) and the relative standard deviation was 3.1% for nine consecutive measurements of 5 ng mL⁻¹ As (III) standard solution. The calibration curve was linear up to 100 ng mL⁻¹. The accuracy of the method was verified by the determination of arsenic in the reference materials GBW08517 (Laminaria Japonica Aresch) and GBW10023 (Porphyra crispata) and the developed method was successfully applied to determine trace amounts of arsenic in edible seaweeds.     

Development and validation of RP‐HPLC‐fluorescence method for quantitative determination of quinidine,a probe substrate for P‐glycoprotein inhibition assay using Caco‐2 cell monolayer

A simple, sensitive and specific reverse‐phase high‐performance liquid chromatographic (RP‐HPLC) method with fluorescence detection was developed for quantitation of quinidine from HBSS buffer. The method was applicable in the bi‐directional transport assay for evaluation of the inhibitory effect of test compounds on P‐glycoprotein‐mediated quinidine transport; quinidine was used as a probe P‐glycoprotein substrate. The calibration curve was linear (correlation coefficient ≥99) in the range 0.30–100.00 nm. The method was validated and is specific and sensitive with limit of quantitation of 300 pm for quinidine. The method was found to be accurate and precise in the working calibration range. Stability studies were carried out at different storage conditions where the analyte was found to be stable. The applicability and reliability of the analytical method was evaluated by successful demonstration of efflux ratio (P_appB → A/P_appA → B) in the Caco‐2 cell monolayer efflux assay. The efflux ratio for quinidine (100 nm) alone was 10.8, which reduced to less than 2 in the presence of the classical P‐gp inhibitors verapamil and ketoconazole (100 μm each). Copyright © 2009 John Wiley & Sons, Ltd.     

Total internal reflectance fluorescence (TIRF) biosensor for environmental monitoring of testosterone with commercially available immunochemistry: antibody characterization, assay development and real sample measurements

Nowadays, little technology exists that can monitor various water sources quickly and at a reasonable cost. The ultra-sensitive, fully automated and robust biosensor River Analyser (RIANA) is capable of detecting multiple organic targets rapidly and simultaneously at a heterogeneous assay format (solid phase: bulk optical glass transducers). Commercialization of such a biosensor requires the availability of commercial high-affinity recognition elements (e.g. antibodies) and suitable commercial haptens (modified target molecules) for surface chemistry. Therfore, testosterone was chosen as model analyte, which is also a task of common analytical methods like gas chromatography-mass spectrometry (GC-MS), because they have to struggle with detecting sub-nanogram per liter levels in environmental samples. The reflectometric interference spectroscopy (RIfS) was used to characterize the commercially available immunochemistry resulting in a high-affinity constant of 2.6 ± 0.3 × 10⁹ mol⁻¹ for the unlabeled antibody. After the labeling procedure, necessary for the TIRF-based biosensor, a mean affinity constant of 1.2 × 10⁹ mol⁻¹ was calculated out of RIfS (1.4 ± 0.4 × 10⁹ mol⁻¹) and TIRF (1.0 ± 0.3 × 10⁹ mol⁻¹) measurements.Thereafter, the TIRF-based biosensor setup was used to determine the steroidal hormone testosterone at real world samples without sample pre-treatment or sample pre-concentration. Results are shown for rapid and ultra-sensitive analyses of testosterone in aqueous samples with at a remarkable limit of detection (LOD) of 0.2 ng L⁻¹. All real world samples, even those containing testosterone in the sub-nanogram per liter range (e.g. 0.9 ng L⁻¹), could be determined with recovery rates between 70 and 120%. Therefore, the sensor system is perfectly suited to serve as a low-cost system for surveillance and early warning in environmental analysis in addition to the common analytical methods. For the first time, commercially available immunochemistry was fully characterized using a label-free detection method (RIfS) and successfully incorporated into a TIRF-based biosensor setup (RIANA) for reliable sub-nanogram per liter detection of testosterone in aqueous environmental samples.     

Preparation of a new core-shell Ag@SiO2 nanocomposite and its application for fluorescence enhancement

Ag@SiO₂ nanoparticles with different shell thicknesses were synthesized via modified Stöber method. Rhodamine B isothiocyanate was covalently bound onto the surface of Ag@SiO₂ nanoparticles to form fluorescent core-shell Ag@SiO₂ nanocomposites. Effects of shell thickness on the fluorescence enhancement were examined using the corresponding nanobubbles prepared by cyanide etching as a control. The result showed that the fluorescence enhanced as the shell thickness increased till the distance between fluorophore and metal core reached about 75 nm with the optimal enhancement factor of ∼5-folds. Further increasing of fluorophore-metal distance caused a decrease in the enhancement factor.     

Analysis of cocaine and two metabolites in dried blood spots by liquid chromatography with fluorescence detection: a novel test for cocaine and alcohol intake

An original HPLC method coupled to spectrofluorimetric detection is presented for the simultaneous analysis in dried blood spots (DBS) of cocaine and two important metabolites, namely benzoylecgonine (its main metabolite) and cocaethylene (the active metabolite formed in the presence of ethanol). The chromatographic analysis was carried out on a C8 column, using a mobile phase containing phosphate buffer (pH 3.0)-acetonitrile (85:15, v/v). Native analyte fluorescence was monitored at 315 nm while exciting at 230 nm. A fast and feasible sample pre-treatment was implemented by solvent extraction, obtaining good extraction yields (>91%) and satisfactory precision values (RSD<4.8%). The method was successfully applied to DBS samples collected from some cocaine users, both with and without concomitant ethanol intake. The results were in good agreement with those obtained from plasma samples subjected to an original solid-phase extraction procedure on C8 cartridges. The method has demonstrated to be suitable for the monitoring of cocaine/ethanol use by means of DBS or plasma testing. Assays are in progress to apply this method on the street, for the control of subjects suspected of driving under the influence of psychotropic substances.     

Quality-by-design-based development and validation of a stability-indicating UPLC method for quantification of teriflunomide in the presence of degradation products and its application to in-vitro dissolution

A systematic design-of-experiments was performed by applying quality-by-design concepts to determine design space for rapid quantification of teriflunomide by the ultraperformance liquid chromatography (UPLC) method in the presence of degradation products. Response surface and central composite quadratic were used for statistical evaluation of experimental data using a Design-Expert software. The response variables such as resolution, retention time, and peak tailing were analyzed statistically for the screening of suitable chromatographic conditions. During this process, various plots such as perturbation, contour, 3D, and design space were studied. The method was developed through UPLC BEH C18 2.1?×?100?mm, 1.7-µ column, mobile phase comprised of buffer (5?mM K₂HPO₄ containing 0.1% triethylamine, pH 6.8), and acetonitrile (40:60 v/v), the flow rate of 0.5?mL?min^?1 and UV detection at 250?nm. The method was developed with a short run time of 1?min. Forced degradation studies revealed that the method was stability-indicating, suitable for both assay and in-vitro dissolution of a drug product. The method was found to be linear in the range of 28–84?µg?mL^?1, 2.8–22.7?µg?mL^?1 with a correlation coefficient of 0.9999 and 1.000 for assay and dissolution, respectively. The recovery values were found in the range of 100.1–101.7%. The method was validated according to ICH guidelines.