Capture and release of DNA using aminosilane-modified bacterial magnetic particles for automated detection system of single nucleotide polymorphisms

Bacterial magnetic particles (BMPs) were modified with 3-[2-(2-aminoethylamino)-ethylamino]-propyltrimethoxysilane (AEEA) to produce a dense amine surface. Modification of BMPs in a toluene solution resulted in an increased amine yield, and approximately 11.3 x 10(4) surface amines were detected on a single particle. The modified BMPs were capable of efficient electrostatic capture of DNA. The maximum amount of DNA captured on 10 microg of aminosilane-modified BMPs was 600 ng. A 10 mM phosphate buffer effectively released the captured DNA. This efficiency was dramatically enhanced by incubation at 80 degrees C and DNA recovery from aminosilane-modified BMPs approached 95%. DNA extraction from whole blood using these modified BMPs, followed by PCR, was successfully performed. Furthermore, automated single nucleotide polymorphism (SNP) detection of the aldehyde dehydrogenase 2 (ALDH2) was demonstrated.     

Evaluation of silica resins for direct and efficient extraction of DNA from complex biological matrices in a miniaturized format

For DNA purification to be functionally integrated into the microchip for high-throughput DNA analysis, a miniaturized purification process must be developed that can be easily adapted to the microchip format. In this study, we evaluate the effectiveness of a variety of silica resins for miniaturized DNA purification and gauge the potential usefulness for on-chip solid-phase extraction. A micro-solid-phase extraction (muSPE) device containing only nanograms of silica resin is shown to be effective for the adsorption and desorption of DNA in the picogram-nanogram mass range. Fluorescence spectroscopy as well as capillary electrophoresis with laser-induced fluorescence detection is employed for the analysis of DNA recovered from solid-phase resins, while the polymerase chain reaction (PCR) is used to evaluate the amplifiable nature of the eluted DNA. We demonstrate that DNA can be directly recovered from white blood cells with an efficiency of roughly 70%, while greater than 80% of the protein is removed with a 500-nl bed volume muSPE process that takes less than 10 min. With a capacity in the range of 10-30 ng/mg of silica resin, we show that the DNA extracted from white blood cells, cultured cancer cells, and even whole blood on the low microliter scale is suitable for direct PCR amplification. The miniaturized format as well as rapid time frame for DNA extraction is compatible with the fast electrophoresis on microfabricated chips.     

Focal extraction of surface-bound DNA from a microchip using photo-thermal denaturation

High-throughput, selective extraction of a particular DNA fragment from a mixture of DNA before PCR amplification is becoming increasingly important in the DNA analysis field. Although the latest microchip technology has enabled real-time DNA expression analysis using hybridization between surface-bound probe DNA and sample DNA, the potential of this technology in purification of a small amount of DNA has not been demonstrated. We report here a method for area-selective release and collection of specific DNA, in which an IR laser beam is focused onto surface-bound sample DNA at the target-spotted area to denature hybridized DNA. First, sample DNA labeled with a fluorescent dye was hybridized to a probe DNA immobilized on a chromium-coated chip. A 1053-nm IR laser beam with an intensity of 10-100 mW was then focused on the target area with a spatial resolution of 10 microns, causing the release of the fluorophore-labeled sample DNA as a result of photo-thermal denaturation. Confirmation of the amount of eluted DNA by PCR amplification after collection indicated that more than 10(-20) mol DNA/micron 2 area was eluted from the microchip, representing more than 70% of the chip-bound sample DNA. These results indicate that this method can be applied to the highly sensitive purification of DNA in microchip technology.     

DNA extraction using modified bacterial magnetic particles in the presence of amino silane compound

Magnetic particles produced by magnetic bacteria have been used to carry out magnetic separation of DNA. Separation was achieved using magnetite coated with 3-aminopropyltriethoxysilane, N-(trimethoxy-silylpropyl) isothiouronium chloride or 3-[2-(2-aminoethyl)-ethylamino]-propyltrimethoxysilane (AEEA). The DNA binding efficiency increased with the number of amino groups present on the silane compounds and was 14 fold higher than with untreated magnetite. Addition of AEEA to aqueous solutions containing coated magnetite increased efficiency due to co-condensation of DNA. From 10(8) Escherichia coli cells, 7.1 microg of DNA was recovered using 100 microg of magnetite. E. coli DNA extracted with modified bacterial magnetite was suitable for restriction enzyme digestion and polymerase chain reaction (PCR). Ten replicate samples of E. coli cells were extracted using an automated magnetic robot and 9.5 microg of DNA was extracted using 100 microg of modified bacterial magnetite and possessed a 1.94 absorbance ratio (260:280 nm).     

DNA提取的应用与相关技术分析

为了分析影响提取DNA的有关因素,采用标准酚—氯仿抽提法和蛋白酶消化法提取DNA。结果表明,平均每mL全血可提取200 ~ 300μgDNA;新鲜组织及OCT包埋冰冻组织提取DNA,平均每0.2g组织可获得200~300μgDNA;直接将石蜡标本提取物制作模版,PCR扩增良好。从4种组织中均获得较为纯净的DNA;OCT对组织DNA无不良影响。 Abstract:To explore influence factors of DNA extraction,the protease and phenol-chloroform method was used to extract DNA in whole blood,fresh tissues,frozen tissues embedding with OCT and tissues embedding in paraffin.It results that 200~300μg DNA was extracted from 1ml whole blood or 0.2g fresh tissues or frozen tissue embedding with OCT.DNA extracted from paraffin specimen can be directly used in PCR amplification.Purity DNA can be extracted from four kinds of different tissues.OCT hasn't harmful effect on tissue DNA extraction.     

High-performance liquid chromatographic assay for the determination of a semisynthetic avermectin analog (eprinomectin) in bovine milk at parts per billion levels--method development and validation

A reversed-phase HPLC method for the quantitative determination of total topotecan in human whole blood and unwashed erythrocytes has been developed and validated in terms of sensitivity, specificity, precision and accuracy. Linear calibration curves were constructed in the range of 0.20 to 50.0 ng/ml. The sample pre-treatment for whole blood involved a two-step extraction with methanol and perchloric acid. Prior to extraction, erythrocytes were separated from other blood components by centrifugation in MESED instruments. Separations were achieved on an Inertsil ODS-80A analytical column (150x4.6 mm, 5 microm particle size), eluted at 50 degrees C and a flow-rate of 1.00 ml/min, with a mixture of 100 mM ammonium acetate (pH 6.0)-tetrahydrofuran (94.6:5.4, v/v). Fluorescence detection was performed using excitation and emission wavelengths of 381 and 525 nm, respectively. With the applied method, 80% of topotecan was extracted out of whole blood. The lower limit of quantitation in whole blood was established at 0.20 ng/ml with within-run and between-run precisions, respectively, ranging from 1.7 to 9.3% and 1.5-6.1%, while the accuracy ranged from 100 to 113%. The described method will be used in clinical studies to explore the role of erythrocytes in the overall kinetic behavior of topotecan.     

Simultaneous determination of selegiline and desmethylselegiline in human body fluids by headspace solid-phase microextraction and gas chromatography-mass spectrometry

A method for the simultaneous determination of selegiline and its metabolite, desmethylselegiline, in human whole blood and urine is presented. The method, which combines a fiber-based headspace solid-phase microextraction (SPME) technique with gas chromatography-mass spectrometry (GC-MS), required optimization of various parameters (e.g., salt additives, extraction temperatures, extraction times and the extraction properties of the SPME fiber coatings). Pargyline was used as the internal standard. Extraction efficiencies for both selegiline and desmethylselegiline were 2.0-3.4% for whole blood, and 8.0-13.2% for urine. The regression equations for selegiline and desmethylselegiline extracted from whole blood were linear (r(2)=0.996 and 0.995) within the concentration ranges 0.1-10 and 0.2-20 ng/ml, respectively. For urine, the regression equations for selegiline and desmethylselegiline were linear (r(2)=0.999 and 0.998) within the concentration ranges 0.05-5.0 and 0.1-10 ng/ml, respectively. The limit of detection for selegiline and desmethylselegiline was 0.01-0.05 ng/ml for both samples. The lower and upper limits of quantification for each compound were 0.05-0.2 and 5-20 ng/ml, respectively. Intra- and inter-day coefficients of variation for selegiline and desmethylselegiline in both samples were not greater than 8.7 and 11.7%, respectively. The determination of selegiline and desmethylselegiline concentrations in Parkinson's disease patients undergoing continuous selegiline treatment is presented and is shown to validate the present methodology.     

Dimethylsulphopropionate (DMSP) and proline from the surface of the brown alga Fucus vesiculosus inhibit bacterial attachment

It was demonstrated previously that polar and non-polar surface extracts of the brown alga Fucus vesiculosus collected during winter from the Kiel Bight (Germany) inhibited bacterial attachment at natural concentrations. The present study describes the bioassay-guided identification of the active metabolites from the polar fraction. Chromatographic separation on a size-exclusion liquid chromatography column and bioassays identified an active fraction that was further investigated using nuclear magnetic resonance spectroscopy and mass spectrometry. This fraction contained the metabolites dimethylsulphopropionate (DMSP), proline and alanine. DMSP and proline caused the anti-attachment activity. The metabolites were further quantified on the algal surface together with its associated boundary layer. DMSP and proline were detected in the range 0.12-1.08 ng cm(-2) and 0.09-0.59 ng cm(-2), respectively. These metabolites were tested in the concentration range from 0.1 to 1000 ng cm(-2) against the attachment of five bacterial strains isolated from algae and sediment co-occurring with F. vesiculosus. The surface concentrations for 50% inhibition of attachment of these strains were always <0.38 ng cm(-2) for DMSP and in four cases <0.1 ng cm(-2) for proline, while one strain required 1.66 ng cm(-2) of proline for 50% inhibition. Two further bacterial strains that had been directly isolated from F. vesiculosus were also tested, but proved to be the least sensitive. This study shows that DMSP and proline have an ecologically relevant role as surface inhibitors against bacterial attachment on F. vesiculosus.     

A label-free biosensor based on gold nanoshell monolayers for monitoring biomolecular interactions in diluted whole blood

Gold nanoshells (GNSs) were self-assembled on the surface of transparent glasses modified with 3-aminopropyltrimethoxysilane (APTES) to form GNS self-assembled monolayers (SAMs). Because the localized surface plasmon resonance (LSPR) of GNSs can be controlled in the near-infrared (NIR) region of the spectrum, where the optical transmission through tissue and whole blood is optimal, GNSs would be used as an effective signal transduction in whole blood. Accordingly, after modified with cystamine and biotin-NHS (N-hydroxy succinimide), GNS SAMs were used as a novel optical biosensor for real-time detection of streptavidin-biotin interactions in diluted human whole blood within short assay time, without any sample purification/separation. An UV-vis-NIR spectrophotometer was used to monitor the absorbance changes at 730 nm as a function of time for different concentrations of streptavidin in 20% whole blood, and the results showed that the biosensor displayed low detection limit of approximately 3 microg/mL and wide dynamic range of approximately 3-50 microg/mL. This approach provides an opportunity to construct LSPR biosensor for protein sensing and cellular analysis in diluted whole blood.     

Liquid chromatography-tandem mass spectrometry method for determination of phencynonate in rat blood and urine

A sensitive and specific high-performance liquid chromatographic assay with electrospray ionization mass spectrometry detection (LC-ESI-MS) has been developed and validated for the identification and quantification of the novel anticholinergic drug phencynonate in rat blood and urine. The sample pretreatment involves basification and iterative liquid-liquid extraction with ethyl ether-dichloromethane (2:1, v/v) solution, followed by LC separation and positive electrospray ionization mass spectrometry detection. The chromatography was on BetaBasic-18 column (150 mm x 2.1mm i.d., 3 microm). The mobile phase was composed of methanol-water (85:15, v/v), containing 0.5 per thousand formic acid, which was pumped at a flow-rate of 0.2 ml/min. Thiencynonate was selected as the internal standard (IS). Simultaneous MS detection of phencynonate and IS was performed at m/z 358.4 (phencynonate), m/z 364 (thiencynonate), and the selected reaction ion monitoring (SRM) of the two compounds was at 156. Phencynonate eluted at approximately 5.25 min, thiencynonate eluted at approximately 5.10 min and no endogenous materials interfered with their measurement. Linearity was obtained over the concentration range of 1-100 ng/ml in rat blood and 1-500 ng/ml in rat urine. The lower limit of quantification (LLOQ) was reproducible at 1 ng/ml in both of rat blood and urine. The precision measured was obtained from 2.92 to 9.76% in rat blood and 4.17 to 9.76% in rat urine. Extraction recoveries were in the range of 69.57-79.49% in blood and 56.85-64.86% in urine. This method was successfully applied to the identification and quantification of phencynonate in pharmacokinetic studies.     

Quantitative determination of Astragaloside IV, a natural product with cardioprotective activity, in plasma, urine and other biological samples by HPLC coupled with tandem mass spectrometry

Astragaloside IV is a novel cardioprotective agent extracted from the Chinese medical herb Astragalus membranaceus (Fisch) Bge. This agent is being developed for treatment for cardiovascular disease. Further development of Astragaloside IV will require detailed pharmacokinetic studies in preclinical animal models. Therefore, we established a sensitive and accurate high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (LC/MS/MS) quantitative detection method for measurement of Astragaloside IV levels in plasma, urine as well as other biological samples including bile fluid, feces and various tissues. Extraction of Astragaloside IV from plasma and other biological samples was performed by Waters OASIS(trade mark) solid phase extraction column by washing with water and eluting with methanol, respectively. An aliquot of extracted residues was injected into LC/MS/MS system with separation by a Cosmosil C18 5 microm, 150 mm x 2.0 mm) column. Acetonitrile:water containing 5 microM NaAc (40:60, v/v) was used as a mobile phase. The eluted compounds were detected by tandem mass spectrometry. The average extraction recoveries were greater than 89% for Astragaloside IV and digoxin from plasma, while extraction recovery of Astragaloside IV and digoxin from tissues, bile fluid, urine and fece ranged from 61 to 85%, respectively. Good linearity (R2>0.9999) was observed throughout the range of 10-5000 ng/ml in 0.5 ml rat plasma and 5-5000 ng/ml in 0.5 ml dog plasma. In addition, good linearity (R2>0.9999) was also observed in urine, bile fluid, feces samples and various tissue samples. The overall accuracy of this method was 93-110% for both rat plasma and dog plasma. Intra-assay and inter-assay variabilities were less than 15.03% in plasma. The lowest quantitation limit of Astragaloside IV was 10 ng/ml in 0.5 ml rat plasma and 5 ng/ml in 0.5 ml dog plasma, respectively. Practical utility of this new LC/MS/MS method was confirmed in pilot pharmacokinetic studies in both rats and dogs following intravenous administration.     

一种适用于双向电泳的水稻磷酸化蛋白富集方法——酚提取法结合固相金属离子亲和层析法

磷蛋白在植物信号传导和胁迫响应中有着非常重要的作用,磷蛋白质组学研究已经成为蛋白质组学研究领域中备受瞩目的一个部分．本研究用酚提取法以水稻日本晴苗期叶片为材料提取叶片总蛋白,提取率达3.5%;用固相金属离子亲和层析柱纯化富集磷蛋白,得到磷蛋白占总蛋白约6.4%．对过柱洗涤液、不同阶段洗脱液等各个组分进行SDS-PAGE,粗略检测其蛋白含量,并根据单向SDS PAGE结果对总蛋白、高峰段磷蛋白、非高峰段磷蛋白以及富集后再纯化的总磷蛋白进行双向电泳,比较其中的蛋白差异．本研究提出的方法和程序可在7 cm聚丙烯酰胺凝胶上检测到多达856个磷蛋白,是一种非常有效的磷蛋白富集、纯化和分离鉴定的方法．     

Analysis of proteolytic degradation of a crude protein mixture using a surface acoustic wave sensor

Degradation of a crude protein mixture by proteases with pH optima from acidic to basic was followed in real time using a surface acoustic wave biosensor in Love-wave geometry. Proteases EC 3.4.23.18 from Aspergillus saitoi, EC 3.4.21.62 from Bacillus licheniformis, and Novozyme from Bacillus sp. have been used. Kinetic constants extracted from the curves resulted for comparable protease concentrations in maximal degradation rates between 1.1 x 10(-2) and 1.5 x 10(-2)s(-1). For the three proteases investigated, similar amounts of up to about two-thirds of the estimated 28 ng/cm2 bound molecules were proteolyzed. The residual material not degraded by the proteases was removed from the surface with 0.5% SDS. The analysis of the sensor signal allows: (1) estimation of the total mass of protein bound to the sensor surface and of the degradable fraction; (2) extraction of the pure mass signal; and (3) kinetic evaluation.     

Rapid, automated nucleic acid probe assays using silicon microstructures for nucleic acid concentration

A system for rapid point-of-use nucleic acid (NA) analysis based on PCR techniques is described. The extraction and concentration of DNA from test samples has been accomplished utilizing silicon fluidic microchips with high surface-area-to-volume ratios. Short (500 bp) and medium size (48,000 bp) DNA have been captured, washed, and eluted using the silicon dioxide surfaces of these chips. Chaotropic (GuHCl) salt solutions were used as binding agents. Wash and elution agents consisted of ethanol-based solutions and water, respectively. DNA quantities approaching 40 ng/cm2 of binding area were captured from input solutions in the 100-1000 ng/mL concentration range. For dilute samples of interest for pathogen detection, PCR and gel electrophoresis were used to demonstrate extraction efficiencies of about 50 percent, and concentration factors of about 10x using bacteriophage lambda DNA as the target. Rapid, multichannel PCR thermal cycling modules with integrated solid-state detection components have also been demonstrated. These results confirm the viability of utilizing these components as elements of a compact, disposable cartridge system for the detection of NA in applications such as clinical diagnostics, biowarfare agent detection, food quality control, and environmental monitoring.     

Reduction of extraction times in liquid-phase microextraction

Recently, we introduced a simple and inexpensive disposable device for liquid-phase microextraction (LPME) based on porous polypropylene hollow fibres. In the present paper, extraction times were significantly reduced by an increase in the surface of the hollow fibres. The model compounds methamphetamine and citalopram, were extracted from 2.5 ml of urine, plasma, and whole blood after dilution with water and alkalisation with 125 μl of 2 M NaOH though a porous polypropylene hollow fibre impregnated with hexyl ether and into an aqueous acceptor phase consisting of 0.1 M HCl. Two commercially available hollow fibres, which differed in surface area, wall thickness and internal diameter, were compared. An increase in the contact area of the hollow fibre with the sample solution by a factor of approximately two resulted in reduction in equilibrium times by approximately the same factor. Thus, the model compounds were extracted to equilibrium within 15 min from both urine and plasma, and within 30 min from whole blood. For the first time LPME was utilised to extract drugs from whole blood, and the extracts were comparable with plasma both with regard to sample clean-up and extraction recoveries. Extraction recoveries for methamphetamine and citalopram varied from 60 to 100% using the two fibres and the different matrices.     

Multisample parallel array fraction collector: application in DNA alkaline elution studies

A fraction collector designed specifically to be compatible with the analytical techniques of alkaline and neutral elution of DNA from carcinogen-treated cells is described. This apparatus, which is operated by microchip controls, permits simultaneous collection of as many as 20 eluted samples directly into standard- or small-size scintillation vials contained in manufacturer's shipping cartons. This instrument saves operator time, offers considerable flexibility, and has several fail-safe features.     

A sensitive high performance liquid chromatography-positive electrospray tandem mass spectrometry method for N7-[2-[(2-hydroxyethyl)thio]-ethyl]guanine determination

A simple, fast, sensitive and robust method for the determination of the sulfur mustard (SM) exposure biomarker-N(7)-[2-[(2-hydroxyethyl)thio]-ethyl]guanine (HETEG) was reported using high-performance liquid chromatography-positive electrospray tandem mass spectrometry (HPLC-ESI-MS/MS), working in multiple reaction monitor (MRM) mode. The method provided limit of detection of 0.330 ng/mL and lower limit of quantitation of 0.940 ng/mL. The method was linearly calibrated from 0.940 ng/mL to 587 ng/mL with precisions of 3.5-14.5%, and accuracies of 88-112%. The recovery varied from 102% to 118%. HETEG spiked in DNA hydrolytes isolated from the human whole blood was stable after five freeze/thaw cycles and 35-day frozen at -20°C. For the exposed biological samples, alkylated DNA was isolated from SM-treated human whole blood, followed by DNA digestion and adducts enrichment, the resulting alkylation base was determined. By the procedure, the HETEG level in DNA hydrolytes isolated from the human whole blood exposure to 312 ng/mL SM was detected successfully.     

Towards a rapid molecular diagnostic for melioidosis: Comparison of DNA extraction methods from clinical specimens

Optimising DNA extraction from clinical samples for Burkholderia pseudomallei Type III secretion system real-time PCR in suspected melioidosis patients confirmed that urine and sputum are useful diagnostic samples. Direct testing on blood remains problematic; testing DNA extracted from plasma was superior to DNA from whole blood or buffy coat.     

Chip PCR. II. Investigation of different PCR amplification systems in microbabricated silicon-glass chips.

We examined PCR in silicon dioxide-coated silicon-glass chips (12 microl in volume with a surface to volume ratio of approximately 17.5 mm(2)/microl) using two PCR reagent systems: (i) the conventional reagent system using Taq DNA polymerase; (ii) the hot-start reagent system based on a mixture of TaqStart antibody and Taq DNA polymerase. Quantitative results obtained from capillary electrophoresis for the expected amplification products showed that amplification in microchips was reproducible (between batch coefficient of variation 7.71%) and provided excellent yields. We also used the chip for PCR directly from isolated intact human lymphocytes. The amplification results were comparable with those obtained using extracted human genomic DNA. This investigation is fundamental to the integration of sample preparation, polynucleotide amplification and amplicate detection on a microchip.     

Sensitive determination of pethidine in body fluids by surface ionization organic mass spectrometry

We have presented a simple and sensitive method for determining pethidine, a narcotic analgesic drug in body fluids by gas chromatography (GC)/surface ionization organic mass spectrometry (SIOMS). Good linearity was obtained in the range of 0.625–25 ng/ml of whole blood and urine by mass chromatography, and in the range of 0.05–2 ng/ml of whole blood by selected ion monitoring (SIM). Pethidine and diphenylpyraline (internal standard) were extracted from body fluids with Bond Elut Certify cartridges; their recoveries were above 95%. The detection limits (signal-to-noise ratio=3) were estimated to be 0.2 ng/ml of whole blood or urine by mass chromatography, 0.02 ng/ml of whole blood by SIM.