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PubMed1995

Sample preparation for Chlamydia pneumoniae PCR.

Fifty-three clinical specimens taken from the retropharyngeal mucosa of patients with longstanding respiratory tract infections were analyzed by polymerase chain reaction using two different methods for sample preparation. All specimens were divided into two aliquots, one treated with proteinase K, and the other with the Amplicor sputum sample preparation kit. All tests were run in parallel, employing a primer pair specific for Chlamydia pneumoniae. Of the samples prepared with the Amplicor kit 20.8% were found to be positive, as compared to 7.5% of the samples prepared with proteinase K. The outcome of C. pneumoniae PCR was improved by treatment involving a more complete lysis of cells derived from the specimen.

Chlamydia Infections↗
PubMed2003

Measurement uncertainty from physical sample preparation: estimation including systematic error.

A methodology is proposed, which employs duplicated primary sampling and subsequent duplicated physical preparation coupled with duplicated chemical analyses. Sample preparation duplicates should be prepared under conditions that represent normal variability in routine laboratory practice. The proposed methodology requires duplicated chemical analysis on a minimum of two of the sample preparation duplicates. Data produced from the hierarchical design is treated with robust analysis of variance (ANOVA) to generate uncertainty estimates, as standard uncertainties ('u' expressed as standard deviation), for primary sampling (ssamp), physical sample preparation (sprep) and chemical analysis (sanal). The ANOVA results allow the contribution of the sample preparation process to the overall uncertainty to be assessed. This methodology has been applied for the first time to a case study of pesticide residues in retail strawberry samples. Duplicated sample preparation was performed under ambient conditions on two consecutive days. Multi-residue analysis (quantification by GC-MS) was undertaken for a range of incurred pesticide residues including those suspected of being susceptible to loss during sample preparation procedures. Sampling and analytical uncertainties dominated at low analyte concentrations. The sample preparation process contributed up to 20% to the total variability and had a relative uncertainty (Uprep%) of up to 66% (for bupirimate at 95% confidence). Estimates of systematic errors during physical sample preparation were also made using spike recovery experiments. Four options for the estimation of measurement uncertainty are discussed, which both include and exclude systematic error arising from sample preparation and chemical analysis. A holistic approach to the combination and subsequent expression of uncertainty is advised.

Calibration↗
PubMed2006

Three-dimensional analysis of single particles by electron microscopy: sample preparation and data acquisition.

Electron microscopy of single particles has recently become a very popular field in both biological and material sciences. It might be difficult for a novice researcher new to this field to know how to start tackling a new project. This chapter is designed to serve as a guideline for anyone starting a new project to determine a three-dimensional structure using single-particle techniques. The chapter describes the basic techniques necessary to prepare the samples and acquire the data to calculate a three-dimensional reconstruction in easy-to-understand, step-by-step instructions. It starts with the basic preparation of support films and the usage of a variety of staining techniques needed to assess the quality of the sample and the viability of the project. It ends with a detailed description of vitreous ice preparations designed to acquire high-resolution structural information. Guidelines and tips are given on how to record the best images with an electron microscope. Although this chapter is geared to researchers new to the field, experts might find it not only useful as a reference but also valuable because of the number of practical tips included.

Coloring Agents↗
PubMed2002

Sample preparation for amino acid determination by integrated pulsed amperometric detection in foods.

This report describes a new sample preparation method for food which allows a complete separation of carbohydrates and amino acids prior to their analysis by anion-exchange chromatography and integrated pulsed amperometric detection. Food samples with high carbohydrate concentrations are applied to solid-phase extraction columns containing a strong cation-exchange resin. Carbohydrates are recovered initially; retained amino acids are eluted with 0.2 M CaC l(2) subsequently. The carbohydrate and the amino acid fractions are analyzed. The recovery calculated for 21 amino acids was in the range from 84 to 126%. The sample preparation was tested for amino acid concentrations between 4.2 and 84.0 nmol of each amino acid (between 2.1 and 42.0 nmol of cystine) and correlation coefficients between 0.84 and 0.99 were obtained. The capacity of the solid-phase extraction columns employed was up to 3.7 micro mol. Sample preparation was evaluated with four different food samples: sourdough, skim milk, lemon juice, and potato.

Absorption↗
PubMed2004

Restricted access materials and large particle supports for on-line sample preparation: an attractive approach for biological fluids analysis.

An analytical process generally involves four main steps: (1) sample preparation; (2) analytical separation; (3) detection; and (4) data handling. In the bioanalytical field, sample preparation is often considered as the time-limiting step. Indeed, the extraction techniques commonly used for biological matrices such as liquid-liquid extraction (LLE) and solid-phase extraction (SPE) are achieved in the off-line mode. In order to perform a high throughput analysis, efforts have been engaged in developing a faster sample purification process. Among different strategies, the introduction of special extraction sorbents, such as the restricted access media (RAM) and large particle supports (LPS), allowing the direct and repetitive injection of complex biological matrices, represents a very attractive approach. Integrated in a liquid chromatography (LC) system, these extraction supports lead to the automation, simplification and speeding up of the sample preparation process. In this paper, RAM and LPS are reviewed and particular attention is given to commercially available supports. Applications of these extraction supports, are presented in single column and column-switching configurations, for the direct analysis of compounds in various biological fluids.

Animals↗
PubMed2005

Optimising uncertainty in physical sample preparation.

Uncertainty associated with the result of a measurement can be dominated by the physical sample preparation stage of the measurement process. In view of this, the Optimised Uncertainty (OU) methodology has been further developed to allow the optimisation of the uncertainty from this source, in addition to that from the primary sampling and the subsequent chemical analysis. This new methodology for the optimisation of physical sample preparation uncertainty (u(prep), estimated as s(prep)) is applied for the first time, to a case study of myclobutanil in retail strawberries. An increase in expenditure (+7865%) on the preparatory process was advised in order to reduce the s(prep) by the 69% recommended. This reduction is desirable given the predicted overall saving, under optimised conditions, of 33,000 pounds Sterling per batch. This new methodology has been shown to provide guidance on the appropriate distribution of resources between the three principle stages of a measurement process, including physical sample preparation.

Data Interpretation, Statistical↗
PubMed2004

Ultrafiltration for proteomic sample preparation.

Proteome analysis represents significant challenges to the existing sample preparation techniques. Traditional methods, such as two-dimensional electrophoresis, typically separate high-molecular-weight proteins while discarding low-molecular-weight species. This approach is well justified considering the complexity of any proteome. However, it is desirable to extract the maximum amount of information from each sample to investigate the entire range of biomolecules. We have demonstrated that ultrafiltration not only improves two-dimensional electrophoresis (2-DE) resolution of the protein fraction but also yields the low-molecular-weight fraction amenable for further analysis by high-resolution mass spectrometry. This approach was successfully adapted to the variety of biological samples including cell and tissue lysates and serum. Therefore, ultrafiltration offers an alternative sample preparation technique that enables more thorough analysis of a proteome.

Animals↗
PubMed2000

A MALDI sample preparation method suitable for insoluble polymers

Polyamides are insoluble or poorly soluble in common organic solvents, which makes normal sample preparation for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry very difficult. An new analytical protocol for MALDI analysis of polyamides or other insoluble samples is described. It consists of pressing a pellet from a solid mixture of the polymer and a matrix, both in the form of finely ground powder. This sample preparation is compared with the common dried droplet sample preparation method and found to perform much better, both in terms of robustness against variation of experimental parameters and high-mass capability.

Journal Article↗
PubMed2001

MEMS-based sample preparation for molecular diagnostics.

Completion of the Human Genome Project is driving the rapid development of molecular diagnostics in the laboratory. To accelerate the penetration of genetic tests and other nucleic acid-based tests into clinical markets, simple, compact, automatic sample-preparation systems for molecular diagnostics must be developed. Microelectromechanical systems (MEMS) is a promising approach for the development of automated sample preparation for the clinical laboratory or point-of-care setting. This review discusses MEMS-based components that could be applied to the different stages of the sample-preparation process such as cell separation, nucleic acid purification, and nucleic acid amplification. Examples of functional component integration are given. Issues discussed include partitioning of functions between the instrument and disposable unit, methods of propulsion of fluids and particles, vapor and liquid barriers, and sample size. Although further evaluation and development are needed to provide practical solutions to some of these issues, we conclude that MEMS-based components might contribute to some components in a sample-preparation system consisting of modular instruments and disposable units, but will not provide a generic or a totally integrated solution.

DNA, Bacterial↗
PubMed2005

Determination of gross alpha, 224Ra, 226Ra, and 228Ra activities in drinking water using a single sample preparation procedure.

The current federal and New Jersey State regulations have greatly increased the number of gross alpha and radium tests for public and private drinking water supplies. The determination of radium isotopes in water generally involves lengthy and complicated processes. In this study, a new approach is presented for the determination of gross alpha, 224Ra, 226Ra, and 228Ra activities in water samples. The method includes a single sample preparation procedure followed by alpha counting and gamma-ray spectroscopy. The sample preparation technique incorporates an EPA-approved co-precipitation methodology for gross alpha determination with a few alterations and improvements. Using 3-L aliquots of sample, spiked with 133Ba tracer, the alpha-emitting radionuclides are isolated by a BaSO4 and Fe(OH)3 co-precipitation scheme. First the gross alpha-particle activity of the sample is measured with a low-background gas-flow proportional counter, followed by radium isotopes assay by gamma-ray spectroscopy, using the same prepared sample. Gamma-ray determination of 133Ba tracer is used to assess the radium chemical recovery. The 224Ra, 226Ra, and 228Ra activities in the sample are measured through their gamma-ray-emitting decay products, 212Pb, 214Pb/214Bi, and 228Ac, respectively. In cases where 224Ra determination is required, the gamma-ray counting should be performed within 2-4 d from sample collection. To measure 226Ra activity in the sample, the gamma-ray spectroscopy can be repeated 21 d after sample preparation to ensure that 226Ra and its progeny have reached the equilibrium state. At this point, the 228Ac equilibration with parent 228Ra is already established. Analysis of aliquots of de-ionized water spiked with NIST-traceable 230Th, 224Ra, 226Ra, and 228Ra standards demonstrated the accuracy and precision of this method. Various performance evaluation samples were also assayed for gross alpha as well as radium isotope activity determination using this procedure and the results were in close agreement with the assigned values. In addition, method comparison results of actual sample analyses agreed well with the ones performed using EPA-approved procedures. With a 3-L sample aliquot and 1,000-min counting time, the average gross alpha minimum detectable concentration (MDC) was about 0.002 Bq L(-1). The average MDC's for 224Ra, 226Ra, and 228Ra were 0.034 Bq L(-1), 0.017 Bq L(-1), and 0.036 Bq L(-1), respectively, based on a 3-L sample aliquot, 85% chemical yield, 40% intrinsic Ge detector, and 1,000-min count time. This method combines and simplifies the analytical procedures and reduces labor while achieving the precision, accuracy, and minimum detection limit requirements of EPA regulations.

Alpha Particles↗
PubMed2002

Effects of the sample preparation temperature on the nanostructure of compression moulded ultrahigh molecular weight polyethylene.

In this study, the effects of the sample sectioning temperature on the surface nanostructure and mechanical response of compression moulded ultrahigh molecular weight polyethylene (UHMWPE) at a nanometer scale (nanomechanical properties) have been characterized. The primary focus of this work was to determine if the sample sectioning temperature significantly changed the nanostructure of UHMWPE, while the secondary focus was to characterize the effect on the mechanical response due to the changes in the sectioned surface nanostructure. The goals of this study were: (a) to investigate the potential possibility of creating surface artefacts by the sample preparation technique by sectioning at different temperatures relative to the published range of glass transition temperatures, Tg, for PE (-12, -80 and -25 degrees C); (b) to determine the possibility of molecular orientation induced by plastic deformation of the UHMWPE sample during the process of sample preparation; (c) to measure the relative difference in nanomechanical properties owing to evolution of different nanostructures as a function of sample sectioning temperature. Field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM) and nanoindentation were used to demonstrate that the sectioning temperature caused a change in nanostructure of the compression moulded UHMWPE sectioned surface, explaining the change in mechanical response to indentation at a nanoscale. In this study, it was demonstrated that significant plastic deformation occurs when a shear stress is applied between the glass or diamond blade and the UHMWPE during sample preparation under ambient conditions at a temperature of 22 degrees C. These results also suggest that an optimum sample sectioning temperature should definitely be below the measured Tg of the polymer.

Artifacts↗
PubMed2002

Development of microwave-assisted drying methods for sample preparation for dried spot micro-X-ray fluorescence analysis.

Although dried spot micro X-ray fluorescence (MXRF) is an effective analytical technique for trace elemental analysis, the sample preparation procedures currently used suffer from a number of drawbacks. These drawbacks include relatively long preparation times, lack of control of the sample preparation environment, and possibility of loss of volatile analytes during the drying process. Microwave-assisted drying offers several advantages for dried spot preparation, including control of the environment and minimized volatility because of the differences between microwave heating and conventional heating. A microwave-assisted drying technique has been evaluated for use in preparing dried spots for trace analysis. Two apparatus designs for microwave drying were constructed and tested using multielement standard solutions, a standard reference material, and a "real-world" semiconductor cleaning solution. Following microwave-assisted drying of these aqueous samples, the residues were redissolved and analyzed by ICPMS. Effective recovery was obtained using the microwave drying methods, demonstrating that the microwave drying apparatus and methods described here may be more efficient alternatives for dried spot sample preparation.

Journal Article↗
PubMed2003

Sample preparation for two-dimensional gel electrophoresis.

The choice of sample preparation protocol is a critical influential factor for isoelectric focusing which in turn affects the two-dimensional gel result in terms of quality and protein species distribution. The optimal protocol varies depending on the nature of the sample for analysis and the properties of the constituent protein species (hydrophobicity, tendency to form aggregates, copy number) intended for resolution. This review explains the standard sample buffer constituents and illustrates a series of protocols for processing diverse samples for two-dimensional gel electrophoresis, including hydrophobic membrane proteins. Current methods for concentrating lower abundance proteins, by removal of high abundance proteins, are also outlined. Finally, since protein staining is becoming increasingly incorporated into the sample preparation procedure, we describe the principles and applications of current (and future) pre-electrophoretic labelling methods.

Buffers↗
PubMed2000

The use of immuno-magnetic separation (IMS) as a tool in a sample preparation method for direct detection of L. monocytogenes in cheese.

A sample preparation procedure was developed for direct detection of L. monocytogenes in cheese. The sample preparation protocol consisted of a 10-fold dilution and homogenization, a centrifugation step to precipitate large food particles, passage of the supernatant over a sieve and through a separatory funnel to further eliminate food particles and fat, a centrifugation step to recover the bacterial pellet and finally enzymatic digestion of the suspension to degrade the remaining small food particles. Recovery of L. monocytogenes was confirmed by plating on Oxford medium and confirmation of suspected colonies. This protocol enabled direct detection (without prior enrichment) of low numbers of L. monocytogenes (0.5-1.5 cfu/g cheese) from different types of cheese. The performance of Dynabeads Anti-Listeria (Dynal, Oslo, Norway) for selective recovery of L. monocytogenes and their applicability in the above mentioned procedure for direct detection of low numbers of L. monocytogenes from cheese was evaluated. IMS could not separate and recover L. monocytogenes from the food particles in the concentrated suspension. The use of IMS after a 24 h enrichment procedure (as recommended by the manufacturer) allowed for the detection of low numbers of L. monocytogenes (< 10 cfu/g). However, experiments in broth cultures showed that although the detection limit of IMS with Dynabeads Anti-Listeria was 40-100 cfu/ml, the ratio of L. monocytogenes to non-Listeria flora was not increased. Thus, selective enrichment or concentration of L. monocytogenes was not obtained.

Animals↗
PubMed1987

Degradation artefacts during sample preparation for sodium dodecyl sulphate polyacrylamide gel electrophoresis.

Preparation of samples for sodium dodecyl sulphate polyacrylamide gel electrophoresis routinely involves heating the protein in solution containing detergent and reducing agent for at least two minutes. Here we show that this treatment causes fragmentation of the protein glycogen phosphorylase, whether purified or as a component of a skeletal muscle preparation. The fragments are detected as minor bands on western blots and represent the products of discrete breakage point in the peptide sequence. Protease inhibitors cannot suppress the fragmentation. Such small amounts of immunoreactive fragments may be incorrectly identified on western blots as contaminants that were originally present in the antigen preparation. They may also be a source of ambiguity in studies that search for degradation intermediates during proteolysis.

Animals↗
PubMedDate not supplied

Automated MALDI-TOF-MS sample preparation in combinatorial polymer research.

A new automated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) sample spotting technique that allows the integration of MALDI sample preparation in the workflow of combinatorial polymer research is described. The technique is performed utilizing a commercially available synthetic robot and was first evaluated with polymer standards of known composition and later on used for the monitoring of the living cationic ring-opening polymerization of 2-ethyl-2-oxazoline. The spotting was carried out as a multiple layer approach, which offers the ability of complex sample preparation without the requirement of premixing the different components. The described technique reduces the time required for sample preparation and offers the possibility of automated sample spotting during polymerization reactions performed in a synthetic robot. This allows the integration of molecular weight screening and polymer end/group determination utilizing MALDI-TOF-MS as a high-throughput tool in combinatorial polymer research.

Journal Article↗
PubMed2006

Biological validation of a sample preparation method for ER-CALUX bioanalysis of estrogenic activity in sediment using mixtures of xeno-estrogens.

The combined estrogenic effects of mixtures of environmental pollutants in the in vitro ER-CALUX (chemical activated luciferase gene expression) bioassaywere examined to biologically validate a sample preparation method for the analysis of estrogenic compounds in sediment. The method used accelerated solvent extraction (ASE) and gel permeation chromatography (GPC) and was validated with respect to recovery of biological response taking mixture effects into account. Four mixtures of three to six xenoestrogenic compounds (bisphenol A, 4-nonylphenol, (4,4'-dichlorodiphenyl)trichloroethane, (2,4'-dichlorodiphenyl)trichloroethane, dieldrin, 4-n-octylphenol, alpha-chlordane, dibutylphthalate, (4,4'-dichlorodiphenyl)dichloroethylene, and 2,4,5-trichlorobiphenyl) were prepared. Experimentally determined mixture effects were well described by the concept of concentration addition (CA), as expected for similarly acting compounds. Observed estradiol equivalence factors of the mixtures (on average 1.2 +/- 0.3) agreed very well with the value predicted according to CA. The sample preparation method was then applied to pure mixtures of standards and to sediment spiked with one of the mixtures. Recoveries of estrogenic compounds were estimated by determination of their mixture potencies in ER-CALUX and compared to the mixture effects predicted by CA. Recoveries of estrogenic activity were between 80 and 129%, indicating that the additive behavior of mixtures of xeno-estrogens is well conserved during sample preparation. Together with an average repeatability of 18.3%, low average limit of detection (2.6 +/- 1.8 pg of EEQ/ g), and coefficient of variance (3.5 +/- 3.3%),this demonstrated the suitability of the sample preparation method for the analysis of mixtures of (xeno-)estrogenic compounds in sediment with the ER-CALUX assay.

Chromatography, Gel↗
PubMed2000

Investigation of Fasciola hepatica sample preparation for two-dimensional electrophoresis.

This paper investigates the preparation of Fasciola hepatica samples for two-dimensional electrophoresis (2-DE). Whole samples were prepared by both hot sodium dodecyl sulfate (SDS) solubilisation and precipitation using trichloroacetic acid (TCA) to remove nonprotein contaminants and to inactivate endogenous proteases. Sample preparation had a marked influence on the 2-DE gel profile. TCA precipitation resulted in no measurable improvement in the profile observed, compared to the untreated control. Solubilisation of sample with hot SDS increased the number of protein spots, as did TCA precipitation with the addition of phosphotungstic acid. The preparation of excretory-secretory (ES) products poses problems due to both high salt concentrations and low protein concentration. All precipitation methods used to overcome this gave similar profiles, except acetone alone, which caused depletion of the larger proteins. TCA in acetone gave the best result, similar to that obtained by centrifugal filtration of the sample. Overcrowding of spots in some regions of the 2-DE gel occurred in the whole Fasciola hepatica sample. This problem was alleviated by differential solubilisation, which also resulted in the enrichment of some proteins.

Animals↗