Automated Sample Preparation System Research Articles

A Study on Trapping CO2 Using Molecular Sieve for 14C AMS Sample Preparation

At the Seoul National University accelerator mass spectrometry (AMS) laboratory, we are planning to develop an automated sample preparation system for higher throughput of radiocarbon dating. This system will consist of several sections, including a combustion line, CO2 trap, graphitization system, and so on. We usually collect CO2 by cryogenic trapping. However, since handling liquid nitrogen is expected to be rather difficult, we are interested in replacing the cryogenic method with the molecular sieve method for the collection of CO2. In this study, we compare the performance of the cryogenic trapping method and molecular sieve method. Zeolite 13X is used as a molecular sieve, and as test samples we use the oxalic acid standard (NIST SRM 4990C), high-purity graphite powder, and archaeological charcoals. The pMC values and the radiocarbon ages (BP) obtained from samples prepared by the above 2 methods are very similar. We especially focused on the memory effect of the molecular sieve, meaning the CO2 contamination from a previous sample, which can cause errors in age determination. To reduce this effect, we flowed He gas through a zeolite container for several minutes at a high temperature before the CO2 was introduced. By the adding this step, we have obtained more reliable results.

A Study on Trapping CO2 by Using a Molecular Sieve for 14C AMS Sample Preparation

At the Seoul National University accelerator mass spectrometry (AMS) laboratory, we are planning to develop an automated sample preparation system for higher throughput of radiocarbon dating. This system will consist of several sections, including a combustion line, CO2 trap, graphitization system, and so on. We usually collect CO2 by cryogenic trapping. However, since handling liquid nitrogen is expected to be rather difficult, we are interested in replacing the cryogenic method with the molecular sieve method for the collection of CO2. In this study, we compare the performance of the cryogenic trapping method and molecular sieve method. Zeolite 13X is used as a molecular sieve, and as test samples we use the oxalic acid standard (NIST SRM 4990C), high-purity graphite powder, and archaeological charcoals. The pMC values and the radiocarbon ages (BP) obtained from samples prepared by the above 2 methods are very similar. We especially focused on the memory effect of the molecular sieve, meaning the CO2 contamination from a previous sample, which can cause errors in age determination. To reduce this effect, we flowed He gas through a zeolite container for several minutes at a high temperature before the CO2 was introduced. By the adding this step, we have obtained more reliable results.

Pre-PCR treatments as a key factor on the probability of detection of Listeria monocytogenes and Salmonella in ready-to-eat meat products by real-time PCR

The aim of this study was to compare the performance of different pre-PCR treatments for the detection of Listeria monocytogenes and Salmonella spp. by real-time PCR in spiked ready-to-eat meat samples (cooked ham, dry-cured ham and fermented sausage). Three different pre-PCR treatments were assayed: (i) PrepMan® Ultra Sample Preparation Reagent, (ii) DNA purification using DNeasy Tissue Kit and QIAcube automated sample preparation system, and (iii) BAX® system. The analysis range was 2 to 2 × 106 CFU/ml. PCR was done in quintuplicate and the experiment was repeated five times. The ‘probability of detection’ was used to compare the efficiency of the pre-PCR treatments and to calculate the theoretical limit of detection of each treatment.The best pre-PCR treatment was DNA purification using DNeasy Tissue kit and QIAcube; it showed the highest detection probability and the lowest limit of detection for each food type and pathogen assayed, followed by PrepMan and BAX treatments. Probability of detection showed differences among food matrices and between L. monocytogenes and Salmonella detection probability. Despite being an expensive method, DNeasy Tissue kit would be the recommended treatment to reduce the risk of false negative results and to improve detection of foodborne pathogens in RTE-meat products by real-time PCR.

A Quantitative Image Cytometry Technique for Time Series or Population Analyses of Signaling Networks

BackgroundModeling of cellular functions on the basis of experimental observation is increasingly common in the field of cellular signaling. However, such modeling requires a large amount of quantitative data of signaling events with high spatio-temporal resolution. A novel technique which allows us to obtain such data is needed for systems biology of cellular signaling.Methodology/Principal FindingsWe developed a fully automatable assay technique, termed quantitative image cytometry (QIC), which integrates a quantitative immunostaining technique and a high precision image-processing algorithm for cell identification. With the aid of an automated sample preparation system, this device can quantify protein expression, phosphorylation and localization with subcellular resolution at one-minute intervals. The signaling activities quantified by the assay system showed good correlation with, as well as comparable reproducibility to, western blot analysis. Taking advantage of the high spatio-temporal resolution, we investigated the signaling dynamics of the ERK pathway in PC12 cells.Conclusions/SignificanceThe QIC technique appears as a highly quantitative and versatile technique, which can be a convenient replacement for the most conventional techniques including western blot, flow cytometry and live cell imaging. Thus, the QIC technique can be a powerful tool for investigating the systems biology of cellular signaling.

An automated sample preparation system with mini-reactor to isolate and process submegabase fragments of bacterial DNA

Existing methods for extraction and processing of large fragments of bacterial genomic DNA are manual, time-consuming, and prone to variability in DNA quality and recovery. To solve these problems, we have designed and built an automated fluidic system with a mini-reactor. Balancing flows through and tangential to the ultrafiltration membrane in the reactor, cells and then released DNA can be immobilized and subjected to a series of consecutive processing steps. The steps may include enzymatic reactions, tag hybridization, buffer exchange, and selective removal of cell debris and by-products of the reactions. The system can produce long DNA fragments (up to 0.5 Mb) of bacterial genome restriction digest and perform DNA tagging with fluorescent sequence-specific probes. The DNA obtained is of high purity and floating free in solution, and it can be directly analyzed by pulsed-field gel electrophoresis (PFGE) or used in applications requiring submegabase DNA fragments. PFGE-ready samples of DNA restriction digests can be produced in as little as 2.1 h and require less than 10 8 cells. All fluidic operations are automated except for the injection of the sample and reagents.

Performance evaluation of the QIAGEN EZ1 DSP Virus Kit with Abbott RealTi me HIV-1, HBV and HCV assays

Background Automated sample preparation systems must meet the demands of routine diagnostics laboratories with regard to performance characteristics and compatibility with downstream assays. Objectives In this study, the performance of QIAGEŃs EZ1 DSP Virus Kit on the BioRobot EZ1 DSP was evaluated in combination with the Abbott RealTi me HIV-1, HCV, and HBV assays, followed by thermalcycling and detection on the Abbott m2000 rt platform. Study design The following performance characteristics were evaluated: linear range and precision, sensitivity, cross-contamination, effects of interfering substances and correlation. Results Linearity was observed within the tested ranges (for HIV-1: 2.0–6.0 log copies/ml, HCV: 1.3–6.9 log IU/ml, HBV: 1.6–7.6 log copies/ml). Excellent precision was obtained (inter-assay standard deviation for HIV-1: 0.06–0.17 log copies/ml (>2.17 log copies/ml), HCV: 0.05–0.11 log IU/ml (>2.09 log IU/ml), HBV: 0.03–0.07 log copies/ml (>2.55 log copies/ml)), with good sensitivity (95% hit rates for HIV-1: 50 copies/ml, HCV: 12.5 IU/ml, HBV: 10 IU/ml). No cross-contamination was observed, as well as no negative impact of elevated levels of various interfering substances. In addition, HCV and HBV viral load measurements after BioRobot EZ1 DSP extraction correlated well with those obtained after Abbott m2000 sp extraction. Conclusions This evaluation demonstrates that the QIAGEN EZ1 DSP Virus Kit provides an attractive solution for fully automated, low throughput sample preparation for use with the Abbott RealTi me HIV-1, HCV, and HBV assays.

Application of Isothermal Helicase-Dependent Amplification with a Disposable Detection Device in a Simple Sensitive Stool Test for Toxigenic Clostridium difficile

Enzyme immunoassays (EIAs) are commonly used for the diagnosis of cases of Clostridium difficile-associated diarrhea (CDAD). However, these EIAs have high false-negative rates, even in patients with severe clinical disease. We have developed an IsoAmp CDAD test using a simple and user-friendly procedure to identify toxigenic C. difficile in feces. After DNA extraction from fecal samples, both the conserved sequence of the 5'-end fragment of the C. difficile tcdA toxin gene and competitive amplification internal control sequence were amplified using helicase-dependent amplification. Amplification products were detected using a novel amplicon-containment detection device. The analytical sensitivity of the assay was 20 copies of C. difficile genomic DNA per reaction. Evaluation of the clinical sensitivity and specificity of the IsoAmp CDAD test versus an EIA method using a PCR method as the reference standard revealed 100% sensitivity and 100% specificity for the IsoAmp CDAD test compared with 90.9% sensitivity and 100% specificity for the EIA method. Because the IsoAmp CDAD test requires no expensive equipments for nucleic acid amplification or detection and can be performed on a random access basis, the test provides a practical alternative to immunoassays for the diagnosis of CDAD with improved sensitivity.

P1732 Evaluation of an automated sample preparation system (MDx) in combination with affigene CMV trender

P1732 Evaluation of an automated sample preparation system (MDx) in combination with affigene CMV trender

Low Temperature Cofired Ceramic Microfluidic Microsystems for High Temperature and High Pressure Applications

As an alternative material to glass, silicon, and plastics, Low Temperature Cofired Ceramic (LTCC) substrate technology is becoming increasingly important for enabling microfluidic microsystems and devices for integrated chemical and biological analysis. LTCC's simple fabrication method and unique ability to withstand high temperatures and high pressures make it well-suited for applications not possible with traditional materials. As part of Sandia's initiative to develop an automated sample preparation system for the μChemlab™ bioagent detector, an integrated microfluidic lyser using LTCC technology has been fabricated, which enables the use of aqueous buffers at high temperatures without boiling by using a pressurized system. Thermal lysing of bacterial spores in a flow-through microfluidic device at temperatures as high as 220°C and pressures up to 10.3 MPa (1,500 psi) represents a new method for solubilizing spore proteins for identification and analysis, eliminating the reliance on harsh chemical reducing agents for lysing. This paper will present results on the development of a microfluidic LTCC-based lyser by taking advantage of the inherent properties of LTCC substrates. Specifically, the performance and implementation of a prototype LTCC lyser for solubilizing bacterial spore proteins will be reported.

Performance of the automated Abbott RealTi me™ HIV-1 assay on a genetically diverse panel of specimens from Brazil

The combination of automated sample preparation and real-time RT-PCR for measurement of HIV-1 viral load has the potential to significantly enhance throughput, reduce operator-associated error, and increase assay sensitivity and dynamic range. In this study, RNA was extracted from the plasma of 91 HIV-1 seropositive Brazilian blood donors using the Abbott m2000 sp™ automated sample preparation system. Viral loads measured using the RealTi me™ HIV-1 (RealTi me HIV-1) assay and the Abbott m2000 rt™ instrument were compared to values obtained in the LCx ® HIV RNA quantitative assay. Subtype was determined for 89 of 91 specimens by sequence/phylogenetic analysis of three genomic regions: gag p24, pol integrase, and env gp41. The panel included 69 subtype B, 1 C, 2 F, and 17 recombinant strains. Eighty-seven specimens were quantified by both assays. Two specimens were quantified only in RealTi me HIV-1. Two additional specimens below the detection limit of both assays were also negative on PCR amplification. Viral load results were highly correlated, and good agreement was observed between assays with 90% of values within 0.5 log 10 copies/ml. The RealTi me HIV-1 assay and m2000 system offer the advantages of automation while providing reliable quantification of diverse HIV strains.

Liquid chromatography–electrospray mass spectrometry determination of a bis-thiazolium compound with potent antimalarial activity and its neutral bioprecursor in human plasma, whole blood and red blood cells

Liquid chromatography–electrospray ionization mass spectrometry methods are described for the simultaneous quantification of a bis-thiazolium compound (T3), its related prodrug (TE3) and an intermediate compound (mTE3) that appeared during the prodrug/drug conversion process, in human plasma, whole blood and red blood cells (RBCs). The methods involve solid phase extraction (SPE) of the compounds and the internal standard (verapamil) from the three different matrices using Oasis ®HLB columns with an elution solvent of 2 × 1 ml of acetonitrile containing 1 ml/l trifluoroacetic acid (TFA). HPLC separation was performed on a C 18 encapped Xterra ® column packed with 3.5 μm particles. The mobile phase used a 8 min gradient, from water containing 1 ml/l TFA to acetonitrile containing 1 ml/l TFA, at a flow rate of 400 μl/min. Verapamil and the TE3 compound were characterized by the protonated molecules at m/ z 455 and m/ z 541, respectively. The mTE3 species was detected through the (M) + ion at m/ z 497. The T3 compound was detected by use of two ions, the quaternary ammonium salt (M 2+/2) at m/ z 227.3 and by the adduct with TFA (M + TFA) + at m/ z 567.3. The drug/internal standard peak area ratios were linked via a quadratic relationship to plasma (or whole blood) concentrations in the tested range of 6.4–1282 μg/l (12.8–2564 μg/kg) for T3, 20–2000 μg/l (40–4000 μg/kg) for mTE3 and 10–2000 μg/l (40–4000 μg/kg) for TE3, and to T3 concentrations in RBCs ranging from 12.8 to 2564 μg/kg. Inter-assay precision (in terms of R.S.D.) was below 13.5% and accuracy ranged from 95.4 to 107%. The dilution of the samples (plasma or whole blood) has no influence on the performance of the methods. The extraction recoveries averaged 87% for T3, 53% for mTE3 and 79% for TE3 in plasma; 79% for T3, 57% for mTE3 and 65% for TE3 in blood; and 93% for T3 in RBCs, and was constant across the calibration range. The lower limits of quantitation were 6.4 μg/l for T3, 20 μg/l for mTE3 and 10 μg/l for TE3 in plasma; 12.8 μg/kg for T3 and 40 μg/kg for mTE3 and TE3 in blood; and 12.8 μg/kg for T3 in RBCs. Stability tests under various conditions were also investigated. The three-step SPE procedure (loading, clean-up, and elution) described in this paper to quantify these new anti-malarial compounds in plasma, whole blood and RBCs, can easily be automated by using either robotisation or an automated sample preparation system.

Quantitation of Melphalan in Human Plasma by Liquid Chromatography and Solid Phase Extraction: Application to Pharmacokinetic Study

A high‐performance liquid chromatographic (HPLC) method for the determination of melphalan in human plasma was developed. This method involved a solid phase extraction (SPE) of melphalan and the internal standard (propylparaben) from plasma, using bond Elut C2 SPE columns with an elution solvent of 0.5 mL of acetonitrile‐purified water (50:50, v/v). Separation of the two analytes was achieved within 15 min, using a reversed‐phase Kromasil C18 analytical column (150 × 4.6 mm I.D., 5 µm particle size) with a mobile phase of methanol–water–acetic acid (48:51:1, v/v). An ultraviolet detector operated at 261 nm was used, with a linear response observed from 10 to 250 ng mL−1. Obtained from the method validation, inter‐assay precision was below 6% and accuracy is near 100%. The extraction efficiency of the assay was approximately 71% and was constant across the calibration range. The lower limit of quantitation was 10 ng mL−1; at this level, precision was 5% and accuracy was 101%. The applicability of this method has been demonstrated by the successful analysis of clinical plasma samples. The SPE procedure developed in this paper, to quantify melphalan in biological samples requiring three steps for sample loading, clean‐up, and elution, can easily be automated by using either a robot or an automated sample preparation system.

The automated sample preparation system MixMaster for investigation of volatile organic compounds with mid-infrared evanescent wave spectroscopy

For efficient development assessment, and calibration of new chemical analyzers a large number of independently prepared samples of target analytes is necessary. Whereas mixing units for gas analysis are readily available, there is a lack of instrumentation for accurate preparation of liquid samples containing volatile organic compounds (VOCs). Manual preparation of liquid samples containing VOCs at trace concentration levels is a particularly challenging and time consuming task. Furthermore, regularly scheduled calibration of sensors and analyzer systems demands for computer controlled automated sample preparation systems. In this paper we present a novel liquid mixing device enabling extensive measurement series with focus on volatile organic compounds, facilitating analysis of water polluted by traces of volatile hydrocarbons. After discussing the mixing system and control software, first results obtained by coupling with an FT-IR spectrometer are reported. Properties of the mixing system are assessed by mid-infrared attenuated total reflection (ATR) spectroscopy of methanol-acetone mixtures and by investigation of multicomponent samples containing volatile hydrocarbons such as 1,2,4-trichlorobenzene and tetrachloroethylene. Obtained ATR spectra are evaluated by principal component regression (PCR) algorithms. It is demonstrated that the presented sample mixing device provides reliable multicomponent mixtures with sufficient accuracy and reproducibility at trace concentration levels.

Fully Automated Nucleic Acid Extraction: MagNA Pure LC

Kinetic PCR analysis by real-time monitoring of DNA amplification was first described 8 years ago (1). Recently, the LightCyclerTM instrument (Roche Molecular Biochemicals, Mannheim, Germany), which allows high-speed thermal cycling by use of air instead of thermal blocks and on-line real-time fluorescence monitoring, was introduced (2). Several reports have been published with regard to utilization of the LightCycler technology for detection of pathogens (3)(4)(5)(6)(7)(8)(9)(10)(11). Real-time PCR has greatly decreased the amount of hands-on time needed to generate and detect amplification products. Before amplification, pathogen-specific DNA or RNA must be extracted from the specimen. This procedure, also called sample preparation, remains the most labor-intensive and time-consuming part of widely automated molecular assays and may be considered the major weakness in most molecular assays today (12)(13). Therefore, a fully automated sample preparation system is urgently needed for the routine diagnostic laboratory. In this study, a fully automated specimen preparation instrument, the MagNA Pure LCTM (Roche) was evaluated. The new instrument was used for extraction of herpes simplex virus (HSV) DNA in combination with real-time PCR on the LightCycler instrument. In the first experiment, the interassay variation and the detection limit were tested. The Second European Union Concerted Action HSV Proficiency Panel, which consists of 12 vials with different concentrations of HSV type 1 (HSV-1), strain MacIntyre (American Type Culture Collection), HSV type 2 (HSV-2), strain MS (American Type Culture Collection), varicella-zoster virus, and negative samples, was used. Samples were analyzed three times on different days. Results were compared with a molecular assay, which consisted of an in-house DNA extraction protocol and real-time PCR. In the second experiment, intraassay variation of the new molecular assay was tested. Plasma was collected from a …

Methods for the Quantitation of Abscisic Acid and Its Precursors from Plant Tissues

Methods are given for the quantitation of the plant stress hormone, abscisic acid (ABA), and its two metabolic precursors, ketone and enolate, that are applicable to all species tested so far. The plant extract is homogenized at neutral pH, hexane-soluble neutrals are extracted and discarded, and then the free ABA and other organic acids are extracted as ion pairs. The remaining aqueous phase is acidified, allowed to stand, neutralized, and extracted to give the ABA ex ketone fraction and then the aqueous phase is treated with base and again extracted to give the ABA ex enolate fraction. Each of these three fractions, free ABA, ABA ex ketone, and ABA ex enolate, along with a deuteriated internal standard, [side-chain- 2H 4]ABA, is then derivatized with pentafluorobenzyl bromide and purified on an automated sample preparation system. The resulting pentafluorobenzyl abscisate samples are then quantified using electron capture negative ionization mass spectrometry with methane as the reagent gas. Using these procedures free ABA, and ABA from its precursors, can be quantified at the level of 100 fg on column. If a large volume injector is used so that the total sample is injected it should be possible to quantify ABA and its precursors in the parts per billion range on a few milligrams of plant tissue.

An Automated Sample Preparation System for Large-Scale DNA Sequencing

Recent advances in DNA sequencing technologies, both in the form of high lane-density gels and automated capillary systems, will lead to an increased requirement for sample preparation systems that operate at low cost and high throughput. As part of the development of a fully automated sequencing system, we have developed an automated subsystem capable of producing 10,000 sequence-ready ssDNA templates per day from libraries of M13 plaques at a cost of $0.29 per sample. This Front End has been in high throughput operation since June, 1997 and has produced > 400,000 high-quality DNA templates.

Multi-pesticide Screening Method for Agricultural Products with an Automated Sample Preparation Instrument

A multi-pesticide screening method for agricultural products was developed using an automated sample preparation instrument in combination with GC/MS by a modification of the previously reported method with two SPE columns. The instrument automatically conducts solid-phase extraction and evaporation. The sample extract (0.2mL) was applied to a PSA cartridge (100mg) in the system, followed by repeated elution with 0.8mL of n-hexane-acetone (1:1) and evaporation three times. The residue was dissolved in 0.2mL of n-hexane-acetone (4:1) for GC/MS. The automated system was tested for 164 pesticides, and recoveries of 56.1-129.5% (160 pesticides) were obtained with a mean CV of 4.1%. Four volatile pesticides gave poor recoveries (18.3-27.6%), but yields of 43.1-46.7% were available with the use of matrices. By using this automated sample preparation system, recoveries of 27 pesticides and 4 metabolites from 3 matrices fortified at 0.1ppm were 105.0% for brown rice, 102.0% for lemon, and 103.0% for spinach. This procedure could be employed as a screening method at the 0.01ppm level. The total time required for one sample preparation was ca. 1.3hr and about 10 samples could be analyzed automatically overnight.

Sensitive high-performance liquid chromatographic determination of nifedipine in dog plasma using an automated sample preparation system with laboratory robot.

Nifedipine, a calcium-channel blocking drug was analysed in dog plasma after oral dosing with two different formulations. Sample preparation was automated with a laboratory robot. Quantitative determination of the drug was performed on a reversed-phase HPLC system with electrochemical detection (ED) using an internal standard. Validation of the analytical method showed that the system is well suited for pharmacokinetic studies on dogs. The assay was linear in the range 1-50 ng/ml. Inter-day and intra-day variability were between 6.43-18.15% C.V. and 1.57-5.53% C.V., respectively.

Automated sample preparation for cholesterol determination in foods

An automated sample preparation system has been developed for the determination of cholesterol in a wide range of matrices. Isolation of cholesterol is performed with a robotic arm coupled with a series of modular stations. Samples are introduced into the system which adds the appropriate reagents, carries out the saponification, pH adjustment, solid-phase extraction and drying steps. This system was evaluated using 15 different food matrices. The average recovery for NIST standards exceeded 97%. A solution of n-hexane-2-propanol was substituted for the traditional methanol-chloroform extraction. Manual pH adjustment was replaced with a buffer. Manual and automated methods were compared and no difference was observed at the 95% confidence level.

On-line dialysis with high-performance liquid chromatography for the automated preparation and analysis of sugars and organic acids in foods and beverages

A quick, simple and robust technique is described for on-line clean-up and analysis of raw liquid food samples containing complex matrices such as dairy products, soft drinks, and fermented beverages. A completely automated sample preparation system (ASTED XL) provides an efficient way of removing macromolecular and microparticulate interferents by high-performance dialysis, prior to HPLC analysis of the sugars, organic acids and related compounds. Processing samples on-line in the concurrent mode permitted both high reproducibility and optimal throughput.