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1.
Anal Chem ; 88(19): 9403-9411, 2016 10 04.
Article in English | MEDLINE | ID: mdl-27264110

ABSTRACT

This paper describes an integrated microsystem for rapid separation, enrichment, and detection of bacteria from blood, addressing the unmet clinical need for rapid sepsis diagnostics. The blood sample is first processed in an acoustophoresis chip, where red blood cells are focused to the center of the channel by an acoustic standing wave and sequentially removed. The bacteria-containing plasma proceeds to a glass capillary with a localized acoustic standing wave field where the bacteria are trapped onto suspended polystyrene particles. The trapped bacteria are subsequently washed while held in the acoustic trap and released into a polymer microchip containing dried polymerase chain reaction (PCR) reagents, followed by thermocycling for target sequence amplification. The entire process is completed in less than 2 h. Testing with Pseudomonas putida spiked into whole blood revealed a detection limit of 1000 bacteria/mL for this first-generation analysis system. In samples from septic patients, the system was able to detect Escherichia coli in half of the cases identified by blood culture. This indicates that the current system detects bacteria in patient samples in the upper part of the of clinically relevant bacteria concentration range and that a further developed acoustic sample preparation system may open the door for a new and faster automated method to diagnose sepsis.


Subject(s)
Blood Culture/methods , Blood/microbiology , Microchip Analytical Procedures/methods , Sepsis/blood , Sepsis/diagnosis , Acoustics , Escherichia coli , Humans , Limit of Detection , Polymerase Chain Reaction , Pseudomonas putida
2.
Int J Food Microbiol ; 125(2): 158-61, 2008 Jul 15.
Article in English | MEDLINE | ID: mdl-18501459

ABSTRACT

We describe a system consisting of rapid sample enrichment and homogeneous end-point PCR analysis that enables the detection of Salmonella in various food matrices in 8 h. Sample preparation starts with 6 h enrichment step in supplemented broth, after which Salmonella cells are collected with immunomagnetic particles. The particles are washed and dispensed to ready-to-use PCR reaction vessels, which contain dried assay-specific reagents and an internal amplification control. PCR is performed with a novel instrument platform utilising the sensitive label technology of time-resolved fluorometry. Qualitative assay results are automatically interpreted and available in 45 min after sample addition. The overall accuracy, sensitivity and specificity of the Magda CA Salmonella system were 99.1%, 98.4% and 100.0%, respectively, based on the evaluation of 107 samples (beef, pork, poultry and ready-to-eat meals) artificially contaminated with sub-lethally injured Salmonella cells.


Subject(s)
Fluorometry/methods , Food Contamination/analysis , Immunomagnetic Separation/methods , Polymerase Chain Reaction/methods , Salmonella/isolation & purification , Automation , Fluorescence , Fluorometry/standards , Gene Amplification , Immunomagnetic Separation/standards , Polymerase Chain Reaction/instrumentation , Polymerase Chain Reaction/standards , Salmonella/classification , Salmonella/immunology , Sensitivity and Specificity , Species Specificity , Time Factors
3.
Anal Biochem ; 374(2): 411-6, 2008 Mar 15.
Article in English | MEDLINE | ID: mdl-18191467

ABSTRACT

We have developed a novel instrument platform, GenomEra, for small-scale analysis of nucleic acids. The platform combines a rapid thermal cycler, an integrated time-resolved fluorescence measurement unit, and user-friendly software for the analysis of results. Disposable low-cost plastic reaction vessels are designed specifically for the instrument and contain all of the assay-specific reagents in dry form. The appropriate assay protocol is specified on barcodes printed under the vessels and is automatically initiated by the software. Detection is based on the use of sequence-specific probes labeled with intrinsically fluorescent europium or terbium chelates and complementary quencher probes, which enable sensitive, homogeneous closed-tube assays without the risk of carryover contamination. The detection limit of the instrument (background + 3 SD) is approximately 20 pmol/L for both chelates with a dynamic range of nearly four orders of magnitude. The functionality of the platform is demonstrated with a dual-label homogeneous polymerase chain reaction (PCR) assay for the detection of Salmonella using a Magda CA Salmonella assay kit. An internal amplification control is included in each reaction to eliminate false negative results caused by PCR inhibition. Qualitative assay results are automatically interpreted by the software and are available 45 min after sample addition.


Subject(s)
Fluorescence , Luminescent Measurements/methods , Polymerase Chain Reaction/instrumentation , Automation , Disposable Equipment , Laboratories , Sensitivity and Specificity , Software , Spectrometry, Fluorescence , Temperature , Time Factors
5.
Thromb Res ; 118(5): 573-85, 2006.
Article in English | MEDLINE | ID: mdl-16040082

ABSTRACT

INTRODUCTION: Normal concentrations of D-Dimer can be used to exclude venous thromboembolism (VTE). However, methods for sensitive and quantitative D-Dimer measurements at the point-of-care (POC) are still limited. MATERIALS AND METHODS: We developed a 10-min, non-competitive immunofluorometric assay for D-Dimer in citrated whole blood and plasma using pre-dispensed reagents dried in single assay wells. The simple, automated assay procedure comprises a 1:50 sample dilution, one-step incubation, washing, and time-resolved fluorometric measurement directly from the wet well surface. RESULTS: The limits of detection (background + 3SD) and quantification (CV <15%) were 0.05 and 0.2 mg/L D-Dimer, respectively, and the assay was linear up to 400 mg/L. Correlations to Roche TinaQuant (r=0.726, n=200) and Biopool Auto.Dimer (r=0.190, n=149) were carried out using citrated plasma. Diagnostic sensitivity, specificity, and negative (NPV) and positive (PPV) predictive values were 98.7%, 64.4%, 99.1% and 55.1%, and 92.2%, 81.0%, 95.9% and 68.3%, respectively, using cut-off values of 0.6 and 1.0 mg/L, respectively, in outpatients with deep vein thrombosis (DVT) and/or pulmonary embolism (PE) (n=77) compared with outpatients with various other diseases (n=174). The within- and between-run CVs near the cut-off values were < or =10% in both whole blood and plasma. The 95th percentile upper range in apparently healthy individuals was 0.68 mg/L of whole blood (n=101). CONCLUSIONS: The high sensitivity and NPV suggest that the rapid immunofluorometric assay could be valuable for rapid exclusion of VTE in outpatients. With appropriate cut-offs, the assay could potentially be used as a stand-alone test or combined with clinical probability assessment, but further studies are required.


Subject(s)
Blood Chemical Analysis/methods , Blood Coagulation Tests/methods , Fibrin Fibrinogen Degradation Products/analysis , Fluoroimmunoassay/methods , Antibodies, Monoclonal , Fibrin Fibrinogen Degradation Products/immunology , Humans , Reproducibility of Results , Sensitivity and Specificity
6.
Clin Biochem ; 38(7): 591-606, 2005 Jul.
Article in English | MEDLINE | ID: mdl-16009140

ABSTRACT

The use of point-of-care (POC) immunoassays has increased significantly and the menu of analytes continues to expand. Most of the rapid immunoassays are currently based on simple manual assay devices such as the immunochromatographic, agglutination, and immunofiltration assays. Although automated readers have recently been introduced at an increasing pace, the major benefit of these genuinely hand-portable assay devices is that they do not usually necessitate instrumentation but can be performed anywhere. Significant advances in assay and detection technologies have, however, recently facilitated the introduction of truly quantitative, sophisticated immunoassay methods to POC settings as well, with the analytical performance characteristics approaching those of conventional laboratory assays. Furthermore, innovative assay technologies such as those based on immunosensors have been introduced to POC testing (POCT) without ever being employed in clinical laboratories. However, further simplification of the assay procedures and analyzers is still feasible, and strong efforts are directed towards the development of miniaturized and simple, yet sensitive and quantitative, novel assay technologies to keep up with the increasing expectations set on future POC immunotesting.


Subject(s)
Immunoassay/standards , Point-of-Care Systems , Clinical Laboratory Techniques/standards , Hemagglutination Tests , Miniaturization , Phlebotomy/methods , Quality Control , Self Care/standards
8.
Clin Chem ; 50(6): 1026-35, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15073089

ABSTRACT

BACKGROUND: Human chorionic gonadotropin (hCG) is among the most common analytes available for point-of-care immunotesting, with most assays currently based on simple manual assay devices. However, as the importance of good analytical performance of rapid assays is increasingly emphasized, more sophisticated immunoassay techniques are needed to meet the future challenges of rapid yet quantitative POC testing. METHODS: We developed a simple, dry-reagent, all-in-one immunoassay for the quantitative measurement of hCG in whole blood, plasma, or serum. The noncompetitive assay equally measures intact, nicked, and hyperglycosylated hCG as well as nonnicked and nicked hCG beta-subunit with a rapid and simple procedure consisting of a 5-min, one-step incubation and, subsequent to washing, the measurement of time-resolved fluorescence directly from a wet well surface. RESULTS: The assay had a detection limit (background + 3 SD) of 0.4 IU/L hCG. The within-run CV was <15% down to 2 IU/L, and the assay was linear to 6000 IU/L. The within- and between-run CVs in heparinized whole blood and plasma were

Subject(s)
Chorionic Gonadotropin/blood , Point-of-Care Systems , Chelating Agents , Chorionic Gonadotropin, beta Subunit, Human/blood , Europium , Female , Fluorescence , Glycosylation , Humans , Immunoassay , Pregnancy , Sensitivity and Specificity
9.
Anal Chem ; 75(13): 3193-201, 2003 Jul 01.
Article in English | MEDLINE | ID: mdl-12964769

ABSTRACT

New labels and assay techniques are needed to improve the sensitivity and quantitativeness of point-of-care immunotesting while sustaining the rapidity and ease of use of the assays. We synthesized a novel, intrinsically fluorescent nonadentate europium chelate with two chromophores and hydrophilic alpha-galactose side groups. The chelate is highly fluorescent, soluble in water, and provides effective shielding of Eu from water. The performance of the nonadentate chelate was compared with a heptadentate chelate in a dry reagent immunoassay for human chorionic gonadotropin (hCG). After 15-min incubation and washing, time-resolved fluorescence was measured directly from a wet or dried well surface. Contrary to the heptadentate label, the effect of aqueous quenching on the nonadentate label was found to be insignificant, with calculated analytical detection limits (background + 3 SD) of 0.9 and 0.7 IU/L hCG for wet and dry measurements, respectively, and a linear range up to 5000 IU/L. The CVs for the new label were <8% at the cutoff of 25 IU/L and above in both whole blood and plasma. The novel nonadentate label facilitates short turnaround times and simple instrumentation due to the absence of all signal development steps, at the same time retaining an excellent immunoassay performance.


Subject(s)
Chelating Agents/chemistry , Europium/chemistry , Fluorescent Dyes/chemistry , Immunoassay/methods , Point-of-Care Systems , Chorionic Gonadotropin/blood , Fluorometry/methods , Humans , Male , Reproducibility of Results , Sensitivity and Specificity
10.
Clin Biochem ; 36(2): 121-8, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12633761

ABSTRACT

OBJECTIVES: Different screening strategies for early diagnosis of hypothyroidism have been discussed increasingly. We demonstrate the applicability of a miniaturized microparticle assay format for rapid and quantitative determination of increased thyrotropin (TSH) concentrations in serum. DESIGN AND METHODS: Porous microparticles were used as solid phase for a noncompetitive, one-step, kinetic immunoassay with varying incubation times and time-resolved fluorescence detection. RESULTS: The analytical (mean of zero + 3 SD) and functional (CV <15%) detection limits were 1.5 and 6.0 mIU/L for 2-min, 0.5 and 1.5 mIU/L for 7-min, and 0.2 and 0.5 mIU/L for 15-min assays, respectively. A good correlation was found with the Chiron Diagnostics ACS:180 assay (slopes 0.885-1.051, y-intercepts < +/- 0.20 mIU/L, S(y logical or, bar below x) 0.98, n = 20). CONCLUSION: The kinetic TSH assay provides reproducible and quantitative information on thyroid status within minutes and is applicable for the detection of hypothyroidism in point-of-care (POC) conditions.


Subject(s)
Hypothyroidism/blood , Thyrotropin/blood , Calibration , Fluorescence Polarization Immunoassay/methods , Fluorescence Polarization Immunoassay/standards , Humans , Hypothyroidism/diagnosis , Kinetics , Linear Models , Particle Size , Point-of-Care Systems , Reproducibility of Results , Sensitivity and Specificity , Statistics as Topic
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