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Biomedical subjects

R A Durst

Publications and source records attributed to R A Durst.

At least 19 recordsLinked to original sources
PubMed2001

Detection of viable Cryptosporidium parvum using DNA-modified liposomes in a microfluidic chip.

This paper describes a microfluidic chip that enables the detection of viable Cryptosporidium parvum by detecting RNA amplified by nucleic-acid-sequence-based amplification (NASBA). The mRNA serving as the template for NASBA is produced by viable C. parvum as a response to heat shock. The chip utilizes sandwich hybridization by hybridizing the NASBA-generated amplicon between capture probes and reporter probes in a microfluidic channel. The reporter probes are tagged with carboxyfluorescein-filled liposomes. These liposomes, which generate fluorescence intensities not obtainable from single fluorophores, allow the detection of very low concentrations of targets. The limit of detection of the chip is 5 fmol of amplicon in 12.5 microL of sample solution. Samples of C. parvum that underwent heat shock, extraction, and amplification by NASBA were successfully detected and clearly distinguishable from controls. This was accomplished without having to separate the amplified RNA from the NASBA mixture. The microfluidic chip can easily be modified to detect other pathogens. We envision its use in mu-total analysis systems (mu-TAS) and in DNA-array chips utilized for environmental monitoring of pathogens.

Animals↗
PubMed2001

Detection of Cryptosporidium parvum using oligonucleotide-tagged liposomes in a competitive assay format.

To meet the technical challenge of accurately and rapidly detecting Cryptosporidium parvum oocysts in environmental water, the authors developed a single-use visual-strip assay. The first step in the overall assay procedure involves extracting C. parvum's mRNA coding for heat-shock protein hsp70, followed by amplification using nucleic acid sequence-based amplification (NASBA) methodology as described previously (Baeumner, A. J.; Humiston, M.; Montagna, R. A.; Durst, R. A. Anal. Chem., in press). Subsequently, generated amplicons are hybridized with dye-entrapping liposomes bearing DNA oligonucleotides (reporter probes) and biotin on their surface. The liposome-amplicon complex is then allowed to migrate upward on a nitrocellulose membrane strip. On the nitrocellulose strip, antisense-reporter probes are immobilized in a capture zone and antibiotin antibodies are immobilized in a second zone above the capture zone. Depending on the presence or absence of amplicon in the sample, the liposomes will bind to the capture zone, or they will be caught via their biotin tag in the second zone. Visual detection or gray-scale densitometry allows the quantification of liposomes that are present in either zone. The detection limit of the assay was determined to be 80 fmol amplicon/test. High accuracy and an internal assay control is established using this competitive format, because the presence or absence of liposomes can be quantified in the two capture zones.

Animals↗
PubMed2001

Detection of viable oocysts of Cryptosporidium parvum following nucleic acid sequence based amplification.

A reliable method using nucleic acid sequence based amplification (NASBA) with subsequent electrochemiluminescent detection for the specific and sensitive detection of viable oocysts of Cryptosporidium parvum in environmental samples was developed. The target molecule was a 121-nt sequence from the C. parvum heat shock protein hsp70 mRNA. Oocysts of C. parvum were isolated from environmental water via vortex flow filtration and immunomagnetic separation. A brief heat shock was applied to the oocysts and the nucleic acid purified using an optimized very simple but efficient nucleic acid extraction method. The nucleic acid was amplified in a water bath for 60-90 min with NASBA, an isothermal technique that specifically amplifies RNA molecules. Amplified RNA was hybridized with specific DNA probes and quantified with an electrochemiluminescence (ECL) detection system. We optimized the nucleic acid extraction and purification, the NASBA reaction, amplification, and detection probes. We were able to amplify and detect as few as 10 mRNA molecules. The NASBA primers as well as the ECL probes were highly specific for C. parvum in buffer and in environmental samples. Our detection limit was approximately 5 viable oocysts/sample for the assay procedure, including nucleic acid extraction, NASBA, and ECL detection. Nonviable oocysts were not detected.

Animals↗
PubMed2001

Electrochemical biosensors: recommended definitions and classification.

Two Divisions of the International Union of Pure and Applied Chemistry (IUPAC), namely Physical Chemistry (Commission 1.7 on Biophysical Chemistry formerly Steering Committee on Biophysical Chemistry) and Analytical Chemistry (Commission V.5 on Electroanalytical Chemistry) have prepared recommendations on the definition, classification and nomenclature related to electrochemical biosensors: these recommendations could, in the future, be extended to other types of biosensors. An electrochemical biosensor is a self-contained integrated device, which is capable of providing specific quantitative or semi-quantitative analytical information using a biological recognition element (biochemical receptor) which is retained in direct spatial contact with an electrochemical transduction element. Because of their ability to be repeatedly calibrated, we recommend that a biosensor should be clearly distinguished from a bioanalytical system, which requires additional processing steps, such as reagent addition. A device that is both disposable after one measurement, i.e. single use, and unable to monitor the analyte concentration continuously or after rapid and reproducible regeneration, should be designated a single use biosensor. Biosensors may be classified according to the biological specificity-conferring mechanism or, alternatively, to the mode of physico-chemical signal transduction. The biological recognition element may be based on a chemical reaction catalysed by, or on an equilibrium reaction with macromolecules that have been isolated, engineered or present in their original biological environment. In the latter cases. equilibrium is generally reached and there is no further, if any, net consumption of analyte(s) by the immobilized biocomplexing agent incorporated into the sensor. Biosensors may be further classified according to the analytes or reactions that they monitor: direct monitoring of analyte concentration or of reactions producing or consuming such analytes; alternatively, an indirect monitoring of inhibitor or activator of the biological recognition element (biochemical receptor) may be achieved. A rapid proliferation of biosensors and their diversity has led to a lack of rigour in defining their performance criteria. Although each biosensor can only truly be evaluated for a particular application, it is still useful to examine how standard protocols for performance criteria may be defined in accordance with standard IUPAC protocols or definitions. These criteria are recommended for authors. referees and educators and include calibration characteristics (sensitivity, operational and linear concentration range, detection and quantitative determination limits), selectivity, steady-state and transient response times, sample throughput, reproducibility, stability and lifetime.

Biosensing Techniques↗
PubMed2000

Immunoliposome sandwich assay for the detection of Escherichia coli O157:H7.

We describe the development of a field-portable colorimetric immunoassay for the detection of Escherichia coli O157:H7, using antibody-directed liposomes (immunoliposomes) encapsulating dye as an analytical reagent. Antibodies (anti-E. coli O157:H7) thiolated by 2-iminothiolane were coupled to malemide-tagged liposomes encapsulating the marker dye, sulforhodamine B. Transmission electron microscopy showed that the immunoliposomes bound only to the serotype without any cross-reactivity with tested negative controls. A wicking reagent containing immunoliposomes and the test sample and a plastic-backed nitrocellulose strip with a measurement zone were used in a sandwich (noncompetitive) assay format. During the capillary migration of the wicking reagent, E. coli, with surface-bound immunoliposomes, was captured at the measurement zone on which antibodies to E. coli O157:H7 were immobilized. The color density of the measurement zone was directly proportional to the amount of E. coli O157:H7 in the sample. The detection limit of the current assay with pure cultures of the serotype was ca. 10(4) colony-forming units (CFU)/mL. The assay, which does not need washing and incubation steps, can be completed in 8 min. These results demonstrate the feasibility of using dye-encapsulating immunoliposomes in microporous membranes for the rapid detection of molecules with multivalent antigenic sites.

Antibodies, Bacterial↗
PubMed2000

Determination of potato glycoalkaloids using a liposome immunomigration, liquid-phase competition immunoassay.

Polyclonal anti-solanine antibodies were raised and used in the assembly of a liposome immunomigration, liquid-phase competition strip immunoassay. In this format, a similar cross-reactivity was observed between the glycoalkaloids alpha-solanine and alpha-chaconine. The strip assay was implemented to quantitate total glycoalkaloids (TGA) from potatoes. Recoveries in spiked potato samples using the strip assay and water:acetic acid:sodium bisulfite extracting solvent were in the range of 84-111%. The values of the TGA quantitations by the strip assay as compared with those obtained by HPLC, in nonspiked tubers coming from cloned potato samples donated by potato breeders, were equivalent and highly correlated (r(2) = 0.91). The strip assay proved advantageous over HPLC for extra-laboratory measurements such as the rapid identification of samples that should be rejected due to an elevated TGA content.

Alkaloids↗
PubMed1999

Liposome dehydration on nitrocellulose and its application in a biotin immunoassay.

The feasibility of utilizing dehydrated liposomes in the development of a simple immunoassay device for point-of-care diagnostics or field assays was demonstrated. The recovery of liposomes after a cycle of dehydration and rehydration was studied using biotin-tagged, dye-loaded liposomes with antibiotin antibodies immobilized in a defined zone on nitrocellulose strips. Liposomes were vacuum-dehydrated on the strip at a location below the antibiotin zone. The strip was placed in a tube containing a carrier solution and capillary action brought the solution to the dehydrated liposomes, rehydrated them, and caused them to migrate to the antibody zone where intact liposomes were captured and measured optically. High concentrations of either trehalose or sucrose external to the liposomes and both polyvinylpyrrolidone and gelatin in the membrane blocking reagent were essential for preservation of the dehydrated/rehydrated liposomes on nitrocellulose. Between 70 and 80% of the liposomes were recovered on the nitrocellulose strips after a cycle of dehydration and rehydration. The dehydrated liposomes on the strips were stable for at least 1 year when stored in vacuum-sealed plastic bags at 4 degrees C. The technique was successfully applied to the development of a rapid one-step strip immunoassay for biotin.

Biotin↗
PubMed1997

Characteristics of DNA-tagged liposomes allowing their use in capillary-migration, sandwich-hybridization assays.

Liposomes that have been labeled externally with a DNA oligomer are used in a capillary-migration, sandwich-hybridization assay for specific DNA target sequences. The liposomes are used in a DNA detection scheme that produces visually observable results in 10 min. The preparation and covalent attachment of a thiol-activated 22-base oligomer to the external surface of dye-containing liposomes is described, and the specificity of the assay toward perfectly complementary target DNA is demonstrated. Several characteristics of DNA-tagged liposomes that allow the use of increased stringency during hybridization are evaluated. These include the effect of temperature, formamide, and salt concentration on both the sandwich-hybridization assay and the liposomes themselves. The effects of several components of a common hybridization solution are determined with regard to both assay performance and liposome stability. Using a solution of 0.02% sodium dodecyl sulfate in 3X standard saline citrate, a visual detection limit of 200 amol of target DNA was obtained.

Buffers↗
PubMed1996

Rapid method for visual identification of specific DNA sequences based on DNA-tagged liposomes.

We describe a rapid method for visually determining specific DNA sequences at femtomole concentrations. Liposomes, encapsulating a red dye and labeled with oligonucleotide, were used in a capillary migration-sandwich hybridization assay. Capture probe was immobilized on nitrocellulose strips, and liposomes, migrating along each strip, formed a visually discernible band in the presence of target DNA. One femtomole of synthetic target sequence could be detected in < 10 min. Sufficiently stringent hybridization conditions can be used to allow the discrimination of a 10% mismatch sequence from perfectly complementary DNA. A 366-base PCR product was detected at 200 fmol.

Base Sequence↗
PubMed1995

Investigation of liposome-based immunomigration sensors for the detection of polychlorinated biphenyls.

The use of immunospecific liposome migration offers many advantages for the extralaboratory detection of environmental contaminants. Devices utilizing this technique are fast, easy to use, and robust and respond to the presence of analyte at low-parts-per-billion concentrations. Investigations have been carried out that determine optimal interactions for key components of these assays, and techniques are presented for the development of generalized liposome immunoassays. Two complementary prototype liposome-based immunomigration techniques have been developed for the detection of polychlorinated biphenyls (PCBs). The liposome immunocompetition assay format measures the competitive reaction between analyte-tagged liposomes and the sample analyte for immobilized antibodies and can detect 0.4 nmol of PCB in less than 8 min. A more sensitive format, the liposome immunoaggregation (LIA) assay detects the inhibition of immunospecific liposome aggregation in solution and can detect 2.6 pmol of PCB in less than 23 min. Laser diffraction particle sizing has been used to study LIA-induced increases in liposome size over time and to determine optimal conditions for the application of this technique. Both formats utilize capillary action to transport liposome-containing solutions along strips of nitrocellulose. Measurement of color intensity is then carried out visually or with a desktop scanner.

Animals↗
PubMed1993

Liposome-based flow-injection immunoassay for determining theophylline in serum.

We developed a method for quantitatively determining theophylline in serum, using a heterogeneous immunoassay called flow-injection immunoanalysis. The reaction involves competition between serum theophylline and theophylline-labeled liposomes. Separation occurs on a solid-phase reactor column containing immobilized antibody to theophylline incorporated in a flow-injection system. Subsequent lysis of the bound liposomes provides sensitive detection of the analyte. Effective regeneration of the immobilized antibody activity allows the reactor to be reused for hundreds of sequential samples. Comparison of the results of the flow-injection immunoassay method with results obtained with a commercially available fluorescence polarization method showed an excellent correlation.

Flow Injection Analysis↗
PubMed1991

Liposome flow injection immunoassay: model calculations of competitive immunoreactions involving univalent and multivalent ligands.

The use of liposomes as detectable reagents in solid-phase immunoassays has been explored in a flow injection immunoanalysis (FIIA) system. Model calculations are presented for FIIA based on the competitive binding of univalent analyte and multivalent liposomes to immobilized antibodies. Parameters such as binding constants, concentrations of liposomes and antibody, and steric hindrance are considered for their relative effects on detectable liposome signal response to analyte concentrations. Qualitative comparisons of the model with the experimental data are made.

Flow Injection Analysis↗
PubMed1991

Immobilization of binding proteins on nonporous supports. Comparison of protein loading, activity, and stability.

Four different nonporous particulate materials, nylon, polystyrene, soda-lime silicate glass, and fused silica glass, have been evaluated for their appropriateness as immobilization supports for immunoglobulins. A method of protein quantitation that is usually applied to solutions, the bicinchoninic acid (BCA) assay, was used successfully to directly measure ng amounts of protein immobilized on the supports. Two proteins, a monoclonal antibody to theophylline and the biotin binding protein avidin, were studied. Radioactive theophylline and radioactive biotin were used to measure the activity of the immobilized protein. Ligand binding capacity per mm2 of support was measured as a function of amount of protein immobilized. By measuring both the amount of protein immobilized and its ligand binding capacity, we have determined that antitheophylline antibody adsorbed on polystyrene balls loses almost 90% of its binding activity after 65 h, although little protein is lost from the balls over this time. Avidin retains nearly full activity for biotin on polystyrene. The binding activity of biotinyl-antibody conjugate immobilized on avidin-adsorbed polystyrene is stable, even when stored for over 22 wk. Antibody covalently immobilized on soda-lime silicate glass beads retains its binding activity over long-term storage, although only 0.1 mol of 3H-theophylline bind per mol of immobilized antibody. Using fused silica glass particles as the solid support, the same antibody binds approx 0.6 mol of ligand per mol of immobilized antibody protein. The structural "softness" of the immunoglobulin requires that interaction with the surface be prevented in order to maintain activity.

Adsorption↗
PubMed1990

Liposome flow injection immunoassay: implications for sensitivity, dynamic range, and antibody regeneration.

We have developed a liposome-based flow injection immunoassay (FIIA) system for quantitation of a clinical analyte, theophylline. With very minor changes in assay format, this procedure can also be used for the quantitation of anti-theophylline. Automated sequential analyses were performed at room temperature with picomole sensitivity and a day-to-day coefficient of variation of less than 5% for aqueous solutions. The system components include liposomes that contain fluorophores in their aqueous centers and an immobilized-antibody reactor column. The immunoreactor was regenerated hundreds of times over 3 months of continuous use with no measurable loss of antibody activity. The two assay formats studied produced distinct dynamic ranges for their respective analytes. The special advantages of using flow injection analysis for immunoassays and of using liposomes in FIIA are discussed.

Antibodies↗
PubMed1989

IFCC document stage 3, draft 1, dated 1989 02 01. An approved IFCC recommendation. IFCC method (1988) for tonometry of blood: reference materials for pCO2 and pO2. International Federation of Clinical Chemistry Scientific Division. Committee on pH, Blood Gases and Electrolytes.

A reference method for tonometry of blood is described. The document covers the theory of tonometry, the materials and equipment needed, and essential aspects of the tonometry procedure for blood. The partial pressures of oxygen and carbon dioxide in tonometered blood are accurately known and therefore this blood is recommended for assessing the accuracy of blood gas analyzers. Tonometry of blood samples from patients may also be used in the determination of acid-base quantities and hemoglobin-oxygen affinity, e.g. p50.

Blood Gas Analysis↗