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PubMed2005

Polyaniline synthesis and its biosensor application.

In this study, five polyaniline compounds were synthesized using different protonic acids and incorporated into a conductometric biosensor used for bovine viral diarrhea virus detection. The biosensor was developed and evaluated by the authors for bacterial pathogen detection in previous studies. The biosensor consisted of two parts: the immunosensor and the electronic data collection system. Liquid sample moved through the immunosensor surface by capillary action. The specificity of the biosensor was based on the unique binding characteristics of the polyclonal and monoclonal antibodies immobilized on the immunosensor. Polyaniline was used in the biosensor architecture as the transducer due to its electronic and bio-molecular properties. Results showed that the biosensor was sensitive at a concentration of 10(3) cell culture infective dose per milliliter (CCID/ml) of BVDV antigens. The promising results on the BVDV detection demonstrated that the conductometric biosensor was interchangeable for different target molecules of detection. Further modification could be implemented to evaluate the biosensor as a rapid diagnostic device to detect other infectious disease outbreaks in livestock population.

Aniline Compounds↗
PubMed2006

Gold nanoparticle-based immunochromatographic test for identification of Staphylococcus aureus from clinical specimens.

BACKGROUND: Staphylococcus aureus is one of the most important human pathogens, causing both nosocomial and community-acquired infections. Therefore, a method for rapidly detecting for S. aureus would be useful. We describe an analytical system of immunochromatographic assay based on gold nanoparticles developed for the detection of S. aureus in patient specimen. METHODS: The assay was in the sandwich format, using anti-protein A IgG with 2 distinct specificities. One anti-protein A IgG was immobilized in a defined detection zone on a porous nitrocellulose membrane, while the other anti-protein A IgG was conjugated with gold nanoparticles. The mixture was then passed along the porous membrane by capillary action past the anti-protein A IgG in the detection zone, binding the particles that to which surface protein A was already bound to their surface, yielding a red color. RESULTS: The sensitivity and specificity in the immunochromatographic test were 100% and 94.7-100% for 130 S. aureus strains and 36 non-S. aureus strains, respectively. The results were comparable to the conventional coagulase test and latex agglutination test of different bacteria. CONCLUSION: This method may be useful for analyzing S. aureus in patient specimen. It was quick, easy to perform, and with a long shelf life at room temperature.

Antibodies↗
PubMed2005

Characterization of flow and voltage profiles in planar electrochromatography.

Planar electrochromatography (PEC) is a new technology for thin layer chromatography (TLC) where the separation is driven by electroosmotic forces, not capillary action. This allows for much faster and more efficient chromatography in a planar format. Care needs to be taken when performing these experiments because voltage and flow characteristics can change through a single run, due to buffer gradients, temperature changes (Joule heating) and localized plate heterogeneity. We have designed a PEC instrument and cover grid to allow investigation of flow and voltage characteristics as solvent moves across a TLC plate. Our unique cover grid allows monitoring voltage at eight discrete points between the positive and negative reservoirs. A linear relationship between voltage and distance should be seen, giving a constant voltage drop across a plate, but this did not occur. This non-linear function changes over time, following the plate equilibration. Once a plate is equilibrated, voltage and flow characteristics remain fairly constant. Theoretical calculations support the physical observations. Larger plate widths (5 cm) were also briefly investigated and it is concluded that large width plates could be easily implemented to maintain multiple sample capability.

Chromatography, Thin Layer↗
PubMed2005

Linear voltage profiles and flow homogeneity in pressurized planar electrochromatography.

Planar electrochromatography (PEC) is an emerging technique for thin-layer chromatography (TLC) where electroosmosis is the driving force for the solvent, not capillary action. This allows for much faster and constant flow rates in turn yielding increased zone capacities and efficiencies. Instrumental designs have changed greatly over the last few years solving many of the initial instrumentation challenges. We have previously shown that low applied pressure (or no applied pressure) PEC instruments do not give linear voltage drops across the separation path length of a TLC plate, which in turn results in non-stable electroosmotic flow (EOF). By the use of our unique reader electrode grid we have the ability to monitor the potential at eight discrete positions throughout the 10-cm separation path length. We now show that high-pressure PEC instruments, most commonly referred to as pressurized planar electrochromatography (PPEC) do show a linear voltage drop and constant EOF. We compare plate equilibration times of PPEC and low-pressure PEC, use of increased field strengths, as well as sample application designs. In addition, we discuss the use of rhodamine B as a visual marker for reproducible migration and calculation of theoretical plates.

Chromatography, Thin Layer↗
PubMed2004

Proposal of blood-collecting needle approach to semi-invasive method.

Many diabetic patients carry a portable self-monitoring of blood glucose (SMBG)-analyzer in order to collect their own blood and examine their glucose levels; this allows them to determine such factors as insulin dose, diet and exercise to stay healthy. However, the test causes physical and mental stress for the subjects. The authors aim to develop a semi-invasive blood-collecting needle which does not need a power source for the pump mechanism. In this study, we fabricated a capillary action needle that can collect the blood sample automatically. A blood-collecting needle was fabricated from 25 gage sized medical needle (diameter of 0.5 mm, stainless steel) by cutting process, and it had a half-opened crevice in the tip. In order to evaluate the physical characteristics of the blood-collecting needle, the relationship between the size and suction time and/or suction volume were measured using an isotonic sodium chloride solution, whole rabbit blood, and whole human blood with anticoagulant. Next, in order to evaluate the degree of invasion, the diameters of erythema in auricles of rabbits were observed for 2 days using a CCD camera-type microscope. The mean suction time of the isotonic sodium chloride solution and the whole rabbit blood were 1.5 s (n = 10) and 9.0 s (n = 5), respectively. Selection of a suitable size of the blood-collecting needle enabled the collection of 0.1 microL of whole human blood in 10 s. Moreover, it was shown, by comparing the observed diameter of the erythema, that the invasiveness of the blood-collecting needle was smaller than for commercial needles of the equal diameter. It became clear that this fulfils the fundamental functions of a semi-invasive blood-collecting needle.

Adult↗
PubMed2004

Colloidal gold-based immunochromatographic assay for detection of botulinum neurotoxin type B.

A rapid immunochromatographic assay was developed to detect botulinum neurotoxin type B (BoNT/B). The assay was based on the sandwich format using polyclonal antibody (Pab). The thiophilic gel purified anti-BoNT/B Pab was immobilized to a defined detection zone on a porous nitrocellulose membrane and conjugated to colloidal gold particles that served as a detection reagent. The BoNT/B-containing sample was added to the membrane and allowed to react with Pab-coated particles. The mixture was then passed along the porous membrane by capillary action past the Pab in the detection zone, which will bind the particles that had BoNT/B bound to their surface, giving a red colour within this detection zone with an intensity proportional to BoNT/B concentration. In the absence of BoNT/B, no immunogold was bound to the solid-phase antibody. With this method, 50 ng/ml of BoNT/B was detected in less than 10 min. The assay sensitivity can be increased by silver enhancement to 50 pg/ml. The developed BoNT/B assay also showed no cross reaction to type A neurotoxin (BoNT/A) and type E neurotoxin (BoNT/E).

Botulinum Toxins↗
PubMed1992

Latex-based thin-layer immunoaffinity chromatography for quantitation of protein analytes.

A rapid immunochromatographic method for qualitative and quantitative analysis of protein antigens is described. The method is based on the "sandwich" assay format using monoclonal antibodies (Mabs) of two distinct specificities. Mabs of one specificity are covalently immobilized to a defined detection zone on a porous membrane while Mabs of the other specificity are covalently coupled to blue latex particles which serve as a label. The sample is mixed with the Mab-coated particles and allowed to react. The mixture is then passed along a porous membrane by capillary action past the Mabs in the detection zone, which will bind the particles which have antigen bound to their surface, giving a blue color within this detection zone with an intensity logarithmetrically proportional to the antigen concentration in the sample. Analysis is complete in less than 10 min, requires a minimum amount of sample (4 microliters), and has a detection limit below the nanomolar range for the antigen we studied, human chorionic gonadotropin.

Antibody Specificity↗
PubMed1983

Scanning electron microscopy of Dalkon Shield tails.

Scanning electron micrographs of Dalkon Shield tails removed from asymptomatic patients show a variety of microbes and debris throughout their entire length. Apparently, even in undamaged tails, bacterial flora thrive in the protein-rich environment within the multifilament tail. The presence of microbes in the portion of the tail beyond the double knot indicates that an alternative mechanism of microbial transport can occur. Since transient endometritis often occurs immediately after insertion of intrauterine devices, microbes may come in contact with both exposed ends of the multifilament tail and be drawn into the tail by capillary action from the uterine environment down the tail toward the double knot as well as upward from the vagina. Such microorganisms could serve as an inoculum for infection.

Disease Reservoirs↗
PubMed2003

A mathematical model for an expanding foam.

A theoretical and numerical model is presented for the shape evolution of the thin liquid films separating the gas bubbles in a foam. The motion is due to capillary action, surface tension gradients, and the overall expansion of the foam. The expansion is the result of the increase in gas content with time. Process modeling is accomplished via the solution of three coupled partial differential equations. Two time scales are included in the model: a process time and a drying or curing time. It is demonstrated that the amount of surfactant is the dominant control mechanism for the final film thickness. If sufficient surfactant is present, the films will be shown to dilate uniformly in space. A number of known features of expanding foams are reproduced by the model.

Journal Article↗
PubMed2002

Colloidal gold-based immunochromatographic assay for detection of ricin.

A rapid immunochromatographic assay was developed to detect ricin. The assay was based on the sandwich format using monoclonal antibodies (Mabs) of two distinct specificities. One anti-ricin B chain Mab (1G7) was immobilized to a defined detection zone on a porous nitrocellulose membrane, while the other anti-ricin A chain Mab (5E11) was conjugated to colloidal gold particles which served as a detection reagent. The ricin-containing sample was added to the membrane and allowed to react with Mab (5E11)-coated particles. The mixture was then passed along the porous membrane by capillary action past the Mab (1G7) in the detection zone, which will bind the particles that had ricin bound to their surface, giving a red color within this detection zone with an intensity proportional to ricin concentration. In the absence of ricin, no immunogold was bound to the solid-phase antibody. With this method, 50 ng/ml of ricin was detected in less than 10 min. The assay sensitivity can be increased by silver enhancement to 100 pg/ml.

Antibodies, Monoclonal↗
PubMed2003

Separation of hydrocarbons and lipid from water using treated bark.

This paper explores the possibility of using treated bark to remove oily compounds from water. Bark was first biologically or chemically treated and saturated with transition metal ions (TMI) to avoid the release of soluble organic compounds from the bark in the treated effluents. Several experimental parameters affecting the oil removal efficiency (RE) were studied (initial oil concentration, temperature, time, etc.). Saturated bark was characterized using Fourier transform infrared (FTIR) spectroscopy and bark wetting index was determined. Results of the retention of lipids suggested that their removal could exceed 95% of initial oil concentration. The uptake of lipid by treated bark varied from 0.2 to 2.0 g of organic oil/g of dry sorbent. No significant chemical modifications of saturated bark were observed in infrared spectroscopy after the sorption of oleic acid on bark treated with transition metal ions. The structure of adsorbed tridimensional layer of oleic acid molecules seemed to take place through the double bond. The hydrocarbon RE exceeded 95% using oil-water mixture with a hydrocarbon/bark ratio of one. The sorption reaction of hydrocarbons and lipids was quasi-instantaneous and seemed to be influenced by the temperature. This indicated that the retention mechanism was related to the capillary action. Results of FTIR spectroscopy suggested that no chemical bonds between barks and oily compounds were established.

Adsorption↗
PubMed1998

Ret oncogene activation in papillary thyroid carcinoma: prevalence and implication on the histological parameters.

The Ret proto-oncogene is known to be rearranged in papillary carcinoma of the thyroid. The aim of this study was to investigate the in situ expression of Ret mRNA in thyroid tumors. Formalin-fixed, paraffin-embedded tissue specimens from 45 thyroid lesions were examined by in situ hybridization using manual capillary action technology (MicroProbe Staining System) and a 52-base synthetic biotinylated oligonucleotide probe complementary to the tyrosine-kinase domain of Ret proto-oncogene. The clinicopathological features of these patients with thyroid lesions also were noted. Ret was noted in 17 (43%) of 40 papillary carcinomas. In contrast, none of the three follicular carcinomas, follicular adenoma, nodular hyperplasia, and normal thyroids, showed evidence of Ret mRNA. Our results showed that, in papillary thyroid carcinoma, there is an important role of Ret activation. The Ret staining could be a useful marker for papillary carcinoma.

Adenocarcinoma, Follicular↗
PubMed2003

Germination and elongation of flax in microgravity.

This experiment was conducted as part of a risk mitigation payload aboard the Space Shuttle Atlantis on STS-101. The objectives were to test a newly developed water delivery system, and to determine the optimal combination of water volume and substrate for the imbibition and germination of flax (Linum usitatissimum) seeds in space. Two different combinations of germination paper were tested for their ability to absorb, distribute, and retain water in microgravity. A single layer of thick germination paper was compared with one layer of thin germination paper under a layer of thick paper. Paper strips were cut to fit snugly into seed cassettes, and seeds were glued to them with the micropyle ends pointing outward. Water was delivered in small increments that traveled through the paper via capillary action. Three water delivery volumes were tested, with the largest (480 microliters) outperforming the 400 microliters and 320 microliters volumes for percent germination (90.6%) and root growth (mean=4.1 mm) during the 34-hour spaceflight experiment. The ground control experiment yielded similar results, but with lower rates of germination (84.4%) and shorter root lengths (mean=2.8 mm). It is not clear if the roots emerged more quickly in microgravity and/or grew faster than the ground controls. The single layer of thick germination paper generally exhibited better overall growth than the two layered option. Significant seed position effects were observed in both the flight and ground control experiments. Overall, the design of the water delivery system, seed cassettes and the germination paper strip concept was validated as an effective method for promoting seed germination and root growth under microgravity conditions.

Equipment Design↗
PubMed2003

Development of a membrane strip immunosensor utilizing ruthenium as an electro-chemiluminescent signal generator.

A photometric immunosensor that can be used for on-site diagnosis has been constructed. The sensor system was assembled by partially superimposing a nitrocellulose membrane strip (the lower) containing an immobilized antigen on the surface with a glass fiber membrane strip (the upper) including two electrodes on the opposite surfaces. To amplify the signal, we introduced a liposome, containing ruthenium molecules trapped in the core, chemically coupled to an antibody specific to the analyte (e.g. Legionella antigen). In the presence of the analyte, immune complexes were formed by antigen-antibody reactions upon addition of the immuno-liposome into a sample. This mixture was then absorbed by the capillary action from the bottom of the membrane strip. The liposome particles in the complexes were carried by a medium through the antigen pad without interaction, while free immuno-liposome was trapped by immune reactions on the pad surfaces. The aqueous medium influx into the glass pad dissolved a detergent pre-located within the compartment and the liposome rupture thereby released ruthenium molecules into the solution. The molecules were oxidized on the electrode surfaces and produced an electro-chemiluminescence (ECL) in proportion to the analyte concentration. The signal generation based on ECL resulted in an exponential dose-response pattern and the analyte detection limit of 2 ng/ml was approximately 10-fold more sensitive than that obtained from a conventional system.

Adsorption↗
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↗
PubMed2002

A strip liposome immunoassay for aflatoxin B1.

A technique has been developed for the preparation of aflatoxin B1 (AFB1)-tagged liposomes encapsulating a visible dye. These liposomes have several useful potential analytical applications, one of which is demonstrated. A simple plastic-backed nitrocellulose strip is the basis for an assay for detecting AFB1. Samples containing aflatoxin B1 are allowed to migrate by capillary action along the strip into a zone containing immobilized antibodies; then aflatoxin B1-tagged, dye-containing liposomes are allowed to migrate into the same area, filling any remaining antibody sites. The liposomes that bound to the antibody zone exhibit an intense purplish pink color whose optical density is inversely proportional to the aflatoxin concentration in the sample. The device is capable of detecting aflatoxin B1 at levels down to 20 ng and could serve as a rapid procedure for visual screening of agricultural and food samples for AFB1 or, with densitometry, as an inexpensive quantitative assay.

Aflatoxin B1↗
PubMed2005

Functional membrane-implanted lab-on-a-chip for analysis of percent HDL cholesterol.

A functional lab-on-a-chip has been developed for simultaneous quantitative analyses of high-density lipoprotein (HDL) cholesterol (HDL-C) and total cholesterol (total-C) in a submicroliter plasma sample. The analytical device was fabricated by placing commercial membranes, traditionally used for rapid diagnostics, within microfluidic channels engraved on the surface of a plastic chip. The concentration of HDL-C was measured using enzymatic reactions to produce a colorimetric signal after separation of the single plasma lipoprotein from a mixture. Two small pieces of different membrane pads were used to provide each group of reagents, for HDL separation and enzyme reactions, deposited within their tiny pores in a dry state. To maintain a connection toward the capillary action of the medium, the pads were arranged in a sequence within the fluidic channel that controlled the inlet and outlet of the flow. Upon the addition of a sample, the fluid was delivered through the pads of the chip and a color signal was subsequently generated in proportion to the concentration of HDL-C. The level of total-C was concurrently determined by following identical processes, except absent HDL separation. The two signals were simultaneously determined by employing optical detectors based on transmittance of a light. Such total analyses were completed within 2 min, and the sample sizes were able to be reduced to 0.4 microL for HDL-C and 0.1 microL for total-C, enough to cover the clinically required dynamic ranges.

Cholesterol↗
PubMed2006

Plastic ELISA-on-a-chip based on sequential cross-flow chromatography.

A plastic chip that can perform immunoassays using an enzyme as signal generator, i.e., ELISA-on-a-chip, was developed by incorporating an immunostrip into channels etched on the surfaces of the chip. To utilize an analytical concept of cross-flow chromatography, the chip consisted of two cross-flow channels in the horizontal and vertical directions. In the vertical channel, we placed a 2-mm-wide immunostrip for cardiac troponin I (cTnI), which was identical to a conventional rapid test kit except for the utilization of an enzyme, horseradish peroxidase (HRP), as tracer. An enzyme substrate supply channel and a horizontal flow absorption pad compartment were transversely arranged on each lateral side of the signal generation pad of the strip, respectively. Upon application of a sample containing cTnI, it migrated vertically through the membrane strip by capillary action, and antigen-antibody binding occurred. After 15 min, the horizontal flow was initiated by the addition of a chromogenic substrate solution for HRP into the supply channel and by partial superimposition of the horizontal flow absorption pad onto the signal generation pad. A color signal proportional to the analyte concentration was produced on this pad, measured after 5 min as optical densities using a digital camera-based detector, and quantified by integration of the densities under the peak after normalization. Its calibration curve indicated that the detection limit of the chip was approximately 0.1 ng/mL and its quantification limit was 0.25 ng/mL. In measuring blindly prepared samples, the chip performance correlated with that of a reference system, Beckman Coulter Access, within 2.5-fold discrepancy at the detection limit.

Antibodies↗