Microbial identification from faces and urine in one step by two-photon excitation assay technique.

Two-photon excitation fluorometry (TPX) is a separation-free bioaffinity assay technique which enables accurate diagnostic testing in microvolumes. The technology is currently commercially applied in an automated mariPOC® test system for rapid phenotypic multi-microbe detection of pathogen antigens. The first TPX applications for diagnostics were intended for respiratory infection testing from nasopharyngeal and oropharyngeal samples. Feces and urine are more complex sample matrices and contain substances that may interfere with immunoassay binding or fluorescence detection. Our objective was to study the suitability of these complex matrices in the TPX technique. As expected, feces and urine elevated fluorescence levels but the methodology has the unique property of compensating for matrix effects. Compensation allows reliable separation of specific fluorescence from the fluorescence caused by the matrix. The studied clinical samples did not contain immunoassay inhibitors. The results suggest that the methodology is robust and may provide reliable testing of feces and urine samples with high accuracy.

Development of a rapid assay methodology for antimicrobial susceptibility testing of Staphylococcus aureus.

Development of a new phenotypic technique for rapid antimicrobial susceptibility testing (AST) of methicillin-resistant Staphylococcus aureus is presented. The new technique combines bacterial culturing and specific immunometric detection in a single separation-free process. The technique uses dry chemistry reagents and the recently developed two-photon excitation detection technology, which allows online detection of bacterium-specific growth. The performance of the new technique was evaluated by monitoring the growth of S. aureus reference strains and determining their susceptibility to oxacillin. In the direct analysis of clinical specimens, method specificity and tolerance to interferences caused by other bacteria present in the sample are pivotal. Other bacteria can compete with the bacteria of interest for nutrients, for example. Specificity and tolerance were studied against Staphylococcus epidermidis reference strains. The results suggest that the new technique could allow rapid AST directly from clinical samples within 6 to 8 h. Such a rapid and simple testing methodology would be a valuable tool in clinical microbiology because it would shorten the turnaround times of microbiologic analyses. Advantages of the new approach in relation to conventional methods are discussed.

Two-photon excitation fluorescence bioassays.

Application of two-photon excitation of fluorescence in microscopy is one of the major discoveries of the "renaissance" of light microscopy that started in the 1980s. The technique derives its advantages from the biologically "smooth" wavelength of the excitation light and the confinement of the excitation. Difficult, and seemingly nontransparent, samples may be imaged with the technique with good resolution. Although the bioresearch has been concentrating mostly on the positive properties of the technique for imaging, the same properties may be applied successfully to nonimaging bioassays. This article focuses on the development path of two-photon excitation-based assay system.

Rapid method for detection of influenza a and B virus antigens by use of a two-photon excitation assay technique and dry-chemistry reagents.

New separation-free assay methods for the rapid detection of influenza A and B virus antigens are presented. The methods employ dry-chemistry reagents and the recently developed two-photon excitation (TPX) fluorescence detection technology. According to the assay scheme, virus antigens are sandwiched by capture antibody onto polymer microspheres and fluorescently labeled antibody conjugate. Consequently, fluorescent immunocomplexes are formed on the surface of microspheres in proportion to the concentration of the analyte in the sample. The fluorescence signal from individual microspheres is measured, separation free, by means of two-photon excited fluorescence detection. In order to demonstrate the applicability of the new assay technique for virus antigen detection, methods for influenza A and B viruses were constructed. The assay method for influenza A virus applied a molecular fluorescent label, whereas the method for influenza B virus required a nanoparticle fluorescent reporter to reach sufficient clinical sensitivity. The new methods utilize a dry-chemistry approach, where all assay-specific reagents are dispensed into assay wells already in the manufacturing process of the test kits. The performance of the assay methods was tested with nasopharyngeal specimens using a time-resolved fluoroimmunoassay as a reference method. The results suggest that the new technique enables the rapid detection of influenza virus antigens with sensitivity and specificity comparable to that of the reference method. The dose-response curves showed linear responses with slopes equal to unity and dynamic assay ranges of 3 orders of magnitude. Applicability of the novel TPX technique for rapid multianalyte testing of respiratory infections is discussed.

Robust estimation of bioaffinity assay fluorescence signals.

In this paper, the challenging problem of robust mean-signal estimation of a single-step microparticle bioaffinity assay is investigated. For this purpose, a density estimation-based robust algorithm (DER) was developed. The DER algorithm was comparatively evaluated with four other parameter estimation methods (mean value, median filtering, least square estimation, Welsch robust m-estimator). Two important questions were raised and investigated: 1) Which of the five methods can robustly estimate the mean bioaffinity signal? and 2) How many microparticles need to be measured in order to obtain an accurate estimate of the mean signal value? To answer the questions, bootstrap and coefficient of variation (CV) analyses were performed. In the CV analysis, the DER algorithm gave the best results: The CV ranged from 0.8% to 4.9% when the number of microparticles used for the mean signal estimation varied from 800 to 30. In the bootstrap analysis of the standard error, the DER algorithm had the smallest variance. As a conclusion, it can be underlined that: 1) of all methods tested, the DER algorithm gave the most consistent and reproducible results according to the bootstrap and CV analysis; 2) using the DER algorithm accurate estimates could be calculated based on 80-100 particles, corresponding to a typical assay measurement time of 1 min; and 3) the investigated bioaffinity signals contained a large number of outliers (observations that severely deviate from the majority of data) and therefore robust techniques were necessary for the mean signal estimation tasks.

Method for localization of sialic acid on cell surface and cell interior in peripheral blood: a pilot study.

This article presents a method for identification and localization of cell surface and intracellular sialoglycoconjugates of peripheral blood cells. To reveal cell surface conjugates, a sample of peripheral blood was incubated with lectin after centrifugation and rinsing. For intracellular localization in leukocytes, RBCs were lysed and the membranes were permeabilized prior to cytochemical reaction. Fluorescein isothiocyanate conjugated lectins were used for visualization in fluorescence microscope. All lectins bound specifically to the surface of erythrocytes. Confocal microscopy showed surface and intracellular labeling of permeabilized leukocytes. A part of the signal in eosinophils originated from binding of anionic fluorophore to cationic granular proteins.

Oleosin expression and trafficking during oil body biogenesis in tobacco leaf cells.

We have established a versatile method for studying the interaction of the oleosin gene product with oil bodies during oil body biogenesis in plants. Our approach has been to transiently express a green fluorescent protein (GFP)-tagged Arabidopsis oleosin gene fusion in tobacco leaf cells containing bona fide oil bodies and then to monitor oleosin-GFP expression using real-time confocal laser scanning microscopy. We show that normally non-oil-storing tobacco leaf cells are able to synthesize and then transport oleosin-GFP fusion protein to leaf oil bodies. Synthesis and transport of oleosin-GFP fusion protein to oil bodies occurred within the first 6 h posttransformation. Oleosin-GFP fusion protein exclusively associated with the endoplasmic reticulum and was trafficked in a Golgi-independent manner at speeds approaching 0.5 microm sec(-1) along highly dynamic endoplasmic reticulum positioned over essentially static polygonal cortical endoplasmic reticulum. Our data indicate that oil body biogenesis can occur outside of the embryo and that oleosin-GFP can be used to monitor early events in oil body biogenesis in real-time.