Biomedical subjects

Aleksi E Soini

Publications and source records attributed to Aleksi E Soini.

15 recordsLinked to original sources

PubMed2005

A novel separation-free assay technique for serum antibodies using antibody bridging assay principle and two-photon excitation fluorometry.

A new technique for separation-free detection of antigen-specific antibodies is presented. The new technique employs antibody bridging assay principle and the recently developed ArcDia TPX fluorescence detection technology. According to the assay scheme, antibody molecules from the sample bind with one arm to an antigen on polymer microspheres and with the other arm to a fluorescently labeled secondary antigen reagent. 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 by means of two-photon excited fluorescence detection. In order to demonstrate the applicability of the new assay technique, an assay for anti-adenovirus antibodies was constructed. The function of the assay method was tested both with monoclonal anti-adenovirus antibody preparation (standard analyte), and with positive serum samples. Standard class-specific ELISA was used as a reference method. The new assay method provides comparable sensitivity and precision, and wider dynamic range for IgG antibodies than the ELISA method. The standard curve showed linear response (R(2)=0.999) with a dynamic range of three orders of magnitude, detection limit (mean+3S.D.) of 8 pM, and intra-assay signal precision of 5%. Applicability of the new method for clinical serodiagnostics is discussed.

Animals↗

PubMed2005

A lab-on-a-chip compatible bioaffinity assay method for human alpha-fetoprotein.

A new lab-on-a-chip compatible binding assay platform is introduced. The platform combines dry-chemistry bioaffinity reagents and the recently introduced ArcDia TPX binding assay technique. The technique employs polymer microspheres as a solid phase reaction carrier, fluorescently labeled antibody conjugates, and detection of fluorescence emission from the surface of individual microspheres by two-photon excitation fluorescence. Signal response of the technique is independent of the reaction volume, thus the technique is particularly well suited for detection of bioaffinity reactions from miniature volumes. Performance of the new assay platform is studied by means of an immunometric assay of human alpha-fetoprotein (hAFP) in 384-plate format, and the results are compared to those of a corresponding wet-chemistry assay method. The results show that the ArcDia TPX detection technique can be combined with dry-chemistry reagents without compromises in assay performance. The microchip field has so far been characterized with a lack of microchip-compatible detection platforms which would allow cost-effective microchip design and sensitive bioaffinity detection. The presented detection technique is expected to provide a solution for this shortage.

Humans↗

PubMed2005

Dipyrrylmetheneboron difluorides as labels in two-photon excited fluorometry. Part I--Immunometric assays.

Seven different two-photon excitable dipyrrylmetheneboron difluoride labels (dipyrrylmethene-BF(2) labels) and a frequently used TAMRA label were conjugated to mouse IgG against alpha-fetoprotein in variable substitution degrees. Altogether 40 IgG conjugates were prepared, and studied with respect to one-photon absorption and emission properties, and two-photon fluorescence efficiency using 1064 nm laser as illumination source. Performance of the IgG conjugates as tracers in a separation-free immunometric assay of alpha-fetoprotein was evaluated using two-photon excitation assay technology, ArcDia TPX. The results show that the dipyrrylmethene-BF(2) labels provide subpicomolar sensitivity, which is an order of magnitude better than that of TAMRA label. The effect of chromophore structure and substitution degree of IgG-label conjugates on the assay performance is discussed.

Journal Article↗

PubMed2005

Dipyrrylmetheneboron difluorides as labels in two-photon excited fluorometry. Part II--Nucleic acid hybridization assays.

Five two-photon excitable dipyrrylmetheneboron difluoride labels (dipyrrylmethene-BF(2) labels) with fluorescence emission maximum between 530 and 590 nm, and a frequently used rhodamine label, TAMRA, were conjugated to aminomodified oligonucleotides. The performance of the labeled oligonucleotides was studied in a separation-free nucleic acid hybridization assay using ArcDia TPX bioaffinity assay technology. The results show that oligonucleotide conjugates of dipyrrylmethene-BF(2) labels provide higher two-photon excited fluorescence yield and better assay sensitivity than corresponding TAMRA conjugate. The effect of conjugation on photophysical properties of the labels and performance of the labeled oligonucleotides in separation-free hybridization assay is discussed.

Journal Article↗

PubMed2004

New separation-free assay technique for SNPs using two-photon excitation fluorometry.

A new separation-free method for detection of single nucleotide polymorphisms (SNPs) is described. The method is based on the single base extension principle, fluorescently labeled dideoxy nucleotides and two-photon fluorescence excitation technology, known as ArcDia trade mark TPX technology. In this assay technique, template-directed single base extension is carried out for primers which have been immobilized on polymer microparticles. Depending on the sequence of the template DNA, the primers are extended either with a labeled or with a non-labeled nucleotide. The genotype of the sample is determined on the basis of two-photon excited fluorescence of individual microparticles. The effect of various assay condition parameters on the performance of the assay method is studied. The performance of the new assay method is demonstrated by genotyping the SNPs of human individuals using double-stranded PCR amplicons as samples. The results show that the new SNP assay method provides sensitivity and reliability comparable to the state-of-the-art SNaPshot trade mark assay method. Applicability of the new method in routine laboratory use is discussed with respect to alternative assay techniques.

DNA Primers↗

PubMed2004

Fluorescent nanoparticles as labels for immunometric assay of C-reactive protein using two-photon excitation assay technology.

We describe the use of fluorophore-doped nanoparticles as reporters in a recently developed ArcDia TPX bioaffinity assay technique. The ArcDia TPX technique is based on the use of polymer microspheres as solid-phase reaction carrier, fluorescent bioaffinity reagents, and detection of two-photon excited fluorescence. This new assay technique enables multiplexed, separation-free bioaffinity assays from microvolumes with high sensitivity. As a model analyte we chose C-reactive protein (CRP). The assay of CRP was optimized for assessment of CRP baseline levels using a nanoparticulate fluorescent reporter, 75 nm in diameter, and the assay performance was compared to that of CRP assay based on a molecular reporter of the same fluorophore core. The results show that using fluorescent nanoparticles as the reporter provides two orders of magnitude better sensitivity (87 fM) than using the molecular label, while no difference between precision profiles of the different assay types was found. The new assay method was applied for assessment of baseline levels of CRP in sera of apparently healthy individuals.

Antibodies, Monoclonal↗

PubMed2004

Syntheses of novel dipyrrylmethene-BF2 dyes and their performance as labels in two-photon excited fluoroimmunoassay.

Two-photon excitation of fluorescence (TPE) has been found a powerful tool in the field of microscopy imaging and recently also in the field of bioanalytics. The recently introduced bioaffinity assay technology, ArcDia TPX, enables separation-free ultra-sensitive immunoassays from microvolumes. This assay technique is based on the use of microspheres as a solid reaction carriers and two-photon excited fluorescence detection. In the ArcDia TPX-technology, the individual microparticles are observed and the number of bound biomolecules on the microparticle surface is quantified by two-photon excited fluorescence. Here we present synthesis and use of a novel dipyrrylmethene-BF2 fluorophore that has been designed to be used as label in ArcDia TPX assay technique. The absorption and emission wavelengths of the label are tuned to allow excitation with a 1064 nm microchip laser. The label contains two-carboxylic residues, one of which is activated as N-hydroxysuccinimide ester to enable labeling of amino residues of biomolecules. The other carboxylic group is in free form to increase solubility in aqueous solutions. This new fluorescent label is tested in a separation-free immunoassay using ArcDia TPX assay technique. The performance of the new label is compared to that of one of the brightest fluorophores available, R-phycoerythrin (RPE). According to the results, the dipyrrylmethene-BF2 label provides significantly better signal-to-background ratio, leading to higher assay sensitivity and broader dynamic range compared to that of RPE. Good solubility to aqueous solutions and high fluorescence quantum efficiency, suggests the dipyrrylmethene-BF2 label is applicable also in other fluorescence-based applications.

Animals↗

PubMed2004

Hydrophilic labeling reagents of dipyrrylmethene-BF2 dyes for two-photon excited fluorometry: syntheses and photophysical characterization.

Recently introduced bioaffinity assay technology, ArcDia TPX, is based on two-photon excited fluorescence (TPE) and it enables separation-free ultra-sensitive immunoassays from microvolumes. Here we present syntheses of novel two-photon excitable fluorescent labeling reagents which have been specially designed to be used as label molecules in the ArcDia TPX assay technique. The labeling reagents are based on dipyrrylmetheneboron difluoride (dipyrrylmethene-BF2) chromophore, which have been substituted with aryl, heteroaryl or arylalkenyl chemical groups to extend the pi-electron conjugation. These substitutions results in a series of dipyrrylmethene-BF2 fluorophores with different photophysical properties. Dipyrrylmethene-BF2 fluorophores have been further substituted with a dipeptide linker unit and finally activated as succinimidyl esters to enable specific coupling with primary amino groups. The dipeptide linker serves as a spacer arm between the label and a target, and enhances the solubility of the label in aqueous solutions. Study of the chemical and photophysical performance of the new labeling reagents is described. The new labeling reagents exhibit high fluorescence quantum yields, and molar absorption coefficients. The results show that the new labels with the hydrophilic dipeptide linker unit provide large two-photon excitation cross-sections, high fluorescence quantum efficiency and good solubility in aqueous solutions. The results suggest that the novel dipyrrylmethene-BF2 labels are highly applicable to bioaffinity assays based on two-photon excitation of fluorescence.

Antibodies, Monoclonal↗

PubMed2003

A new technique for multiparameter imaging microscopy: use of long decay time photoluminescent labels enables multiple color immunocytochemistry with low channel-to-channel crosstalk.

In this report, we describe luminescence imaging microscopy using five different photoluminescent dyes in a single specimen. We combined the long decay time luminophores, europium(III) chelate, terbium(III) chelate, palladium(II) coproporphyrin, and platinum(II) coproporphyrin, with a green nuclear stain, Syto 25 trade mark, that emits conventional fast decaying fluorescence. The luminescence emissions from the five different luminophores were separated from each other by the differences in spectra and decay times using time-resolved detection. Applicability of this dye-combination for multiparameter analysis of a biological object was verified in a mixed population of peripheral blood leukocytes. Leukocyte cytocentrifugates were incubated in one step with a cocktail of luminophore-conjugated antibodies recognizing neutrophil- and lymphocyte-specific markers, followed by rapid staining with a mixture of nuclear stain and Pt-porphyrin as an eosinophil stain. The results show that multiple luminescent dyes with long decay time can be used together, and in combination with a conventional fluorophore. The separation of the signals of the long decay time labels was distinctive and enabled reliable identification of different leukocyte types, as well as an automated cell count. The long decay time luminophores together with time-resolved luminescence imaging microscopy (TR-LIM) provide a unique tool for studies of simultaneous expression of multiple antigens at the level of a single cell. In comparison with other multiparameter imaging techniques, the described technique offers increased accuracy of results, simplification of preparation procedure, and dramatic shortening of the total processing time. To our knowledge, this is the first time that simultaneous fivefold labeling/staining and analysis in a single specimen has been performed in the field of immunocytochemistry.

Fluorescent Dyes↗

PubMed2003

Evaluation of the phosphorescent palladium(II)-coproporphyrin labels in separation-free hybridization assays.

Palladium(II)-coproporphyrin label and a set of corresponding monofunctional labeling reagents with different linker arms were evaluated for labeling of oligonucleotides and subsequent use in hybridization assays. The properties of resulting oligonucleotide probes including phosphorescence spectra, quantum yields, lifetimes, and labeling yields were examined as functions of the label and oligonucleotide structures. Upon hybridization with complementary sequences bearing dabcyl, QSY-7, and rhodamine green dyes, the probes displayed strong quenching due to close proximity effects. Intensity and lifetime changes of the phosphorescence, distance, and temperature dependences were investigated in detail. The potential of the new label and probes for sensitive and separation-free hybridization assays was discussed.

Biological Assay↗

PubMed2003

Fluorescence-based cell viability screening assays using water-soluble oxygen probes.

A simple luminescence-based assay for screening the viability of mammalian cells is described, based on the monitoring of cell respiration by means of a phosphorescent water-soluble oxygen probe that responds to changes in the concentration of dissolved oxygen by changing its emission intensity and lifetime. The probe was added at low concentrations (0.3 microM to 0.5 nM) to each sample containing a culture of cells in the wells of a standard 96-well plate. Analysis of oxygen consumption was initiated by applying a layer of mineral oil on top of each sample followed by monitoring of the phosphorescent signal on a prompt or time-resolved fluorescence plate reader. Rates of oxygen uptake could be determined on the basis of kinetic changes of the phosphorescence (initial slopes) and correlated with cell numbers (10(5) to 10(7) cells/mL for FL5.12 lymphoblastic cell line), cell viability, or drug/effector action using appropriate control samples. The assay is cell noninvasive, more simple, robust, and cost-effective than existing microplate-based cell viability assays; is compatible with existing instrumentation; and allows for high-throughput analysis of cell viability.

Apoptosis↗

PubMed2002

Performance evaluation of the phosphorescent porphyrin label: solid-phase immunoassay of alpha-fetoprotein.

Phosphorescent conjugates of antibodies, neutravidin, and biotin (pentylamine derivative) were synthesized using previously described monofunctional labeling reagent of platinum(II) coproporphyrin-I with isothiocyanate reactive group (PtCP-NCS). These conjugates, which can be considered as standard reagents for a range of bioanalytical applications, were evaluated in solid-phase immunoassay schemes with the clinical analyte a-fetoprotein (AFP). A custom-designed time-resolved phosphorescence plate reader based on a compact and low-cost 532-nm laser and optimized for measurement of porphyrin labels was used. Using optimized tracers, instrumentation and assay protocols, subpicomolar detection limits were obtained both for PtCP label in solution and for AFP in solid-phase immunoassay. This sensitivity is comparable with standard time-resolved fluorescence immunoassays with lanthanide labels. The performance of metalloporphyrin labels, instrumentation, and solid-phase immunoassays as an alternative to the established detection platforms is discussed.

Biotin↗

PubMed2002

Synthesis and evaluation of phosphorescent oligonucleotide probes for hybridisation assays.

Monofunctional, p-isothiocyanatophenyl-derivatives of platinum (II)-coproporphyrin-I (PtCP-NCS) were evaluated as phosphorescent labelling reagents for synthetic oligonucleotides containing a 3'- or 5'-amino modification. Synthesis and purification conditions were optimised to generate high yields and purity of PtCP-labelled oligonucleotide probes. Phosphorescent properties of the PtCP label have been shown to be largely unaffected by conjugation to oligonucleotides of various length, GC composition and label attachment site. 5'-PtCP-labelled oligonucleotides were shown to work efficiently as primers in a standard PCR. A dedicated 532 nm laser-based time-resolved fluorescence plate reader enabled highly sensitive detection of PtCP-labelled oligonucleotides and PCR products, both in solution and in agarose gels, with limits of detection in the order of 0.3 pM. A model system employing two complementary oligonucleotides labelled with PtCP and QSY 7 dye (dark quencher) showed strong (approximately 20-fold) and specific proximity quenching of PtCP label upon hybridisation in solution. The potential applications of PtCP-labelled probes in hybridisation assays were discussed.

Luminescent Measurements↗

PubMed2002

Phosphorescent metalloporphyrins as labels in time-resolved luminescence microscopy: effect of mounting on emission intensity.

In this study, we present an investigation of the effects of mounting media on the phosphorescence of metalloporphyrin stained microscopy samples. The samples were: (1) Platinum(II) coproporphyrin (=PtCP) stained porous Sephadex beads; (2) compact polystyrene microspheres coated with IgG-PtCP conjugate; and (3) immunocytochemically labeled human peripheral blood neutrophils. The human neutrophils in a mixed leukocyte population were fixed, permeabilized, and then immunolabeled with PtCP conjugate of monoclonal mouse IgG directed to the intracellular antigen myeloperoxidase. The samples were mounted in twelve different mounting media and studied with quantitative time-resolved luminescence imaging microscopy with respect to the intensity and stability of the phosphorescence signal. The results indicate that microscopy samples stained with PtCP exhibit the brightest phosphorescence emission in non-mounted form or when mounted in non-aqueous permanent mounting media.

Animals↗

PubMedDate not supplied

Influence of linker unit on performance of palladium(II) coproporphyrin labelling reagent and its bioconjugates.

In this paper we describe the preparation of a series of new phosphorescent labelling reagents, based on monosubstituted palladium(II) coproporphyrin-I and the isothiocyanato reactive group. The labelling reagents differ with respect to the chemical composition of the linker unit that combines the reactive group and the porphyrin chromophore. Altogether, seven different labelling reagents are prepared. The new labelling reagents are conjugated with monoclonal mouse IgG to yield label conjugates with variable degrees of conjugation. The effect is studied of linker unit on: (a) the conjugation reaction kinetics; (b) the biological activity of the resulting IgG conjugates; and (c) the efficiency of phosphorescence emission. The results show that an increase in the length of the linker unit has a positive effect on both the reactivity of the label and the biological activity of the resulting conjugates. In addition, the results indicate that the labels with the most hydrophilic linker units exhibit the highest phosphorescence emission efficiencies.

Animals↗