Monofunctional derivatives of coproporphyrins for phosphorescent labeling of proteins and binding assays.

p-Isothiocyanatophenyl derivatives of Pt(II)- and Pd(II)-coproporphyrin I are described as stable monofunctional reagents which enable simple covalent labeling of proteins and other biomolecules under mild conditions in aqueous solutions. Labeling procedure was optimized for antibodies, avidin, and neutravidin. Photophysical properties of resulting conjugates important for their use in binding assays based on time-resolved phosphorescence detection were studied. The functional activity and long-term storage stability of antibody conjugates were assessed in comparison with unmodified proteins. The new labels and their conjugates were evaluated in the solid-phase immunoassays using commercial time-resolved phosphorescence readers Victor(2) and Arcus-1230 (Wallac). Potential applications of these reagents in in vitro diagnostics are discussed.

Two-photon fluorescence excitation in detection of biomolecules.

Two-photon fluorescence excitation has been found to be a very powerful method for enhancing the sensitivity and resolution in far-field light microscopy. Two-photon fluorescence excitation also provides a substantially background-free detection on the single-molecule level. It allows direct monitoring of formation of labelled biomolecule complexes in solution. Two-photon excitation is created when, by focusing an intensive light source, the density of photons per unit volume and per unit time becomes high enough for two photons to be absorbed into the same chromophore. In this case, the absorbed energy is the sum of the energies of the two photons. In two-photon excitation, dye molecules are excited only when both photons are absorbed simultaneously. The probability of absorption of two photons is equal to the product of probability distributions of absorption of the single photons. The emission of two photons is thus a quadratic process with respect to illumination intensity. Thus in two-photon excitation, only the fluorescence that is formed in the clearly restricted three-dimensional vicinity of the focal point is excited. We have developed an assay concept that is able to distinguish optically between the signal emitted from a microparticle in the focal point of the laser beam, and the signal emitted from the surrounding free labelled reagent. Moreover, the free labels outside the focal volume do not contribute any significant signal. This means that the assay is separation-free. The method based on two-photon fluorescence excitation makes possible fast single-step and separation-free immunoassays, for example, for whole blood samples. Since the method allows a separation-free assay in very small volumes, the method is very useful for high-throughput screening assays. Consequently we believe that two-photon fluorescence excitation will make a remarkable impact as a research tool and a routine method in many fields of analysis.

Phosphorescent porphyrin probes in biosensors and sensitive bioassays.

Platinum(II) and palladium(II) complexes of porphyrins and related tetrapyrrolic pigments emit strong phosphorescence at room temperatures, which is characterized by long lifetimes falling into the sub-millisecond range and long-wave spectral characteristics. These features make the dyes useful as probes for a number of bioanalytical applications, particularly those employing time-resolved fluorescent detection. They can provide high sensitivity and selectivity, together with rather simple instrumental set-up. A number of analytical systems are now under development that are based on the use of phosphorescent porphyrin probes. Experimental results are presented on the following systems: (i) fibre-optic phosphorescence lifetime-based oxygen sensor on the basis of hydrophobic platinum-porphyrins and development of advanced sensing materials and prototype instrumentation; (ii) practical applications of the optical oxygen sensor, including a sensitive immunosensor that employs glucose oxidase labels, a rapid screening method for cell viability in microtitre-plate format, non-destructive measurement of oxygen in packaged foods and reagentless biosensors for metabolites (glucose, lactate); and (iii) the use of water-soluble platinum- and palladium-porphyrins as labels for ultra-sensitive time-resolved phosphorescence immunoassays.

A new microvolume technique for bioaffinity assays using two-photon excitation.

Bioaffinity binding assays such as the immunoassay are widely used in life science research. In an immunoassay, specific antibodies are used to bind target molecules in the sample, and quantification of the binding reaction reveals the amount of the target molecules. Here we present a method to measure bioaffinity assays using the two-photon excitation of fluorescence. In this method, microparticles are used as solid phase in binding the target molecules. The degree of binding is then quantified from individual microparticles by use of two photon excitation of fluorescence. We demonstrated the effectiveness of the method using the human alpha-fetoprotein (AFP) immunoassay, which is used to detect fetal disorders. The sensitivity and dynamic range we obtained with this assay indicate that this method can provide a cost-effective and simple way to measure various biomolecules in solution for research and clinical applications.