The invention relates to a separation free bioanalytical
assay method for measuring an
analyte from a
biological fluid or suspension comprising of microparticles as a bioaffinity binding
solid phase, a biospecific secondary
reagent labelled with a two-
photon fluorescent dipyrrometheneboron
difluoride dye, focusing the
laser into the reaction suspension, measuring two-
photon excited
fluorescence from single microparticles when they randomly float or are guided by the
radiation pressure of the excitation
laser through the
focal volume of the
laser beam using a two-
photon fluorescent dipyrrometheneboron
difluoride dye. The dye has the structure (II): Either at least one of groups R1, R2, R3, R4, R5, R6 and R7 is a substituted or unsubstituted phenyl, thienyl, pyrrolyl, furanyl, oxazolyl, isoxazolyl oxadiazolyl, imidazolyl, benzoxazolyl, benzothiazolyl, benzimidazolyl, benzofuranyl, indolyl, conjugated ethenyl, dienyl or trienyl group, and at least one of the groups R1, R2, R3, R4, R5, R6 or R7 is substituted to yield a chemically reactive group that can be used for selective covalent linkage to other molecules, and at least one of the groups R1, R2, R3, R4, R5, R6 or R7 is substituted to yield a water-solubilizing group, and the remaining groups of R1, R2, R3, R4, R5, R6 and R7 are each independently selected from the group consisting of
hydrogen,
halogen,
alkyl, cyano, carboxy, each of which can optionally be substituted; or groups R1, R2, R3, R5, R6 and R7 are substituted or unsubstituted
alkyl groups, R4 is a
hydrogen or a substituted or unsubstituted
alkyl, and at least one of the groups R1, R2, R3, R4, R5, R6 or R7 is substituted to yield a chemically reactive group that can be used for selective covalent linkage to other molecules; and at least one of the groups R1, R2, R3, R4, R5, R6 or R7 is su