System and method of quantitatively determining a biomolecule, system and method of detecting and separating a cell by flow cytometry, and fluorescent silica particles for use in the same, and kit comprising plural kinds of the silica particles in combination

A method of quantitatively determining a target biomolecule in flow cytometry, comprising the steps of:

• providing plural kinds of target biomolecules;
• providing plural kinds of different fluorescent silica particles having an average diameter of 300 nm to 1200 nm, wherein the different fluorescent silica particles are optionally substantially uniform in particle diameter,
• wherein each kind of fluorescent silica particles is capable of emitting individual fluorescence B, detectable in a flow cytometer, each of the plural kinds of silica particles having dyes different in kinds and/or ratios differing in fluorescence emission spectrum and/or having a fluorescence intensity in plural phases, wherein each kind of the fluorescent silica particles is surface-coated with a binding substance capable of distinguishably immobilizing each kind of the target biomolecules;
• providing fluorescent-labeled probes, each labeled probe capable of emitting fluorescence A for quantitative determination of the target biomolecules, the fluorescent-labeled probes specifically capable of binding to the plural kinds of target biomolecules respectively, wherein fluorescence A emits fluorescence at a different wavelength from fluorescence B emitted by each of the fluorescent silica particles;
• immobilizing each kind of the target biomolecules on the surface of the corresponding kind of fluorescent silica particles;
• mixing the fluorescent-labeled probes with the fluorescent silica particles;
• and
• distinguishably detecting signals from each one of the individual plural kinds of fluorescent silica particles emitting fluorescence B, and detecting an intensity of the fluorescence A from each of the fluorescent-labeled probes to determine a quantity of each kind of target biomolecule, by using the flow cytometer.

A method of quantitatively determining a biomolecule, comprising the steps of:

• providing plural kinds of target biomolecules;
• providing plural kinds of different fluorescent silica particles having an average diameter of 300 nm to 1200 nm, wherein the different fluorescent silica particles are optionally substantially uniform in particle diameter,
• wherein each kind of fluorescent silica particles is capable of emitting individual fluorescence B, detectable in a flow cytometer, each of the plural kinds of silica particles having dyes different in kinds and/or ratios differing in fluorescence emission spectrum and/or having a fluorescence intensity in plural phases, wherein each kind of the fluorescent silica particles is surface-coated with a binding substance capable of distinguishingly immobilizing each kind of the target biomolecules;
• providing at least two kinds of molecular recognition substances capable of specifically binding to the plural kinds of target biomolecules respectively;
• providing fluorescent-labeled probes, each labeled probe capable of emitting fluorescence A for quantitative determination of the target biomolecules, the fluorescent-labeled probes specifically capable of binding to the at least two molecular recognition substances respectively, wherein fluorescence A emits fluorescence at a different wavelength from fluorescence B emitted by each of the fluorescent silica particles;
• mixing the molecular recognition substances with the plural kinds of fluorescent silica particles;
• mixing the fluorescent-labeled probes with the plural kinds of fluorescent silica particles;
• and
• distinguishably detecting signals from each one of the individual plural kinds of fluorescent silica particles emitting fluorescence B, and detecting an intensity of the fluorescence A from each of the fluorescent-labelled probes to determine a quantity of each kind of target biomolecule, by using the flow cytometer.