Method and device for determining biological analytes
The invention relates to a method for quantitatively determining biological analytes in an aqueous solution in the presence of one or more functionalised surfaces, wherein the aqueous solution comprises at least one type of biological analyte and at least one type of fluorescence marker, characterised in that the quantity and/or concentration of the biological analyte or analytes is determined by measuring the fluorescence emission of the unbound fluorescence markers, as well as to a devices for carrying out said method.
1. Method for quantitatively determining biological analytes in an aqueous solution in the presence of one or more functionalised surfaces, comprising
(a) introducing at least one type of functionalised particle into a measuring chamber which has a detection region which is accessible to light through the bottom of the measuring chamber, and a separation region which is not accessible to light through the bottom of the measuring chamber;
(b) introducing a sample comprising at least one type of biological analyte into the measuring chamber;
(c) introducing at least one type of fluorescence marker into the measuring chamber;
(c′) mixing the at least one type of functionalised particle, the sample and the at least one type of fluorescence marker in the measuring chamber;
(c″) separating the unbound fluorescence markers from the bound fluorescence markers so that the bound fluorescence markers are located in the separation region;
(d) measuring the fluorescence emission of the unbound fluorescence markers in the detection region; and
(e) determining the quantity and/or concentration of the biological analyte or analytes using the measured fluorescence emission from (d),
wherein the functionalised surfaces comprise the functionalised particles, which are particles of polymer or a polymer mixture, on the surface of which there are catcher molecules which bind to the biological analyte or analytes and/or to the fluorescence marker or markers, and
wherein the functionalised particles have a mean diameter in a range of from about 20 to about 200 μm.
2. The method according to claim 1 , wherein (a), (b) and (c) are carried out sequentially or at least two of (a)-(c) are carried out simultaneously.
3. The method according to claim 1 , wherein (a) and/or (c) are carried out before remaining steps and with a time interval of greater than 12 hours between (a) and/or (c).
4. The method according to claim 1 , wherein the method is a direct immunoassay, sandwich immunoassay, displacement immunoassay, competitive immunoassay or secondary immunoassay.
5. The method according to claim 1 , wherein the functionalised particles have a mean diameter in a range of from about 80 to about 200 μm.
6. The method according to claim 1 , wherein the functionalised particles have a binding capacity of more than 200 mg IgG or analyte per mL.
7. The method according to claim 1 , further comprising:
(a′) treating at least one surface inside the measuring chamber with a coating buffer that promotes binding of a catcher molecule, and adding at least one type of catcher molecule.
8. The method according to claim 7 , wherein in (a′), the at least one type of catcher molecule is added in solution.
9. The method according to claim 1 , wherein the at least one type of functionalised particle is not a magnetic particle.