Array analysis for online detection

A method for performing the analysis of a microarray surface having immobilized capture molecule for performing real time PCR amplification of a polynucleotide molecule being present in a solution, said method comprising the steps of:

• a) providing a closed device comprising a transparent solid support with a surface having capture polynucleotide molecules immobilized thereon in at least one particular surface location;
• b) introducing a solution containing said polynucleotide molecule into said closed device, reagents for polynucleotide amplification, at least one label being a fluorophore and at least one light absorbing molecule as a light absorbing agent which has an absorption band which overlaps the emission and/or absorption band of the fluorophore and is not a quencher molecule of said at least one fluorescent label;
• c) submitting the solution to at least 2 thermal cycles having at least 2 and preferably 3 different temperature steps in order to obtain labelled target polynucleotide molecules by PCR amplification;
• d) measuring at least twice the obtained labelled target polynucleotide molecules present in said solution after or during at least two thermal cycles in the following way:
• incubating said labelled target polynucleotide molecules under conditions allowing a specific binding between said labelled target polynucleotide molecules and corresponding capture molecules,
• illuminating the surface of the support on which the labelled target polynucleotide molecules are bound using an excitation light reaching said surface through the support, in the presence of the solution containing the labelled target polynucleotide molecules thereby causing excitation of the said labelled target polynucleotide molecules,
• detecting the light emitted from the said surface locations through said support;
• e) analyzing the surface of the support in order to discriminate between the locations of the micro-array having bound targets and the locations deprived of target molecules wherein the discrimination is perfect so that no signal is attributed as positive signal to location(s) deprived of target molecules and to associate a positive signal value at the locations related to bound labelled target polynucleotide molecules;
• f) Processing the signal value data obtained after or during at least one thermal cycle in order to detect and/or quantify the target polynucleotide molecule present in the solution during and/or before the amplification.

A kit for detection and/or quantification of one or multiple polynucleotide target molecules being present in a solution by the analysis of a micro-array having immobilized capture molecules specific of said targets comprising:

• a) an hybridization composition comprising:
• a salt composed of a cation and an anion, wherein the said anion has preferably two carboxylic groups and one amine group, wherein the salt concentration in the composition is comprised between 10 mM and 400 mM and an exclusion agent from 1% to 20% by weight,
• a fluorescent label,
• a light absorbing agent wherein said light absorbing agent is a light absorbing molecule which has an absorption band which overlaps the emission and/or absorption band of the fluorophore and is not a quencher molecule of said fluorescent label;
• b) a device comprising:
• a transparent solid support having refractive index higher than 1.33 and a thickness of at least 0.5 mm and better at least 3 mm and even better 5 mm with a surface that comprises at least four capture molecules present at particular locations on said solid support surface,
• a chamber being formed on the surface of the said solid support covering the capture molecules having a thickness of 2 mm or lower;
• c) a software system comprising program instruction for position configuration indicating the position of the grid point within said frame with the two dimensional array of grid comprising the same number of spots as the array and a data storage having the identification of the grid points according to their position information on the array and their corresponding target molecules;
• preferably further comprising a composition for PCR amplification:
• at least one nucleic acid primer pair, a thermostable DNA polymerase and a plurality of dNTPs.