Analysis Chip With Reference Range, Kits and Methods of Analysis

• US 20080254448A1
• Filed: 12/23/2004
• Published: 10/16/2008
• Est. Priority Date: 12/29/2003
• Status: Active Grant

• Current Assignee: Commissariat a L'Energie Atomique (AES Corporation), Biomerix Corp.
• Original Assignee: Commissariat a L'Energie Atomique (AES Corporation), Biomerix Corp.
• Inventors: Mallard, Frederic, Ginot, Frédéric, Novelli-Rousseau, Armelle, Ricoul, Florence
• Granted Patent: US 8,129,113 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2545/101 with an internal standard/control
• CPC Class Codes: C12Q 2545/113 with an external standard/control, i.e. control reaction is separated from the test/target reaction

Abstract

Analysis chip of at least one analyte, said chip comprising at least one analysis spot for recognition and immobilization specific to the analyte; and a reference range (G) comprising several reference spots each arranged on said chip in a defined manner and independently of one another, each reference spot of this range comprising, immobilized on its surface and in a defined proportion P that is different and known for each spot relative to the other reference spots of said range: a probe reference molecule (PRM) permitting recognition and hybridization specifically to a defined target reference molecule (TRM), and/or an inert oligonucleotide molecule (IM) incapable of recognition and hybridization with said PRM, these molecules both being unable to immobilize said analyte or analytes. The method of the invention comprises the use of said chip with its reference range for analysis of said, at least one, analyte.

Claims

1. An analysis chip of at least one analyte present in a liquid sample, said chip comprising:

(a) at least one analysis spot of said, at least one, analyte, said analysis spot being arranged on the chip in such a way as to permit the recognition and immobilization specific to the analyte; and

(b) a reference range (G), said reference range comprising several reference spots each arranged on said chip in a defined manner and independently of one another, each reference spot of this range comprising, immobilized on its surface and in a defined proportion P different for each spot relative to the other reference spots of said range;

(i) at least one probe reference molecule (PRM) for recognizing and specifically immobilizing a defined target reference molecule (TRM), and/or

(ii) at least one inert molecule (IM) incapable of recognizing and attaching to said target reference molecule, the probe reference molecule and the inert molecule both being unable to recognize and immobilize said analyte or said analytes;

with

2. A chip according to claim 1, in which the analyte and the target reference molecule are of the same nature.

3. A chip according to claim 2, in which the analyte and the target reference molecule are oligonucleotides or antibodies.

4. A chip according to claim 1, in which the probe reference molecule, the target reference molecule and the inert molecule are oligonucleotides.

5. A chip according to claim 1, in which the, at least one, analyte is a nucleic acid, and in which the, at least one, analysis spot is a spot functionalized by a nucleic acid complementary to the latter.

6. A chip according to claim 1, in which the spots of the reference range are disposed on the chip linearly and are arranged as a function of the proportion P of each spot.

7. A diagnostic kit comprising a chip according to claim 1.

8. An analysis kit comprising a chip according to claim 1.

9. A method for monitoring variations in gene expression in cells or tissues in vitro, which comprises the use of a chip according to claim 1.

10. A method of genotyping in vitro, which comprises the use of a chip according to claim 1.

11. A method of analysis in vitro of at least one analyte that may be present in a liquid sample comprising the following steps:

(a) optionally, immobilization of a first detecting means on the analyte;

(b) addition, to the sample to be analyzed, comprising the optionally labelled analyte, of a target reference molecule (TRM) on which a second detecting means, identical to or different from the first detecting means, has optionally been immobilized, said TRM being capable of recognizing and of binding specifically to the probe reference molecule (PRM) of an analysis chip according claim 1, said TRM being added to said sample in a sufficient amount to saturate the PRM of the reference range (G) of said chip, thus creating a reference range that is a function of the defined proportion P which is different for each reference spot;

(c) bringing the sample to be analyzed comprising the analyte and the TRM, optionally labelled, into contact with said analysis chip according to claim 1 comprising at least one analysis spot of said at least one analyte in physicochemical conditions such that;

on the one hand, the analyte to be analyzed, if it is present, binds to its analysis spot on the chip; and

on the other hand, the TRM specifically recognizes the PRM and binds to the latter on the various reference spots of the reference range of the chip;

(d) determination of a reference signal emitted, if applicable owing to the second detecting means, by each reference spot of the reference range, said signal being a function of the amount of target reference molecule immobilized on the latter; and

(e) determination of an analysis signal emitted, if applicable owing to the first detecting means, by said at least one analysis spot and being a function of the amount of analyte immobilized by the analysis spot, and comparison with the signals determined in step (d) for expressing this analysis signal as a function of P.

12. A method according to claim 11, in which the analyte and the target reference molecule are of the same nature.

13. A method according to claim 12, in which the analyte and the target reference molecule are oligonucleotides or antibodies.

14. A method according to claim 11, in which the probe reference molecule, the target reference molecule and the inert molecule are oligonucleotides.

15. A method according to claim 11, in which the at least one analyte is a nucleic acid, and in which the at least one analysis spot is a spot functionalized by a nucleic acid complementary to this nucleic acid.

16. A method according to claim 11, in which the first and the second detecting means involve respectively a first marker and a second marker.

17. A method according to claim 16, in which the first and the second markers are selected independently from the group consisting of fluorescent markers, radioactive markers, enzymatic markers, latex particles, photonic crystals and colloidal gold.

18. A method according to claim 11, in which the determination of the signal of each spot of the reference range and/or of each analysis spot is performed by a method selected from the group consisting of methods of detection by surface plasmon resonance, methods of photothermal detection, ellipsometric techniques and methods of photometric detection.

19. A method according to claim 11, in which the analysis is a detection or a quantitative analysis.