التفاصيل البيبلوغرافية
| العنوان: |
DETECTION AND/OR QUANTIFICATION METHOD OF TARGET MOLECULES ON A SOLID SUPPORT |
| Document Number: |
20110281775 |
| تاريخ النشر: |
November 17, 2011 |
| Appl. No: |
13/107353 |
| Application Filed: |
May 13, 2011 |
| مستخلص: |
The present invention relates to a method and device for detecting and/or quantifying one or multiple target molecules present in a solution by quantifying online their binding on specific capture molecules immobilized at different locations (spots) of a surface of an optically transparent solid support without substantial detection of target molecules present in solution. The present invention allows multiple target assays to be performed in a simultaneous detection. More particularly, the invention comprises detecting in real-time the hybridization between capture DNA molecules present on a micro-array and target polynucleotides present in solution. The invention is also related to real-time PCR of multiple targets on a micro-array. |
| Inventors: |
ALEXANDRE, ISABELLE (Haltinne, BE); Koehn, Heinz (Hamburg, DE); Remacle, Jose (Jambes, BE); De Roeck, Sven (Bruxelles, BE) |
| Assignees: |
EPPENDORF AG (Hamburg, DE) |
| Claim: |
1. A device for detection and/or quantification of different target molecules, said device comprising: (a) an optically transparent solid support having refractive index higher than 1.33 and a thickness of at least 1 mm that comprises at least 4 target molecules bound on capture molecules present at defined locations on said solid support surface in the form of an array, wherein said solid support comprises two surfaces inclined relative to the surface of the support on which the capture molecules are bound, a first surface being optically transparent and inclined by an angle of between 90 and 60° relative to the surface of the support on which the capture molecules are bound and a second surface opposite to the first surface being black or covered with a color being black or covered with a color having an absorption corresponding to the wavelength of the emitted light; (b) a chamber being formed on the surface of the said solid support covering the bound capture molecules having a thickness of 1 mm or lower; and (c) a closing system. |
| Claim: |
2. The device according to claim 1, comprising two interconnected chambers separated by a spur and an injection chamber having a close system preferably a screw cap. |
| Claim: |
3. The device according to claim 1, wherein the optical block comprises a ridge channel on the opposite side of the support compared to the observation area. |
| Claim: |
4. A kit for PCR amplification and detection of a target nucleic acid sequences comprising: (a) an amplification composition having at least one primer pair, a thermostable DNA polymerase, a hot start PCR amplification system, a plurality of dNTPs, and (b) a device according to any one of claims 1 to 3 comprising a micro-array having at least four capture probes having sequences corresponding to the said target nucleic acid sequences to be detected. |
| Claim: |
5. The kit according to claim 4, wherein the composition also contains a salt composed of a cation and an anion, wherein the anion has two carboxylic groups and one amine group, and 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. |
| Claim: |
6. An apparatus for detection and/or quantification of different target molecules being bound at different locations (spots) of a surface of an optically transparent solid support, said apparatus comprising: (a) an optically transparent solid support having refractive index n1 that comprises at least four target molecules bound on capture molecules present at defined locations on said solid support surface in the form of an array and a chamber being formed on the surface of the said solid support forming a device covering the bound capture molecules and wherein the refractive index of the solid support is higher than 1.33, (b) a light source to produce a light beam that reaches the surface of the support having immobilized capture molecules with an angle of 90° plus or minus 10°, said light beam being capable of homogeneous excitation of the surface of the support on which the target molecules are bound, (c) a lens located on the emitted light path between the surface of the support on which the target molecules are bound and the detector, and (d) a detector for measuring light emitted from the target molecules in at least four different locations as an image of at least 4 different locations that are spatially discriminated is simultaneously obtained onto the surface of detector, said emitted light being collected through a side of said support which is inclined relative to the surface of the support on which the target molecules are bound, said detector being positioned at an observation angle θobin relative to the normal of said solid support surface, such that 90°>θobin>sin−1(n2/n1) and wherein said emitted light is focused on the detector surface and is in focus for at least 4 different locations. |
| Claim: |
7. The apparatus of claim 6, wherein the detector has at least 10 pixels and the surface corresponding to a first location has at least 1 pixel difference from a second location. |
| Claim: |
8. The apparatus of claim 6, wherein the detector is a Camera or a planned detector having at least 10 pixels on which the image is projected. |
| Claim: |
9. The apparatus of claim 6, wherein the image of the overall surface of the support having bound targets is in focus and projected at once onto the detector surface so that pixels data is attributed to the different locations having bound targets. |
| Claim: |
10. The apparatus of claim 6, wherein a surface opposite to the side of the support which is inclined relative to the surface of the support on which the target molecules are bound is black or covered with a color being black or covered with a color having an absorption corresponding to the wavelength of the emitted light. |
| Claim: |
11. The apparatus of claim 6, comprising a device according to any one of claims 1 to 3. |
| Claim: |
12. The apparatus of claim 6, further comprising an automated thermal cycler capable of alternatively heating and cooling, and adapted to receive at least one closed reaction chamber containing said immobilized capture molecules, and reagents for nucleic acid amplification. |
| Claim: |
13. The apparatus of claim 6, further comprising means for correcting image distortion |
| Claim: |
14. The apparatus of claim 6, further comprising means for increasing the depth of focus, said means comprising a mechanical tilt of the captor surface of the detector relative to the emitted light direction. |
| Current U.S. Class: |
506/39 |
| Current International Class: |
40 |
| رقم الانضمام: |
edspap.20110281775 |
| قاعدة البيانات: |
USPTO Patent Applications |
