Method of sampling, amplifying and quantifying segment of nucleic acid, polymerase chain reaction assembly having nanoliter-sized sample chambers, and method of filling assembly

• US 20020164820A1
• Filed: 04/25/2002
• Published: 11/07/2002
• Est. Priority Date: 04/17/1997
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated), United States Department Of Health And Human Services
• Original Assignee: Genomic Nanosystems, LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Brown, James F.
• Granted Patent: US 7,459,315 B2
• Time in Patent Office: Days
• US Class Current: 436/180
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00644 the porous medium being present in discrete locations, e.g. gel pads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00677 Ex-situ synthesis followed by deposition on the substrate
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 2200/06 Fluid handling related problems
• CPC Class Codes: B01L 2200/0642 Filling fluids into wells by specific techniques
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2200/16 Reagents, handling or storing thereof
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2400/0409 centrifugal forces
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: B01L 3/5085 for multiple samples, e.g. microtitration plates
• CPC Class Codes: B01L 3/50851 specially adapted for heating or cooling samples laboratory heating apparatus B01L7/00; incubators C12M
• CPC Class Codes: B01L 3/5088 confining liquids at a location by surface tension, e.g. virtual wells on plates, wires B01L3/50857 takes precedence
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6844 : Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 : Polymerase chain reaction [PCR]
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: Y10S 436/805 : Optical property
• CPC Class Codes: Y10S 436/809 : Multifield plates or multicontainer arrays
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

A miniaturized assembly is provided whereby a fluid sample can be divided into a plurality of sample portions in retaining wells and the sample fluid can be displaced from open ends of the wells while simultaneously being sealed in the wells. A method of dividing a fluid sample using the assembly is also provided.

Claims

1. A miniaturized assembly comprising:

a substrate;

a plurality of portion retaining means in or on the substrate and each including an open end having a dimension of 2 millimeters (mm) or less;

means for loading a chemical or biological fluid sample into said plurality of portion retaining means;

means for displacing said fluid sample away from the open ends of said plurality of portion retaining means; and

means for sealing at least the open end of each of said plurality of portion retaining means while simultaneously displacing sample fluid away from said open end.

2. The miniaturized assembly of claim 1, wherein said open end has a diameter of 500 μ

m or less.

3. The miniaturized assembly of claim 1, wherein said open ends are defined by a surface of the substrate and said surface is hydrophobic.

4. The miniaturized assembly of claim 1, wherein said open ends are defined by a surface of the substrate and said surface is adhesive.

5. The miniaturized assembly of claim 1, wherein said means for displacing comprises a curable adhesive.

6. The miniaturized assembly of claim 1, wherein said substrate comprises a flexible material.

7. The miniaturized assembly of claim 1, wherein said means for displacing and said means for sealing both comprise a single surface of a seal.

8. The miniaturized assembly of claim 7, wherein said single surface is hydrophobic.

9. The miniaturized assembly of claim 7, wherein said single surface comprises a flexible material.

10. The miniaturized assembly of claim 7, wherein said single surface is adhesive.

11. The miniaturized assembly of claim 1, wherein said means for displacing is hydrophobic.

12. The miniaturized assembly of claim 1, wherein said means for displacing includes a liquid that is immiscible with the fluid sample.

13. The miniaturized assembly of claim 1, wherein said means for displacing is adhesive.

14. The miniaturized assembly of claim 1, wherein each of said plurality of retaining means is hydrophilic.

15. The miniaturized assembly of claim 1, wherein each of said plurality of retaining means comprises a retaining well having a volume of about 1 microliter (μ

l) or less.

16. The miniaturized assembly of claim 1, wherein said plurality of retaining means includes retaining means having different sizes.

18. The method of claim 17, wherein the open end of each of said plurality of portion retaining means has a dimension of 500 μ

m or less.

19. The method of claim 17, wherein said displacing includes displacing the sample fluid with a curable adhesive.

20. The method of claim 19, further comprising curing the curable adhesive after said displacing.

21. The method of claim 17, wherein said displacing includes displacing the sample fluid with a flexible material.

22. The method of claim 17, wherein said displacing includes displacing the sample fluid with a hydrophobic surface.

23. The method of claim 17, wherein said displacing includes displacing the sample fluid with an adhesive surface.

17. A method for dividing a chemical or biological fluid sample into a plurality of sample portions and simultaneously sealing said plurality of sample portions against contamination and evaporation, said method comprising:

introducing a chemical or biological fluid sample into an assembly having a substrate and a plurality of portion retaining means formed in or on the substrate, each portion retaining means having a dimension 2 millimeters (mm) or less and including an open end;

displacing said fluid sample away from the open end of each of said plurality of portion retaining means and simultaneously sealing the open end of each of said plurality of portion retaining means.