Diagnostic microarray and method of use thereof

• US 20030148362A1
• Filed: 02/04/2003
• Published: 08/07/2003
• Est. Priority Date: 02/07/2002
• Status: Abandoned Application

• Current Assignee: The Medical College of Hampton Roads
• Original Assignee: The Medical College of Hampton Roads
• Inventors: Luka, Janos
• Application Number:
  US10/357,756
• Publication Number:
  US 20030148362A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: G01N 27/44726 using specific dyes, markers or binding molecules
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings

Abstract

A microarray device for the analysis of biological samples is provided. The device includes a liquid permeable layer having a plurality of microregions, each including a plurality of probe-labeled microbeads embedded in the liquid permeable layer. The microbeads in a given microregion include a plurality of the same target probes on their surfaces. The target probes are capable of specifically binding to one or more particular target molecules (e.g., nucleic acid, polypeptide, small molecule antigen). The device typically has the capability of inducing a sample solution to move through the liquid permeable layer under the influence of an applied voltage. Kits which include the device and methods of simultaneously detecting a plurality of different target molecules in a sample solution are also provided.

Claims

1. A microarray device for the analysis of biological samples comprising:

a liquid permeable layer including a plurality of microregions, each microregion including a plurality of microbeads embedded in the liquid permeable layer;

wherein the microbeads in a given microregion have a plurality of target probes on their surfaces.

2. The device of claim 1 wherein all the microbeads in a given microregion have a plurality of a single target probe on their surfaces.

3. The device of claim 1 wherein the liquid permeable layer comprises agarose, polyacrylamide, cellulose or gelatin.

4. The device of claim 3 wherein the liquid permeable layer comprises about 0.1 to 2.0 wt. % agarose.

5. The device of claim 1 wherein the microregions have a largest dimension of no more than about 10 microns.

6. The device of claim 1 wherein the liquid permeable layer comprises about 250 to 2500 of the microregions per mm².

7. The device of claim 1 further comprising a first liquid chamber in fluid connection with the liquid permeable layer;

wherein the first liquid chamber includes an electrode.

8. The device of claim 7 further comprising a second liquid chamber in fluid connection with the liquid permeable layer;

wherein the second liquid chamber includes an electrode.

9. The device of claim 1 comprising a set of at least about 10 different lots of probe-labeled microbeads, each different lot of probe-labeled microbeads being present in at least one separate microregion;

wherein all the microbeads in a given lot have the same target probes on their surfaces.

10. The device of claim 1 wherein the target probes are covalently bound to the surfaces of the microbeads.

11. The device of claim 1 wherein the target probes include at least one target probe which is a polypeptide.

12. The device of claim 11 wherein the polypeptide includes an antibody Fab fragment.

13. The device of claim 1 wherein the target probes include at least one nucleic acid probe capable of specifically binding to a target nucleic acid.

14. The device of claim 13 wherein the nucleic acid probe is a DNA molecule.

15. The device of claim 13 wherein the nucleic acid probe is a modified nucleotide.

16. The device of claim 13 wherein the target probes include oligonucleotides capable of specifically binding to a nucleic acid from at least one of HIV, HHV, HSV, EBV, HCV, CMV, VZV, HPV, Hu, B19, and Ch1.

17. The device of claim 13 wherein the target probes include at least one probe selected from the group consisting of oligonucleotides capable of specifically binding to a nucleic acid from at least one of HHV-6, HHV-7 or HHV-8.

18. The device of claim 1 wherein the target probes include at least one probe capable of specifically binding to a target polypeptide.

19. The device of claim 1 wherein the liquid permeable layer has a volume of about 100 to 200 microliters.

20. The device of claim 1 wherein the liquid permeable layer has a thickness of about 5 to 20 microns.

21. The device of claim 1 wherein the microbeads are about 50 to 200 nm in size.

22. A method of detecting one or more target molecules in a sample solution, the method comprising:

(a) electrophoretically transporting the sample solution through a liquid permeable layer, wherein the liquid permeable layer includes at least one microregion having a plurality of microbeads embedded in the liquid permeable layer;

the microbeads having a plurality of target probes on their surfaces;

wherein the target probes are capable of specifically binding to designated target molecules to form target probe/target molecule complexes; and

(b) detecting the target probe/target molecule complexes.

23. The method of claim 22 wherein detecting the probe/target complexes includes (i) electrophoretically transporting a probe solution including visualization probes through the liquid permeable layer, wherein a given visualization probe is capable of specifically binding to a target probe/target molecule complex to form a bound visualization probe;

and (ii) detecting the bound visualization probe.

24. The method of claim 22 wherein detecting the probe/target complexes includes (i) electrophoretically transporting a probe solution including labeled target molecules through the liquid permeable layer, wherein the labeled target molecules are capable of specifically binding to complementary target probes to form labeled target molecule/target probe complexes;

and (ii) detecting the labeled target molecule/target probe complexes.

25. The method of claim 22 wherein the one or more target molecules are nucleic acids which have been purified prior to introduction into the liquid permeable layer.

26. The method of claim 22 wherein electrophoretically transporting the sample solution through a liquid permeable layer comprises applying a current of about 50 to 100 microamperes to the liquid permeable layer.

27. A method of detecting a target molecule in a sample comprising:

(a) introducing a first low conductivity buffer solution including the sample into a liquid chamber;

(b) electrophoretically transporting the first low conductivity buffer solution through a liquid permeable layer which is in fluid connection with the liquid chamber;

wherein the liquid permeable layer includes at least one microregion having a plurality of microbeads embedded in the liquid permeable layer;

the microbeads having a plurality of a target probe on their surfaces;

the target probe being capable of specifically binding to the target molecule to form a target molecule/target probe complex;

(c) introducing a second low conductivity buffer solution into the liquid chamber;

wherein the second low conductivity buffer solution includes a fluorescently labeled target molecule;

(d) electrophoretically transporting the second low conductivity buffer solution through the liquid permeable layer to form a fluorescent target molecule/target probe complex; and

(e) detecting the fluorescent target molecule/target probe complex.

28. The method of claim 27 wherein the first and second low conductivity buffer solutions are mixed together prior to being electrophoretically transported through the liquid permeable layer.

29. A kit for the analysis of biological samples comprising:

(a) a microarray device comprising a liquid permeable layer including a plurality of microregions, each microregion including a plurality of microbeads embedded in the liquid permeable layer;

wherein the microbeads have a plurality of target probes on their surfaces and the microbeads in a given microregion have a plurality of the target probes on their surfaces;

(b) a low conductivity buffer solution; and

(c) a buffer solution including a set of visualization probes.

30. The kit of claim 29 wherein the low conductivity buffer has a conductivity of about 5 to 50 μ

S/cm.

31. The kit of claim 29 wherein the low conductivity buffer has an inorganic salt content of no more than about 10 mM.

32. The kit of claim 29 wherein the low conductivity buffer includes lysine or histidine.

33. The kit of claim 29 wherein the low conductivity buffer includes barbituric acid, barbital, or a mixture thereof.

34. The kit of claim 29 wherein the visualization probes include fluorescent-labeled target molecules.

35. The kit of claim 34 wherein the target probes are capable of complementary binding to specific nucleic acid target molecules and the visualization probes include fluorescent-labeled nucleic acids capable of hybridizing to one of the specific nucleic acid target molecules.

36. A microarray device for the analysis of biological samples comprising:

a liquid permeable layer including at least one microregion which includes a plurality of microbeads embedded in the liquid permeable layer;

wherein the microbeads have a plurality of target probes on their surfaces.

37. The device of claim 36 wherein the liquid permeable layer includes at least 10 of the microregions and a low conductivity buffer having a conductivity of no more than about 50 μ

S/cm;

each microregion having a maximum dimension of no more than about 10 microns; and

the microbeads are about 50 to 200 nm in size and all the microbeads in a given microregion have a plurality of a single target probe on their surfaces.

38. A method of detecting a target molecule in a sample comprising:

(a) introducing a plurality of a visualization probe into a low conductivity solution including the sample to form a labeled solution;

wherein the visualization probe is capable of specifically binding to the target molecule to form a labeled target molecule;

(b) electrophoretically transporting the labeled solution through a liquid permeable layer;

wherein the liquid permeable layer includes at least one microregion having a plurality of microbeads embedded in the liquid permeable layer;

the microbeads having a plurality of a target probe on their surfaces;

wherein the target probe is capable of specifically binding to the labeled target molecule to form a labeled target molecule/target probe complex on a microbead surface; and

(c) detecting the bound labeled target molecule/target probe complex.

39. The method of claim 38 wherein the target molecule is a nucleic acid;

the target probe is capable of hybridizing to the target molecule; and

the visualization probe is capable of hybridizing to the target molecule.

40. The method of claim 39 wherein the visualization probe is a fluorescent-labeled nucleic acid.