초록

Methods of detecting target molecules using electrophoresis and media containing immobilized capture are described.

대표청구항 ▼

What is claimed is: 1. A method of detecting the presence or absence of one or more target molecules in a test sample, wherein the one or more target molecules has a defined mobility in an appropriate electrophoretic medium when the medium is exposed to an electric field and the target molecule can

What is claimed is: 1. A method of detecting the presence or absence of one or more target molecules in a test sample, wherein the one or more target molecules has a defined mobility in an appropriate electrophoretic medium when the medium is exposed to an electric field and the target molecule can form binding complexes with capture probes, comprising the steps of: a) copolymerizing one or more capture probes with a material capable of forming a matrix suitable for electrophoresis to form an electrophoretic medium containing immobilized capture probes covalently attached to the electrophoretic medium, wherein said one or more capture probes are modified with a 5'-acrylamide moiety and are selected from the group consisting of a nucleic acid, modified nucleic acid and a nucleic acid analog, and wherein said one or more capture probes are immobilized in a two dimensional array within the electrophoretic medium; b) introducing a test sample into the electrophoretic medium; c) subjecting the electrophoretic medium to an electric field resulting in the electrophoretic migration of the test sample into the region or regions of the electrophoretic medium containing immobilized capture probes, under conditions suitable for the target molecules to bind to the immobilized probes; and d) detecting the presence, or absence of target molecules or target molecule/capture probe complexes immobilized in the medium, thereby detecting the presence or absence of one or more target molecules in the test sample. 2. The method of claim 1, wherein the medium is a gel formed from at least one type of polymer. 3. The method of claim 2, wherein the electrophoretic medium is formed using a polymer of polyacrylamide. 4. The method of claim 1, wherein the capture probes are attached to the acrylamide via a polyethylene glycol spacer group. 5. The method of claim 1, wherein the capture probes are immobilized in a discrete region or regions of the electrophoretic medium. 6. The method of claim 1, wherein the electrophoretic medium contains multiple classes of capture probes, and wherein each class of capture probes is immobilized in a discrete region of the electrophoretic medium. 7. The method of claim 1, wherein the target molecule is selected from the group consisting of: nucleic acids, modified nucleic acids, nucleic acid analogs, proteins, carbohydrates, polysaccharides and small organic molecules. 8. The method of claim 1, wherein the target molecule is either a single-stranded or double-stranded nucleic acid molecule. 9. The method of claim 1, wherein the capture probe is about 5-50 nucleotides in length. 10. A method for purifying or concentrating target molecules from a complex test sample, comprising: a) copolymerizing one or more capture probes modified with a 5'-acrylamide moiety and selected from the group consisting of a nucleic acid, modified nucleic acid and a nucleic acid analog with a material capable of forming a matrix suitable for electrophoresis to form an electrophoretic medium containing immobilized capture probes covalently attached to the electrophoretic medium, wherein said one or more capture probes are immobilized in a two dimensional array within the electrophoretic medium; b) introducing a test sample into the electrophoretic medium; c) subjecting the electrophoretic medium to an electric field resulting in the electrophoretic migration of the test sample through the medium, under conditions and time sufficient for the target molecules in the test sample to specifically bind to the capture probes, thereby forming target molecule/capture probe complexes, and for non-target molecules to migrate through and elute from the medium, wherein only target molecules bind to the capture probes and are immobilized in the medium; d) treating the electrophoretic medium to release at least one of the following: i) the one or more capture probes, ii) the target molecules, or iii) the target/capture probe binding complexes; and e) eluting the target molecules or target/capture probe complexes from the medium, whereby the released and eluted target molecules are substantially pure. 11. The method of claim 10, wherein the releasing treatment is accomplished by raising the temperature of the medium to a temperature sufficient to denature the target/capture probe complexes. 12. The method of claim 10, wherein the releasing treatment is accomplished by chemical cleavage of the chemical linkage which immobilizes the capture probe within the medium, and the released target molecule/capture probe complex is eluted from the medium. 13. The method of claim 10, wherein the releasing treatment is accomplished by photochemical cleavage of the chemical linkage which immobilizes the capture probe within the medium, and the released target molecule/capture probe complex is eluted from the medium. 14. The method of claim 10, wherein the releasing treatment is accomplished by increasing the electrophoretic field strength. 15. The method of claim 10, wherein the target molecule is selected from the group consisting of: nucleic acids, modified nucleic acids, nucleic acid analogs, proteins, carbohydrates, polysaccharides and small organic molecules. 16. The method of claim 10, wherein the capture probe is about 5-50 nucleotides in length. 17. A method of detecting one or more target molecules in a test sample, comprising the steps of: a) copolymerizing one or more nucleic acid aptamer capture probes modified with a 5'-acrylamide moiety with a material capable of forming a matrix suitable for electrophoresis to form an electrophoretic medium containing at least one immobilized nucleic acid aptamer capture probe covalently attached to the electrophoretic medium, wherein the aptamer capture probe is a thrombin-binding DNA aptamer; b) introducing a test sample into the electrophoretic medium; c) subjecting the electrophoretic medium to an electric field resulting in the electrophoretic migration of the test sample into the electrophoretic medium under conditions suitable for the target molecules to bind to the at least one immobilized aptamer capture probe, wherein the target molecule is human thrombin and is SEQ ID No.1; and d) detecting the presence or absence of target molecule/aptamer capture probe complexes immobilized in the medium, thereby detecting the presence or absence of one or more target molecules in the test sample. 18. The method of claim 17, wherein the concentration of the aptamer capture probe immobilized in the electrophoretic medium is from about 10 to about 100 μM. 19. The method of claim 17, wherein the aptamer capture probe is attached to the acrylamide moiety via a polyethylene glycol spacer group. 20. A method of detecting one or more target molecules in a test sample, comprising the steps of: a) copolymerizing one or more nucleic acid aptamer capture probes modified with a 5'-acrylamide moiety with a material capable of forming a matrix suitable for electrophoresis to form an electrophoretic medium containing at least one immobilized nucleic acid aptamer capture probe covalently attached to the electrophoretic medium; b) introducing a test sample into the electrophoretic medium: c) subjecting the electrophoretic medium to an electric field resulting in the electrophoretic migration of the test sample into the electrophoretic medium under conditions suitable for the target molecules to bind to the at least one immobilized aptamer capture probe, wherein the target molecule is selected from the group consisting of: SEQ ID No.2 and SEQ ID No.3; and d) detecting the presence or absence of target molecule/aptamer capture probe complexes immobilized in the medium, thereby detecting the presence or absence of one or more target molecules in the test sample. 21. A method for purifying molecules in a complex test sample comprising target molecules and non-target molecules, comprising: a) copolymerizing one or more capture probes modified with a 5'-acrylamide moiety and selected from the group consisting of a nucleic acid, modified nucleic acid and a nucleic acid analog with a material capable of forming a matrix suitable for electrophoresis to form an electrophoretic medium containing immobilized capture probes covalently attached to the electrophoretic medium, wherein said one or more capture probes are immobilized in a two dimensional array within the electrophoretic medium; b) introducing a test sample into the electrophoretic medium; c) subjecting the electrophoretic medium to an electric field resulting in the electrophoretic migration of the test sample through the medium, under conditions and time sufficient for the target molecules in the test sample to specifically bind to the capture probes, thereby forming target molecule/capture probe complexes and allowing non-target molecules to migrate through and elute from the medium, wherein only target molecules bind to the capture probes and are immobilized in the medium, thereby subtracting out the target molecule from the sample preparation and purifying molecules in the complex test sample. 22. A method of detecting the presence or absence of one or more target molecules in a test sample, wherein the one or more target molecules have a defined mobility in an appropriate electrophoretic medium when the medium is exposed to an electric field and the one or more target molecules can form binding complexes with capture probes, comprising the steps of: a) copolymerizing one or more capture probes with a material capable of forming a matrix suitable for electrophoresis, wherein said copolymerization forms an electrophoretic medium containing immobilized capture probes covalently attached to the electrophoretic medium, and wherein said one or more capture probes are modified with a monomer capable of forming a matrix suitable for electrophoresis, and wherein said capture probes are selected from the group consisting of a nucleic acid, modified nucleic acid and a nucleic acid analog, and wherein one or more capture probes are immobilized in a two dimensional array within the electrophoretic medium, b) introducing a test sample into the electrophoretic medium; c) subjecting the electrophoretic medium to an electric field resulting in the electrophoretic migration of the test sample into the region or regions of the electrophoretic medium containing immobilized capture probes, under conditions suitable for the target molecules to bind to the immobilized probes; and d) detecting the presence or absence of target molecules or target molecule/capture probe complexes immobilized in the medium, thereby detecting the presence or absence of one or more target molecules in the test sample. 23. The method of claim 22, wherein the medium is formed from at least one type of polymer. 24. The method of claim 23, wherein the electrophoretic medium is formed using at least one class of polymers selected from the group consisting of: polyacrylamide, agarose and starch. 25. The method of claim 22, wherein the capture probes are immobilized in a discrete region or regions of the electrophoretic medium. 26. The method of claim 22, wherein the electrophoretic medium contains multiple classes of capture probes, and wherein each class of capture probes is immobilized in a discrete region of the electrophoretic medium. 27. The method of claim 22, wherein the target molecule is selected from the group consisting of: nucleic acids, modified nucleic acids, nucleic acid analogs, proteins, carbohydrates, polysaccharides and small organic molecules. 28. The method of claim 22, wherein the target molecule is either a single-stranded or double-stranded nucleic acid molecule. 29. The method of claim 22, wherein the capture probe is about 5-50 nucleotides in length. 30. A method for purifying or concentrating target molecules from a complex test sample, comprising: a) copolymerizing one or more capture probes with a material capable of forming a matrix suitable for electrophoresis, wherein said copolymerization forms an electrophoretic medium containing immobilized capture probes covalently attached to the electrophoretic medium, and wherein said one or more capture probes are modified with a monomer capable of forming a matrix suitable for electrophoresis, and wherein said capture probes are selected from the group consisting of a nucleic acid, modified nucleic acid and a nucleic acid analog, and wherein one or more capture probes are immobilized in a two dimensional array within the electrophoretic medium, b) introducing a test sample into the electrophoretic medium; c) subjecting the electrophoretic medium to an electric field resulting in the electrophoretic migration of the test sample through the medium, under conditions and time sufficient for the target molecules in the test sample to specifically bind to the capture probes, thereby forming target molecule/capture probe complexes, and for non-target molecules to migrate through and elute from the medium, wherein only target molecules bind to the capture probes and are immobilized in the medium; d) treating the electrophoretic medium to release at least one of the following: i) the one or more capture probe, ii) the target molecules, or iii) the target/capture probe binding-complexes; and e) eluting the target molecules or target/capture probe complexes from the medium, whereby the released and eluted target molecules are substantially pure. 31. The method of claim 30, wherein the releasing treatment is accomplished by raising the temperature of the medium to a temperature sufficient to denature the target/capture probe complexes. 32. The method of claim 30, wherein the releasing treatment is accomplished by chemical cleavage of the chemical linkage which immobilizes the capture probe within the medium, and the released target molecule/capture probe complex is eluted from the medium. 33. The method of claim 30, wherein the releasing treatment is accomplished by photochemical cleavage of the chemical linkage which immobilizes the capture probe within the medium, and the released target molecule/capture probe complex is eluted from the medium. 34. The method of claim 30, wherein the releasing treatment is accomplished by increasing the electrophoretic field strength to a level sufficient to disrupt target/capture probe complexes. 35. The method of claim 30, wherein the target molecule is selected from the group consisting of: nucleic acids, modified nucleic acids, nucleic acid analogs, proteins, carbohydrates, polysaccharides and small organic molecules. 36. The method of claim 30, wherein the capture probe is about 5-50 nucleotides in length. 37. A method of detecting one or more target molecules in a test sample, comprising the steps of: a) copolymerizing one or more aptamer capture probes with a material capable of forming a matrix suitable for electrophoresis, wherein said copolymerization forms an electrophoretic medium containing immobilized aptamer capture probes covalently attached to the electrophoretic medium, and wherein said one or more aptamer capture probes are modified with a monomer capable of forming a matrix suitable for electrophoresis, and wherein the aptamer capture probe is a thrombin-binding DNA aptamer; b) introducing a test sample into the electrophoretic medium; c) subjecting the electrophoretic medium to an electric field resulting in the electrophoretic migration of the test sample into the electrophoretic medium under conditions suitable for the target molecules to bind to the at least one immobilized aptamer capture probe, wherein the target molecule is human thrombin and is SEQ ID No.1; and d) detecting the presence or absence of target molecule/aptamer capture probe complexes immobilized in the medium, thereby detecting the presence or absence of one or more target molecules in the test sample. 38. The method of claim 37, wherein the concentration of the aptamer capture probe immobilized in the electrophoretic medium is from about 10 to about 100 μM. 39. A method of detecting one or more target molecules in a test sample, comprising the steps of: a) copolymerizing one or more aptamer capture probes with a material capable of forming a matrix suitable for electrophoresis, wherein said copolymerization forms an electrophoretic medium containing immobilized aptamer capture probes covalently attached to the electrophoretic medium, and wherein said one or more aptamer capture probes are modified with a monomer capable of forming a matrix suitable for electrophoresis; b) introducing a test sample into the electrophoretic medium; c) subjecting the electrophoretic medium to an electric field resulting in the electrophoretic migration of the test sample into the electrophoretic medium under conditions suitable for the target molecules to bind to the at least one immobilized aptamer capture probe, wherein the target molecule is selected from the group consisting of: SEQ ID No.2 and SEQ ID No.3; and d) detecting the presence or absence of target molecule/aptamer capture probe complexes immobilized in the medium, thereby detecting the presence or absence of one or more target molecules in the test sample. 40. A method for purifying molecules in a complex test sample comprising target molecules and non-target molecules, comprising: a) copolymerizing one or more capture probes with a material capable of forming a matrix suitable for electrophoresis, wherein said copolymerization forms an electrophoretic medium containing immobilized capture probes covalently attached to the electrophoretic medium, and wherein said one or more capture probes are modified with a monomer capable of forming a matrix suitable for electrophoresis, and wherein said capture probes are selected from the group consisting of a nucleic acid, modified nucleic acid and a nucleic acid analog, and wherein one or more capture probes are immobilized in a two dimensional array within the electrophoretic medium; b) introducing a test sample into the electrophoretic medium; c) subjecting the electrophoretic medium to an electric field resulting in the electrophoretic migration of the test sample through the medium, under conditions and time sufficient for the target molecules to specifically bind to the capture probes, thereby forming target molecule/capture probe complexes, and allowing non-target molecules to migrate through and elute from the medium, wherein only target molecules bind to the capture probes and are immobilized in the medium, thereby subtracting out the target molecule from the sample preparation and purifying molecules in the complex test sample. 41. A method of detecting the presence or absence of one or more target molecules in a test sample, wherein the one or more target molecules has a defined mobility in an appropriate electrophoretic medium when the medium is exposed to an electric field and the target molecule can form binding complexes with capture probes, comprising the steps of: a) copolymerizing one or more capture probes with a material capable of forming a matrix suitable for electrophoresis to form an electrophoretic medium containing immobilized capture probes covalently attached to the electrophoretic medium, wherein said one or more capture probes are modified with a 5'-acrylamide moiety and are selected from the group consisting of a nucleic acid, modified nucleic acid and a nucleic acid analog, and wherein said one or more capture probes are immobilized in a three dimensional array within the electrophoretic medium; b) introducing a test sample into the electrophoretic medium; c) subjecting the electrophoretic medium to an electric field resulting in the electrophoretic migration of the test sample into the region or regions of the electrophoretic medium containing immobilized capture probes, under conditions suitable for the target molecules to bind to the immobilized probes; and d) detecting the presence, or absence of target molecules or target molecule/capture probe complexes immobilized in the medium, thereby detecting the presence or absence of one or more target molecules in the test sample. 42. The method of claim 41, wherein the medium is a gel formed from at least one type of polymer. 43. The method of claim 42, wherein the electrophoretic medium is formed using a polymer of polyacrylamide. 44. The method of claim 41, wherein the capture probes are attached to the acrylamide via a polyethylene glycol spacer group. 45. The method of claim 41, wherein the capture probes are immobilized in a discrete region or regions of the electrophoretic medium. 46. The method of claim 41, wherein the electrophoretic medium contains multiple classes of capture probes, and wherein each class of capture probes is immobilized in a discrete region of the electrophoretic medium. 47. The method of claim 41, wherein the target molecule is selected from the group consisting of: nucleic acids, modified nucleic acids, nucleic acid analogs, proteins, carbohydrates, polysaccharides and small organic molecules. 48. The method of claim 41, wherein the target molecule is either a single-stranded or double-stranded nucleic acid molecule. 49. The method of claim 41, wherein the capture probe is about 5-50 nucleotides in length. 50. A method for purifying or concentrating target molecules from a complex test sample, comprising: a) copolymerizing one or more capture probes modified with a 5'-acrylamide moiety and selected from the group consisting of a nucleic acid, modified nucleic acid and a nucleic acid analog with a material capable of forming a matrix suitable for electrophoresis to form an electrophoretic medium containing immobilized capture probes covalently attached to the electrophoretic medium, wherein said one or more capture probes are immobilized in a three dimensional array within the electrophoretic medium; b) introducing a test sample into the electrophoretic medium; c) subjecting the electrophoretic medium to an electric field resulting in the electrophoretic migration of the test sample through the medium, under conditions and time sufficient for the target molecules in the test sample to specifically bind to the capture probes, thereby forming target molecule/capture probe complexes, and for non-target molecules to migrate through and elute from the medium, wherein only target molecules bind to the capture probes and are immobilized in the medium; d) treating the electrophoretic medium to release at least one of the following: i) the one or more capture probes, ii) the target molecules, or iii) the target/capture probe binding complexes; and e) eluting the target molecules or target/capture probe complexes from the medium, whereby the released and eluted target molecules are substantially pure. 51. The method of claim 50, wherein the releasing treatment is accomplished by raising the temperature of the medium to a temperature sufficient to denature the target/capture probe complexes. 52. The method of claim 50, wherein the releasing treatment is accomplished by chemical cleavage of the chemical linkage which immobilizes the capture probe within the medium, and the released target molecule/capture probe complex is eluted from the medium. 53. The method of claim 50, wherein the releasing treatment is accomplished by photochemical cleavage of the chemical linkage which immobilizes the capture probe within the medium, and the released target molecule/capture probe complex is eluted from the medium. 54. The method of claim 50, wherein the releasing treatment is accomplished by increasing the electrophoretic field strength. 55. The method of claim 50, wherein the target molecule is selected from the group consisting of: nucleic acids, modified nucleic acids, nucleic acid analogs, proteins, carbohydrates, polysaccharides and small organic molecules. 56. The method of claim 50, wherein the capture probe is about 5-50 nucleotides in length. 57. A method for purifying molecules in a complex test sample comprising target molecules and non-target molecules, comprising: a) copolymerizing one or more capture probes modified with a 5'-acrylamide moiety and selected from the group consisting of a nucleic acid, modified nucleic acid and a nucleic acid analog with a material capable of forming a matrix suitable for electrophoresis to form an electrophoretic medium containing immobilized capture probes covalently attached to the electrophoretic medium, wherein said one or more capture probes are immobilized in a three dimensional array within the electrophoretic medium; b) introducing a test sample into the electrophoretic medium; c) subjecting the electrophoretic medium to an electric field resulting in the electrophoretic migration of the test sample through the medium, under conditions and time sufficient for the target molecules in the test sample to specifically bind to the capture probes, thereby forming target molecule/capture probe complexes and allowing non-target molecules to migrate through and elute from the medium, wherein only target molecules bind to the capture probes and are immobilized in the medium, thereby subtracting out the target molecule from the sample preparation and purifying molecules in the complex test sample. 58. A method of detecting the presence or absence of one or more target molecules in a test sample, wherein the one or more target molecules have a defined mobility in an appropriate electrophoretic medium when the medium is exposed to an electric field and the one or more target molecules can form binding complexes with capture probes, comprising the steps of: a) copolymerizing one or more capture probes with a material capable of forming a matrix suitable for electrophoresis, wherein said copolymerization forms an electrophoretic medium containing immobilized capture probes covalently attached to the electrophoretic medium, and wherein said one or more capture probes are modified with a monomer capable of forming a matrix suitable for electrophoresis, and wherein said capture probes are selected from the group consisting of a nucleic acid, modified nucleic acid and a nucleic acid analog, and wherein one or more capture probes are immobilized in a three dimensional array within the electrophoretic medium, b) introducing a test sample into the electrophoretic medium; c) subjecting the electrophoretic medium to an electric field resulting in the electrophoretic migration of the test sample into the region or regions of the electrophoretic medium containing immobilized capture probes, under conditions suitable for the target molecules to bind to the immobilized probes; and d) detecting the presence or absence of target molecules or target molecule/capture probe complexes immobilized in the medium, thereby detecting the presence or absence of one or more target molecules in the test sample. 59. The method of claim 58, wherein the medium is formed from at least one type of polymer. 60. The method of claim 59, wherein the electrophoretic medium is formed using at least one class of polymers selected from the group consisting of: polyacrylamide, agarose and starch. 61. The method of claim 58, wherein the capture probes are immobilized in a discrete region or regions of the electrophoretic medium. 62. The method of claim 58, wherein the electrophoretic medium contains multiple classes of capture probes, and wherein each class of capture probes is immobilized in a discrete region of the electrophoretic medium. 63. The method of claim 58, wherein the target molecule is selected from the group consisting of: nucleic acids, modified nucleic acids, nucleic acid analogs, proteins, carbohydrates, polysaccharides and small organic molecules. 64. The method of claim 58, wherein the target molecule is either a single-stranded or double-stranded nucleic acid molecule. 65. The method of claim 58, wherein the capture probe is about 5-50 nucleotides in length. 66. A method for purifying or concentrating target molecules from a complex test sample, comprising: a) copolymerizing one or more capture probes with a material capable of forming a matrix suitable for electrophoresis, wherein said copolymerization forms an electrophoretic medium containing immobilized capture probes covalently attached to the electrophoretic medium, and wherein said one or more capture probes are modified with a monomer capable of forming a matrix suitable for electrophoresis, and wherein said capture probes are selected from the group consisting of a nucleic acid, modified nucleic acid and a nucleic acid analog, and wherein one or more capture probes are immobilized in a three dimensional array within the electrophoretic medium, b) introducing a test sample into the electrophoretic medium; c) subjecting the electrophoretic medium to an electric field resulting in the electrophoretic migration of the test sample through the medium, under conditions and time sufficient for the target molecules in the test sample to specifically bind to the capture probes, thereby forming target molecule/capture probe complexes, and for non-target molecules to migrate through and elute from the medium, wherein only target molecules bind to the capture probes and are immobilized in the medium; d) treating the electrophoretic medium to release at least one of the following: i) the one or more capture probe, ii) the target molecules, or iii) the target/capture probe binding-complexes; and e) eluting the target molecules or target/capture probe complexes from the medium, whereby the released and eluted target molecules are substantially pure. 67. The method of claim 66, wherein the releasing treatment is accomplished by raising the temperature of the medium to a temperature sufficient to denature the target/capture probe complexes. 68. The method of claim 66, wherein the releasing treatment is accomplished by chemical cleavage of the chemical linkage which immobilizes the capture probe within the medium, and the released target molecule/capture probe complex is eluted from the medium. 69. The method of claim 66, wherein the releasing treatment is accomplished by photochemical cleavage of the chemical linkage which immobilizes the capture probe within the medium, and the released target molecule/capture probe complex is eluted from the medium. 70. The method of claim 66, wherein the releasing treatment is accomplished by increasing the electrophoretic field strength to a level sufficient to disrupt target/capture probe complexes. 71. The method of claim 66, wherein the target molecule is selected from the group consisting of: nucleic acids, modified nucleic acids, nucleic acid analogs, proteins, carbohydrates, polysaccharides and small organic molecules. 72. The method of claim 66, wherein the capture probe is about 5-50 nucleotides in length. 73. A method for purifying molecules in a complex test sample comprising target molecules and non-target molecules, comprising: a) copolymerizing one or more capture probes with a material capable of forming a matrix suitable for electrophoresis, wherein said copolymerization forms an electrophoretic medium containing immobilized capture probes covalently attached to the electrophoretic medium, and wherein said one or more capture probes are modified with a monomer capable of forming a matrix suitable for electrophoresis, and wherein said capture probes are selected from the group consisting of a nucleic acid, modified nucleic acid and a nucleic acid analog, and wherein one or more capture probes are immobilized in a three dimensional array within the electrophoretic medium; b) introducing a test sample into the electrophoretic medium; c) subjecting the electrophoretic medium to an electric field resulting in the electrophoretic migration of the test sample through the medium, under conditions and time sufficient for the target molecules to specifically bind to the capture probes, thereby forming target molecule/capture probe complexes, and allowing non-target molecules to migrate through and elute from the medium, wherein only target molecules bind to the capture probes and are immobilized in the medium, thereby subtracting out the target molecule from the sample preparation and purifying molecules in the complex test sample.