Kit, Device and Method For Analyzing Biological Substance
First Claim
1. An analytical kit comprising:
- i) an analytical device comprising a passage allowing a liquid to flow through the same, formed by bonding together a first member having a groove, 1 μ
m to 5 mm width and 1 μ
m to 750 μ
m depth in cross-section, and a second member covering the groove, and a first nucleic acid (N1) having an arbitrary base sequence and immobilized in a capturing zone provided in the passage on the first member and/or second member prior to bonding the first member and second member together;
ii) a reagent A containing (1) a conjugate (N2-L1) composed of a second nucleic acid (N2) having a sequence at least complementary to the base sequence of the first nucleic acid and (2) a first ligand (L1) capable of specifically binding to a biological substance (O) to be assayed;
iii) a reagent B containing a conjugate (L2-M) resulting from binding of a marker (M) to a second ligand (L2) capable of specifically binding to the biological substance (O) to be assayed.
1 Assignment
0 Petitions
Accused Products
Abstract
The invention provides an analytical device insusceptible to inactivation or other influences even when exposed to a thermal load or organic compounds contained in an adhesive in the process for manufacturing the same and, more over, allowing an immunological substance or the like to be readily immobilized at a site in the microchannel passage therein.
The analytical kit is a combination of the analytical device and a reagent or reagents. The analytical device used in the analytical kit comprises a passage 2, 1 μm-5mm width and 1 μm-750 μm depth in cross-section formed therein and belongs to the category of the so-called microfluidic systems suited for analyzing very small amounts of liquid samples; thus, it is suited for analyzing biological substances. The analytical device 1 to be used in the analytical kit is prepared by forming a groove not wider than 5 mm on a first member 5 and/or second member 6, immobilizing a nucleic acid(s) at a part (capturing zone 7) of a place to become the channel 2 after joining the two members together and joining the two members together. The reagent(s) is (are) used after joining of the two members of the analytical device 1 and therefore will not be influenced by the fusion or adhesive.
25 Citations
57 Claims
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1. An analytical kit comprising:
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i) an analytical device comprising a passage allowing a liquid to flow through the same, formed by bonding together a first member having a groove, 1 μ
m to 5 mm width and 1 μ
m to 750 μ
m depth in cross-section, and a second member covering the groove, and a first nucleic acid (N1) having an arbitrary base sequence and immobilized in a capturing zone provided in the passage on the first member and/or second member prior to bonding the first member and second member together;ii) a reagent A containing (1) a conjugate (N2-L1) composed of a second nucleic acid (N2) having a sequence at least complementary to the base sequence of the first nucleic acid and (2) a first ligand (L1) capable of specifically binding to a biological substance (O) to be assayed; iii) a reagent B containing a conjugate (L2-M) resulting from binding of a marker (M) to a second ligand (L2) capable of specifically binding to the biological substance (O) to be assayed. - View Dependent Claims (11, 12, 13, 14, 28, 29)
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2. An analytical kit comprising:
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i) an analytical device comprising a passage allowing a liquid to flow through the same, formed by bonding together a first member having a groove, 1 μ
m to 5 mm width and 1 μ
m to 750 μ
m depth in cross-section, and a second member covering the groove, and a first nucleic acid (N1) having an arbitrary base sequence and immobilized in a capturing zone provided in the passage on the first member and/or second member prior to bonding the first member and second member together;ii) a reagent A containing a conjugate (N2-L1) composed of (1) a second nucleic acid (N2) having a sequence at least complementary to the base sequence of the first nucleic acid and (2) a first ligand (L1) capable of specifically binding to a biological substance (O) to be assayed; iii) a reagent B′
containing a second ligand (L2) capable of specifically binding to the biological substance (O) to be assayed; andiv) a reagent C containing a conjugate (L3-M) composed of a third ligand (L3) capable of specifically binding to the second ligand (L2) and a marker (M). - View Dependent Claims (30, 31, 32, 33)
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3. An analytical kit containing no marker and comprising:
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i) an analytical device comprising a passage allowing a liquid to flow through the same, formed by bonding together a first member having a groove, 1 μ
m to 5 mm width and 1 μ
m to 750 μ
m depth in cross-section, and a second member covering the groove, and a first nucleic acid (N1) having an arbitrary base sequence and immobilized in a capturing zone provided in the passage on the first member and/or second member prior to bonding the first member and second member together; andii) a reagent A containing a conjugate (N2-L1) composed of (1) a second nucleic acid (N2) having a sequence at least complementary to the base sequence of the first nucleic acid (N1) immobilized in the capturing zone of the analytical device and (2) a first ligand (L1) capable of specifically binding to a biological substance (O) to be assayed.
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4. An analytical kit comprising:
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i) an analytical device comprising a passage allowing a liquid to flow through the same formed by bonding together a first member having a groove, 1 μ
m to 5 mm width and 1 μ
m to 750 μ
m depth in cross-section, and a second member covering the groove, a first nucleic acid (N1) having an arbitrary base sequence and immobilized in a capturing zone provided in the passage on the first member and/or second member prior to bonding the first member and second member together, and a conjugate (N2-L1) composed of a first ligand (L1) capable of specifically binding to a biological substance (O) to be assayed and a second nucleic acid (N2) having a base sequence at least complementary to the immobilized first nucleic acid and immobilized in the capturing zone in the form of a conjugate (N1-N2-L1) by specific binding between the first nucleic acid (N1) and second nucleic acid (N2); andii) a reagent B containing a conjugate (L2-M) resulting from binding between a second ligand (L2) capable of specifically binding to the biological substance (O) to be assayed and a marker (M). - View Dependent Claims (34, 35)
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5. An analytical kit comprising the reagent B′
- , reagent C and analytical device specified below in combination;
i) an analytical device comprising a passage allowing a liquid to flow through the same, formed by bonding together a first member having a groove, 1 μ
m to 5 mm width and 1 μ
m to 750 μ
m depth in cross-section, and a second member covering the groove, a first nucleic acid (N1) having an arbitrary base sequence and immobilized in a capturing zone provided in the passage on the first member and/or second member prior to bonding the first member and second member together, and a conjugate (N2-L1) composed of a first ligand (L1) capable of specifically binding to a biological substance (O) to be assayed and a second nucleic acid (N2) having a base sequence at least complementary to the immobilized first nucleic acid (N1) and immobilized in the capturing zone in the form of a conjugate (N1-N2-L1) by specific binding between the first nucleic acid (N1) and second nucleic acid (N2); andii) a reagent B′
containing a second ligand (L2) capable of specifically binding to the biological substance (O) to be assayed; andiii) a reagent C containing a conjugate (L3-M) composed of a third ligand (L3) capable of specifically binding to the second ligand (L2) and a marker (M). - View Dependent Claims (36, 37)
- , reagent C and analytical device specified below in combination;
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6. An analytical kit comprising:
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i) an analytical device comprising a passage allowing a liquid to flow through the same, formed by bonding together a first member having a groove, 1 μ
m to 5 mm width and 1 μ
m to 750 μ
m depth in cross-section, and a second member covering the groove, and a plurality of first nucleic acid species (N1g;
g being an integer), each having an arbitrary base sequence and immobilized independently, from species to species, in a capturing zone provided in the passage on the first member and/or second member prior to bonding the first member and second member together;ii) a reagent A solution containing a plurality of conjugate species (N2h-L1i;
h and i each independently being an integer), each composed of (1) one of a plurality of second nucleic acid species (N2h;
h being an integer) and each having a sequence at least complementary to the base sequence of one of the plurality of first nucleic acid species and (2) one of a plurality of first ligand species (L1i;
i being an integer) which is capable of specifically binding to the corresponding one of biological substance species (Ok;
k being an integer) to be assayed; andiii) a reagent B containing conjugate species (L2j -M1;
j and 1 each independently being an integer) resulting from binding between one or more second ligand species (L2j;
j being an integer) capable of specifically binding to corresponding one of biological substance species to be assayed and one or more marker species (M1;
1 being an integer). - View Dependent Claims (38, 39)
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7. An analytical kit comprising:
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i) an analytical device comprising a passage allowing a liquid to flow through the same, formed by bonding together a first member having a groove, 1 μ
m to 5 mm width and 1 μ
m to 750 μ
m depth in cross-section, and a second member covering the groove, and a plurality of first nucleic acid species (N1g;
g being an integer) each having an arbitrary base sequence and immobilized independently, from species to species, in a capturing zone provided in the passage on the first member and/or second member prior to bonding the first member and second member together;ii) a reagent A solution containing a plurality of conjugate species (N2h-L1i;
wherein h and i are integers, each composed of (1) one of a plurality of second nucleic acid species (N2h;
h being an integer), each having a sequence at least complementary to the base sequence of one of the plurality of first nucleic acid species and (2) one of a plurality of first ligand species (L1i;
i being an integer) which is capable of specifically binding to one of biological substance species (Ok;
k being an integer) to be assayed;iii) a reagent B′
containing one or more second ligand species (L2j;
j being an integer), each capable of specifically binding to one of the one or more biological substance species to be assayed; andiv) a reagent C containing conjugate species (L3m-M1;
wherein m and 1 are integers composed of one or more third ligand species (L3m;
m being an integer) capable of specifically binding to a corresponding one of the one or more second ligand species and one or more marker species (M1;
1 being an integer). - View Dependent Claims (40, 41)
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8. An analytical kit comprising:
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i) an analytical device comprising a passage allowing a liquid to flow through the same and formed by bonding together a first member having a groove, 1 μ
m to 5 mm width and 1 μ
m to 750 μ
m depth in cross-section, and a second member capable of covering the groove, and a plurality of first nucleic acid species (N1g;
g being an integer), each having an arbitrary base sequence and immobilized independently, from species to species, in a capturing zone provided in the passage on the first member and/or second member prior to bonding the first member and second member together;ii) a reagent A containing a plurality of conjugate species (N2h-L1i;
h and i each independently being an integer), each composed of one of a plurality of second nucleic acid species (N2h;
h being an integer), each having a sequence at least complementary to the base sequence of the corresponding one of the plurality of first nucleic acid species and one of a plurality of first ligand species (L1i;
i being an integer) which is capable of specifically binding to a corresponding one of biological substance species (Ok;
k being an integer) to be assayed. - View Dependent Claims (42, 43)
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9. An analytical kit comprising the reagent B and analytical device specified below in combination:
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i) an analytical device comprising a passage allowing a liquid to flow through the same and formed by bonding together a first member having a groove, 1 μ
m to 5 mm width and 1 μ
m to 750 μ
m depth in cross-section, and a second member covering the groove, and a plurality of first nucleic acid species (N1g;
g being an integer), each having an arbitrary base sequence and immobilized independently, from species to species, in a capturing zone provided in the passage on the first member and/or second member prior to bonding the first member and second member together, and conjugate species (N2h-L1i;
wherein h and i are each an integer), each composed of one of a plurality of first ligand species (L1i;
i being an integer), which is capable of specifically binding to a corresponding one of biological substance species (Ok;
k being an integer) to be assayed, and one of a plurality of second nucleic acid species (N2h;
h being an integer) and which has a base sequence at least complementary to one of the first nucleic acid species immobilized in the capturing zone in the form of conjugate species (N1g-N2h-L1i;
g, wherein h and i are each an integer) by specific binding between the first nucleic acid species and second nucleic acid species; andii) a reagent B containing conjugate species (L2j-M1;
wherein j and 1 are each an integer) resulting from binding between one or more second ligand species (L2j;
j being an integer) respectively capable of specifically binding to the corresponding one biological substance species to be assayed and one or more marker species (M1;
1 being an integer). - View Dependent Claims (44, 45)
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10. An analytical kit comprising:
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i) an analytical device comprising a passage allowing a liquid to flow through the same, formed by bonding together a first member having a groove, 1 μ
m to 5 mm width and 1 μ
m to 750 μ
m depth in cross-section, and a second member covering the groove, a plurality of first nucleic acid species (N1g;
g being an integer), each having an arbitrary base sequence and immobilized independently, from species to species, in a capturing zone provided in the passage on the first member and/or second member prior to bonding the first member and second member together, and conjugate species (N2h-L1i;
h and i each independently being an integer), each composed of one of a plurality of first ligand species (L1i;
i being an integer), which is capable of specifically binding to a corresponding one of the one or more biological substance species (Ok;
k being an integer) to be assayed, and one of a plurality of second nucleic acid species (N2h;
h being an integer), which has a base sequence at least complementary to a corresponding one of the immobilized first nucleic acid species (N1g;
g being an integer), each conjugate species (N2h-L1i) independently immobilized in the capturing zone in the form of conjugate species (N1g-N2h-L1i;
wherein g, h and i are each an integer) by specific binding between the first nucleic acid species and second nucleic acid species; andii) a reagent B′
containing one or more second ligand species (L2j;
j being an integer) capable of specifically binding to a corresponding one of the biological substance species to be assayed;iii) a reagent C containing conjugate species (L3m-M1;
wherein m and 1 are each an integer) derived from one or more third ligand species (L3m;
m being an integer) capable of specifically binding to corresponding one of the second ligand species (L2j;
j being an integer) and one or more marker species (M1;
1 being an integer). - View Dependent Claims (46, 47)
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15. (canceled)
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17. (canceled)
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18. An analytical device comprising a passage allowing a liquid to flow through the same, formed by bonding together a first member having a groove, 1 μ
- m to 5 mm width and 1 μ
m to 750 μ
m depth in cross-section, and a second member covering the groove to form the passage, a first nucleic acid (N1) having an arbitrary base sequence immobilized in a capturing zone provided in the passage prior to bonding the first member and second member together, and a conjugate (N2-L1) composed of a first ligand (L1) capable of specifically binding to a biological substance (O) to be assayed and a second nucleic acid (N2) having a base sequence at least complementary to the immobilized first nucleic acid (N1) and immobilized in the capturing zone by specific binding between the first nucleic acid (N1) and second nucleic acid (N2). - View Dependent Claims (48)
- m to 5 mm width and 1 μ
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19. An analytical device comprising a passage allowing a liquid to flow through the same formed by bonding together a first member having a groove, 1 μ
- m to 5 mm width and 1 μ
m to 750 μ
m depth in cross-section, and a second member covering the groove to form the passage, a plurality of first nucleic acid species (N1g;
g being an integer), each having an arbitrary base sequence and immobilized independently, from species to species, in a capturing zone provided in the passage on the first member and/or second member prior to bonding the first member and second member together, conjugate species (N2h-L1i;
h and i each being an integer), each conjugate species being composed of one of a plurality of a first ligand species (L1i;
i being an integer), which is capable of specifically binding to a corresponding one of biological substance species (Ok;
k being an integer) to be assayed, and one of a plurality of second nucleic acid species (N2h;
h being an integer), each of which has a base sequence at least complementary to a corresponding one of the immobilized first nucleic acid species and which is immobilized independently, from species to species, in the capturing zone by specific binding between the first nucleic acid species and second nucleic acid species. - View Dependent Claims (49)
- m to 5 mm width and 1 μ
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20. (canceled)
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24. An analytical method comprising:
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preparing an analytical device, comprising a passage allowing a liquid to flow through the same, by bonding together a first member having a groove, 1 μ
m to 5 mm width and 1 μ
m to 750 μ
m depth in cross-section, and a second member covering the groove;immobilizing a first nucleic acid (N1), having an arbitrary base sequence, in a capturing zone provided in the passage on the first member and/or second member prior to bonding the first member and second member together; preparing a reagent A containing a conjugate (N2-L1) resulting from binding of a first ligand (L1), capable of specifically binding to a biological substance to be assayed, to a second nucleic acid (N2) having a sequence at least complementary to the base sequence of the first nucleic acid (N1); mixing a liquid sample suspected of containing the biological substance to be assayed and the reagent A, either after conjugate formation or while allowing conjugate formation to form a mixture; introducing the mixture into the passage in the analytical device to immobilize the conjugate within the passage; and assaying the immobilized conjugate.
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25. An analytical method comprising:
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preparing an analytical device, comprising a passage allowing a liquid to flow through the same, by bonding together a first member having a groove, 1 μ
m to 5 mm width and 1 μ
m to 750 μ
m depth in cross-section, and a second member covering the groove;immobilizing a first nucleic acid (N1), having an arbitrary base sequence, in a capturing zone provided in the passage on the first member and/or second member prior to bonding the first member and second member together; preparing a reagent A containing a conjugate (N2-L1) resulting from binding of a first ligand (L1), capable of specifically binding to a biological substance to be assayed, to a second nucleic acid (N2) having a sequence at least complementary to the base sequence of the first nucleic acid (N1); separately introducing a liquid sample suspected to contain the biological substance to be assayed and the reagent A, without preliminary mixing of the liquid sample and reagent A, into the passage in the analytical device to immobilize the conjugate within the passage; and assaying the immobilized conjugate.
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26. An analytical method comprising:
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preparing an analytical device, comprising a passage allowing a liquid to flow through the same, by bonding together a first member having a groove, 1 μ
m to 5 mm width and 1 μ
m to 750 μ
m depth in cross-section, and a second member covering the groove;immobilizing a plurality of first nucleic acid species (N1g;
g being an integer), each having an arbitrary base sequence, independently from species to species, in a capturing zone provided in the passage on the first member and/or second member prior to bonding the first member and second member together;preparing a reagent A containing a plurality of conjugate species (N2h-L1i;
h and i each being an integer), each resulting from binding of (1) one of a plurality of first ligand species (L1i;
i being an integer), capable of specifically binding to a corresponding one of biological substance species (Ok;
k being an integer) to be assayed, to (2) one of a plurality of second nucleic acid species (N2h;
h being an integer), each having a sequence at least complementary to the base sequence of a corresponding one of the plurality of first nucleic acid species;mixing a liquid sample, suspected of containing one or more of the biological substance species to be assayed, and the reagent A to form a mixture; introducing the mixture, either after conjugate formation or while allowing conjugate formation, into the passage in the analytical device to immobilize one or more of the conjugate species within the passage; and assaying the immobilized conjugate(s).
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27. An analytical method comprising:
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preparing an analytical device, comprising a passage allowing a liquid to flow through the same, by bonding together a first member having a groove, 1 μ
m to 5 mm width and 1 μ
m to 750 μ
m depth in cross-section, and a second member covering the groove;immobilizing a plurality of first nucleic acid species (N1g;
g being an integer), each having an arbitrary base sequence, independently from species to species, in a capturing zone provided in the passage on the first member and/or second member prior to bonding the first member and second member together;preparing a reagent A containing a plurality of conjugate species (N2h-L1i;
h and i each being an integer) each resulting from binding of (1) one of a plurality of first ligand species (L1i;
i being an integer), capable of specifically binding to one of biological substance species (Ok;
k being an integer) to be assayed, to (2) one of a plurality of second nucleic acid species (N2h;
h being an integer), each having a sequence at least complementary to the base sequence of a corresponding one of the plurality of first nucleic acid species;separately introducing (1) a liquid sample suspected of containing one or more of the biological substances to be assayed and (2) the reagent A into the passage in the analytical device to immobilize the resulting one or more conjugate species within the passage; and assaying the immobilized conjugate(s).
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50. (canceled)
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51. A method of preparing an analytical device comprising:
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preparing a first member having a groove, 1 μ
m to 5 mm width and 1 μ
m to 750 μ
m depth in cross-section, and a second member capable of covering the groove, wherein the groove forms a portion of a passage upon joining the first member and second member together and one of the first member and second member or both have a passage inlet and a passage outlet,immobilizing a nucleic acid (N), having an arbitrary base sequence, at a site on a portion the first member and/or second member forming the passage, to form a zone for capturing a biological substance to be assayed, then joining the first member and second member together by thermal fusion or with an adhesive to give an assembly with the passage formed therein, introducing into the passage a reagent A containing a conjugate (N2-L1) composed of a second nucleic acid (N2) having a base sequence at least complementary to the base sequence of the first nucleic acid (N1) which is immobilized in the capturing zone and a first ligand (L1) capable of specifically binding to a biological substance to be assayed, and allowing the conjugate (N2-L1) to specifically bind, for immobilization thereof, to the first nucleic acid (N1) in the capturing zone. - View Dependent Claims (54)
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52. A method of preparing an analytical device comprising:
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preparing a first member having a groove, 1 μ
m to 5 mm width and 1 μ
m to 750 μ
m depth, and a second member capable of covering the groove, wherein the groove forms a portion of a passage upon joining the first member and second member together and one of the first member and second member or both have a passage inlet and a passage outlet,immobilizing a plurality of first nucleic acid species (N1g;
g being an integer) each having an arbitrary base sequence at independent sites forming a zone within the passage for capturing one or more biological substance species to be assayed,then joining the first member and second member together by thermal fusion or with an adhesive to give an assembly with the passage formed therein, introducing into the passage a reagent A containing conjugate species (N2h-L1i;
wherein h and i are each an integer), each composed of one of a plurality of second nucleic acid species (N2h;
h being an integer), each second nucleic acid species having a base sequence at least complementary to the base sequence of a corresponding species of the plurality of first nucleic acid species (N1g;
g being an integer) immobilized in the capturing zone, and one of a plurality of first ligand species (L1i;
i being an integer), each first ligand species being capable of specifically binding to a corresponding species of the one or more biological substance species to be assayed, andallowing the plurality of conjugate species N2h-L1i to specifically bind, for immobilization thereof, to the plurality of first nucleic acid species previously immobilized in the capturing zone.
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53. (canceled)
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55. (canceled)
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56. (canceled)
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57. (canceled)
Specification