Biochemical examination method

• US 20020168665A1
• Filed: 03/15/2002
• Published: 11/14/2002
• Est. Priority Date: 03/21/2001
• Status: Active Grant

• Current Assignee: Olympus Optical Company Ltd. (Olympus Corporation)
• Original Assignee: Olympus Optical Company Ltd. (Olympus Corporation)
• Inventors: Okawa, Kaneyasu
• Granted Patent: US 7,027,932 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings

Abstract

According to the present invention, there is provided a biochemical examination method of supplying a solution of a first biochemical material into an array of which respective second biochemical materials are held, and using a marker by a luminescent molecule to detect a luminescent intensity of the luminescent molecule for each probe array element of the array with an array type detector, so that a reaction state of the first biochemical material with the second biochemical materials is examined for each probe array element, the method comprising: irradiating a reference array by which the luminescent molecule is held with an excitation lighting to photograph a reference light image; irradiating the array for biochemical examination with the same excitation lighting to photograph a sample light image; and correcting the sample light image with the reference light image.

Claims

1. A biochemical examination method of supplying a solution of a first biochemical material to an array for biochemical examination in which respective second biochemical materials peculiarly reacting with the first biochemical material are held on a surface, and using a marker by a luminescent molecule excited by a light to emit the light to detect a luminescent intensity of said luminescent molecule for each probe array element of said array for biochemical examination with an array detector, so that a reaction state of said first biochemical material with the second biochemical materials peculiarly reacting with the first biochemical material is examined for said each probe array element, said method comprising:

irradiating a reference array holding said luminescent molecule with an excitation lighting and photographing a first light image as a reference light image;

irradiating said array for biochemical examination with the same excitation lighting and photographing a second light image as a sample light image; and

correcting the sample light image with said reference light image.

2. The biochemical examination method according to claim 1, wherein said array for biochemical examination is configured by supplying a solution of a probe into different positions of the surface of a reaction carrier having a solid structure formed of a porous or fibrous material or a molded material, and holding the probe contained in the solution inside said porous or fibrous material or the molded material, and a solution of a sample to be examined containing said first biochemical material is supplied to said array for biochemical examination.

3. The biochemical examination method according to claim 1, further comprising:

using a mixture solution of a plurality of samples containing the first biochemical material labeled by a plurality of different luminescent molecules excited by the light to emit the light as the solution of the sample to be examined; and

using said reference array in which said plurality of different luminescent molecules are mixed and held at a constant ratio to examine the reaction state of the probe in said each probe array element with said each sample.

4. The biochemical examination method according to claim 1, wherein said reference array is configured by holding the same number of said luminescent molecules in an array element corresponding to said each probe array element in the same format as that of said array for biochemical examination.

5. The biochemical examination method according to claim 1, wherein said reference array is configured by holding the luminescent molecules on a substrate with a homogeneous distribution.

9. The biochemical examination method according to claim 1, further comprising:

dividing a light image into a plurality of divided images by a unit of said probe array element;

designating a measurement region and a local background region with respect to each divided image;

detecting a local background from the local background region; and

using a detected amount to correct a detected amount from said measurement region.

12. The biochemical examination method according to claim 1, further comprising:

detecting said luminescent intensity corresponding to a standard area of said probe array element or an average luminescent intensity per unit area.

16. The biochemical examination method according to claim 1, further comprising:

setting a nonlinear coefficient as a parameter for determining a luminescent function; and

using the nonlinear coefficient to obtain the number of luminescent molecules present in said each probe array element.

19. The biochemical examination method according to claim 1, further comprising:

assuming that P_Mk is a luminescent intensity of a luminescent molecule M in said probe array element k, n_RM is a quantity of the luminescent molecules M in an element k of said reference array, and P_RMK is a luminescent intensity of the luminescent molecule M in said reference array element k; and

obtaining a quantity n_Mk of luminescent molecules M in said probe array element k by the following equation;

6. A biochemical examination method of supplying a solution of a first biochemical material to an array for biochemical examination in which respective second biochemical materials peculiarly reacting with the first biochemical material are held on a surface, and using a marker by a luminescent molecule excited by a light to emit the light to detect a luminescent intensity of said luminescent molecule for each probe array element of said array for biochemical examination with an array detector, so that a reaction state of said first biochemical material with the second biochemical materials peculiarly reacting with the first biochemical material is examined for said each probe array element, said method comprising:

dividing a light image of said array for biochemical examination with the solution of said biochemical material supplied thereto into a plurality of divided images by a unit of said probe array element;

designating a measurement region and a local background region with respect to each divided image;

detecting a local background from the local background region; and

using a detected amount to correct a detected amount from said measurement region.

7. The biochemical examination method according to claim 6, wherein said array for biochemical examination is configured by supplying a solution of a probe into different positions of the surface of a reaction carrier having a solid structure formed of a porous or fibrous material or a molded material, and holding the probe contained in the solution inside said porous or fibrous material or the molded material, and a solution of a sample to be examined containing said first biochemical material is supplied to said array for biochemical examination.

8. The biochemical examination method according to claim 6, further comprising:

using a mixture solution of a plurality of samples containing the biochemical material labeled by a plurality of different luminescent molecules excited by the light to emit the light as the solution of the sample to be examined; and

using said reference array in which said plurality of different luminescent molecules are mixed and held at a constant ratio, so that the reaction state of the probe in said each probe array element with said each sample is examined.

34. The biochemical examination method according to claim 6, further comprising:

forming a shape of said divided image as a parallelogram shape.

35. The biochemical examination method according to claim 6, further comprising:

setting a boundary of said measurement region to be the same as a boundary of said local background region.

36. The biochemical examination method according to claims 6, further comprising:

detecting a dark region width of a compressed image of an array line or an array row which forms a gradient with respect to an x-axis or a y-axis of a binarized image of the light image obtained by said array type detector; and

obtaining the gradient of said array line or the gradient of the array row when the dark region width is broadest as the gradient of a line dividing line or a row dividing line of said divided image.

42. The biochemical examination method according to claim 6, further comprising:

binarizing said divided image with a middle value of an output in the divided image;

obtaining a rectangle as a boundary of said local background region by expanding a rectangle circumscribed by the binarized image by a constant amount; and

obtaining the outside of the boundary in the divided image as the local background region.

43. The biochemical examination method according to claim 6, further comprising:

binarizing said divided image with a middle value of an output in the divided image;

obtaining a rectangle as a boundary of said measurement region by compressing a rectangle inscribed by the binarized image by a constant amount; and

obtaining the inside of the boundary as the measurement region.

44. The biochemical examination method according to claim 42, further comprising:

assuming that λ

is a fluorescent wavelength to be detected, a is a numerical aperture on a sample side, and Δ

is a defocus amount; and

determining said amount of expansion δ

by the following equation;

δ

=1.619×

λ

/a+a|Δ

|.

45. The biochemical examination method according to claim 43, further comprising:

assuming that X is a fluorescent wavelength to be detected, a is a numerical aperture on a sample side, and A is a defocus amount; and

determining said amount of compression 6 by the following equation;

÷

=1.619×

λ

/a+a|Δ

|.

46. The biochemical examination method according to claim 6, further comprising:

detecting an intensity obtained by subtracting an average luminescent intensity per unit area of said local background region from an average luminescent intensity per unit area of said measurement region as a reaction intensity with each probe.

10. A biochemical examination method of supplying a solution of a sample to be examined containing a first biochemical material to an array for biochemical examination configured by supplying a solution of a probe into different positions of the surface of a reaction carrier having a solid structure formed of a porous or fibrous material or a molded material and holding the probe contained in the solution inside said porous or fibrous material or the molded material, and using a marker by a luminescent molecule to detect a luminescent intensity of said luminescent molecule for each probe array element of said array for biochemical examination with an array detector so that a reaction state of said first biochemical material with a second biochemical material peculiarly reacting with the first biochemical material is examined for said each probe array element, said method comprising:

detecting said luminescent intensity corresponding to a standard area of said probe array element or an average luminescent intensity per unit area.

11. The biochemical examination method according to claim 10, further comprising:

using a mixture solution of a plurality of samples containing the first biochemical material labeled by a plurality of different luminescent molecules excited by the light to emit the light as the solution of the sample to be examined; and

using said reference array in which said plurality of different luminescent molecules are mixed and held at a constant ratio, so that the reaction state of the probe in said each probe array element with said each sample is examined.

14. The biochemical examination method according to claim 13, wherein said array for biochemical examination is configured by supplying a solution of a probe into different positions of the surface of a reaction carrier having a solid structure formed of a porous or fibrous material or a molded material and holding the probe contained in the solution inside said porous or fibrous material or the molded material, and a solution of a sample to be examined containing said first biochemical material is supplied to said array for biochemical examination.

15. The biochemical examination method according to claim 13, further comprising:

using a mixture solution of a plurality of samples containing the first biochemical material labeled by a plurality of different luminescent molecules excited by the light to emit the light as the solution of the sample to be examined; and

using a reference array in which said plurality of different luminescent molecules are mixed and held at a constant ratio as said luminescent function, so that the reaction state of the probe in said each probe array element with said each sample is examined.

17. The biochemical examination method according to claim 13, further comprising:

assuming that n_Mk is a quantity of luminescent molecules M in said probe array element k, J is the number of types of luminescent molecules, I_Mk is a luminescent intensity per one molecule of the luminescent molecules M in said probe array element k, ρ

is a nonlinear coefficient, and P_Mk is a luminescent intensity of the luminescent molecule M in said probe array element k; and

obtaining said luminescent function by the following equation;

18. The biochemical examination method according to claim 17, wherein said I_Mk is obtained by the following equation:

20. The biochemical examination method according to claim 13, further comprising:

assuming that ρ

is a nonlinear coefficient, n_RM is a quantity of luminescent molecules M in an element k of said reference array, P_RMk is a luminescent intensity of the luminescent molecule M in said reference array element k, P_Mk is a luminescent intensity of the luminescent molecule M in said probe array element k,

and n_Mk⁽ⁱ⁾ is an i-th operation value of a quantity n_Mk of luminescent molecules M in said probe array element k;

using an initial value n_Mk⁽⁰⁾=0, the following successive operation equation

13. A biochemical examination method of supplying a solution of a first biochemical material to an array for biochemical examination in which respective second biochemical materials peculiarly reacting with the first biochemical material are held on a surface, and using a marker by a luminescent molecule excited by a light to emit the light to detect a luminescent intensity of said luminescent molecule for each probe array element of said array for biochemical examination with an array detector, so that a reaction state of said first biochemical material with the second biochemical materials peculiarly reacting with the first biochemical material is examined for said each probe array element, said method comprising:

setting a nonlinear coefficient as a parameter for determining a luminescent function; and

using the nonlinear coefficient to obtain the number of luminescent molecules present in said each probe array element.

21. A biochemical examination method of supplying a solution of a first biochemical material to an array for biochemical examination in which respective second biochemical materials peculiarly reacting with the first biochemical material are held on a surface, and using a marker by a luminescent molecule excited by a light to emit the light to detect a luminescent intensity of said luminescent molecule for each probe array element of said array for biochemical examination with an array detector, so that a reaction state of said first biochemical material with the second biochemical materials peculiarly reacting with the first biochemical material is examined for said each probe array element, said method comprising:

obtaining an abnormal region designated with respect to a light image of said array for biochemical examination with the solution of said first biochemical material supplied thereto as a non object region.

22. The biochemical examination method according to claim 21, wherein said array for biochemical examination is configured by supplying a solution of a probe to different positions of a surface of a reaction carrier having a solid structure formed of a porous or fibrous material or a molded material, and holding the probe contained in the solution inside said porous or fibrous material or the molded material, and a solution of a sample to be examined containing said first biochemical material is supplied to said array for biochemical examination.

23. The biochemical examination method according to claim 21, further comprising:

obtaining a mixture solution of a plurality of samples containing the first biochemical material labeled with a plurality of different luminescent molecules excited by the light to emit the light as the solution of the sample to be examined; and

supplying the solution of the sample to be examined to said array for biochemical examination.

24. The biochemical examination method according to claims 21, further comprising:

acquiring a foreign particle image of said array for biochemical examination beforehand; and

adding a foreign particle region of the foreign particle image to said abnormal region to obtain a non object region.

26. The biochemical examination method according to claim 25, wherein said array for biochemical examination is configured by supplying a solution of a probe into different positions of the surface of a reaction carrier having a solid structure formed of a porous or fibrous material or a molded material, and holding the probe contained in the solution inside said porous or fibrous material or the molded material, and a solution of a sample to be examined containing said first biochemical material is supplied to said array for biochemical examination.

27. The biochemical examination method according to claim 25, further comprising:

obtaining a mixture solution of a plurality of samples containing the biochemical material labeled with a plurality of different luminescent molecules excited by the light to emit the light as the solution of the sample to be examined; and

supplying the solution of the sample to be examined to said array for biochemical examination.

29. The biochemical examination method according to claim 28, wherein said array for biochemical examination is configured by supplying a solution of a probe into different positions of the surface of a reaction carrier having a solid structure formed of a porous or fibrous material or a molded material, and holding the probe contained in the solution inside said porous or fibrous material or the molded material, and a solution of a sample to be examined containing said first biochemical material is supplied to said array for biochemical examination.

30. The biochemical examination method according to claim 28, further comprising:

obtaining a mixture solution of a plurality of samples containing the biochemical material labeled with a plurality of different luminescent molecules excited by the light to emit the light as the solution of the sample to be examined; and

supplying the solution of the sample to be examined to said array for biochemical examination.

25. A biochemical examination method of supplying a solution of a first biochemical material to an array for biochemical examination in which respective second biochemical materials peculiarly reacting with the first biochemical material are held on a surface, and using a marker by a luminescent molecule excited by a light to emit the light to detect a luminescent intensity of said luminescent molecule for each probe array element of said array for biochemical examination with an array detector, so that a reaction state of said first biochemical material with the second biochemical materials peculiarly reacting with the first biochemical material is examined for said each probe array element, said method comprising:

obtaining an average image of a plurality of light images of said array type detector having the same accumulated time as an object image.

28. A biochemical examination method of supplying a solution of a first biochemical material to an array for biochemical examination in which respective second biochemical materials peculiarly reacting with the first biochemical material are held on a surface, and using a marker by a luminescent molecule excited by a light to emit the light to detect a luminescent intensity of said luminescent molecule for each probe array element of said array for biochemical examination with an array detector, so that a reaction state of said first biochemical material with the second biochemical materials peculiarly reacting with the first biochemical material is examined for said each probe array element, said method comprising:

using an averaging operator to subject a light image obtained by said array type detector to a smoothing processing.

31. A biochemical examination method of supplying a solution of a first biochemical material to an array for biochemical examination in which respective second biochemical materials peculiarly reacting with the first biochemical material are held on a surface, and using a marker by a luminescent molecule excited by a light to emit the light to detect a luminescent intensity of said luminescent molecule for each probe array element of said array for biochemical examination with an array detector, so that a reaction state of said first biochemical material with the second biochemical materials peculiarly reacting with the first biochemical material is examined for said each probe array element, said method comprising:

obtaining a minimum detected amount in an object region of a light image obtained by said array type detector as a fluorescent background; and

obtaining a light image as an object image by subtracting the fluorescent background from said light image.

32. The biochemical examination method according to claim 31, wherein said array for biochemical examination is configured by supplying a solution of a probe into different positions of the surface of a reaction carrier having a solid structure formed of a porous or fibrous material or a molded material, and holding the probe contained in the solution inside said porous or fibrous material or the molded material, and a solution of a sample to be examined containing said first biochemical material is supplied to said array for biochemical examination.

33. The biochemical examination method according to claim 31, further comprising:

obtaining a mixture solution of a plurality of samples containing the biochemical material labeled with a plurality of different luminescent molecules excited by the light to emit the light as the solution of the sample to be examined; and

supplying the solution of the sample to be examined to said array for biochemical examination.

37. The biochemical examination method according to claim 36, further comprising:

binarizing the light image with a middle value of an output in an object region of the light image obtained by said array type detector; and

obtaining said binarized image.

38. The biochemical examination method according to claim 36, further comprising:

obtaining an interval among said line dividing lines or among said row dividing lines by a plurality of points configured by middle positions of respective dark regions or respective bright regions of a compressed image of said array line or said array row.

39. The biochemical examination method according to claim 38, further comprising:

comparing said continuous bright regions with arrangement conditions of said probe array elements;

replacing a region having no validity with the dark region based on the arrangement conditions of said probe array elements, when there is a region having no validity in said each bright region; and

obtaining the interval among said line dividing lines or said row dividing lines.

40. The biochemical examination method according to claim 36, further comprising:

obtaining the interval among said line dividing lines or said row dividing lines in accordance with the arrangement conditions of said probe array elements.

41. The biochemical examination method according to claim 36, further comprising:

detecting a center position of an array element for position detection disposed in said array for biochemical examination; and

obtaining an initial position of said line dividing line or said row dividing line based on the center position.