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Apparatus and method for reading fluorescence

外国特許コード F110005640
整理番号 RX08P12WO
掲載日 2011年9月9日
出願国 アメリカ合衆国
出願番号 52778007
公報番号 20100140503
公報番号 8384044
出願日 平成19年8月27日(2007.8.27)
公報発行日 平成22年6月10日(2010.6.10)
公報発行日 平成25年2月26日(2013.2.26)
国際出願番号 JP2007000910
国際公開番号 WO2008102417
国際出願日 平成19年8月27日(2007.8.27)
国際公開日 平成20年8月28日(2008.8.28)
優先権データ
  • 特願2007-038706 (2007.2.19) JP
  • 2007JP000910 (2007.8.27) WO
発明の名称 (英語) Apparatus and method for reading fluorescence
発明の概要(英語) It is an objection of the present invention to provide a fluorescence reading apparatus in view of the influence of fluorescence derived from a fluorescence substance that is not involved with an interaction between a probe substance and a target substance.
The above-mentioned problem is solved by a fluorescence reading apparatus 12 comprising an incidence angle adjusting means 10 for adjusting an incidence angle when light from a light source 7 is incident on a substrate 2; and a controller 11 that controls an amount of the incidence angle adjusted by the incidence angle adjusting means 10 and also comprises a means for receiving information on the incidence angle and information on the fluorescence intensities or fluorescence images at a plurality of incidence angles and obtaining the penetration depths of the evanescent fields with respect to the plurality of incidence angles from the information on the incidence angles; and a means for obtaining information on the fluorescence intensities in the obtained plurality of penetration depths.
従来技術、競合技術の概要(英語) BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a fluorescence reading apparatus in view of the influence of fluorescence derived from a fluorescence substance that is not involved with an interaction between a probe substance and a target substance.
More particularly, the invention relates to a microarray reading apparatus and a reading method, in which a penetration depth of an evanescent field is changed to obtain fluorescence intensities in a plurality of penetration depths, allowing an accurate determination whether the probe substance and the target substance have interacted with each other.
2. Description of the Related Art
In a DNA microarray, DNAs such as gene segments are arranged in and fixed to a substrate.
For example, a plurality of gene segments are respectively fixed to spots to prepare a probe, and mRNA extracted from a human cell is converted into cDNA by a reverse transcriptase to obtain a target substance.
In a DNA micro array reading apparatus, hybridization of the target substance and the probe fixed to the substrate is observed to analyze various genetic information.
For example, a fluorescent substance is used in order to detect the interaction between the target substance and the probe substance.
The fluorescent substance may be added to the target substance, or the fluorescent substance may generate fluorescence by the influence of the interaction between the target substance and the probe substance.
As a fluorescence reading apparatus such as the microarray reading apparatus that observes the fluorescence derived from the fluorescent substance, there is an apparatus that generates the evanescent field in a region where the probe substance is fixed to excite the fluorescent substance, for example.
In such case, it takes a long time to hybridize the target substance and the probe substance.
Therefore, there is a demand for a real-time microarray reading apparatus that can detect a state of the interaction between the target substance and the probe substance.
To this end, for example, Japanese Patent Application Laid-Open No. 2006-38816 (Patent Document 1 below) discloses a microarray reading apparatus that detects the specific interaction between the probe substance and the target substance when a sample containing at least the fluorescent substance and the target substance is brought into contact with the substrate to which the probe substance is fixed.
The microarray reading apparatus includes a light irradiation means for irradiating light; a light incident means for causing the light irradiated by the irradiation means to be incident on the substrate such that the evanescent field is generated in a surface of the substrate to which the probe substance is fixed; and a light detecting means for detecting the fluorescence generated from the fluorescent substance contained in the sample excited by the evanescent field.
In the microarray reading apparatus, the light detecting means includes an optical lens that acts as an objective lens, and the optical lens acts as the light incident means.
The microarray reading apparatus disclosed in Japanese Patent Application Laid-Open No. 2006-38816 (Patent Document 1 below) is excellent in rapidly and accurately observing the hybridization of the target substance and the probe substance.
However, disadvantageously the microarray reading apparatus observes not only the emission derived from the hybridization of the target substance and the probe substance but also a noise such as the fluorescence derived from the fluorescent substance flowing in a flow-through cell.
That is, in the conventional real-time microarray reading apparatus, the evanescent field having intensity enough to excite the luminous material is generated in a region up to about 100 nm from a substrate surface.
In the region within 100 nm from the substrate surface, there are many luminous materials that emit light irrespective of the interaction between the target substance and the probe substance because the sample containing the fluorescent substance is continuously brought into contact with the probe substance.
Therefore, in the conventional microarray reading apparatus, because strong fluorescence is often observed even when the target substance and the probe substance do not hybridize with each other, a determination whether the target substance and the probe substance have interacted with each other cannot correctly be made.
Further, in the conventional micro array reading apparatus, because the emission caused only by the interaction between the target substance and the probe substance cannot be observed, an amount of the target substance interacting with the probe substance cannot quantitatively be evaluated.
Furthermore, in the conventional microarray reading apparatus, a distribution of the fluorescent substance generating the fluorescence, which indicates at which positions from the substrate the target substance and the probe substance have interacted with each other, is not recognized.
Japanese Patent Application Laid-Open No. 2006-189741 (Patent Document 2 below) discloses an invention in which an incidence angle adjusting means is controlled such that a leach-through depth of evanescent light is set to a desired amount (see, for example, claim 12).
However, in the invention disclosed in this document, an incidence angle is controlled in order to keep the leach-through depth of evanescent light constant even if the laser beam sources are changed.
That is, because the technique is essentially directed to keep the penetration depth of the evanescent field as constant as possible, the technique does not inspire fluorescence measurement in various penetration depths of the evanescent field.
Japanese Patent Application Laid-Open No. 2001-194310 (Patent Document 3 below) relates to a surface plasmon spectral apparatus in which a plurality of samples are measured at a time although only one objective lens is used.
U.S. Patent Application Publication No. 2003/0205681 (Patent Document 4 below) discloses a fluorescence analyzer in which a microarray is used.
In the fluorescence analyzer, two penetration depths of evanescent fields are obtained using two light beams (paragraph [0069], claim 41).
However, this document fails to disclose that the fluorescence is observed in a number of penetration depths of evanescent fields to effectively observe the presence or absence of the hybridization.
Patent Document 1: Japanese Patent Application Laid-Open No. 2006-38816
Patent Document 2: Japanese Patent Application Laid-Open No. 2006-189741
Patent Document 3: Japanese Patent Application Laid-Open No. 2001-194310
Patent Document 4: U.S. Patent Application Publication No. 2003/0205681

特許請求の範囲(英語) [claim1]
1. A fluorescence reading apparatus 12 that detects a specific interaction between a probe substance and a target substance, comprising: a substrate 2 to which a probe substance 1 is fixed;
a sample chamber 6 that accommodates the probe substance 1 and also accommodates a sample 5 that contains a fluorescent substance 3 and a target substance 4 such that the sample is brought into contact with the probe substance 1;
a light source 7;
an optical system 8 that guides the light from the light source 7 to the substrate 2 to generate an evanescent field; and
a fluorescence detecting unit 9 that detects a fluorescence intensity or a fluorescence image generated by the fluorescent substance 3 excited by the evanescent field,
wherein the optical system 8 comprises an incidence angle adjusting means 10 for adjusting an incidence angle when the light from the light source 7 is incident on the substrate 2;
and a controller 11 that controls an amount of the incidence angle adjusted by the incidence angle adjusting means 10; wherein the controller 11 comprises a means for receiving information on the incidence angle adjusted by the incidence angle adjusting means 10 and information on the fluorescence intensities or fluorescence images at a plurality of incidence angles detected by the fluorescence detecting unit 9 and obtaining the penetration depths of the evanescent fields with respect to the plurality of incidence angles from the information on the incidence angles; and a means for obtaining information on the fluorescence intensities in the obtained plurality of penetration depths; and
wherein the controller comprises a means for obtaining a graph, using the relationships between the penetration depths of the evanescent fields and the fluorescence intensities with respect to the plurality of penetration depths, where one axis represents the fluorescence intensity while the other axis represents the penetration depth of the evanescent field.
[claim2]
2. The fluorescence reading apparatus 12 as claimed in claim 1, wherein the optical system 8 comprises an optical element 13 on which the light from the light source 7 is incident; and an objective lens 14 on which the light transmitted through the optical element is incident; and
wherein the incidence angle adjusting means 10 comprises an optical element moving means for moving the optical element 13 such that the position relative to the objective lens 14 is changed.
[claim3]
3. The fluorescence reading apparatus 12 as claimed in claim 1, comprising a substrate moving means 15 for moving the position of the substrate 2.
[claim4]
4. The fluorescence reading apparatus 12 as claimed in claim 1, wherein the controller further comprises: a means for obtaining a hypothetical fluorescence intensity, using each point on the graph, when the penetration depth of the evanescent field is zero;
a means for comparing a set threshold and the hypothetical fluorescence intensity when the penetration depth of the evanescent field is zero; and
a means for determining whether the probe substance 1 and the target substance 4 have interacted with each other using the comparison result.
[claim5]
5. The fluorescence reading apparatus 12 as claimed in claim 1, wherein the controller comprises a means for observing elapsed time.
[claim6]
6. The fluorescence reading apparatus 12 as claimed in claim 1, wherein not only a connecting pipe, that connects the sample chamber and a sample storage storing the target substance, is connected to the uppermost-stream region of the sample chamber, but also one or more connecting pipes are connected to the midstream region of the sample chamber.
[claim7]
7. The fluorescence reading apparatus 12 as claimed in claim 1, comprising a plurality of objective lenses and also comprising an optical system that guides light to the plurality of objective lenses.
[claim8]
8. The fluorescence reading apparatus 12 as claimed in claim 1, comprising a sample storage storing the target substance and a connecting pipe that connects the sample storage and a plurality of sample chambers, wherein the connecting pipe has a branch portion, and the downstream portions of each connecting pipe branched by the branch portion of the connecting pipe are connected to the plurality of sample chambers respectively.
[claim9]
9. A fluorescence reading apparatus 12 that detects a specific interaction between a probe substance and a target substance, comprising: a substrate 2 to which a probe substance 1 is fixed;
a sample chamber 6 that accommodates the probe substance 1 and also accommodates a sample 5 that contains a fluorescent substance 3 and a target substance 4 such that the sample is brought into contact with the probe substance 1;
a light source 7; an optical system 8 that guides the light from the light source 7 to the substrate 2 to generate an evanescent field; and
a fluorescence detecting unit 9 that detects a fluorescence intensity or a fluorescence image generated by the fluorescent substance 3 excited by the evanescent field,
wherein the optical system 8 comprises an incidence angle adjusting means 10 for adjusting an incidence angle when the light from the light source 7 is incident on the substrate 2;
and a controller 11 that controls an amount of the incidence angle adjusted by the incidence angle adjusting means 10, wherein the controller 11 comprises a means for receiving information on the incidence angle adjusted by the incidence angle adjusting means 10 and information on the fluorescence intensities or fluorescence images at a plurality of incidence angles detected by the fluorescence detecting unit 9 and obtaining the penetration depths of the evanescent fields with respect to the plurality of incidence an angles from the information on the incidence angles; and a means for obtaining information on the fluorescence intensities in the obtained plurality of penetration depths; and
wherein the substrate 2 comprises a spot 21 to which the probe substance 1 is fixed;
wherein the fluorescence detecting unit 9 obtains the fluorescence image generated by the fluorescent substance 3; and
wherein the controller 11 comprises a means for scanning the fluorescence image to compute a boundary 21 at which the fluorescence intensity is changed; a means for grasping a region 23 inside the spot and a region 24 outside the spot in the fluorescence image from the shape of the boundary; and a means for obtaining the fluorescence intensity inside the spot.
[claim10]
10. A fluorescence reading apparatus 12 that detects a specific interaction between a probe substance and a target substance, comprising: a substrate 2 to which a probe substance 1 is fixed;
a sample chamber 6 that accommodates the probe substance 1 and also accommodates a sample 5 that contains a fluorescent substance 3 and a target substance 4 such that the sample is brought into contact with the probe substance 1;
a light source 7;
an optical system 8 that guides the light from the light source 7 to the substrate 2 to generate an evanescent field; and
a fluorescence detecting unit 9 that detects a fluorescence intensity or a fluorescence image generated by the fluorescent substance 3 excited by the evanescent field,
wherein the optical system 8 comprises an incidence angle adjusting means 10 for adjusting an incidence angle when the light from the light source 7 is incident on the substrate 2; and a controller 11 that controls an amount of the incidence angle adjusted by the incidence angle adjusting means 10; and
wherein the controller 11 comprises a means for receiving information on the incidence angle adjusted by the incidence angle adjusting means 10 and information on the fluorescence intensities or fluorescence images at a plurality of incidence angles detected by the fluorescence detecting unit 9 and obtaining the penetration depths of the evanescent fields with respect to the plurality of incidence angles from the information on the incidence angles; and a means for obtaining information on the fluorescence intensities in the obtained plurality of penetration depths; and
wherein the concentration of the probe substance 1 is increased toward the downstream region of the sample chamber in the substrate 2 to which the probe substance is fixed.
[claim11]
11. A fluorescence reading method using a fluorescence reading apparatus that detects a specific interaction between a probe substance and a target substance, comprising: a substrate 2 to which a probe substance 1 is fixed;
a sample chamber 6 that accommodates the probe substance 1 and also accommodates a sample 5 that contains a fluorescent substance 3 and a target substance 4 such that the sample is brought into contact with the probe substance 1;
a light source 7;
an optical system 8 that guides the light from the light source 7 to the substrate 2 to generate an evanescent field; and
a fluorescence detecting unit 9 that detects a fluorescence intensity or a fluorescence image generated by the fluorescent substance 3 excited by the evanescent field,
characterized in that a step of changing the penetration depth of the evanescent field and a step of obtaining the fluorescence intensity after the penetration depth of the evanescent field is changed are repeatedly performed to obtain the fluorescence intensities in a plurality of penetration depths of the evanescent fields,
wherein a graph is obtained where one axis represents the fluorescence intensity while the other axis represents the penetration depth of the evanescent field, using the relationships between the penetration depths of the evanescent fields and the fluorescence intensities with respect to the plurality of penetration depths.
[claim12]
12. A fluorescence reading method as claimed in claim 9, wherein, in the step of changing the penetration depth of the evanescent field, the penetration depth of the evanescent field is changed by adjusting the incidence angle of the light incident on the substrate 2.
[claim13]
13. A fluorescence reading method as claimed in claim 9, wherein the substrate (2) comprises a spot 21 to which the probe substance 1 is fixed; and
wherein the relationships between the penetration depths of the evanescent fields and the fluorescence intensities with respect to the plurality of penetration depths are obtained for a certain spot 21 and then the relationships between the penetration depths of the evanescent fields and the fluorescence intensities with respect to the plurality of penetration depths are obtained for the spot 21 after a predetermined time has elapsed.
[claim14]
14. A fluorescence reading apparatus 12 that detects a specific interaction between a probe substance and a target substance, comprising: a substrate 2 to which a probe substance 1 is fixed;
a sample chamber 6 that accommodates the probe substance 1 and also accommodates a sample 5 that contains a fluorescent substance 3 and a target substance 4 such that the sample is brought into contact with the probe substance 1;
a light source 7;
an optical system 8 that guides the light from the light source 7 to the substrate 2 to generate an evanescent field; and
a fluorescence detecting unit 9 that detects a fluorescence intensity or a fluorescence image generated by the fluorescent substance 3 excited by the evanescent field,
characterized in that the concentration of the probe substance 1 is increased toward the downstream region of the sample chamber in the substrate 2 to which the probe substance is fixed.
  • 発明者/出願人(英語)
  • SUGIURA TADAO
  • MORI MASATO
  • INAMOTO EIJI
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
  • NARA INSTITUTE OF SCIENCE AND TECHNOLOGY
国際特許分類(IPC)
米国特許分類/主・副
  • 250/459.1
  • 250/361.000R
  • 250/458.1
  • 250/483.1
  • 250/484.2
  • 359/385
  • 359/388
  • 435/287.2
  • 435/288.7
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