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