WO2008029591A1 - Backgroud light reducing method in evanscent wave exciting fluorescent oservation, and component therefor - Google Patents

Abstract

In an evanescent wave exciting fluorescent observation, stray light arriving at a probe solution layer on an upper layer of a fixing region of molecules under examination in a reaction chamber is effectively absorbed by disposing a stray light absorptive region on a waveguide substrate, so that the stay light is suppressed to enter into the reaction chamber and the scattering stray light is suppressed to occur at an edge surface of the waveguide substrate made by applying a laser cutting process to the edge surface of the waveguide substrate on which light is incident. With the structure, critical background fluorescence at the evanescent wave exciting fluorescent observation is significantly reduced.

Description

 Specification

 Background light reduction method and member in evanescent wave excitation fluorescence observation

 The present invention relates to a method for reducing background fluorescence in evanescent wave excitation fluorescence observation and a member used in the method.

 Background art

 [0002] The fluorescence observation method using the evanescent wave excitation method is a technology that has achieved high results in recent years, such as enabling single-molecule observation of proteins in the field of microscopy.

 [0003] The fluorescence observation method based on such an evanescent wave excitation method is limited to a few hundreds of neighborhoods when light is totally reflected in a substrate in contact with a fluorescently labeled probe solution on the substrate. This is based on the fact that the fluorescently labeled probe molecules near the substrate-liquid layer interface are selectively excited by the evanescent wave. In other words, incident light is incident from the end face of the slab waveguide substrate and repeatedly reflected and totally reflected in the slide substrate to generate an evanescent wave, and the fluorescence emitted by the probe molecules excited by the evanescent wave is observed. By doing so, the interaction between the molecules immobilized on the slide substrate surface and the probe molecules can be observed (Fig. 1).

 [0004] Such fluorescence observation by evanescent wave excitation is particularly suitable for observing the binding state between the above-mentioned molecule and a fluorescent probe in a microarray in which a large number of test molecules are immobilized. Such an evanescent wave excitation method is used. The microarray technology used is, for example, injecting a fluorescent probe sample solution into a reaction vessel installed on a substrate having a slab waveguide installed in a horizontal direction, thereby introducing the slab type guide into the fluorescent probe sample. It is configured to contact the wave path, and by introducing incident light at an angle in the waveguide, it is possible to selectively excite the probe molecules in the vicinity of the interface by the evanescent wave generated on the waveguide. (See Patent Document 1), and the inventor has filed several applications (PCT-JP2004009600, PCT_JP2004019333, Japanese Patent Application 2005-184171)

[0005] However, the selective excitation of the evanescent wave excitation method is theoretically a phenomenon that depends on the function of the distance from the interface, not the selectivity for the interface-bound molecules, and is far above the interface. Does not completely eliminate the excitation of non-bonded molecules present in In reality, there is stray light that deviates from the total reflection condition that occurs non-ideally in the device. In actual observation, the phenomenon that the probe solution layer above the evanescent region shines may be observed. It was known. Such stray light or light that does not satisfy the total reflection condition while traveling in the waveguide reaches the upper layer of the probe liquid and directly excites the probe molecules, resulting in increased background light intensity, resulting in a large S / N ratio. Cause a significant decline. For this reason, it has been difficult to achieve a sufficient S / N ratio and sensitivity with the conventional power evanescent excitation method. In addition, when the background light is strong, the use of high-concentration probe solution becomes a limiting factor, which makes it difficult to detect weak interactions.

 [0006] On the other hand, as a countermeasure against stray light entering the reaction tank on the substrate in the evanescent wave excitation fluorescence detection, the present inventors disperse the colloid by adding a colloid solution to the probe liquid layer, and stray light. We have developed a method that allows the generated fluorescence to reach the detector even when the upper layer of the probe solution is excited by (Japanese Patent Application 2005-006298). Although this method can easily attenuate background light, the equilibrium reaction may change depending on the contents of the colloidal solution added, and the probe molecules may collide with the colloidal particles. In some cases, it is not sufficient because it is unsuitable for adsorbing cases.

 [0007] Further, the present inventors have advanced one step further, as a countermeasure against stray light entering the reaction vessel on the substrate in evanescent wave excitation fluorescence detection, by reducing the thickness of the probe solution layer extremely, A technology has been developed to reduce the absolute number of fluorescent molecules in the upper layer of the probe solution that is excited when stray light passes through the reaction chamber (Japanese Patent Application 2006-203257). However, both of these technologies are methods that suppress the effects of stray light that does not exist in a method that deals with the source of stray light that excites fluorescent molecules in the upper layer of the probe solution. Therefore, there is a natural lower limit to the background light reduction. Also, if the existence of stray light itself is removed from the reaction tank! /, The fundamental countermeasure technology has never been reported.

 Patent Document 1: Japanese Translation of Special Publication 2003-521684

 Disclosure of the invention

Problems to be solved by the invention [0008] An object of the present invention is that stray light generated under optically non-ideal conditions is observed in a reaction tank on a substrate when evanescent wave excitation fluorescence observation is performed using a substrate having a slab waveguide. This method achieves fluorescence observation at a high S / N ratio by effectively suppressing the phenomenon of reaching the upper part of the evanescent field, and the above stray light can be suppressed easily and inexpensively. In addition, it is to provide a new means for suppressing stray light that can be applied to fully automated analysis equipment.

 Means for solving the problem

 [0009] The present inventor has proposed a slab-type waveguide substrate that remains in the evanescent wave excitation fluorescence observation apparatus and that is not ideal optical stray light and is totally incident upon the inside of the waveguide and repeatedly undergoes total reflection. Focusing on the fact that stray light that deviates from the total internal reflection condition reaches the upper layer of the fluorescent probe solution on the substrate and excites fluorescent molecules, this is a major cause of background light. The stray light absorption region is effectively placed on the substrate as a means to suppress reaching the upper reaction vessel, thereby suppressing the amount of stray light reaching the upper layer of the probe solution layer and evanescent wave excitation fluorescence. The background light during observation was greatly reduced and the present invention was completed.

 That is, the present invention is as follows.

 (1) A probe solution containing a fluorescently labeled probe molecule is introduced into a reaction vessel having a waveguide substrate with a test molecule immobilized on the bottom, and the immobilized molecule and the probe molecule are bound to each other. When introducing light into a waveguide substrate and performing evanescent wave excitation fluorescence observation, it is a method of suppressing background light in the evanescent wave excitation fluorescence observation by suppressing the incidence of stray light into the reaction vessel, The background light described above, wherein a stray light absorption region having a width of at least 3 mm in the direction of the incident light is provided on the upper or lower surface of the waveguide substrate between the incident light incident end and the test molecule fixing position. Reduction method.

 (2) The background light reducing method according to the above (1), wherein the stray light absorption regions are provided on both upper and lower surfaces with the waveguide substrate interposed therebetween.

 (3) The background light reduction method according to (1) or (2) above, wherein the stray light absorption region is formed by adhesion of a light absorption member.

(4) The incident light incident end face of the waveguide substrate is laser-cut! / The background light reduction method according to any one of the above (1) to (3).

 (5) The background light reduction method according to (1) to (4) above, characterized in that the reaction vessel is formed by a light absorbing member.

 (6) A waveguide substrate having a reaction vessel with a test molecule fixed at the bottom and used for observation of evanescent wave excitation fluorescence, the upper surface of the waveguide substrate between the incident light incident end and the test molecule fixing position or The waveguide substrate according to claim 1, wherein a stray light absorption region having a width of at least 3 mm in the incident light direction is provided on the lower surface.

 (7) The waveguide substrate according to (6) above, wherein stray light absorption regions are provided on both upper and lower surfaces with the waveguide substrate interposed therebetween.

 (8) The waveguide substrate according to (6) or (7) above, wherein the stray light absorption region is formed by adhesion of a light absorption member.

 (9) The waveguide substrate according to any one of the above (6) to (8), wherein the incident light incident end face of the waveguide substrate is laser-cut! /.

 (10) The waveguide substrate according to any one of (6) to (9) above, wherein a reaction vessel is formed by a light absorbing member.

 (11) An evanescent wave excitation characterized by a waveguide substrate used for observation of evanescent wave excited fluorescence having a reaction vessel with a test molecule fixed on the bottom, wherein the incident light incident end face is laser-cut. A waveguide substrate used for fluorescence observation. The invention's effect

 According to the present invention, by providing the stray light absorption region at an effective position on the waveguide substrate, optically non-ideal stray light is absorbed before entering the reaction vessel, and the slab type waveguide is obtained. When performing evanescent wave-excited fluorescence observation using, the generation of background fluorescence, which is the biggest obstacle to observation, is suppressed, thereby enabling fluorescence observation with a high S / N ratio. In addition, the stray light suppressing means of the present invention can be implemented easily and inexpensively, and in addition, by adapting in combination with other methods for reducing the influence of stray light (liquid layer thickness control method), the background fluorescence can be reduced. Further reduction is possible.

 Brief Description of Drawings

[0012] [Fig.1] Selective excitation near the solid-liquid interface in conventional evanescent wave excitation fluorescence observation It is a figure which shows the principle of.

 FIG. 2 is a diagram showing a substrate and its characteristics that have been used for conventional evanescent wave excitation fluorescence observation.

 FIG. 3 is a diagram showing the characteristics of a waveguide substrate equipped with stray light suppression means used for evanescent wave excitation fluorescence observation invented this time.

 FIG. 4 is a diagram showing the result of observing the relationship between the width of the light absorbing member in the incident direction of incident light and the fluorescence intensity of the upper layer.

 FIG. 5 is a diagram showing the relationship between the width of the light absorbing member in the incident direction of incident light and the fluorescence intensity of the upper layer.

FIG. 6 is a diagram showing the arrangement of members on a substrate provided with a light absorbing member in a stray light absorbing region on a waveguide substrate provided with the reaction vessel of the present invention.

 FIG. 7 is a diagram showing the arrangement of members on a substrate provided with a light absorbing member in a stray light absorbing region on a waveguide substrate provided with the reaction vessel of the present invention.

 FIG. 8 is a diagram showing dimensions of a light absorbing member designed in the practice of the present invention.

 FIG. 9 is a diagram showing dimensions of a light absorbing member designed in the practice of the present invention.

 FIG. 10 is a diagram showing the results of an experiment comparing the background light attenuation effect of the stray light absorption method of the present invention with the background light attenuation effect of a conventional substrate.

 FIG. 11 is a diagram showing experimental results observing the background light reduction effect by the combination of the stray light absorption method and the liquid layer thickness control method.

 FIG. 12 is a view showing an arrangement form of an accessory modifier on a substrate provided with a light absorbing member in a stray light absorbing region on a waveguide substrate provided with the reaction vessel of the present invention.

 FIG. 13 is a view showing a more preferable usage form in which an accessory modifier is attached to a substrate equipped with a light absorbing member in a stray light absorbing region on a waveguide substrate provided with the reaction vessel of the present invention. BEST MODE FOR CARRYING OUT THE INVENTION

 The present invention relates to an evanescent wave excited fluorescence observation method using a slide substrate itself as a slab type waveguide.

As shown in Fig. 1, in the observation of evanescent wave excitation light, the probe solution 14 is brought into contact with the waveguide substrate 5 on which the test molecule 19 is fixed, and then the binding reaction is sufficiently advanced. After that, incident light enters from the end face of the waveguide substrate 5 and is repeatedly totally reflected in the substrate, thereby generating an evanescent wave 15 on the waveguide. As a result, the fluorescently labeled probe molecule 13 bound to the test molecule 19 is excited by an evanescent wave to emit fluorescence, and the fluorescence is detected by the detector 16 to identify the test molecule bound to the probe molecule on the substrate. it can.

 [0014] At this time, the intensity of the evanescent field that exudes from the interface has the property of exponentially decaying from the substrate interface in the vertical direction, and the region involved in the excitation of the fluorescent molecule is about several hundred nm in the vertical direction from the interface. Since it is known, even if the remaining probe solution layer is thick, in principle, the probe molecules 13 bound to the test molecules 19 on the waveguide substrate 5 are selectively detected. However, in an actual optical system, light that satisfies the total reflection condition (broken line in Fig. 2) among the light incident on the slide substrate, which is a waveguide, travels inside the substrate, whereas all light enters or reflects during the process of incidence. The S / N ratio is used to generate background light by directly exciting fluorescently labeled probe molecules in the upper layer of the probe solution where a part of the light (hereinafter referred to as stray light) that no longer meets the reflection conditions (solid line in Fig. 2) remains. There was a problem that a good image could not be obtained with good sensitivity due to a decrease in the image quality.

 [0015] In contrast, the stray light suppressing means of the present invention is a waveguide substrate for evanescent wave excitation fluorescence observation. In the waveguide substrate between the incident light incident end and the region where the test molecule is fixed, the incident direction A stray light absorption region having a width of at least 3 mm is provided.

 [0016] By providing the stray light absorption region only on the upper surface or the lower surface of the substrate or on both the upper and lower surfaces, stray light that deviates from the total reflection condition and escapes above the test molecule fixing region in the reaction tank. It is a supplement (Figure 3). This suppresses the conventional process in which the stray light generated in the optical system escapes upward in the test molecule fixing region in the reaction tank on the substrate, and the phenomenon that the fluorescent molecules in the upper layer of the probe solution are directly excited by stray light. Suppress. In addition, the incident end face of the incident light on the waveguide substrate is laser-cut to suppress the generation of scattered stray light on the end face. For this reason, by using the substrate equipped with the stray light suppression method of the present invention, it becomes possible to significantly reduce the background light during evanescent wave excitation fluorescence observation, and a good image with improved S / N ratio can be obtained. can get.

As a means for forming the stray light absorption region of the present invention, for example, black such as carbon black Examples thereof include means for applying a paint containing a color pigment, or means for adhering a light absorbing member made of a molded product such as rubber or resin containing a black pigment to a waveguide substrate. The form of the waveguide substrate provided with the light absorbing member of the present invention is shown in FIG. In order to form the stray light absorbing region 6 by the upper and lower light absorbing members 3 in each reaction tank formed by dividing the light absorbing member of this form by the upper waterproof partition material 1 on the waveguide substrate. Each light absorbing member is designed so as to correspond to the position of each reaction tank. The upper waterproof partition member 1 and the upper light absorbing member 3 can be attached to each other with an adhesive or the like. As shown in FIG. 7, the upper waterproof partition member using the material constituting the upper light absorbing member 3 is used. And the upper light absorbing member may be integrally formed, and the reaction vessel may be formed using the light absorbing member as a constituent member of the reaction vessel side wall. This method is more advantageous in terms of cost because it can save the manufacturing cost of the upper waterproof partition member. The light absorbing member of the present invention can form a stray light absorbing region 6 having a width of at least 3 mm or more in the incident light direction on the waveguide substrate between the incident light incident end and the region where the test molecule is fixed. Any shape is possible! /.

 As is clear from the experimental example shown in the examples described later, the background light reduction effect is not improved so much as the width of the stray light absorbing region is increased beyond 5.4 mm. The thickness of the waveguide substrate used in this experimental example is the usual lmm force S, and even if it is thicker than this, it is necessary to provide a stray light absorption region at least 3 mm wide or 3 to 6.5 mm wide Background light can be effectively reduced.

Regarding the material of the light absorbing member to be arranged on the substrate, in this embodiment, a force S using silicon rubber 20 ° (black), or a material (resin / sealing agent, etc.) that adheres to another slide glass may be used. Furthermore, the color is preferably black, but may be changed. The adhesive shape can also be adhered by the adhesiveness of the material itself, and an adhesive having as low autofluorescence as possible in the incident light wavelength region can be used. The thickness of the light-absorbing member layer is not particularly limited. However, the thickness may be increased as long as it is for the convenience of operation which is preferably 1 mm or more. As a preferable example of such a waveguide substrate, the reaction center disposed on the waveguide substrate has a center interval in the vertical direction of 8.4 to 9.6 mm, and is disposed between the stray light absorption regions on the left and right sides of the substrate. (Figure 8). [0019] In addition, the arrangement method of the reaction tank is not limited to one horizontal row, but may be two or more horizontal rows! As an example, the reaction tank can be divided into two horizontal rows in the reaction tank area excluding the stray light absorption areas on the left and right sides of the substrate (Fig. 9). 8 and 9 are provided with 5.4 mm stray light absorption regions on both the left and right sides in the longitudinal direction of the substrate.

 [0020] The waveguide substrate combined with the effect of reducing background light in the stray light suppression method of the present invention may be any light-transmitting substrate, but preferably has as little autofluorescence as possible and the end face shape is optical. In particular, it should be processed so that it does not generate scattered light. Examples of such a plate-like material include borosilicate glass, white plate glass, quartz glass, synthetic quartz, and other optical use glasses (BK7, SF03, etc.). As a more preferable form, in order to reduce generation of stray light due to scattering of incident light on the substrate end surface, use of a substrate subjected to laser cutting treatment on the substrate end surface can be mentioned (FIG. 10). In addition, this substrate has the correspondence that can be observed with a scanner such as a confocal fluorescence observation scanner other than the evanescent wave excitation fluorescence scanner. After sufficiently reacting the probe sample solution with the immobilized molecules on the substrate, It is also possible to remove the light absorbing member and observe with a confocal fluorescence observation scanner.

[0021] Further, the upper modified member and the lower modifying member may be provided so as to overlap the light absorbing member, thereby improving the strength and the convenience of operation during sample injection (FIG. 12). In addition, the upper protective seal can be used to protect against physical contact from the top surface of the substrate and to prevent evaporation of the probe sample solution. Protection from contact can be achieved.

In Fig. 12! /, The upper modifying member 8 · the upper protective seal 11 is arranged on the upper light absorbing member 3 and the lower modifying member 9 · the lower protective seal 12 is arranged below the lower light absorbing member 4 The force indicating the case where there is no special inconvenience even if all or part of the protective seal is not provided. However, when the upper modifying member 8 is provided on the upper light absorbing member 3 on the waveguide substrate 5 as shown in FIG. 13, the operability at the time of injecting the probe solution is greatly improved. At this time, by providing the upper protective seal 11 on the upper light absorbing member 3, the convenience of handling is improved by preventing evaporation of the probe sample solution. Bottom protection By attaching to the lower modifying member 9 or the lower light absorbing member 4, the role 12 serves to prevent contamination of the back surface of the waveguide in the reaction tank due to physical contact from the lower surface. In addition, the upper protective seal 11 and the lower protective seal 12 are made of a light-shielding material or a wavelength-selective light-transmitting material, thereby fading the photochromic molecules in the fluorescent probe solution due to prolonged exposure to light (photo bleaching). ) Can be added.

Example

 Examples of the present invention are shown below, but the present invention is not limited to these examples.

 (1) Preparation of fluorescently labeled protein probe

 Fluorescently labeled protein probes are Cy3 Mono-reactive, a fluorescent dye that has absorption maximum wavelength around 550 nm for cashier fetuin (ASF) (SIGMA) or ushi serum albumin (BSA) (SIGMA). It was prepared by fluorescent labeling using Dye (Amersham Almasia, hereinafter referred to as Cy3). The procedure is to prepare ASF and BSA in phosphate buffered saline, pH 7.4 (hereinafter PBS) to a final concentration of 1 mg / mL, and then mix 1 mL with 1.0 mg Cy 3 powder. The reaction was carried out at a certain place with timely stirring. Next, unreacted Cy3 molecules were removed by gel filtration chromatography using S-marked hadex G_25 (Amersham Pharmacia) as a carrier, and purified as a Cy3-labeled protein probe preparation.

(2) Examination of installation position and optimum dimension width of light absorbing member

 In order to design the shape of the black light absorbing member that absorbs stray light more effectively, it is necessary to know what is the best balance between the spot area and the background light reduction effect. For this study, we used a substrate with the same material and shape as the actual experimental system, and experimented under different conditions. Examples are shown below.

Black silicon rubber 20 ° light-absorbing material (thickness lmm) punched to improve adhesion surface smoothness is cut and the distance between the absorbent material ends and the slide end is kept constant at 6 mm. The width of the tank was changed to 3, 4, and 6 mm, and the reactor was coated with 3_glycidoxypropyltrimethoxysilane (Shin-Etsu Silicone, hereinafter GTMS). It was fabricated on a white glass slide substrate having a thickness of 25.4 mm (± 0.05 mm) and a thickness of 1.0 mm (± 0.05 mm). Next, add purified water with 100 ng / mL Cy3_BSA dissolved on the back of the slide substrate. A spot sample for evaluating fluorescence intensity by evanescent wave excitation was prepared by spot-drying with mL. Next, fill the reaction vessel with 100% PBS solution containing 1% BSA, and leave it in a storage container kept at a humidity of 90% or more for 25 hours at 25 ° C. Probe sample on top of slide substrate in reaction vessel A blocking operation was performed to suppress nonspecific adsorption of molecules. In each reaction vessel on this substrate, 5 mg / mL Cy3_BSA was added and allowed to stand for 1 hour.

 As a result, it was observed that the background light decreased with the same probe solution of the same volume as the width of the rubber in the incident direction of the incident light increased (Fig. 4). From this result, it was confirmed that the width of the rubber in the incident direction of the incident light is effective in reducing the background light. In addition, in this experimental result, there was no significant change in the signal luminance value of the spot in each reaction tank. Therefore, in the reaction tank where the background light decreased, the total excitation light was not lowered. It can be seen that the background fluorescence from the upper layer of the solution was selectively reduced.

 In this experiment, it was found that the background light intensity decreases as the width of the light-absorbing member in the incident direction of the incident light becomes wider (Fig. 4). Furthermore, when plotting this relationship, it was shown that there is a curvilinear relationship between the two (Figure 5). In other words, it can be seen that the background reduction effect is not improved so effectively no matter how much the width is increased after the width of the light absorbing member in the incident light incident direction exceeds 5.4 mm. Based on these results, in this experimental system, the width of the light-absorbing member in the incident light incident direction was estimated to be about 5.4 mm as the most well-balanced dimension between the spot area and the background light reduction effect.

 (3) Reduction of background light in evanescent wave excitation fluorescence observation

Conducted by white glass glass coated with 3-glycidoxypropyltrimethoxysilane (Shin-Etsu Silicone) 76.2 mm long x 25.4 mm wide (± 0.05 mm) and 1.0 mm thick (± 0.05 mm) By repeating the dispensing operation of lectins on a waveguide substrate using a non-contact spotter, a protein array was prepared in which 43 spots of 43 proteins with a center-to-center spacing of 3 spots were spotted. Here, a reaction tank forming rubber integrally formed with a light absorbing member made of black silicon rubber was adhered to form a reaction tank capable of filling eight liquids on the substrate. Next, fill the reaction vessel with 100 L of PBS solution containing 1% BSA at a time and keep the humidity at 90% or more in a storage container at 25 ° C for 1 hour, and place the probe sample on the top surface of the slide substrate in the reaction vessel. Min A blocking operation was performed to suppress nonspecific adsorption of the children.

 Add Cy3_ASF solution prepared at a concentration of 100 g / mL, which is 1000 times higher than normal use, as a fluorescently labeled protein probe to the microarray in the reaction vessel prepared through the above steps, and shield it from light. It was allowed to stand in an incubator set at 20 ° C for 3 hours to react with the protein immobilized on the array. The slide substrate was observed under four different conditions to verify the effect of the stray light suppression method. When the light absorbing member was attached to the slide substrate, an experiment was conducted to compare the difference in background light between the case where only the upper surface was attached and the case where the light absorbing member was attached to the upper and lower surfaces. Using the slide after probing the entire reaction vessel with a 100 ng / mL Cy3_ASF probe, the probe solution in the reaction vessel was replaced with a Cy3_glycine solution, and then scanned. For scanning, an evanescent wave excitation type microarray scanner (GTMAS Scan IV) was used, and evanescent wave excitation fluorescence observation was performed. The scanning parameters were set to Gain “4000 times”, integration number “4 times”, and exposure time “34 msec”. Array-Pro Analyzer (version 4.0 for Windows Media and Ybernetics, Inc.), a commercially available microprecise angle detector for microarrays, was used to quantify the brightness of the scanned image.

 As a result, when the intensity of the background light of the upper layer of the probe solution was observed with the absorbent material attached only on the upper surface and the upper and lower surfaces, the light absorbing member was attached to the upper and lower surfaces of the slide substrate. Compared to the method of attaching only the upper surface to the method, the effect of greatly reducing the fluorescence derived from the upper layer of the probe solution was higher and the result was more effective for background light removal (Fig. 10).

 (4) Observing the effect of the combined use of the stray light suppression method and the liquid layer thickness control method

In contrast to the system in which the background light derived from the probe solution upper layer is suppressed by using the light absorbing member, a borosilicate glass plate is set in the reaction vessel as the liquid layer thickness control member, so that The background light intensity of the upper layer of the probe solution was observed when the liquid layer thickness control method (Japanese Patent Application No. 2006-203257), which is a background light reduction method, was applied. Remove the probe solution from the substrate probed with 100 ng / mL Cy3_ASF from the reaction vessel and deliberately introduce a new high-concentration fluorescent-labeled glycoprotein probe solution (lO ^ g / mL Cy3_BSA). Was injected into a 100 11 L reaction vessel and observed with an evanescent wave excitation fluorescence scanner. Result, stray light For the system in which the background light derived from the upper layer of the probe solution was suppressed by the suppression method, a further background light attenuation effect was observed by controlling the liquid layer thickness using a liquid layer thickness controller. From the results of this experiment, it was found that the absorbent layer can be used together with the liquid layer thickness control device by attaching the absorbent to the lower surface of the substrate, and both methods enhance the effect of attenuating background fluorescence derived from the upper layer of the probe solution. (Figure 11).

Claims

The scope of the claims  [1] After introducing a probe solution containing a fluorescently labeled probe molecule into a reaction vessel having a waveguide substrate having a test molecule immobilized on the bottom, and binding the immobilized molecule and the probe molecule, the waveguide When performing evanescent wave excitation fluorescence observation by introducing light into a substrate, it is a method for suppressing background light in the evanescent wave excitation fluorescence observation by suppressing the incidence of stray light to the reaction vessel. The background light reduction method described above, wherein a stray light absorption region having a width of at least 3 mm or more in the direction of incident light is provided on the upper surface or the lower surface of the waveguide substrate between the end and the test molecule fixing region. [2] The stray light absorption region is provided on both upper and lower sides with the waveguide substrate interposed therebetween. The background light reducing method according to claim 1. [3] The background light reduction method according to claim 1 or 2, wherein the stray light absorption region is formed by adhesion of a light absorption member. [4] The background light reduction method according to any one of [1] to [3] above, wherein the incident light incident end face of the waveguide substrate is laser-cut! /. [5] The background light reducing method according to any one of [1] to [4] above, wherein a reaction tank is formed by the light absorbing member. [6] A waveguide substrate used for evanescent wave excitation fluorescence observation having a reaction vessel with a test molecule fixed at the bottom, and an upper surface of the waveguide substrate between the incident light incident end and the test molecule fixing region Alternatively, the waveguide substrate according to claim 1, wherein a stray light absorption region having a width of at least 3 mm in the incident direction is provided on the lower surface. [7] The stray light absorption region is provided on both upper and lower sides with the waveguide substrate interposed therebetween. The waveguide substrate according to claim 6. 8. The waveguide substrate according to claim 6, wherein the stray light absorption band is formed by adhesion of a light absorbing member. [9] The waveguide substrate according to any one of [6] to [8], wherein an incident light incident end face of the waveguide substrate is laser-cut! /. [10] The waveguide substrate according to any one of [6] to [9], wherein the reaction vessel is formed of a light absorbing member. An evanescent wave excited fluorescence characterized by a waveguide substrate used for observation of evanescent wave excited fluorescence having a reaction vessel with a test molecule fixed at the bottom, wherein the incident light incident end face is laser-cut and processed. A waveguide substrate used for observation.