JP6533603B2 - Laser beam irradiation method - Google Patents

Description

  The present invention relates to a microchip for highly sensitively detecting fluorescent substances present in a plurality of channels provided in the interior, a method of manufacturing the same, and a multichannel fluorescence detection apparatus for analyzing a sample using the microchip.

  Research and development of microchips, which constitute channels of micrometre size channels and reaction vessels on the chip and analyze samples such as biological substances, have been vigorously carried out for the past 20 years, and their commercialization is in progress . The microchip is often made of transparent glass or resin as a member, and the size varies from several mm to several tens cm, and the thickness is smaller than the above size. With a microchip, it is possible to conveniently analyze a small amount of sample in a short time on the spot. Examples of microchips that have already been put to practical use include PCR, real-time PCR, digital PCR, electrophoresis analysis, immunoassay (immunoassay), flow cytometer (cell sorter), single cell analysis, microreactor, and so on. A microchip that integrates the steps of sample introduction, extraction, mixing with reagents, and analysis, including reactions, is called a micro TAS (Total Analysis System) or Lab on a Chip, and has various problems for practical use. R & D to be solved continues to be popular. As a measuring means of a microchip, optical measurement which can measure the substance which exists in the inside of a channel non-contact in many cases is used. For example, after labeling a fluorescent substance on a biological substance in a channel and removing an unlabeled fluorescent substance, the emitted fluorescence is measured by irradiating a laser beam. Alternatively, the biological material is observed with an optical microscope, and the shape and number thereof are measured, etc.

  Resin-made microchips can be manufactured by processing techniques such as injection molding and nanoimprinting, and can be mass-produced at low cost, so they can be disposable. Such disposable microchips are particularly important in fields such as medical diagnostics and food testing where it is strongly required to avoid contamination other than the sample to be analyzed. Also, configuring a large number of channels on a single chip and measuring them in parallel is used to analyze multiple items of one sample in parallel, or to measure multiple samples in parallel, and measure them using these. It is important to improve the throughput of and reduce the analysis cost per analysis. Alternatively, by measuring multiple points of a single channel in parallel, it is possible to analyze time-series changes in reaction and separation.

  Here, how to efficiently perform laser irradiation fluorescence detection on a plurality of channels provided in a microchip is a major problem, and the conventional method is classified into the following (1) to (5). In either method, laser irradiation parts of a plurality of channels are arranged in parallel on the same plane in the chip. Hereinafter, this plane is called an array plane. When multiple points of a single channel are measured in parallel, the points to be measured of the channels are arranged in parallel on the same plane in the chip by turning back the channels multiple times.

(1) Beam expanding method: A laser beam is expanded and irradiated simultaneously so as to straddle multiple channels, and fluorescence from multiple channels is simultaneously detected. When a laser beam is expanded in a line shape and plural channels are irradiated simultaneously. In some cases, the laser beam is expanded in a circle to simultaneously irradiate a plurality of channels. Compared with the case where the laser beam is focused to a single channel and irradiated, when the N channels are simultaneously irradiated, the laser beam intensity density is expanded in a circle like (1 / N) or less when expanded in a line shape Then it decreases to (1 / N ² ) or less. Therefore, the fluorescence detection sensitivity of each channel is reduced. As one form of the beam expansion method, the case where the laser beam is divided into a plurality of beams and each of them is irradiated to each channel can be considered, and has the same problem as described above.

(2) Scanning method: A system for detecting the fluorescence from the same channel by irradiating a single channel with a laser beam is narrowed and irradiated to a plurality of channels by irradiating the laser beam to a single channel for scanning When the N channels are serially irradiated by scanning, the effective density of the laser beam intensity is reduced to (1 / N) or less, and the fluorescence detection sensitivity of each channel is reduced, as compared with the case where no light is detected. In addition, the time resolution of each channel is also less than (1 / N), which may be disadvantageous in measurement. Furthermore, since the scanning mechanism is required, the size of the apparatus is increased, the cost is increased, and the number of failures is increased.

(3) Independent irradiation detection system method: A system for detecting fluorescence from the same channel by narrowing and irradiating a laser beam to a single channel is installed in the same number for a plurality of channels Optimal laser and detection for each channel If one can use the instrument, high fluorescence detection sensitivity can be obtained in any of the channels, but in that case the cost of the instrument becomes very high. On the other hand, since a plurality of channels which can be laid out on the same chip must be close to each other, it is physically difficult to provide a highly sensitive laser-irradiated fluorescence detection system for each channel. Therefore, it is necessary to adopt a compact and low-cost laser-irradiated fluorescence detection system which is relatively insensitive.

(4) Optical waveguide method: A plurality of channels are irradiated with an evanescent wave through a laser beam to an optical waveguide adjacent to a plurality of channels, and fluorescence from multiple channels is simultaneously detected. The evanescent wave can make the laser beam irradiation volume very small. By reducing the background light derived from the solution in the channel, it is advantageous, for example, in the case of sensitively detecting fluorescence derived from a single fluorescent molecule. However, the target substance to be detected by the microchip is often a large number of molecules rather than such a small number of molecules. In such a case, if the laser beam irradiation volume is too small, the sensitivity will be reduced.

(5) Transverse incidence method: A laser beam is emitted across multiple channels along the array plane from the side of the chip plane, and fluorescence from multiple channels is detected simultaneously from the direction perpendicular to the array plane in the simplest configuration. Although the highest sensitivity can be expected, it is difficult to efficiently irradiate a plurality of channels because the laser beam is refracted at the interface of each channel. When the laser beam width is expanded to be larger than the channel width, the laser beam intensity density decreases and the fluorescence detection sensitivity decreases. In Patent Document 1, a refracted laser beam can be condensed by inserting a lens or a mirror between the channels, and a plurality of channels can be penetrated while the laser beam is narrowed, which is highly sensitive. Fluorescence detection is possible. On the other hand, Patent Document 2 shows a lateral incidence method in the case where not a plurality of channels on a microchip but a plurality of capillaries are arranged on the same plane. By inserting a rod lens between a plurality of capillaries, it is possible to condense a refracted laser beam, to penetrate a plurality of capillaries while the laser beam is narrowed, and to detect fluorescence with high sensitivity. It is possible.

JP 2011-59095 A Unexamined-Japanese-Patent No. 9-288088 gazette

  With the laser beam narrowed, for example, with the laser beam width narrowed to less than or equal to the channel width, along the array plane in which the major axes of the plurality of channels are arranged in parallel, the major axis of each channel The lateral incidence method of vertically introducing and penetrating a plurality of channels and irradiating a laser beam simultaneously is the most efficient laser irradiation fluorescence detection method of a plurality of channels, and is a method which enables the highest sensitivity. Here, the central axis of the laser beam in the case where the introduced laser beam travels straight through the plurality of channels without being refracted will be hereinafter referred to as a horizontal incident axis. Compared with other conventional methods, the lateral incidence method has extremely high utilization efficiency of the laser beam, the ratio of the laser beam directly or indirectly entering the detector due to reflection, etc., and the micro It has the feature that Rayleigh scattering, Raman scattering, fluorescence, etc. emitted from the member of the tip by laser beam irradiation have a very small proportion of the fluorescence emitted from the channel being mixed with the fluorescence to be measured. Both contribute to the realization of highly sensitive fluorescence detection with a simple configuration.

  In Patent Document 1, a lens or a mirror is inserted between the channels, and a laser beam which is refracted and deviates from the transverse incident axis when passing through the channel is condensed, and the laser beam is returned to the transverse incident axis. The lateral incidence method is realized by passing this channel and repeating this. However, it is difficult to actually arrange a lens or the like between the channels. First, it is necessary to form a space for inserting a lens or the like into the microchip. For example, after manufacturing a microchip having a plurality of channels, it is necessary to cut a hole of a size that can penetrate the microchip and accommodate a lens or the like. Next, it is necessary to insert a lens or the like into this space and fix the lens or the like in a state in which the optical central axis of the lens or the like coincides with the transverse incident axis. Here, the central axis of an optical system such as a lens must be aligned with micrometer accuracy with respect to both the long axis direction of the channels and the direction perpendicular to the array plane of the channels. In addition, such high-precision alignment must be performed for all of a plurality of lenses or the like disposed between the channels. Since it is extremely difficult to obtain such positional accuracy only with the mechanical accuracy when inserting a lens or the like into the hole provided in the microchip, for example, after inserting each lens or the like between the channels, It is necessary to adjust the position finely and fix it. If the central axis of the lens or the like deviates from the horizontal incident axis, the laser beam is deflected from the horizontal incident axis, and simultaneous irradiation of a plurality of channels becomes impossible. The above alignment takes time and effort, and additionally requires a mechanism for fine adjustment of alignment, leading to a rise in the manufacturing cost of the microchip. This is particularly disadvantageous in the case of disposable use of the microchip.

  Further, in Patent Document 1, since the positions of the respective lenses are fixed to the microchip while the central axis of the introduced laser beam and the central axes of the respective lenses coincide with each other, the central axis of the introduced laser beam It can not move freely in the longitudinal direction of each channel. If the laser beam is deviated from the central axis of the lens or the like, the laser beam is deflected from the transverse incident axis. This means that, for example, the horizontal incident axis can be set to avoid the position where flaws and dust are present in the channel, and adjustment can not be performed so as to maximize detection sensitivity. Similarly, the laser beam is expanded only in the major axis direction of each channel and laterally incident, or a plurality of transverse incident axes shifted in position in the major axis direction of each channel are set, and a plurality of different lasers are respectively set It can not irradiate the beam. These mean that the behavior of the fluorescent substance present in each channel can not be captured in a two-dimensional image, and fluorescence emissions of different types of fluorescent substances can not be detected independently and with high sensitivity.

  Furthermore, in Patent Document 1, in order to insert a lens or the like between channels and align it with high accuracy, it is necessary to take a large distance between adjacent channels, and it is necessary to provide them in a single microchip There is a problem that the number of channels that can be made is smaller than that of the conventional (1) beam expansion method and (2) scan method.

  On the other hand, in Patent Document 2, a plurality of glass capillaries and a plurality of glass rod lenses are alternately arranged in the same plane in water, and the laser beam is parallel to the arrangement plane and with respect to the major axis of each capillary. Is introduced vertically to realize the transverse incidence method. The capillary in water suppresses the reflection of the laser beam on the outer surface compared to the air, but acts as a concave lens so that the laser beam tries to diverge from the transverse incident axis. However, the rod lenses arranged next to each other act as convex lenses even in water, so they refocus the diverging laser beam. This repetition enables simultaneous laser beam irradiation and high sensitivity fluorescence detection of a plurality of capillaries.

  In this method, since the outer diameters of the capillary and the rod lens can be made uniform, for example, by sandwiching the alternately arranged capillary and the rod lens with two flat plates, the central axes of each capillary and each rod lens can be relatively compared. It is possible to easily arrange at high density in parallel and on the same plane. This means that the transverse incident system can be simply realized by making the transverse incident axis coincide with the central axis of the rod lens, and simultaneous detection of a large number of capillaries becomes possible. In addition, since the rod lens has the same optical characteristics with respect to its axial direction, even if the irradiation position of the laser beam is shifted in the axial direction of the capillary and rod lens, the horizontal incident axis is similarly shifted. realizable. In other words, set the horizontal incident axis that maximizes sensitivity, expand the laser beam only in the axial direction of the capillary to capture a two-dimensional fluorescence image, or shift different laser beams in the axial direction of the capillary It is possible to independently measure emission fluorescence of different fluorescent substances at a plurality of transverse incident axes.

  However, this method is possible because capillaries and rod lenses of the same outer diameter are used, and when the capillaries are replaced with channels provided in the microtip, the central axes of the rod lenses coincide with the plane of arrangement of the channels It becomes very difficult to arrange the rod lenses in the same way as the arrangement of lenses etc. in Patent Document 1. It takes much time and effort for preparation, it can not be avoided that it becomes a very expensive microchip, and it can not be used disposable.

  In the present invention, a laser beam which solves the above-mentioned problems of the conventional method of the lateral incidence method and which has a simple configuration but narrows the same degree to the channel width to a plurality of channels provided in a single microchip We present a method to detect fluorescence with high sensitivity by irradiating simultaneously with the lateral incidence method. At the same time, the irradiation position of the laser beam and the transverse incident axis can be moved in the long axis direction of each channel, and the laser beam can be expanded and irradiated in the long axis direction of each channel. The method which enables also making it possible to shift and irradiate a horizontal incident axis to an axial direction is shown. As a microchip member, not only glass but also resin with low unit price is targeted. The manufacturing methods for microchips are not only methods that require time and cost, such as cutting, optical shaping, and semiconductor processing, but also methods that are excellent in low cost and mass productivity, such as injection molding and nanoimprinting. For example, although injection molding using a resin material is excellent in low cost and mass productivity, a method for realizing a lateral incidence method is presented using a microchip that can be used in such a disposable manner.

Microchip according to the present invention, a plurality of channels are provided in the transparent solid member having a refractive index n _1, a plurality of channels long axis of each channel are arranged parallel to each other in the same plane at least part of the region, the plurality of channels, a first channel the first member of the refractive index n ₂ in the interior filled, a second channel second member having a refractive index n ₃ is satisfied are mixed, n _It satisfies the relationship ₂ <n ₁ <n ₃ At least a part of the area includes an area where the measurement laser beam is incident.

  As an example, the first channel and the second channel are alternately arranged in the arrangement direction of the plurality of channels.

  The second member may be a liquid, in which case the second channel is preferably sealed to prevent the second member from coming off.

  As one example, the plurality of channels have a circular cross-sectional shape perpendicular to the major axis in at least a part of the area.

  As one example, the plurality of channels have a trapezoidal cross-sectional shape perpendicular to the long axis in at least a part of the area.

Multichannel fluorescence detection apparatus according to the present invention, a microchip long axis of each channel are arranged parallel to one another in the same plane at least part of the region is a plurality of channels in the interior of the transparent solid member having a refractive index n _1, A laser light source, an irradiation optical system for causing laser light generated from the laser light source to be perpendicularly incident on long axes of a plurality of channels arranged parallel to one another along the same plane from the side surface of the microchip; And each of the plurality of channels of the microchip is filled with a member of refractive index n ₂ to be detected. A first channel containing a phosphor and a second channel filled with a second member of refractive index n ₃ are mixed, and n ₂ <n ₁ <n _It satisfies the relationship of ₃ .

  A plurality of laser beams may be provided, and the plurality of laser beams may be incident on different positions in the long axis direction of the plurality of channels.

  As an example, the fluorescent substance to be detected is a fluorescent substance labeled to a sample derived from a living body, and fluorescence emitted from a plurality of first channels upon irradiation of a laser beam is simultaneously detected from the direction perpendicular to the same plane Configure to

Further, the method of manufacturing a microchip according to the present invention is a method of manufacturing a microchip in which a plurality of channels are arranged in the same plane parallel to each other in at least a partial region of the transparent solid member. Preparation Te, by injection molding, the first plate-like transparent solid member having a refractive index n ₁ which is formed so as to be parallel to each other a plurality of grooves in said at least part of the region which is a trapezoidal cross-sectional shape on the surface A second plate-like transparent solid member having a refractive index n1 laminated on a _first plate-like transparent solid member to form a plurality of channels by a plurality of grooves, and a plurality of channels a out and filling a predetermined member having a refractive index n ₃ a plurality of channels, the refractive index n ₁ and n ₃ is characterized by satisfying the relation of n ₁ <n _3.

As an example, members of the refractive index n ₃ is a liquid, after filling the member of the refractive index n _3, a step of sealing the channel filled with the member.

Also has a step of filling a medium for electrophoresis of the refractive index n ₂ in the channels other than the channel filled with members of the refractive index n _3, the refractive index n _2, the relation of n ₂ <n ₁ <n ₃ May be satisfied.

  According to the present invention, a laser beam can be efficiently simultaneously irradiated to a plurality of channels provided in a single microchip by the lateral incidence method. Thus, a system that achieves high-sensitivity fluorescence detection of a plurality of channels by exciting the fluorescent substance present inside each channel and collectively measuring the emitted fluorescence from the direction perpendicular to the array plane of each channel. Can be configured. The microchip used in this case can be manufactured inexpensively by a mass-producible processing method such as injection molding, and it is also possible to make the microchip disposable. In addition, the optical system used for detection can be simplified, and the entire system can be compact and inexpensive.

  Problems, configurations, and effects other than those described above will be clarified by the description of the embodiments below.

The figure which shows the structural example of the microchip containing several channel with a circular cross section. The figure which shows the structural example of the microchip containing several channel whose cross section is a square. The figure which shows the definition of the refraction angle of the laser beam which injects into a triangular prism. FIG. 7 is a process diagram showing a manufacturing process of a microchip. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic view showing an example of a multi-channel fluorescence detection apparatus according to the present invention. Schematic cross-sectional view showing the ends sealing method of a channel filled with members m _3. Schematic which shows the structural example of a microchip. The figure which shows the ray-tracing simulation result of microchip structure a and a transverse incident laser beam. The figure which shows the microchip structure b and the ray tracing simulation result of a transverse incident laser beam. The figure which shows the microchip structure c and the ray tracing simulation result of a transverse incident laser beam. The figure which shows the microchip structure d and the ray tracing simulation result of a transverse incident laser beam. The figure which shows the microchip structure e and the ray tracing simulation result of a transverse incident laser beam. The figure which shows the microchip structure f and the ray tracing simulation result of a transverse incident laser beam. The figure which shows the ray-tracing simulation result of microchip structure f 'and transversal incidence laser beam. The figure which shows the microchip structure g and the ray tracing simulation result of a transverse incident laser beam. The figure which shows the microchip structure g 'and the ray tracing simulation result of a transverse incident laser beam. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic view showing an example of a multi-channel fluorescence detection apparatus according to the present invention. The figure which shows the ray-tracing simulation result of microchip structure h and a transverse incident laser beam. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic view showing an example of a multi-channel fluorescence detection apparatus according to the present invention. FIG. 17 shows ray tracing simulation results for a laterally incident laser beam of microchip configuration h ′.

  1 and 2 are schematic cross-sectional views showing a basic configuration example of the microchip of the present invention. FIG. 1 shows an example of the configuration of a microchip including a plurality of channels having a circular cross section, and FIG. 2 shows an example of the configuration of a microchip including a plurality of channels having a square cross section.

  These figures are cross-sectional views perpendicular to the long axes of the plurality of channels, including the lateral incidence axis of the microchip. That is, at a position where a plurality of channels are arranged in parallel and on the same plane with the long axes of the channels inside the microchip, the cross section is perpendicular to the long axes of the channels and along the array plane of each channel The cross section includes the central axis of the laser beam emitted perpendicularly to the long axis of each channel. It goes without saying that the configurations shown in these figures are merely representative examples representing the basic idea of the present invention, and other configurations based on the same idea are also the subject of the present invention.

Structure a in FIG. 1, the microchip 1 is constituted by a member m ₁ of refractive index n _1, the cross-section of each channel 2 is circular with the same shape, each channel 2 are arranged at equal pitches, each channel 2 is filled with members m ₂ of refractive index n _2. Member m ₂ were medium existing the target substance to be analyzed in the microchip, an analysis of the target substance using the channel 2 of member m ₂ is filled. Here, the member m ₁ is a transparent solid member such as glass or resin, while the member m ₂ is a liquid or gel-like material such as an aqueous solution, and in many cases n ₂ <n ₁ . At this time, each channel 2 functions as a concave lens with respect to the introduced laser beam 4 and the laser beam 4 diverges from the transverse incident axis as it travels along the transverse incident axis. I can not irradiate well. Conversely, it is also possible to select a resin material with a low refractive index such that n ₁ <n ₂ , in which case each channel 2 functions as a convex lens, so that the laser beam 4 is collected horizontally A plurality of channels 2 can be efficiently irradiated while traveling along the incident axis. For example, the the use of fluorine-based resin as a member m ₁ by reducing the n _1, can be n ₁ <n _2. Hereinafter, the configuration example of the present invention will be described for the case of more general n ₂ <n ₁ .

Structure b in FIG. 1 are filled with members m ₃ of refractive index n ₃ a plurality of channels every one in the configuration a. Here, by setting n ₂ <n ₁ <n ₃ , for the laser beam 4, the channel 2 filled with the member m ₂ functions as a concave lens, and the channel 3 filled with the member m ₃ functions as a convex lens. Member m ₃ satisfies the refractive index conditions, a transparent, member absorption of the laser beam 4 is preferably small. Member m ₃ may be a liquid, it is possible to easily fill after microchip having a plurality of channels are prepared. As a result, the convex lens condenses the portion where the laser beam 4 diverges from the horizontal incident axis by the concave lens, returns it to the horizontal incident axis, and it is possible to efficiently irradiate the plurality of channels 2 by repeating this. Here, the cross sections of the channel 2 and the channel 3 are circular with the same shape, and have the same outer diameter.

This configuration b has the following differences and advantages as compared to the conventional method. In Patent Document 1, it was considered difficult to insert a lens or the like between channels and to make the central axis of the lens or the like coincide with the horizontal incident axis with an accuracy of micrometer level. In the present invention, some channels are lensed by filling a member m ₃ of refractive index n ₃ satisfying n ₂ <n ₁ <n ₃ , which is usually not selected, by sacrificing part of a plurality of channels. It is converted to Since the plurality of channels 2 and channels 3 are manufactured together by the same processing process, the respective channels should be arranged on the same plane inside the microchip 1, that is, the central axis of each channel is aligned with the transverse incident axis It is easy to do so and there is no concern that the relative position between the channels changes, and it is possible to efficiently irradiate a plurality of channels simultaneously by the lateral incidence method. In addition, since the channel 3 having a convex lens function manufactured in this manner is optically equivalent in the long axis direction, for example, the different laser beams are positioned in the long axis direction of the channel 2 and the channel 3 It is also possible to illuminate simultaneously for a plurality of offset transverse axes.

Patent Document 2 has a surface similar to the configuration b in that the concave lens function of a plurality of capillaries is offset by the convex lens function of a plurality of capillaries instead of a plurality of channels provided in a microchip. The means based on the optical property conditions for realizing the method are different. The capillary is a cylindrical tube made of quartz glass having a refractive index n ₃ = 1.46, and the inside is filled with, for example, an electrophoretic separation medium having a refractive index n ₂ = 1.38. The rod lens is made of quartz glass having a refractive index n ₃ = 1.46. On the other hand, the periphery of the plane in which the plurality of capillaries and rod lenses are arrayed is water having a refractive index n ₁ = 1.33. The reason why the surrounding area is not air but water is to reduce the reflection loss of the laser beam on the outer surface of the capillary and to improve the utilization efficiency of the laser beam. However, if the refractive index of the surrounding area is further increased to further reduce the reflection loss, the concave lens action of the capillary is enhanced, and the convex lens action of the rod lens can not suppress the divergence of the laser beam from the transverse incident axis. Can not be irradiated simultaneously. That is, in Patent Document 2, it is inevitable that the optical characteristic condition of n ₁ <n ₂ <n ₃ is satisfied, and the most effective effect is exhibited when this condition is satisfied. Whereas the present invention, under conditions that higher refractive index n ₁ external than the refractive index n ₂ of the internal channel 2, than the refractive index n _2, a higher refractive index than n ₁ n channel 3 filled with ₃ parts m _3, a structure to mix the channel 2 filled with members m ₂ of refractive index _{n 2, n 2 <n 1} < be optical characteristics satisfying the n ₃ Inevitably, it is most effective when this condition is met. This configuration is not assumed in Patent Document 2, and in fact, it is difficult to configure using a capillary and a rod lens.

  In configuration b, since the outer diameters of the cross sections of channel 2 and channel 3 are equal, when the concave lens function of channel 2 is large, laser beam 4 diverges more than the cross sectional outline of channel 3 and enters adjacent channel 3 The portion which was not present continues to diverge because it is not subjected to the convex lens action by the channel 3. In this case, the intensity of the laser beam 4 is attenuated as it travels in the transverse incident axis, making it difficult to efficiently irradiate the plurality of channels 2 simultaneously.

Therefore, the structure c in FIG. 1, unlike the configuration b, than the outer diameter r ₂ of the cross section of the channel 2 which member m ₂ is filled, the cross-section of the channel 3 where the member m ₃ is filled in the outer diameter r ₃ Is larger (r ₂ <r ₃ ). This makes it possible to reduce the ratio of the portion where the diverging laser beam 4 does not enter the adjacent channel 3 due to the concave lens action of the channel 2 and allows simultaneous laser beam irradiation of a plurality of channels 2 more efficiently than the configuration b. It becomes.

Configuration d in FIG. 1 changes the cross-sectional shape of the channel 2 in configuration c from circular to square. As a result, the concave lens action or refraction action of the laser beam 4 of the channel 2 is reduced, so that the ratio of the portion where the laser beam 4 diverged by the concave lens action or refraction action of the channel 2 does not enter the adjacent channel 3 is further reduced. This enables simultaneous laser beam irradiation of a plurality of channels 2 more efficiently than configuration c. Like the configuration c, by increasing the diameter r ₃ of the channel 3 than the diameter r ₂ of the cross section of the channel 2, it is possible to realize a lateral incidence method of more efficiently laser beam.

  As mentioned above, although the cross-sectional shape of the channel 2 and the channel 3 is made circular or a square in the structure shown in FIG. 1, of course, the same effect is acquired also with other shapes. For example, instead of a circle, it may be an ellipse, a semicircle or a part of an ellipse, and instead of a square, it may be a rectangle. Also, although the channels are arranged at equal intervals, the same effect can be obtained even if the channels are not arranged at equal intervals. Furthermore, in configurations b to d, although channel 2 and channel 3 are alternately arranged, it is not necessary to do so, and at least one or more channel 2 and channel 3 are arranged on the same microchip It is necessary to

  Although FIG. 1 deals with channels 2 and 3 having a circular cross-sectional shape, it is difficult to manufacture a microchip having a plurality of such channels at low cost and with high accuracy. For example, if a processing method such as stereolithography is used, the configuration shown in FIG. 1 can be manufactured in a single process, and the cross-sectional shape of the channel can be made circular, but such a process requires processing time As a result, mass productivity tends to be low and manufacturing costs tend to be high.

On the other hand, in the case of using a manufacturing method with high mass productivity such as injection molding, in the configuration of FIG. 1, the microchip 1 is a boundary plane including the central axes of the channels 2 and 3 (not shown in FIG. 1) It can be manufactured by assembling the two upper and lower parts and bonding these two parts together at the interface. Here, a plurality of grooves each having a semicircular cross-sectional shape are formed on the interface between the two parts. The reason why the above processing method is adopted is that, in injection molding, since a process of removing the mold is necessary after pouring and solidifying a member m ₁ such as resin in the mold, the cross-sectional shape of the groove that can be formed is the bottom of the groove It is necessary for the width to expand from the center to the boundary surface. Here, the relative positional accuracy when bonding the two parts of the microchip 1 becomes an issue. When the bonding positions of the two parts shift in the transverse incident axis direction, the cross-sectional shapes formed in the two parts are joined with the relative positions of the semicircular grooves shifted, and the cross-sectional shape of the resulting channel is circular It is because it is not. Of course, if the mutual position is adjusted with high positional accuracy, it is possible to make the cross-sectional shape circular, but it becomes a factor to increase processing time and manufacturing cost.

  In the configuration example shown in FIG. 2, the cross-sectional shape of each of the channels 2 and 3 is a quadrangle to cope with the above problem. In the configuration shown in FIG. 2, the microchip 1 is composed of two upper and lower parts, with the plane including the upper end face of each channel 2 and 3 as the boundary surface 5 (indicated by a dotted line in FIG. 2) It can manufacture by pasting together at the said interface and integrating. Bonding of the upper and lower parts is carried out by a method such as thermocompression bonding, and the interface 5 is made optically transparent so as not to include air or adhesive layers. Here, a plurality of grooves having a rectangular cross section are provided in the boundary surface 5 of the lower part of the boundary surface 5, and the boundary surface of the upper part of the boundary surface 5 is a plane in which no groove is provided. Therefore, the shapes and positions of the channels 2 and 3 are not affected even if the pasting positions of the upper and lower parts shift. Therefore, the microchip 1 shown in FIG. 2 can be manufactured by a processing method which is excellent in mass productivity such as injection molding and a simple laminating process which does not require positional accuracy.

In the configuration e of FIG. 2, the cross-sectional shape of each channel 2 provided in the microchip 1 made of the member m ₁ is a square having the same shape. Member m ₂ is filled in the channel 2. Here, in FIG. 2, the upper and lower surfaces of the square are parallel to the boundary surface 5 and the transverse incident axis, and the two side surfaces of the square are perpendicular to these. At this time, since the laser beam 4 is always incident perpendicularly to these side surfaces for each channel 2, it is expected that the laser beam 4 does not receive any refraction in principle and travel straight along the transverse incident axis. That is, the configuration e can simultaneously irradiate the plurality of channels 2 very efficiently by the laser beam 4 and can detect the fluorescent substance present inside each channel 2 with high sensitivity.

However, making the cross-sectional shape of each channel 2 a square as in the configuration e involves difficulties in manufacturing by a processing method such as injection molding. Because, as described above, in the injection molding, since the mold requires a process to extract the mold after solidifying by pouring member m ₁ such as a resin, the cross-sectional shape of the formed can groove is directed from the bottom of the groove at the interface It is necessary to expand the width according to If the cross-sectional shape is a square, the width at the bottom of the groove and the width at the interface are equal, and if the mold is not removed carefully, the mold or microchip may be damaged.

Therefore, in configuration f of Fig. 2, the cross-sectional shape of each channel 2 provided in the microchip 1 consisting of members m ₁ was isosceles trapezoid. The upper base of the isosceles trapezoid is a part of the boundary surface 5 and the lower base is a bottom of a groove provided in the component below the boundary surface 5 of the microchip, and the width of the upper base> the width of the lower base. With such a shape, it is possible to facilitate the process of removing the mold in the processing method such as injection molding and to increase the mass productivity. Of the base angles of the isosceles trapezoid, the part exceeding 90 degrees is called the draft. That is, when the draft angle is D degrees, the base angle of the isosceles trapezoid is 90 + D degrees. The draft angle D is 0 degrees <D <90 degrees, and the larger the value of D, the easier the process of removing the mold. However, it is desirable that the cross-sectional shape of each channel be uniform. It is desirable to set D> 2 degrees in consideration of processing accuracy.

However, when the laser beam 4 is laterally incident on the microchip 1 of the configuration f, the laser beam 4 passes from each channel 2 to the side opposite to the boundary surface 5 from the transverse incident axis, in FIG. It deflects downward from the horizontal incident axis. This phenomenon can be explained as follows. Although isosceles trapezoid is a cross-sectional shape of each channel 2 can be regarded as a part of a cross section of an isosceles triangle of the prism, the refractive index of the surrounding of the prism, i.e. than the refractive index n ₁ of the member m ₁ microchip 1 Also, since the refractive index of the prism, ie, the refractive index n ₂ of the member m ₂ filled in each channel 2 is smaller, the laser beam 4 incident on the prism is the apex on the opposite side to the base of the isosceles triangle. It refracts to the side of the corner.

FIG. 3 schematically illustrates this phenomenon in an easily understandable manner. Some members m ₁ refractive index n _1, consists member m ₂ of refractive index n _2, cross-section is triangular prism apex angle A, horizontally base, it is located in a vertical angle downward. As defined in FIG. 3, the incident angle on the incident surface is α, the refraction angle is β, and the incident angle on the emission surface when a laser beam of virtually zero width is horizontally incident on this prism. the gamma, the refraction angle [delta], the angle of refraction net when the laser beam passes through the prism and epsilon _2. alpha, beta, gamma, [delta] is a positive value between 0 and 90 degrees either but, epsilon ₂ is -90 degrees <epsilon ₂ <90 degrees, code, so that the laser beam of FIG. 3 The case of refraction to the base side is positive, and the case of refraction to the apex side as in the configuration f is negative. ε ₂ is the angle of refraction that the laser beam 4 receives as it passes through the channel 2 in FIG. Here, according to Snell's law and geometrical relations, the following holds.

n ₁ * sin α = n ₂ * sin β (1)
n ₂ * sin γ = n ₁ * sin δ (2)
γ = A−β (3)
ε ₂ = α + δ−A (4)
Also, assuming that the draft of injection molding is D,
A = 2 * D (5)
Because the incident laser beam and the base are parallel,
α = D (6)
Is true. From the above,
ε ₂ = sin ⁻¹ [sin {2 * D-sin ⁻¹ (sin D * n ₁ / n ₂ )} * n ₂ / n ₁ ] -D (7)
It is expressed as In the configuration f, since n ₂ <n ₁ , the refraction angle becomes ε ₂ <0, and as described above, the laser beam 4 is refracted in the apex angle side and away from the horizontal incident axis when passing through the channel 2 Do. Further, when passing through a plurality of channels 2, the above-described refraction angle is integrated, so that the laser beam 4 deviates rapidly from the transverse incident axis. Therefore, the configuration f can be said to be an inappropriate configuration for laterally irradiating a laser beam and simultaneously irradiating a plurality of channels 2.

Configuration g of 2, in order to solve the above problems of the configuration f, a plurality of channels 2 member m ₂ of refractive index n ₂ in the microchip 1 is filled consisting member m _1, the refractive index n ₃ a plurality of channels 3 which member m ₃ is filled alternately arranged, arrangement b shown in FIG. 1, c, similarly to the d, and _{n 2 <n 1 <n 3} . At this time, the refraction angle ε ₃ received when the laser beam 4 passes through the channel 3 is derived by the same derivation as the refraction angle ε _{2 received} when passing the channel 2 as described above,
ε ₃ = sin ^-1 [sin {2 * D-sin ^-1 (sin D * n ₁ / n ₃ )} * n ₃ / n ₁ ] -D (8)
It is expressed as Here, since n ₂ <n ₁ <n ₃ , ε ₃ > 0 for ε ₂ <0, and channel 3 refracts laser beam 4 in the direction opposite to channel 2, that is, channel 2 Thus, it is possible to refract the laser beam 4 refracted in the direction away from the transverse incident axis back to the transverse incident axis by the channel 3.

The balance of ε ₂ and ε ₃ is important to make the lateral incidence system work well, at least by making the absolute value of the net angle of refraction smaller by the addition of channel 3 to channel 2, ie,
| Ε ₂ + ε ₃ | <| ε ₂ | (9)
It is effective to satisfy Substituting equations (7) and (8) into equation (9),
| Sin ^-1 [sin {2 * D-sin ^-1 (sin D * n ₁ / n ₂ )} * n ₂ / n ₁ ]-D
^{+ Sin -1 [sin {2 *} D-sin -1 (sinD * n 1 / n 3)} * n 3 / n 1] -D |
<| Sin ⁻¹ [sin {2 * D-sin ⁻¹ (sin D * n ₁ / n ₂ )} * n ₂ / n ₁ ] -D | (10)
It becomes. More ideally,
| Ε ₂ + ε ₃ | ≒ 0 (11)
Is valid. Similarly, substituting equation (7) and equation (8) into equation (10),
| Sin ^-1 [sin {2 * D-sin ^-1 (sin D * n ₁ / n ₂ )} * n ₂ / n ₁ ]-D
^{+ Sin -1 [sin {2 *} D-sin -1 (sinD * n 1 / n 3)} * n 3 / n 1] -D | ≒ 0 (12)
It becomes. When these relationships are satisfied, the laser beam 4 can be made to travel along the transverse incident axis, and the channels 2 can be efficiently irradiated simultaneously by the transverse incident method.

FIG. 4 is a process diagram showing a process of manufacturing the microchip 1 having the configuration g of FIG. 2 by injection molding in a schematic cross-sectional view. Relative to the mold 100 shown in (a), a transparent resin member m ₁ in which heated and melted by injection injected as in (b), however, allowed to cool and solidify. Next, the mold 100 is removed to obtain a molded part of the transparent solid member as the part 101 having the channel of the microchip 1 as shown in (c), that is, the part 101 below the interface 5 of the configuration g. . The lower part 101 is formed by forming a plurality of grooves having a trapezoidal cross-sectional shape on the surface of a plate-like transparent solid member. The grooves are arranged parallel to one another in at least part of the area.

On the other hand, a plate-like transparent solid member is separately prepared as the component 102 not having the channel of the microchip 1, ie, the component 102 above the interface of the configuration g, and as shown in (d) At 5 and bonding together by heat welding etc., the microchip 1 shown to (e) is obtained. That is, in this process, a plurality of grooves formed on the surface of the component 101 constitute a channel of the microchip. In this state, the inside of each channel is filled with air. Finally, as shown in (f), the member m ₃ is filled inside the desired one of the plurality of channels to make the channel 3. The microchip 1 is distributed to the user, for example, in the state of (f), and the user fills the other channel with, for example, a member m ₂ consisting of a migration medium for electrophoresis before starting analysis. And make it into the state of configuration g. Of course, it may be distributed to the user after completing the configuration g in advance.

Although the case where the cross-sectional shape of the channels 2 and 3 is an isosceles trapezoid was considered above, the case of a trapezoid which is not isopod can also be considered similarly. Two base angles of the trapezoid 90 + D _L level (0 degrees <D _L <90 degrees) and 90 + D _R of (0 ° <D _R <90 degrees) when a, be and _{D = (D L + D R} ) / 2 For example, the relational expressions from the expressions (1) to (12) may be applied as they are.

The same effect can be obtained even if the cross-sectional shapes of the channels 2 and 3 are other than trapezoidal. For example, it is provided on the microchip 1 of the refractive index n ₁ even if the trapezoid which is not isopod, parallelogram, triangle, or each side is not a straight line but an arc shape or corners are rounded. a refractive action opposite the laser beam 4 with the channel 3 of the refractive action as the refractive index n ₃ of the laser beam 4 having a channel 2 having a refractive index n _2, weakening the refractive effect of the net by offsetting each other Is a feature of the present invention, and typical conditions are n ₂ <n ₁ <n ₃ . Also, although the channels are arranged at equal intervals, the same effect can be obtained even if the channels are not arranged at equal intervals. Furthermore, in configuration g, channel 2 and channel 3 are alternately arranged, but it is not necessary to configure as such, and at least one or more channel 2 and channel 3 are arranged on the same microchip Need to be For example, when the refraction of two channels 2 and the refraction of one channel 3 are well balanced, the numbers of channels 2 and 3 may be arranged in a ratio of 2: 1.

  Hereinafter, embodiments of the present invention will be described.

Example 1
FIG. 5 is a schematic explanatory view showing an example of the multi-channel fluorescence detection device according to the present invention. This example shows a system for performing electrophoresis analysis of DNA contained in a biological sample, where (a) is a bird's-eye view of the microchip 1, (b) contains the transverse incident axis of the laser beam 4 to the microchip 1 constituting the system. A cross section, a cross section of a fluorescence detection optical system, and a data analysis device are shown, and (c) shows a two-dimensional fluorescence image obtained by the two-dimensional sensor 12.

Figure 5 (a), the the microchip 1, each channel 3 is alternately each channel 2 and the members m ₃ which member m ₂ was filled is filled, parallel to each other, arranged on the same plane It is done. An inlet port 6 and an outlet port 7 are provided in each of the channels 2 respectively. Each of the channels 2 in the vicinity of the inlet port 6 is provided with a cross injection part or a T injection part for introducing a sample, but it is omitted in FIG. The DNA in the sample is previously amplified in a region of interest and labeled with a fluorophore. After sample introduction, by applying voltage to both ends of each channel 2 with the inlet port 6 as the negative electrode and the outlet port 7 as the positive electrode, the fluorescently labeled DNA contained in the sample is electrophoretically separated from the inlet port 6 to the outlet port 7 Do.

On the other hand, each channel 3 is provided with an inlet port 8 and an outlet port 103 respectively. May remain member m ₃ to be filled in each channel 3 in the case of solid opens the inlet port 8 and outlet port 103, but in the case of liquid, so as not to come out the member m ₃ from the channel 3 by evaporation and the pressure difference It is necessary to Here, after filling the member m ₃ in channel 3, sealing the inlet port 8 and an outlet port 103. FIG. 6 is a schematic cross-sectional view of the microchip 1 taken along one channel 3 of FIG. 5 (a). First, as shown in FIG. 6 (a), the inlet port 8, the channel 3, although both the outlet port 103 air is filled, a member m ₃ inside the channels 3, as shown in FIG. 6 (b) It was filled. Subsequently, as shown in FIG. 6C, a silicone rubber plug 107 was inserted into each of the inlet port 8 and the outlet port 103 so that the member m3 inside the channel ₃ would not move. Here, the same effect can be obtained by setting the inlet port 8 and the outlet port 103 with an adhesive or resin instead of the rubber plug. The internal space of the channel 3 which is sealed at both ends with a rubber stopper 107 is preferably filled with only member m ₃ of air not mixed. However, the small amount of movement of the member m ₃ if air contamination suppression can be controlled so that at least a laser beam irradiation position of the channel 3 is always filled with the member m ₃ during the measurement.

  As shown in FIG. 5 (b), the laser beam 4 emitted from the laser light source 111 is introduced from the side of the microchip 1 by the illumination optical system including the lens and the side of the microchip 1, and arranged in the portion where each channel 2 and 3 are arranged. Irradiate along planar and transverse incident axes and perpendicular to the long axis of each channel 2 and 3 and penetrate through each channel 2 and 3 simultaneously. The fluorescently labeled DNA electrophoresed in each channel 2 is excited by the laser beam and emits fluorescence when crossing the transverse incident axis. The fluorescence emitted from each channel 2 is detected by a fluorescence detection optical system. That is, the light is collimated by the common condenser lens 9, transmitted through the filter and the diffraction grating 10, and imaged on the sensor surface of the two-dimensional sensor 12 by the imaging lens 11. A filter is provided to block the wavelength of the laser beam serving as background light in fluorescence detection, and a diffraction grating is provided to perform wavelength dispersion of fluorescence to detect polychromatic light. It should be noted that since the channel 2 and the channel 3 perform the same lens action at a wide position in the long axis direction of the channel like a cylindrical lens, even if the incident position of the laser beam 4 to the microchip 1 shifts slightly, fluorescence detection There is no impact on accuracy.

  FIG. 5C is a schematic view showing a two-dimensional fluorescence image 104 obtained by the two-dimensional sensor 12. The direction of wavelength dispersion is perpendicular to the long axis direction of each channel 2 (direction perpendicular to the cross-sectional view in FIG. 5B), that is, the arrangement direction of the plurality of channels 2. The wavelength dispersive images are independently measured without overlapping each other. Here, a wavelength dispersion image 105 of laser light scattering and fluorescence which can not be removed by a filter is obtained from each channel 2, and a wavelength dispersion image 106 of laser light scattering which can not be removed by a filter is obtained from each channel 3 . The fluorescence signal measured in this manner is analyzed by the data analyzer 13 to analyze the sample introduced to each channel 2.

  FIG. 7 is a schematic view showing a configuration example of the microchip 1 different from that of FIG. 5A. Although the plurality of channels 2 and 3 are alternately arranged, they are the same, but the arrangement interval is narrowed near the laser beam irradiation position, and a common outlet port 113 exists for each channel 2 and 3 Is different. This is because the sample introduction side requires a space for forming the inlet port 6 and the sample introduction mechanism by cross injection independently for each channel 2, but the arrangement interval is narrow at the laser beam irradiation position. This is because it is advantageous to the lateral incidence method. Here, since the common outlet port 113 is downstream of the laser beam irradiation position in the electrophoresis analysis, there is no problem even if it is made common. The total length of the channel 3 is shorter than that of the channel 2 because the channel 3 only needs to be at the laser beam irradiation position.

  FIG. 5 shows a part of an embodiment common to the present invention. However, in some embodiments, channel 3 shown in FIG. 5 may not exist, in which case it is assumed that channel 3, inlet port 8 and outlet port 103 have been deleted from FIG. good. Further, the numbers and the cross-sectional shapes of the channels 2 and 3 are merely examples, and the present invention is not limited thereto.

In the present invention, in order to demonstrate the effect, ray tracing simulation of the laser beam laterally incident on a plurality of channels provided in the microchip is carried out, and for the total intensity of the laser beam before being laterally incident. The ratio of the intensity of the laser beam passing through the inside of each channel, that is, the laser beam irradiation efficiency for each channel, was determined, and it was evaluated how many channels can be cross incident with what efficiency. The laser beam irradiation efficiency for each channel obtained from such ray tracing simulation has been proved to be in good agreement with the fluorescence intensity ratio for each channel obtained in the experiment, as shown in Patent Document 2 It is a highly reliable evaluation method. In the present invention, as a three-dimensional ray tracing simulator using lighting design analysis software LightTool ^TM and (Synopsys' Optical Solutions Group).

  FIG. 8 is a view showing a configuration a, which is an embodiment of the microchip 1 of the present invention, and a result of ray tracing simulation of the laser beam 4 incident to the side thereof.

  FIG. 8A is a view showing a cross section perpendicular to each channel 2 including the transverse incident axis of the microchip 1. A total of 24 channels 2 from # 1 to # 24 are arranged on the same plane. Here, # 1 is at the end to which the laser beam 4 is introduced, and indicates the number of the channel 2 to which the laser beam 4 is first irradiated. Hereinafter, # 2, # 3,..., # 24 indicate the numbers sequentially assigned to the respective channels 2 in accordance with the traveling direction of the laser beam 4. FIG. 8 (b) is an enlarged view of parts # 1 to # 4 in FIG. 8 (a). FIG. 8 is represented by a yz plane composed of the y-axis and the z-axis, but the origin is the central axis of # 1, and the z-axis coincides with the horizontal incident axis.

The configuration of the microchip 1 shown in FIG. 8 follows the configuration a of FIG. The microchip 1 was placed in the air. Member m ₁ of the microchip 1 was a ZEONOR ^TM (ZEONOR, Nippon Zeon). Zeonor is a cycloolefin polymer (COP) resin and is often used for microchip members due to its high transparency and low hygroscopicity. The refractive index of Zeonor is n ₁ = 1.53. The cross-sectional shape of the channel 2 was a circle with a diameter of 50 μm, and 24 channels 2 were arranged on the same plane at an interval of 300 μm. That is, the distance between the central axis of # 1 and the central axis of # 24 is 6.9 mm. Inside each channel 2, was charged with 3500 / 3500xL POP-7 ^TM polymers (Life Technologies). POP-7 is an aqueous solution containing 8 M urea and a polymer as an electrophoretic separation medium, and its refractive index is n ₂ = 1.41 due to the effect of 8 M urea.

  FIGS. 8 (c) and 8 (d) show the results of ray tracing simulation of the laterally incident laser beam 4 in FIGS. 8 (a) and 8 (b), respectively. The laser beam 4 was introduced from the left side of FIG. 8 and was incident perpendicularly to the left side surface of the microchip 1 to irradiate the channel 2 of # 1. The laser beam 4 before entering the microchip 1 was a parallel beam with a wavelength of 505 nm and a diameter of 50 μm, and its central axis was aligned with the transverse incident axis. The laser beam 4 is composed of 300 infinitely narrow beam elements, and the positions of these beam elements are uniformly and randomly arranged within a diameter of 50 μm. Furthermore, the total intensity of the laser beam 4 is 1.0, and each beam element is equally given an intensity of 1/300. In ray tracing simulation, Snell's law and Fresnel at each position where the refractive index of the entrance surface to the microchip 1, the entrance surface to the channel 1, the exit surface from the channel 1, etc. changes for each beam element The law of L is applied to track the traveling direction and intensity of refracted light. However, when the beam element is totally reflected at the position where the refractive index changes, the traveling direction and intensity of the reflected light are tracked. Figures 8 (c) and (d) show the optical paths of the 300 beam elements so calculated. FIG. 8 (e) extracts the beam element transmitted through the inside for each channel among the 300 beam elements thus calculated, and sums the intensities at those positions as the laser beam irradiation efficiency. It is represented as

As shown in FIGS. 8C and 8D, the laterally incident laser beam 4 has a concave lens function for each channel 2 by n ₂ (= 1.41) <n ₁ (= 1.53). In order to have it, it diverged from the horizontal incident axis. As shown in FIG. 8 (e), it was found that the laser beam irradiation efficiency was sharply attenuated, and only one or at most two channels 2 could be irradiated with the laser beam. Therefore, it has become clear that the configuration a of the microchip of the present example shown in FIG. 8 is not suitable for efficient simultaneous irradiation of a large number of channels by the lateral incidence method.

  The ray tracing simulation described above was performed on a three-dimensional model, but FIGS. 8A to 8D are two-dimensional images projected on a plane perpendicular to the central axis of each channel 2. FIG. 8E shows the result of calculation in three dimensions.

  FIG. 9 is a diagram showing a configuration b, which is an embodiment of the microchip 1 of the present invention, and a result of ray tracing simulation of the laser beam 4 transversely incident thereon. The configuration of this microchip 1 conforms to the configuration b of FIG. Hereinafter, the configuration b will be described. However, unless otherwise noted, it may be considered that the same description as that of the configuration a holds. As shown in FIGS. 9A and 9B, the difference between the configuration b and the configuration a is that among the 24 channels, the odd # remains as channel 2 but the even # as channel 3 The channel 2 and the channel 3 are alternately arranged. Here, in FIGS. 9A and 9B, in order to make the channel 3 more visible, the channel 3 is outlined.

Member m ₂ to be filled in member m ₁ and channel 2 of the microchip 1 was the same as the configuration a. Member m ₃ for filling the channel 3, of Cargill standard refractive liquid Standard Group Combined Set (shot Moritex), the refractive index is a standard refractive liquid n ₃ = 1.60. This standard refractive liquid set is commercially available with refractive index range 1.400 to 1.700, refractive index 0.002 steps, tolerance ± 0.0002. After the channel 3 was filled with the standard refractive liquid, the both ends of the channel 3 were sealed to prevent the standard refractive liquid from coming out of the channel 3 due to evaporation, pressure or the like. In this way, it becomes possible to distribute to the market the microchip in which the channel 3 is filled with the standard refractive liquid in advance, and it is possible to save the user the trouble of filling the channel 3 with the standard refractive liquid. At this time, the relationship of n ₂ (= 1.41) <n ₁ (= 1.53) <n ₃ (= 1.60) is satisfied, and while each channel 2 exhibits a concave lens action, each channel 3 shows a convex lens action.

  As shown in FIGS. 9 (c) and 9 (d), unlike FIGS. 8 (c) and 8 (d), a part of the laser beam 4 incident on the side is not diverged from the side incident axis, Multiple channels 2 and 3 could be transmitted along the transverse incident axis. This is because the convex lens action of the channel 3 partially condenses the laser beam 4 expanded by the concave lens action of the channel 2. However, among the laser beams 4 diverged by the channel 2 of # 1, the beam element expanded so as not to be incident on the channel 3 of # 2 was not received by the condensing by the channel 3 and continued diverging as it was.

  As a result, as shown in FIG. 9 (e), the laser beam irradiation efficiency decreases rapidly from channel 1 of # 1 to channel 3 of # 2, but the attenuation is very high up to channel 3 of # 24 thereafter. It became smaller. The change in laser beam irradiation efficiency from # 1 to # 2 is similar to that in FIG. 8 (e), but from # 3 to # 24 is significantly different from that in FIG. 8 (e). Since the laser beam irradiation efficiency of # 24 in FIG. 9 (e) was over 20%, from the experience, the fluorescence detection sensitivity is slightly reduced, but simultaneous fluorescence detection and simultaneous electrophoresis analysis using 12 channels 2 of odd # Was found to be possible. Although the laser beam irradiation efficiency differs greatly between channels # 1 and # 2 and thereafter, it is possible to simultaneously analyze different samples using a plurality of channels even with this difference. However, in the case where fluorescence intensity is to be compared between channels or quantitative property is required for the fluorescence intensity, it is desirable that the laser beam intensities between the channels be uniform. In such a case, it is better to use channel 2 after # 3 for analysis without using # 1 for analysis.

  FIG. 10 is a diagram showing a configuration c which is an embodiment of the microchip 1 of the present invention, and the result of ray tracing simulation of the laser beam 4 incident on the configuration c. The configuration of this microchip 1 conforms to the configuration c of FIG. The configuration c will be described below, but it may be considered that the same description as that of the configuration b holds unless otherwise noted. The difference between the configuration c and the configuration b is that, as shown in FIGS. 10A and 10B, the cross-sectional shape of the odd # channel 3 is a circle with a diameter of 100 μm which is twice as large. Here, in FIGS. 10A and 10B, in order to make the channel 3 easy to see, the channel 3 is outlined.

Member m ₃ to be filled in each channel 3, of the same Cargill standard refractive liquid set, the refractive index is a standard refractive liquid n ₃ = 1.68. In FIGS. 9 (c) and 9 (d), the diameter of the circle of the cross section of channel 3 is enlarged so that it does not enter channel 3 of # 2 among laser beams 4 diverged by channel 2 of # 1. To reduce the proportion of beam elements. On the other hand, an increase in the circular diameter of the cross section of the channel 3 means that the curvature of the surface of the channel 3 becomes smaller, that is, the convex lens action becomes weaker. Therefore, by making the internal refractive index higher than in the case of the configuration b, it is devised that the convex lens action of the channel 3 is maintained. The relationship of n ₂ (= 1.41) <n ₁ (= 1.53) <n ₃ (= 1.68) is satisfied, and the concave lens function of each channel 2 and the convex lens function of each channel 3 are balanced.

  As shown in FIGS. 10 (c) and 10 (d), unlike FIGS. 9 (c) and 9 (d), more of the laterally incident laser beam 4 diverges from the transverse incident axis. In addition, a large number of channels 2 and 3 can be transmitted along the transverse incident axis. This is because the laser beam 4 expanded by the concave lens action of the channel 2 is more efficiently condensed by the convex lens action of the channel 3 having a large cross section. As a result, as shown in FIG. 10 (e), the attenuation of the laser beam irradiation efficiency from channel 2 of # 1 to channel 3 of # 2 is significantly suppressed as compared with FIG. 9 (e), and thereafter The attenuation up to channel 3 of # 24 was also kept very small. Overall, the laser beam irradiation efficiency was improved about twice as compared with the case of FIG. 9 (e). From this, it was found that simultaneous fluorescence detection and simultaneous electrophoresis analysis using 12 channels 2 of odd # are possible.

  FIG. 11 is a view showing a configuration d which is an embodiment of the microchip 1 of the present invention, and a result of ray tracing simulation of the laser beam 4 transversely incident thereon. The configuration of this microchip 1 is in accordance with the configuration d of FIG. Hereinafter, the configuration d will be described. However, unless otherwise noted, it may be considered that the same description as that of the configuration c holds. The difference between the configuration d and the configuration c is that, as shown in FIGS. 11A and 11B, the cross-sectional shape of the odd # channel 2 is changed from a circle with a diameter of 50 μm to an isosceles trapezoid is there. The isosceles trapezoid has a lower base of 50 μm, a height of 50 μm, a base angle of 92 degrees, and an upper base of about 53.5 μm. Here, in FIGS. 11A and 11B, in order to make the channel 3 easy to see, the channel 3 is outlined.

The reason for making the cross-sectional shape into a square is to weaken the refraction of each channel 2. Among the squares, the isosceles trapezoidal shape is the mass productivity of the microchip 1, as will be described later in the configuration f of FIG. To improve the Member m ₂ to be filled in each channel 2 is the same as structure c, member m ₃ to be filled in each channel 3, of the same Cargill standard refractive liquid set, a refractive index of n ₃ = 1.66 standard It was a refractive liquid. Was slightly lowered than constituting c the refractive index n ₃ of the member m ₃ to be filled in each channel 3, the above changes, also good laser beam 4 dropped the convex lens action than the channels 3 in the configuration c It was thought that it could be focused on the The relationship of n ₂ (= 1.41) <n ₁ (= 1.53) <n ₃ (= 1.66) is satisfied, and the refraction of each channel 2 and the convex lens of each channel 3 are balanced.

  As shown in FIGS. 11 (c) and (d), unlike in FIGS. 10 (c) and (d), all beam elements of the laser beam 4 incident on the side are not diverged from the side incident axis. In addition, a large number of channels 2 and 3 can be transmitted along the transverse incident axis. This is because, in addition to the weak refracting action of the channel 2, the convex lens action of the channel 3 having a large cross section efficiently collects light.

As a result, as shown in FIG. 11 (e), the laser beam irradiation efficiency was able to obtain a high laser beam irradiation efficiency of at least 85% for all channels 2 and 3 from # 1 to # 24. . Since all beam elements of the laser beam 4 can be utilized, the slight attenuation of the laser beam irradiation efficiency with the channel # is determined by the member m _{1 of the} microchip 1 of the laser beam 4 and the member m _{2 of} each channel ₂ or each channel The reflection loss at the boundary of the _third member m3 is described. The reason why the # 1 laser beam irradiation efficiency is about 90. 5 minutes is a reflection loss when the laser beam 4 is incident on the microchip 1. This indicates that highly sensitive simultaneous fluorescence detection and simultaneous electrophoresis analysis using 12 channels 2 of odd # are possible.

Example 2
In the present embodiment, differences from the first embodiment will be mainly described, and it may be considered that the same description as that of the first embodiment holds unless otherwise described. The basic difference from the first embodiment is that the cross-sectional shape of each channel is not circular but square.

FIG. 12 is a diagram showing a configuration e, which is an embodiment of the microchip 1 of the present invention, and a result of ray tracing simulation of the laser beam 4 laterally incident thereon. The configuration of this microchip 1 is in accordance with the configuration e of FIG. As shown in FIGS. 12 (a) and 12 (b), all the channels # 1 to # 24 were channels 2, the cross-sectional shape was a regular square with a diameter of 50 μm, and 24 channels 2 were arranged on the same plane at a spacing of 300 μm. . As in Example 1, member m ₁ is ZEONOR (n ₁ = 1.53) of the microchip 1, member m ₂ to be filled within each channel 2 3500 / 3500xL POP-7 ^TM polymers (n ₂ = 1 .41).

  As shown in FIGS. 12C and 12D, the laterally incident laser beam 4 travels straight along the transverse incident axis without being refracted by each channel 2. This is because the cross sectional shape of each channel 2 is a square, the upper and lower sides are parallel to the horizontal incident axis, and the left and right sides are perpendicular to the horizontal incident axis. The emission angle is always at a right angle, and the laser beam 4 is not refracted. Since all 300 beam elements constituting the laser beam 4 contribute to the irradiation of all the channels 2, as shown in FIG. 12 (e), the laser beam irradiation efficiency is set to all the channels 2 of # 1 to # 24. Thus, it was possible to obtain a high laser beam irradiation efficiency of 80. 5 minutes or more. The result of FIG. 12 (e) is almost the same as the result of FIG. 11 (e), and both can realize the ideal lateral incidence method of the laser beam 4. In configuration e, since all of # 1 to # 24 were channel 2, it was found that highly sensitive simultaneous fluorescence detection and simultaneous electrophoresis analysis using 24 channels 2 were possible.

  FIG. 13 is a view showing a configuration f which is one form of the microchip 1 and the result of ray tracing simulation of the laser beam 4 incident to the side thereof. The configuration of this microchip 1 conforms to the configuration f of FIG. Although the configuration f will be described below, it may be considered that the same description as that of the configuration e holds unless otherwise noted.

The difference between the configuration f and the configuration e is that, as shown in FIGS. 13A and 13B, the cross-sectional shape of each channel 2 is changed from a square to an isosceles trapezoid. In order to improve the mass productivity of the microchip 1, specifically, this change is made to be easily manufacturable by a processing method such as injection molding. Member m ₂ to fill the inside of the member m ₁ and the channel 2 of the microchip 1 was equivalent to configuration e. The isosceles trapezoid had a lower base of 50 μm, a height of 50 μm, a base angle of 92 degrees, and an upper base of about 53.5 μm. That is, the draft angle in the case of producing an isosceles trapezoidal groove by injection molding is 2 degrees. At this time, the refraction angle of the laser beam 4 by the channel 2 is calculated by the equation (7) as ε ₂ = −0.31 degrees, and in FIGS. 13A and 13B, the laser beam 4 is directed downward from the transverse incident axis. Under the influence of refraction.

  As shown in FIGS. 13 (c) and 13 (d), as the transversely incident laser beam 4 passes through each channel 2, it is gradually deflected downward from the transverse incident axis and sharply from the transverse incident axis. I deviated. This is because the refraction by each channel 2 is accumulated according to the number of channels 2 through which the laser beam 4 passes. From channel 2 of # 9 onwards, laser beam 4 deviates completely from the array of channel 2. As shown in FIG. 13 (e), the laser beam irradiation efficiency is rapidly attenuated at channel 2 of # 4 and later, and becomes zero at channel 2 and later of # 9. According to the configuration f, it was found that the number of channels 2 which can be simultaneously irradiated with the laser beam 4 efficiently by the lateral incidence method is only 6 to 7.

  FIG. 14 is a view showing a configuration f ′ which is one form of the microchip 1 and the result of ray tracing simulation of the laser beam 4 incident on the configuration f ′. The configuration f 'of the microchip 1 conforms to the configuration f of FIG. Hereinafter, the configuration f 'will be described. However, unless otherwise noted, it may be considered that the same description as the configuration f holds.

The difference between the configuration f ′ and the configuration f is only the cross-sectional shape of each channel 2. The cross-sectional shape of each channel 2 of the configuration f ′ is an isosceles trapezoid, with a lower base of 50 μm, a height of 50 μm, a base angle of 94 °, and an upper base of about 57.0 μm. That is, the draft angle in the case of producing an isosceles trapezoidal groove by injection molding is 4 degrees. The reason why the draft angle of the structure f ′ is twice as large as that of the structure f is to further improve the mass productivity by processing methods such as injection molding. At this time, the refraction angle of the channel 2 of the laser beam 4 is calculated by equation (7) to ε ₂ = −0.63 degrees, and in FIGS. 13A and 13B, the laser beam 4 is directed downward from the transverse incident axis. Is about twice as large as in the case of configuration f.

  As shown in FIGS. 14 (c) and (d), as the transversely incident laser beam 4 passes through each channel 2, it is larger from the transverse incident axis than in the cases of FIGS. 13 (c) and (d). It is deflected downward and deviates more rapidly from the transverse incident axis. This is because the angle of refraction of each channel 2 of configuration f 'is greater than that of configuration f. From channel 2 of # 6 onward, laser beam 4 deviates completely from the array of channel 2. As shown in FIG. 14 (e), the laser beam irradiation efficiency is rapidly attenuated at channel 2 of # 4 and later, and becomes zero at channel 2 and later of # 6. According to the configuration f ', it is found that the number of channels 2 which can be simultaneously irradiated with the laser beam 4 efficiently by the lateral incidence method is only 4 to 5.

  FIG. 15 is a view showing a configuration g which is an embodiment of the microchip 1 of the present invention, and a result of ray tracing simulation of the laser beam 4 transversely incident thereon. The configuration of this microchip 1 is in accordance with the configuration g of FIG. The configuration g will be described below, but if there is no particular notice, it may be considered that the same description as the configuration f holds. As shown in FIGS. 15A and 15B, the difference between the configuration g and the configuration f is that among the 24 channels, the odd # remains as channel 2 but the even # as channel 3 The channel 2 and the channel 3 are alternately arranged. Here, in FIGS. 15 (a) and 15 (b), in order to make the channel 3 more visible, the channel 3 is outlined.

Member m ₂ to be filled in member m ₁ and channel 2 of the microchip 1 was the same as the structure f. Member m ₃ for filling the channel 3, of Cargill standard refractive liquid set, the refractive index is a standard refractive liquid n ₃ = 1.66. After the channel 3 was filled with the standard refractive liquid, both ends of the channel 3 were sealed to prevent the standard refractive liquid from coming out of the channel 3 by evaporation, pressure or the like. At this time, the relationship of n ₂ (= 1.41) <n ₁ (= 1.53) <n ₃ (= 1.66) is satisfied, and the refraction of each channel 2 and the refraction of each channel 3 are I made it balanced. The refraction angle of the laser beam 4 by the channel 2 is calculated by equation (7) as ε ₂ = −0.31 degree, while the refraction angle of the laser beam 4 by the channel 3 is as ε ₃ = 0 by the equation (8) Calculated at 34 degrees. As ε ₂ + ε ₃ = 0.03 degrees, equation (9) holds. That is, the magnitude of the net refraction angle of the laser beam 4 from the pair of channel 2 and channel 3 is smaller than the magnitude of the refraction angle of the laser beam 4 from channel 2 alone.

  As shown in FIGS. 15 (c) and (d), most of the laterally incident laser beam 4 penetrates channels 2 and 3 and is different from those in FIGS. 13 (c) and (d). It was well irradiated simultaneously. This is because the refraction due to channel 2 which is apparent in configuration f is offset by the refraction due to channel 3 added in configuration g. As shown in FIG. 15 (e), the laser beam irradiation efficiencies of all the channels # 1 to # 24 were maintained high at over 80%. This result is comparable in level to the ideal result of configuration e of FIG. 12 (e). Therefore, the configuration g is a configuration for efficiently simultaneously irradiating the plurality of channels 2 and 3 provided in the microchip 1 with the laser beam 4 by the lateral incidence method, and high sensitivity simultaneous by the 12 channels 2 of odd # It has been found that fluorescence detection and simultaneous electrophoresis analysis are possible.

  FIG. 16 is a view showing a configuration g 'which is an embodiment of the microchip 1 of the present invention, and the result of ray tracing simulation of the laser beam 4 incident to the side thereof. The configuration g 'of the microchip 1 conforms to the configuration g of FIG. Hereinafter, the configuration g ′ will be described, but it may be considered that the same description as the configuration g holds, unless otherwise noted. The difference between the configuration g ′ and the configuration g is only the cross-sectional shape of each channel 2 and channel 3.

As shown in FIGS. 16 (a) and 16 (b), the cross-sectional shape of each channel 2 and channel 3 of configuration g 'is an isosceles trapezoid, lower 50 μm, height 50 μm, base angle 94 °, upper base Was about 57.0 μm. That is, in the configuration g ′, in the configuration f ′, the odd number # of the 24 channels remains the channel 2, but the even number # is the channel 3 and the channel 2 and the channel 3 are alternately arranged and expressed You may. Here, in FIGS. 16A and 16B, in order to make the channel 3 easy to see, the channel 3 is outlined. At this time, the refraction angle of the laser beam 4 by the channel 2 is calculated by equation (7) as ε ₂ = −0.63 degrees, while the refraction angle of the laser beam 4 by the channel 3 is as ε ₃ = by the equation (7) Calculated at 0.68 degrees. As ε ₂ + ε ₃ = 0.05 degrees, equation (9) holds. That is, the magnitude of the net refraction angle of the laser beam 4 from the pair of channel 2 and channel 3 is smaller than the magnitude of the refraction angle of the laser beam 4 from channel 2 alone.

  As shown in FIGS. 16 (c) and (d), most of the laterally incident laser beam 4 penetrates channels 2 and 3 and is different from those in FIGS. 14 (c) and (d). It was well irradiated simultaneously. This is because the refraction due to channel 2 which is apparent in configuration f 'is offset by the refraction due to channel 3 added in configuration g'. As shown in FIG. 16 (e), the laser beam irradiation efficiencies of all the channels # 1 to # 24 were maintained high at 70% or more. Although this result is slightly inferior to the configuration g of FIG. 15 (e), it is a level sufficient for highly sensitive fluorescence detection. Therefore, the configuration g ′ is a configuration for efficiently simultaneously irradiating the plurality of channels 2 and 3 provided in the microchip 1 with the laser beam 4 by the lateral incidence method, and high sensitivity by the 12 channels 2 of odd # It was found that simultaneous fluorescence detection and simultaneous electrophoresis analysis were possible.

[Example 3]
FIG. 17 is a schematic explanatory view showing an example of the multi-channel fluorescence detection device according to the present invention. This example shows a system for performing electrophoresis analysis of DNA contained in a biological sample using a plurality of laser beams, (a) shows a bird's-eye view of the microchip 1, (b) shows the laser beam of the microchip 1 constituting the system 4 shows a cross section including the transverse incident axis of the laser beam 108, a cross section of a fluorescence detection optical system, and a data analysis device, and (c) shows a two-dimensional fluorescence image obtained by the two-dimensional sensor 12. The following description will be made focusing on differences from FIG. 5 of FIG.

  As shown in FIG. 17 (a), the laser beam 4 and the laser beam 108 are parallel to each other along the arrangement plane of the plurality of channels 2 and 3 and perpendicular to the long axis of each channel, and the long axis direction of each channel They were irradiated at intervals. The laser beam 4 has a wavelength of 505 nm while the laser beam 108 has a wavelength of 635 nm. All were made into the parallel beam of diameter 50micrometer. At this time, as shown in FIG. 17A, one horizontal incident axis exists for each laser beam. Since the conditions of the cross-sectional shape, arrangement interval, refractive index, etc. of each channel are the same for any horizontal incident axis, it is possible to realize the same horizontal incident method. Generally, the refractive index of each member varies depending on the wavelength, which may affect the performance of the lateral incidence method, but the wavelength dependency of the refractive index of each member used in the present invention is small, so the effect is small.

  FIG. 17 (b) shows a cross-sectional view including the transverse incident axis of the laser beam 4 or the laser beam 108, which are not different from each other. A laser beam 108 is emitted from a laser light source 112. FIG. 17 (c) shows a two-dimensional fluorescence image obtained. In addition to the wavelength dispersion image 105 of laser light scattering and fluorescence from each channel 2 by excitation of the laser beam 4 and the wavelength dispersion image 106 of laser light scattering from each channel 3, from each channel 2 by excitation of the laser beam 108 The laser beam scattering and fluorescence wavelength dispersion image 109 of the laser light and the wavelength dispersion image 110 of the laser light scattering from each channel 3 are measured independently of each other. With the above configuration, it is possible to identify a minute amount of fluorescence by increasing the number of types of fluorescence that can be simultaneously detected in each channel 2 or by separating and detecting different fluorescences with high accuracy. In this example, different samples were labeled with different fluorophores and analyzed simultaneously in the same channel to improve throughput. In the following description, the laser beam 4 will be described, but the same description can be applied to the laser beam 108.

  FIG. 18 is a view showing a configuration h which is an embodiment of the microchip 1 of the present invention, and a result of ray tracing simulation of the laser beam 4 transversely incident thereon. The configuration h is a modification of the cross-sectional shape of the channel 3 of the configuration g 'of FIG. As shown in FIGS. 18A and 18B, the cross-sectional shape of odd-numbered channel 2 is not changed, but the cross-sectional shape of even-numbered channel 3 is the height without changing the base angle of the isosceles trapezoid It was increased from 50 μm to 100 μm. The upper base of each channel 2 and each channel 3 was coplanar, i.e. coincident with the interface 5, as in the case of the configuration g '. Meanwhile, the lower base of each channel 2 and the lower base of each channel 3 are arranged on different planes. That is, the cross-sectional shape of the channel 3 of the structure h is the same upper base as the cross-sectional shape of the structure g ', and is the shape obtained by doubling the depth with the same slope. That is, the cross-sectional shape of the channel 3 was about 43.0 μm in the lower base, 100 μm in height, 94 ° in base angle, and about 57.0 μm in the upper base. As in the case of the second embodiment, the microchip 1 having such a configuration can be easily manufactured by bonding two upper and lower parts with the upper bottom of each channel as the boundary surface 5.

Member m ₁ of the microchip 1, member m ₂ to be filled in the channel 2, member m ₃ for filling the channel 3 was equivalent to a configuration g 'either. At this time, the refraction angles of the laser beam 4 by the channel 2 and the channel 3 are the same as the configuration g ′, and ε ₂ = −0.63 degrees, ε ₃ = 0.68 degrees. On the other hand, since the depth of the channel 3 of the configuration h is twice as large as that of the configuration g ′, of the beam elements of the laser beam 4 from the horizontal incident axis to the opposite side to the boundary surface 5, that is, FIG. And g) can be deflected in the direction of the transverse incident axis in arrangement h, in arrangement g '.

  As shown in FIGS. 18 (c) and (d), more parts of the laterally incident laser beam 4 pass through channel 2 and channel 3 compared to FIGS. 16 (c) and (d), They were simultaneously irradiated efficiently. This is because the beam element deviated from each channel in the configuration g 'can be used for irradiation of the subsequent channel without deviation in the configuration h. As a result, as shown in FIG. 18E, the laser beam irradiation efficiencies of all the channels # 1 to # 24 were maintained high at over 80%. Therefore, the configuration h is a configuration for efficiently simultaneously irradiating the plurality of channels 2 and 3 provided in the microchip 1 with the laser beam 4 by the lateral incidence method, and high sensitivity simultaneous by the 12 channels 2 of odd # It has been found that fluorescence detection and simultaneous electrophoresis analysis are possible.

  FIG. 19 is a schematic explanatory view showing an example of the multi-channel fluorescence detection device according to the present invention. This example shows a microchip electrophoresis analyzer that irradiates a two-divided laser beam from both sides, (a) is a bird's-eye view of the microchip 1, (b) is a laser beam 4 or laser beam 108 of the microchip 1 constituting the system. 8A shows a cross section including the horizontal incident axis of the cross section of the fluorescence detection optical system, and a data analysis device.

  In the configuration h ′, the laser beam 4 and the laser beam 108 are divided into two by using the half mirror 114 and a plurality of mirrors 115 are used as shown in FIG. Then, they were made to face each other from both sides of the array plane of each channel 2 and 3, and the central axis of each divided beam was made to coincide with the respective transverse incident axes. The two-dimensional fluorescence image obtained at this time is the same as that shown in FIG. 17 (c). Other conditions were the same as in the configuration h. In the following description, the laser beam 4 will be described, but the same description can be applied to the laser beam 108.

  FIG. 20 shows the laser beam irradiation efficiency for each channel in the structure h ', and it was found that the laser beam irradiation efficiency between the channels can be made uniform as compared with that of the structure h. Here, the total intensity of the split beams is 0.5. The standard deviation of the laser beam irradiation efficiency between the channels could be significantly reduced to 0.01 in configuration h 'whereas it was 0.04 in configuration h. As a result, the fluorescence emitted from each channel can be detected with high sensitivity and uniformity, and it becomes possible to effectively expand the fluorescence detection dynamic range.

In the present invention, it is important to irradiate with the central axis of the laser beam 4 aligned with the horizontal incident axis, but as a means for realizing this reliably, a member m filled in each channel 2 or 3 Raman scattering by irradiation of ₂ or m ₃ laser beams 4 and fluorescence as an index, relative position of laser beam 4 and microchip 1 or array plane plane so that they are detected with maximum or separation between channels It is effective to fine-tune the relationship. In order to this method more effective, the member m _3, which is filled into the channels 3 are mixed fluorescent material, while more clearly the indication of the above, the fluorescence detection of the samples from each channel 2 It is possible not to affect.

  Although all of the above-described embodiments have exemplified electrophoresis analysis using a microchip, it is of course possible to apply the present invention to other analysis using a microchip. For example, simultaneous fluorescence detection can be performed by performing PCR of a plurality of samples in a plurality of different channels and irradiating the laser beam laterally to these channels, thereby quantifying a target DNA sequence contained in the plurality of samples with high sensitivity . In addition, it is also possible to integrate the pretreatment process on a microchip to which the present invention is applied to make a micro TAS or a lab on a chip. For example, after a human blood sample is injected into a microchip, blood cell separation, genome extraction, etc. are performed in the microchip, and it is divided and introduced into a plurality of channels, and a specific disease in each channel. The present invention can be applied to a system in which the presence of a plurality of related DNA sequences is quantified with high sensitivity by PCR, and a gene diagnosis of a specific disease is performed based on these results. In such applications, it is necessary to be able to mass-produce microchips at low cost and to be disposable in order to prevent contamination between samples, and the effects of the present invention are particularly exhibited. Besides, it is possible to apply the present invention to various applications performed on a microchip, such as an immunoassay, a flow cytometer, a single cell analysis, a microreactor, and so on.

  In this embodiment, as shown in FIG. 17 and FIG. 19, the emission fluorescence from each channel 2 was measured by a common fluorescence detection system, but for each channel from the direction perpendicular to the array plane of each channel An independent fluorescence detection system may be constructed. Such a configuration makes it possible to further reduce crosstalk between channels. In addition, a light reflection preventing film may be formed on the outer surface of the microchip 1 in the direction opposite to the direction in which fluorescence detection is performed with respect to the array plane of each channel. The antireflective film does not necessarily have to be directly bonded to the outer surface of the microchip 1 and, for example, a light absorbing member may be disposed in contact with the outer surface of the microchip 1. With such a configuration, among the fluorescence emitted from each channel, the component advancing in the direction opposite to the fluorescence detection system is reflected on the outer surface or the outside of the microchip 1 and the reflected light is fluorescence. Detection by a detection system makes it possible to reduce the possibility of crosstalk.

  The present invention is not limited to the embodiments described above, but includes various modifications. For example, the embodiments described above are described in detail in order to explain the present invention in an easy-to-understand manner, and are not necessarily limited to those having all the configurations described. Also, part of the configuration of one embodiment can be replaced with the configuration of another embodiment, and the configuration of another embodiment can be added to the configuration of one embodiment. In addition, with respect to a part of the configuration of each embodiment, it is possible to add, delete, and replace other configurations.

1 microchip m ₁ microchip 1 member n ₁ microchip 1 member refractive index 2 channel m ₂ channel 2 internal member n ₂ channel 2 internal member refractive index r ₂ channel 2 diameter 3 channel m ₃ channel 3 inner member n ₃ channel 3 inner member refractive index r ₃ channel 3 diameter 4 laser beam 5 interface 6 channel 2 inlet port 7 channel 2 outlet port 8 channel 3 inlet port 9 collection Light lens 10 filter and diffraction grating 11 imaging lens 12 two-dimensional sensor 13 data analysis device 103 outlet port 104 of channel 3 two-dimensional fluorescent image 105 wavelength dispersed image of laser light and fluorescence from channel 2 laser from channel 3 Wavelength dispersion image 107 of scattered light Rubber stopper 108 Laser beam 111 Laser light source 112 Source 113 Channel 2 and Channel 3 common exit port 114 half mirror 115 mirror

Claims (3)

A plurality of channels are provided inside the transparent solid member having a refractive index n ₁ , and in the plurality of channels, the major axes of the channels are arranged parallel to each other in the same plane in at least a partial region, the lengths of the plurality of channels A width of a cross-sectional shape perpendicular to the axis extends toward one side perpendicular to the same plane, and the plurality of channels is a first channel filled with a first member of refractive index n ₂ inside And a second channel filled with a second member of refractive index n ₃ , and a laser beam is applied to the side surface of the microchip for the microchip satisfying the relationship of n ₂ <n ₁ <n ₃ Perpendicularly from the long planes of the plurality of channels arranged parallel to one another along the same plane from the
The laser beam and the array plane of the microchip or the plurality of channels with reference to Raman scattering or fluorescence by irradiation of the laser beam of the first member or the second member filled in the plurality of channels, and Adjusting the relative positional relationship of
A laser beam irradiation method having: The laser beam irradiation method according to claim 1, wherein the relative positional relationship is adjusted such that the Raman scattering or fluorescence is maximized or separated between the plurality of channels.   The laser beam irradiation method according to claim 1, wherein a fluorescent substance is mixed in the second member.

Images