JP2009063470A - Biological material detecting substrate and biological material detecting apparatus using same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biological material detecting substrate capable of preventing detection signals from being lowered even if biological material detecting substrates having large size variations are used, and to provide a biological material detecting apparatus using the same. <P>SOLUTION: The biological material detecting substrate determines biological materials through the use of transport reaction acquired when the biological materials are moved through a detection channel filled with a buffer agent and includes a positioning marker having a rotationally symmetrical shape and at least one detection channel to emit fluorescence in the vicinity of the detection channel. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a biological material detection substrate for detecting DNA, protein, and other biological materials, and a biological material detection device using the same, and more particularly, a biological material detection substrate that is detachable and has a high measurement system, and The present invention relates to a biological material detection apparatus using the same.

  In recent years, a fluorescence detection method using a fluorescent label has been adopted as a technique for detecting biological substances such as DNA and proteins. Fluorescence detection methods include enzyme immunoassay, electrophoresis, and confocal scanning because biological units can be measured safely and inexpensively without using radioisotopes using an optical unit consisting of an excitation light source, optical filter, and light receiving element. It is applied to the detection of various biological materials such as scanning fluorescence microscopy.

  The fluorescence detection method is a method of measuring the presence / absence or amount of a biological material by detecting a fluorescent signal from a fluorescent label that is labeled on the biological material by irradiating excitation light. For example, Cy5 is a fluorescent label that emits fluorescence with a wavelength of 670 nm with respect to excitation light with a wavelength of 635 nm. In order to detect a biological substance labeled with Cy5, the sample is irradiated with excitation light of 635 nm, and the light from the sample is detected by a light receiving element through an optical filter that transmits only light near 670 nm. There are various types of fluorescent labels. By selecting an excitation light source and an optical filter corresponding to the excitation wavelength and the fluorescence wavelength of the fluorescent label, biological substances labeled with various fluorescent labels can be detected.

  When performing fluorescence detection, a substrate or cell that supports the sample is required, but there are devices that perform detection using a substrate with a fine flow path so that measurement can be performed even when the amount of sample is small. ing. In this device, after a sample labeled with a fluorescent label and a buffering agent are filled in the flow path, the sample is electrophoresed by applying a predetermined potential gradient to the buffer, and the sample being migrated is irradiated with excitation light to fluoresce. By detecting the intensity distribution, it is possible to observe the migration state of the sample (see, for example, Patent Document 1).

  In addition, the conventional biological material detection substrate is optically opaque except for the flow path portion on the substrate, and has an optically transparent position detection hole at a certain distance from the detection portion. FIG. 7 shows a conventional biological substance detection substrate. The sample flow path 206 and the migration flow path 207 are arranged on the glass-made biological material detection substrate 200 so as to intersect at the cross portion 208. At both ends of the sample flow path 206, liquid reservoirs of the sample injection section 202 and the sample discharge section 203 are provided, respectively. At both ends of the migration flow path 207, liquids of the negative electrode section 204 and the positive electrode section 205 are respectively provided. A reservoir is provided.

  First, by injecting a buffer into the negative electrode unit 204 and applying pressure with an external pump, the negative electrode unit 204 passes through the migration channel 207 to the positive electrode unit 205, the migration channel 207 and the sample channel 206. All the flow paths from the cross section 208 through the sample flow path 206 to the sample injection section 202 and the sample discharge section 203 are filled with a buffer.

Next, after injecting a sample modified with a fluorescent label into the sample injection unit 202, a voltage is applied to the sample injection unit 202 and the sample discharge unit 203, thereby passing through the sample channel 206 to the sample discharge unit 203. The sample channel 206 is filled with the sample. At this time, since the sample is present only in the cross portion of the sample flow channel 206 on the migration flow channel 207, the sample is quantified by this cross portion. Thereafter, the quantified sample starts electrophoresis by applying a voltage to the negative electrode portion 204 and the positive electrode portion 205. The sample can be analyzed by irradiating the sample during electrophoresis with excitation light and detecting the intensity distribution of the fluorescence emitted from the sample. At this time, the position detection hole 201 is detected to obtain the position of the position detection hole 201, and the detection signal is detected by the detection unit 209 at a certain distance from the position detection hole 201 to improve measurement accuracy (for example, (See Patent Document 2).
JP 2005-064339 gazette JP 2001-305050 A

  However, in the conventional configuration, since the biological material detection substrate is made of glass, thermal contraction is small, and the position of the position detection hole and the migration channel can be kept constant. However, if the material for the biological material detection substrate is made of resin instead of glass, the substrate contracts due to thermal stress before and after molding, and the position of the position detection hole and the migration channel varies. When a biological material detection substrate having such variations is mounted on the biological material detection device, the relative position between the biological material detection substrate and the detection optical unit inside the biological material detection device varies, and the detection signal is reduced. Had problems.

  The present invention solves the above-described conventional problems, and even when a biological material detection substrate having a large dimensional variation is used, a biological material detection substrate capable of preventing a decrease in detection signal and a biological body using the same An object is to provide a substance detection device.

  In order to solve the above-described conventional problems, the biological material detection substrate of the present invention and the biological material detection device using the same are obtained when the detection material flow path is filled with the biological material in the buffer. In the biological material detection substrate that performs the determination of the biological material using the transport reaction, the substrate has at least one detection channel, and has a rotationally symmetric shape that emits fluorescence in the vicinity of the detection channel. A positioning marker is provided.

  Furthermore, the biological material detection substrate of the present invention and the biological material detection device using the biological material detection substrate according to claim 1 and the positioning marker are irradiated with excitation light to detect fluorescence by the excitation light. An optical unit for driving the optical unit, a driving unit for moving the optical unit from the center of the biological material detection substrate toward the outer periphery, and an irradiation optical axis of the optical unit according to the fluorescence detected by the optical unit; And a signal analysis unit that instructs the drive unit so that the center of the positioning marker coincides with the center of the positioning marker.

  According to the biological material detection substrate and the biological material detection apparatus using the biological material detection substrate of the present invention, the detection optical unit is inspected by providing a positioning through-hole in the vicinity of the detection flow path of the biological material detection substrate. Since it can be easily matched with the path, it is possible to realize a highly sensitive and highly accurate biological substance detection performance without causing a decrease in the detection signal.

Embodiments of a biological material detection substrate and a biological material detection device using the same according to the present invention will be described below in detail with reference to the drawings.

(Embodiment 1)
FIG. 1A is a plan view showing a biological substance detection substrate 1, and FIG. 1B is a diagram showing one electrophoresis unit 3 in an enlarged view thereof.

  The biological material detection substrate 1 will be described with reference to FIG. The material for the biological substance detection substrate 1 is an acrylic resin and has a thickness of 2 mm. A channel having a depth of 50 μm and a groove serving as a reservoir are dug on the channel forming surface, and an acrylic film having a thickness of 50 μm is adhered to the upper surface to form a sealed channel. A hole for fixing the biological material detection substrate 1 to the biological material detection device is provided in the center 2 of the biological material detection substrate 1. In the present embodiment, a configuration in which eight migration units 3 are provided on the biological material detection substrate 1 is taken as an example.

  The migration unit 3 of the biological material detection substrate 1 will be described in detail with reference to FIG.

  In the electrophoresis unit 3, a buffer injection part 4 for injecting a buffer and a sample injection part 5 for injecting a biological substance are formed. The sample injection unit 5 is connected to the sample holding unit 7 through a flow path 6, and the sample holding unit 7 is connected to the buffer unit 9 through a flow path 8. The positive electrode 11 is connected to the buffer injection part 4 by the flow path 10 and is further connected to the sample holding part 7 by the flow path 14. The negative electrode 12 is connected to the buffer injection part 4 by the flow path 10 and further connected to the sample holding part 7 by the flow path 13.

  The biological material is a single-stranded DNA having a length of about 60 bases including the SNPs site to be distinguished, and is labeled with a fluorescent material (Cy5). The biological material injected into the sample injection unit 5 moves through the flow path 6 to the sample holding unit 7, and a certain amount of biological material is held in the sample holding unit 7. The biological material exceeding a certain amount moves through the flow path 8 to the buffer unit 9.

  A DNA conjugate is prepared as a buffer for electrophoresis. The DNA conjugate is obtained by covalently bonding a high molecular linear polymer to the 5 'end of a 6 to 12 base long single-stranded DNA. Furthermore, DNA is a sequence that is complementary to the normal type but not complementary to the mutant type, has a strong binding force to the normal type DNA, and a weak binding force to the mutant type DNA. There are characteristics. In addition, when electrophoresed, it bound to the 5 'end. There is also a characteristic that the migration speed is considerably slow due to the weight of the linear polymer. The buffer injected into the buffer injection unit 4 moves from the flow path 10 to the positive electrode 11 and the negative electrode 12 and further fills the flow path 13 and the flow path 14.

  A voltage is applied between the positive electrode 11 and the negative electrode 12 in order to perform electrophoresis of the biological material. The voltage is applied in a state where needle-like electrodes are inserted from the positive electrode 11 and the negative electrode 12 so as to come into contact with the buffer. By applying a voltage, an electric field is generated in the flow path 13 and the flow path 14, and the biological material held in the sample holding unit 7 is electrophoresed in the flow path 14.

  The detection of DNA was performed by exciting the DNA modified with the fluorescent label (Cy5) with light of 635 nm and detecting the state of the DNA sample to be electrophoresed in the flow path 14 by light detection around 670 nm. Further, the distribution of the DNA sample to be electrophoresed in the flow path 14 is measured by rotating the biological material detection substrate 1 by making the flow path 14 for electrophoresis circular.

  Next, the positioning marker provided on the biological material detection substrate 1 will be described. The positioning marker is provided at a position indicated by 15 between the migration unit 3 on the biological material detection substrate 1 and the adjacent migration unit. In the present embodiment, the positioning marker has a circular shape of φ0.5 mm, and is arranged so that the flow path 14 and the center of the positioning marker are concentric. FIG. 2 shows a cross-sectional view of the electrophoresis unit 3 at point A and point B in FIG. Here, the surface on which the flow path 14 is formed is defined as a-plane, and the opposite surface is defined as b-plane. The positioning marker shown in FIG. 2 includes a cylindrical through hole 15 and a fluorescent sheet 16, and the fluorescent sheet 16 is attached to the b-surface side so as to cover the through hole 15. Here, the size of the through hole 15 was set to φ0.5 mm.

  The fluorescent sheet has a characteristic of emitting fluorescence around a wavelength of 670 nm by excitation light having a wavelength of 635 nm, and the amount of light is similar to that of a DNA sample. When excited by 0.5 μW, the fluorescent sheet emits fluorescence of several tens of pW. The reason is that if the amount of light is too large, the range becomes overrange and positioning cannot be performed.

  Since the through-hole 15 and the flow path 14 are concentric, the through-hole 15 and the flow path 14 are concentric even if shrinkage after molding of the biological material detection substrate 1 and thermal shrinkage at the time of film application occur. Located on the top. Therefore, by detecting the position of the through-hole 15 and matching the relative position of the fluorescence detection device and the through-hole 15, the position of the fluorescence detection device and the flow path 14 in the inner and outer peripheral directions can be matched. In addition, the fluorescence detection signal of the DNA sample to be electrophoresed in the flow path 14 can be detected without lowering.

  As described above, in the first embodiment, the through hole 15 is provided concentrically with the flow path 14 and on the extension line of the flow path 14, and is formed on the opposite surface of the biological material detection substrate 1 to the flow path forming surface. By attaching a fluorescent sheet and detecting the position of the through hole 15 with a fluorescence detection device, the biological material detection substrate and the fluorescence are detected even when a biological material detection substrate with large dimensional variation is used. Variations in relative position with the apparatus can be prevented.

(Embodiment 2)
FIG. 3 is a cross-sectional view of the electrophoresis unit 3 in FIG. In FIG. 3, the flow path 14 and the through-hole 15 are provided, and the acrylic film 17 is adhered to the a surface. The difference from the configuration of the first embodiment is that an ink 18 that emits fluorescence is applied to the through-hole 15 portion of the acrylic film 17. The ink 18 is applied from the b-surface side, and the area to be applied covers the through hole 15. The advantage of applying the ink 18 from the b surface is that when the through hole 15 is covered by the application of the ink 18, the position and size of the ink 18 coincide with the through hole 15 from the a surface side. For example, when applying from the flow path forming surface of the biological detection substrate, it is necessary to recognize and print the position of the positioning marker, and the print position may deviate from the position of the positioning marker. However, by applying ink from the b-side, it is possible to easily form a positioning marker with high positional accuracy. In addition, the ink 18 has a property of emitting fluorescence around a wavelength of 670 nm by excitation light having a wavelength of 635 nm, and the amount of light is the same as that of the DNA sample.

  By detecting the position of the through-hole 15 and matching the relative position between the fluorescence detection device and the through-hole 15, the fluorescence detection device and the position of the flow path 14 in the inner and outer peripheral directions can be matched. Detection can be performed without reducing the fluorescence detection signal of the DNA sample to be electrophoresed in the channel 14.

  As described above, in the second embodiment, the through hole 15 is provided concentrically with the flow channel 14 and on the extension line of the flow channel 14, and is the opposite surface of the biological material detection substrate 1 from the flow channel formation surface. A fluorescent sheet 16 is affixed to the optical material 41, and the position of the through hole 15 is detected by the optical unit 41, so that the biological material detection substrate and the fluorescence can be detected even when a biological material detection substrate with large dimensional variation is used. It is possible to prevent variations in relative position with respect to the device to be detected.

(Embodiment 3)
FIG. 4 is a configuration diagram showing a biological material detection apparatus that performs fluorescence detection and positioning for DNA sample discrimination according to Embodiment 1 of the present invention.

  In FIG. 4, 41 is an optical unit composed of the following parts. As the light source 30, a laser diode having a wavelength of 635 nm is used. This light source may be any excitation power that can excite the fluorescent substance Cy5 in the absorption wavelength region of the fluorescent substance Cy5 that labels DNA. The dichroic mirror 31 has a characteristic of reflecting light having a wavelength of about 635 nm and transmitting light having a wavelength of about 670 nm. Therefore, the excitation light is reflected by the dichroic mirror 31. The reflected excitation light is collimated by the objective lens 32 and applied to the flow path 33 on the biological material detection substrate 34. The fluorescent substance labeled on the DNA sample in the flow path 33 receives the excitation light irradiated from the objective lens 32 and emits fluorescence. This fluorescence passes through the objective lens 32, the dichroic mirror 31, and the condenser lens 35, and is received by the photodiode 36.

  Next, a method for positioning the fluorescence detection flow path 14 and the optical unit 41 will be described in detail. As the biological material detection substrate 34 used in this example, the biological material detection substrate provided with the fluorescent sheet 16 in the through hole 15 shown in the first embodiment is used. However, the biological material detection substrate shown in the second embodiment is used. The same positioning can be performed even if is used.

  When the biological material detection substrate 34 is first attached to the biological material detection device, the drive unit 37 is composed of a stepping motor, and moves the biological material detection substrate 34 to the initial position. The detection of the initial position may be performed by attaching a reflection plate to the rear surface of the tray on which the biological material detection substrate 34 is generally performed, and detecting the reflected light by a reflection photosensor. Next, in order to make the position of the positioning marker substantially coincide with the optical axis emitted from the optical unit 41, the biological material detection substrate 34 is rotated by a preset number of steps with reference to the initial position.

  Thereafter, the excitation light emitted from the optical unit 41 is irradiated onto the fluorescent sheet 16 attached to the through hole 15, and the fluorescence emitted from the fluorescent sheet 16 is detected by the optical unit 41. The drive circuit 39 drives the actuator 40 that moves the optical unit 41 in the inner and outer peripheral directions, and moves the optical unit 41 in the inner and outer peripheral directions with respect to the biological material detection substrate 34, while the signal analyzing unit 38 removes from the photodiode 36. The detection signal of the obtained fluorescent sheet 16 is monitored.

  FIG. 5 shows the relationship between the fluorescence value (detection signal) from the fluorescent sheet 16 obtained when the optical unit 41 passes through the through hole 15 and the position of the optical unit 41. As shown in FIG. 5, when the positions of the through hole 15 and the optical unit 41 coincide with each other, the detection signal becomes the maximum value 50, and otherwise, the detection signal becomes small. Therefore, the position of the optical unit 41 and the through hole 15 can be detected by monitoring the detection signal of the fluorescent sheet 16 obtained from the photodiode 36 by the analysis unit 38.

  Furthermore, the positioning method of the flow path 14 and the optical unit 41 is demonstrated. The fluorescence from the fluorescent sheet 16 is detected, and the optical unit 41 is moved by the actuator 40. The position of the through hole 15 and the optical unit 41 can be matched by obtaining the maximum value of the detection signal by using the detection signal and the position information as a set.

FIG. 6 is a flowchart showing a signal analysis flow when positioning the positioning marker and the optical unit 41. The position of the optical unit 41 in the y direction is defined as y. A signal of light received by the photodiode 36 is input to the signal analysis unit 38 as a signal I _y . The actuator 40 moves the position of the optical unit 41 in the y direction by +0.1 mm, and inputs the light signal received by the photodiode 36 to the signal analysis unit 38 as a signal I _{y +0.1} . A magnitude comparison of the signal I _y and the signal I _{y + 0.1,} when the signal I _{y + 0.1} is large dy = + 0.1 mm, when the signal I _{y + 0.1} is smaller is defined as dy = -0.1 mm.

The signal I _n is defined as I _{y +0.1+ (dy * n),} and the initial value of n is 0. The actuator 40 moves the position of the optical unit 41 by + dy, and inputs the light signal received by the photodiode 36 to the signal analysis unit 39 as the signal In _{+ 1} . If the signal I large signal I _{n + 1} than _n, by an actuator 40 to move the optical unit 41 + dy only repeats the comparison of the signal I _n before the movement and the light receiving signal I _{n + 1} of the photodiode 36 . If the signal I _{n + 1} signal I _n is larger than the, by the actuator 40 to move the position of the optical unit 41 by -dy. The position can be determined as the position where the light received by the photodiode 36 has the maximum intensity, and the position y + 0.1 + n * dy can be determined as the position of the through hole.

  The measurement of the DNA sample is performed by rotating the biological material detection substrate 34 by the drive unit 37 and scanning the fluorescence of the flow path 14. The through-hole 15 and the flow path 14 are arranged concentrically, and detects the position of the through-hole 15 and aligns the relative position of the device that detects fluorescence and the through-hole 15 to detect fluorescence. The positions in the inner and outer peripheral directions of the flow channel 14 can be matched, and detection can be performed without reducing the fluorescence detection signal of the DNA sample that is electrophoresed in the flow channel 14.

  As described above, in the third embodiment, the flow path 14 is obtained by aligning the positions of the through hole 15 on the biological material detection substrate 1 and the optical unit using the fluorescence detection device for biological material discrimination. And the optical unit 41 can be aligned. As a result, even if a biological material detection substrate having large dimensional variations is used, variations in relative positions between the biological material detection substrate and the fluorescence detection device can be prevented.

  The biological material detection substrate and the biological material detection device using the same according to the present invention can detect the detection channel position of the biological material detection substrate without separately providing an optical unit for detecting the positioning marker. Therefore, it is useful as positioning of the biological material detection substrate.

(A) Configuration diagram of biological substance detection substrate according to the first embodiment of the present invention (b) Enlarged view showing the electrophoresis unit according to the first embodiment of the present invention Sectional drawing of the biological material detection board | substrate in Embodiment 1 of this invention. Sectional drawing of the biological material detection board | substrate in Embodiment 2 of this invention. Configuration diagram of biological material detection apparatus according to Embodiment 3 of the present invention The figure which shows the detection waveform of the positioning marker in Embodiment 3 of this invention. Flowchart of positioning method in Embodiment 3 of the present invention Configuration diagram of conventional biological substance detection substrate

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Biological substance detection board | substrate 2 Center 3 Electrophoresis unit 3 Buffer injection part 4 Sample injection part 14 Channel 15 Through-hole 16 Fluorescent sheet 18 Ink 30 Light source 31 Dichroic mirror 32 Objective lens 35 Condensing lens 36 Photodiode 38 Signal analysis part 39 Drive circuit 40 Actuator

Claims (8)

In the biological material detection substrate for distinguishing the biological material using a transport reaction obtained when the detection flow path filled with the biological material in the buffer is moved,
A biological substance detection substrate comprising at least one detection channel and a rotationally symmetric positioning marker that emits fluorescence in the vicinity of the detection channel. The positioning marker is
The biological substance detection substrate according to claim 1, wherein the biological material detection substrate is disposed between the detection flow path and the detection flow path so that the symmetry point substantially coincides with the center of the detection flow path. The positioning marker is
The biological material detection substrate according to claim 2, wherein a through hole is provided in the biological material detection substrate, and a fluorescent sheet is attached to a surface opposite to the detection flow path forming surface so as to cover the through hole. The biological substance detection substrate according to claim 3, wherein the fluorescent sheet has a characteristic of emitting fluorescence having a wavelength of about 670 nm by excitation light having a wavelength of 635 nm. The positioning marker is
A through-hole is provided in the biological material detection substrate, a resin film is attached to the detection flow path forming surface so as to cover the through-hole, and fluorescent ink is applied only to the bottom surface of the through-hole formed by the resin film. The substrate for detecting a biological material according to claim 2, to which is applied. The biological substance detection substrate according to claim 5, wherein the fluorescent ink has a characteristic of emitting fluorescence having a wavelength of about 670 nm by excitation light having a wavelength of 635 nm. The biological substance detection substrate according to claim 1;
An optical unit for irradiating the positioning marker with excitation light and detecting fluorescence by the excitation light;
A drive unit for moving the optical unit from the center of the biological material detection substrate toward the outer periphery;
A biological substance detection apparatus comprising: a signal analysis unit that instructs the drive unit so that an irradiation optical axis of the optical unit and a center of the positioning marker coincide with each other according to fluorescence detected by the optical unit. The signal analysis unit
8. The drive unit is instructed to move to the position to obtain the position where the fluorescence value detected by the optical unit is maximized while moving the optical unit in the inner and outer peripheral directions by the drive unit. The biological substance detection apparatus described.

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