JP2006119065A - Diagnostic device and diagnostic method using biochip - Google Patents

Abstract

PROBLEM TO BE SOLVED: To detect a biomolecule without using a conventional repetitive work or a conventional work time by using an apparatus having a simple configuration, and further, based on the detected biomolecule data, a disease can be detected. A diagnostic device using a biochip capable of performing the above diagnosis and a diagnostic method therefor are provided.
SOLUTION: A diagnostic device using a biochip, in which excitation light irradiation means for irradiating excitation light to one or more probes labeled or stained with a fluorescent material of the biochip, and responding to the excitation light A fluorescence detection means for detecting fluorescence emitted from the probe, a disease data storage means for storing disease data associated with the fluorescence, and a disease for which a disease is determined for each probe based on the detected fluorescence and disease data Provided are a diagnostic device including a determination unit and a determination result notification unit for reporting a determination result of a disease, and a diagnosis method thereof.
[Selection] Figure 1

Description

  The present invention relates to a diagnostic apparatus for identifying the type of a biomolecule by using hybridization of a probe provided on a biochip and diagnosing infection of a disease, and a diagnostic method thereof.

  As a method for specifying the type of biomolecules such as artificial nucleic acids such as DNA, RNA and PNA, proteins and peptides, a detection method using hybridization of a probe fixed on a biochip is known.

  Here, the biochip is obtained by fixing a reference biomolecule having a known sequence as a probe on a substrate such as glass, silicon, or plastic. For detection, whether a target biomolecule labeled with a fluorescent substance was administered to this probe, and the target biomolecule was bound to the probe (complementary binding in the case of DNA, RNA, artificial nucleic acid, or affinity binding in the case of protein) Determine whether or not. Specifically, when the target biomolecule is bound to the probe, the fluorescent substance is immobilized together with the probe, so that the fluorescent substance is excited by the excitation light from the light source and emits light. In addition, since no fluorescent substance is present in the probe that does not bind to the target biomolecule, it does not emit light by excitation light. Therefore, by detecting this fluorescence, it can be determined whether or not the target biomolecule is bound (hybridized) to the probe.

  Biomolecule detection devices using biochip hybridization include an excitation light source that irradiates the probe on the biochip with excitation light, a filter that transmits only light in the fluorescent wavelength region generated from the probe, and this filter. 2D optical sensor using a CCD (Charge Coupled Device) or the like that detects fluorescence that has passed through and a control device that creates and displays image data and read data based on the 2D optical sensor. Molecular detection devices are known.

In addition, regarding biomolecule detection devices using hybridization, inventions as shown in Patent Documents 1 and 2 have been proposed in order to solve various problems that occur during detection. Patent Document 1 discloses an invention that can detect hybridization without drying a probe by further including an ammeter for detecting the charge of a probe excited by excitation light in a scanner device that detects fluorescence. It is disclosed. Further, Patent Document 2 discloses an invention that enables image measurement in a bright state over a wide measurement region of a biochip by moving the light detection unit stepwise and combining the images taken by moving the light detection unit. Is disclosed.
JP 2002-181777 A JP 2004-191160 A

However, the invention disclosed in Patent Document 1 and Patent Document 2 is the same as the conventional method for detecting the fluorescence generated from the probe. In general, the fluorescence emitted from the probe by excitation light is excited. Although it has a wavelength shifted to a longer wavelength side than the wavelength of light, this shift width is small, so in order to detect only the fluorescence from the probe without being affected by the excitation light, the fluorescence of It is necessary to install a dedicated filter that matches the wavelength.
Therefore, the detection device becomes a dedicated and expensive device, and it is necessary to replace the filter for each fluorescence to be measured. Therefore, it takes a lot of work steps and a lot of work time to complete the measurement of the entire biochip. It will be.

As for biomolecule detection devices using hybridization, many devices for reading fluorescence emitted from a probe have been proposed. However, judgment processing such as diagnosis is further performed using the fluorescence data. No device has been proposed.
Therefore, for example, in order to diagnose a disease infection using a biochip, it is necessary for a doctor or other person to make a judgment based on fluorescence data detected by a fluorescence reader.

  Therefore, the object of the present invention is to solve the above-mentioned problems, and it is possible to detect biomolecules without using a repetitive work and a long work time as in the past, using an apparatus with a simple configuration. It is an object of the present invention to provide a diagnostic device using a biochip capable of performing disease diagnosis and the like based on the detected biomolecule data, and a diagnostic method therefor.

  In order to achieve the above-mentioned object, as a first embodiment of a diagnostic device using a biochip of the present invention, a diagnostic device using a biochip, which is labeled or stained with a fluorescent substance on the biochip Excitation light irradiation means for irradiating one or more probes with excitation light, fluorescence detection means for detecting fluorescence emitted from the probe in response to the excitation light, and disease data associated with the fluorescence are stored Disease data storage means, based on the detected fluorescence and the disease data, a disease determination means for determining a disease for each probe, a determination result notification means for notifying the determination result of the disease, Can be considered.

Here, the “biochip” is a substrate in which a reference biomolecule having a known sequence is fixed as a “probe”. As a substrate material, glass, silicon, or the like can be used as long as it has a predetermined strength. Any material including plastic can be used. In addition, as the “fluorescent substance” for labeling or staining the probe, any substance can be used as long as it emits fluorescence in response to the irradiation of excitation light. For example, a quantum dot can also be used. It is.
Note that “labeling” refers to covalently binding a fluorescent substance to a probe. In addition, “staining” is performed in order to enhance the detection efficiency by further strengthening only the hybridized mode with an intercalator, a group binder, etc., against the hybridized state of the reference biomolecule and the target biomolecule. Is the method.
The “excitation light” emitted by the “excitation light irradiation means” is not limited to visible light, but includes light of all wavelengths including the ultraviolet region and the infrared region. Further, the excitation light includes light having a single wavelength, and also includes light in which light having a plurality of wavelengths is mixed.

  The “fluorescence detection means” includes any detection means as long as fluorescence can be detected in a manner capable of being compared with “disease data”. For example, it is possible to use a two-dimensional optical sensor such as a CCD, and not only a dedicated device for fluorescence detection but also a general-purpose image reader represented by a monochrome / color scanner or a CCD camera. included. Further, not only those that perform control processing by converting fluorescence into electrical signals, but also those that detect fluorescence using a scientific film or the like.

Regarding the “fluorescence” emitted in response to the irradiation of the excitation light, one type of fluorescence can be used, or a plurality of fluorescences can be used. In addition, fluorescence having a single wavelength can be used, and fluorescence in which light having a plurality of wavelengths is mixed can also be used.
In addition, as a method of associating a fluorescent probe with a disease, the fluorescent material and the disease are associated in advance, and the disease is recognized by identifying the color and wavelength of the fluorescence emitted by the excitation light irradiation. It is also possible to recognize the disease by associating the position of the probe to be fixed with the disease and identifying the position of the fluorescent probe. In the latter case, it is possible to identify a plurality of diseases with one kind of fluorescent substance.

  The “disease data” includes any data as long as it can identify a predetermined disease based on the fluorescence detected by the fluorescence detection means. In addition, the disease data may include accompanying data such as detailed explanation data of the disease and data on how to deal with the disease.

  The “disease determination unit” includes any unit that can determine the disease by comparing the fluorescence detected by the fluorescence detection unit with the disease data. In this disease determination means, it is possible to use a dedicated control means, or to use a function of a general-purpose control device represented by a personal computer.

  The “determination result notifying unit” may notify the determination result by image display, may be notified by voice, or a combination thereof. Any other means can be considered as long as the determination result can be notified to the user.

  In this embodiment relating to the diagnostic apparatus, not only the fluorescence from the probe subjected to hybridization can be detected, but also the disease can be determined with respect to the biomolecule contained in the probe. Regarding the diagnosis of a disease that had to be examined by going to the clinic, the user can easily and quickly obtain a diagnosis result at a low cost in a home or public facility.

  As another embodiment of the diagnostic apparatus using the biochip of the present invention, as the fluorescent material for labeling or staining the probe, a fluorescent material in which two or more fluorescent materials emitting fluorescence of different colors are mixed at a predetermined ratio is used. A diagnostic apparatus can be considered in which the disease determination means determines the disease based on the ratio of the fluorescence of the different colors contained in the detected fluorescence.

  “Fluorescence of different colors” in the present embodiment includes not only light having a single wavelength but also mixed light having a plurality of wavelengths and wavelength bands as long as the light can be identified by the fluorescence detection means. . For example, when the fluorescence detection means includes the optical sensor 1 having the filter 1 and the optical sensor 2 having the filter 2, the light 1 that passes through the filter 1 but does not pass through the filter 2 is transmitted through the filter 2. The light 2 that does not pass through the filter 1 is light of a different color, and the light 1 and the light 2 do not need to be light having a single wavelength.

  In this embodiment, for example, even when two types of filters are used, identification is possible by mixing two color fluorescent materials that pass only through each filter and changing the mixing ratio to various ratios. Many fluorescent materials can be obtained. Therefore, in the past, since it was necessary to replace many filters according to the color of the fluorescent substance, a dedicated device for fluorescence reading is necessary, and the reading operation is repeated while replacing the filter many times. Although it was necessary, in this embodiment, a general-purpose color scanner or the like can also be used, and a fluorescence reading operation can be performed at once without using a low-cost apparatus and replacing a filter. it can. Accordingly, it is possible to reduce the manufacturing cost of the diagnostic device, simplify and simplify the diagnostic work, and reduce the diagnostic cost.

As another embodiment of the diagnostic apparatus using the biochip of the present invention, two or more probes that bind to different structural parts of the same target biomolecule are used, and based on the detected fluorescence from each of the probes. Thus, a diagnostic apparatus for determining the specificity for the target biomolecule can be considered.
According to this embodiment, it is possible to quantitatively determine the specificity related to the identification of biomolecules that are generally difficult to determine.

As another embodiment of the diagnostic apparatus using the biochip of the present invention, an image reading unit that reads an image of a predetermined region including one or more biochips based on data detected by the fluorescence detection unit And an image analyzing means for analyzing the image based on the read image, and a positioning marker that emits light in response to the excitation light is predetermined on the biochip with respect to the position of the probe. Provided in position,
The image analysis means determines the position of the probe based on the position of the positioning marker in the read image, and the disease determination means determines the disease based on the fluorescence emitted from the probe whose position is determined. A diagnostic device that performs the determination is conceivable.

The “image reading unit” in the present embodiment is a control unit that creates image data of a predetermined region including the biochip based on the data detected by the fluorescence detection unit, and a dedicated control unit can also be used. In addition, the function of a general-purpose image reading apparatus such as a scanner can be used.
The “image analysis means” is a control means for creating fluorescence data in such a manner that the disease determination means can determine the disease using the image data of the predetermined area created by the image reading means. It is also possible to use these control means, and it is also possible to use functions of a general-purpose control device such as a personal computer.

  Any “positioning marker” may be used as long as it emits light in response to excitation light, including a substance similar to a fluorescent substance that labels or stains a probe. Further, as a method for the image analysis means to identify the positioning marker, it is possible to identify the positioning marker by arranging each positioning marker in a predetermined positional relationship on the biochip. For ease of use, it is also possible to use a fluorescent material of a different color (monochromatic or mixed color) from the fluorescent material that labels or stains the probe.

  In this embodiment, since the position of each probe on the image data can be recognized in advance, it is not necessary to perform all image processing that is generally performed, and the fluorescence of each probe can be identified with less control processing in a shorter time than in the past. can do. Also, the control device can be simplified, and the data capacity can be reduced even when data transmission is performed.

  As a first embodiment of a diagnostic method using a biochip of the present invention, a diagnostic method using a biochip, which excites one or more probes labeled or stained with a fluorescent substance on the biochip A step of irradiating light, a step of detecting fluorescence emitted from the probe in response to the excitation light, and a disease for each probe based on the detected fluorescence and the disease data associated with the fluorescence. A diagnostic method including the step of performing the determination and the step of notifying the determination result of the disease is conceivable.

  As another embodiment of the diagnostic method using the biochip of the present invention, the fluorescent material for labeling or staining the probe is a fluorescent material in which two or more fluorescent materials emitting fluorescence of different colors are mixed at a predetermined ratio. Thus, a diagnostic method for determining a disease based on the ratio of the fluorescence of the different colors included in the detected fluorescence can be considered.

  As another embodiment of the diagnostic method using the biochip of the present invention, based on the fluorescence from each probe detected using two or more probes that bind to different structural parts of the same target biomolecule. Thus, a diagnostic method for judging the specificity for the target biomolecule can be considered.

  As another embodiment of the diagnostic method using the biochip of the present invention, a positioning marker that emits light in response to the excitation light is provided on the biochip at a position predetermined with respect to the position of the probe. A step of reading an image of a predetermined region including one or more of the biochips, and a step of determining the position of the probe based on the position of the positioning marker in the read image. A diagnostic method for determining the disease based on the determined fluorescence of the probe is conceivable.

  In the diagnostic method of the present invention as described above, not only a predetermined diagnostic apparatus is used, but also, for example, a mode such as a diagnostic system that performs diagnosis using an electric communication line or the like, A mode in which a color is judged and a diagnosis is performed is also included.

  According to the diagnostic apparatus of the present invention, it is possible not only to detect the fluorescence from the probe, but also to determine the disease for the biomolecule contained in the probe. Diagnosis results of diseases can be obtained at a low cost.

  Further, by using a fluorescent material in which two or more fluorescent materials that emit fluorescence of different colors are mixed at a predetermined ratio, the manufacturing cost of the diagnostic apparatus is reduced, the diagnostic work is simplified, facilitated, and diagnosed. Cost reduction is possible.

  In addition, by providing a positioning marker on the biochip, the fluorescence of each probe can be identified in a short time with less control processing than in the past.

  Furthermore, in the diagnostic method of the present invention, not only a predetermined diagnostic apparatus is used, but also a diagnostic system for performing a diagnosis using an electric communication line or the like, and a person makes a diagnosis by judging the color of fluorescence using the naked eye. It can also be applied to such a diagnostic method.

Embodiments of a diagnostic device and a diagnostic method using the biochip of the present invention will be described below in detail with reference to the drawings.
(Description of the entire diagnostic device)
First, the outline | summary of one embodiment of the diagnostic apparatus of this invention is demonstrated using the schematic diagram of the diagnostic apparatus shown in FIG. FIG. 1 (a) shows an embodiment of an integrated diagnostic apparatus that performs all processing by a single device, and FIG. 1 (b) shows a reading unit that performs fluorescence detection processing, and diagnostic processing. An embodiment of a separation type diagnostic apparatus in which a control unit for performing the above and the like is performed by another apparatus is shown.

First, an embodiment of an integrated diagnostic apparatus will be described with reference to FIG. A reading bed 12 is provided on the upper surface of the diagnostic apparatus 1, and the biochip 2 is placed on the reading bed 12 to read fluorescence. In the present embodiment, a plurality of biochips 2 can be placed on the reading bed 12 and fluorescence can be read simultaneously.
Here, FIG. 11 shows an example of a biochip. In the biochip 2 of FIG. 11, PNA is immobilized as a probe as a reference biomolecule on a plastic substrate. In diagnosis, target biomolecules collected from a user's body fluid are labeled or stained with a predetermined fluorescent substance, and the target biomolecules labeled or stained with the fluorescent substance and a plastic substrate on which the probe is fixed are placed on a high level. Hybridization treatment is performed in the hybridization solution.

As shown in FIG. 11, in the biochip 2, 4 × 5 rows = 20 probes 3 (probe numbers J = 1 to 20) can be fixed on one chip. Further, positioning markers A to C and a bar code are provided outside the 20 probes 3. Details of the biochip 2 will be described later.
By adopting a PNA chip as a biochip as in this embodiment, it becomes easy to use a plastic substrate, and the risk of target biomolecule infection due to damage to the biochip being handled can be reduced. it can. Moreover, since it can be handled at room temperature by using PNA, the user can easily diagnose.

  In the present embodiment, a fluorescent material using quantum dots is used. Fluorescent materials using quantum dots generate extremely bright fluorescence over a long period of time, and are characterized by a large difference (Stokes shift or half width) between the wavelength of the excitation light and the wavelength of the fluorescence emitted in response to the excitation light. Have.

Here, returning to the description of FIG. 1A, a moving carriage 8 including an excitation light irradiation lamp 4 and an optical sensor 5 is installed in the diagnostic apparatus 1 in a movable state. The movable carriage 8 is driven by a drive motor 11 (see FIG. 3, not shown in FIG. 1).
As shown by the arrow in FIG. 1A, excitation light is irradiated from the excitation light irradiation lamp 4 to the biochip 2. In this embodiment, since a fluorescent material to which a quantum dot having a large wavelength shift is applied is used, ultraviolet light is used as excitation light, and visible light can be used as fluorescence emitted in response to this excitation light. Can be detected simultaneously using a single light source. Therefore, unlike the prior art, it is not necessary to repeat the measurement by exchanging the filters on both the excitation light source side and the fluorescence detection side.

  The excitation light irradiation lamp 4 has a length that covers the width direction of the reading bed 12, and is placed on the reading bed 12 by the carriage 8 moving in the direction of the arrow by the entire length of the reading bed 12. All the biochips 2 can be irradiated with excitation light. Therefore, among the probes 3 of the biochip 2, the probe in which the reference biomolecule and the target biomolecule are bound (hybridized) emits fluorescence corresponding to the fluorescent material labeled or stained.

  The fluorescence emitted from the probe 3 on the biochip 2 is detected by the optical sensor 5 as shown by the arrow in FIG. In this photosensor 5, photodetection elements such as CCDs are arranged in N lines so as to cover the width direction of the reading bed 12. The N light detecting elements can convert the light energy of the incident fluorescence into an electric signal, and the signal processing circuit 120 in the control device 6 electrically connected to the light sensor 5 (see FIG. 3). Are output as N sequential voltage signals and used for subsequent control processing. Further, when the carriage 8 moves in the direction of the arrow by the entire length of the reading bed 12, the fluorescence emitted from all the biochips 2 placed on the reading bed 12 is detected by the N photodetecting elements. be able to.

  In the photosensor 5 of the present embodiment, as with a general-purpose color scanner, three photodetecting element lines including blue (B), green (G), and red (R) color filters are provided. FIG. 2 schematically shows the structure of some of the N photodetecting elements provided in a line (for three colors). As shown in FIG. 2, a light detection element provided with a blue filter that transmits only blue light on the light receiving surface side of the light detection element, and a green filter that transmits only green light on the light reception surface side of the light detection element And a photodetecting element provided with a red filter that transmits only red light on the light receiving surface side of the photodetecting element. These color filters have the same spectral transmittance as the color filters used in general-purpose color scanners.

  The light sensor 5 detects the intensity of each of the blue, green, and red light contained in the fluorescence emitted from the probe 2 and, as described above, from the signal processing circuit 120 (see FIG. 3) in the control device 6. The output voltage signal can be used for control processing. Based on this fluorescence detection data, biomolecules (including pathogenic viruses, pathogenic bacteria, and the like) contained in each probe are determined and diagnostic processing is performed. A diagnosis result, which is output data of the diagnosis process, can be displayed on the image display device 7 and printed by a printer as necessary. The control of these diagnoses will be described later.

Next, an embodiment of the separation type diagnostic apparatus shown in FIG. The basic equipment configuration is the same as that of the integrated diagnostic apparatus, but the movable carriage 8 having the reading bed 12 on the upper surface and the excitation light irradiation lamp 4 and the optical sensor 5 inside is movable. The difference is that the reading unit 1A installed is separated from a control unit 1B that performs diagnostic control based on detection data from the reading unit 1A. The image display device 7 and the printer 9 can be configured as separate devices or as a part of the control unit 1B.
In this separation type diagnostic apparatus, for example, it is possible to use as a reading unit 1A only by changing the light source of a general-purpose color scanner to an excitation light irradiation lamp, and the control unit 1B includes a general-purpose personal computer. Can be used.

As described above, in the diagnostic apparatus of the present invention as shown in FIGS. 1A and 1B, it is necessary to provide a large number of special filters having a small wavelength bandwidth as in the conventional fluorescence reading apparatus of a biochip. Therefore, the manufacturing cost of the diagnostic device can be reduced. In addition, in the conventional reader, it is necessary to replace the filter for each labeled fluorescent substance and repeat the detection operation. However, in the diagnostic device of the present invention, it is possible to perform all detections with one reading. In addition, a plurality of biochips can be detected at a time. Accordingly, the processing time for reading the fluorescence is greatly shortened, and the user can easily make a diagnosis, and the diagnosis cost can be reduced.
Furthermore, the separation-type diagnostic apparatus shown in FIG. 1B can be configured using a general-purpose scanner and a general-purpose personal computer, and can be expected to further reduce manufacturing costs.

(Description of control device)
Next, the control device 6 that performs control processing for diagnosis will be described in detail with reference to FIGS. FIG. 3 shows a block diagram showing the contents of the control device 6, and FIG. 4 shows a functional block diagram related to diagnostic control.

<Explanation of block diagram>
The block diagram of FIG. 3 shows a control device corresponding to the integrated diagnostic device of FIG. 1A, but the apparatus configuration of the separated diagnostic device shown in FIG. 1B is the same. The control device 6 includes a calculation unit 104 having a CPU 101, a ROM (Read Only Memory) 102, and a RAM (Random Access Memory) 103, and performs a predetermined calculation process to perform a control process for diagnosis. .
In addition, these devices receive a signal from an external device via the interface circuit 110 and transmit the signal to the external device.

  First, a signal received by the control device 6 from an external device will be described. The intensity of blue, green, and red light detected by the optical sensor 5 is converted into a voltage signal by the signal processing circuit 120, and the interface circuit 110. Is output. Although not shown in FIGS. 1A and 1B, a signal from the operating device 10 is output to the interface circuit 110 via the signal processing circuit 122.

  Next, a signal transmitted from the control device 6 to an external device will be described. When an irradiation start signal is transmitted to the lamp driving circuit 124 based on a signal from the operating device 10 based on a user operation or a preset program, the excitation light irradiation lamp 4 is turned on by the lamp driving circuit 124, Irradiate with excitation light. Also, by transmitting a drive start signal to the motor drive circuit 126, the drive motor 11 is turned on by the motor drive circuit 126, and the carriage 8 on which the excitation light irradiation lamp 4 and the optical sensor 5 are mounted can be driven.

  Further, when diagnostic control is performed by the arithmetic unit 104 described above and predetermined diagnostic data is created, an image display start signal is transmitted to the display drive circuit 128 to display a predetermined image on the image display device 7. can do. Similarly, a predetermined diagnosis result can be printed by the printer 9 by transmitting a print start signal to the printer drive circuit 130.

<Explanation of functional block diagram>
Next, with reference to FIG. 4, a functional block diagram relating to diagnostic control processing according to the present invention will be described. Explaining in the order of diagnosis using the diagnostic apparatus 1, first, the excitation light irradiation unit 310 including the excitation light irradiation lamp 4 and the lamp driving circuit 124 is operated by a signal from the operating device 10 or a predetermined program. When the transmitted irradiation start signal is received, the excitation light irradiation lamp 4 is turned on to irradiate the excitation light. Next, the fluorescence detection means 320 including the optical sensor 5 and the signal processing circuit 120 detects fluorescence emitted from the biochip 2 in response to the irradiated excitation light. The image reading unit 210 creates image data of the entire reading bed 12 based on the signal detected by the fluorescence detecting unit 320, and the image analyzing unit 220 further determines the positioning markers A to C based on the image data. Is used to recognize the position of each biochip and the position of each probe on the biochip, and to generate detected fluorescence data for each probe of each biochip.

  Next, the disease determination unit 240 reads the association table between the fluorescence and the disease stored in the disease data storage unit 230 (specifically, the area of the RAM 103), and analyzes the association table between the fluorescence and the disease, and image analysis. The determination processing of the disease contained in each probe is performed by comparing the detection fluorescence data for each probe of each biochip created by the means 220.

Then, the determination result notification unit 250 transmits image data based on the determination result to the image display unit 330. The image display unit 330 including the image display device 7 and the display drive circuit 128 displays a predetermined diagnostic image on the image display device 7 based on the image data received from the determination result notification unit 250. Further, the diagnosis result can be printed by the printer 9 as necessary.
In addition, description about the control means regarding the operating device 10, the printer 9, and the drive motor 11 shown in the block diagram of FIG. 3 is omitted in this functional block diagram.

(Description of biochip)
Next, the biochip 2 according to the present invention will be described in detail.
In the biochip 2 of the present embodiment, probes having probe numbers J = 1 to 20 (hereinafter referred to as probes #J (J = 1 to 20)) made of PNA are fixed on a plastic substrate. As shown in FIG. 11, the arrangement is 4 × 5 rows from the rows of probes # 1 to # 4 to the rows of # 17 to # 20. Positioning markers A to C are provided at the three corners of the outer biochip 2, and a barcode for identifying each biochip 2 is described with a fluorescent material between the positioning marker A and the positioning marker B. Has been.

  In addition, the relationship between the fluorescent substance and the reference biomolecule (disease) corresponding to the fluorescent substance in this embodiment is shown in the fluorescence-disease control table of FIG. In this table, fluorescent materials with fluorescent material numbers K = 1 to 21 (hereinafter referred to as fluorescent materials #K (K = 1 to 21)) are shown in the vertical column. Disease A to Disease T correspond, and the fluorescent material # 21 corresponds to a positioning marker or a barcode. Quantum dots are used as the fluorescent material.

  Specifically, for example, for a probe to which a reference biomolecule having the same sequence as that of disease A is immobilized, a target biomolecule collected from a user's body fluid or the like is labeled or stained with fluorescent substance # 1. Then, a hybridization process is performed. Similarly, a probe in which a reference biomolecule having the same sequence as disease B to disease T is immobilized is obtained by labeling or staining fluorescent substances # 2 to 20 on the target biomolecule collected from the body fluid of the user, etc. And a hybridization treatment is performed. In the present embodiment, the fluorescent material # 21 is used for providing a positioning marker or a barcode at a predetermined position of the biochip. As will be described in detail below, in this embodiment, the number of fluorescent substances corresponding to the disease is 20, and the number of probes fixed to the biochip is also 20. Therefore, the probe number J (J = 1 to 20) ) And the fluorescent substance number K (K = 1 to 20) correspond to each other, but it is not always necessary to match the two.

  The fluorescent materials # 1 to 20 to be labeled or stained will be described as a single substance of three fluorescent materials of blue, green, and red, or a fluorescent material obtained by mixing these fluorescent materials. These three fluorescent substances are a blue fluorescent substance that emits blue fluorescence having a wavelength of 450 nm by ultraviolet irradiation, a green fluorescent substance that emits green fluorescence having a wavelength of 530 nm by ultraviolet irradiation, and an ultraviolet irradiation. It is a fluorescent material of three colors, a red fluorescent material that emits red fluorescence having a wavelength of 650 nm. In this embodiment, the wavelengths of blue, green, and red fluorescence coincide with the peak wavelengths of the blue, green, and red color filters provided in the optical sensor 5 of the diagnostic apparatus. However, it is not always necessary to match the wavelength of light of each color with the peak wavelength of the color filter. Even if it does not match the peak wavelength, the light intensity of each detected color is corrected to perform appropriate analysis processing. Can be done. Accordingly, light of any wavelength can be used as long as the blue, green, and red light have wavelengths that transmit only the blue, green, and red color filters, respectively.

  As shown in the fluorescence-disease control table of FIG. 8, for example, in the fluorescent substance # 1, the reference biomolecule having the same structure as that of the disease A is fixed, and only the blue fluorescent substance (blue 100%, green 0%, red 0%) is fluorescent or # 1 labeled or stained. Further, for example, in the fluorescent material # 5, the reference biomolecule having the same structure as the disease E is fixed, and the fluorescent material # 5 is mixed with the blue fluorescent material 60%, the green fluorescent material 20%, and the red fluorescent material 20%. Is labeled or stained. Similarly in other probes, in this embodiment, the mixed ratio of the fluorescent materials of blue, green, and red is changed by 20% to form a total of 21 types of mixed fluorescent materials. However, in this embodiment, the fluorescent material # 21 containing only the red fluorescent material (blue 0%, green 0%, red 100%) is used for the positioning markers A to C and the barcode. Thereby, the positioning marker and the barcode can be easily identified by identifying the fluorescent color. However, even if fluorescence of the same color as that of other probes is used, it is also possible to identify them by positioning markers, barcode arrangement (for example, distance between markers), or the like.

Next, the fluorescence emitted when the probe is irradiated with ultraviolet excitation light will be described by taking fluorescent substance # 5 as an example. If the reference biomolecule and the target biomolecule of the probe labeled or stained with the fluorescent substance # 5 bind (hybridize), that is, if the body fluid of the user contains the disease E, the fluorescent substance # 5 is excited. When excited by light, it emits fluorescence. This fluorescence basically emits fluorescence at a ratio corresponding to the mixing ratio of the fluorescent substances of the respective colors (blue, green, red). Specifically, the ratio of the light intensity of the blue fluorescent light (light transmitted through the blue color filter) emitted from the blue fluorescent material having a mixing ratio of 60% is the value 60% of the reference light intensity LB (5) plus or minus the fluctuation range DB. The ratio of the light intensity of the green fluorescent light (light transmitted through the green color filter) emitted from the green fluorescent material with the mixing ratio of 20% is the value _DB5 of (5), and the value of the reference light intensity LG (5) is 20%. a value D _G5 plus or minus variation width DG (5), the light intensity ratio of the red fluorescence emitted from the mixing ratio of 20% red fluorescent substance (light transmitted through the red color filter), the reference light intensity LR (5 ) Value 20% plus or minus fluctuation range DR (5) value _DR5 .

These fluctuation range values D _B5 , D _G5 , and D _R5 can be set to appropriate values by conducting a repeated test in advance. Depending on the probe, it is conceivable that the overall fluorescence intensity is different. However, by changing the fluorescence intensity and conducting a test repeatedly in advance, an appropriate variation width value D _B5 in any fluorescence intensity is considered. , D _G5 , D _R5 can be set. Further, in this embodiment, the mixing ratio of the fluorescent materials of the respective colors (blue, green, red) is changed by 20%, but when the value of the fluctuation range of these fluorescences is sufficiently smaller than 20%. It is also possible to make the change in the mixing ratio even smaller (for example, by 10%), and in this case, more mixed colors can be used.
As described above, the fluorescence emitted from the fluorescent material has been described by taking the fluorescent material # 5 as an example. However, in other probes, the intensity of the fluorescence corresponding to the mixing ratio of the fluorescent materials of the respective colors can be obtained in the same manner. It is possible to identify the fluorescent material by recognizing the ratio.

However, in the diagnostic apparatus and diagnostic method of the present invention, the fluorescence of each basic color is not limited to blue, green, and red, and any color can be determined according to the transmission wavelength of the filter provided in the optical sensor 5. Is possible. Also, any kind of fluorescent material can be used depending on the number of filters. Also, it is possible to use a monochromatic fluorescent material without mixing the fluorescent materials, and for example, it is possible to identify a disease by the position of the probe. In this case, it may be considered that no special filter is provided in the optical sensor 5.
Furthermore, in the biochip according to the present invention, strong fluorescence can be obtained by using quantum dots as the fluorescent material. Therefore, it is conceivable that the fluorescence is captured with the naked eye and the diagnosis is made by judging the color with the human eye. . It is also conceivable to make a diagnosis by taking fluorescence with a chemical film and determining the color based on the photograph.

(Description of diagnostic control processing)
Next, diagnosis control processing by the diagnosis apparatus of the present invention will be described in detail using the flowcharts shown in FIGS.
In the control processing shown in the flowcharts of FIGS. 5 to 7, the biochip 2 subjected to the hybridization processing between the target biomolecule collected from a human body fluid or the like and each probe is used as the diagnostic device 1 of the present invention. It is placed on the reading bed 12 and is ready for diagnostic control processing by the diagnostic apparatus 1. Here, as long as the biochip 2 is within a predetermined readable area of the reading bed 12, an arbitrary number of biochips 2 can be mounted and fluorescence can be read simultaneously. Moreover, the biochip 2 may be placed at any position and orientation as well. When the excitation light is irradiated, fluorescence of a predetermined color (red in the present embodiment) is emitted from the positioning markers A to C attached to each biochip 2, so that each biomarker is detected by detecting the fluorescence of the positioning marker. The position of the chip 2 can be recognized. As described above, the probe 3 in which the target biomolecule and the reference biomolecule are bound (hybridized), that is, the probe 3 in which a biomolecule related to a certain disease exists is fluoresced in a predetermined color in response to irradiation with excitation light. To emit.

<Description of main routine>
In FIG. 5, first, an irradiation start signal is transmitted to the excitation light irradiation means 310, the excitation light irradiation lamp 4 is turned on, and irradiation of excitation light (ultraviolet rays in the present embodiment) is started. A detection start signal is transmitted to turn on the optical sensor 5 to enable detection of fluorescence from the biochip probe (step S10). Next, a drive start signal is transmitted to start the rotation of the drive motor 11, and the movement of the carriage 8 on which the excitation light irradiation lamp 4 and the optical sensor 5 are mounted is started (step S12). As the carriage 8 moves, all the biochips 2 on the reading bed 12 are irradiated with excitation light, fluorescence emitted from a probe in which the reference biochip and the target biomolecule are bound (hybridized), a positioning marker, Fluorescence emitted from the barcode is detected by the optical sensor 5 (step S14). As described above, the fluorescence detection by this optical sensor detects the intensity of light for each of blue, green, and red. When the carriage 8 moves the entire length of the reading bed 12 and reaches the end position, the carriage 8 is stopped, excitation light irradiation is stopped, and detection by the optical sensor 5 is stopped. Then, a process for returning the carriage 8 to the original start position is performed (step S16).

Based on the fluorescence detected by each color (blue, green, red) optical sensor 5, the image reading means 210 creates image data of the entire image bed 12 (step S18). Then, based on this image data, the image analysis means 220 performs control processing from step S20 to step S26.
First, the fluorescent light emitting a single red color is identified to recognize the positioning marker and the barcode. Then, from the information read from the barcode, the biochip number I is identified, and from the positional relationship between the positioning marker and the barcode, the biochip of each biochip number I (hereinafter referred to as biochip #I), The positions of the positioning markers A to C of the biochip #I are recognized (step S20).

  Based on the positions of the positioning markers A to C, the predicted positions of the probes # 1 to # 20 of each biochip #I can be calculated. Using the calculated predicted position data of each probe, the position of each probe #J can be recognized (step S22). For example, in the process of identifying the intensity of the detected fluorescent light for each pixel unit (unit of each light detection element of the light sensor) based on the image data of the entire reading bed 12, the position of the positioning markers A to C is used. Of course, it is possible to omit this identification process for an area where it is apparent that there is no probe, and in some areas, it may be possible to thin out pixels for this process. In that case, the identification processing time can be greatly shortened.

  When the reference biomolecule to be fixed and the position of the probe are fixed as in this embodiment, even if all the probes are labeled or stained with the same color fluorescent substance, From the location, it is possible to identify each reference biomolecule. However, in the present embodiment, in order to ensure correct diagnosis even when the reference biomolecule is mistakenly fixed at a different probe position, and any reference biomolecule is labeled on any probe. Alternatively, in order to be able to cope with a free process of staining, the detected fluorescence color is discriminated, and the process of discriminating the reference biomolecule based on the discriminated color difference is performed. Details of this processing will be described later.

  Further, the total number BCTOTAL of the biop 2 that has been read is recognized from the detected positioning markers A to C and the barcode information, and stored in the RAM 103 (step S24). Then, for each probe #J of each biochip #I, the detected fluorescence light intensity is detected as blue fluorescence detection light intensity MLB (J), green fluorescence detection light intensity MLG (J), and red fluorescence. A detection fluorescence data table is created for each of the detected light intensities MLR (J) (step S26).

  Here, FIG. 9 shows an example of the detected fluorescence data table. In this embodiment, fluorescence readings are simultaneously performed for biochips # 1 to # 3. For each biochip, the detection light intensity for each of the probes # 1 to # 20 is shown in the vertical column of the table shown in FIG. 9. For the same probe #J, the blue detection light intensity MLB (J ), Green detection light intensity MLG (J) and red detection light intensity MLR (J). In the column of the table, the column indicated as NONE indicates that no fluorescence was detected.

  Biochip # 1 shows that fluorescence was detected with probes # 5, # 12, # 14, and # 18, and biochip # 2 showed fluorescence with probes # 2, # 3, # 17, and # 18 Biochip # 3 shows that fluorescence was detected with probes # 4, # 8, and # 13.

  Subsequently, the process proceeds to step S28 shown in FIG. 6, and the fluorescence-disease reference table stored in the disease data storage unit 230 (specifically, the area of the RAM 103) is read. As detailed above, an example of a fluorescence-disease reference table is shown in FIG. Based on this table, it is possible to perform a diagnosis determination process for determining the disease A to the disease T from the ratios of the detected blue, green, and red light intensities.

  Next, the disease determination means 240 performs a disease determination process for each biochip I by comparing the detected fluorescence intensity of each probe J with the fluorescence-disease contrast table. First, 1 is input as the value of the biochip number I (step S30), and 1 is input as the value of the probe number J (step S32). And it is judged whether fluorescence is detected about probe #J of this biochip #I (step S34). If it is determined in this determination that fluorescence is detected (YES), a disease determination subroutine (step S36) is performed, and the process proceeds to step S38. Details of this disease determination subroutine will be described later.

  If it is determined in step S34 that no fluorescence is detected (NO, if blue, green, and red are described as NONE in the table of FIG. 8), the disease determination subroutine is not performed and step S38 is performed. Proceed to In step S38, it is determined whether or not the value of the probe number J is greater than or equal to the number of biochip probes (20 in this embodiment). In this determination, if it is determined that the value of J is smaller than the number of probes of the biochip (NO), 1 is added to the value of J (step S40), and the processing from step S34 to step S38 is repeated.

  If it is determined in step S38 that the value of J is equal to or greater than the number of probes on the biochip (YES), the disease determination process for all probes on one biochip is completed. It is determined whether the value of the chip number I is equal to or greater than the total number of biochips BCTOTAL (3 in this embodiment). In this determination, if it is determined that the value of the biochip number I is smaller than the BCTOTAL (NO), 1 is added to the value of the biochip number I (step S44), and the next biochip I is set to step S32. To S42 are repeated. If it is determined in step S42 that the value of the biochip number I is greater than or equal to BCTOTAL (YES), the process proceeds to step S46.

<Description of disease determination subroutine>
Here, the disease determination subroutine shown in step S36 will be described in detail with reference to the flowchart of FIG.
In this subroutine, first, 1 is input as the value of the fluorescent substance number K (step S100). Then, the light intensity MLB (J), MLG (J), MLR (J) for each color of the detected fluorescence (blue, green, red) is compared with the light intensity of the fluorescence emitted from the fluorescent material #K. Do. Specifically, first, the light intensity MLB (J) of the detected blue component of the fluorescence is larger than the reference light intensity LB (K) −the fluctuation range DB (K) of the blue component of the fluorescent material #K. It is smaller than the intensity LB (K) + the fluctuation range DB (K), that is, whether the detected light intensity MLB (J) is within the range of the reference light intensity LB (K) plus or minus the fluctuation range DB (K). Judgment is made (step S102). If it is determined that it is not within this range (NO), the process proceeds to step S110 as it is.

  If it is determined in step S102 that MLB (J) is within the range of LB (K) plus / minus DB (K) (YES), the light intensity MLG of the detected green component of the fluorescence is next detected. (J) is larger than the reference light intensity LG (K) of the green component of the fluorescent material # K−the fluctuation range DG (K) and smaller than the reference light intensity LG (K) + the fluctuation range DG (K). It is determined whether or not the light intensity MLG (J) is within the range of the reference light intensity LG (K) plus / minus fluctuation range DG (K) (step S104). If it is determined that it is not within this range (NO), the process proceeds to step S110 as it is.

  If it is determined in step S104 that MLG (J) is within the range of LG (K) plus or minus DG (K) (YES), then the light intensity MLR of the detected red component of the fluorescence (J) is larger than the reference light intensity LR (K) −variation width DR (K) of the red component of the fluorescent material #K and smaller than the reference light intensity LR (K) + variation width DR (K), that is, detected. It is determined whether or not the light intensity MLR (J) is within the range of the reference light intensity LR (K) plus or minus fluctuation range DR (K) (step S106). If it is determined that it is not within this range (NO), the process proceeds to step S110 as it is.

  If it is determined in step S106 that MLR (J) is within the range of LR (K) plus or minus DR (K) (YES), the detected fluorescence is detected in all blue, green, and red light components. Thus, it can be determined that the fluorescent material #K emits fluorescence. Accordingly, it is determined that the biomolecule of the disease corresponding to the fluorescent material #K exists in the probe #J, and the value of this J, the value of K, and the corresponding disease name (for example, the fluorescent material # 5 is the disease. E) is stored in the RAM 103 (step S108).

  Then, it is determined whether or not the value of K is 20 or more, which is the maximum number of fluorescent substances (step S110). If it is determined in this determination that the value of K is smaller than 20 (NO), 1 is added to the value of K (step S112), and the processing from step S102 to step S110 is repeated again. If it is determined in step S110 that the value of K is 20 or more (YES), this subroutine is terminated.

<Description of main routine (re)>
Returning to the description of the main routine again, after performing disease determination based on the detected fluorescence for all probes #J of all biochips #I by the disease determination processing of step S30 to step S44 by the disease determination means 240, A diagnostic result table is created for each probe #J of biochip #I and stored in the RAM 103 (step S46).

  Here, FIG. 10 shows an example of a diagnosis result table. This example relates to biochip # 1. The name of the disease in which the biomolecule was detected is described together with the probe number J. Moreover, it is also possible to add explanation of each disease, treatment method, etc. as needed. Then, the determination result notifying unit 250 transmits image data corresponding to the diagnosis result table to the image display unit 330 and displays a predetermined image on the image display device 7 (step S48). The user can know an appropriate diagnosis result from this image. Therefore, according to the diagnostic apparatus of the present invention, it is possible to easily diagnose a disease without taking the trouble of going to a hospital or clinic and receiving an examination. The diagnosis result can be displayed not only on the image display device but also on a printer or the like.

(Other embodiments)
<Embodiment for determining specificity>
In the above-described embodiment, one probe corresponds to one biomolecule, but a plurality of probes having reference biomolecules that bind to different structural parts are used for the same target biomolecule. It is also conceivable to detect fluorescence. In this embodiment, the specificity for the target biomolecule can be quantitatively determined based on the fluorescence of each detected probe.

<Embodiment for Determining Probability of Disease>
An embodiment is also conceivable in which the fluorescence of a plurality of probes having the same reference biomolecule is detected, and the probability that the biomolecule exists, that is, has a disease, is determined according to the ratio of the fluorescent probe. It is done. In addition, for one probe, the probability that a biomolecule is present (affected by a disease) is determined based on the ratio between the area where the fluorescent substance is fixed and the area of the image (pixel) where fluorescence is detected. Forms are also conceivable.

  Furthermore, the diagnostic apparatus and diagnostic method of the present invention are not limited to the above-described embodiments, and include various other embodiments.

It is a schematic diagram showing one embodiment of a diagnostic device of the present invention, (a) shows an embodiment of an integrated diagnostic device, and (b) shows an embodiment of a separated type diagnostic device. It is a perspective view which shows the structure of the light beam 5 with which the diagnostic apparatus shown in FIG. 1 was equipped. It is a block diagram which shows one Embodiment of the control apparatus of the diagnostic apparatus of this invention. It is a functional block diagram regarding the diagnostic control processing of the present invention. It is a flowchart which shows the main routine regarding the control processing of the diagnostic apparatus of this invention, and a diagnostic method. It is a flowchart which shows the main routine regarding the control processing of the diagnostic apparatus of this invention, and a diagnostic method. It is a flowchart which shows the detail of the disease determination subroutine shown in the flowchart shown in FIG. It is a table | surface which shows the Example of the fluorescence-disease contrast table used for the control processing of the diagnostic apparatus of this invention, and a diagnostic method. It is a table | surface which shows the Example of the detection fluorescence data table used for the control processing of the diagnostic apparatus of this invention, and a diagnostic method. It is a table | surface which shows the Example of the diagnostic result table | surface used for the control processing of the diagnostic apparatus of this invention, and a diagnostic method. It is a schematic diagram which shows the Example of the biochip used for the diagnostic apparatus and diagnostic method of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Diagnostic apparatus 1A Reading part 1B Control part 2 Biochip 3 Probe 4 Excitation light irradiation lamp 5 Optical sensor 6 Control apparatus 7 Image display apparatus 8 Carriage 9 Printer 10 Operation apparatus 11 Drive motor 12 Reading bed 101 CPU
102 ROM
103 RAM
104 arithmetic unit 110 interface circuit 120 signal processing circuit 122 signal processing circuit 124 lamp drive circuit 126 motor drive circuit 128 display drive circuit 130 printer drive circuit 200 control means 210 image reading means 220 image analysis means 230 disease data storage means 240 disease determination means 250 Determination result notifying means 310 Excitation light irradiation means 320 Fluorescence detection means 330 Image display means

Claims (8)

A diagnostic device using a biochip,
Excitation light irradiation means for irradiating one or more probes labeled or stained with a fluorescent substance on the biochip with excitation light;
Fluorescence detection means for detecting fluorescence emitted from the probe in response to the excitation light;
Disease data storage means for storing disease data associated with the fluorescence;
A disease determination means for determining a disease for each probe based on the detected fluorescence and the disease data;
Determination result notifying means for notifying the determination result of the disease;
A diagnostic device comprising:   As the fluorescent material for labeling or staining the probe, a fluorescent material in which two or more fluorescent materials emitting different colors of fluorescence are mixed at a predetermined ratio, and the ratio of the fluorescence of the different colors included in the detected fluorescence is set. The diagnosis apparatus according to claim 1, wherein the disease determination means determines a disease based on the disease.   Using two or more of the probes that bind to different structural parts of the same target biomolecule, and determining the specificity for the target biomolecule based on the fluorescence from each detected probe The diagnostic device according to claim 1 or 2. Image reading means for reading an image of a predetermined region including one or more of the biochips based on the data detected by the fluorescence detection means;
Image analysis means for analyzing the image based on the read image,
On the biochip, a positioning marker that emits light in response to the excitation light is provided at a position predetermined with respect to the position of the probe,
The image analysis means determines the position of the probe based on the position of the positioning marker in the read image,
The diagnostic apparatus according to any one of claims 1 to 3, wherein the disease determination unit determines the disease based on the fluorescence emitted from the probe whose position is determined. A diagnostic method using a biochip,
Irradiating one or more probes labeled or stained with a fluorescent substance on the biochip with excitation light;
Detecting fluorescence emitted from the probe in response to the excitation light;
Determining disease for each probe based on the detected fluorescence and the disease data associated with the fluorescence;
Notifying the determination result of the disease;
A diagnostic method comprising the steps of:   The fluorescent substance that labels or stains the probe is a fluorescent substance in which two or more fluorescent substances that emit fluorescence of different colors are mixed at a predetermined ratio, and the ratio of the fluorescence of the different colors included in the detected fluorescence is 6. The diagnosis method according to claim 5, wherein disease is determined based on the diagnosis.   Using two or more of the probes that bind to different structural parts of the same target biomolecule, and determining the specificity for the target biomolecule based on the fluorescence from each detected probe The diagnostic method according to claim 6. On the biochip, a positioning marker that emits light in response to the excitation light is provided at a position predetermined with respect to the position of the probe,
Reading an image of a predetermined area containing one or more of the biochips;
Determining the position of the probe based on the position of the positioning marker in the read image;
Further including
The diagnosis method according to claim 5, wherein the disease is determined based on the fluorescence of the probe whose position is determined.

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