JP2012255772A - Sample analyzing disk - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable the concentration of an antigen or an antibody contained in a sample to be analyzed to be detected with high quantitativity in a wide range from low concentration to high concentration.SOLUTION: In a sample analyzing disk 100 for measuring labeling beads with biopolymers bound immobilized on a track area 105 having a structure provided with groove structures or pits consisting of grooves and lands provided on a disk surface, quantitatively by optical reading means: injection holes 101 for dropping a sample containing the biopolymers and flow channels 102 continuing to the injection holes 101 respectively and used for reaction between the labeling beads and the biopolymers are provided on the inner circumferential side of the sample analyzing disk 100; and detection areas 104 continuing to the flow channels 102 respectively and provided with the grooves or pits each having a width equal to the diameter of the labeling bead are provided on the outer circumferential side of the sample analyzing disk 100.

Description

  The present invention relates to a sample analysis disk for analyzing a biopolymer such as an antigen or an antibody contained in a biological sample such as blood by an optical method.

  In recent years, immunoassays for detecting antigens and antibodies from biological samples contained in blood or the like have been used in various situations for the purpose of early diagnosis of diseases in disease diagnosis and health checkup. An immunoassay is a method in which a measurable label is attached to an antibody (or antigen) that specifically binds to a specific antigen (or antibody) contained in a biological sample, and the result of the reaction of both is quantified. In this method, the concentration of the antigen (or antibody) contained in the sample is determined to determine the disease.

  Since this method is relatively sensitive and easy to operate, various labeling methods have been developed. For example, there are RIA method (radioinom assay) using radioisotope for labeling, EIA method (enzyme immunoassay) using enzyme, FIA method using fluorescent label, CLIA method using chemiluminescence.

  Among them, an ELISA method (Enzyme-Linked ImmunoSorbent Assay), which is one of the methods using an enzyme, is widely and widely used as a test method using a microplate. The method using a microplate can measure a large number of samples at a time using a 96-well plate and a general-purpose microplate reader, and is relatively inexpensive. In addition, compared to the RIA method that uses radioactive materials, there are advantages such as fewer restrictions on the place to use. However, pretreatment to fix the antibody on the plate, reaction time to react the antibody and antigen, B / F (bond / free) separation to wash away the unreacted label, enzyme reaction of the label, etc. And the total inspection time is from several hours to one day.

  For this reason, in order to shorten the inspection time, an analysis chip (Lab on a chip) has been developed by using microfabrication technology and replacing the operation procedure in each well of the microplate on a chip of several centimeters square. Yes. The processing time is shortened by preparing antibodies on the chip and immobilizing antibodies and preparing pre-treatments for general ELISA in advance. In addition, the reaction time is shortened by performing the antigen-antibody reaction in a narrow channel of several tens of micrometers to several millimeters. This makes it possible to shorten the inspection time by several tens of minutes by using a dedicated analysis chip for each inspection. Conventionally, after taking a patient sample, it took a long time to send it to the laboratory, but in some examinations such as myocardial markers, examinations in the examination room or the patient's bedside, so-called Devices capable of POCT (Point of Care Test) are being developed.

Special table 2004-533606 gazette JP-T-2006-521558 JP-T-2002-530786

  Patent Document 1 and Patent Document 2 disclose a disc-shaped analysis device having a feature that combines the advantages of a microplate capable of simultaneously testing multiple specimens and the advantage of an analysis chip that can obtain test results in a short time. Analytical devices have been proposed. Specifically, a plurality of microchannel structures are provided in a disk-shaped device, and a solid phase substance for reacting a substance contained in a sample is filled in a part of the channel. The measurement is performed by detecting the intensity of fluorescence obtained by exciting a label such as a fluorescent dye with a laser with a photodetector. Further, Patent Document 3 discloses a method using a structure of an optical disk by using a disk. A method is shown in which a biological sample or particles are attached to the same surface as the surface on which the track shape of an optical disk is formed, and a change in signal is detected using an optical pickup.

  In the methods of Patent Document 1 and Patent Document 2, the result of developing a sample in a flow path provided on a disk and reacting with a reagent is detected as a change in fluorescence intensity or absorbance, and the amount of antigen or antibody contained in the sample is determined. Measuring. The detection method is the same in principle as the method using a well plate, and the concentration of the labeling substance contained in the solution is measured by fluorescence or absorbance. Therefore, it is necessary to prepare a plurality of samples diluted in several stages for a sample having a narrow concentration range in which quantitative measurement is possible and a high concentration. On the other hand, as a method for detecting a low-concentration sample with high sensitivity, Patent Document 3 discloses a detection method in which latex beads or magnetic beads are bound to the focus surface of an optical disc. As a result, beads are aggregated over a wide area of the optical disc, and it becomes impossible to discriminate surface scratches and signal surface defects as optical disc signals, and the number of beads cannot be detected.

  The present invention has been made in view of such problems, and is capable of highly quantitative detection in a wide range from the low concentration to the high concentration of the antigen or antibody contained in the sample to be analyzed. The purpose is to provide a disc.

  In order to achieve the above object, the sample analysis disk (100) according to the first invention is provided with a groove structure or pits (109) comprising grooves (107) and lands (108) provided on the disk surface. In the sample analysis disk (100) for measuring the quantity of the labeled beads (110) to which the biopolymer is bound, which are immobilized on the track region (105) having the above structure, by an optical reading means An injection hole (101) for dropping the sample containing the biopolymer to the inner peripheral side of the sample analysis disk (100), and the bead for labeling (110) and the living body continuous to the injection hole (101) A flow path (102) for a polymer to react is provided, connected to the flow path (102) on the outer peripheral side of the sample analysis disk (100), and the label beads (1 Comprising the groove (107) or the comprising a pit (109) detects a region having a diameter equal to the width 0) (104), characterized in that.

  In the sample analysis disk (100) according to the second invention, in the first invention, the area of the detection region (104) is larger than the area of the flow path (102) connected to the detection region (104). It is characterized by that.

  In the sample analysis disk (100) according to the third aspect of the present invention, in the first or second aspect of the invention, the detection area (104) is formed by moving the detection area (104) toward the outer periphery of the sample analysis disk (100). 104) is wide.

  In the sample analysis disk (100) according to the fourth invention, in any one of the first to third inventions, the detection area (104) differs depending on a distance from a center of the sample analysis disk (100). The labeling beads (110) to which various kinds of biopolymers are bound are fixed.

  The sample analysis disk (100) according to a fifth aspect of the present invention is the sample analysis disk (100) according to any one of the first to fourth aspects, wherein the detection region (104) is the groove (107) in a portion not connected to the flow path (102). ) Or the pit (109) is recorded with an address signal for specifying the position of the detection area (109).

  According to the sample analysis disk of the present invention, detection can be performed with the same configuration as an optical pickup for reproducing information on an optical disk, and the apparatus can be reduced in size and price. Further, since the labeling beads can be counted one by one at high speed, it is possible to quantitatively measure from a low concentration sample to a high concentration sample.

Conceptual block diagram of a sample analysis disk reader according to the present invention. Configuration conceptual diagram of a sample analysis disk according to the present invention Schematic cross-sectional view of a track region in a sample analysis disk according to the present invention Schematic diagram showing the cross-sectional structure of the sample analysis disk according to the present invention and the reading state of the beads Schematic diagram showing sample reaction on sample analysis disk Schematic diagram showing sample reaction on sample analysis disk

  FIG. 1 is a configuration block diagram of a reading apparatus 1 that optically reads a sample analysis disk 100 according to the present invention and detects antibodies and antigens.

  The configuration of the reading device 1 is the same as that of a device that reads an optical disc on which data and content information are recorded. The reading device 1 includes a spindle motor 2, an objective lens 3, an actuator 4, a beam splitter 5, a laser oscillator 6, a collimator lens 7, a condenser lens 8, a light detection unit 9, and a control unit 10.

  The control unit 10 includes a rotation control unit 11, a measurement unit 12, and a focus / tracking control unit 13. The configuration of the reading device 1 may include other elements as necessary in addition to the above.

  The spindle motor 2 rotates the sample analysis disk 100 at a predetermined rotation speed under the control of the rotation control unit 11. The objective lens 3 is an objective lens having a numerical aperture (NA) of 0.85, for example, and condenses the laser beam output from the laser oscillator 6 on the reading surface of the sample analysis disk 100.

  The actuator 4 is, for example, a biaxial actuator that performs adjustment for condensing the laser light output from the laser oscillator 6 on the reading surface of the sample analysis disk 100 under the control of the focus / tracking control unit 13. . The beam splitter 5 reflects the laser light output from the laser oscillator 6 in the direction of the objective lens 3 and guides the reflected light from the sample analysis disk 100 to the light detection unit 9.

  The laser oscillator 6 is a semiconductor laser oscillator having the same wavelength of 405 nm as that for reproducing a Blu-ray (BD) disc, for example. The collimator lens 7 converts the laser beam output from the laser oscillator 6 into a parallel light beam.

  The condenser lens 8 guides the light guided from the beam splitter 5 to the light detection unit 9. The light detection unit 9 is a photodiode, and outputs a detection signal corresponding to the amount of light reflected from the sample analysis disk 100 to the measurement unit 12 and the focus / tracking control unit 13.

  The control unit 10 includes, for example, a CPU (Central Processing Unit) and a DSP (Digital Signal Processor), and includes a storage unit (not shown). The control unit 10 performs various controls according to operations by an operation unit (not shown), various controls based on detection signals output from the light detection unit 9, and the like. Among the various types of control described above, control necessary for the description of the present invention includes control by the rotation control unit 11, control by the measurement unit 12, and control by the focus / tracking control unit 13. The rotation control unit 11, the measurement unit 12, and the focus / tracking control unit 13 are realized by processing, a program, and the like in the control unit 10.

  The rotation control unit 11 controls the spindle motor 2 to rotate the sample analysis disk 100 at a predetermined number of rotations. The measurement unit 12 generates an RF signal from the detection signal output from the light detection unit 9, and counts the labeling beads 110 immobilized on the groove 107 and the pit 109 by the generated RF signal.

  The focus / tracking control unit 13 generates an FE (focus error) signal and a TE (tracking error) signal from the detection signal output from the light detection unit 9, and controls the actuator 4 and the like according to the value of each generated signal. Then, control for condensing light on the reading surface of the sample analysis disk 100 and control for following the track are performed.

  Next, the structure of the sample analysis disk 100 of the present invention will be described with reference to FIGS. FIG. 2 is a conceptual diagram of a sample analysis disk 100 according to the present invention.

  The sample analysis disc 100 has a disk shape with the same diameter as, for example, a CD (Compact Disc), a DVD (Digital Versatile Disc), and the like, and a similar material such as polycarbonate is used.

  The sample analysis disk 100 is provided with a plurality of injection holes 101 on the inner peripheral side as a rotation target with respect to the center O of the sample analysis disk 100. The injection hole 101 is a hole for dropping a sample to be analyzed, and its volume is sized to measure a sample necessary for inspection. Specifically, the sample can inject the volume of the injection hole 101, and the injected sample is transmitted through the flow path 102 described later by the rotation of the sample analysis disk 101, and the labeling beads are detected in the detection region 104. 110 is an appropriate amount to be detected.

  Further, flow paths 102 through which the dropped sample flows from each of the injection holes 101 are provided radially when viewed from the center O of the sample analysis disk 100. Furthermore, a detection region 104 is provided which is connected to each of the flow paths 102 and located on the outer periphery of the sample analysis disk 100. In addition, a part of the channel 102 is provided with a bead filling unit 103 filled with a predetermined amount of labeling beads 110 modified with an antibody that binds to a specific antigen contained in the sample.

  The area of the detection region 104 is larger than the area of the connected flow channel 102 so that the sample flowing through the flow channel 102 spreads evenly in the detection region 104 and is reliably detected. .

  Further, a track area 105 is formed on the outer periphery of the sample analysis disk 100. The track area 105 is formed with a spiral groove 107 or pit 109 having the same structure as the signal surface of the optical disk so as to include the detection area. . Since the groove 107 is formed in the track area 105, the groove 107 and the land 108 which become a groove exist on the reading surface of the sample analysis disk 100 as shown in FIG. When pits 109 are formed in the track area 105, the pits 109 are formed in a spiral shape as shown in FIG.

  Therefore, in FIG. 3A, the groove 107 is the concave portion 120 and the land 108 is the convex portion 121. In FIG. 3B, the pit 109 is a concave portion 120 and the portions other than the pit 109 are convex portions 121.

  FIG. 4 is a schematic diagram showing the cross-sectional structure of the sample analysis disk 100 and the reading state of the labeling beads 110. An injection hole 101 and a channel 102 having a depth of 100 μm to 500 μm continuous to the injection hole 101 are formed on the inner peripheral side of the sample analysis disk 100, and a channel 102 through which a sample to be analyzed passes. A bead filling unit 103 is provided in which a part of the bead is filled with a labeling bead 110 modified with a specific antibody. A protective layer 106 is provided on the reading surface side of the sample analysis disk 100. The protective layer 106 may be provided so as to cover the flow path 102, the bead filling unit 103, and the detection region 104. In FIG. 4, grooves 107 or pits 109 are formed above the detection area 104.

  The antigen-antibody reaction reacts when the antigen and the antibody come into contact with each other in the solution, and the labeling beads 110 and the antigen to be analyzed bind. In general, the concentration of an antigen targeted in an immunoassay in a solution is very low. Therefore, the size of the flow path 102 is reduced, and a large number of labeling beads 110 are filled in a narrow region to increase the reaction efficiency, so that the labeling beads 110 are bound in a short time.

  The labeling beads 110 that have reacted with the sample in the bead filling unit 103 flow further to the outer region by the centrifugal force generated by the rotation of the sample analysis disk 100 and are developed in the detection region 104. In the detection area 104, grooves 107 and pits 109 are provided as in the optical disc. In the case of BD, a standard is set so that a signal is read through a protective layer 106 having a thickness of 0.1 mm. Many of the objective lenses 3 of a general BD optical pickup are designed with a distance (working distance) to the protective layer 106 of about 0.3 mm to 0.4 mm. Therefore, in consideration of the design margin of the apparatus, it is preferable to make the space in the thickness direction in which the sample is developed in the detection region 104 with respect to the depth of the flow path 102 thinner. In FIG. 4, the depth of the flow path 102 and the depth of the detection region 104 are configured to be stepwise different, but the flow path 102 may be gradually thinned.

  Further, in order to count the labeling beads 110 one by one, it is necessary that the labeling beads 110 filled in the flow channel 102 have a sufficient area that does not overlap in the thickness direction. The area of the detection region 104 is set so that the area of the groove 107 or the pit 109 is larger than the area obtained by multiplying the area determined by the number of the labeling beads 110 filled in the flow path 102 and the diameter of the labeling beads 110. It has been. When magnetic beads are used as the labeling beads 110, the reaction time can be shortened by guiding the labeling beads 110 to the surface on which the grooves 107 and pits 109 are formed by magnetism.

  When the structure of the sample analysis disk 100 is a groove structure or a pit structure, when the spot light collected by the objective lens 3 passes through the position of the labeling bead 110 captured in the detection region 104, the reflected light is reflected. The phase of light changes, and the amount of detection signal obtained by the light detection unit 9 changes. When the diameter of the labeling bead 110 is substantially equal to the shortest pit length of the BD, it is possible to detect from one labeling bead 110 on the track.

  The position where the labeling beads 110 are detected is the detection region 104 of the sample analysis disk 100, that is, the portion connected to the flow path 102 at the portion having the groove 107 and the pit 109 as the track region 105. In the track area 105, information in the radial direction and the track direction can be obtained by previously recording an address signal in a portion not connected to the flow path 102. For this reason, the position where the bead 110 for labeling was detected can be specified based on the recorded address information.

  As another method, in the case of the groove structure, the sample analysis disk 100 is manufactured so that undulation is generated in the radial direction of the disk when the groove 107 is cut, the fluctuation is detected from the TE signal, and the frequency is measured from the disk. Wobble detection, which is a method for calculating the address, may be used.

  In addition, information can be additionally recorded on the signal surface by using a phase change material or a dye material similar to that of a recording or write-once disc such as BD-R or BD-RW. For example, by adding the ID number of the dropped sample, the sample analysis disk 100 is once taken out from the apparatus and used for specifying the sample when it is read again by the apparatus for analysis. Further, it is possible to prevent another sample from being accidentally dropped onto the detection region 104 of the sample analysis disk 100 that has already been used.

  Next, analysis of an analysis sample using the sample analysis disk 100 of the present invention will be described with reference to FIGS.

  When a sample is injected from the injection hole 101 and the sample analysis disk 100 is rotated, the dropped sample is guided to the flow path 102 for antigen-antibody reaction by centrifugal force. A bead filling unit 103 connected to the flow channel 102 is preliminarily filled with a predetermined amount of labeling beads 110 modified with an antibody that binds to a specific antigen contained in the sample.

  As the labeling beads 110, polymer particles having a diameter of about 100 nm to 1 μm, or magnetic beads containing a magnetic material such as ferrite inside are used. It is also possible to use metal fine particles such as gold colloid or silica beads. The diameter of the labeling bead 110 is determined by the optical system of the reader 1 used for detection, and the optimum size for detection is determined. The reader 1 includes components used for existing optical disc drives, particularly optical pickups. There is an advantage that can be used.

  Among the optical disk readers currently in widespread use, the highest resolution is Blu-ray Disc (BD), which collects laser light with a wavelength of 405 nm on the disk with an objective lens with a numerical aperture (NA) of 0.85. Can be lighted. The shortest pit length of the disc signal is about 0.14 μm. Therefore, when the BD optical system is used, the signal can be detected as a signal up to the diameter of the labeling bead 110 of about 140 nm.

  An antibody 210 that specifically binds to the antigen 200 contained in the sample is bound to the surface of the labeling bead 110 in advance. There are various types of antibodies 210. For example, in the case of testing for hepatitis B, the HBsAg monoclonal antibody that specifically binds to the HBs antigen contained in the blood is modified to the labeling beads 110. The type of antibody 210 to be modified is selected to specifically bind to the type of antigen 200 to be detected.

  When the sample is mixed with the labeling bead 110 modified with the antibody 210 at the bead filling portion 103 in the channel 102, the antigen 200 contained in the sample binds to the antibody 210 modified on the surface of the labeling bead 110. A complex of the labeling bead 110, the antibody 210 and the antigen 200 is formed.

  At this stage, depending on the amount of the antigen 200 contained in the sample, the labeling beads 110 of the complex in which the antigen 200 and the antibody 210 have reacted and the labeling beads 110 that have not reacted with the antigen 200 are constant. Present in proportion. Furthermore, the solution reacted in the flow path 102 is developed in the detection region 104 on the outer periphery by the centrifugal force accompanying the rotation of the sample analysis disk 100.

  Similar to the signal surface of the optical disc, the upper surface of the detection area 104 has a spiral groove structure or pit structure. In the case of the groove structure, the width of the groove 107 is preferably substantially the same as the diameter of the labeling beads 110 used as the label. In the case of a pit structure, it is preferable that the width of each pit 109 is substantially the same as the diameter of the marker bead 110.

  In these detection regions 104, an antibody 210 of the same type as that modified on the surface of the labeling bead 110 is fixed by a method such as silane coupling. The antigen 200 contained in the solution developed in the detection region 104, that is, the complex that has reacted with the labeling beads 110 modified with the antibody 210 in the flow channel 102 is further immobilized on the detection region 104. 210 to form a sandwich-type complex consisting of the labeling beads 110, the antibody 210, the antigen 200, and the antibody 210 (also referred to as a solid-phased antibody) immobilized on the detection region, and immobilized on the substrate of the detection region 104 It becomes. The bead 110 for labeling that has not reacted with the antigen 200 is not limited to the detection area 104 but is further sent to the outer periphery of the disk by the centrifugal force accompanying the rotation of the sample analysis disk 100, and outside the detection area 104, specifically the sample. It is discharged out of the analysis disk 100.

  FIG. 5 is a diagram schematically showing the structure of the composite after the antigen 200 and the antibody 210 react on the sample analysis disk 100. In FIG. 5 (a), a labeling bead 110, which is a measurable label, is attached to the antigen 200 contained in the sample according to the principle of the sandwich method using the same antigen-antibody reaction as that performed in the well plate. In this state, the detection area 104 is fixed. The sandwich method is often used as a method that facilitates quantitative measurement because an output is obtained linearly with respect to the absolute amount of a detection target. The immobilization method is not limited to this, and other methods such as a competition method can also be used.

  When the detection target is not the antigen 200 but the antibody 210, as shown in FIG. 5B, the antigen 200 that specifically binds to the antibody 210 is immobilized on the detection region 104, and the secondary antibody 213 is fixed. In the same manner as the detection of the antigen 200, the antibody 210 can be measured by the sandwich method by the method of binding the labeling beads 110 by the above method.

  FIG. 6 illustrates the detection region 104 in the case where different antibodies are attached for each predetermined range with respect to the radial direction of the sample analysis disk 100. Monoclonal antibodies 211A to 211C that specifically bind to a plurality of types of antigens 200A to 200C contained in a sample to be analyzed are fixed to a track divided into a plurality of regions in the sample analysis disk 100.

  The labeling beads 110 are modified with a polyclonal antibody 212 that binds to multiple types of antigens. The labeling beads 110 developed in the detection region 104 react with different types of antigens depending on the radial position of the sample analysis disk 100 and bind to the groove 107 or the pit 109.

    In FIG. 6, a monoclonal antibody 211A that specifically binds to the antigen 200A is fixed within a predetermined range from the inner periphery side of the detection region 104. Further, a monoclonal antibody 211B that specifically binds to the antigen 200B is fixed in the next predetermined range, and a monoclonal antibody 211C that specifically binds to the antigen 200C is fixed in the next predetermined range.

  The range where each type of monoclonal antibody 211 is fixed can be specified by an address signal recorded in a portion of the track area 105 that is not connected to the flow path 102.

  Usually, in order to detect multiple types of antigens simultaneously by fluorescence or enzymatic reaction, multiple types of labels are used to detect differences in fluorescence wavelength or color development. A combination of a light source, a wavelength filter, and a photodetector is required. In the method shown in FIG. 6, by obtaining the radial position of the sample analysis disk 100 from the address, a plurality of types of antigens can be measured almost simultaneously by the same detection method. Furthermore, even in the case of analysis methods that statistically determine the possibility of disease from the ratio of multiple types of antigens, the same sample can be measured under the same conditions, and errors when measured separately It is possible to reduce the measurement variation compared to.

  1: Reading device, 2: Spindle motor, 3: Objective lens, 4: Actuator, 5: Beam splitter, 6: Laser oscillator, 7: Collimator lens, 8: Condensing lens, 9: Light detection unit, 10: Control unit , 100: Sample analysis disk, 101: Injection hole, 102: Flow path, 103: Bead filling section, 104: Detection area, 105: Track area, 106: Protection layer, 107: Groove, 108: Land, 109: Pit 110: Labeling beads, 200: Antigen, 210: Antibody

Claims (5)

The number of labeling beads to which biopolymers are bound, fixed to the track area having a groove structure or pit structure consisting of grooves and lands provided on the disk surface, is measured by optical reading means. In the disk for sample analysis to measure,
On the inner peripheral side of the sample analysis disk, an injection hole for dropping the sample containing the biopolymer, and a flow path for allowing the labeling beads and the biopolymer to react continuously with the injection hole,
On the outer peripheral side of the sample analysis disk, provided with a detection region provided with the groove or the pit connected to the flow path and having a width equivalent to the diameter of the labeling bead.
A sample analysis disc characterized by the above. The area of the detection region is larger than the area of the flow path connected to the detection region,
The sample analysis disk according to claim 1. The detection area is characterized in that the width of the detection area is widened toward the outer periphery of the sample analysis disk.
The disk for sample analysis according to claim 1 or 2. In the detection region, the labeling beads to which different types of biopolymers are bound depending on the distance from the center of the sample analysis disk are fixed.
The sample analysis disk according to any one of claims 1 to 3. In the detection area, an address signal for specifying a position of the detection area is recorded in the groove or the pit in a portion not connected to the flow path,
The sample analysis disk according to any one of claims 1 to 4.

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