JP2009210316A - Emission measuring device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an emission measuring device can improve detection sensitivity by increasing a light quantity condensed by an objective lens and allowed to enter a photomultiplier. <P>SOLUTION: This emission measuring device for measuring a biosubstance measuring chip having a working electrode having a recessed part or a projection part formed on a center part in order to measure a biosample, is equipped with: a lens unit arranged so that the center of the recessed part or the projection part agrees with an optical axis; a reflecting mirror arranged between the lens unit and the working electrode, and having a hole and a focal point passed by the optical axis; and a light receiving sensor arranged on a position where light emitted from the lens unit is received. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a luminescence measuring apparatus for measuring the luminescence amount of a luminescent labeling agent. More specifically, the present invention relates to a technique for improving detection sensitivity of a luminescence measuring device.

  A conventional luminescence measuring apparatus measures the amount of DNA or protein by the luminescence amount of a luminescent labeling agent. A DNA or protein labeled with a luminescent labeling agent is specifically adsorbed to the electrode, and the luminescent labeling agent in the vicinity of the electrode is caused to emit light by an electrochemical reaction. The electrochemiluminescence is received by an optical sensor, and the amount of light is converted into an electrical signal and output as emission amount data.

  FIG. 16 shows a conventional luminescence measuring apparatus. The chip substrate 200 is covered with an optically transparent cover 204 to form a flow path 203 for flowing a sample solution and a reagent. On the surface of the chip substrate in the flow path 203, two types of electrodes for performing an electrochemical reaction, a working electrode 201 and a counter electrode 202 are formed. When the protein is allowed to undergo an antigen-antibody reaction by the sandwich method, the procedure is as follows. A primary antibody that specifically binds to the target protein is immobilized on the working electrode 201 in advance.

When the sample solution is passed through the flow path 203, only the primary antibody and the specific protein bind on the working electrode 201. Next, when a secondary antibody that specifically binds to the target protein and is labeled with a luminescent labeling agent is passed through the flow path 203, the secondary antibody binds only when the target protein is present on the working electrode 201. When unnecessary secondary antibody in the flow path 203 is washed away with a buffer solution and a voltage is applied between the working electrode 201 and the counter electrode 202, the working electrode 201 is bonded to the working electrode 201. It emits light above and does not emit light when the secondary antibody is not bound. The light emission 205 on the working electrode 201 is guided to the light receiving surface 209 of the photomultiplier tube 208 by the objective lens 206 and the condenser lens 207, and the light emission amount is measured by the photon counting method. Based on the amount of luminescence, the amount and presence of the target protein are determined (for example, see Patent Document 1).
Japanese Patent Laid-Open No. 6-34548

  However, in the conventional configuration, since light emitted from the luminescent labeling agent on the biological material detection chip is irradiated radially, only a part of the light incident on the objective lens is used, and other light is used. It is thrown away without being. For this reason, there is a problem that the detection sensitivity cannot be improved because the amount of light collected by the objective lens and incident on the photomultiplier tube is small.

  SUMMARY OF THE INVENTION The present invention solves the above-described conventional problems, and an object thereof is to provide a luminescence measuring apparatus capable of improving detection sensitivity by increasing the amount of light collected by an objective lens and incident on a photomultiplier tube.

  In order to solve the above-mentioned conventional problems, the luminescence measuring apparatus of the present invention is a luminescence measuring apparatus for measuring a biological material measuring chip having a working electrode in which a concave portion or a convex portion is formed at a central portion in order to measure a biological sample. A lens unit disposed so that the optical axis coincides with the center of the concave or convex portion, and a reflection having a hole and a focal point through which the optical axis disposed between the lens unit and the working electrode passes. The apparatus includes a mirror and a light receiving sensor disposed at a position for receiving the light emitted from the lens unit.

  According to the luminescence measuring apparatus of the present invention, among the luminescence of the luminescent labeling agent, luminescence that could not be incident on the objective lens can be incident, and the amount of light incident on the photomultiplier tube is increased. Therefore, high detection sensitivity can be realized.

  Hereinafter, embodiments of the luminescence measuring apparatus of the present invention will be described in detail with reference to the drawings.

(Embodiment 1)
FIG. 1 shows an overall configuration diagram of a luminescence measuring apparatus according to Embodiment 1 of the present invention. In FIG. 1, a biological material labeled with a luminescent label is dropped at the center of the biological material detection chip 7. The biological material detection chip 7 is fixed onto the biological material detection chip fixing base 41, and light emission from the central portion of the biological material detection chip 7 is detected by the light receiving unit 40. The biological material detection chip 7 is regulated by a chip alignment protrusion 48 and fixed so that the light receiving unit optical axis 15 that is the optical axis of the light receiving unit 40 and the light emission center of the biological material detection chip 7 are positioned. The light receiving unit 40 includes a lens unit including the objective lens 11 and the condenser lens 12, a reflecting mirror 20, and a photomultiplier tube unit 13 as a light receiving sensor.

  In the light receiving unit 40, light emitted from the biological material detection chip 7 is reflected by the reflecting mirror 20 and is condensed on the light receiving surface 14 of the photomultiplier tube unit 13 by a lens unit constituted by the objective lens 11 and the condenser lens 12. . The optical axis 15 of the light receiving unit is the same as the optical axis of the lens unit constituted by the objective lens 11 and the condenser lens 12 and is perpendicular to the biological material detection chip 7. The reflecting mirror 20 is a spherical mirror that is symmetrical around the optical axis 15 of the light receiving unit, and has a shape in which the center is hollowed out. The reason for hollowing out is to prevent the light incident on the objective lens 11 from being blocked by the light emitted from the biological material detection chip 7.

  The center of the spherical surface of the reflecting mirror 20 is substantially coincident with the light emission center of the biological material detection chip 7. By setting the reflecting mirror 20 in such a shape and arrangement, it is possible to reflect light rays that are not directly incident on the objective lens 11 and return them to the biological material detection chip 7. There is an electrode made of a metal reflecting film at the center of the biological material detection chip 7, and reflects the light returned from the reflecting mirror 20. Since the electrode made of a metal reflection film has a concave shape, the direction of reflection is the direction of the objective lens 11.

  By configuring the light receiving unit 40 and the biological material detection chip 7 in this manner, the light emitted from the biological material detection chip 7 is incident on the lens unit, and the light reflected by the reflecting mirror 20 is transferred onto the biological material detection chip 7. Then, the light is further reflected by the biological material detection chip 7 and enters the lens unit. Therefore, since the direct light and the reflected light from the reflecting mirror 20 are added to the lens unit and incident, the detected light amount is doubled, and as a result, the detection sensitivity is doubled. Details of this mechanism will be described later.

  Application of a voltage for causing the biological material detection chip 7 to emit light is performed as follows. A voltage application profile is set from the host PC (not shown) to the controller 46 via the host PC interface 47. This voltage application profile is, for example, a step of starting from 0 volt, rising to 1.3 V after 1 second, holding 1.3 V for 2 seconds, and then becoming 0 volt. The voltage application unit 42 generates a voltage based on the voltage application profile, and is connected to the biological material detection chip 7 via an electrode disposed on the biological material detection chip fixing base 41. Any connection method may be used, but in order to allow the biological material detection chip 7 to be detached, the electrical connection to the biological material detection chip 7 is configured to hold the biological material detection chip 7 with a spring electrode. Is good.

  The signal processing of the detected light amount received by the light receiving unit 40 is performed as follows. The photomultiplier tube unit 13 is of a photon count method and outputs a pulse signal according to the received light quantity. The pulse signal is counted by the counter 43 at regular time units, and is sequentially stored in the count value memory 44. The timing generation unit 45 generates a timing signal for controlling counting in a fixed time unit and storage of the count value. The start / stop of timing signal generation of the timing generation unit 45 is controlled by the controller 46. The controller 46 transfers count value data from the count value memory 44 to a host PC (not shown) via the host PC interface 47. Here, the constant time unit is 10 ms, and the number of pulses generated in 10 ms after counting in 10 ms units is used as the count value data. The pulse width resolution which is the minimum period of the output pulse of the photomultiplier tube unit 13 is 70 ns. At this time, the maximum number of pulses for 10 ms is 142,857, so the counter 43 has 18 bits. The thing was adopted. Since the measurement time of the light emission amount is 3 seconds, the number of memories in the count value memory 44 is 301.

  The operation of measuring the amount of luminescence is performed in synchronization with voltage application to the biological material detection chip 7 and signal processing of the detected light amount received by the light receiving unit 40. First, a measurement start command is issued from the host PC (not shown) to the controller 46 via the host PC interface 47. In response to the measurement start command, the controller 46 outputs a voltage profile to the voltage application unit 42 and applies a voltage based on the voltage profile to the biological material detection chip 7. At the same time, the timing generator 45 is started, and signal processing of the detected light amount received by the light receiving unit 40 is started. When a predetermined measurement time elapses, the voltage profile to the voltage application unit 42 stops outputting, and the voltage application to the biological material detection chip 7 is stopped. At the same time, the timing generation unit 45 is stopped to stop the signal processing of the detected light amount. Thereafter, the count value data in the count value memory 44 is transferred to the host PC (not shown) via the host PC interface 47 as the detected light quantity data, and the measurement is completed.

  FIG. 2 shows a block diagram of the voltage application unit in the first embodiment of the present invention. Voltage data is sequentially sent from the voltage application profile storage unit 30 to the D / A converter 31 to generate a voltage based on the voltage application profile. The operational amplifier 32 applies a voltage to the counter electrode contact 33 so that the voltage of the reference electrode contact 34 becomes equal to the output voltage of the D / A converter 31. The working electrode contact 35 is grounded to GND. The working electrode contact 35 is connected to the working electrode, the reference electrode contact 34 is connected to the reference electrode, and the counter electrode contact 33 is connected to the counter electrode. The voltage application profile described above of 1.3 V is defined by the potential of the working electrode with respect to the reference electrode. Here, since the working electrode is grounded to GND and the potential is zero, the potential of the counter electrode is set in the minus direction so that the potential of the reference electrode becomes −1.3V. That is, the output voltage of the D / A converter is opposite in sign to the voltage application profile, and is −1.3 V here.

  FIG. 3 shows a top view of the biological material detection chip according to the first embodiment of the present invention. In this embodiment, the chip substrate 4 is made of glass and has a thickness of 1 mm and a size of 20 mm square, and three electrodes of a working electrode 1, a counter electrode 2, and a reference electrode 3 are formed on the chip substrate 4. These three electrodes are electrodes for performing electrochemiluminescence by a three-electrode method. A working electrode 1 serving as a light emitting portion of the light emitting label is formed in a circular shape at the center of the chip substrate 4, a counter electrode 2 is disposed so as to surround the working electrode 1, and a reference electrode 3 is provided in a gap between the working electrode 1 and the counter electrode 2. It is arranged. In this embodiment, the diameter of the circular part of the working electrode 1 is 3 mm, the gap to the counter electrode 2 is 1 mm, the diameter of the outer periphery of the circular part of the counter electrode 2 is 7 mm, and the line width of the reference electrode 3 in the gap is 0.2 mm. did. The three electrodes extend toward the end of the chip substrate 4, which is a contact portion for connecting to the voltage application portion.

  The working electrode 1, the counter electrode 2, and the reference electrode 3 were formed by sputtering gold as a material that reflects the light emitted from the luminescent label. In order to reflect the light from the above-described reflecting mirror to the light receiving unit on the chip, the electrode material needs to have a high reflectance. Examples of the electrode material include noble metals such as gold, platinum, palladium, and rhodium. The light emitted from the light-emitting label is emitted in all directions, but light reflected downward from the chip can be emitted upward due to the reflection of the electrodes, and there is an advantage that the amount of light collection can be increased. The reservoir rubber 5 is arranged so as to surround the central portion where the three electrodes are gathered. The external shape of the liquid reservoir rubber 5 should be smaller than that of the chip substrate 4 so that the contact portions at the ends of the chip substrate 4 of the three electrodes are exposed. In the present embodiment, the diameter of the droplet portion of the reservoir rubber 5 is 7.5 mm.

  FIG. 4 is a cross-sectional view taken along line A-A ′ in FIG. 3 of the biological material detection chip according to the first embodiment of the present invention. A working electrode 1 is provided at the center of the chip substrate 4, and the center of the working electrode has a concave conical shape. The apex of the cone coincides with the center of the circular portion of the working electrode 1 at the center of the tip shown in FIG. In this embodiment, the angle formed by the side surface of the cone with respect to the perpendicular of the chip substrate 4, more precisely, the angle formed by the generatrix of the cone is 62.5 degrees in this embodiment. Therefore, the apex angle of this cone is 125 degrees. This apex angle was determined by ray tracing so that the reflected light of the reflecting mirror 20 of the light emission measuring device described above was incident on the objective lens 11.

  The counter electrode 2 is provided so as to surround the working electrode 1, and the liquid reservoir rubber 5 is disposed on the outer side of the counter electrode 2, so that the electrolyte can be stored on all the electrodes like the droplet 6. Since the liquid storage rubber 5 needs to prevent liquid droplets 6 from leaking, silicon rubber is preferable as a material having high adsorptivity to the chip substrate 4 and high water repellency. In this embodiment, since the amount of the electrolyte solution of the droplet 6 is 80 μl, the thickness of the silicon rubber is 1 mm so that the 80 μl droplet does not overflow.

  FIG. 5 shows a configuration for condensing light emitted from the biological material detection chip according to the first embodiment of the present invention onto a photomultiplier tube. The biological material detection chip 7 has a working electrode 1 at the center on the chip substrate 4 and a counter electrode 2 around it. Further, a reservoir rubber 5 is disposed on the outside, and the electrolytic solution is stored like droplets 6. A biological substance labeled with a luminescent label is dropped on the working electrode 1, and when a voltage is applied between the working electrode 1 and the counter electrode, the luminescent label emits light on the working electrode 1.

  Light emitted from the biological material detection chip 7 is collected by the objective lens 11 and further collected by the condenser lens 12 onto the light receiving surface 14 of the photomultiplier tube unit 13. The light receiving unit optical axis 15, which is the optical axis of the objective lens 11, the condenser lens 12, and the photomultiplier tube unit 13, is arranged at the center of the working electrode 1. In this embodiment, the focal length of the objective lens 11 is 40 mm, the effective diameter is 30 mm, and the convergence angle U is 22 degrees. The convergence angle U of the objective lens 11 is an angle formed by the outermost ray and the optical axis among the rays that are spread from the emission center and enter the objective lens.

  In the present embodiment, the same condenser lens 12 as the objective lens 11 is used, and the distance between the objective lens 11 and the condenser lens 12 is set to 1 mm in order to shorten the overall length of the light receiving unit 40. The objective lens 11 is arranged so that the front focal position of the objective lens 11 is the center of the working electrode 1, and the photomultiplier tube unit 13 is arranged so that the rear focal position of the condenser lens 12 is the light receiving surface 14. . Thereby, an image of the working electrode 1 is formed on the light receiving surface 14.

  In this embodiment, the reflecting mirror 20 reflects a light beam having a radius of curvature of 40 mm, an effective diameter of 79 mm, a central bore hole diameter of 40 mm, and a convergence angle V of 30 to 80 degrees. The optical axis of the reflecting mirror 20 is aligned with the optical axis 15 of the light receiving unit, and the objective lens 11 is located at the center of the bored hole so as to surround it. The reflecting mirror 20 is arranged so that the focal position of the reflecting mirror 20 comes to the center of the working electrode 1 with respect to the positional relationship with the biological material detection chip 7. The reflecting mirror 20 was a surface reflecting mirror in which aluminum was deposited on a glass concave surface. If it is a surface reflecting mirror, the material need not be glass and may be metal or resin, or conversely, a reflecting mirror in which aluminum is deposited on a convex surface made of glass may be attached in a convex direction away from the electrode.

  The focal length of the objective lens 11 and the radius of curvature of the reflecting mirror 20 are preferably 10 times or more than the radius of the working electrode 1. This is because when the focal length and the radius of curvature are small, the influence of aberration increases, and the light emitting component near the outer periphery of the working electrode 1 does not reach the light receiving surface 14. Making it too large is not optically problematic, but is not practical, for example, because the size of the apparatus becomes large.

  Next, the optical path of the light reflected by the reflecting mirror 20 will be described. As shown in FIG. 5, the light beam 16 that exits from the right side surface of the working electrode 1 and travels toward the reflecting mirror 20 is reflected by the right surface of the reflecting mirror 20 to become a light beam 17. Thereafter, the light is reflected from the left side surface of the working electrode 1 to become a light beam 18, and reaches the light receiving surface 14 through the objective lens 11 and the condenser lens 12. Since the reflecting mirror 20 is arranged symmetrically around the light receiving unit optical axis 15, the light emitted from the side surface on the opposite side of the working electrode follows a symmetrical optical path.

  Light exiting from the left side surface of the working electrode 1 and traveling toward the left side surface of the reflecting mirror 20 is reflected by the reflecting mirror 20 and returns to the right side surface of the working electrode 1. The returned light is reflected by the working electrode in the direction of the objective lens 11 and finally reaches the light receiving surface 14. Since the reflecting mirror 20 reflects the light beam of 30 to 80 degrees, the angle formed by the center of the light beam toward the reflecting mirror 20 and the light receiving unit optical axis 15 is 55 degrees. When the light beam is reflected by the working electrode 1 so as to be directed toward the center of the objective lens 11, the reflected light can be guided most efficiently. For this purpose, the angle formed between the normal of the working electrode and the light receiving unit optical axis 15 is preferably 27.5 degrees, which is half of 55 degrees. Accordingly, the apex angle of the cone of the working electrode 1 is 125 degrees. In this way, light emitted from the working electrode 1 is reflected by the reflecting mirror 20 and further reflected by the working electrode 1, and reaches the light receiving surface 14 through the objective lens 11 and the condenser lens 12.

  FIG. 6 is a tracking diagram of light rays that are directly incident on the photomultiplier tube according to the first embodiment of the present invention. Light rays reaching the light receiving surface 14 from the lower surface of the working electrode at the center of the droplet 6 through the objective lens 11 and the condenser lens 12 are shown.

  FIG. 7 shows a tracking diagram of light rays passing through the reflecting mirror in the first embodiment of the present invention. From the lower surface of the working electrode 1 at the center of the droplet 6 toward the reflecting mirror 20, reflected by the reflecting mirror 20 and reflected again by the upper surface of the working electrode, and passes through the objective lens 11 and the condenser lens 12 to receive light. The light rays reaching 14 are shown. As described above, the reflected light from the reflecting mirror 20 as shown in FIG. 7 is incident on the light receiving surface 14 in addition to the light beam of FIG. it can.

  Now, an example of DNA detection using the luminescence measuring apparatus according to Embodiment 1 of the present invention will be described. First, a DNA sample is extracted from a biochemical sample. Extract necessary samples from sputum, blood, feces, semen, saliva, cultured cells, tissue cells, and other biochemical samples containing DNA using physical means such as ultrasound and shaking, and chemical means using DNA extraction solutions To do. The extracted long double-stranded DNA is cleaved to an arbitrary length by a restriction enzyme or physical means such as ultrasonic waves. The cut double-stranded DNA is separated into single-stranded DNA by heat treatment or alkali denaturation. A DNA sample is obtained by these steps.

  Next, a DNA probe having a site capable of specifically binding to the DNA to be detected is immobilized on the surface of the magnetic fine particle. Magnetic fine particles are obtained by dispersing a magnetizable substance such as iron oxide in a polymer such as polystyrene or dextran, which is an electrically insulating material. The particle size of the magnetic fine particles can be selected from a wide range of 10 nm to 10 μm, and is not particularly limited, but is preferably 300 nm to 5 μm from the viewpoint of dispersibility in solution, separation and recovery.

  Furthermore, a labeling agent is prepared. The labeling agent used in the present invention is one that binds to a luminescent label and a DNA probe that is a site capable of specifically binding to the DNA to be detected. The site capable of specifically binding to DNA is single-stranded DNA having a base sequence complementary to the DNA sequence to be detected, like the above-described DNA probe immobilized on magnetic fine particles.

  Examples of the luminescent label include a metal complex having a heterocyclic compound as a ligand. In particular, a complex in which the central metal is ruthenium or osmium has good electrochemiluminescence properties, and examples of the material having such good electrochemiluminescence properties include ruthenium bipyridine complexes, ruthenium phenanthroline complexes, and osmium. Examples include bipyridine complexes and osnium phenanthroline complexes.

  The DNA sample obtained as described above, the magnetic fine particles to which the DNA probe is immobilized, and the labeling agent are mixed in a reaction vessel and hybridized. When the DNA sequence to be detected is present in the DNA sample, the labeling agent forms a complex bound to the magnetic fine particles through the DNA sample.

  After hybridization, non-target samples and unreacted substances that have not bound are removed by B / F separation. B / F separation is performed by placing a magnet in a part of the reaction vessel, fixing the magnetic fine particles by magnetic force, and then removing the solution. By this B / F separation, the DNA sequence to be detected and the labeling agent bound to the magnetic fine particles can be selectively obtained.

  After B / F separation, the luminescent label is separated from the complex. The luminescent label may be separated as long as at least the electrochemiluminescent substance can be separated from the magnetic fine particles, and the DNA probe may be in a cleaved and decomposed state. Further, it may be separated from the magnetic fine particles together with the DNA probe or DNA sample of the labeling agent and the DNA probe immobilized on the magnetic fine particles. Examples of a method for separating such a luminescent label include alkali denaturation, heat denaturation, physical destruction, and enzymatic degradation.

  After separating the luminescent label in this way, a solution containing the luminescent label separated from the complex is dropped onto the working electrode 1 of the biological material detection chip 7 and then fixed on the working electrode by drying.

  Next, the electrolytic solution is dropped onto the biological material detection chip 7. Here, the electrolytic solution is a solution containing a supporting electrolyte and a reducing agent, and a known one can be used. Examples of the supporting electrolyte include substances that give ionic conductivity to the solution, for example, salts such as sodium phosphate and sodium chloride. Examples of the reducing agent include substances that reduce an electrochemically oxidized electrochemiluminescent substance to an excited state, such as triethylamine, tripropylamine, and oxalic acid.

  A potential is applied to the biological material detection chip 7 to cause electrochemiluminescence. The photomultiplier tube 13 detects the amount of electrochemiluminescence emission during potential application, and the measured value is transferred by the controller 46 to a host PC (not shown).

  FIG. 8 shows a specific example of the voltage waveform and the light emission waveform in the first embodiment of the present invention. The upper graph shows the voltage waveform to be applied, with the horizontal axis representing time and the vertical axis representing voltage value. The lower graph is a light emission waveform, which is a count value per unit time of the photomultiplier tube in which the horizontal axis indicates time and the vertical axis indicates the light emission amount. In this embodiment, as shown in the voltage waveform 100, the applied voltage was t1 for 1 second, swept to 1.3V in 1 second, and maintained at 1.3V for 2 seconds. Further, the unit time for detecting the light emission amount was 10 ms, and the pulse from the photomultiplier tube every 10 ms was plotted as a count value. The light emission waveform 101 is a light emission waveform according to the prior art, and the light emission waveform 102 is a light emission waveform according to the present embodiment. The light emission detection by this prior art used the light-receiving unit which is not equipped with the reflecting mirror. In FIG. 8, in the case of the light emission waveform 102 according to the present embodiment, a value approximately twice as high as that of the light emission waveform 101 according to the prior art is obtained. This difference is an effect of reflection by the reflecting mirror and the working electrode described in FIG. 7, and shows the result that the present invention is effective for highly sensitive detection.

  So far, the shape of the concave portion has been described as a cone. In either case, the apex angle is preferably set to 125 degrees, and the amount of detected light can be increased by reflecting on the opposing electrode surfaces in the same manner as in the case of the cone.

(Embodiment 2)
In the first embodiment, the working electrode has only one concave portion, but a plurality of concave portions can be similarly realized. The shape of the concave portion may be a cone or a polygonal pyramid. The difference from the configuration of the first embodiment is that the concave portion of the working electrode is composed of a plurality of concave portions, and the other biological material measurement chip and the configuration of the luminescence measurement apparatus using the same are the same as those of the first embodiment. It is. A configuration example of a working electrode having a plurality of recesses will be described with reference to FIGS. In each figure, the left half of the figure is a front view of the working electrode 1 and the right half is a cross-sectional view taken along the line AA ′. FIG. 9 shows a case where the working electrode 1 is composed of a plurality of cones. Each white outline in the front view is a conical recess. FIG. 10 shows a case where the working electrode 1 is composed of a plurality of triangular pyramids. Each white outline in the front view is a triangular pyramid-shaped recess.

  FIG. 11 shows a case where the working electrode 1 is composed of a plurality of quadrangular pyramids. Each white outline in the front view is a quadrangular pyramid-shaped recess. FIG. 12 shows a case where the working electrode 1 is composed of a plurality of hexagonal pyramids. Each white outline in the front view is a hexagonal pyramid-shaped recess. In either case, the amount of detected light can be increased by reflection at the opposing working electrode. The mechanism for increasing the detected light quantity is the same as in the first embodiment. In addition, the detection example of DNA using the biological material measurement chip and the luminescence measurement apparatus using the same according to the second embodiment of the present invention can be performed in the same manner as in the first embodiment, and the effect is also shown in FIG. As shown in FIG. 2, the emission waveform is about twice as high.

(Embodiment 3)
In the first embodiment, the working electrode has a concave shape, but a convex shape can be similarly realized. The difference from the configuration of the first embodiment is that the concave portion of the working electrode is formed of a convex portion, and the other biomaterial measurement chip and the configuration of the luminescence measurement device using the same are the same as those of the first embodiment. is there. A configuration example in the case of a working electrode having a conical shape protruding toward the objective lens 11 will be described with reference to FIGS.

  The light emitted directly toward the objective lens 11 is exactly the same as in the first embodiment. The optical path of light reflected by the reflecting mirror 20 is different, and a reflection surface opposite to that in the first embodiment is used. As shown in FIG. 13, the light beam 66 that exits from the right side surface of the working electrode 1 and travels toward the reflecting mirror 20 is reflected by the left surface of the reflecting mirror 20 to become a light beam 67. Thereafter, the light is reflected on the left side surface of the working electrode 1 to become a light beam 68 and reaches the light receiving surface 14 through the objective lens 11 and the condenser lens 12. Since it is symmetrical around the optical axis 15 of the light receiving unit, the light emitted from the side surface on the opposite side of the working electrode follows a symmetrical optical path.

  Light exiting from the left side surface of the working electrode 1 and traveling toward the right side surface of the reflecting mirror 20 is reflected by the reflecting mirror 20 and returns to the right side surface of the working electrode 1. The returned light is reflected by the working electrode in the direction of the objective lens 11 and finally reaches the light receiving surface 14. Since the reflecting mirror 20 reflects the light beam of 30 to 80 degrees, the angle formed by the center of the light beam toward the reflecting mirror 20 and the light receiving unit optical axis 15 is 55 degrees. When the light beam is reflected by the working electrode 1 so as to be directed toward the center of the objective lens 11, the reflected light can be guided most efficiently. For this purpose, the angle formed between the normal of the working electrode and the light receiving unit optical axis 15 is preferably 27.5 degrees, which is half of 55 degrees. Accordingly, the apex angle of the cone of the working electrode 1 is 125 degrees. In this way, light emitted from the working electrode 1 is reflected by the reflecting mirror 20 and further reflected by the working electrode 1, and reaches the light receiving surface 14 through the objective lens 11 and the condenser lens 12.

  FIG. 14 is a tracking diagram of light rays that are directly incident on the photomultiplier tube according to the third embodiment of the present invention. Light rays reaching the light receiving surface 14 from the lower surface of the working electrode at the center of the droplet 6 through the objective lens 11 and the condenser lens 12 are shown. FIG. 15 is a tracking diagram of light rays passing through the reflecting mirror in the first embodiment of the present invention. From the lower surface of the working electrode 1 at the center of the droplet 6 toward the reflecting mirror 20, reflected by the reflecting mirror 20 and reflected again by the upper surface of the working electrode, and passes through the objective lens 11 and the condenser lens 12 to receive light. The light rays reaching 14 are shown. As described above, in addition to the light beam of FIG. 14 without the reflecting mirror 20, the reflected light from the reflecting mirror 20 as shown in FIG. it can.

  Further, the detection example of DNA using the biological material measurement chip according to the third embodiment of the present invention and the luminescence measurement apparatus using the same can be performed in the same manner as in the first embodiment, and the effect thereof is also shown in FIG. As shown in FIG. 2, the emission waveform is about twice as high.

  Here, the shape of the convex portion has been described as a cone, but it may be a polygonal pyramid such as a triangular pyramid, a quadrangular pyramid, or a hexagonal pyramid. In either case, the apex angle is preferably set to 125 degrees, and the amount of detected light can be increased by reflecting on the opposite electrode surface in the same manner as in the case of the cone.

(Embodiment 4)
In the third embodiment, there is only one convex portion of the working electrode, but a plurality of convex portions can be similarly realized. The shape of the convex portion may be a cone or a polygonal pyramid. The difference from the configuration of the first embodiment is that the convex portion of the working electrode is composed of a plurality of convex portions, and the configuration of the other biological material measurement chip and the luminescence measurement apparatus using the same are described in the first embodiment. Is the same. In the configuration example of the working electrode having a plurality of convex portions, only the concave portions in FIGS. 9 to 12 shown in the second embodiment are changed to convex portions. Each white outline in the front view of the left half is a convex portion, and the AA ′ cross-sectional view of the right half has a reverse relationship between the convex and concave portions. In either case, the amount of detected light can be increased by reflection at the opposing working electrode. The mechanism for increasing the detected light quantity is the same as in the third embodiment. Further, the detection example of DNA using the biological material measurement chip and the luminescence measurement apparatus using the same according to the fourth embodiment of the present invention can be performed in the same manner as in the first embodiment, and the effect is also shown in FIG. As shown in FIG. 2, the emission waveform is about twice as high.

  As described above, as shown in the present invention, a concave or convex portion is provided on the working electrode 1 of the biological material detection chip 7, and the reflecting mirror 20 that is hollowed out so as not to block the light beam to the objective lens 11 has a spherical center. By arranging so as to be substantially coincident with the center of the working electrode, the light emitting component that is not directly incident on the objective lens 11 is reflected by the reflecting mirror 20, and is further reflected by the working electrode 1 to be incident on the objective lens 11, thereby detecting sensitivity. Can be improved.

  The luminescence measuring apparatus according to the present invention can also enter the luminescence of the luminescent labeling agent that could not be incident on the objective lens conventionally, and increase the amount of light incident on the photomultiplier tube. Therefore, it has the effect of realizing high detection sensitivity, and is useful as a use for condensing light and increasing the amount of light.

1 is an overall configuration diagram of a luminescence measuring apparatus according to Embodiment 1 of the present invention. The block diagram of the voltage application part in Embodiment 1 of this invention Top view of biological material detection chip in Embodiment 1 of the present invention Sectional drawing of the biological material detection chip in Embodiment 1 of this invention The figure which shows the structure which condenses light emission of the biological material detection chip in Embodiment 1 of this invention to a photomultiplier tube. Tracking diagram of light rays directly incident on the photomultiplier tube in the first embodiment of the present invention Tracking diagram of light rays passing through reflecting mirror in Embodiment 1 of the present invention The figure which shows the voltage waveform and light emission waveform in Embodiment 1 of this invention. The figure which comprised the working-electrode surface in Embodiment 2 of this invention with many cones The figure which comprised the working electrode surface in Embodiment 2 of this invention with many triangular pyramids The figure which comprised the working-electrode surface in Embodiment 2 of this invention with many square pyramids The figure which comprised the working-electrode surface in Embodiment 2 of this invention with many hexagonal pyramids The figure which shows the structure which condenses light emission of the biological material detection chip in Embodiment 3 of this invention to a photomultiplier tube. Tracking diagram of light beam directly incident on photomultiplier tube in embodiment 3 of the present invention Tracking diagram of light ray passing through reflecting mirror in Embodiment 3 of the present invention The figure which shows the structure of the condensing of the conventional light emission measuring device

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Working electrode 2 Counter electrode 3 Reference electrode 4 Chip substrate 5 Liquid reservoir 6 Droplet 7 Biological substance detection chip 11 Objective lens 12 Condensing lens 13 Photomultiplier tube unit 14 Light receiving surface 15 Light receiving unit Optical axis 20 Reflector 40 Light receiving Unit 41 Biological substance detection chip fixing table 42 Voltage application unit 43 Counter 44 Count value memory 45 Timing generation unit 46 Controller 48 Chip alignment protrusion

Claims (11)

In a luminescence measuring device for measuring a biological material measuring chip having a working electrode in which a concave portion or a convex portion is formed in the central portion in order to measure a biological sample,
A lens unit arranged so that the optical axis coincides with the center of the concave or convex part,
A reflector having a hole and a focal point through which the optical axis is disposed, disposed between the lens unit and the working electrode;
A light receiving sensor disposed at a position for receiving light emitted from the lens unit;
A luminescence measuring device comprising: The luminescence measuring apparatus according to claim 1, wherein the reflecting mirror is arranged so that a focal point of the reflecting mirror coincides with a center of a concave portion or a convex portion of the working electrode. The light emission measuring device according to claim 2, wherein the working electrode side surface of the reflecting mirror is a concave surface on which a metal reflecting film is formed. The luminescence measuring apparatus according to claim 2, wherein the reflecting mirror is formed of a light transmissive member, and a surface opposite to the working electrode is a convex surface on which a metal reflecting film is formed. The luminescence measuring apparatus according to claim 1, wherein the concave portion or the convex portion of the working electrode has a conical shape. The luminescence measuring apparatus according to claim 1, wherein the concave or convex portion of the working electrode has a polygonal pyramid shape. The lens unit includes an objective lens and a condensing lens, and the objective lens and the condensing lens are arranged such that a light emitting surface of the working electrode and a light receiving surface of the light receiving sensor are in an imaging relationship. Item 2. The luminescence measuring device according to Item 1. The luminescence measuring apparatus according to claim 7, wherein both the focal length of the objective lens and the radius of curvature of the spherical mirror are 10 times or more the maximum width of the central portion of the working electrode of the biological material measuring chip. The light emission measuring device according to claim 1, wherein the working electrode has a plurality of concave portions or convex portions. The luminescence measuring apparatus according to claim 9, wherein all the concave portions or the convex portions have the same conical shape. The luminescence measuring apparatus according to claim 9, wherein all the concave portions or the convex portions have the same polygonal pyramid shape.

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