JP2010117287A - Fluorescence polarization detection device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fluorescence polarization detection device capable of performing accurate and precise measurement by suppressing generation of a noise or by improving an S/N ratio, and having a simple constitution not including a movable part. <P>SOLUTION: This fluorescence polarization detection device includes: a light emitting system including a laser diode for emitting excitation light, a dichroic mirror for directing the excitation light emitted from the laser diode to a sample, and an objective lens for condensing the excitation light directed to the sample; a sample holder having a penetration hole for holding the sample by a surface tension; and a light receiving system including the objective lens and the dichroic mirror for allowing transmission of fluorescence generated from the sample held by the sample holder by the excitation light, a polarization beam splitter for splitting the fluorescence transmitted through the dichroic mirror, and a photodetector for receiving each split fluorescence respectively. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a fluorescence polarization detection apparatus, and more particularly to a fluorescence polarization detection apparatus that measures fluorescence polarization degree, fluorescence anisotropy, fluorescence depolarization property, and the like.

  A conventional fluorescence polarization detector is disclosed in Japanese Patent Application Laid-Open No. 11-31603. In the fluorescence polarization detection apparatus according to the invention described in this publication, a condenser lens is installed in front of the light emitting direction of the light source, and the light beam converted into parallel rays by the lens passes through a filter installed in front of the lens. The light beam that has passed through the filter becomes light of a predetermined wavelength, and further passes through an excitation-side polarizer installed in front. The light beam that has passed through the polarizer becomes linearly polarized light in a predetermined polarization direction, and further enters from a side surface of a square cell (sample holder) installed in front. When fluorescence is emitted from the specimen (fluorescent substance) in the cell, the fluorescence L2 obtained from the direction orthogonal to the excitation light L1 passes through the second filter, and the light flux that has passed through the second filter has a predetermined wavelength. The light passes through a fluorescent-side polarizer installed further forward. This polarizer passes the horizontal component and the vertical component alternately with respect to the excitation-side polarizer in a plane perpendicular to the traveling direction of the fluorescence L2. The light that has passed through the fluorescence side polarizer enters a photodetector installed in front, and is detected by the photodetector.

  Here, fluorescence is emitted by the specimen in the cell, but in addition to that, fluorescence that becomes noise is emitted from the cell. Further, a weak fluorescent signal emitted from a light emitting element (laser diode) used in the light emitting system becomes noise. In addition, the fluorescence side polarizer is rotated (rotated) in order to pass the horizontal component and the vertical component alternately with respect to the excitation side polarizer in a plane perpendicular to the traveling direction of the fluorescence L2. An objective lens is used between the polarizer and the filter on both the light emitting side and the light receiving side before and after the cell.

References related to the fluorescence polarization detection apparatus or fluorescence polarization detection method include the following.
(1) Overview of measurement of biological substances by fluorescence polarization method
(2) Measurement of nucleic acid hybridization (nucleic acid hybridization reaction) If this apparatus is used, nucleic acid (DNA or RNA) can be measured or detected by a nucleic acid hybridization reaction.

Non-patent documents 4-6
(3) Measurement of antigen-antibody reaction If this apparatus is used, protein or biochemical related substance can be measured or detected by antigen-antibody reaction.

Non-Patent Document 7
(4) Measurement of Ligand / Receptor Reaction By using this apparatus, proteins and biochemical related substances can be measured or detected by the ligand / receptor reaction.

Non-Patent Document 8
JP-A-11-316033 Biosensor / Chemical sensor encyclopedia; Techno system; Optical device; Shared writing; 2007; 108-113. Biosensors and bioelectronics; Asakura Shoten; Biosensors using optical technology; Division writing; 1994; 62-78. Chemistry and education; Chemical Society of Japan; Rapid detection of pathogenic Escherichia coli O-157-DNA hybridization reaction basics and application; Tsuruoka, Fukuhara, Karabe; 1997; 45; 392-393. Combinatorial Chemistry & High Throughput Screening; Bentham Publishers; Rapid hybridization at high salt concentration and detection of bacterial DNA using fluorescence polarization; Tsuruoka, M. and Karube, I .; 2003; 6; 225-234. Biosensors &Bioelectronics;Elsevier; The extremely rapid oligonucleotide hybridization and high throughput detection of microbial gene sequences using fluorescence polarization; Tsuruoka, M., Murano, S., Okada, M., Ohiso, I. and Fujii, T .; 2001; 16; 695- 699. Nucleic Acids Symposium Series; Oxford University Press; Rapid and specific detection of RNA base sequence using fluorescence polarization; Tsuruoka, M. and Fujii, T; 1999; 42; 239-240. Biosensors &Bioelectronics;Elsevier; Fluorescence polarization immunoassay compressing antibody; Tsuruoka, M., Tamiya, E. and Karube, I .; 1991; 6; 501- 505. Analytical Sciences; Analytical Society of Japan; Weak Interaction between Inhibition Peptides and a Soluble Receptor of Fusion Protein in the Liquid Phase; Shimizu, M., Yoshiaki, Y., Sato, J. and Tsuruoka, M .; 2006; 22 (9 ); 1185-1188.

  As described above, fluorescence is emitted by the specimen in the cell, but in addition to that, fluorescence that becomes noise is emitted from the cell, and precise detection cannot be performed. In addition, a component having the same wavelength as the weak fluorescent signal emitted from the light emitting element used in the light emitting system becomes noise, and precise detection cannot be performed. In addition, since the fluorescent side polarizer is rotated (rotated) in order to alternately pass the horizontal component and the vertical component with respect to the excitation side polarizer in a plane perpendicular to the traveling direction of the fluorescence L2, the cause of failure or the like is caused. There will be a movable part that tends to become. In addition, an objective lens is used between the polarizer and the filter on both the light-emitting side and the light-receiving side before and after the cell, which makes the configuration complicated and increases the manufacturing cost.

  Accordingly, an object of the present invention is to detect fluorescence polarization with a simple structure, which can perform accurate and precise measurement by suppressing the generation of noise or improving the S / N ratio, including no moving parts. To provide an apparatus.

  A fluorescence polarization detection apparatus of the present invention includes a laser diode that emits excitation light, a dichroic mirror that directs excitation light emitted by the laser diode toward a sample, and an objective lens that collects the excitation light directed to the sample A sample holder having a through-hole that holds the sample by surface tension, and the objective lens, the dichroic mirror, and the dichroic mirror that transmit fluorescence generated from the sample held by the sample holder by the excitation light. A polarization beam splitter that divides the transmitted fluorescence according to polarization; and a light receiving system that includes a light receiver that receives each of the separated fluorescence.

According to the present invention, the following effects can be obtained.
(1) The fluorescence polarization detection device can be significantly reduced in size compared to conventional devices.
(1-1) An optical system using a semiconductor laser is configured, and the system using a conventional xenon lamp or the like is excellent in miniaturization and easy to carry.

(1-2) The objective lens that irradiates the sample with excitation light and the lens that condenses the fluorescence generated by the excitation light are the same, and the apparatus can be miniaturized.
(2) Measurement with a smaller amount of sample is possible than before.

  (2-1) An optical system that collects coherent light is realized, excitation light can be reduced to the diffraction limit, and even if the measurement sample is a trace amount, the sample is irradiated without reducing the energy density of the excitation light. be able to.

  (2-2) Light with the same wavelength as the fluorescent light existing in a minute amount in the excitation light is completely removed by an optical system using a dispersion prism, and leakage into the fluorescent detection system is eliminated, thereby reducing noise. Make it possible.

(2-3) The fluorescence emitted from the sample can be efficiently collected by a high NA objective lens or a cone mirror to detect the fluorescence from a very small amount of the sample.
(3) A high-performance fluorescence polarization detector can be realized at a low price.

  (3-1) A low-cost blue semiconductor laser can be used as an excitation light source, and the price of the apparatus can be reduced.

(3-2) It is possible to reduce the price by using an optical system that requires only one expensive band-pass filter, which is conventionally required.
(4) Others (4-1) It is possible to realize a highly reliable fluorescence polarization detection apparatus that does not have a movable part in the apparatus and is unlikely to fail.

  (4-2) It is possible to provide a fluorescence polarization detection device that uses a semiconductor laser as excitation light and a high-sensitivity PIN photodiode as a light receiving element, can suppress current consumption, and can be driven by a battery.

  (4-3) Since the sample and the reagent liquid are not separated by a liquid holding wall such as glass or plastic, they are not affected optically by the holding wall when irradiated with excitation light in fluorescence measurement.

The fluorescence polarization detection apparatus of the present invention is
A light emitting system including a laser diode that emits excitation light, a dichroic mirror that directs the excitation light emitted by the laser diode to the sample, an objective lens that collects the excitation light directed to the sample, and
A sample holder having a through hole for holding the sample by surface tension;
The objective lens that transmits the fluorescence generated from the sample held on the sample holder by the excitation light, the dichroic mirror, a polarization beam splitter that divides the fluorescence transmitted through the dichroic mirror by polarization, and a light reception that receives each of the divided fluorescence A light receiving system including a detector,
Is provided.

Next, embodiments of the present invention will be described with reference to the drawings.
Example 1
FIG. 1 is a diagram illustrating an internal configuration of a fluorescence polarization detection apparatus according to a first embodiment of the present invention. In broad terms, it can be divided into a light emitting system (1-6) for causing excitation light to enter the sample and a light receiving system (5-12) for taking in light emitted by the sample.

  The light emitting system includes a laser diode 1 that emits excitation light for exciting a sample, a lens 2 that collimates the laser light to make it parallel light, a polarizing plate 3 that defines a polarization direction, and only near the wavelength of the laser light. It comprises a bandpass filter 4 that passes through, a dichroic mirror 5 that reflects and guides the excitation light toward the sample, and an objective lens 6 (about NA 0.2) that condenses the laser of the excitation light and irradiates a minute sample.

  The light receiving system is disposed in the same housing as the light emitting system, and includes an objective lens 6 that captures fluorescence emitted from the sample, a dichroic mirror 5 that transmits the fluorescence wavelength, a bandpass filter 7 that transmits only the vicinity of the fluorescence wavelength, A polarization beam splitter 8 that separates the polarization components of the fluorescence, condensing lenses 9 and 10 that collect the fluorescence separated for each polarization component and guide it to the light receiver, and receive the weak fluorescent signal that has been collected. It consists of light receivers 11 and 12 that convert signals.

  As a preferable feature of the fluorescence polarization detection apparatus having the above-described configuration, the objective lens that irradiates the sample with excitation light and the lens that condenses the fluorescence generated by the excitation light are the same (indicated by reference numeral 6 in the figure), and the size of the apparatus is small. Can be realized. In addition, the dichroic mirror 5 is used to separate excitation light and fluorescence on the optical path, and mutual interference is eliminated by utilizing the difference in wavelength between excitation light and fluorescence. Specifically, as a function of the dichroic mirror, the light is reflected for the wavelength of the excitation light of the laser diode, the excitation light is guided to the objective lens 6, and the fluorescence emitted from the sample is collected by the objective lens 6. The transmitted fluorescence wavelength is transmitted to the light receivers 11 and 12.

One of the major problems of the fluorescence polarization detection apparatus is separation of excitation light and fluorescence. When excitation light is mixed in the light receiving part, it appears as noise in the measurement of fluorescence intensity, making it difficult to measure the correct fluorescence. In order to prevent the excitation light from leaking into the light receiving section, in the first embodiment, not only the separation of the excitation light and the fluorescence by the dichroic mirror 5 described above, but also a band pass filter 7 that transmits only the vicinity of the fluorescence wavelength. Is installed between the dichroic mirror 5 and the light receivers 11 and 12 to prevent the excitation light from leaking into the light receiving unit.
(Example 2)
FIG. 2 is a diagram illustrating an internal configuration of the fluorescence polarization detection apparatus according to the second embodiment of the present invention. Further, in Example 2, it is possible to prevent the excitation light from leaking into the light receiving unit, and to guide more fluorescence signals to the light receiving unit as compared with Example 1, and in FIG. A semiconductor laser diode 14 that emits a wavelength of light that is desirable as excitation light for exciting the sample, a lens 15 that collimates the laser light to make it parallel light, and an extra wavelength component proceeds using the refractive index difference of the light. A wavelength dispersion prism 16 for changing the direction, a mirror 17 for bending light for miniaturization, beam shaping prisms 18 and 19 for reducing the beam diameter of the excitation light to about 1/3, and a traveling direction thereof are changed. A light blocking plate 21 that blocks light other than the wavelength of the excitation light, a dichroic mirror 24 that reflects and guides the excitation light toward the sample, and a high NA that collects the excitation light laser and irradiates a minute sample. Made from the objective lens 22.

  Further, a beam splitter indicated by 20 is included in the optical path, and a part of the excitation light is condensed by the condenser lens 32 and received by the light receiving element 33. This purpose is used as a power monitor to stabilize the light emission of the semiconductor laser. When there is a fluctuation in the light emission amount, the change is transmitted to the laser driver 34, and the light emission amount of the semiconductor laser is controlled by feedback control in the laser driver. It is a mechanism to keep it constant.

  The light receiving system has substantially the same configuration as that of the first embodiment, and includes a high NA objective lens 22 that captures fluorescence emitted from the sample, a dichroic mirror 24 that transmits the fluorescence wavelength, and a bandpass filter 26 that transmits only the vicinity of the fluorescence wavelength. , A polarization beam splitter 27 that separates the polarization components of the fluorescence, condensing lenses 28 and 30 that collect the fluorescence separated for each polarization component and guide it to a light receiver, and receive the weak fluorescent signal that has been collected. It consists of light receivers 29 and 31 that convert signals.

  Here, reference is also made to electrical circuit configurations other than the detection system and the light receiving system. The laser driver 34 drives the semiconductor laser 14, detects output fluctuation from the voltage change of the power monitor mentioned above, suppresses output fluctuation, and oscillates the semiconductor laser 14 with a stable and constant output. The fluorescence emitted from the sample is converted into an electrical signal by the light receivers 30 and 31, and then amplified to a voltage that allows easy signal processing later by the amplifier 35, and A / D conversion is performed by the repeater 36. Measurement, measurement, etc. are performed with a personal computer.

  As a feature of this configuration, when a semiconductor laser is used for the excitation light, the light oscillated by the semiconductor laser contains the same component as the wavelength of the fluorescence obtained as a signal although being weak, in addition to the wavelength of the excitation light. Instead of removing it with an optical filter, the prism 16 using highly dispersible glass whose refractive index changes depending on the wavelength of light, changes the traveling direction of light other than the desired wavelength component, and blocks with a light shielding plate 21 Thus, it does not leak into the detection system. The optical filter has a slight disadvantage that it passes light other than the desired wavelength component and enters the detection system as noise. In this apparatus, since light other than the desired wavelength component can be physically cut, a fluorescence detection system with less noise can be configured.

As another feature, the excitation light laser beam is made thinner by about 1/3 of the objective lens diameter by the beam shaping prisms 18 and 19, and the excitation light is incident on the sample through the vicinity of the center of the high NA objective lens 22. It is mentioned. With this method, the excitation light only uses a part of the high NA objective lens and prevents the sample from becoming too small with respect to the size of the sample when connecting the focused spot to the sample. Can shine light. The fluorescence generated there is captured in a range of 74 ° when viewed from the objective lens because the objective lens 22 has a high NA (for example, NA 0.6), and compared with the first embodiment (the NA of the objective lens is 0. 0). Since the angle is about 3 times, the light collecting performance can be expected to be 9 times as large as the capturing area, and measurement can be performed with higher sensitivity.
(Example 3)
FIG. 3 is an enlarged view showing a sample holder used in the apparatus of the present invention as the third embodiment. Furthermore, as another feature of the present fluorescence polarization detection apparatus common to the configurations of Examples 1 and 2, as shown in the enlarged view of FIG. 3 at the position of the sample that emits fluorescence when irradiated with excitation light. The sample holder 13 having a minute hole for holding the sample in the center is configured to hold the liquid sample without flowing out due to surface tension in the minute space that penetrates the sample holder 13. Can be mentioned. The effect is that no other substances are present between the excitation light and the sample, and the collected excitation light can be directly irradiated to capture the fluorescence emitted from the sample, avoiding the influence of substances other than the sample. Is possible.

In addition, in the fluorescence polarization detectors of Embodiments 1 and 2 of the present invention, the fluorescence signal detector also has a feature, and as a means for detecting the difference in the polarization component of the fluorescence emitted from the sample, polarized light is detected. When a beam splitter (indicated by reference numeral 8 in FIG. 1 and by reference numeral 27 in FIG. 2) is arranged and the polarization component of the fluorescence changes, the amount of light received by one light receiver decreases and the light received by the other light receiver. The configuration is such that the amount of light increases. With this configuration, compared to the conventional method of reading the change in polarization direction by rotating the analyzer or the like, the apparatus has no movable part, and high reliability and downsizing can be realized. In addition, since both the laser diode and the light receiver can select parts with low power consumption, battery driving is possible, and portability that is easy to carry can be realized.
Example 4
FIG. 4 is a diagram illustrating an internal configuration of the fluorescence polarization detection apparatus according to the fourth embodiment of the present invention. As an internal configuration of the fluorescence polarization detection apparatus of the present invention, it is possible to adopt a configuration as shown in FIG. 4 as a configuration example different from the first and second embodiments, so that fluorescence polarization detection with higher sensitivity can be performed. Become. With reference to FIG. 4, its preferred features are shown.

  This configuration is divided into a light emitting system and a light receiving system as in the first and second embodiments. The light emitting system has a laser diode 38, a lens 39 for condensing it toward the sample, and an optical axis bent by 90 degrees. It consists of a rod mirror 40 that directs laser light toward the sample. The light receiving system includes a cone mirror 42 that collects fluorescence emitted from the sample, a condensing lens 43 that guides the collected light to the light receiver as focused light, a polarization beam splitter 44 that separates the polarization components of the fluorescence, and separates each polarization component. It comprises light receivers 45 and 46 that receive the received fluorescence and convert it into electrical signals. In this configuration, the sample holding container 41 is a pipe-shaped one made of a transparent material such as a hollow glass tube, and the hollow portion is filled with a liquid sample.

  When excitation light enters the sample and fluorescence is emitted from the sample, the fluorescence does not diverge only in a specific direction when viewed from the sample, but diverges in all directions. Sensitivity can be increased by capturing more. As a preferable feature of this configuration, the cone mirror 42 is disposed so as to cover the sample position, and most of the fluorescence generated from the sample placed in a transparent container 41 such as a hollow glass tube is efficiently collected by the cone mirror 42. There is something you can do.

  The fluorescence reflected by the cone mirror 42 is collected into a convergent light by the condenser lens 43, and when the polarization component of the fluorescence changes through the polarization beam splitter 44, the amount of light received by one of the light receivers decreases, A configuration in which the amount of light received by the other light receiver is increased is converted into an electric signal by the light receiver, and a desired signal can be obtained with high sensitivity.

By adopting such a configuration, as another advantage, the number of parts can be further reduced, and the manufacturing cost can also be reduced.
(Example 5)
FIG. 5 is a view showing a sample holder used in the apparatus of the present invention as Example 5. FIG. As shown in FIG. 5, the sample holder of this embodiment is formed with a large number of sample holding holes (through holes for holding the sample with surface tension) arranged in the vertical and horizontal directions. A large number of samples can be measured in a short time by holding a large number of samples in the sample holding holes of the sample holder and moving the sample holder in the vertical direction and the horizontal direction sequentially.
(Experimental example)
Verotoxin type I gene derived from enterohemorrhagic E. coli O-157 was amplified by asymmetric PCR. 1 μL of this amplification product and 1 μL of a reagent for measurement (probe DNA) were mixed, the solution was set in a sample holder of the same type as 13 in FIG. 3, and the degree of fluorescence polarization was measured with a prototype apparatus having the configuration shown in FIG. The measurement results are shown in FIG. From FIG. 6, the fluorescence polarization degree for the asymmetric PCR product of verotoxin type I gene showed a higher value than the fluorescence polarization degree for negative control 1 and negative control 2, and synthetic complementary DNA. This means that the gene has been sufficiently amplified and the measurement reagent has been combined with the amplified gene by a hybridization reaction. As shown in this reference example, the fluorescence polarization detection apparatus of the present invention can measure DNA that is a biological substance.

  The horizontal axis of FIG. 6 shows the measurement object, “positive” indicates the amplification product of the above gene by asymmetric PCR, and “synthesis” has the same chain length and complementary sequence to the probe DNA. Synthetic DNA, “Negative C1” indicates the product (negative control 1) that did not contain the above gene and was subjected to the same asymmetric PCR as above, and “Negative C2” does not contain any DNA. Liquid (negative control 2) is shown. The vertical axis in FIG. 6 indicates the degree of polarization, and the value obtained by multiplying the degree of polarization, which is usually unitless, by 1000 is plotted and expressed as “mP” as a convenient unit. In FIG. 6, that is, each bar of the bar graph indicates the average value of the values measured three times from immediately after mixing each measurement object and the measurement reagent to 5 minutes later, and the error bar indicates the sampling standard deviation. This gene amplification and a reagent for detecting the amplification product are described in detail in Non-Patent Document 5.

It is a figure which shows the internal structure of the fluorescence polarization detection apparatus of Example 1 of this invention. It is a figure which shows the internal structure containing the signal processing system of the fluorescence polarization detection apparatus of Example 2 of this invention. 10 is an enlarged view showing a sample holder used in the present invention as Example 3. FIG. It is a figure which shows the internal structure of the fluorescence polarization detection apparatus of Example 4 of this invention. 6 is a view showing a sample holder used in the present invention as Example 5. FIG. It is a graph which shows the result of the experiment example of this invention.

Explanation of symbols

1 to 6 Light emitting system 5 to 12 Light receiving system 1 Laser diode 2 Lens 3 Polarizing plate 4 Band pass filter 5 Dichroic mirror 6 Objective lens 7 Band pass filter 8 Polarizing beam splitter 9, 10 Condensing lens 11, 12 Light receiver 13 Sample holding Body 14 laser diode 15 lens 16 wavelength dispersion prism 17 mirror 18, 19 beam shaping prism 21 light shielding plate 22 objective lens 24 dichroic mirror 26 band pass filter 27 polarization beam splitter 28, 30 condenser lens 29, 31 light receiver 38 laser diode 39 Lens 40 Rod mirror 41 Sample holding container 42 Cone mirror 43 Lens 44 Polarizing beam splitters 45 and 46

Claims (6)

A light emitting system including a laser diode that emits excitation light, a dichroic mirror that directs excitation light emitted from the laser diode toward the sample, an objective lens that collects the excitation light directed to the sample, and
A sample holder having a through hole for holding the sample by surface tension;
The objective lens that transmits the fluorescence generated from the sample held on the sample holder by the excitation light, the dichroic mirror, a polarization beam splitter that divides the fluorescence transmitted through the dichroic mirror by polarization, and receives each of the divided fluorescence A light receiving system including a light receiver;
A fluorescence polarization detection apparatus comprising:   2. The fluorescence polarization detection apparatus according to claim 1, wherein a removing means removes excitation light having a wavelength other than a predetermined wavelength for the laser diode to generate fluorescence of the sample between the laser diode and the dichroic mirror. A fluorescence polarization detection apparatus comprising:   3. The fluorescence polarization detection apparatus according to claim 2, wherein the removing means disperses light from a laser diode, a mirror that disperses the wavelength dispersion prism and changes the direction of transmitted light, and the mirror. A fluorescent polarization detection device comprising a molding prism for molding reflected light from the light.   4. The fluorescence polarization detection apparatus according to claim 3, wherein the removing unit further includes a light shielding plate that blocks light having a wavelength other than a predetermined wavelength included in the light from the molding prism. apparatus.   The fluorescence polarization detection apparatus according to any one of claims 1 to 4, wherein the sample holder has a plurality of through holes for holding a plurality of samples by surface tension. A light emitting system including a laser diode that emits excitation light, a dichroic mirror that directs excitation light emitted from the laser diode toward the sample, an objective lens that collects the excitation light directed to the sample, and
A sample holding container for holding the sample that generates fluorescence by excitation light;
A cone mirror that directs the fluorescent light generated from the sample by the excitation light and dispersed in the radial direction to the objective lens;
The objective lens that transmits the direct fluorescence from the sample and the fluorescence from the cone mirror, the dichroic mirror, a polarization beam splitter that divides the fluorescence transmitted through the dichroic mirror by polarization, and a receiver that receives each of the divided fluorescence Including a light receiving system;
A fluorescence polarization detection apparatus comprising: