JP2015203654A - Biomolecule count measurement device and biomolecule count measurement method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a biomolecule count measurement device and biomolecule count measurement method capable of measuring a count of biomolecules in sample solution accurately.SOLUTION: A biomolecule count measurement device is provided that has a light irradiation unit for irradiating a biomolecule as a measuring object with excitation light, a first detector and a second detector for detecting a single photon emitted from a single biomolecule irradiated with the excitation light, and a detection unit that includes a half mirror that makes the light emitted from the biomolecule irradiated with the excitation light branch and enter one or both of the first detector or the second detector.

Description

  The present technology relates to a molecular number measuring apparatus and a molecular number measuring method. More specifically, the present invention relates to a technique for optically measuring the number of biomolecules contained in a sample solution.

  As a method for measuring the concentration of a biomolecule contained in a sample solution, for example, there is a method in which fine holes are formed in a matrix on a substrate, and the biomolecule to be measured is captured and detected through the holes (Patent Document). 1). The inventor has also proposed a method for measuring the concentration of a component in blood using Raman spectroscopy (see Patent Document 2).

Special table 2013-521500 gazette JP 2014-18599 A

  However, the method described in Patent Document 1 requires processing to form fine holes in the substrate, and the sensitivity is determined by the number of holes. For example, when measuring a dilute solution, a very large number of holes are required. There is a problem that it is necessary to form. In addition, since the number of biomolecules existing in the measurement unit space (including two dimensions) of the sample solution follows the Poisson distribution represented by the following formula 1, when the average concentration of the sample solution increases, two or more in the measurement unit space The probability that a biomolecule exists is increased. In Equation 1 below, λ is the number of measurement unit space average molecules, and n is the number of measured molecules.

  On the other hand, in the conventional measurement method, it is assumed that the number of molecules existing in the measurement unit space is 0 or 1. Therefore, the conventional biomolecule concentration measurement method described in Patent Document 1 is a sample solution. In the case of a high concentration, there is a problem that a measurement error becomes large.

  Therefore, the main object of the present disclosure is to provide a molecular number measurement apparatus and a molecular number measurement method that can accurately measure the number of biomolecules contained in a sample solution.

The molecular number measurement apparatus according to the present disclosure includes a light irradiation unit that irradiates a biomolecule to be measured with excitation light, and a first detection that detects a single photon emitted from the single biomolecule irradiated with the excitation light. Detector and second detector, and a detector having a half mirror that diverges light emitted from the biomolecule irradiated with the excitation light and makes it incident on the first detector or the second detector or both And having.
In the molecular number measurement apparatus of the present disclosure, the first detector and the second detector are arranged at positions where the optical path lengths from the half mirror are the same, and are output from the first detector. You may have the determination part which determines whether the light which injected into the said half mirror is a single photon by comparing a detection signal and the detection signal output from the said 2nd detector.
In that case, for example, when the first detector detects photons when the first detector detects photons, the light incident on the half mirror is derived from a plurality of biomolecules. When it is determined that a plurality of emitted photons are included and only one of the first detector and the second detector detects a photon, the light incident on the half mirror is A single photon emitted from one biomolecule is determined.
In addition, the light irradiation unit irradiates a single measurement point with excitation light a plurality of times, and the determination unit determines whether or not it is a single photon based on a plurality of detection results associated with a plurality of excitations. Can also be determined.
The molecular number measurement apparatus according to the present disclosure includes an analysis unit that irradiates the excitation light while the light irradiation unit scans two-dimensionally and calculates the number of biomolecules per unit volume based on a determination result in the determination unit. It may be.
Further, the light irradiation unit may perform pulse irradiation of the excitation light.
In that case, the pulse width of the excitation light can be made equal to or less than the relaxation time of the excitation relaxation process of the biomolecule to be measured.

  The number-of-molecules measurement method according to the present disclosure includes a light irradiation step of irradiating a biomolecule to be measured with excitation light, a light emitted from the biomolecule irradiated with the excitation light by a half mirror, and a single photon And a detection step of making the light incident on one or both of the first detector and the second detector capable of detecting the first detector.

  According to the present disclosure, the number of biomolecules contained in the sample solution can be accurately measured. Note that the effects described here are not necessarily limited, and may be any of the effects described in the present disclosure.

It is a figure showing an example of composition of a molecule number measuring device of a 1st embodiment of this indication. It is an enlarged view which shows the structure of the detection part 20 shown in FIG. It is a figure which shows the detection result in the detection part. It is a figure which shows the relationship between the density | concentration of a sample solution, and the probability of detecting multiple molecules. It is a figure which shows the structural example of the molecular number measuring apparatus of 2nd Embodiment of this indication.

Hereinafter, modes for carrying out the present disclosure will be described in detail with reference to the accompanying drawings. In addition, this indication is not limited to each embodiment shown below. The description will be given in the following order.

1. First Embodiment (Example of a molecular number measuring apparatus that irradiates excitation light from the back side of a substrate)
2. Second Embodiment (Example of a molecular number measuring apparatus that irradiates excitation light from the substrate surface side)

(1. First embodiment)
First, the molecular number measurement apparatus according to the first embodiment of the present disclosure will be described.

  Conventionally, in a concentration measurement method for counting the bright spots of a measurement image, a single bright spot is not always caused by a single molecule. Therefore, in the molecular number measurement apparatus of this embodiment, it is compensated that a single bright spot is caused by a single molecule. It is known that fluorescent molecules having a single emission center and quantum dots emitting excitons emit a single photon in one excitation relaxation process. Hereinafter, this phenomenon is referred to as “single photon emission”. Therefore, in this embodiment, by confirming that such a single fluorescent molecule or a single molecule labeled with a quantum dot emits a single photon, a single bright spot of the measurement image is obtained. To compensate for the presence of a single molecule.

  FIG. 1 is a diagram illustrating a configuration example of a molecular number measurement apparatus according to the present embodiment, and FIG. 2 is an enlarged view illustrating a configuration of the detection unit 20. The molecular number measurement apparatus 1 of the present embodiment includes a light irradiation unit 10 that irradiates a biomolecule to be measured with excitation light, and a detection unit that detects a single photon emitted from a single biomolecule irradiated with the excitation light. 20. In addition, the molecular number measurement apparatus 1 of the present embodiment is provided with a determination unit 30, an analysis unit 40, a focus adjustment unit 50, and the like as necessary.

[Light irradiation unit 10]
The light irradiation unit 10 irradiates, for example, the excitation light 3 onto a sample solution containing a biomolecule held on the substrate 2 or the like. And this light irradiation part 10 is provided with the light source 11, the isolator 12, the anamorphic prism 13, the optical amplifier 14, the acousto-optic modulation element 15, the mirror 16, etc., for example.

(Light source 11)
The light source 11 can be appropriately selected according to the wavelength of the excitation light 3. For example, a mode-locked semiconductor laser, a mode-locked titanium sapphire laser, a self-excited oscillation semiconductor laser, a pulse-driven semiconductor laser, a Q-switch laser, or the like is used. can do. In order to obtain single photon emission, the excitation light 3 emitted from the light source 11 is preferably pulsed light. In that case, it is preferable that the pulse width of the excitation light 3 is equal to or less than the relaxation time of the excitation relaxation process of the biomolecule to be measured. Thereby, single photon emission can be obtained with high accuracy.

(Isolator 12)
The isolator 12 prevents reflected light generated on the surface of each optical component from returning to the laser constituting the light source 11. When such return light is incident on the laser, laser oscillation becomes unstable and output fluctuation occurs. However, by placing an isolator 12 immediately in front of the light source 11 in the traveling direction of the return light, the laser oscillation is stabilized. Can be

(Anamorphic prism 13)
The anamorphic prism 13 forms an elliptical beam into a circular shape.

(Optical amplifier 14)
The optical amplifier 14 amplifies and outputs incident light with almost no change in wavelength according to the strength of the incident laser.

(Acousto-optic modulation element 15)
The acousto-optic modulation element 15 is a light modulation element that uses primary refracted light from ultrasonic waves.

(Mirror 16)
The mirror 16 reflects the excitation light 3 toward a sample solution containing biomolecules held on the substrate 2 or the like.

[Detection unit 20]
The detection unit 20 detects a single photon emitted from a single biomolecule irradiated with excitation light, and includes two detectors (a first detector 21a and a second detector) that detect the single photon. 21b) and a half mirror 22. In addition, the detection unit 20 may be provided with various optical components such as an objective lens 23, a dichroic mirror 24, a band pass filter 25, lenses 26a and 26b, and a pinhole 27 as necessary.

(Detectors 21a and 21b)
The first detector 21a and the second detector 21b only need to be able to detect a single photon emitted from a single biomolecule irradiated with the excitation light 3. For example, the optical path length from the half mirror 22 is long. Arranged at the same position. When a detector that performs avalanche amplification is used as the first detector 21a and the second detector 21b, the timing is set so that the next photon is detected after the amplified carrier has sufficiently disappeared. .

(Half mirror 22)
The half mirror 22 divides the light emitted from the biomolecule irradiated with the excitation light 3 and makes it incident on the first detector 21a and / or the second detector 21b, and the transmittance and reflectance. Are 50% each.

(Objective lens 23)
The objective lens 23 collects light emitted from biomolecules. The objective lens 23 can be an oil immersion lens or a water immersion lens.

(Dylock mirror 24)
The dichroic mirror 24 reflects light of a specific wavelength and transmits light of other wavelengths. In the case of the molecular number measuring apparatus 1 of the present embodiment, the dichroic mirror 24 reflects light emitted from a biomolecule. Use other materials that transmit light. That is, in the molecular number measurement apparatus 1 of the present embodiment, the dichroic mirror 24 causes the excitation light 3, the fluorescence emitted from the biomolecule, and the focus servo light (near infrared) used in the focus adjustment unit 50 described later. Separated from light 4).

(Band pass filter 25)
The band pass filter 25 is a filter that transmits only light of a specific wavelength, and in the case of the molecular number measuring apparatus 1 of the present embodiment, a filter that transmits fluorescence emitted from a biomolecule and attenuates other light is used. To do. That is, in the molecular number measurement apparatus 1 of this embodiment, the excitation light 3 and the focus servo light (near infrared light 4) are removed by the bandpass filter 25, and only the fluorescence emitted from the biomolecule is extracted.

(Lens 26a, 26b)
The lenses 26a and 26b form a conjugate image of the sample and collimate the beam behind it.

(Pinhole 27)
The pinhole 27 spatially filters incident light, and is arranged to remove stray light and improve the S / N ratio (signal noise ratio).

[Determining unit 30]
The determination unit 30 compares the detection signal output from the first detector 21a with the detection signal output from the second detector 21b to determine whether the light incident on the half mirror 22 is a single photon. Determine. For example, when the second detector 21b also detects a photon when the first detection 21a detects a photon, the light incident on the half mirror 22 includes a plurality of photons emitted from a plurality of biomolecules. Is determined. On the other hand, when only one of the first detector 21a and the second detector 21b detects a photon, the light incident on the half mirror 22 is a single photon emitted from a single biomolecule. Is determined.

  In addition, whether or not the photon is a single photon is determined based on a plurality of detection results associated with a plurality of excitations by irradiating the single measurement point with the excitation light 3 a plurality of times by the light irradiation unit 10. Is preferred. Thereby, detection accuracy can be further improved.

[Analysis unit 40]
In the molecular number measurement apparatus of this embodiment, the light irradiation unit 10 irradiates the excitation light 3 while scanning in two dimensions, and the analysis unit 40 determines the number of biomolecules per unit volume based on the determination result in the determination unit 30 ( Biomolecule concentration) can also be calculated.

[Focus adjustment unit 50]
The molecular number measurement apparatus of this embodiment may further include a focus adjustment unit 50 that adjusts the position of the substrate 2 with respect to the objective lens 23. The focus adjustment unit 50 can be constituted by, for example, a near-infrared semiconductor laser 51, a polarization beam splitter 52, a quarter wavelength plate 53, a lens 54, a cylindrical lens 55, a quadrant detector 56, and the like. As the focus servo system, for example, an astigmatism method can be used.

[Method for measuring the number of molecules]
Next, a method for measuring the number of biomolecules using the molecular number measurement apparatus 1 of the present embodiment will be described taking as an example the case where the biomolecule is a fluorescently labeled troponin that is a myocardial inflammation marker. In the method for measuring the number of molecules according to the present embodiment, the light irradiation step of irradiating the biomolecule to be measured with the excitation light 3, the light emitted from the biomolecule irradiated with the excitation light 3 is branched by the half mirror 22, A detection step of entering one or both of the first detector 21a and the second detector 21b is performed.

  In the light irradiation step, for example, when the biomolecule to be measured is dispersed on the substrate 2 and this dispersed surface is the surface of the substrate 2, the excitation light 3 is irradiated from the back surface of the substrate 2 under total reflection conditions. . Here, as the excitation light 3, for example, a pulse laser having a wavelength of 405 nm, a pulse energy of 500 pJ, a pulse width of 2 ps, and a repetition frequency of 1 GHz generated by the method described in Applied Physics Express 5 (2012) 022702 is used. it can. Then, the excitation light 3 is irradiated on the back surface of the substrate 2 after the frequency is lowered to 10 MHz using the acoustooptic modulator 15.

  At this time, since the excitation relaxation time of the troponin to be measured is approximately 1 ns, the excitation relaxation process is not repeated twice or more during one pulse irradiation time, and the fluorescence from a single molecule is 1 for one pulse. Limited to photon emission. In this way, by using the excitation light 3 as pulse light and further setting the pulse width to be equal to or less than the relaxation time of the excitation relaxation process of the biomolecule to be measured, it is possible to suppress the emission of a plurality of photons from a single molecule. it can.

  On the other hand, in the detection step, first, photons emitted from the biomolecules are captured by the objective lens 23 having a numerical aperture (NA) of 0.9, for example, arranged on the biomolecule dispersion surface (surface of the substrate 2) to be measured. To do. Next, the excitation light 3 is removed from the photons captured by the objective lens 23 using, for example, dichroic mirrors 24a and 24b having cutoff wavelengths of 510 nm and 700 nm and a bandpass filter 25 having a peak value of 535 nm and a bandwidth of 50 nm, for example. Thereafter, an image is formed by the imaging lens 26a so as to be 10 times the image plane.

  For example, a pinhole 27 having an opening having a diameter of 10 μm is arranged at the position of the image so that photons from a range of 1 μm diameter can be captured on the biomolecule dispersion surface. Then, the light is again converted into parallel light by the lens 26b, introduced into the Hanbury Brown Twiss interference system shown in FIG. 2, and detected by the first detector 21a and the second detector 21b.

  R. Hanbury Brown and RQ Twiss (1958). "Interferometry of the intensity fluctuations in light. II. An experimental test of the theory for partially coherent light". Proceedings of the Royal Society A 243 (1234): 291-319) According to this, photons that are elementary particles cannot be divided into two by a half mirror. In the molecular number measurement apparatus of the present embodiment, a Hanbury Brown Twiss interference system is prepared in the detection unit, and a photodetector that can obtain a sufficient signal even with the intensity of a single photon is installed. It is possible to accurately determine whether or not the photon is one photon.

  Here, the position of the substrate 2 with respect to the objective lens 23 is focused by the focus adjustment unit 50 using an astigmatism method using the laser light 4 of the near infrared wavelength separately from the excitation light 3 and the fluorescence emitted from the biomolecule. Servo can be performed. In this case, it is preferable to use an objective lens 23 that has been subjected to chromatic aberration correction with respect to the wavelength of light used for focus servo.

  Furthermore, in the method for measuring the number of molecules of the present embodiment, the detection result of the detection unit 20 may be input to the determination unit 40 in synchronization with the repetition of the pulsed laser beam to obtain the autocorrelation. FIG. 3 is a diagram illustrating a detection result in the detection unit 20. As shown in FIG. 3, when the autocorrelation is shot noise level at correlation time 0, it can be confirmed that the photon emission is single photon, and the bright spot under observation is due to a single molecule. it can.

  By observing all of the measurements in the region where the biomolecules on the substrate 2 are dispersed, the number of molecules to be measured bonded to the substrate 2 can be counted. That is, the sample solution is obtained by irradiating the excitation light while two-dimensionally scanning the dispersion region of the substrate 2 by the light irradiation unit 10 and calculating the number of biomolecules per unit volume based on the determination result in the determination unit 40. The concentration of can also be calculated. In that case, a bright spot that could not be confirmed as single photon emission can be counted as, for example, emission by two molecules.

  As described above in detail, in the molecular number measurement apparatus of the present embodiment, the light irradiation unit 10 irradiates the single measurement point with the excitation light 3 a plurality of times, and the determination unit 40 performs the excitation for the plurality of times. It can also be determined whether or not it is a single photon based on a plurality of detection results. Thereby, detection sensitivity can be improved and an error can be reduced.

  FIG. 4 is a diagram showing the relationship between the concentration of the sample solution and the probability of detecting a plurality of molecules. When the number of molecules is measured by the conventional measuring method, detection is performed in the range of the number of molecules 0 to 1 shown in FIG. 4, but in the method of measuring the number of molecules of the present embodiment, the number of molecules 0 to 2 shown in FIG. It is possible to detect within the range. As shown in FIG. 4, as the average number of molecules (concentration) increases, the observation probability of two molecules increases. However, the molecular number measurement method of this embodiment has a higher concentration side measurement than the conventional measurement method. Since the limit can be increased, the detection accuracy in a high concentration sample solution can be improved.

  As described above, since the molecular number measurement apparatus of the present embodiment detects single photons emitted from a single biomolecule irradiated with excitation light, the number of biomolecules contained in the sample solution is accurately determined. It can be measured. In addition, the molecular number measurement apparatus of the present embodiment can make the measurement limit higher than in the prior art when measuring the number and concentration of biomolecules.

(2. Second Embodiment)
Next, a molecular number measurement apparatus according to the second embodiment of the present disclosure will be described. FIG. 5 is a diagram illustrating a configuration example of a molecular number measurement apparatus according to the second embodiment of the present disclosure. In FIG. 5, the same components as those of the molecular number measuring apparatus according to the first embodiment shown in FIG. 1 are denoted by the same reference numerals, and detailed description thereof is omitted.

[Device configuration]
As shown in FIG. 5, the molecular number measurement apparatus 100 according to the present embodiment is configured to irradiate the excitation light 3 from the surface side of the substrate 20, except for the molecular number measurement apparatus according to the first embodiment described above. It is the same. Thus, when it is set as the structure which irradiates the excitation light 3 from the surface side of the board | substrate 20, since an optical system can be simplified, an apparatus can be made more compact.

[Method for measuring the number of molecules]
Next, a method for measuring the number of biomolecules using the molecular number measurement apparatus 100 of the present embodiment will be described by taking the case where the biomolecule is a cancer marker molecule as an example. In the molecular number measurement apparatus 100 of the present embodiment, for example, a pulse laser is used as the light source 11, and the polarization can be linearly polarized in a direction parallel to the paper surface.

  The excitation light 3 emitted from the light source 11 passes through the dichroic mirror 24 arranged in front of the objective lens 23 and focuses on the focal position of the objective lens 23. That is, in the molecular number measurement apparatus 100, the optical system of the light irradiation apparatus 10 is arranged in a confocal relationship with the positions of the first detector 21a and the second detector 21b. The excitation light 3 transmitted through the polarizing beam splitter 52 is circularly polarized by the quarter wavelength plate 53 at a position where the biological sample to be measured is present, and is perpendicular to the paper surface at the position of the polarizing beam splitter 52 in the return optical path. The polarized light.

  Thereby, the return light to the light source 11 can be reduced, and the return light reflected by the polarization beam splitter 52 can be used for the focus servo. Then, focus servo is performed in the same manner as in the first embodiment, and the photons emitted from the biomolecules are detected using the dichroic mirror 24 and the bandpass filter 25 while keeping the distance between the substrate 20 and the objective lens 23 constant. It is guided to the Hanbury Brown Twiss interference system and detected by the first detector 21a and the second detector 21b. Further, the determination unit 40 compares the autocorrelation of the signals from the two detectors 21a and 21b, and determines whether or not the light incident on the half mirror 22 is a single photon.

  Since the molecular number measurement apparatus of the present embodiment also detects single photons emitted from a single biomolecule irradiated with excitation light, it can accurately measure the number of biomolecules contained in the sample solution. it can.

In addition, the present disclosure can take the following configurations.
(1)
A light irradiation unit for irradiating the biomolecule to be measured with excitation light;
A first detector and a second detector for detecting a single photon emitted from a single biomolecule irradiated with the excitation light, and a light emitted from the biomolecule irradiated with the excitation light; A detector comprising a half mirror that is incident on the first detector or the second detector or both;
A molecular number measuring apparatus having
(2)
The first detector and the second detector are arranged at positions where the optical path lengths from the half mirror are the same,
A determination unit that determines whether the light incident on the half mirror is a single photon by comparing the detection signal output from the first detector with the detection signal output from the second detector. The number-of-molecules measurement apparatus according to (1), comprising:
(3)
In the case where the second detector also detects photons when the first detector detects photons, the determination unit includes a plurality of light incident on the half mirror emitted from a plurality of biomolecules. If only one of the first detector and the second detector detects a photon, light incident on the half mirror is emitted from the single biomolecule. The number-of-molecules measuring apparatus according to (2), wherein the molecule number is determined to be a single photon.
(4)
The light irradiation unit irradiates excitation light multiple times to a single measurement point,
The said determination part is a molecular number measuring apparatus as described in (2) or (3) which determines whether it is a single photon based on the several detection result accompanying multiple times of excitation.
(5)
The light irradiation unit irradiates the excitation light while scanning in two dimensions,
The molecular number measurement apparatus according to any one of (2) to (4), further including an analysis unit that calculates the number of biomolecules per unit volume based on a determination result in the determination unit.
(6)
The said light irradiation part is a molecular number measuring apparatus in any one of (1)-(5) which irradiates the said excitation light with a pulse.
(7)
The number-of-molecules measuring apparatus according to (6), wherein a pulse width of the excitation light is equal to or less than a relaxation time of an excitation relaxation process of a biomolecule to be measured.
(8)
A light irradiation step of irradiating the biomolecule to be measured with excitation light;
A detection step in which light emitted from the biomolecule irradiated with the excitation light is split by a half mirror and is incident on one or both of the first detector and the second detector capable of detecting a single photon;
A method for measuring the number of molecules.

  Note that the effects described in the present specification are merely examples and are not limited, and other effects may be obtained.

DESCRIPTION OF SYMBOLS 1,100 Molecular number measuring apparatus 2 Substrate 3 Excitation light 4 Near infrared light 10 Light irradiation part 20 Detection part 30 Judgment part 40 Analysis part 50 Focus adjustment part

Claims (8)

A light irradiation unit for irradiating the biomolecule to be measured with excitation light;
A first detector and a second detector for detecting a single photon emitted from a single biomolecule irradiated with the excitation light, and a light emitted from the biomolecule irradiated with the excitation light; A detector comprising a half mirror that is incident on the first detector or the second detector or both;
A molecular number measuring apparatus having The first detector and the second detector are arranged at positions where the optical path lengths from the half mirror are the same,
A determination unit that determines whether the light incident on the half mirror is a single photon by comparing the detection signal output from the first detector and the detection signal output from the second detector. The molecular number measuring apparatus according to claim 1, comprising:   In the case where the second detector also detects photons when the first detector detects photons, the determination unit includes a plurality of light incident on the half mirror emitted from a plurality of biomolecules. If only one of the first detector and the second detector detects a photon, light incident on the half mirror is emitted from the single biomolecule. The molecular number measuring apparatus according to claim 2, wherein the molecular number measuring apparatus is determined to be a single photon. The light irradiation unit irradiates excitation light multiple times to a single measurement point,
The molecular number measuring apparatus according to claim 3, wherein the determination unit determines whether or not the photon is a single photon based on a plurality of detection results accompanying a plurality of excitations. The light irradiation unit irradiates the excitation light while scanning in two dimensions,
The molecular number measurement apparatus according to claim 2, further comprising an analysis unit that calculates the number of biomolecules per unit volume based on a determination result in the determination unit.   The molecular number measurement apparatus according to claim 1, wherein the light irradiation unit performs pulse irradiation of the excitation light.   The molecular number measurement apparatus according to claim 6, wherein a pulse width of the excitation light is equal to or less than a relaxation time of an excitation relaxation process of a biomolecule to be measured. A light irradiation step of irradiating the biomolecule to be measured with excitation light;
A detection step in which light emitted from the biomolecule irradiated with the excitation light is split by a half mirror and is incident on one or both of the first detector and the second detector capable of detecting a single photon;
A method for measuring the number of molecules.

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