WO2013031141A1 - Molecule detecting apparatus, molecule detecting method, and cartridge for detecting molecules - Google Patents

Abstract

A molecule detecting apparatus comprises: a semiconductor laser (214) that emits a light; first (306) and second (307) condenser lenses that condense the light, which is emitted from the semiconductor laser (214), into a hold space; an aperture (304) that limits the incidence angle of the light from the second condenser lens (307) into the hold space; and a light detector (215) that detects a reflected light from the hold space. The aperture (304) limits the incidence angle θ of the light through a transparent member (216) into the hold space in such a manner that satisfies a formula, n1/n2 < sin θ ≤ NA/n2, where: the refractive index of the hold space is n1; the refractive index of the transparent member (216) existing between the second condenser lens (307) and the hold space is n2; n1 < n2; and the numerical aperture that defines the maximum value of the incidence angle of the light that enters the hold space is NA.

Description

MOLECULE DETECTION DEVICE, MOLECULE DETECTION METHOD, AND MOLECULE DETECTION CARTRIDGE

The present invention relates to a molecular detection apparatus, a molecular detection method, and a molecular detection cartridge for detecting ultra-trace amounts of molecules, and mainly used in biomedical research, medical diagnosis, preventive diagnosis, biotechnology, odor detection and the like in the past. Therefore, the present invention provides a molecular detection device, a molecular detection method, and a molecular detection cartridge that detect the presence or concentration of an extremely small amount of molecules that are difficult to detect.

With the development of biotechnology, many biomarker candidates such as sRNA (soluble RNA), miRNA (micro-RNA), antibody protein, and odorant molecule have been discovered. These biomarkers are expected to be applied to gene expression in cells, protein synthesis processes, diagnosis of diseases such as cancer, or treatment of diseases, and energetic research continues. In addition to biological applications, biomarkers can also be used as probes for detecting trace amounts of molecules, and for example, applications outside the bio field, such as odor detection, are also being studied.

A detection method using a microchip is widely used as a method for detecting the type or amount of such specifically bound molecules. Since the microchip was reported as a DNA microchip in 1995, it is not limited to basic medical fields such as DNA base sequence analysis or RNA expression analysis as a tool that can comprehensively analyze many types of biomolecules at once. It is also spreading in the fields of drug discovery and pharmaceutical applications.

Hereinafter, the principle of the DNA microchip will be briefly described by taking the case of detecting miRNA with the DNA microchip as an example. In order to detect miRNA, several hundred to several thousand DNAs having a base sequence that specifically binds to the base sequence of the miRNA to be detected are immobilized on the substrate in advance as a probe. A fluorescent dye is bound to DNA produced by reverse transcriptase from miRNA extracted from cells or samples. When this DNA is hybridized on the microchip, only the DNA that has been hybridized in a complementary relationship with the DNA probe on the microchip is bound.

Bound DNA is labeled with a fluorescent dye. Therefore, if the DNA microchip is scanned while shining light with a scanner, fluorescence is generated from the fluorescent dye at the bonded position, so that the bonded position can be specified. The position on the DNA microchip and the type of the DNA probe have a one-to-one correspondence, and it is possible to specify which type of marker has reacted from the position on the DNA microchip. Further, by measuring the intensity of fluorescence, the relative amount of hybridization can be measured.

DNA microchips are beginning to be widely used in the bioresearch field. However, for use in actual medical diagnosis sites, food quality inspections, or security inspections assuming bioterrorism, it is required to shorten inspection time, improve detection sensitivity, and improve reproducibility. Several detection methods have been proposed for these improvements.

For example, Patent Document 2 discloses a method for measuring the time course of hybridization in real time. Patent Document 2 discloses a method for improving detection sensitivity by using evanescent light to prevent noise fluorescence from a fluorescent dye that has not caused hybridization.

The DNA microarray detection apparatus of Patent Document 1 will be described with reference to FIG. FIG. 12 is a diagram showing a configuration of a conventional DNA microarray detection apparatus. In FIG. 12, a DNA microchip fluorescence detection apparatus has a laser 801 for irradiating excitation light, a lens 802 for condensing the laser, a mirror 803 for changing the direction of the optical path, and for making the laser light incident under total reflection conditions. In addition, the lens 804 for changing the optical axis, the microchip 805, the base material 806 for fixing the microchip 805, the lens 807 for collecting the fluorescent light 810, the sensor 808 for receiving the fluorescent light 810, and the excitation light are cut. An optical filter 809 is provided.

The laser light emitted from the laser 801 is collected by the lens 802, reflected by the mirror 803, and enters the end of the lens 804. When light enters the end of the lens 804, the incident angle of light on the microchip 805 can be increased.

The microchip 805 is usually used in an aqueous solution. Since the refractive index of water is 1.33, assuming that the refractive index of the substrate of the microchip 805 is 1.5, the condition for total reflection of light is satisfied if light is incident at an incident angle of 62 degrees or more. To do. At this time, the light reaching the microchip 805 in the aqueous solution is totally reflected between the substrate 806 and the aqueous solution. However, in a very narrow range of the boundary between the substrate 806 and the aqueous solution, light called evanescent light oozes out to a depth of about a fraction of the wavelength of the excitation light. By using the evanescent light, it is possible to irradiate the excitation light only in a limited range on the microchip 805 in the depth direction. By utilizing such an effect of evanescent light, Patent Document 1 suppresses the background noise of fluorescence and improves the S / N of detection.

However, the detection sensitivity is not sufficient for using a conventional DNA microarray detection apparatus in actual medical diagnosis, inspection at an agricultural test site, security inspection, food inspection, and the like. This problem of detection sensitivity is a factor that hinders the practical use of DNA microarray detection devices, and it is required to detect with good sensitivity in a short time. In particular, miRNA, which is thought to bring about a revolutionary revolution in disease diagnosis and treatment in recent years, has a very low concentration in blood, and a detection device with greatly improved detection sensitivity than before has been demanded. Yes. If such a detection device with extremely high detection sensitivity can be realized, an odor detector comparable to the olfactory sense of a dog can be realized, and therefore its application range is expected to be diverse.

JP 2006-38816 A US Patent Application Publication No. 2010-0140503

The present invention has been made to solve the above-described problems, and an object of the present invention is to provide a molecular detection device, a molecular detection method, and a molecular detection cartridge that can improve detection sensitivity.

A molecular detection device according to an aspect of the present invention is a molecular detection device that detects molecules existing in a holding space, and includes a light source that emits light, and the light emitted from the light source is collected in the holding space. A condensing lens, an aperture for limiting an incident angle of the light emitted from the condensing lens and entering the holding space, and a photodetector for detecting reflected light from the holding space. The refractive index of the space is n1, the refractive index of the transparent member existing between the condenser lens and the holding space is n2, n1 <n2, and the maximum incident angle of light incident on the holding space is When the defined numerical aperture is NA, the aperture limits the incident angle θ of light that passes through the transparent member and enters the holding space so as to satisfy the following formula (1).

N1 / n2 <sin θ ≦ NA / n2 (1)

According to this configuration, the light source emits light. The condensing lens condenses the light emitted from the light source in the holding space. The aperture limits the incident angle of light emitted from the condenser lens and entering the holding space. The photodetector detects reflected light from the holding space. The refractive index of the holding space is n1, the refractive index of the transparent member existing between the condenser lens and the holding space is n2, and n1 <n2, and the maximum incident angle of light incident on the holding space is defined. When the numerical aperture is NA, the aperture limits the incident angle θ of the light that passes through the transparent member and enters the holding space so as to satisfy the above formula (1).

According to the present invention, the incident angle θ of the light that passes through the transparent member and enters the holding space is limited so as to satisfy the above formula (1), so that the molecular unit has a minute size in the holding space. A three-dimensional irradiation space can be formed, one molecule in the minute three-dimensional irradiation space can be detected, and detection sensitivity can be improved.

The objects, features and advantages of the present invention will become more apparent from the following detailed description and the accompanying drawings.

It is a figure which shows the structure of the molecule | numerator detection system in embodiment of this invention. It is a top view which shows the structure of the detection cartridge and molecule | numerator detector in embodiment of this invention. It is a side view which shows the structure of the detection cartridge and molecule | numerator detector in embodiment of this invention. It is a figure which shows the detailed optical structure of the molecular detector shown in FIG. It is a figure which expands and shows the condensing point of the light condensed by the 2nd condensing lens. It is a figure which shows the relationship between a gap signal and a gap space | interval. It is a figure which shows the other example of the 2nd condensing lens in embodiment of this invention. (A) is a figure which shows the fluorescent substance detection probe before a molecule | numerator couple | bonds in embodiment of this invention, (B) is the fluorescent substance after a molecule | numerator couple | bonded in embodiment of this invention. It is a figure which shows a detection probe. It is a figure for demonstrating the wavelength dependence of the fluorescence intensity before a molecule | numerator couple | bonds, and the fluorescence intensity after a molecule | numerator couple | bonds. It is a flowchart for demonstrating the molecule | numerator detection method in embodiment of this invention. It is a figure which shows an example of the fluorescence light signal detected by the photodetector. It is a figure which shows the structure of the conventional DNA microarray detection apparatus.

Hereinafter, embodiments of the present invention will be described with reference to the drawings. The following embodiments are examples embodying the present invention, and do not limit the technical scope of the present invention.

FIG. 1 is a diagram showing a configuration of a molecular detection system according to an embodiment of the present invention. In FIG. 1, the molecule detection system 10 includes a molecule detection device 100 and a detection cartridge 101. The detection cartridge 101 holds an inspection sample. The molecule detection device 100 detects molecules existing in a holding space for holding molecules.

The molecular detection apparatus 100 includes a holding base 102, a control contact 103, a molecular detector 104, a flow controller 105, a laser controller 106, a spot controller 107, a fluorescent light signal processor 108, a system controller 109, and a host controller 110. Prepare.

The holding base 102 holds the detection cartridge 101. The control contact 103 transmits and receives control signals to and from the detection cartridge 101. The molecule detector 104 detects molecules. The flow controller 105 controls the operation of the detection cartridge 101. The laser controller 106 controls the laser light source of the molecular detector 104. The spot controller 107 controls the position of the irradiation light of the molecular detector 104. The fluorescent light signal processor 108 detects the fluorescent light signal from the molecule detector 104 and performs signal processing on the detected fluorescent light signal. The system controller 109 controls the operation of each block of the molecular detection device 100 in an integrated manner. The host controller 110 communicates with the system controller 109 to instruct display of detection results, input of operation modes, or execution of inspection.

Hereinafter, the operation of the molecule detection system 10 shown in FIG. 1 will be briefly described. The detection cartridge 101 is set on the holding base 102. The molecular measurement operation starts in accordance with an instruction from the host controller 110. A signal from the flow controller 105 is sent to the detection cartridge 101 through the control contact 103, and various operations of the detection cartridge 101 are controlled. Details of the control of the detection cartridge 101 will be described later in detail using a flowchart.

A molecule detector 104 is disposed below the detection cartridge 101. The molecular detector 104 detects fluorescent light generated by irradiating light to molecules in the detection cartridge 101. The molecule detector 104 includes a semiconductor laser as a light source for irradiating light to molecules. The laser controller 106 controls the emission intensity of the semiconductor laser and the on / off of the semiconductor laser in accordance with an instruction from the system controller 109.

Also, the molecular detector 104 needs to improve the detection accuracy by irradiating light to a predetermined position of the detection cartridge 101 stably to reduce detection noise. For this reason, the spot controller 107 controls the light irradiation position in accordance with an instruction from the system controller 109. The fluorescent light signal processor 108 performs signal processing on the fluorescent light signal corresponding to the fluorescent light detected by the molecular detector 104. Thereby, various detections are performed. These operations are sequentially performed by an automatic process manually or programmed by the host controller 110.

The operation of the molecular detection device of the present embodiment will be described in detail. Hereinafter, the detection cartridge 101, the molecular detector 104, the flow controller 105, the laser controller 106, the spot controller 107, and the fluorescent light signal processor 108 will be described.

2 is a top view showing the configurations of the detection cartridge 101 and the molecular detector 104 in the embodiment of the present invention, and FIG. 3 shows the configurations of the detection cartridge 101 and the molecular detector 104 in the embodiment of the present invention. FIG.

2 and 3, the detection cartridge 101 includes a test solution tank 202, a flow tube 203, a micro pump 204, phosphor detection probe storage units 205 to 209, a probe valve 210, a test solution valve 211, and a transparent member 216.

The test solution tank 202 stores a test sample (molecule test solution) containing molecules to be tested. The flow tube 203 holds molecules. A molecular test solution flows in the flow tube 203. The micropump 204 is composed of, for example, a piezoelectric element, and causes a solution to flow through the flow tube 203. The micropump 204 moves molecules in the flow tube 203.

Fluorescent substance detection probe storage units 205 to 209 store fluorescent substance detection probes (fluorescent substances having probes that bind to specific molecules) corresponding to the molecules desired to be detected. The phosphor detection probe storage units 205 to 209 hold in advance a phosphor (phosphor detection probe) that binds to a molecule in the flow tube 203. The probe valve 210 inputs each phosphor detection probe stored in the phosphor detection probe storage units 205 to 209 into the flow tube 203. The test solution valve 211 puts the molecular test solution stored in the test solution tank 202 into the flow tube 203. The transparent member 216 transmits light incident on the flow tube 203.

The molecular detector 104 includes an actuator 212, a fluorescent light detection optical unit 213, a semiconductor laser 214, and a light detector 215. The actuator 212 moves the irradiation light in a three-dimensional direction. The semiconductor laser 214 emits light for irradiating molecules. The photodetector 215 detects fluorescent light generated by irradiating light to molecules. The fluorescent light detection optical unit 213 guides the light from the semiconductor laser 214 to the actuator 212 and guides the fluorescent light to the photodetector 215.

The molecular detection device of the present embodiment detects a specific molecule with an accuracy of one molecule by irradiating the flow tube 203 of the detection cartridge 101 with the laser light from the molecular detector 104 to detect fluorescent light. be able to. As a first point, the optical configuration of the molecular detector 104 has a great feature. First, the configuration of the molecule detector 104 capable of detecting molecules with very high accuracy will be described, and then the specific operation of the detection cartridge 101 will be described.

FIG. 4 is a diagram showing a detailed optical configuration of the molecular detector 104 shown in FIG. In FIG. 4, the same components as those in FIGS. 1, 2, and 3 are denoted by the same reference numerals. In FIG. 4, the molecular detector 104 includes an actuator 212, a fluorescence light detection optical unit 213, a semiconductor laser 214, and a light detector 215.

The actuator 212 includes a first condenser lens 306, a second condenser lens 307, and a condenser lens actuator 308. The fluorescent light detection optical unit 213 includes a collimating lens 302, a collimating lens actuator 303, an aperture 304, a half mirror 305, a gap signal detector 309, a fluorescent light collimating lens 310, and a triangular prism 311.

The collimating lens 302 converts the laser light 301 emitted from the semiconductor laser 214 into substantially parallel light. The collimating lens actuator 303 moves the collimating lens 302 in the front-rear direction (optical axis direction). The collimating lens actuator 303 corrects the spherical aberration included in the light condensed on the flow tube 203 (holding space). The aperture 304 has a circular shape and shields the central portion of the laser beam 301. The aperture 304 limits the incident angle of the light emitted from the second condenser lens 307 and entering the holding space (flow tube 203). The half mirror 305 reflects the laser light 301 whose central portion is shielded and transmits the fluorescent light 313 generated from the molecules.

The first condenser lens 306 condenses the laser light 301 whose central portion is shielded from light. The second condenser lens 307 further condenses the laser light 301 collected by the first condenser lens 306. The first condenser lens 306 and the second condenser lens 307 collect light emitted from the semiconductor laser 214 in the holding space (flow tube 203). The condenser lens actuator 308 moves the first condenser lens 306 and the second condenser lens 307 in the vertical direction (optical axis direction) and the horizontal direction (direction perpendicular to the optical axis). The condenser lens actuator 308 moves the first condenser lens 306 and the second condenser lens 307 together. The spot controller 107 controls the condensing lens actuator 308 so that the condensing position of the light condensed by the first condensing lens 306 and the second condensing lens 307 is constant.

The gap signal detector 309 detects light reflected at the boundary between the detection cartridge 101 and the second condenser lens 307. The gap signal detector 309 detects the reflected light from the surface of the detection cartridge 101 having the holding space (flow tube 203), and controls the gap between the second condenser lens 307 and the detection cartridge 101. Output a signal. The spot controller 107 controls the condenser lens actuator 308 based on the gap control signal output from the gap signal detector 309 so that the distance between the second condenser lens 307 and the cartridge 101 is constant.

The fluorescent light collimating lens 310 converts the fluorescent light 313 generated from the molecules to be detected into parallel light. The triangular prism 311 decomposes the incident fluorescent light 313 into light for each wavelength. The triangular prism 311 separates the fluorescent light from the holding space for each wavelength corresponding to the fluorescent light. The photodetector 215 detects fluorescent light for each wavelength. The photodetector 215 detects reflected light from the holding space (flow tube 203). The photodetector 215 detects the intensity of the fluorescent light separated by the triangular prism 311.

The semiconductor laser 214 emits blue-violet laser light having a wavelength of 405 nm. The emission position of the laser beam 301 of the semiconductor laser 214 substantially coincides with the focal position of the collimating lens 302. As a result, the laser beam 301 that has passed through the collimating lens 302 is converted into substantially parallel light. The central portion of the laser beam 301 that has passed through the collimating lens 302 is shielded by a circular aperture 304. The laser beam 301 that has passed through the aperture 304 is guided to the first condenser lens 306 and the second condenser lens 307 by the half mirror 305.

The molecular detector according to the present embodiment has a great feature in that the central portion of the light is blocked by the aperture 304 and the light is incident on the outer peripheral portion of the condenser lens having a high numerical aperture. In the present embodiment, a condensing optical system with a high numerical aperture is realized by combining the first condenser lens 306 and the second condenser lens 307. The light condensed by the first condenser lens 306 and the second condenser lens 307 is designed to be condensed on the boundary surface between the transparent member 216 of the detection cartridge 101 and the flow tube 203.

The inside of the flow tube 203 is filled with an inspection sample. The refractive index of the inspection sample is n1. When the inspection sample is a normal aqueous solution, the main component of the inspection sample is almost moisture, so that the refractive index n1 is about 1.33. This molecule detection apparatus detects molecules contained in a test sample having a very low concentration. Therefore, when the test sample is an aqueous solution, the refractive index n1 is approximately 1.33.

FIG. 5 is an enlarged view showing a condensing point of light collected by the second condenser lens.

Here, the minimum incident angle of light that passes through the aperture 304 and is collected at the focal point (light that passes through the transparent member 216 and enters the flow tube 203) is δ, and the condensed beam of the detection cartridge 101 passes. The refractive index of the portion (the transparent member 216 existing between the second condenser lens 307 and the flow tube 203) is n2. By changing the size of the aperture 304, the minimum incident angle δ of light at the condensing portion can be changed. The minimum incident angle δ increases as the size of the aperture 304 increases, and the minimum incident angle δ decreases as the size of the aperture 304 decreases. In the present embodiment, the size of the aperture 304 is set so as to satisfy the condition of the following formula (2).

Sinδ> n1 / n2 (2)

When the minimum incident angle δ satisfies the condition of the above formula (2), the total reflection condition is satisfied at the boundary surface between the detection cartridge 101 and the flow tube 203, and all the collected light is reflected at the boundary surface. Will be. However, the light that satisfies the total reflection condition cannot be led to a desired condensing position only under the above conditions. This is because the refractive index of the air layer between the second condenser lens 307 and the detection cartridge 101 is 1, and thus the value of n2 / 1 (the refractive index of the air layer) is 1 or more. At this time, since the total reflection condition is satisfied at the boundary surface between the second condenser lens 307 and the air layer, light cannot be guided into the detection cartridge 101.

Therefore, in the molecular detection device of the present embodiment, this problem is solved by bringing the second condenser lens 307 close to the surface of the detection cartridge 101 to a distance equal to or shorter than the wavelength of light. Thus, the light can be guided to a position where it is desired to be collected without being totally reflected at the boundary surface between the detection cartridge 101 and the air layer. By using the condensing optical system configured as described above, light does not pass toward the flow tube 203 at the condensing point 401 shown in FIG. 5 because the incident light satisfies the total reflection condition. However, at the condensing point 401 that satisfies the total reflection condition, an evanescent effect occurs in which light oozes out at a distance that is a fraction of the wavelength of the light. Moreover, since the incident light is condensed by using the first condenser lens 306 and the second condenser lens 307, a three-dimensional minute light irradiation space is formed at the condensing point 401. The

In the present embodiment, the numerical aperture NA of the condenser lens including the first condenser lens 306 and the second condenser lens 307 is 1.6, and the refractive index n2 of the transparent member 216 of the detection cartridge 101 is 1. Eight. Since the inspection sample is an aqueous solution, the refractive index n1 is 1.33. The refractive index n1 is smaller than the refractive index n2. In this case, sin δ in the above equation (2) is 0.83. As a result, light incident at an angle where sin δ is in the range of 0.83 to 0.88 (= NA / n2) is collected at the condensing point 401. When the light condensed at the condensing point 401 is incident at an incident angle θ in a range satisfying the following expression (3), the light forms a minute three-dimensional condensing spot.

N1 / n2 <sin θ ≦ NA / n2 (3)

That is, the refractive index of the flow tube 203 is n1, the refractive index of the transparent member 216 existing between the second condenser lens 307 and the flow tube 203 is n2, and n1 <n2, and enters the flow tube 203. When the numerical aperture that defines the maximum value of the incident angle of light is NA, the aperture 304 is incident on the flow tube 203 through the transparent member 216 so as to satisfy the above equation (3). Limit θ. Further, the incident angle θ of the light that passes through the transparent member 216 and enters the flow tube 203 satisfies the above-described formula (3).

Therefore, in order to guide more light to the condensing point 401, it is desirable that the numerical aperture NA be as large as possible than the refractive index n1. In a normal molecular detection device, it is necessary to use as light for collecting 10% or more of the output power of the semiconductor laser. Therefore, it is desirable that the numerical aperture NA satisfies the condition of the following formula (4).

In this embodiment, since the numerical aperture NA is 1.6 and the wavelength of the laser beam is 405 nm, a minute three-dimensional irradiation space of about 100 nm × 100 nm × 100 nm is formed at the condensing point 401.

However, it is assumed that there is an error in the thickness of the transparent member 216 between the surface of the detection cartridge 101 and the flow tube 203 through which the light emitted from the second condenser lens 307 passes. If the detection cartridge 101 is produced with high accuracy, the necessary accuracy can be ensured for the thickness of the transparent member 216, but this leads to an increase in the cost of the detection cartridge 101. If the thickness error of the transparent member 216 can be corrected, the thickness accuracy of the transparent member 216 of the detection cartridge 101 can be relaxed, which is very effective in practice.

If there is an error in the thickness of the transparent member 216 of the detection cartridge 101 through which the collected light passes, spherical aberration occurs and the light collection performance deteriorates. In order to avoid this problem, the molecular detection device of the present embodiment includes a collimating lens actuator 303 as a mechanism for correcting spherical aberration. The collimating lens actuator 303 is composed of, for example, a stepping motor, and moves the collimating lens 302 in the front-rear direction (optical axis direction). By moving the collimating lens 302 in the optical axis direction, the light that has passed through the collimating lens 302 can be slightly shifted from the parallel light, thereby generating spherical aberration. The spherical aberration generated by the thickness error of the transparent member 216 of the detection cartridge 101 can be canceled out by the spherical aberration generated by the collimating lens 302. Therefore, a highly accurate molecular detection device can be realized with an inexpensive configuration.

Furthermore, a desirable configuration of the present embodiment is that the molecular detection device moves the first condenser lens 306 and the second condenser lens 307 in the vertical direction (optical axis direction) and the horizontal direction (direction perpendicular to the optical axis). And a condensing lens actuator 308 to be moved. In order to stably maintain a condensing space on the condensing point 401, the distance between the second condensing lens 307 and the detection cartridge 101 (hereinafter referred to as a gap) is 405 nm, which is the wavelength of the irradiation light. It is necessary to keep it at a fraction of the time. If the distance between the second condenser lens 307 and the detection cartridge 101 fluctuates, the intensity of light reflected at the boundary between the second condenser lens 307 and the detection cartridge 101 changes, and light detection is performed. It becomes noise of the detection signal of the device 215.

In order to suppress the fluctuation of the gap, it is necessary to keep the gap constant by controlling the positions of the first condenser lens 306 and the second condenser lens 307 in the optical axis direction. In order to control the positions of the first condenser lens 306 and the second condenser lens 307, a gap detection signal for detecting the gap amount is required. Therefore, the molecular detection device in the present embodiment includes a gap signal detector 309 that detects a gap signal.

The gap signal from the gap signal detector 309 is a signal as shown in FIG. 6 with respect to the gap interval. FIG. 6 is a diagram illustrating the relationship between the gap signal and the gap interval. When the gap interval is wide, the reflected light increases because all the light is reflected at the front interface (surface) of the detection cartridge 101. On the other hand, when the gap interval approaches the wavelength of the incident light, the reflected light decreases because the light enters the detection cartridge 101. By controlling the reflected light from the gap surface (surface) of the detection cartridge 101 to be a constant control target value, the gap interval can be kept constant.

Further, in order to stabilize the condensed light in this embodiment, the condenser lens actuator 308 also moves the positions of the first condenser lens 306 and the second condenser lens 307 in the direction perpendicular to the optical axis. Control. If the first condenser lens 306 and the second condenser lens 307 move in a direction perpendicular to the optical axis, the thickness of the transparent member 216 of the detection cartridge 101 changes, and aberration occurs. Therefore, the gap signal detector 309 includes two light receiving portions that receive reflected light from the surface of the detection cartridge 101 and are disposed at positions facing each other across the optical axis. The spot controller 107 controls the condenser lens actuator 308 so that the signal intensities of the two light receiving units of the gap signal detector 309 are equal.

In the above embodiment, a hemispherical lens is used as the second condenser lens 307, but the present invention is not particularly limited to this. FIG. 7 is a diagram showing another example of the second condenser lens in the embodiment of the present invention. As shown in FIG. 7, the second condenser lens 501 may have a shape that is shorter in the optical axis direction than the hemisphere. In this case, a function similar to that of a hemispherical lens can be realized. The second condenser lens may have a super hemispherical shape that is longer in the optical axis direction than the hemisphere. In either case, it can be realized in the same manner as a hemispherical lens, and the second condenser lens is not limited to a hemispherical lens. If the second condenser lens is a part of a spherical shape, the function as a condenser lens can be easily realized. In the present embodiment, the condenser lens is composed of two lenses (a first condenser lens and a second condenser lens), but the present invention is not particularly limited thereto, You may comprise with one lens with a high refractive index.

The fluorescent light collimating lens 310, the triangular prism 311, and the photodetector 215 are configured to detect fluorescent light generated from molecules, but the operation of these is specifically described using the detection cartridge 101 with respect to the principle of molecular detection. This will be explained later.

2 and 3 show a schematic configuration of the detection cartridge of the molecular detection device of the present embodiment. The detection cartridge 101 of the present embodiment includes phosphor detection probe storage units 205 to 209 that store five types of phosphor detection probes, respectively. By using this detection cartridge 101, five types of specific molecules can be detected with very high sensitivity. The phosphor detection probes stored in the phosphor detection probe storage units 205 to 209 and the principle of molecular detection will be described with reference to FIGS. 8A and 8B.

FIG. 8 (A) and FIG. 8 (B) are diagrams showing the phosphor detection probe used in the embodiment of the present invention. FIG. 8A is a diagram showing a phosphor detection probe before molecules are bound in the embodiment of the present invention, and FIG. 8B is a diagram showing how molecules are bound in the embodiment of the present invention. It is a figure which shows the fluorescent substance detection probe after.

8A, the phosphor detection probe includes a quantum dot 601 and a probe 602. The quantum dot 601 is composed of GdSe fine particles, and has a characteristic of emitting fluorescent light when irradiated with light. In the present embodiment, quantum dots are used as the phosphor. A probe 602 that binds complementarily to a specific molecule is bound to the quantum dot 601. The probe 602 may take any form as long as it has the ability to bind complementarily to a specific molecule. For example, in order to detect DNA having a specific sequence, the probe 602 becomes a single-stranded DNA having a sequence paired with the DNA. Similarly, in the case of RNA, a single-stranded RNA complementary to the RNA to be detected is selected as the probe 602. In addition to this, when detecting an antigen, an antibody that complementarily binds to the antigen to be detected is selected as the probe 602. Further, when detecting odor, a receptor corresponding to the odor molecule to be detected is selected as the probe 602.

By using the phosphor detection probe in which the probe 602 is bonded to the quantum dot 601, it is possible to easily detect whether or not a complementary molecule is bonded by changing the fluorescence wavelength. This is due to the fact that the fluorescence wavelength of the quantum dot 601 composed of fine particles such as GdSe depends on the band gap of the quantum dot 601. When the size of the quantum dot 601 is changed, the band gap width is easily changed, and the fluorescence wavelength is continuously changed.

As described above, the band gap energy that is very sensitive to the change in size greatly changes under the influence of a specific molecule binding to the probe 602 in a complementary manner. That is, the fluorescence wavelength or fluorescence intensity of the phosphor detection probe changes when a specific molecule is bound to the probe 602.

FIG. 9 is a diagram for explaining the wavelength dependence of the fluorescence intensity 701 before the molecules are bonded and the fluorescence intensity 702 after the molecules are bonded. As shown in FIG. 9, it can be seen that the fluorescence wavelength is greatly changed by the detection molecule 603 binding to the probe 602.

The phosphor detection probe shown in FIG. 8 (A) can be easily produced simply by coupling the probe 602 to the quantum dot 601 of a specific size, and mass production can be easily realized. In the conventional DNA microchip, it is necessary to arrange a specific probe according to the position on the chip, and mass production is difficult. Furthermore, it is necessary to label a molecule to be detected with a fluorescent dye in advance. When a molecule to be detected is labeled with a fluorescent dye in advance, pretreatment for binding the fluorescent dye to the molecule to be detected is necessary, and it is necessary to confirm in advance whether the fluorescent dye can be accurately bound to the molecule to be detected. For this reason, it has been difficult to achieve downsizing and simple operability with the conventional molecular detection apparatus.

On the other hand, the molecular detection device of the present embodiment can detect molecules with high accuracy simply by mixing a mass-produced phosphor detection probe with a test sample and measuring the wavelength of fluorescent light. Can be realized without processing.

Also, in the detection method using the conventional DNA microchip, the detection sensitivity is also a problem. In the detection method using a DNA microchip, a fluorescent dye is bound to a test sample in advance, and hybridization with a DNA probe on the DNA microchip is performed. The bound DNA is labeled with a fluorescent dye. Therefore, if the DNA microchip is scanned while shining light with a scanner, fluorescence is generated from the fluorescent dye at the bonded position, so that the bonded position can be specified.

However, the sensitivity of the scanner is low and it is difficult to detect unless a certain amount of fluorescent dye is bound. The problem of this decrease in detection sensitivity is a fundamental problem of the detection method in which a plurality of binding molecules are irradiated with light and fluorescence light is detected simultaneously. A large number of detection probes are distributed in the depth side and in the surface in the detection region for one molecule of the bound DNA microchip, and it is necessary to uniformly irradiate the detection region with scanning light. There is. The fluorescent dyes spatially distributed in response to the scanning light generate fluorescence independently. However, the scanning light is not applied only to the fluorescent dye of the detection probe, but is applied to the entire detection region. For example, the scan light is also applied to a base holding the detection probe or a fixing agent for fixing the detection probe. Since scanning light is reflected also from these materials and unnecessary fluorescence is generated from the materials themselves, it is theoretically difficult to increase the detection sensitivity. Further, since fluorescence is generated in a non-correlated manner from a plurality of fluorescent dyes, the phases of the light are not aligned, and the light intensity may be reduced.

In the molecular detection device of the present embodiment, the above-described minute three-dimensional irradiation space is formed in a detection solution (including a gas sample or the like), and fluorescent light generated in the minute three-dimensional irradiation space is detected. This solves the problem related to detection sensitivity that could not be overcome. Conventionally, the irradiation region of the irradiation light irradiated in the detection solution is wide, and there are a plurality of phosphors in the irradiation region, so it is difficult to measure the fluorescent light generated from one molecule. When multiple phosphors exist in the irradiation space, there are a mixture of phosphors that are bound to the molecules that are to be detected and phosphors that are not bound to the molecules, and each phosphor generates fluorescence. The separation of fluorescent light becomes impossible.

However, since the molecular detector of this embodiment and the photodetector used in the molecular detector can realize a very small three-dimensional irradiation space, it is possible to detect the presence of a small amount of molecules, which was difficult with the conventional method. .

In this embodiment, the molecular detection device 100 corresponds to an example of a molecular detection device, the semiconductor laser 214 corresponds to an example of a light source, and the first condenser lens 306 and the second condenser lens 307 are collected. It corresponds to an example of an optical lens, the aperture 304 corresponds to an example of an aperture, the photodetector 215 corresponds to an example of a photodetector, the triangular prism 311 corresponds to an example of a fluorescent light separation unit, and the first collection The optical lens 306 corresponds to an example of a first condenser lens, the second condenser lens 307 corresponds to an example of a second condenser lens, and the gap signal detector 309 corresponds to an example of a gap detector. The condensing lens actuator 308 corresponds to an example of a condensing lens actuator, the spot controller 107 corresponds to an example of a gap controller and a condensing position controller, and a collimating lens actuator The screen 303 corresponds to an example of a spherical aberration correction unit, the spot controller 107 corresponds to an example of a spherical aberration control unit, the flow controller 105 corresponds to an example of a sample loading instruction unit, and the fluorescent light signal processor 108 This corresponds to an example of a molecular concentration calculator.

In the present embodiment, the detection cartridge 101 corresponds to an example of a molecular detection cartridge, the flow tube 203 corresponds to an example of a holding space, the phosphor detection probe corresponds to an example of a phosphor, and the micropump 204. Corresponds to an example of the moving unit, the transparent member 216 corresponds to an example of the transparent member, the phosphor detection probe storage units 205 to 209 correspond to an example of the phosphor holding unit, and the quantum dot 601 corresponds to an example of the quantum dot. The probe 602 corresponds to an example of the probe.

Hereinafter, a molecular detection method in the molecular detection system shown in FIGS. 1 to 4 will be described with reference to FIG. Note that FIG. 10 illustrates an example of detecting an extremely small amount of miRNA.

FIG. 10 is a flowchart for explaining the molecular detection method according to the embodiment of the present invention.

First, in step S1, the detection cartridge 101 is attached to the molecule detection apparatus 100.

Next, in step S2, the flow controller 105 receives the cartridge information from the control contact 103, and identifies the cartridge type based on the received cartridge information. Based on the cartridge type identification, measurement is started by an instruction from the host controller 110. The cartridge information is stored in the detection cartridge 101 in advance. The cartridge information includes a cartridge type for specifying what molecule the detection cartridge 101 is a detection target.

Next, in step S3, the flow controller 105 activates the micropump 204 composed of a piezoelectric element. Thereby, the buffer solution circulates in the flow tube 203. In the present embodiment, since a very small amount of miRNA is detected, a buffer solution is inserted into the flow tube 203 as a solution in which miRNA is stably present in advance.

Next, in step S4, the laser controller 106 turns on the semiconductor laser 214 which is an irradiation light source. At this time, the laser controller 106 preferably determines the light emission power of the semiconductor laser 214 based on the cartridge information acquired in advance. This is because there are cases where the sensitivity of quantum dots varies depending on the cartridge type, or there is an upper limit in the power of laser light that can be irradiated depending on the type of sample to be detected. When a laser beam is irradiated to the same place of a stationary sample for a long time, the sample may be damaged by the laser beam. Therefore, as described above, it is more desirable to turn on the semiconductor laser 214 after circulating the buffer solution.

In the present embodiment, the laser controller 106 emits laser light with a power of 2.1 mW based on the cartridge information, and the laser light emitted with a power of 2.1 mW is emitted from the condensing point 401 of the cartridge. Is irradiated. Although not shown in FIG. 4, the molecular detection device further includes a sensor that detects a part of the light emitted from the semiconductor laser 214, and the laser controller 106 is based on a detection signal from the sensor. It is more desirable to perform feedback control of the laser beam. In this case, the semiconductor laser can emit light with stable power.

The cartridge information includes the light emission power of the semiconductor laser 214. In this case, the laser controller 106 acquires the light emission power from the detection cartridge 101 via the control contact 103. Further, the laser controller 106 may store in advance a table in which the cartridge type and the light emission power of the semiconductor laser 214 are associated with each other. In this case, the laser controller 106 determines the light emission power corresponding to the acquired cartridge type by referring to the table.

Next, in step S <b> 5, the spot controller 107 sets the positions of the first condenser lens 306 and the second condenser lens 307 so that a minute three-dimensional irradiation space is formed at the position of the condenser point 401. Control is performed by a condensing lens actuator 308. As described above, the gap signal from the gap signal detector 309 is used for this control.

The spot controller 107 controls the positions of the first condenser lens 306 and the second condenser lens 307 in the optical axis direction so that the intensity of the gap signal is constant. Further, the spot controller 107 is perpendicular to the optical axes of the first condenser lens 306 and the second condenser lens 307 so that the intensities of the gap signals from the two light receiving portions of the gap signal detector 309 are equal. Control the position in any direction.

Next, in step S6, the flow controller 105 controls the opening and closing of the probe valve 210, and puts the phosphor detection probe into the flow tube 203 from the phosphor detection probe storage units 205 to 209. The probe valve 210 is a valve composed of a piezoelectric element. Therefore, the flow controller 105 can accurately control the input amount of the phosphor detection probe by controlling the voltage pulse applied to the probe valve 210. Note that the flow controller 105 desirably determines the amount of the phosphor detection probe input based on the cartridge information described above. Thereby, measurement can be performed easily.

That is, the cartridge information includes the input amount of the phosphor detection probe. In this case, the flow controller 105 acquires the input amount of the phosphor detection probe from the detection cartridge 101 via the control contact 103. Further, the flow controller 105 may store in advance a table in which the cartridge type and the input amount of the phosphor detection probe are associated with each other. In this case, the flow controller 105 determines the input amount of the phosphor detection probe corresponding to the acquired cartridge type by referring to the table.

Here, the phosphor detection probe used for detecting miRNA in the present embodiment will be described. The quantum dot 601 which is a fluorescent portion of the phosphor detection probe is composed of GdSe fine particles as shown in FIG. The size of the quantum dot is changed for each solution in the phosphor detection probe storage units 205 to 209. The phosphor detection probe storage unit 205 stores a phosphor detection probe having a size of 2.0 nm and having green fluorescent quantum dots. The phosphor detection probe storage unit 206 stores a phosphor detection probe having a size of 2.9 nm and having yellow-green fluorescent quantum dots. The phosphor detection probe storage unit 207 stores a phosphor detection probe having a size of 4.1 nm and having orange fluorescent quantum dots. The phosphor detection probe storage unit 208 stores a phosphor detection probe having a size of 5.9 nm and having red fluorescent quantum dots. The phosphor detection probe storage unit 209 stores a phosphor detection probe having a size of 6.5 nm and having dark red fluorescent quantum dots.

The phosphor detection probes obtained by adding probes 602 that specifically bind to five different types of miRNAs to the quantum dots 601 are stored in the phosphor detection probe storage units 205 to 209. In this embodiment, the probe 602 specifically binds to five types of miRNAs, miR-1, miR-20a, miR-27a, miR-34a, and miR-423-5p, which are used as cancer biomarkers. Use a probe that

The five types of phosphor detection probes thrown into the flow tube 203 are diffused into the flow tube 203 by the micropump 204. Therefore, the phosphor detection probe is minute with a predetermined probability determined by the amount of the phosphor detection probe, the volume of the buffer solution in the flow tube 203, and the volume of the minute three-dimensional irradiation space formed at the condensing point 401. It passes through the three-dimensional irradiation space.

When the phosphor detection probe passes through the minute three-dimensional irradiation space, the quantum dot generates fluorescent light. As shown in FIG. 4, the generated fluorescent light passes through the first condensing lens 306, the second condensing lens 307, the half mirror 305, the fluorescent light collimating lens 310, and the triangular prism 311. At this time, the triangular prism 311 separates the incident fluorescent light into fluorescent light having a wavelength corresponding to each quantum dot. Then, the photodetector 215 detects the intensity of the fluorescent light for each wavelength. That is, the photodetector 215 can independently detect the presence or absence of a plurality of phosphor detection probes for each wavelength. The fluorescent light collimating lens 310, the triangular prism 311 and the photodetector 215 correspond to a fluorescent light intensity detector that detects fluorescent light for each wavelength.

At the center of the half mirror 305, a wavelength selection filter 314 that blocks excitation light having a wavelength of 405 nm is formed. As a result, stray light that hinders detection of weak fluorescent light can be eliminated, noise caused by excitation light can be reduced, and S / N can be improved.

In order to avoid stray light reflected at the boundary between the second condenser lens 307 and the detection cartridge 101, the refractive index of the second condenser lens 307 and the refractive index of the transparent member 216 of the detection cartridge 101 should be close to each other. Good. That is, it is preferable that the refractive index n3 of the condenser lens is substantially the same as the refractive index n2 of the transparent member 216. In this case, since no reflection occurs at the boundary between the second condenser lens 307 and the detection cartridge 101, it is effective against stray light. In this embodiment, two groups of condensing lenses are used. Basically, the refractive index n3 of the condensing lens is made substantially equal to the refractive index n2 of the transparent member 216 of the detection cartridge 101, thereby eliminating stray light. The effect can be realized. Furthermore, stray light can be removed by increasing the distance between the condensing point 401 and the boundary between the detection cartridge 101 and the second condensing lens 307.

FIG. 11 is a diagram showing an example of a fluorescent light signal detected by the photodetector. In FIG. 11, the vertical axis represents fluorescence intensity, and the horizontal axis represents time (ns). Further, FIG. 11 shows a fluorescent light signal for the fluorescent substance detection probe stored in the fluorescent substance detection probe storage unit 205. The fluorescent light has a wavelength of 510 nm and the fluorescent light has a green color.

As shown in FIG. 11, the fluorescent light signal is detected at a predetermined period, and each fluorescent light signal corresponds to one fluorescent substance detection probe. Thus, by appropriately selecting the concentration of the phosphor detection probe, the sample solution, and the volume of the minute three-dimensional irradiation space, the number of the phosphor detection probes that pass through the minute three-dimensional irradiation space at a time is stochastically determined. Can be almost one. Therefore, a single molecule can be easily detected by a change in the intensity of fluorescent light.

That is, when the volume of the minute three-dimensional irradiation space formed at the condensing point of the condensing lens is V1, and the volume of the flow tube 203 (holding space) is V2, the phosphor detection probe to be put into the flow tube 203 The number n of (phosphor) satisfies the following formula (5).

N <V2 / V1 (5)

The flow controller 105 adjusts the number n of the phosphor detection probes so as to satisfy the above formula (5).

This is realized for the first time by the molecular detection apparatus of the present embodiment that can form a minute three-dimensional irradiation space in a sample, and a conventional detection method cannot capture a signal of one molecule. It was very difficult. Similarly, other phosphor detection probes in the phosphor detection probe storage units 206 to 209 can be similarly detected by paying attention to the wavelength at which each quantum dot fluoresces.

Furthermore, in the molecular detection device of the present embodiment, since the fluorescence wavelength is changed for each quantum dot, a plurality of phosphor detection probes can be detected simultaneously.

Returning to FIG. 10, in step S7, the spot controller 107 corrects the spherical aberration by moving the collimating lens 302. Aberration optimization adjustment by the spot controller 107 is performed using the time width or fluorescence intensity of the fluorescent light signal shown in FIG. A small three-dimensional irradiation space formed at the condensing point 401 is preferably small in order to improve detection sensitivity. However, as described above, the size of the minute three-dimensional irradiation space increases due to the spherical aberration of the condensing optical system.

Spherical aberration can be corrected by moving the collimating lens 302. To perform this correction, it is necessary to detect the optimal position of the collimating lens 302. If the minute three-dimensional irradiation space is expanded due to spherical aberration, the intensity of the fluorescent light signal shown in FIG. 11 decreases. Further, since the passage time is long, the pulse width of the fluorescent light signal is widened. By using such a signal characteristic of the fluorescent light signal, the collimating lens 302 can be moved to an optimum position. Specifically, the spot controller 107 controls the collimating lens actuator 303 so that the amplitude of the fluorescent light signal output from the photodetector 215 is maximized or the pulse width of the fluorescent light signal is minimized. This aberration optimization adjustment process is a process for correcting the spherical aberration shown in step S7 of FIG.

Next, in step S8, the fluorescent light signal processor 108 measures the number of fluorescent substance detection probes. The number of fluorescent light signal pulses shown in FIG. 11 within a predetermined time is a number proportional to the concentration of the input phosphor detection probe. The fluorescent light signal processor 108 counts the number of pulses of the fluorescent light signal for a predetermined time, and stores the count number N1 in a memory (not shown).

Next, in step S <b> 9, the flow controller 105 controls opening and closing of the test solution valve 211, and a test sample is put into the flow tube 203 from the test solution tank 202. If any of the biomarkers miR-1, miR-20a, miR-27a, miR-34a, and miR-423-5p, which are the inspection targets of the present embodiment, is included in the input inspection sample The biomarker specifically binds to the corresponding fluorophore detection probe.

Next, in step S10, the fluorescent light signal processor 108 counts the number of fluorescent substance detection probes to which molecules are not bonded. As shown in FIG. 9, when a molecule is bound to the phosphor detection probe, the fluorescence wavelength is shifted to the longer wavelength side. In the present embodiment, since a small amount of miR-1 is contained in the test sample, miR-1 is bound to the phosphor detection probe from the phosphor detection probe storage unit 205. Due to the binding of miR-1, the fluorescence wavelength of the phosphor detection probe is shifted from 510 nm to 545 nm.

The fluorescent light signal processor 108 counts the number of fluorescent light signals corresponding to a wavelength of 510 nm during the same predetermined time as when the number of fluorescent substance detection probes was measured before the test sample was introduced, Count the number of fluorophore detection probes to which no molecules are bound. The fluorescence signal processor 108 counts the number of pulses of the fluorescence signal corresponding to the fluorescence wavelength of the phosphor detection probe to which no molecule is bound for a predetermined time, and stores the count number N2 in a memory (not shown). . The fluorescent light signal processor 108 can specify the relative number of molecules (miRNA) bound by calculating the difference between the count number N1 and the count number N2. Further, the fluorescent light signal processor 108 can also measure the number of directly bound molecules (miRNA) by counting the number of fluorescent light signals corresponding to a wavelength of 545 nm during a predetermined time.

As described above, the molecular detection device according to the present embodiment can count the number of molecules in units of molecules, so that it can easily detect a very low concentration of molecules that could not be detected until now. be able to.

Next, in step S11, the fluorescent light signal processor 108 calculates the concentration of molecules. The more excellent point of the molecular detection device of the present embodiment is that the concentration of the detection molecule in the test sample can be specified. The volume of the minute three-dimensional irradiation space formed at the condensing point of the condenser lens is V1 [ml], the volume of the flow tube 203 is V2 [ml], and the phosphor detection probe before the inspection sample is introduced And N1> N2, and the flow rate of the solution moving through the flow tube 203 is V3 [N2]. ml / s], the measurement time of the count number N1 and the count number N2 is T [s], and the amount of the input test sample is V4 [ml], the fluorescent light signal processor 108 Based on the equation (5), the concentration of molecules in the test sample is calculated.

The memory (not shown) provided in the molecular detection device includes a volume of a minute three-dimensional irradiation space, a volume of the flow tube 203, a flow rate of a solution moving through the flow tube 203, a measurement time of the count number N1 and the count number N2, and The amount of the test sample that has been input is stored in advance. The fluorescent light signal processor 108 includes the volume of the minute three-dimensional irradiation space, the volume of the flow tube 203, the flow rate of the solution moving through the flow tube 203, the measurement time of the count number N1 and the count number N2, and the inspection sample that has been input. Is read from the memory, and the concentration of molecules in the test sample is calculated.

It should be noted that the flow rate of the solution moving through the flow tube 203, the measurement time of the count number N1 and the count number N2, and the amount of the test sample input may be actually measured.

The cartridge information acquired from the detection cartridge 101 includes the volume of the minute three-dimensional irradiation space, the volume of the flow tube 203, the flow rate of the solution moving through the flow tube 203, the measurement time of the count number N1 and the count number N2, and the input. The amount of the test sample may be included. In this case, the fluorescent light signal processor 108 includes the volume of the minute three-dimensional irradiation space, the volume of the flow tube 203, the flow rate of the solution moving through the flow tube 203, the count number N1 and the count number N2 included in the acquired cartridge information. The concentration of molecules in the test sample is calculated based on the measurement time and the amount of the test sample input.

As described above, the concentration of molecules in the test sample can be specified based on the count number N1 and the count number N2, but the detection error increases as the count number N2 approaches zero. Therefore, it is more desirable for the fluorescent light signal processor 108 to adjust the input amount of the test sample so that the count number N2 can ensure 10% or more of the count number N1. In the present embodiment, it was confirmed that the concentration of molecules of several moles that could not be detected by a conventional DNA microchip could be detected.

As described above, the present embodiment provides an excellent molecular detection device capable of detecting one molecule in principle, and is a highly sensitive biomarker detection device, odor detection device, and A security monitoring device can be provided.

In the above embodiment, each of the phosphor detection probe storage units 205 to 209 stores one type of phosphor detection probe, but a plurality of types of phosphor detection probes are stored in one phosphor detection probe. You may mix and store in a part. Even in such a configuration, it is possible to detect the fluorescent light from the individual fluorescent substance detection probes for each fluorescent wavelength, so that the same performance as described above can be realized. In the present embodiment, each phosphor detection probe is simultaneously inserted from the phosphor detection probe storage units 205 to 209. However, each phosphor detection probe may be individually input, and in particular, the fluorescence wavelength of the phosphor. This method is effective when separation is difficult due to overlapping.

In addition, the molecular detection device of this embodiment includes a triangular prism 311 for separating fluorescent light having different fluorescent wavelengths, but instead of the triangular prism 311, a dichroic filter that separates light of a specific wavelength is used. You may prepare. Further, the light separated by the dichroic filter can be further finely separated by the triangular prism, and the molecular detection device of the present embodiment does not depend on the fluorescence light separation method.

In this embodiment, an example in which miRNA is detected is described. However, the probe 602 of the phosphor detection probe is changed to various probes such as an antibody or a molecular receptor that can specifically bind to a molecule. By doing so, it is possible to detect at a sensitivity corresponding to one molecule as described above.

In this embodiment, quantum dots are used as the phosphor. However, the quantum dots are not necessarily used as long as the phosphors change in fluorescence characteristics at a specific wavelength by binding molecules to the probe. The same effects as described above can be obtained.

The specific embodiments described above mainly include inventions having the following configurations.

A molecular detection device according to an aspect of the present invention is a molecular detection device that detects molecules existing in a holding space, and includes a light source that emits light, and the light emitted from the light source is collected in the holding space. A condensing lens, an aperture for limiting an incident angle of the light emitted from the condensing lens and entering the holding space, and a photodetector for detecting reflected light from the holding space. The refractive index of the space is n1, the refractive index of the transparent member existing between the condenser lens and the holding space is n2, n1 <n2, and the maximum incident angle of light incident on the holding space is When the defined numerical aperture is NA, the aperture limits the incident angle θ of light that passes through the transparent member and enters the holding space so as to satisfy the following formula (6).

N1 / n2 <sin θ ≦ NA / n2 (6)

According to this configuration, the light source emits light. The condensing lens condenses the light emitted from the light source in the holding space. The aperture limits the incident angle of light emitted from the condenser lens and entering the holding space. The photodetector detects reflected light from the holding space. The refractive index of the holding space is n1, the refractive index of the transparent member existing between the condenser lens and the holding space is n2, and n1 <n2, and the maximum incident angle of light incident on the holding space is defined. When the numerical aperture is NA, the aperture restricts the incident angle θ of the light that passes through the transparent member and enters the holding space so as to satisfy the above formula (6).

Therefore, since the incident angle θ of the light passing through the transparent member and entering the holding space is limited so as to satisfy the above formula (6), a minute three-dimensional irradiation space having a molecular unit size in the holding space. And one molecule in the minute three-dimensional irradiation space can be detected, and the detection sensitivity can be improved.

In the molecular detection device, the molecules in the holding space are bound to a phosphor, and fluorescent light is generated by irradiating the phosphor with light, and the fluorescent light from the holding space is generated. It is preferable that the apparatus further includes a fluorescent light separating unit that separates each wavelength corresponding to the fluorescent light, and the photodetector detects the intensity of the fluorescent light separated by the fluorescent light separating unit.

According to this configuration, molecules in the holding space are bound to the phosphor. Fluorescent light is generated when the phosphor is irradiated with light. The fluorescent light separation unit separates the fluorescent light from the holding space for each wavelength corresponding to the fluorescent light. The photodetector detects the intensity of the fluorescent light separated by the fluorescent light separation unit.

Therefore, the fluorescent light from the holding space is separated for each wavelength corresponding to the fluorescent light, and the intensity of the separated fluorescent light is detected, so that a plurality of molecules that bind to different phosphors can be detected simultaneously. .

In the molecular detection device, the condensing lens includes a first condensing lens that condenses the light, and a second condensing the light collected by the first condensing lens. It is preferable that a condensing lens is included.

According to this configuration, since the condensing lens is composed of two groups of lenses, a condensing optical system having a large numerical aperture can be easily realized.

Further, in the above molecular detector, when the refractive index of the condenser lens is n3, it is preferable that the refractive index n3 is substantially the same as the refractive index n2.

According to this configuration, since the refractive index n3 of the condenser lens is substantially the same as the refractive index n2 of the transparent member, stray light generated at the boundary between the transparent member and the condenser lens can be reduced. .

Further, in the above molecular detector, a gap detector that detects reflected light from the surface of the cartridge including the holding space and outputs a gap control signal for controlling the interval between the condenser lens and the cartridge; The distance between the condensing lens and the cartridge is made constant based on the condensing lens actuator that moves the condensing lens in the optical axis direction and the gap control signal output from the gap detector. It is preferable to further include a gap controller for controlling the condenser lens actuator.

According to this configuration, the gap detector detects the reflected light from the surface of the cartridge having the holding space, and outputs a gap control signal for controlling the interval between the condenser lens and the cartridge. The condenser lens actuator moves the condenser lens in the optical axis direction. The gap controller controls the condenser lens actuator based on the gap control signal output from the gap detector so that the distance between the condenser lens and the cartridge is constant.

Therefore, since the distance between the condensing lens and the cartridge is controlled to be constant, noise generated when the distance between the condensing lens and the cartridge varies can be reduced, and the S / N can be improved. be able to.

Further, in the above-described molecular detection device, a condensing lens actuator that moves the condensing lens in an optical axis direction, and the condensing lens so that a condensing position of light condensed by the condensing lens is constant. It is preferable to further include a spot controller that controls the actuator.

According to this configuration, the condenser lens actuator moves the condenser lens in the optical axis direction. The spot controller controls the condensing lens actuator so that the condensing position of the light collected by the condensing lens is constant.

Therefore, since the condensing position of the light condensed by the condensing lens is controlled to be constant, noise generated when the condensing position fluctuates can be reduced, and S / N is improved. be able to.

Further, in the above-described molecular detection device, it is preferable to further include a spherical aberration correction unit that corrects a spherical aberration included in the light condensed in the holding space.

According to this configuration, since the spherical aberration included in the light condensed in the holding space is corrected, when the thickness of the transparent member changes, the spherical aberration generated due to the change in the thickness of the transparent member is corrected. And detection accuracy can be improved.

In the above-described molecular detection device, a spherical aberration control unit that controls the spherical aberration correction unit so that the amplitude of the signal output from the photodetector is maximized or the pulse width of the signal is minimized. It is preferable to further provide.

According to this configuration, since the spherical aberration correction unit is controlled so that the amplitude of the signal output from the photodetector is maximized or the pulse width of the signal is minimized, the signal output from the photodetector Can be used to easily correct spherical aberration.

In the molecule detection apparatus, the molecule in the holding space is bonded to a phosphor, and a volume of a minute three-dimensional irradiation space formed at a condensing point of the condenser lens is set to V1, and the holding space is set. When the volume of V is V2, it is preferable that the number n of the phosphors put into the holding space satisfies the following formula (7).

N <V2 / V1 (7)

According to this configuration, molecules in the holding space are bound to the phosphor. When the volume of the minute three-dimensional irradiation space formed at the condensing point of the condenser lens is V1, and the volume of the holding space is V2, the number n of phosphors put into the holding space is expressed by the above equation (7). ) Is satisfied.

Therefore, it is possible to make one phosphor present in the minute three-dimensional irradiation space, and it is possible to specify the number of molecules by counting the fluorescence light.

Further, in the above-described molecular detection device, the molecule in the holding space binds to a phosphor, and a sample insertion instruction unit that instructs to input a sample into the holding space; and a condensing point of the condenser lens The volume of the minute three-dimensional irradiation space formed on the substrate is V1, the volume of the holding space is V2, the count number of the phosphor before the sample is charged is N1, and the sample is charged after the sample is charged The count number of the phosphors to which no molecules are bonded is N2, N1> N2, the flow rate of the solution moving through the holding space is V3, and the measurement time of the count number N1 and the count number N2 is T. It is preferable to further include a molecular concentration calculation unit that calculates the concentration of the molecule based on the following formula (8) when the amount of the sample introduced is V4.

According to this configuration, molecules in the holding space are bound to the phosphor. The sample loading instruction unit instructs to load a sample into the holding space. The volume of the minute three-dimensional irradiation space formed at the condensing point of the condenser lens is V1, the volume of the holding space is V2, and the phosphor count number before the sample is charged is N1, and the sample is loaded. After that, the count number of the phosphor not bound to the molecule is N2, N1> N2, the flow rate of the solution moving through the holding space is V3, and the measurement time of the count number N1 and the count number N2 is T. When the amount of the sample obtained is V4, the molecular concentration calculation unit calculates the molecular concentration based on the above equation (8).

Therefore, it is possible to easily calculate the concentration of molecules based on the count number N1 of the phosphor before the sample is charged and the count number N2 of the phosphor to which no molecules are bonded after the sample is charged. Can do.

A molecule detection method according to another aspect of the present invention is a molecule detection method for detecting molecules existing in a holding space, and includes a light emission step of emitting light from a light source, and collecting the light emitted from the light source. A condensing step for condensing light into the holding space by an optical lens; a light limiting step for restricting the incident angle of the light emitted from the condensing lens and entering the holding space by an aperture; and reflection from the holding space A light detection step of detecting light with a photodetector, wherein the refractive index of the holding space is n1, the refractive index of the transparent member existing between the condenser lens and the holding space is n2, and n1 < When the numerical aperture that defines the maximum value of the incident angle of the light incident on the holding space is NA, the aperture passes through the transparent member and satisfies the following expression (9). Limiting the incident angle θ of the light incident into the holding space.

N1 / n2 <sin θ ≦ NA / n2 (9)

According to this configuration, light is emitted from the light source in the light emission step. In the condensing step, the light emitted from the light source is condensed in the holding space by the condensing lens. In the light limiting step, the incident angle of the light emitted from the condenser lens and entering the holding space is limited by the aperture. In the light detection step, the reflected light from the holding space is detected by the photodetector. The refractive index of the holding space is n1, the refractive index of the transparent member existing between the condenser lens and the holding space is n2, n1 <n2, and the maximum value of the incident angle of light incident on the holding space is When the defined numerical aperture is NA, the aperture limits the incident angle θ of light that passes through the transparent member and enters the holding space so as to satisfy the above formula (9).

Therefore, since the incident angle θ of the light that passes through the transparent member and enters the holding space is limited so as to satisfy the above formula (9), a minute three-dimensional irradiation space having a molecular unit size in the holding space. And one molecule in the minute three-dimensional irradiation space can be detected, and the detection sensitivity can be improved.

A cartridge for detecting molecules according to another aspect of the present invention includes a holding space for holding molecules, a moving unit for moving the molecules in the holding space, and a transparent member that transmits light incident on the holding space. When the refractive index of the holding space is n1, the refractive index of the transparent member is n2, n1 <n2, and the numerical aperture that defines the maximum incident angle of light incident on the holding space is NA The incident angle θ of light that passes through the transparent member and enters the holding space satisfies the following formula (10).

N1 / n2 <sin θ ≦ NA / n2 (10)

According to this configuration, the holding space holds molecules. The moving unit moves molecules in the holding space. The transparent member transmits light incident on the holding space. When the refractive index of the holding space is n1, the refractive index of the transparent member is n2, n1 <n2, and the numerical aperture that defines the maximum angle of incidence of light incident on the holding space is NA, the transparent member The incident angle θ of light that passes through and enters the holding space satisfies the above equation (10).

Therefore, since the incident angle θ of light that passes through the transparent member and enters the holding space is limited so as to satisfy the above formula (10), a minute three-dimensional irradiation space having a molecular unit size in the holding space. And one molecule in the minute three-dimensional irradiation space can be detected, and the detection sensitivity can be improved.

In addition, it is preferable that the molecule detection cartridge further includes a phosphor holding unit that holds in advance a phosphor that binds to the molecule in the holding space.

According to this configuration, since the phosphor that binds to the molecule in the holding space is held in advance, a desired molecule can be easily detected using the held phosphor.

In the molecular detection cartridge, the molecule in the holding space is bonded to a phosphor, and the phosphor is connected to the quantum dot and the quantum dot, and specifically to a specific molecule. And a probe that binds.

According to this configuration, one molecule can be detected by measuring a change in fluorescent light due to binding.

In the above-described molecular detection cartridge, the fluorescent wavelength or fluorescent intensity of the phosphor is preferably changed by binding a specific molecule to the probe.

According to this configuration, the fluorescence wavelength or fluorescence intensity of the phosphor changes when a specific molecule binds to the probe, so that only a specific molecule can be detected by measuring the fluorescence wavelength or fluorescence intensity. Can do.

It should be noted that the specific embodiments or examples made in the section for carrying out the invention are merely to clarify the technical contents of the present invention, and are limited to such specific examples. The present invention should not be interpreted in a narrow sense, and various modifications can be made within the spirit and scope of the present invention.

The molecular detection device, the molecular detection method, and the molecular detection cartridge according to the present invention can improve the detection sensitivity, and are useful for the molecular detection device, the molecular detection method, and the molecular detection cartridge that detect molecules, and have high sensitivity. The present invention can be applied to biomarker detection devices, odor detection devices, security monitoring devices, and the like.

Claims (15)

A molecular detection device for detecting molecules existing in a holding space,
A light source that emits light;
A condenser lens that condenses the light emitted from the light source in the holding space;
An aperture for limiting an incident angle of the light emitted from the condenser lens and incident on the holding space;
A photodetector for detecting reflected light from the holding space;
The refractive index of the holding space is n1, the refractive index of the transparent member existing between the condenser lens and the holding space is n2, n1 <n2, and the maximum incident angle of light incident on the holding space When the numerical aperture that defines the value is NA,
The aperture detector limits the incident angle θ of light that passes through the transparent member and enters the holding space so as to satisfy the following expression (1).
n1 / n2 <sin θ ≦ NA / n2 (1) The molecules in the holding space bind to a phosphor;
When the phosphor is irradiated with light, fluorescent light is generated,
A fluorescent light separation unit that separates the fluorescent light from the holding space for each wavelength corresponding to the fluorescent light;
The molecular detector according to claim 1, wherein the photodetector detects an intensity of the fluorescent light separated by the fluorescent light separation unit. The condensing lens includes a first condensing lens that condenses the light and a second condensing lens that further condenses the light collected by the first condensing lens. The molecular detection device according to claim 1 or 2, characterized in that When the refractive index of the condenser lens is n3,
The molecular detection device according to any one of claims 1 to 3, wherein the refractive index n3 is substantially the same as the refractive index n2. A gap detector that detects reflected light from the surface of the cartridge including the holding space and outputs a gap control signal for controlling the interval between the condenser lens and the cartridge;
A condenser lens actuator for moving the condenser lens in the optical axis direction;
And a gap controller that controls the condenser lens actuator based on the gap control signal output from the gap detector so that a distance between the condenser lens and the cartridge is constant. The molecular detection device according to any one of claims 1 to 4. A condenser lens actuator for moving the condenser lens in the optical axis direction;
6. A condensing position controller that controls the condensing lens actuator so that a condensing position of light condensed by the condensing lens is constant. The molecular detector according to 1. The molecular detection device according to any one of claims 1 to 6, further comprising a spherical aberration correction unit that corrects a spherical aberration included in the light condensed in the holding space. The apparatus further comprises a spherical aberration control unit that controls the spherical aberration correction unit so that an amplitude of a signal output from the photodetector is maximized or a pulse width of the signal is minimized. 8. The molecular detection device according to 7. The molecules in the holding space bind to a phosphor;
When the volume of the minute three-dimensional irradiation space formed at the focal point of the condenser lens is V1, and the volume of the holding space is V2,
The molecular detection device according to any one of claims 1 to 8, wherein the number n of the phosphors put into the holding space satisfies the following formula (2).
n <V2 / V1 (2) The molecules in the holding space bind to a phosphor;
A sample loading instruction unit for instructing to load a sample into the holding space;
The volume of the minute three-dimensional irradiation space formed at the condensing point of the condenser lens is V1, the volume of the holding space is V2, and the count number of the phosphor before the sample is charged is N1; The count number of the phosphor to which no molecules are bonded after the sample is introduced is N2, N1> N2, the flow rate of the solution moving through the holding space is V3, the count number N1 and the count number A molecular concentration calculation unit that calculates the concentration of the molecule based on the following equation (3), where T is the measurement time of N2 and V4 is the amount of the sample charged: The molecular detection device according to any one of claims 1 to 9.

A molecular detection method for detecting molecules existing in a holding space,
A light emitting step for emitting light from the light source;
A condensing step of condensing the light emitted from the light source into the holding space by a condensing lens;
A light limiting step of limiting an incident angle of the light emitted from the condenser lens and entering the holding space with an aperture;
A light detection step of detecting reflected light from the holding space by a photodetector,
The refractive index of the holding space is n1, the refractive index of the transparent member existing between the condenser lens and the holding space is n2, n1 <n2, and the maximum incident angle of light incident on the holding space When the numerical aperture that defines the value is NA,
The molecular detection method, wherein the aperture limits an incident angle θ of light that passes through the transparent member and enters the holding space so as to satisfy the following expression (4).
n1 / n2 <sin θ ≦ NA / n2 (4) A holding space for holding molecules;
A moving unit for moving the molecules in the holding space;
A transparent member that transmits light incident on the holding space;
When the refractive index of the holding space is n1, the refractive index of the transparent member is n2, n1 <n2, and the numerical aperture that defines the maximum incident angle of light incident on the holding space is NA,
The molecular detection cartridge, wherein an incident angle θ of light passing through the transparent member and entering the holding space satisfies the following expression (5).
n1 / n2 <sin θ ≦ NA / n2 (5) 13. The molecular detection cartridge according to claim 12, further comprising a phosphor holder that holds in advance a phosphor that binds to the molecule in the holding space. The molecules in the holding space bind to a phosphor;
The cartridge for molecular detection according to claim 12 or 13, wherein the phosphor includes a quantum dot and a probe that is connected to the quantum dot and specifically binds to a specific molecule. 15. The molecular detection cartridge according to claim 14, wherein the fluorescence wavelength or fluorescence intensity of the phosphor changes when a specific molecule binds to the probe.

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