Particle-in-liquid analysis system

Abstract

To improve the detection accuracy of fluorescence compared to the particle-in-liquid analysis system described in patent document 1.SOLUTION: A particle-in-liquid analysis system (1) is provided with an integrated circuit chip (13), a flow path (10) through which fine particles flow, a light source (semiconductor laser 17) for irradiating the flow path (10) with pulse-like excitation light, and a control unit (15) for controlling the state of a single-photon avalanche diode (14A) to either a light-receiving state or a standby state. The control unit (15) selects a delay time from the irradiation timing of the light source (semiconductor laser 17) until the single-photon avalanche diode (14A) is brought into the light-receiving state from a first delay time and a second delay time, detects pulse-like photons in response to an avalanche current in the single-photon avalanche diode (14A) in the light-receiving state, and repeatedly determines the number of times of detection of photons using the first delay time as a first detection number and the number of times of detection of photons using the second delay time as a second detection number, thereby detecting the fluorescent substance accompanying the fine particles.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a liquid-submerged particle analysis system and a liquid-submerged particle analysis apparatus for optically analyzing particles in a liquid. The present invention relates to a method for analyzing particles in the body. [Background technology]

[0002] One of the testing methods used in research and clinical testing in biology and medicine is the use of fluorescence. Fluorescence is light with a longer wavelength than the excitation light that is emitted when a molecule or ion that has absorbed excitation light (e.g., ultraviolet or visible light) and gone from the ground state to an excited state and then dropped to an intermediate excited state returns to the ground state. Fluorescence testing methods for living or non-living samples have been successfully applied in a wide range of fields, including staining of cellular components, gene expression, and observation of the localization and movement of dynamic components.

[0003] The most commonly used fluorescence inspection method is to irradiate an object with excitation light and detect only the emitted fluorescence. One method for separating the fluorescence emitted from the object and the excitation light reflected by the object is to use an optical filter to separate the two, taking advantage of the difference in wavelength between the fluorescence and the excitation light.

[0004] On the other hand, another method for separating fluorescence and excitation light is to use the fluorescence lifetime. The fluorescence lifetime is the average time it takes for an object to return from an excited state to the ground state. For many types of molecules, the fluorescence lifetime is on the order of subnanoseconds to a few nanoseconds, and varies depending on the type of molecule. By using the fluorescence lifetime, it is possible to detect the fluorescence emitted after the excitation light is quenched.

[0005] Patent Document 1 describes an example of a device that detects fluorescence using the fluorescence lifetime, with the configuration shown in Figures 1 and 2. The device described in Patent Document 1 is configured to make a single photon avalanche diode (SPAD) in a state capable of detecting photons after the excitation light is quenched, and to count pulses due to an avalanche current that flows when the SPAD receives a fluorescent photon. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 6860890 (registered March 31, 2021) Summary of the Invention [Problem to be solved by the invention]

[0007] Incidentally, in the liquid-submerged particle analysis system of Patent Document 1, when a particle flowing through the flow path is present near the single-photon avalanche diode, the excitation light may be scattered or refracted by the particle and enter the SPAD. In this case, the amount of photons entering the single-photon avalanche diode increases compared to when the particle flowing through the flow path is not present near the single-photon avalanche diode. As a result, in a liquid-submerged particle analysis system that detects fluorescence using the fluorescence lifetime, it may be difficult to separate the excitation light and the fluorescence according to the detection result of the SPAD.

[0008] The present invention has been made in consideration of the above-mentioned problems, and has an object to improve the detection accuracy of fluorescence compared to the liquid-submerged particle analyzing system described in Patent Document 1. [Means for solving the problem]

[0009] In order to solve the above-mentioned problems, a liquid-borne particle analysis system according to a first aspect of the present invention comprises an integrated circuit chip including a single-photon avalanche diode, a flow path through which particles flow formed on the side of the single-photon avalanche diode of the integrated circuit chip, a light source that irradiates the flow path with pulsed excitation light, and a control unit that controls the state of the single-photon avalanche diode to either a light-receiving state or a standby state.

[0010] In this liquid-submerged particle analysis system, the control unit selects a delay time from the irradiation timing at which the light source irradiates the excitation light to the time at which the single-photon avalanche diode is put into the light-receiving state from a first delay time and a second delay time longer than the first delay time, and after the delay time has elapsed from the irradiation timing, puts the single-photon avalanche diode into the light-receiving state for a predetermined light-receiving period, and in the light-receiving state, detects photons as pulses of avalanche current in the single-photon avalanche diode, repeats the detection of the photons using the first delay time a fixed number of times, with the number of times the photons are detected during the repetitions being the first detection number, repeats the detection of the photons using the second delay time a fixed number of times, with the number of times the photons are detected during the repetitions being the second detection number, and detects fluorescent substances associated with the particles by repeatedly determining the first detection number and the second detection number. Effect of the Invention

[0011] According to the fluorescence inspection system according to one aspect of the present invention, it is possible to improve the detection accuracy of the fluorescence compared to the liquid-borne particle analysis system described in Patent Document 1. [Brief description of the drawings]

[0012] [Figure 1] 1 is a schematic diagram showing a schematic configuration of a liquid-submerged particle analysis system according to an embodiment of the present invention; [Diagram 2]2 is a schematic diagram showing a schematic configuration of an integrated circuit chip provided in the liquid-borne particle analysis system shown in FIG. 1. FIG. [Diagram 3] 2 is a timing chart showing the timing of the irradiation operation of a light source provided in the liquid-submerged particle analysis system shown in FIG. 1 and the first delay measurement and the second delay measurement performed by a control unit provided in the liquid-submerged particle analysis system shown in FIG. 1. [Figure 4] 13 is a graph showing the output of a first delay measurement, the background time average of the first delay measurement, the output of a second delay measurement, the background time average of the second delay measurement, the normalized output of the first delay measurement and the output of the second delay measurement, and the ratio of the normalized output of the first delay measurement to the normalized output of the second delay measurement. [Diagram 5] 1 is a timing chart showing the irradiation operation of a light source provided in a liquid-submerged particle analysis system according to one embodiment of the present invention, and the timing of a first delay measurement, a second delay measurement, a third delay measurement, and a fourth delay measurement performed by a control unit provided in the liquid-submerged particle analysis system. [Figure 6] This is a graph showing the response time distribution of a SPAD in a liquid-submerged particle analysis system of embodiment 2 of the present invention, which shows the response time distribution of the SPAD when no particles are flowing through the flow path and the response time distribution of the SPAD when particles accompanied by fluorescent substances are flowing through the flow path. [Figure 7] This is a graph showing the response time distribution of a SPAD in a liquid-borne particle analysis system according to one embodiment of the present invention, which shows the response time distribution of the SPAD when no particles are flowing through the flow path, and an exponential decay curve with a time constant equal to the fluorescence lifetime of a fluorescent substance associated with the particles. [Figure 8] 11 is a timing chart showing the irradiation timing of the light source and the light receiving state of the SPAD when a delay time of 0 nsec, 1 nsec, and 2 nsec is used. [Figure 9] FIG. 1 is a schematic diagram of a liquid-borne particle analysis system showing how photons scattered by particles flowing through a flow path are detected by a SPAD. [Figure 10]This is a graph showing the response time distribution of the SPAD in a liquid-submerged particle analysis system according to one embodiment of the present invention and a conventional liquid-submerged particle analysis system, which shows (1) the response time distribution of the SPAD when no particles are flowing through the flow path, (2) the response time distribution of the SPAD when particles without fluorescent substances are flowing through the flow path, and (3) the response time distribution of the SPAD when particles with fluorescent substances are flowing through the flow path. [Figure 11] 10 is a timing chart showing an irradiation operation of a light source provided in a conventional liquid-submerged particle analysis system. [Figure 12] 1 is a graph showing the output of a SPAD in a conventional liquid-borne particle analysis system. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] [Embodiment 1] Hereinafter, the first embodiment of the present invention will be described in detail.

[0014] <Configuration of liquid-borne particle analysis system 1> Fig. 1 is a schematic diagram showing the schematic configuration of a liquid-submerged particle analyzing system 1 according to a first embodiment of the present invention, and is a schematic diagram showing a side view of the liquid-submerged particle analyzing system 1. Fig. 2 is a schematic diagram showing the schematic configuration of an integrated circuit chip 13, and is a schematic diagram showing the integrated circuit chip 13 as viewed from above.

[0015] 1, the liquid-submerged particle analyzing system 1 includes a control unit 15, an integrated circuit chip 13, and a light source control circuit 16. The control unit 15 is a calculation device having a function of comprehensively controlling each part of the liquid-submerged particle analyzing system 1. The control unit 15 controls each component of the liquid-submerged particle analyzing system 1 by, for example, one or more processors (e.g., CPU, etc.) executing programs stored in one or more memories (e.g., RAM, ROM, etc.).

[0016] The integrated circuit chip 13 includes a flow path 10 and a photon measurement unit 14. The integrated circuit chip 13 may have a configuration in which the flow path 10, which is a fluid circuit unit that flows a liquid, and the photon measurement unit 14, which is an electronic circuit unit, are built into a semiconductor integrated circuit using silicon such as CMOS. For example, the integrated circuit chip 13 may have a configuration in which the flow path 10 and the photon measurement unit 14 are integrally formed using so-called MEMS technology. The flow path 10 is provided on one of a pair of main surfaces of the integrated circuit chip 13, which is the main surface on the side of a single photon avalanche diode (SPAD) described later (in FIG. 1, the main surface above the SPAD).

[0017] Furthermore, the integrated circuit chip 13 may have a configuration in which the flow path 10 and the photon measurement unit 14 are superimposed and integrated together. For example, the integrated circuit chip 13 may have a configuration in which the flow path 10, which is a minute flow path formed in a light-transmitting member such as a glass substrate and silicone resin, is placed on a silicon integrated circuit in which the photon measurement unit 14 is built in.

[0018] Integrated circuit chip 13 has flow channel 10 on the surface side facing semiconductor laser 17, and photon measurement unit 14 is provided below flow channel 10. Flow channel 10 has injection hole 11 and discharge hole 12. Flow channel 10 is used as a flow channel for liquid containing fine particles that flows from injection hole 11 to discharge hole 12.

[0019] The photon measurement unit 14 has a single-photon avalanche diode (SPAD) 14A and a photon counting circuit 14B. The photon counting circuit 14B controls the SPAD 14A in response to a control signal supplied from the control unit 15, and processes the output signal of the SPAD 14A.

[0020] The photon counting circuit 14B has a function of measuring the number of electrical pulses generated by the incidence of photons on the SPAD 14A.

[0021] The light source control circuit 16 controls the light emission drive of the semiconductor laser 17 in response to a control signal supplied from the control unit 15, and performs an irradiation operation of irradiating an irradiation target with an excitation light pulse 18 from the semiconductor laser 17. Note that, although an example in which the control unit 15 and the light source control circuit 16 are separate from the integrated circuit chip 13 is shown in FIG. 1, the control unit 15 and the light source control circuit 16 may be configured to be formed within the integrated circuit chip 13.

[0022] In the liquid-submerged particle analyzing system 1, while liquid is flowing through the flow path 10, the light source control circuit 16 causes the semiconductor laser 17 to emit pulsed light to irradiate excitation light. Within the irradiation range of the semiconductor laser 17, the fluorescent molecules FM in the liquid in the flow path 10 are excited by irradiation of the excitation light pulses 18 from the semiconductor laser 17. The photon measurement unit 14 performs a photon detection operation to detect fluorescent photons generated by the excitation of the fluorescent molecules FM.

[0023] The irradiation operation of the semiconductor laser 17 by the light source control circuit 16 and the photon measurement operation of the photon measurement unit 14 are synchronously controlled by the control unit 15. Fig. 8 is a timing chart showing an example of the timing of the control of the irradiation operation of the excitation light pulse 18 and the photon measurement operation of the photon measurement unit 14 based on the control of the control unit 15. The excitation light pulse 18 is emitted at the rising edge of the irradiation trigger 30 of the pulsed laser, and a pulse can be generated by the avalanche current due to the photons incident on the SPAD during the SPAD light receiving period 35.

[0024] <Method of analyzing particles in liquid using the liquid-submerged particle analysis system 1> In the synchronous control diagram of the pulsed laser and the SPAD 14A in FIG. 8, SPAD control timings 32, 33, and 34 with different delay times from the rising edge of the pulsed laser irradiation trigger 30 to the start of the SPAD light receiving period 35 are shown. The control unit 15 in the first embodiment can change the delay time in this way. FIG. 3 is a diagram showing a measurement method in the first embodiment. The unit measurement sequence 100 is composed of a first delay measurement 101 in which the control unit 15 sets the delay from the rising edge of the pulsed laser irradiation trigger 30 to the start of the SPAD light receiving period 35 to the first delay time 72 in FIG. 10, repeats the irradiation of the pulsed laser 65536 times, and counts the output pulses of the SPAD during that time, and a second delay measurement 102 in which the control unit 15 sets the delay from the rising edge of the pulsed laser irradiation trigger 30 to the start of the SPAD light receiving period 35 to the first delay time 72 in FIG. 10, repeats the irradiation of the pulsed laser 65536 times, and counts the output pulses of the SPAD. FIG. 10 will be described later in the section on problems with conventional liquid-submerged particle analysis systems.

[0025] An output 111 in FIG. 4 is a time series plot of the output of the first delay measurement obtained by repeating the unit measurement sequence 100, and represents the number of times a photon is detected (first detection number) during the repetition of the first delay measurement 101. An output 113 in FIG. 4 is a time series plot of the second delay measurement, and represents the number of times a photon is detected (second detection number) during the repetition of the second delay measurement 102. 115 is a time series plot of the value (first normalized detection number) of the output of the first delay measurement normalized by the background time average 112 of the first delay measurement, and the value (second normalized detection number) of the output of the second delay measurement normalized by the background time average 114 of the second delay measurement. 116 is a time series plot of the ratio of the normalized first delay measurement value to the normalized second delay measurement value in the same unit measurement sequence (the ratio of the first normalized detection number to the second normalized detection number). In this measurement, when the fluorescence lifetime of the fluorescence to be observed is longer than 0.2 nsec, the distribution of the time response of the SPAD is as shown in Fig. 10, and when the measurement at the first delay time 72 is compared with the measurement at the second delay time 73, the ratio of the response 71 when fluorescence is added to the response 61 when only direct incident light is present is larger at the second delay time 73 than at the first delay time 72. Also, the ratio of the response 62 when the incident light is increased due to scattering or refraction to the response 61 when only direct incident light is present is the same at both the first delay time 72 and the second delay time 73. Therefore, in Fig. 4, in the section 117 where the signal changes due to fluorescence, the ratio of the normalized first delay measurement value to the normalized second delay measurement value in the same unit measurement sequence of 116 changes to become larger than 1, and in the section 118 where the signal changes due to scattering or refraction, the ratio of the normalized first delay measurement value to the normalized second delay measurement value in the same unit measurement sequence of 116 does not change much from 1. Therefore, it is possible to detect the passage of a particle accompanied by the fluorescent molecule FM to be observed by observing the time series plot of 116. Furthermore, if the time series plot of 116 does not change when a change in the output 111 or 113 is detected, it is possible to detect that a change in the amount of incident light has occurred due to scattering or refraction caused by the passage of a particle.

[0026] [Embodiment 2] The second embodiment will be described with reference to Fig. 5. The liquid-submerged particle analysis system in this embodiment is equipped with two lasers with different wavelengths, a first laser and a second laser. The unit measurement sequence in Fig. 5 is composed of a first laser emission period 121 and a second laser emission period 122. Furthermore, the first laser emission period 121 is composed of a first delay measurement 101 in which the control unit 15 sets the delay from the rising edge of the irradiation trigger 30 of the pulsed laser 1 to the start of the SPAD light receiving period 35 to a first delay time, repeats the irradiation of the pulsed laser 65,536 times, and counts the output pulses of the SPAD during that time, and a second delay measurement 102 in which the control unit 15 sets the delay from the rising edge of the irradiation trigger of the pulsed laser 2 to the start of the SPAD light receiving period to a third delay time, repeats the irradiation of the pulsed laser 65,536 times, and counts the output pulses of the SPAD during that time, and a fourth delay measurement 124 in which the control unit 15 sets the delay from the rising edge of the irradiation trigger of the pulsed laser 2 to the start of the SPAD light receiving period to a third delay time, repeats the irradiation of the pulsed laser 65,536 times, and counts the output pulses of the SPAD during that time. The fluorescent molecules FM to be measured are a first fluorescent molecule that is excited by a first laser but not by a second laser, and a second fluorescent molecule that is not excited by the first laser but is excited by the second laser.

[0027] By observing a change in the ratio of the first normalized delay measurement to the second normalized delay measurement within the same unit measurement sequence, the passage of a particle carrying a first fluorescent molecule can be detected, and by observing a change in the ratio of the third normalized delay measurement to the fourth normalized delay measurement within the same unit measurement sequence, the passage of a particle carrying a second fluorescent molecule can be detected.

[0028] [Embodiment 3] The third embodiment will be described with reference to Figs. 6 and 7. The response time distribution 131 of the SPAD when only direct incident light is present in the liquid-submerged particle analysis system of the first embodiment can be measured by sweeping the delay time. 131 is the delay time dependency of the number of photon detections (background detection number) during the repetition of sweeping the delay time and repeating the detection of photons using each swept delay time a certain number of times in a state where no particles are flowing through the flow path. It is assumed that the response when fluorescence is added due to the passage of a particle accompanied by a fluorescent molecule FM to be measured will be as shown by 141 in Fig. 6. In order to detect the response when fluorescence is added with high sensitivity in the measurement of the first embodiment, it is desirable to measure with a delay time that increases the ratio of 141 to 131. Here, the amount of increase in the output pulse due to fluorescence at a delay time of 0 is denoted as A, the fluorescence lifetime is denoted as τ, the delay time is denoted as t, and the response when only direct incident light is present is denoted as BG(t), and the response 141 when fluorescence is added is expressed by Equation 1. Therefore, the ratio to the background 131 is calculated as in Equation 2. It can be seen that in order to maximize the value of Equation 2, it is sufficient to use the optimal delay time (152 in FIG. 7) that maximizes the ratio (Equation 3) between the response time distribution 131 obtained by sweeping the delay time and the exponential decay function (151 in FIG. 7) with the fluorescence lifetime τ as the time constant. The numerator of Equation 3 can be calculated if the fluorescence lifetime is known, and the denominator can be calculated from measurements obtained by sweeping the delay time, so Equation 3 can be evaluated and the optimal delay time 152 can be derived during implementation.

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[0029] The most commonly used fluorescence inspection method is to irradiate an object with excitation light and detect only the emitted fluorescence. One method for separating the fluorescence emitted from the object and the excitation light reflected by the object is to use an optical filter to separate the two, taking advantage of the difference in wavelength between the fluorescence and the excitation light.

[0030] On the other hand, another method for separating fluorescence and excitation light is to use the fluorescence lifetime. The fluorescence lifetime is the average time it takes for an object to return from an excited state to the ground state. For many types of molecules, the fluorescence lifetime is on the order of subnanoseconds to a few nanoseconds, and varies depending on the type of molecule. By using the fluorescence lifetime, it is possible to detect the fluorescence emitted after the excitation light is quenched.

[0031] Patent Document 1 describes a liquid-submerged particle analysis system as an example of a device that detects fluorescence using the fluorescence lifetime. The liquid-submerged particle analysis system of Patent Document 1 has a structure similar to that of the liquid-submerged particle analysis system 1 shown in Figures 1 and 2. Like the liquid-submerged particle analysis system 1, the liquid-submerged particle analysis system of Patent Document 1 is configured to put the SPAD into a photon detection state after the excitation light is extinguished, and to count pulses due to an avalanche current that flows when the SPAD receives a fluorescent photon.

[0032] On the other hand, as described in M. Ghioni, A. Gulinatti, I. Rech, F. Zappa and S. Cova, "Progress in Silicon Single-Photon Avalanche Diodes," in IEEE Journal of Selected Topics in Quantum Electronics, vol. 13, no. 4, pp. 852-862, 2007, it is known that the probability distribution of the time from when a photon enters the depletion layer to when the avalanche phenomenon occurs in a SPAD includes a distribution of decay characteristics due to several time constants. In other words, in addition to the case where the avalanche occurs almost simultaneously with the photon incidence, the avalanche current can occur with a delay from the photon incidence.

[0033] Here, an example of the response characteristic from when a photon is incident on the SPAD to when an avalanche current is generated will be described with reference to Figs. 8 to 10. Fig. 8 is a diagram showing an example of the relationship between the periodic irradiation timing 31 of the SPAD with a pulsed laser having a half-width of about several tens of psec and the operation of the SPAD, which are controlled in synchronization with each other. The SPAD light-receiving period 35 in Fig. 8 is a period in which the SPAD is in a state in which the reverse bias of the SPAD is raised above the breakdown voltage and the SPAD is in a state in which the SPAD can generate an avalanche current by using the incidence of a photon as a trigger. The SPAD control timing 32 with a delay of 0 nsec shows control in which the SPAD light-receiving period starts simultaneously with the irradiation of the pulsed laser. Moreover, the SPAD control timing 33 with a delay of 1 nsec shows control in which the SPAD light-receiving period starts 1 nsec after the irradiation of the pulsed laser. Furthermore, the SPAD control timing 34 with a delay of 2 nsec shows control in which the SPAD light-receiving period starts 2 nsec after the irradiation of the pulsed laser. Measurement at each delay time is performed by detecting whether or not a pulse due to an avalanche current occurs within each light-reception start period, repeating this measurement a certain number of times, and integrating the number of times the pulse occurs.

[0034] Figure 10 shows the results of measurements taken at two different delay times, varying the delay time from the irradiation of the pulsed laser to the start of light reception by the SPAD from 0 nsec to 5 nsec in increments of 0.02 nsec, using the control shown in Figure 8. 16 This is an example of a graph in which the number of pulses generated is approximated by a combination of exponential functions and plotted on a graph. The vertical axis of the graph in Figure 10 is displayed on a logarithmic scale. Since the number of measurements at each delay time is 65536, the maximum value of the pulse count is saturated at 65536.

[0035] In the saturated region 41 in Fig. 10, a pulse is generated by the avalanche current almost immediately after the photon of the pulsed laser light is incident. Since the number of measurement repetitions at each delay time is 65536, and pulses are counted in all repeated measurements, the pulse count is saturated at 65536. Between the delay times of 0.1 nsec and 1.5 nsec, there is a region of fast tail 42 that decays with a time constant of 0.2 nsec, and after the delay time of 1.3 nsec, there is a region of slow tail 43 that decays with a time constant of 20 nsec. The exponential decrease in the pulse count in the fast tail 42 and slow tail 43 is thought to be due to the statistical fluctuation in the time it takes for the charge carriers generated by the photon incident on the SPAD to reach the edge of the depletion layer by diffusion and cause an avalanche. In addition, the existence of the two decay characteristics of the fast tail 42 and the slow tail 43 is thought to be due to the difference in the position where the photon is absorbed. FIG. 10 is a schematic example of the SPAD data used in the experiment, and it is known that the response is actually approximated by various combinations of exponentially decreasing components.

[0036] In view of the characteristics of the response time distribution in the SPAD described above, the method of detecting fluorescence using the fluorescence lifetime shown in Patent Document 1 can be understood to count pulses due to the avalanche current of the SPAD in the fast tail or slow tail region, and to detect fluorescence when the count number increases from the count number when there is no fluorescence (background).

[0037] In the integrated circuit chip 57 of the liquid-submerged particle analysis system (denoted by reference numeral 50 in FIG. 9) of Patent Document 1, wiring metals 54 and 55 that do not transmit light are provided in the area other than the light receiving surface of the SPAD 56. In the integrated circuit chip 57 configured in this way, the excitation light 53 is scattered or refracted by the particles 52 flowing in the liquid in the direction of the arrow 51 as shown in FIG. 9, and the amount of excitation light incident on the SPAD 56 may increase compared to when the particles 52 are not present near the SPAD 56. The response characteristic of the SPAD 56 in such a situation increases in proportion to the increase in the amount of excitation light incident, as shown in response 62 in FIG. 10, compared to the response characteristic when only direct incident light is received, and becomes a characteristic shown in response 62 when the incident light increases. On the other hand, when the particles 52 with fluorescent molecules pass over the SPAD, the response characteristic of the SPAD 56 in a state in which the fluorescent molecules are excited by the excitation light and the fluorescence is incident on the SPAD becomes a response 71 when fluorescence is added in FIG. 10. Here, it is assumed that the fluorescence lifetime of the fluorescent molecules is about 2 nsec. In the liquid-submerged particle analysis system of Patent Document 1, if a cell X1 without fluorescent molecules passes near the SPAD 56 in the same state as response 62, and then a cell X2 with fluorescent molecules passes near the SPAD 56 whose response characteristics are expected to be response 71, by repeatedly irradiating the pulsed laser as shown in Figure 11, photon measurements are performed 65,536 times at a delay from the pulse oscillation, for example, the first delay time 72 in Figure 10, and the output pulses of the SPAD are counted, a time series graph of pulse count 90 as shown in Figure 12 is obtained.

[0038] As shown in FIG. 12, in this situation, a signal change 92 due to an increase in excitation light and a signal change 91 due to fluorescence are observed in sequence. However, there is a problem in that it is difficult to determine whether these signal changes are due to fluorescence or not from this observation data alone.

[0039] 〔summary〕 The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. The technical scope of the present invention also includes embodiments obtained by appropriately combining the technical means disclosed in the different embodiments. Furthermore, new technical features can be formed by combining the technical means disclosed in the respective embodiments.

[0040] [Additional Notes] A liquid-borne particle analysis system according to a first aspect of the present invention comprises an integrated circuit chip including a single-photon avalanche diode, a flow path through which particles flow formed on the side of the integrated circuit chip on the single-photon avalanche diode, a light source that irradiates the flow path with pulsed excitation light, and a control unit that controls the state of the single-photon avalanche diode to either a light-receiving state or a standby state.

[0041] In this liquid-submerged particle analysis system, the control unit selects a delay time from the irradiation timing at which the light source irradiates the excitation light to the time at which the single-photon avalanche diode is put into the light-receiving state from a first delay time and a second delay time longer than the first delay time, and after the delay time has elapsed from the irradiation timing, puts the single-photon avalanche diode into the light-receiving state for a predetermined light-receiving period, and in the light-receiving state, detects pulsed photons in accordance with the avalanche current in the single-photon avalanche diode, repeats the detection of the photons using the first delay time a fixed number of times, with the number of times the photons are detected during the repetitions being the first detection number, repeats the detection of the photons using the second delay time a fixed number of times, with the number of times the photons are detected during the repetitions being the second detection number, and detects fluorescent substances associated with the particles by repeatedly determining the first detection number and the second detection number.

[0042] As shown in FIG. 3, in the first delay measurement 101, the measurement at the first delay time 72 in FIG. 10 is repeated 65532 times to count the output pulses of the SPAD, and in the second delay measurement 102, the measurement at the second delay time 73 in FIG. 10 is repeated 65532 times to count the output pulses of the SPAD. By repeating the first delay measurement and the second delay measurement as a unit measurement sequence 100, the output 111 of the first delay measurement and the output 113 of the second delay measurement in FIG. 4 are obtained. When the output 111 of the first delay measurement is normalized by the background time average 112 of the first delay measurement, and the output 113 of the second delay measurement is normalized by the background time average 114 of the second delay measurement, a normalized graph 115 by the background time average is obtained. Furthermore, when the ratio of the normalized first delay measurement value to the normalized second delay measurement value in the same unit measurement sequence is plotted, a graph 116 is obtained.

[0043] In the graph of 116, only the output change due to the fluorescence appears, and no change is seen in response to the change in the amount of incident excitation light. Therefore, it is possible to detect the fluorescence based on the time series data of 116. That is, according to the above configuration, the detection accuracy of the fluorescence can be improved compared to the liquid-submerged particle analysis system described in Patent Document 1.

[0044] This result can be explained as follows. The SPAD output observed at the first and second delays when there is no change in the amount of excitation light incident or when fluorescence photons are incident is based on the response of the fast tail 131 in FIG. 6 and has an exponential decay characteristic with a time constant of 0.2 nsec. Here, when the amount of excitation light incident on the SPAD increases due to scattering or refraction, the output of the fast tail region also increases in proportion to the increase rate. Therefore, the rate of change of the normalized value in the first delay measurement is the same as the rate of change of the normalized value in the second delay, and the ratio of these two values ​​in the same unit measurement sequence shown in 116 is approximately 1, and no change occurs. On the other hand, the response characteristic of the SPAD when fluorescence photons of a fluorescence whose fluorescence lifetime is longer than the decay time constant of the fast tail are incident is shown by response 71 in FIG. 10, and is the output of response 61 or response 62 to which an exponential decay amount with the fluorescence lifetime (assumed to be 2 nsec here) is added. Since the decay of the fast tail region of response 61 or response 62 has a shorter time constant and is more rapid than the decay of fluorescence, the ratio of the value of response 71 (response when fluorescence is added) to the background (response 61) at the second delay time 73 is greater than the ratio of the value of response 71 (response when fluorescence is added) to the background (response 61) at the first delay time 72, resulting in a change such as 116.

[0045] In a liquid-submerged particle analysis system according to a second aspect of the present invention, in addition to the configuration of the liquid-submerged particle analysis system according to the first aspect described above, a configuration is adopted in which the light source irradiates excitation light of a plurality of different wavelengths at different irradiation timings, and the control unit performs the detection by repeatedly determining each of the first detection count and the second detection count for each of the excitation light.

[0046] According to the above configuration, it is possible to detect fluorescent substances associated with particles using excitation light of different wavelengths. Therefore, by appropriately selecting the fluorescent substances associated with the particles and the wavelength of the excitation light that causes the fluorescent substances to fluoresce, it is possible to detect the fluorescence of each fluorescent substance separately from the fluorescence of other fluorescent substances. More specifically, it is possible to distinguish between multiple fluorescent substances by selecting multiple fluorescent substances whose absorption spectra do not overlap with each other and using excitation light of a wavelength included in the absorption spectrum for each of the selected fluorescent substances.

[0047] In a liquid-submerged particle analysis system according to a third aspect of the present invention, in addition to the configuration of the liquid-submerged particle analysis system according to the first or second aspect described above, a configuration is adopted in which the control unit calculates a first average value which is the time average value of the first detection count and a second average value which is the time average value of the second detection count when no particles are flowing through the flow path, normalizes the first detection count by the first average value to form a first normalized detection count, normalizes the second detection count by the second average value to form a second normalized detection count, and performs the detection according to the ratio between the first normalized detection count and the second normalized detection count.

[0048] Among the photons detected by the single-photon avalanche diode, photons caused by the fluorescence of the fluorescent material tend to react sensitively to changes in delay time. On the other hand, among the photons detected by the single-photon avalanche diode, photons caused by scattering and refraction by fine particles tend to react insensitively to changes in delay time. Therefore, according to the above configuration, photons are detected according to the ratio between the first normalized detection count and the second normalized detection count, so that photons caused by fluorescence that are sensitive to changes in delay time can be reliably detected.

[0049] In a liquid-submerged particle analysis system according to a fourth aspect of the present invention, in addition to the configuration of the liquid-submerged particle analysis system according to the third aspect described above, a configuration is adopted in which the particle is detected in response to a change in at least one of the first normalized detection number and the second normalized detection number, the change being due to at least one of scattering and refraction by the particle.

[0050] According to the above-mentioned configuration, since it is possible to detect photons caused by at least one of scattering and refraction by the particles, it is possible to detect the passage of the particles through the flow path located near the single-photon avalanche diode, and therefore it is possible to reliably detect the emission of fluorescence from the fluorescent substance associated with the particles.

[0051] In a liquid-submerged particle analyzing system according to a fifth aspect of the present invention, in addition to the configuration of the liquid-submerged particle analyzing system according to any one of the first to fourth aspects described above, the control unit sweeps the delay time when no particles are flowing through the flow path, repeats the detection of the photons using each swept delay time a fixed number of times, determines the number of photon detections during the repetitions as the background detection number, obtains the delay time dependence of the background detection number, determines an optimal delay time that maximizes the ratio of the delay time dependence of the background detection number to the delay time dependence of the exponential decay amount calculated from the fluorescence lifetime of the fluorescent substance, and uses the optimal delay time as either the first delay time or the second delay time.

[0052] According to the above configuration, an optimal delay time calculated from the fluorescence lifetime of a known fluorescent substance associated with the microparticles can be used as either the first delay time or the second delay time, thereby further improving the detection accuracy of the fluorescence compared to the liquid-submerged microparticle analysis system described in Patent Document 1. [Explanation of symbols]

[0053] 1. Liquid-borne particle analysis system 10 Flow Path 11 Injection hole 12 Discharge hole 13 Integrated Circuit Chips 14A Single Photon Avalanche Diode (SPAD) 14B Photon measurement circuit 15 Control section 16 Light source control circuit 17 Semiconductor laser (light source) 18 Excitation light pulse (excitation light) FM fluorescent molecules X1 Cells without fluorescent molecules X2 Cells with fluorescent molecules X3 Next cell to be observed 72 First Delay Time 73 Second Delay Time 111 First detection count 113 Second detection count 115 First normalized detection count and second normalized detection count 116 Ratio of the first normalized detection count to the second normalized detection count 131 background detections 151 Exponential decay calculated from the fluorescence lifetime of fluorescent substances 152 Optimal Delay Time

Claims

1. an integrated circuit chip including a single photon avalanche diode; a flow path through which particles flow, the flow path being formed on the side of the single-photon avalanche diode of the integrated circuit chip; a light source that irradiates the flow channel with pulsed excitation light; a control unit that controls a state of the single-photon avalanche diode to either a light receiving state or a standby state; The control unit is a delay time from the timing at which the light source irradiates the excitation light to the timing at which the single-photon avalanche diode is brought into the light-receiving state is selected from a first delay time and a second delay time longer than the first delay time; After the delay time has elapsed from the irradiation timing, the single-photon avalanche diode is set in the light-receiving enabled state for a predetermined light-receiving period; In the light receiving state, detecting pulsed photons in response to an avalanche current in the single-photon avalanche diode; repeating the detection of the photon using the first delay time a certain number of times, and defining the number of times the photon is detected during the repetition as a first detection number; repeating the detection of the photon using the second delay time the certain number of times, and defining the number of times the photon is detected during the repetition as a second detection number; The first detection count and the second detection count are repeatedly obtained to detect the fluorescent substance associated with the fine particles. A liquid-borne particle analysis system comprising:

2. the light source irradiates excitation light of a plurality of wavelengths different from each other at the irradiation timings different from each other, the control unit performs the detection by repeatedly determining each of the first detection count and the second detection count for each of the excitation light.

2. A system for analyzing particles in a liquid according to claim 1.

3. The control unit is calculating a first average value which is a time average value of the first detection count and a second average value which is a time average value of the second detection count in a state in which no particles are flowing through the flow path; The first detection count is normalized by the first average value to obtain a first normalized detection count; The second detection count is normalized by the second average value to obtain a second normalized detection count; performing the detection according to a ratio between the first normalized detection number and the second normalized detection number; 3. A system for analyzing particles in a liquid according to claim 1 or 2.

4. detecting the particle according to a change in at least one of the first normalized detection number and the second normalized detection number, the change being caused by at least one of scattering and refraction by the particle; 4. A system for analyzing particles in a liquid according to claim 3.

5. The control unit is In a state where no particles are flowing through the flow path, the delay time is swept, and the detection of the photons using each swept delay time is repeated a certain number of times, and the number of times the photons are detected during the repetition is defined as a background detection number, and the delay time dependency of the background detection number is obtained; determining an optimal delay time that maximizes the ratio of the delay time dependency of the number of background detections to the delay time dependency of an exponential decay amount calculated from the fluorescence lifetime of the fluorescent substance; using the optimum delay time as either the first delay time or the second delay time; 3. A system for analyzing particles in a liquid according to claim 1 or 2.

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