Concentration measurement method and concentration measurement device

Abstract

The invention relates to a concentration determination method and a concentration determination device, which are suitable for various different shapes of liquid to be detected and can accurately determine the concentration of a component to be detected. The concentration determination method is used for determining the concentration of a to-be-detected component in a to-be-detected liquid, and comprises the following steps: an excitation step: irradiating exciting light towards the to-be-detected liquid, keeping the to-be-detected liquid in a keeping unit through surface tension, enabling the exciting light to pass through a part of the to-be-detected liquid and then reach a light collection area of a to-be-detected area, after leaving the light collection area, the liquid passes through the other part of the liquid to be detected and leaves the liquid to be detected, and the light collection area is an area for collecting elastic scattered light and inelastic scattered light generated by irradiation in the liquid to be detected; a collection and detection step of collecting and detecting elastic scattered light and inelastic scattered light generated by irradiation of the light collection region; and a concentration determination step: determining the concentration of the component to be detected according to the collected elastic scattered light and inelastic scattered light.

Description

Technical Field

[0001] This application relates to the field of optical detection, and more specifically to a concentration measurement method and a concentration measurement device. Background Technology

[0002] It is known that optical detection techniques can be used to determine the concentration of liquids such as reagents or pharmaceuticals. Currently, fluorescence devices are used as a detection method to determine the concentration of these liquids. Typically, in existing fluorescence-based detection techniques, the liquid to be tested (hereinafter referred to as the test liquid) is placed in a test tube or cuvette. An excitation light is shone onto the test tube or cuvette containing the test liquid using a light source, while a light-receiving device receives the fluorescence produced by the test liquid under the excitation light. After obtaining the fluorescence produced by the test liquid under the excitation light, the concentration of the test liquid is determined based on the intensity of the excitation light and the intensity of the fluorescence.

[0003] Figure 1 A schematic diagram illustrating the specific structure of a concentration measuring device A0 in the prior art is shown. (See diagram for example.) Figure 1 As shown, the concentration measuring device A0 mainly includes a light source 1, a first filter 2, a beam splitter 3, a first light receiving part 4, a first slit 5, a second slit 6, a second filter 7, and a second light receiving part 8. This concentration measuring device A0 is used to measure the concentration of a component to be tested in a test liquid (e.g., a protein preparation) contained in a test tube 9. The excitation light emitted from the light source 1 propagates in the air until it reaches the test tube 9, and it can be assumed that the excitation light does not experience energy attenuation during propagation in the air. Therefore, if the luminous intensity of the excitation light just reaching the test tube 9 (i.e., the luminous intensity of the excitation light received by the first light receiving part 4) is set to I0, the optical path length of the excitation light within the test tube 9 is set to L, and the luminous intensity of the inelastic scattered light generated by the test liquid in the test tube 9 under the irradiation of the excitation light (i.e., the luminous intensity of the inelastic scattered light received by the second light receiving part 8) is set to I... flour Then, the concentration (molar concentration) C of the analyte in the liquid to be tested can be expressed by the following mathematical formula:

[0004]

[0005] Where ε represents the extinction coefficient (unit: L) 2 / mol), C represents the molar concentration of the liquid being tested (unit: mol / L). 3 ).

[0006] When using test tubes or cuvettes to hold the liquid to be tested, sufficient volume of the liquid needs to be placed in the container to ensure the accuracy of the results. However, in practice, problems may arise such as the difficulty in obtaining samples or the high cost of reagents.

[0007] Therefore, a high-precision method is needed to determine the concentration of the analyte in a trace amount of the test liquid, so as to ensure the accuracy and reliability of the test results while reducing the amount of test liquid used, and preferably be applicable to test liquids with different fluid properties. Summary of the Invention

[0008] This application was developed to solve the aforementioned technical problems, and its purpose is to provide a concentration determination method applicable to various different forms of liquids to be tested, and capable of accurately determining the concentration of the analyte in the test liquid. Based on this, the present invention also provides a concentration determination device.

[0009] One technical solution of this application provides a concentration determination method for measuring the concentration of an analyte in a liquid. The concentration determination method includes an excitation step, a collection and detection step, and a concentration determination step. In the excitation step, excitation light is irradiated toward the liquid to be tested. The liquid is held in a holding unit by surface tension. The excitation light passes through a portion of the liquid to reach a light-collecting region, and after leaving the light-collecting region, it passes through another portion of the liquid and exits. The light-collecting region is the area in the liquid to be tested where elastically scattered and inelastically scattered light generated by the irradiation is collected. In the collection and detection step, the elastically scattered and inelastically scattered light generated by the irradiation in the light-collecting region are collected and detected separately. In the concentration determination step, the concentration of the analyte is determined based on the collected elastically scattered and inelastically scattered light.

[0010] According to the concentration determination method described in this technical solution, elastically scattered light and inelastically scattered light generated when the light-collecting area of ​​the liquid to be tested is irradiated are collected and detected separately. The concentration of the analyte in the liquid to be tested is determined based on the luminous intensity of the separately detected elastically scattered light and inelastically scattered light. Since the liquid column shape exhibited by the liquid to be tested when held in the holding unit is mainly affected by the sample volume of the liquid to be tested, different sample volumes will result in different liquid column shapes. The luminous intensity of the excitation light emitted from the light source will attenuate to varying degrees as it passes through the liquid to be tested due to the different liquid column shapes. As a result, the parameters of the light actually irradiating the light-collecting area (e.g., luminous intensity) may be difficult to obtain, leading to a decrease in the accuracy of concentration determination. In other words, the luminous intensity of the light irradiating the light-collecting area is not equivalent to the luminous intensity of the excitation light emitted from the light source; for example, the excitation light may attenuate after entering the liquid to be tested but before reaching the light-collecting area. By correcting the influence of liquid column shape changes on the concentration results using relevant information of elastically scattered light, higher accuracy can be obtained.

[0011] Optionally, the collection and detection step further includes collecting elastically scattered light and inelastically scattered light at the same location for separate detection.

[0012] According to the concentration determination method described in this technical solution, the complexity of the system can be reduced by collecting elastic and inelastic scattered light at the same location, and the elastic and inelastic scattered light collected at the same location have good correlation, which can improve the accuracy of the measurement results.

[0013] Optionally, before the excitation step, the method includes: holding the liquid to be detected at the contact surface of the holding unit, the contact surface being used to provide surface tension to hold the liquid to be detected. The collection detection step further includes collecting elastically scattered and inelastically scattered light generated by irradiation of the light collection area through a receiving optical fiber, the light collection area being determined at least by the numerical aperture of the receiving optical fiber and the irradiation range of the excitation light.

[0014] The concentration determination method described in this technical solution can conveniently collect elastically scattered light and inelastically scattered light.

[0015] Optionally, the holding unit has multiple holding portions, and the liquid to be detected is in direct contact with a receiving optical fiber at least partially disposed inside one of the multiple holding portions, or one of the multiple holding portions near the receiving optical fiber includes a light-transmitting layer.

[0016] Optionally, an optical element is disposed between the light-transmitting layer and the receiving optical fiber.

[0017] Alternatively, the optical element may be any of a spherical mirror, a hemispherical mirror, or a lens group.

[0018] According to the concentration measurement method described in this technical solution, by making the receiving optical fiber in direct contact with the liquid to be tested, the loss of optical signal can be minimized, thereby improving the light collection efficiency. Furthermore, by placing an optical element between the receiving optical fiber and the liquid to be tested, the generated elastically scattered and inelastically scattered light can be focused, thereby improving the intensity and quality of the collected optical signal.

[0019] Optionally, the concentration determination step includes: adjusting the luminescence intensity of the excitation light based on the luminescence intensity of the detected elastic scattered light; and collecting and detecting the adjusted inelastic scattered light generated by the liquid to be tested under the irradiation of the adjusted excitation light, and determining the concentration of the analyte based on the detected adjusted inelastic scattered light and the luminescence intensity of the adjusted excitation light.

[0020] According to the concentration determination method described in this technical solution, the luminous intensity of the excitation light can be dynamically adjusted based on the detection results, which helps to avoid optical signal distortion or noise increase caused by excessively strong or weak excitation light.

[0021] Optionally, the concentration determination step includes: estimating relevant information of inelastic scattered light in the light collection area based on the luminescence intensity of the detected elastic scattered light and a pre-set estimation model; and determining the concentration of the analyte based on the luminescence intensity of the detected inelastic scattered light and the relevant information of the inelastic scattered light.

[0022] According to the concentration determination method described in this technical solution, by using the relevant information of inelastic scattered light to replace the relevant information of excitation light for determining the concentration of the analyte in the liquid to be tested, the influence of excitation light attenuation caused by changes in the shape of the liquid column on the concentration determination is eliminated, which can improve the accuracy of concentration determination of the analyte in the liquid to be tested and reduce the measurement repeatability.

[0023] Optionally, the relevant information includes the calculated luminous intensity of the inelastic scattered light.

[0024] Optionally, the presumed model is a learned model trained on historical data of the luminescence intensity of elastically scattered light.

[0025] Optionally, the concentration of the analyte is a molar concentration, which can be expressed by the following mathematical formula:

[0026]

[0027] Where C represents the molar concentration, μ represents the fluorescence quantum efficiency, ε represents the extinction coefficient, L represents the equivalent distance of light passing through the light collection region, and I flour I represents the luminous intensity of the inelastic scattered light.excit This represents the luminous intensity of the elastically scattered light. The estimated model representing the luminous intensity of light reaching the light-collecting region is a function of the luminous intensity of the elastically scattered light.

[0028] Optionally, the estimation model is an Nth-degree polynomial of the luminous intensity of the elastically scattered light, where N is a natural number, expressed by the following mathematical formula:

[0029]

[0030] Among them, a i It is a set of coefficients obtained in advance through training.

[0031] The concentration determination method described in this technical solution can reduce the measurement repetition rate.

[0032] Optionally, the luminous intensity of the elastically scattered light and the luminous intensity of the inelastically scattered light are, respectively, the luminous intensity of the inelastically scattered light after normalization.

[0033] According to the concentration determination method described in this technical solution, the luminescence intensity of inelastic scattered light is normalized so that the estimation model can be applied to samples / analytes of different concentrations, thereby further improving the concentration calculation of the analyte in the liquid to be tested.

[0034] Furthermore, this application also provides a concentration measuring device for detecting the concentration of an analyte in a liquid to be tested. The concentration measuring device includes a holding unit, an excitation light source, a light collection unit, a detection unit, and a determination unit. The holding unit holds and positions the liquid to be tested. The excitation light source illuminates the liquid column with excitation light, causing the excitation light to irradiate the liquid to be tested. The light collection unit collects elastically scattered and inelastically scattered light generated when the light collection area of ​​the liquid to be tested is irradiated. The detection unit detects the elastically scattered and inelastically scattered light collected by the light collection unit, respectively. The determination unit determines the concentration of the analyte based on the detection results from the detection unit.

[0035] Optionally, the holding unit has multiple holding portions, each having a contact surface for providing surface tension to hold the liquid to be tested. The light collecting unit includes a receiving optical fiber, through which elastically scattered and inelastically scattered light generated by irradiation of the light collecting region is collected. The light collecting region is defined at least by the numerical aperture of the receiving optical fiber and the irradiation range of the excitation light.

[0036] Optionally, the receiving optical fiber is at least partially disposed inside one of the plurality of holding portions to be in direct contact with the liquid to be detected, or one of the plurality of holding portions near the receiving optical fiber includes a light-transmitting layer.

[0037] Optionally, an optical element is disposed between the light-transmitting layer and the receiving optical fiber.

[0038] Alternatively, the optical element may be any of a spherical mirror, a hemispherical mirror, or a lens group.

[0039] Optionally, the light collection unit collects both elastically scattered and inelastically scattered light at the same location.

[0040] Optionally, the detection unit includes a first light detection unit and a second light detection unit. The first light detection unit is used to detect elastically scattered light, and the second light detection unit is used to detect inelastically scattered light. The detection unit and the light collection unit are configured in a way that allows them to move relative to each other, so that the first light detection unit or the second light detection unit can detect the elastically scattered light or inelastically scattered light collected by the light collection unit.

[0041] Optionally, the detection unit further includes an optical path switching mechanism or a dichroic mirror. The optical path switching mechanism is used to switch between the first optical detection unit and the second optical detection unit so that the first optical detection unit detects elastically scattered light or the second optical detection unit detects inelastically scattered light. The dichroic mirror is used to distribute elastically scattered light and inelastically scattered light to the first optical detection unit and the second optical detection unit, respectively. Attached Figure Description

[0042] Figure 1 This is a schematic diagram showing the specific structure of a concentration measuring device in the prior art.

[0043] Figure 2 This is a schematic diagram showing the specific configuration of the concentration measuring device according to one embodiment of this application.

[0044] Figure 3 This is a schematic diagram showing the main structure of the first example of the light collection unit of the concentration measuring device according to an embodiment of this application.

[0045] Figure 4 This is a schematic diagram showing the main structure of a second example of the light collection unit of the concentration measuring device according to an embodiment of this application.

[0046] Figure 5 This is a schematic diagram showing the main structure of a third example of the light collection unit of the concentration measuring device according to an embodiment of this application.

[0047] Figure 6 This is a schematic diagram showing the state of the excitation light before and after passing through the light collection region.

[0048] Figure 7 It is a graph showing the relationship between the sample loading volume of the liquid column and the luminescence intensity of the generated elastically scattered light and inelastically scattered light.

[0049] Figure 8 It is a graph showing the fitted curves that represent the relationship between the luminous intensity of elastically scattered light and the luminous intensity of attenuated excitation light.

[0050] Symbol Explanation

[0051] A0, A concentration measuring device

[0052] 1. Light source section

[0053] 2 First filter section

[0054] 3 beam splitters

[0055] 4 First light-receiving section

[0056] 5 First slit

[0057] 6 Second slit section

[0058] 7 Second filter section

[0059] 8 Second light-receiving section

[0060] 9 test tubes

[0061] 10 Holding Units

[0062] 10A First Holding Section

[0063] 10B Second Holding Section

[0064] 11. Optical fiber (receiving optical fiber)

[0065] 11A fiber core

[0066] 11B cladding

[0067] NA (Numerical Aperture)

[0068] CR light collection area

[0069] U1, U2, U3 detection units

[0070] 13 Optical switching mechanism

[0071] 14. Spectrometer

[0072] 15. Fluorescent filter

[0073] 16. Photodiodes for fluorescent applications

[0074] O1 First Hole

[0075] O2 Second hole

[0076] 14A Elastic Light Scattering Filter

[0077] 14B Photodiode for Elastic Light Scattering

[0078] 13A Dichroic Mirror Detailed Implementation

[0079] In this invention, the term "light collection region" refers to the area located in the liquid to be detected, where the liquid is irradiated to generate elastically and inelastically scattered light, which can then enter and be collected by the light collection unit / light collection component (e.g., optical fiber). More specifically, in this invention, the "light collection region" is determined at least by the numerical aperture of the light collection unit and the irradiation range of the excitation light. In a common and ideal scenario, the light source typically has a relatively large irradiation angle, thereby illuminating the entire liquid column of the liquid to be detected. That is, the irradiation range of the excitation light includes the area illuminating the entire liquid column. In this case, it can be considered that the determination of the light collection region can disregard the irradiation range of the excitation light, and only at least consider the numerical aperture of the light collection unit.

[0080] In this invention, the term "elastically scattered light" refers to scattered light in which photons of incident light collide elastically with particles whose diameter is larger than their wavelength, causing the photons' direction of travel to be deflected and the emitted photons to have the same wavelength, frequency, and energy as the photons before incident light.

[0081] In this invention, the term "inelastic scattered light" refers to scattered light in which photons of incident light collide with particles larger than their own wavelength in an inelastic manner, resulting in energy exchange and causing changes in the wavelength, frequency, and energy of the emitted photons relative to the photons before they were incident.

[0082] In this invention, the term "concentration" can be molar concentration, but is not limited to it; it can also be mass concentration, mass-volume concentration, or equivalent concentration, and can also be expressed as volume fraction, mass fraction, mole fraction, and volume ratio, etc. However, for ease of explanation, molar concentration is used as an example in the following embodiments.

[0083] In this invention, the term "presumed model" refers to a model for estimation based on historical data, which can be in the form of a function or a learned model. For example, in the following embodiments, the predicted model is a polynomial function fitted at least based on historical data of the luminescence intensity of elastically scattered light, or a learned model trained at least using historical data of the luminescence intensity of elastically scattered light as training data.

[0084] In this invention, the term "numerical aperture" is a dimensionless number used in optical systems to measure the angular range of light that can be collected. The precise definition of "numerical aperture" varies slightly across different fields of optics. In optical microscopy, "numerical aperture" describes the size of the objective lens's cone angle. In fiber optics, "numerical aperture" describes the cone angle at which light enters and exits the fiber.

[0085] The following is for reference Figure 2 The main components of the concentration measuring device A according to one embodiment of this application will be described below. It should be noted that, in order to avoid repetition, only the differences between the concentration measuring device A and the concentration measuring device A0 described above will be described below.

[0086] like Figure 2 As shown, with Figure 1 The difference from the existing examples shown is that, in this embodiment, instead of using containers such as test tubes or cuvettes to hold the liquid to be tested, a holding unit 10 holds the liquid between them. Specifically, in this embodiment, the holding units 10 are arranged at certain intervals in the vertical direction, and the liquid to be tested is held between the holding units 10 by its surface tension. The holding unit 10 includes a first holding part 10A and a second holding part 10B, as shown below. Figure 2 As shown, the first holding part 10A is located above, and the second holding part 10B is located below. In this embodiment, an optical fiber 11 is used as the light collection unit, such as... Figure 2 As shown, fiber 11 along Figure 2 The first holding part 10A and the second holding part 10B are arranged vertically inside the second holding part 10B. A small amount of the liquid to be tested is placed between the first holding part 10A and the second holding part 10B, both of which have contact surfaces for providing surface tension to hold the liquid. Since the liquid to be tested is held at the contact surfaces with the first holding part 10A and the second holding part 10B by surface tension, the liquid is held between the holding units 10 and forms a liquid column. The interval between the first holding part 10A and the second holding part 10B is, for example, 1000 μm. Furthermore, the term "small amount" can be 5 μL or less, preferably in the range of 1 μL to 3 μL. It should also be noted that this embodiment shows an example where the holding unit 10 includes two holding parts, namely the first holding part 10A and the second holding part 10B, but it is not limited to this; the holding unit 10 may also include three or more holding parts.

[0087] The receiving optical fiber 11 disposed inside the second holding part 10B includes a core 11A and a cladding 11B. Furthermore, as... Figure 2 As shown, one end of the receiving optical fiber 11 ( Figure 2The upper end of the receiving optical fiber 11 extends to the contact surface of the second holding portion 10B and is flush with that contact surface. Thus, the liquid to be detected directly contacts the end face of the receiving optical fiber 11. The receiving optical fiber 11 has a numerical aperture NA (see [reference]). Figure 6 As mentioned above, in the field of optical fibers, numerical aperture (NA) is an important parameter describing the taper angle of light entering and exiting the fiber, and thus the light-collecting capability of the receiving fiber 11. Furthermore, from... Figure 2 It can be observed that only light located within the numerical aperture NA can be collected by the receiving fiber 11. Specifically, as... Figure 2 As shown (and see also) Figure 6 In the liquid to be tested, the region of a cone-shaped area with a point (preferably the center point of the end face) of the fiber core 11A as its vertex, which overlaps with the irradiation range of the excitation light emitted by the light source unit 1, constitutes the light collection region CR. Figure 2 The light collection region CR (enclosed by the thick dashed line in the diagram) is the area where light (including elastically scattered and inelastically scattered light) generated by illumination in the liquid to be tested is collected. In other words, the light collection region CR is determined at least by the numerical aperture of the receiving fiber 11 and the illumination range of the excitation light emitted from the light source 1. The illumination range of the excitation light needs to be determined by comprehensively considering parameters such as the geometry of the light source, light intensity distribution, illumination depth, divergence angle, and uniformity, describing the illuminated area formed by the excitation light in space. It should be noted that, in common and ideal cases, the light source usually has a relatively large illumination angle, thereby illuminating the entire liquid column of the liquid to be tested. That is, the illumination range of the excitation light includes the area that illuminates the entire liquid column. In this case, it can be considered that the determination of the light collection region can disregard the illumination range of the excitation light, and only at least consider the numerical aperture of the receiving fiber 11. Figure 2 As can be seen, relative to the receiving optical fiber 11, the liquid column to be detected has a light-collecting region CR and a non-collecting region located outside the light-collecting region CR. The non-collecting region is the area where elastically scattered light and inelastically scattered light generated by light irradiation are not collected by the receiving optical fiber 11. It should be noted that after the material and structure parameters of the receiving optical fiber 11 are set, the value of its numerical aperture, which is one of its optical parameters, is also determined.

[0088] The above description uses the example of a liquid to be tested being held between the contact surfaces of the holding unit 10 by surface tension, with the receiving optical fiber 11 disposed inside the second holding part 10B and one end flush with the contact surface. However, the specific implementation is not limited to this. For example, one end of the receiving optical fiber 11 may protrude from the contact surface of the second holding part 10B or be located inside the second holding part 10B relative to the contact surface, as long as light can be collected.

[0089] Furthermore, if the receiving optical fiber 11 is disposed inside the second holding part 10B and one end is flush with the contact surface, a light-transmitting layer may be provided in at least a portion of the contact surface of the second holding part 10B, including the end face of the receiving optical fiber 11 that is flush with the contact surface.

[0090] Furthermore, when the receiving optical fiber 11 is disposed inside the second holding portion 10B and the aforementioned end is also located inside the second holding portion 10B, a light-transmitting layer flush with or located on the contact surface of the second holding portion 10B at the portion corresponding to the aforementioned end of the receiving optical fiber 11 can be provided. In other words, the light-transmitting layer is provided only in the area corresponding to the end face of the receiving optical fiber 11.

[0091] When a light-transmitting layer is provided as described above, an optical element for converging the generated elastically and inelasticly scattered light can be provided between the light-transmitting layer and the receiving optical fiber 11. The optical element can be, for example, a spherical mirror, a hemispherical mirror, or a lens group, or other types of elements. When an optical element is provided, the determination of the light collection region CR takes into account not only the numerical aperture of the receiving optical fiber 11 and the irradiation range of the excitation light, but also the optical characteristics of the optical element (e.g., focusing performance).

[0092] Alternatively, the receiving optical fiber 11 may be disposed inside the first holding part 10A located above, rather than inside the second holding part 10B located below.

[0093] Furthermore, it should be noted that, preferably, in this embodiment, the receiving optical fiber 11 and the second holding portion 10B that houses the receiving optical fiber 11 are movable as a unit relative to the first holding portion 10A. Specifically, by moving the second holding portion 10B that houses the receiving optical fiber 11 relative to the support base, the receiving optical fiber 11 can be moved closer to or further away from the detection units U1, U2, and U3 described later.

[0094] In addition, it should be noted that the receiving optical fiber 11 includes not only the fiber core 11A and cladding 11B, but also the outer sheath, filler, etc.

[0095] Furthermore, the concentration measuring device A described in this embodiment also includes detection units U1, U2, and U3, which are used to detect the luminous intensity of elastically scattered light and inelastically scattered light collected by the receiving optical fiber 11, respectively. Hereinafter, in conjunction with... Figures 3-5 The structures of the first, second, and third examples of the detection unit of the concentration measuring device A will be described.

[0096] Figure 3 A schematic diagram of the main structure of the first example of the detection unit of the concentration measuring device A described in this embodiment, namely the detection unit U1, is shown. Figure 3 As shown, the detection unit U1 mainly includes an optical switching mechanism 13, a spectrometer 14 (an example of a first optical detection unit), and a fluorescence filter 15 and a fluorescence photodiode 16 (an example of a second optical detection unit). The optical switching mechanism 13 is movable relative to a second holding portion 10B on which a receiving optical fiber 11 is provided, in a direction orthogonal to the axis of the second holding portion 10B. A first aperture O1 and a second aperture O2 are formed through two parts of the optical switching mechanism 13. The first aperture O1 is connected to the spectrometer 14, and the second aperture O2 is connected to the fluorescence filter 15 and the fluorescence photodiode 16. As an example of the first optical detection unit, the spectrometer 14 receives elastically scattered light collected and transmitted from the light collection region CR of the liquid to be detected via the receiving optical fiber 11, thereby determining the luminous intensity I of the elastically scattered light. excit As an example of the second optical detection unit, the fluorescent filter 15 first filters the inelastic scattered light collected and transmitted from the light collection region CR of the liquid to be detected via the receiving optical fiber 11. The filtered inelastic scattered light is then received by the fluorescent photodiode 16 to determine the luminous intensity I of the inelastic scattered light. flour Specifically, the optical switching mechanism 13 switches the connection between the first aperture O1 and the second optical fiber 10B and the connection between the second aperture O2 and the second optical fiber 10B in a time-division manner.

[0097] Figure 4 A schematic diagram of the main structure of the detection unit U2, a second example of the concentration measuring device A according to an embodiment of this application, is shown. The difference between this second example and the first example is only the use of an elastic scattering light filter 14A and an elastic scattering light photodiode 14B. Specifically, the elastic scattering light 14A filters the elastic scattering light collected and transmitted from the light collection area CR of the liquid to be detected via the receiving optical fiber 11. The filtered elastic scattering light is then received by the elastic scattering light photodiode 14B, thereby determining the luminous intensity I of the elastic scattering light. excit .

[0098] Figure 5This diagram shows a third example of the detection unit of the concentration measuring device A according to an embodiment of this application, namely the main structure of the detection unit U3. Unlike the first and second examples described above, the detection unit U3 uses a dichroic mirror 13A instead of the optical path switching mechanism 13. When collecting scattered light, the dichroic mirror 13A is positioned below the second holding portion 10B where the receiving optical fiber 11 is provided, splitting the scattered light transmitted via the receiving optical fiber 11 into two paths: one path is elastic scattered light, and the other is inelastic scattered light. Under the action of the dichroic mirror 13A, the elastic scattered light is allocated to the photodiode 14B for elastic scattered light, and the inelastic scattered light is allocated to the photodiode 16 for fluorescence.

[0099] It should be noted that the first and second examples above illustrate cases involving various types of filters, but are not limited to these, and these filters may also be omitted.

[0100] Furthermore, the concentration measuring device A also includes a determination unit, which determines the concentration of the analyte in the liquid to be tested based on the luminous intensity of the detected elastic and inelastic scattered light. Specifically, the unit calculates relevant information about the inelastic scattered light using a pre-set estimation model and based on the luminous intensity of the collected elastic scattered light, and then determines the concentration of the analyte based on the luminous intensity of the collected inelastic scattered light and the calculated relevant information about the inelastic scattered light. This relevant information includes, for example, the calculated luminous intensity, which is the calculated value of the luminous intensity of the inelastic scattered light estimated from the luminous intensity of the collected elastic scattered light and the pre-set estimation model. It should be noted that this calculated luminous intensity is not the luminous intensity of the detected inelastic scattered light, but rather represents the attenuated excitation light (i.e., attenuated excitation light) emitted from the light source unit 1 and reaching the boundary of the light collection region CR of the liquid column of the liquid to be tested. The estimation model can be pre-stored in the determination unit or the concentration measuring device A that includes the determination unit before it leaves the factory, or it can be constructed, set and stored based on new detection results during the use of the determination unit and the concentration measuring device A.

[0101] Furthermore, in the concentration measuring device A described in this embodiment, the light collecting unit 11 is also movable. More specifically, the second holding part 10B, on which the light collecting unit 11 is provided, is movable. In addition, the detection units U1, U2, and U3 are also movable as a whole. That is, the detection units U1, U2, and U3 are configured to move relative to the light collecting unit 11, so that the first light detection unit or the second light detection unit can detect the elastically scattered light or inelastically scattered light collected by the light collecting unit 11.

[0102] Next, the specific steps of the concentration measurement method according to an embodiment of this application will be described in conjunction with the concentration measuring device A described in this embodiment.

[0103] First, in step ST1 (i.e., the excitation step), an excitation light is irradiated in one direction toward a small amount of the liquid to be tested, which is held in the holding unit 10 by surface tension. The excitation light passes through a portion of the liquid to be tested and reaches the light collection region CR of the liquid, and after leaving the light collection region, it passes through another portion of the liquid to be tested and leaves the liquid. As described above, the light collection region CR is the region in which elastically scattered light and inelastically scattered light generated by irradiation in the liquid to be tested are collected. Specifically, when the holding unit 10 employs a first holding part 10A and a second holding part 10B, the liquid to be tested is placed and held between the first holding part 10A and the second holding part 10B. Since the liquid to be tested is in contact with the contact surfaces of the first holding part 10A and the second holding part 10B, under the action of surface tension, the liquid to be tested is held between the first holding part 10A and the second holding part 10B in a manner forming a liquid column.

[0104] Next, in step ST2 (i.e., the collection and detection step), the elastically scattered light and inelastically scattered light generated when the light collection region CR is irradiated are collected and detected separately. In this embodiment, the elastically scattered light and inelastically scattered light generated when the light collection region CR is irradiated can be collected at the same location using the receiving optical fiber 11. In this embodiment, the receiving optical fiber 11 can be as follows: Figure 2 The straight optical fiber shown can also be a Y-shaped optical fiber. When the receiving optical fiber 11 is a straight optical fiber, elastically scattered light and inelastically scattered light propagate from one end of the straight optical fiber to the other end for collection. On the other hand, when the receiving optical fiber 11 is a Y-shaped optical fiber, it includes a straight section and a pair of bifurcated sections connected to the straight section. Each bifurcated section is provided with a filter element that allows only elastically scattered light and inelastically scattered light to pass through. Thus, elastically scattered light and inelastically scattered light enter from the straight section of the Y-shaped optical fiber and then propagate through one end of one bifurcated section to the other end of the other bifurcated section, thereby completing the collection. Furthermore, when an optical element is provided between the receiving optical fiber 11 and the liquid to be detected, the elastically scattered light and inelastically scattered light converge to the same position through the optical element and then propagate from one end of the receiving optical fiber 11 to the other end, thereby completing the collection. After the collection of elastically scattered light and inelastically scattered light is completed, the luminous intensity of the collected elastically scattered light and the luminous intensity of the inelastically scattered light are detected separately. Specifically, this can be achieved by using... Figures 3-5Any one of the detection units U1, U2, and U3 shown can separately detect the luminous intensity of the collected elastically scattered light and the luminous intensity of the inelastically scattered light. As described above, detection units U1 and U2 include an optical path switching mechanism 13, which can separately detect the luminous intensity of the elastically scattered light and the inelastically scattered light collected through the receiving optical fiber 11 in a time-division manner. Detection unit U3 includes a dichroic mirror 13A, which can distribute the collected elastically scattered light and the inelastically scattered light to each optical detection unit for separate detection. After completing step ST2, proceed to step ST3.

[0105] In step ST3 (i.e., the concentration determination step), the concentration of the analyte in the liquid to be tested is determined based on the luminescence intensity of the detected elastic scattered light and the luminescence intensity of the inelastic scattered light.

[0106] Figure 6 A schematic diagram shows the state of the excitation light before and after passing through the light collection region CR. Figure 6 In this equation, I0 represents the luminous intensity of the excitation light reaching the surface of the liquid column of the liquid to be detected, and L represents the equivalent distance of the light passing through the light-collecting region CR. When the light-collecting region CR can be considered as a frustum cone, if the upper diameter of the light-collecting region CR is set to L2 and the lower diameter to L1, then the equivalent distance L can be approximately represented by the following mathematical formula (2):

[0107]

[0108] Where NA is the value of the numerical aperture.

[0109] It should be noted that the equivalent distance can also be expressed by other mathematical formulas besides the above mathematical formula (2) according to the actual application.

[0110] In this case, the luminous intensities of the inelastic scattered light and the elastic scattered light generated by irradiating the CR liquid in the light-collecting region are respectively set as I. flour and I excit Then the inelastic scattered light I flour It can be expressed by the following mathematical formula:

[0111] I f1our =μεCL×I′0 (3)

[0112] Where μ represents the fluorescence quantum efficiency (a dimensionless parameter), and ε represents the extinction coefficient (unit: L). 2 / mol), C represents the molar concentration of the analyte in the liquid to be tested (unit: mol / L). 3 ), where I′0 is the luminescence intensity of the attenuated excitation light, which will be described later.

[0113] Regarding the luminescence intensity I′0 of the attenuated excitation light, analogous to the mathematical formula (1) used in the prior art, it can be understood as the luminescence intensity of the excitation light after eliminating the influence of the change in the shape of the liquid column of the liquid to be detected. Figure 6 The I′0 shown is a diagram of the above understanding, which can be understood as the attenuated excitation light reaching the boundary of the light collection region CR.

[0114] Then, by transforming mathematical formula (3), the molar concentration of the component to be tested in the liquid can be expressed by the following mathematical formula (4):

[0115]

[0116] Therefore, the molar concentration of the analyte in the liquid to be tested is determined.

[0117] In different tests, the shape of the sample liquid column formed by the liquid to be tested in the holding unit 10 varies, and the shape of the non-collection area also differs. Therefore, the luminescence intensity I′0 of the attenuated excitation light is not constant and changes with the shape of the liquid column. Thus, if the relationship between the luminescence intensity I′0 of the attenuated excitation light and the above-mentioned changes can be determined, the measurement error caused by the shape change of the liquid column can be eliminated, and the test repeatability can be reduced.

[0118] In response, the inventors of this application, through careful study, discovered that the inelastic and elastic scattered light exhibit the same trend in response to changes in the shape of the liquid column in the liquid being tested. This phenomenon indicates a correlation between the luminescence intensity of the elastic or inelastic scattered light and the luminescence intensity of the attenuated excitation light. Furthermore, the luminescence intensity I′0 of the attenuated excitation light and the luminescence intensity I of the collected elastic scattered light... excit There is some kind of relationship between them, for example, it can be defined as Therefore, the luminous intensity I of elastically scattered light can be used as a metric. excit Let I′0 represent the luminescence intensity of the decaying excitation light. Specifically, the luminescence intensity I′0 of the decaying excitation light can be represented by the following mathematical formula (5):

[0119]

[0120] in, It is an estimated model representing the luminous intensity of light reaching the boundary of the light-collecting region CR, and is an Nth-degree polynomial in terms of the luminous intensity of the elastically scattered light, where N is a natural number and a i It is a set of coefficients (e.g., regression coefficients) obtained by performing numerical analysis (e.g., regression analysis) on experimental data.

[0121] The following section uses regression analysis as an example to explain how to determine the coefficient α. i Please provide an explanation.

[0122] First, a liquid column of the liquid to be tested is formed between the holding units 10 using the surface tension of the liquid to be tested. Different sample volumes result in different liquid column shapes. For samples of the same concentration, under constant conditions in the light source unit 1, the luminescence intensity of elastically scattered and inelasticly scattered light generated by liquid columns with different sample volumes is collected using a micro-fluorometer to be calibrated. Specifically, the test conditions are as follows:

[0123] The test sample was Invitrogen Qubit dsDNA BR Buffer;

[0124] The light source unit 1 uses an LED light source with a wavelength of 470nm and a power supply current of 50mA;

[0125] The elastic light scattering filter 14A uses a 463nm excitation filter.

[0126] The fluorescent filter 15 uses a 545nm fluorescent filter.

[0127] The spectrometer 14 uses a Thermofisher spectrometer (CGS) to measure the luminescence intensity of elastically scattered light;

[0128] Figure 7 A graph showing the relationship between the luminous intensity of elastically scattered light and the luminous intensity of inelastically scattered light generated by the liquid column under the above test conditions is presented. Table 1 shows fifteen sets of data on the luminous intensity of elastically scattered light and the luminous intensity of inelastically scattered light of the sampled test samples.

[0129] Table 1

[0130] serial number 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Elastic scattered light intensity 6.033449 6.085557 6.036439 6.234001 6.43118 6.660809 6.918998 6.867284 7.107417 7.329746 7.552441 8.045818 8.375188 9.255059 10.44336 Inelastic scattered light intensity 0.1589 0.1683 0.17 0.1857 0.1915 0.191 0.1731 0.1798 0.1917 0.1999 0.2028 0.2121 0.241 0.2678 0.3078

[0131] To facilitate data processing and improve computational accuracy, the luminous intensity of inelastic scattered light is at least normalized to the mean. Here, only the luminous intensity of inelastic scattered light is normalized to the mean, resulting in Table 2.

[0132] Table 2

[0133]

[0134] Then, construct the above mathematical formula (5), assuming N = 4, and perform least squares fitting based on the luminous intensity of elastically scattered light shown in Table 2 to obtain the regression coefficient a. i Therefore, it was determined that mathematical expression (5) can be specifically expressed as the following mathematical expression (6):

[0135]

[0136] Figure 8 The fitted curve corresponding to the above mathematical formula (6) is shown.

[0137] Therefore, based on the pre-set estimation model (e.g., the nonlinear regression function model shown in the above mathematical formula (6)) and the collected luminescence intensity I of the elastic excitation light, it can be concluded that... excit The calculated luminous intensity of the inelasticly scattered light can be estimated, and this calculated luminous intensity can be understood as a light intensity correction factor. Furthermore, as mentioned above, since the estimated calculated luminous intensity of the inelasticly scattered light can characterize the luminous intensity of the attenuated excitation light, mathematically speaking, the numerical value of the calculated luminous intensity can be equivalent to the numerical value of the luminous intensity of the attenuated excitation light. Then, the calculated luminous intensity, which characterizes the magnitude of the luminous intensity I′0 of the attenuated excitation light, and the luminous intensity I of the collected inelasticly scattered light are compared... flour Substituting these values ​​into the mathematical formula (4) above, the molar concentration of the analyte in the liquid to be tested can be determined. In other words, in this example, the luminous intensities of multiple sets of elastically scattered light obtained through experimental measurement are input to a pre-defined function with unknown coefficients (in this example, an Nth-degree polynomial with unknown coefficients) to determine these coefficients, thereby obtaining an empirical formula, which is then stored in advance. When determining the concentration of the analyte in the liquid to be tested, the luminous intensity of the currently collected elastically scattered light is input into the above empirical formula to obtain the luminous intensity of the attenuated excitation light. Then, by combining the luminous intensity of the collected inelasticly scattered light and the calculated luminous intensity of the attenuated excitation light, the molar concentration of the analyte in the liquid to be tested can be determined.

[0138] As shown in Table 3, by comparing the measured values ​​before and after calibration, it can be seen that the measurement repeatability of the microfluorescence meter decreased significantly. Specifically, the measurement repeatability decreased from 19.35% to 7.33%.

[0139] Table 3

[0140]

[0141] According to the concentration determination method described in this embodiment, instead of excitation light that propagates in the air and reaches the liquid to be tested, elastically scattered light generated by the liquid to be tested being irradiated by light irradiated into the light-collecting region CR is collected, and inelasticly scattered light (i.e., fluorescence) generated by the liquid to be tested being irradiated by light irradiated into the light-collecting region CR is also collected. The concentration (more precisely, molar concentration) of the analyte in the liquid to be tested is determined based on the elastically scattered light and inelasticly scattered light collected in the light-collecting region CR. This avoids the reduction in concentration measurement accuracy caused by differences in the liquid column shape of the liquid to be tested.

[0142] It should be noted that in the above example, the luminescence intensity I′0 of the attenuated excitation light and the luminescence intensity I of the collected elastically scattered light are compared. excit There exists a certain correlation between them, which is defined as a functional relationship. The specific expression of the function is obtained through numerical analysis of experimental data. However, the correlation between the two is not limited to being presented and determined as a function. For example, a machine learning learning completion model can also be used to construct the relationship between the luminescence intensity I′0 of the decaying excitation light and the luminescence intensity I of the collected elastically scattered light. excit The relationship between them. In other words, a learned model, trained at least on historical data of the luminescence intensity of elastically scattered light, can be used as an inference model to determine the concentration of the component to be measured.

[0143] Furthermore, in this embodiment, the example described is that the excitation light emitted from the light source 1 irradiates the liquid to be detected in a direction orthogonal to the direction in which the holding unit 10 holds the liquid to be detected, but this is not a limitation. For example, the light may also irradiate the liquid to be detected in a direction that is not orthogonal to the direction in which the holding unit 10 holds the liquid to be detected, but intersects with it. However, compared to the non-orthogonal case, orthogonality can reduce the difference between the luminous intensity of the collected elastically scattered light and the luminous intensity of the inelastically scattered light.

[0144] Alternatively, the intensity of the excitation light emitted from the light source unit 1 is adjusted based on the intensity of the detected elastic scattered light, the adjusted inelastic scattered light generated by the liquid to be tested under the irradiation of the adjusted excitation light is collected and detected, and the concentration of the analyte in the liquid to be tested is determined based on the intensity of the detected adjusted inelastic scattered light and the intensity of the adjusted excitation light.

[0145] The foregoing description has already given many features and advantages, including various alternative implementations, as well as details of the structure and function of the apparatus and methods. This document is intended to be exemplary and is not exhaustive or limiting.

[0146] It will be apparent to those skilled in the art that various modifications can be made within the full scope indicated by the broad superordinate meaning of the terms expressed in the appended claims, particularly in terms of structure, materials, elements, components, shapes, dimensions, and arrangements of components, including combinations of these aspects within the scope of the principles described herein. Such various modifications are intended to be included herein, provided they do not depart from the spirit and scope of the appended claims.

Claims

1. A concentration determination method for determining the concentration of a component to be tested in a liquid, characterized in that, comprises: an excitation step in which excitation light is irradiated toward a liquid to be detected held by a holding unit by surface tension, the excitation light reaching a light collection region of the liquid to be detected after passing through a portion of the liquid to be detected and exiting the liquid to be detected after passing through another portion of the liquid to be detected after exiting the light collection region, the light collection region being a region in which elastically scattered light and inelastically scattered light generated by the irradiation of the liquid to be detected are collected; a collection detection step in which the elastically scattered light and the inelastically scattered light generated by the irradiation of the light collection region are collected and detected separately; a concentration determination step in which the concentration of the component to be detected is determined based on the detected elastically scattered light and the detected inelastically scattered light.

2. The concentration measurement method according to claim 1, wherein The collection detection step further comprises - collecting the elastically scattered light and the inelastically scattered light at the same position for separate detection.

3. The concentration measurement method according to claim 1, wherein The excitation step further comprises: - holding the liquid to be detected at a contact surface of the holding unit, the contact surface being configured to provide surface tension to hold the liquid to be detected, The collection detection step further comprises - collecting the elastically scattered light and the inelastically scattered light generated by the irradiation of the light collection region by a receiving optical fiber, The light collection region is determined by at least a numerical aperture of the receiving optical fiber and an irradiation range of the excitation light.

4. The concentration measurement method according to claim 3, wherein the holding unit has a plurality of holding portions, the liquid to be detected is in direct contact with the receiving optical fiber disposed at least partially inside one of the plurality of holding portions, or one of the plurality of holding portions that is close to the receiving optical fiber includes a light-transmissive layer.

5. The concentration measurement method according to claim 4, wherein An optical element is disposed between the light-transmissive layer and the receiving optical fiber.

6. The concentration measurement method according to claim 5, wherein the optical element is any one of a spherical mirror, a semi-spherical mirror, and a lens group.

7. The concentration measurement method according to claim 1, wherein the concentration determination step includes: - adjusting the light emission intensity of the excitation light based on the light emission intensity of the detected elastically scattered light; and - collecting and detecting adjusted inelastically scattered light generated by the irradiation of the liquid to be detected by the adjusted excitation light, and determining the concentration of the component to be detected based on the detected adjusted inelastically scattered light and the light emission intensity of the adjusted excitation light.

8. The concentration measurement method according to claim 1, wherein the concentration determination step includes: - estimating information related to the inelastically scattered light of the light collection region based on the light emission intensity of the detected elastically scattered light and a predetermined estimation model; - determining the concentration of the component to be detected based on the light emission intensity of the detected inelastically scattered light and the information related to the inelastically scattered light.

9. The concentration measurement method according to claim 8, wherein The information related to the inelastically scattered light includes a calculated light emission intensity of the inelastically scattered light.

10. The concentration measurement method according to claim 8, wherein The estimation model is a learning-completed model trained using historical data of the emission intensity of the elastically scattered light.

11. The concentration measurement method according to claim 8, wherein The concentration of the component to be measured is a molar concentration, and the molar concentration is represented by the following mathematical expression: where C denotes molar concentration, μ denotes fluorescence quantum efficiency, ε denotes extinction coefficient, L denotes an equivalent distance of light passing through the light collection area, I flour represents the luminescence intensity of the non-elastically scattered light, I excit represents the luminescence intensity of the elastically scattered light, is the estimation model representing the luminescence intensity of light reaching the light collection area, and is a function with respect to the luminescence intensity of the elastically scattered light.

12. The concentration measurement method according to claim 11, wherein The estimation model is an Nth degree polynomial with respect to the emission intensity of the elastically scattered light, and N is a natural number, and is represented by the following mathematical expression: where a i is a set of coefficients obtained in advance by training.

13. The concentration measurement method according to any one of claims 1 to 12, wherein In the emission intensity of the elastically scattered light and the emission intensity of the inelastically scattered light, the emission intensity of the inelastically scattered light is an emission intensity after normalization processing.

14. A concentration measuring device (A) for detecting the concentration of a component to be measured in a liquid, characterized in that, including: a holding unit (10) for placing and holding the liquid to be measured; an excitation light source (1) for irradiating excitation light toward the liquid to be measured so that the excitation light irradiates the liquid to be measured; a light collecting unit (11) for collecting the elastically scattered light and the inelastically scattered light generated by irradiation of a light collecting region of the liquid to be measured; a detection unit (U1, U2, U3) for detecting the elastically scattered light and the inelastically scattered light collected by the light collecting unit, respectively; and a determination unit for determining the concentration of the component to be measured based on the detection results of the detection unit.

15. The concentration measurement device (A) according to claim 14, wherein the holding unit has a plurality of holding portions having a contact surface for providing surface tension to hold the liquid to be measured, the light collecting unit includes a receiving optical fiber through which the elastically scattered light and the inelastically scattered light generated by irradiation of the light collecting region are collected, the light collecting region is determined by at least a numerical aperture of the receiving optical fiber and an irradiation range of the excitation light.

16. The concentration measurement device (A) according to claim 15, wherein the receiving optical fiber is at least partially disposed inside one of the plurality of holding portions to directly contact the liquid to be measured, or one of the plurality of holding portions near the receiving optical fiber includes a light-transmissive layer. An optical element is disposed between the light-transmissive layer and the receiving optical fiber.

17. The concentration measuring apparatus (A) according to claim 16, characterized in that 18. The concentration measurement method according to claim 17, wherein the optical element is any one of a spherical mirror, a semi-spherical mirror, and a lens group.

19. The concentration measurement device (A) according to claim 14, wherein the light collecting unit collects the elastically scattered light and the inelastically scattered light at the same position.

20. The concentration measurement device (A) according to claim 14, wherein the detection unit includes: a first light detection portion for detecting the elastically scattered light; and a second light detection portion for detecting the inelastically scattered light, ​ The detection unit and the light collection unit are configured to be relatively movable so that the first light detection portion or the second light detection portion detects the elastically scattered light or the inelastically scattered light collected by the light collection unit.

21. The concentration measuring apparatus (A) according to any one of claims 14 to 20, wherein The detection unit further includes a light path switching mechanism (13) or a dichroic mirror (13A), The light path switching mechanism (13) is configured to switch the first light detection portion and the second light detection portion so that the first light detection portion detects the elastically scattered light or the second light detection portion detects the inelastically scattered light, The dichroic mirror (13A) is configured to distribute the elastically scattered light and the inelastically scattered light to the first light detection portion and the second light detection portion, respectively.

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