A rotating disk and device for high-throughput measurement of fluorescence phosphorescence lifetime

Abstract

A turntable and device for measuring the fluorescence phosphorescence lifetime can be used for high-throughput fluorescence phosphorescence lifetime measurement. The measurement method is to focus the excitation light into a beam and illuminate a point at the edge of the turntable. When the turntable is driven to rotate by a motor, the optical channel on the turntable can introduce the excitation light irradiated on the edge of the turntable into the sample slot for exciting the sample. After the samples in each sample slot are excited, they rotate in space, so that the luminescent signals with different delay times are separated in space. The fluorescence phosphorescence lifetime of the sample can be obtained by taking pictures and analyzing with a camera. This method realizes the simultaneous measurement of the fluorescence phosphorescence lifetime of multiple samples using a continuous light source; since the excitation light has a high power density, low-concentration samples and weakly luminescent samples can be measured; limiting the excitation light to be transmitted inside the optical channel can significantly reduce the light source scattering background. In addition, this method does not require expensive pulse light sources, high-speed detectors and complex phase-locked control, and has the characteristics of low cost and simple and portable instruments.

Description

Technical Field

[0001] The invention relates to a photoluminescence lifetime measurement method and the manufacture and application of related instruments and devices, belonging to the field of optical instrument manufacturing and instrument analysis. Background Art

[0002] The properties of substances such as light absorption, photoluminescence, and chemiluminescence have many applications in the field of analytical detection. Most of the principles are based on the fact that changes in certain factors in the environment will lead to changes in the absorbance and luminescence intensity of the substance, and the changes in certain factors are judged by detecting changes in light intensity. However, in actual measurements, whether it is the measurement of absorbance or luminescence intensity, it has a lot to do with the sensitivity of the detector; in the detection of photoluminescence, the intensity of the excitation light directly affects the intensity of fluorescence and phosphorescence. In addition, many factors such as the instrument's light collection efficiency, interference from ambient light, and luminescence from impurities will affect the measurement results. Therefore, the results obtained from measuring the same sample using different instruments will be different.

[0003] In order to eliminate the above-mentioned errors, standard samples are often used to compare the results obtained under the same measurement conditions with the samples to be tested to eliminate instrument errors, or to prepare working curves to achieve quantitative analysis of the samples to be tested. In addition, due to factors such as instrument aging (such as weakening of the light source), the instrument often needs to be calibrated repeatedly, which directly leads to cumbersome operation processes, increases human errors, and reduces the repeatability of measurement results. On the other hand, most of the instruments relied on by the above-mentioned tests are large instruments, which are not convenient for point-of-care testing (POCT). Although many portable instruments and equipment have been developed in recent years, and even many tests can be implemented on smart hardware such as mobile phones, the essence of detecting light intensity has not changed, so there is still the problem of the need for instrument calibration.

[0004] Time-resolved luminescence detection also has many applications in analytical detection (see patents CN201210215872.0, CN201610166288.9, CN201610416029.7). Among them, time-gated detection detects the luminescence intensity after a delay after the end of excitation. This method can remove short-lived background fluorescence interference and has a high signal-to-noise ratio. However, its essence is still to detect light intensity, and the instrument still needs to be calibrated.

[0005] Another time-resolved detection is to measure the lifetime of fluorescence or phosphorescence, that is, the lifetime of the excited state, and to determine the change of a certain factor in the environment by detecting the lifetime of the luminescence. One method of detecting the luminescence lifetime is to use a pulsed light source to excite the sample, and use a detector to detect the change of the sample's luminescence intensity over time; analyze the curve of the light intensity decaying over time to obtain the length of the lifetime. In a single exponential decay curve, the length of the lifetime is defined as the time required to decay to 1 / e of the original (reference Anal. Chem., 1990, 62, 270A–277A; Pure Appl. Chem. 2014,86, 1969–1998.).

[0006] The lifetime of the excited state is an inherent property of the molecule. In most cases, the lifetime of the excited state is independent of the intensity of the excitation light. Therefore, the lifetime values ​​measured by different instruments for the same sample are basically the same, so there is no need to calibrate the instrument separately. Compared with the detection of light intensity, using the excited state lifetime as the basis for detection can eliminate the errors caused by instrument differences and has a very high reliability.

[0007] Existing lifetime detection has been applied in fluorescence lifetime imaging (reference Anal. Chem., 2017, 89,8104–8111), and can be used to distinguish the structure of cells and tissues. However, lifetime detection has not been widely used in the analysis and detection of substances. The main reason is that the existing instruments for detecting lifetime are expensive, such as pulse light sources, time-correlated single photon counters, streak cameras and other detectors (reference Chinese patents CN201410353200.5, CN201310694918.6, CN201310027775.3). On the other hand, the above-mentioned various devices for measuring lifetime are difficult to be small and portable, which limits the application and development of lifetime detection.

[0008] In addition, in existing lifetime detection, one measurement can often only detect the lifetime of one sample, and cannot be used for high-throughput fluorescence lifetime detection. This is mainly because existing lifetime detectors such as single-photon counters and streak cameras are difficult to distinguish different samples in the spatial domain. In recent years, some special image sensors have been developed, such as single-photon avalanche diode (SADP) arrays and CMOS sensors that can record phases (references M. Gersbach, et al., Proc. SPIE2010, 7780, 77801H; R. Franke, GA Holst, Proc. SPIE 2015, 9328, 93281K), which can measure the fluorescence lifetime of multiple pixels at the same time and have a faster imaging speed in global fluorescence lifetime imaging. However, these detectors also need to be used with pulsed light sources and phase-locked control.

[0009] The inventor disclosed a method for measuring lifetime using an image sensor in a previous patent (CN201711110647.X), which uses a motor to drive the excited sample to rotate in space, so that the luminescent signals with different delay times are separated in space, and the time domain information is converted into spatial domain information for detection, thereby realizing the synchronous measurement of the fluorescence lifetime of multiple samples. This method does not require pulse and phase-locked control and has ultra-low cost. However, in high-throughput lifetime measurement, in order to achieve multi-point excitation, multiple laser spots or divergent LED lights need to be focused on the turntable. On the one hand, this will cause scattering interference; on the other hand, for samples that require strong light excitation, the dispersed laser or LED beam directly reduces the excitation light power density, making it difficult to detect low-concentration or trace samples. Although the use of multiple laser point-to-point excitation light can ensure the intensity of the excitation light, it will increase the complexity and cost of the instrument. Summary of the invention

[0010] This patent is an important improvement of the patent (CN201711110647.X). Both use motors to drive the excited samples to rotate in space, so that the luminescent signals with different delay times are separated in space, and the time domain information is converted into spatial domain information for detection. The improvement of this patent is to focus the excitation light into one beam, so that the excitation light beam excites the sample from the parallel direction of the sample rotation plane, so that the rotation trajectory of all samples will intersect with the excitation light beam, that is, the sample will be excited by the excitation light during the rotation process. This method uses only one light source, which not only ensures the energy density of the light beam, but also can be used for high-throughput fluorescence lifetime measurement, which is a significant improvement over previous patents.

[0011] To achieve the above purpose, this patent discloses a turntable for measuring the fluorescence phosphorescence lifetime, which is characterized by:

[0012] There are multiple sample slots on the turntable, and the distances between each sample slot and the center of the turntable are different. Each sample slot is connected to an optical channel. All optical channels are distributed on the turntable, and each optical channel is independent and does not interfere with each other.

[0013] The other end of the optical channel is connected to the edge of the turntable, so as to introduce the excitation light irradiated on the edge of the turntable into the optical channel and excite the sample in the sample slot;

[0014] The medium constituting the optical channel is selected from air, optical fiber, and capillary.

[0015] In a preferred turntable, the optical channel medium is a multimode optical fiber.

[0016] In another preferred turntable, the medium of the light channel is air, and the inner wall of the light channel is provided with a reflective film.

[0017] In another preferred turntable, the optical channel medium is a transparent capillary tube, and the capillary tube also serves as a sample slot.

[0018] Based on the above-mentioned various rotating disks, this patent discloses a fluorescence phosphorescence lifetime measurement device, which is characterized by:

[0019] It includes any one of the above-mentioned various turntables, and also includes a motor, a light source, and a camera.

[0020] The turntable is driven by a motor shaft, the light emitted by the light source is irradiated on a certain point on the edge of the turntable, and the lens of the camera is parallel to the turntable for shooting the image of the turntable.

[0021] A preferred lifetime measurement device further includes a condenser lens between the light source and the turntable, which is used to focus the light from the light source onto a certain point on the edge of the turntable, thereby improving the utilization rate of the excitation light and enhancing the fluorescence signal.

[0022] A preferred lifetime measurement device is characterized in that the light source is selected from a laser diode or an LED light source.

[0023] A preferred lifetime measurement device is characterized in that: the optical channel medium is a capillary; when measuring a liquid sample, the liquid sample is placed in the capillary and can be centrifuged first and then detected, thereby realizing the integration of centrifugal separation and lifetime detection.

[0024] Another preferred life measurement device further includes an imaging lens between the camera and the turntable, which is used to magnify the image of the turntable and then take a photo.

[0025] Preferably, in all the above-mentioned measuring devices, a filter is also provided in front of the camera lens to filter out the excitation light.

[0026] All of the above devices can be used to detect fluorescence and phosphorescence lifetime.

[0027] The principle of measuring fluorescence lifetime using the above-mentioned devices is as follows:

[0028] The light from the light source shines on a certain point on the edge of the turntable. When the turntable rotates, the light will enter each light channel of the turntable and stimulate the sample in the sample slot at the end of the light channel to emit light.

[0029] Since the turntable is rotating continuously, the sample in the sample slot can only be excited when the end of the light channel turns to the illumination area; as the turntable continues to rotate, the end of the light channel turns outside the illumination area, and the sample is no longer excited by the light, but the luminescence of the sample will decay over time; if the turntable rotates at a constant speed, the luminescence of the sample will form a light arc on the turntable, and the arc length is proportional to the delay time.

[0030] Since there are multiple light channels on the turntable, multiple samples can be stimulated to form multiple light arcs. If the turntable is photographed, the images of the light arcs emitted by all samples can be recorded. Each light arc represents the curve of the luminescence decay of a sample over time.

[0031] According to the rotation speed of the turntable, the delay time of each point on the light arc can be calculated;

[0032] According to the brightness or gray value of the image pixel, the relative luminous intensity of each point on the light arc can be calculated;

[0033] In this way, a curve showing the decay of the luminous intensity of the sample over time is obtained, and the lifetime can be obtained by fitting the curve.

[0034] Such a method can obtain the luminescence lifetime of multiple samples with just one photo, thus enabling high-throughput lifetime measurement.

[0035] Compared with the method in the previous patent (CN201711110647.X), the advantages of this method are:

[0036] Focusing the light from a single light source to one point ensures the power density of the excitation light while performing high-throughput measurements. This eliminates the need for multiple light sources, significantly simplifies the instrument and reduces costs.

[0037] Since the excitation light has a high power density, low-concentration samples and weakly luminescent samples can be measured;

[0038] Limiting the excitation light to be transmitted inside the optical channel can significantly reduce the light source scattering background;

[0039] The use of multimode optical fiber to transmit the excitation light allows each sample to be excited by light of approximately equal intensity, facilitating the testing of multiple parallel samples.

[0040] Compared with the existing fluorescence and phosphorescence lifetime measurement devices, the method and device disclosed in this patent realizes the measurement of the lifetime of the excited state with a steady-state light source, which has significant advantages:

[0041] No expensive pulse light source and detector are required, and phase-locked control is not required, which greatly reduces the cost of lifetime measurement;

[0042] All components can be miniaturized and made into portable micro devices for instant detection applications;

[0043] The luminescence lifetime of multiple samples can be detected simultaneously, which improves the detection throughput and efficiency and helps to reduce system errors. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1The invention is a life measurement device, wherein 101 is a turntable, 102 is a sample slot, 103 is an optical channel, 104 is a motor, 105 is a light source, and 106 is a camera.

[0045] Figure 2 The invention is a life measurement device, wherein 201 is a turntable, 202 is a sample slot, 203 is an optical channel, 204 is a light blocking ring, 205 is a motor, 206 is a light source, 207 is a reflector, 208 is a lens, 209 is a lens, 210 is a filter, and 211 is a mobile phone.

[0046] Figure 3 It is a schematic diagram of a top view of a turntable, 301 is a sample slot, and 302 is a light channel.

[0047] Figure 4 It is a schematic diagram of a turntable in side view and top view, 401 is the turntable, 402 is a transparent capillary, 403 is a sample adding slot, and 404 is a light hole.

[0048] Figure 5 It is a schematic diagram of a turntable in side view and top view, 501 is the turntable, 502 is the capillary, and 503 is the light hole. DETAILED DESCRIPTION

[0049] In order to illustrate the principles and advantages of the present invention, the present invention is further described below through specific examples, the purpose of which is to help better understand the content of the present invention, but these specific implementation schemes do not limit the protection scope of the present invention in any way.

[0050] Example 1: a turntable and a life measuring device

[0051] As attached Figure 1 As shown, there are sample slots 102 on the turntable 101, each sample slot is connected to an optical channel 103, the optical channel is an air duct, the laser light source 105 emits laser light parallel to the turntable 101, and when the turntable rotates, the laser light can enter each optical channel in turn and excite the sample in the sample slot. The turntable 101 is driven to rotate by the motor 104 shaft.

[0052] In the working state, the turntable rotates at a constant speed and the laser is turned on. With each rotation, the samples in all the sample slots are excited by the laser once, and the fluorescence or phosphorescence of the samples is released during the rotation to form a light arc. The rotation radius of each sample slot is different, so the light arcs of each sample will not overlap. The light arcs are photographed using the camera 106 to record the luminescence information.

[0053] By processing the image, the luminescence lifetime can be obtained. The processing method is consistent with the patent (CN201711110647.X), as follows:

[0054] The grayscale value of the pixel point of the light arc is proportional to the luminous intensity and can be used as the relative luminous intensity.

[0055] Under the condition of uniform rotation, the arc length is proportional to the delay time. Knowing the motor speed, the delay time of each pixel can be calculated.

[0056] The luminescence decay curve can be obtained by plotting the pixel grayscale value against the delay time, and the luminescence lifetime value can be obtained by fitting the curve according to formulas and methods known in the art.

[0057] In this embodiment, optical fiber may also be used as the optical channel.

[0058] Based on the same principle, the number of sample slots and optical channels can be increased on the turntable to achieve simultaneous measurement of more samples.

[0059] Compared with the method of the previous patent (CN201711110647.X), this method concentrates the excitation light energy in one place, has a higher power density, can excite samples with lower concentrations and weaker luminescence, and each sample is excited in turn with each rotation, which improves the utilization rate of the excitation light. Moreover, the excitation light intensity received by each sample slot is roughly equal, and each sample can be compared. In addition, the optical channel is pre-buried in the turntable, which greatly reduces the interference of scattered light.

[0060] Example 2: a turntable and a life measuring device

[0061] As attached Figure 2 As shown, there are sample slots 202 on the turntable 201, and the rotation radius of each sample slot is different. Each sample slot is connected to an optical channel 203, which is a multi-mode optical fiber and leads to the edge of the turntable. The light emitted by the light source 206 is reflected by the reflector 207 and then focused on the edge of the turntable 201 by the lens 208; 204 is a light blocking ring for blocking the scattering of the excitation light; the turntable 201 is driven to rotate by the shaft of the motor 205; the lens 209, the filter 210, and the mobile phone 211 constitute a photographing system for taking pictures of the light arc.

[0062] The method and principle of measuring life of the device are consistent with those of implementation example 1.

[0063] Lens 209 can be used to magnify the local image of the turntable, that is, to magnify the light arc. Since the length of the light arc represents the delay time, at the same rotation speed, the magnified light arc is recorded by more pixels, so it has a higher time resolution and can measure a shorter luminous lifetime.

[0064] The filter 210 can filter out scattered light and light of non-measurement wavelengths, thereby enhancing the signal-to-noise ratio of arc imaging.

[0065] The entire device can be miniaturized, making it convenient to take photos using a mobile phone.

[0066] Implementation Example 3, a turntable

[0067] The main structure of the turntable is shown in the top view. Figure 3 As shown, 301 is a sample slot, 302 is an optical channel, the optical channel is composed of optical fiber, connecting the sample slot and the edge of the turntable, and can guide the light at the edge of the turntable into the sample slot. The turntable can replace the turntable in Examples 1 and 2 for life measurement.

[0068] The turntable takes full advantage of the flexibility of optical fiber and can establish a non-linear optical path between any point on the turntable and the edge of the turntable.

[0069] Implementation Example 4, a turntable

[0070] As attached Figure 4 As shown, the turntable 401 is divided into two layers, 402 is a transparent capillary, 403 is a sample loading slot, and 404 is a light hole. A plurality of transparent capillaries 402 are distributed in the inner layer of the turntable. Each capillary is similar to an S-shape, connecting the sample loading slot 403 and the edge of the turntable. Due to the capillary action, the liquid sample can be automatically sucked into the capillary. The capillary itself can be used as a waveguide to guide the light from the edge of the turntable to the location of the liquid sample and excite the sample to emit light.

[0071] During the rotation of the turntable, the liquid sample is confined to the turning corner of the capillary due to centrifugal force; the upper layer of the turntable is opaque, but has multiple light-transmitting holes 404, each corresponding to a turning corner of a capillary, so that the light emitted by the liquid sample can form a light arc, and the rotation radius of the turning corners of each capillary is different, so the light arcs do not overlap.

[0072] This turntable can replace the turntable in Implementation Examples 1 and 2 for life measurement.

[0073] This implementation example fully utilizes the waveguide characteristics and capillary principle of the capillary tube, and also utilizes the centrifugal force.

[0074] This method can be used for centrifugal separation and lifetime measurement of mixtures.

[0075] Implementation Example 5, a turntable

[0076] As attached Figure 5As shown, the turntable 501 is divided into two layers, the upper layer is opaque and has multiple light holes 503; the lower layer is buried with a U-shaped transparent capillary 502, the opening of the U-shaped capillary points to the center of the turntable, and the bottom leads to the edge of the turntable, which is used to guide the light at the edge of the turntable into the capillary, so that the sample in the tube can be excited; each light hole corresponds to a capillary, so that a small section of light from the capillary can pass through the turntable, so that when the turntable rotates, a luminous arc can be formed, and because the rotation radius of each light hole is different, the trajectories of the light arcs will not overlap, so it can be used for lifetime luminescence lifetime detection of multiple samples.

[0077] This turntable can replace the turntable in Implementation Examples 1 and 2 for life measurement.

[0078] In the turntable, the capillary tube serves as both a sample slot and a light guide; and when the turntable rotates, centrifugal force is generated, so that different components of some samples can be separated in the U-shaped capillary tube, thereby achieving the purpose of separation and detection.

[0079] Like implementation example 4, this implementation example fully utilizes the waveguide characteristics and capillary principle of the capillary tube, and also utilizes centrifugal force.

[0080] This method can be used for centrifugal separation and lifetime measurement of mixtures.

Claims

1. A turntable for measuring fluorescent phosphorescence lifetime, characterized in that: There are multiple sample slots on the turntable, and the distances between each sample slot and the center of the turntable are different. Each sample slot is connected to an optical channel. All optical channels are pre-buried in the turntable. Each optical channel is independent and does not interfere with each other. The other end of the optical channel is connected to the edge of the turntable, and is used to introduce excitation light in a direction parallel to the sample rotation plane into the optical channel and excite the sample in the sample slot; The medium constituting the optical channel is selected from air, optical fiber, and capillary.

2. The turntable according to claim 1, characterized in that: The optical channel medium is multimode optical fiber.

3. The turntable according to claim 1, characterized in that: The medium of the optical channel is air, and the inner wall of the optical channel is provided with a reflective film.

4. The turntable according to claim 1, characterized in that: The optical channel medium is a transparent capillary, which also serves as a sample slot.

5. A fluorescence phosphorescence lifetime measurement device, characterized in that: A turntable comprising the turntable according to any one of claims 1 to 4, further comprising a motor, a point light source, and a camera; The turntable is driven by a motor shaft, and the light emitted by a point light source is focused on the edge of the turntable, so that the excitation beam excites the sample from a direction parallel to the sample rotation plane. The camera lens is parallel to the turntable and is used to capture the image of the turntable.

6. The life measurement device according to claim 5, characterized in that: A condenser lens is also included for focusing the light from the light source onto a certain point on the edge of the turntable.

7. The life measurement device according to claim 5, characterized in that: The light source is selected from a laser diode or an LED light source.

8. The life measurement device according to claim 5, characterized in that: The optical channel medium is a capillary; when measuring a liquid sample, the liquid sample is placed in the capillary, centrifuged first, and then detected, thereby realizing the integration of centrifugal separation and life detection.

9. The life measurement device according to claim 5, characterized in that: It also includes an imaging lens for magnifying the image of the turntable and then taking a photo.

10. The life measurement device according to claim 5, characterized in that: A filter is also included in front of the camera lens to filter out the excitation light.

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