Microfluidic polymerase chain reaction detection device and system
By using snapshot hyperspectral cameras and mosaic filters in microfluidic PCR detection, the problem of spectral overlap in multi-color microfluidic PCR detection is solved, efficient detection of 7 colors and above is achieved, and cost and use complexity is reduced.
Patent Information
- Application Number
- CN202421581266.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Utility models(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-04
- Publication Date
- 2025-06-20
- Estimated Expiration
- 2034-07-04
AI Technical Summary
The existing multicolor microfluidic PCR technology is difficult to effectively solve the problem of overlapping fluorescence signal spectrums of more than 7 targets, resulting in insufficiency of detection. The filter needs to be replaced every time the fluorescent dye is replaced, which increases cost and inconvenience of use.
A snapshot hyperspectral camera is used to combine mosaic filters and microlens arrays to draw the spectral curve of multi-color mixed fluorescence signal through 20 to over a thousand spectral channels, and demixed to obtain the relative intensity of each monochromatic fluorescence signal, achieving 7 colors and above microfluidic PCR detection.
Multi-color microfluidic PCR detection is realized, especially 7 colors and above detection, which improves detection efficiency, avoids spectral overlap problems, and does not need to replace filters, reducing cost and complexity of use.
Smart Images
Figure CN223006033U_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the technical field of fluorescence detection, and particularly to a microfluidic polymerase chain reaction detection device and system. Background Art
[0002] Microfluidic PCR (Polymerase Chain Reaction) is a nucleic acid molecule quantification technique that can perform quantitative analysis on sample nucleic acid molecules. The most common method is to add a fluorescent dye or a fluorescent probe into a reaction unit containing nucleic acid molecules (DNA) to be detected. The fluorescent dye is represented by SYBR Green (a nucleic acid dye). When the dye is free, its fluorescence is weak, but once it binds to double-stranded DNA, the fluorescence is greatly enhanced. The fluorescent probe utilizes the phenomenon of fluorescence resonance energy transfer. There is a pair of groups on the probe that can generate fluorescence resonance energy transfer. During processes such as enzymatic cleavage and hybridization that occur in the PCR reaction, the distance between the two groups changes, causing the fluorescence intensity to also change. Each fluorescent dye and fluorescent probe will only bind to some specific nucleic acid molecule targets. Therefore, the fluorescence intensity in the reaction unit is directly proportional to the number of double-stranded DNA containing the corresponding targets. Thus, the types and quantities of double-stranded DNA generated in the test tube can be calculated by detecting the fluorescence intensity.
[0003] Parallel amplification and detection of multiple nucleic acid molecule targets in a single microfluidic reaction unit is called multi-color microfluidic PCR. The demand for multi-color microfluidic PCR in scientific research and clinical applications is increasing. For example, HPV detection requires quantitative analysis of 14 typing targets. Currently, in a single unit of multi-color microfluidic PCR technology, at most only 4 - 6 targets can be detected. Because when the number of targets exceeds 7, more than 7 fluorescent signals are required, and inevitable spectral overlap will cause serious crosstalk problems, which are difficult to eliminate with existing technologies. At the same time, in existing multi-color microfluidic PCR, a filter is configured separately for each fluorescent signal. When the fluorescent dye is replaced, the corresponding filter needs to be replaced simultaneously, resulting in inconvenience in use and an increase in cost. Summary of the Utility Model
[0004] In view of this, the purpose of the present application is to provide a microfluidic polymerase chain reaction detection device and system, which can achieve multi-color, even 7-color and above dPCR detection with high detection efficiency.
[0005] According to one aspect of the present application, the present application provides a microfluidic polymerase chain reaction detection device, which includes:
[0006] A microfluidic chip module and a temperature control module. The microfluidic chip module is used for quantitatively injecting and flowing a nucleic acid molecule sample, and the temperature control module is used for controlling the temperatures of different regions on the microfluidic chip module and forming a low-temperature annealing region, a medium-temperature extension region, and a high-temperature denaturation region on the microfluidic chip module to perform amplification processing on the nucleic acid molecule sample; wherein, the medium-temperature extension region includes a plurality of detection points, and each of the detection points corresponds to the amplification of different cycles of the nucleic acid molecule sample.
[0007] An illumination module, which is used for generating excitation light and irradiating the excitation light onto the nucleic acid molecule sample in each of the detection points to excite the nucleic acid molecule sample to generate a fluorescence signal.
[0008] A collection module, including a collection component and a snapshot hyperspectral camera. The collection component is used for collecting the fluorescence signal and converging the fluorescence signal to the snapshot hyperspectral camera, and the snapshot hyperspectral camera is used for converting the fluorescence signal into an image signal.
[0009] In one implementation, the illumination module includes:
[0010] A light source, a filter element, a first converging element, and a plurality of illumination optical fibers. The illumination optical fibers are arranged in one-to-one correspondence with the detection points. The filter element filters the light emitted by the light source to obtain excitation light with a corresponding wavelength. The first converging element converges the excitation light with the corresponding wavelength to each of the illumination optical fibers, and the excitation light emitted from each of the illumination optical fibers is incident on the corresponding detection point.
[0011] In one implementation, the illumination module further includes:
[0012] A plurality of second converging elements. The second converging elements are arranged in one-to-one correspondence with the illumination optical fibers. Each of the second converging elements is located between the corresponding illumination optical fiber and the detection point, and the second converging element is used for converging the excitation light emitted from the illumination optical fiber to the corresponding detection point.
[0013] In one implementation, the first converging element is a convex lens or a Fresnel lens.
[0014] In one implementation, the filter element is a band-pass filter or a beam splitter prism.
[0015] In one implementation, the light source is an LED, a mercury lamp, a laser, a halogen lamp, or a xenon lamp.
[0016] In one implementation, the first converging element is a convex lens or a Fresnel lens.
[0017] In one embodiment, the acquisition component includes:
[0018] A plurality of acquisition optical fibers and a third converging element. Each acquisition optical fiber acquires the fluorescence signal generated by the nucleic acid molecule sample at the corresponding detection point. The third converging element is located between the acquisition optical fiber and the snapshot hyperspectral camera, and the third converging element converges and couples the fluorescence signal into the snapshot hyperspectral camera.
[0019] In one embodiment, the third converging element is a convex lens or a Fresnel lens.
[0020] In one embodiment, the snapshot hyperspectral camera includes a mosaic filter, a microlens array, and an image sensor.
[0021] According to another aspect of the present application, the present application provides a microfluidic polymerase chain reaction detection system, including a computer and the above-mentioned microfluidic polymerase chain reaction detection device. The computer is connected to the snapshot hyperspectral camera, and the computer receives and records the image signal output by the snapshot hyperspectral camera.
[0022] The microfluidic polymerase chain reaction detection device disclosed in the present application applies a snapshot hyperspectral camera to microfluidic PCR detection. By using the 20 to thousands of spectral channels in the snapshot hyperspectral camera, the spectral curve of the detected multi-color mixed fluorescence signal can be drawn, and then the relative intensity of each monochromatic fluorescence signal can be obtained. It can realize multi-color, even 7-color and above microfluidic PCR detection, and has the advantages of miniaturization and low cost. Description of the Drawings
[0023] Figure 1 Schematic diagram of a microfluidic polymerase chain reaction detection device provided by an embodiment of the present application;
[0024] Figure 2 Schematic diagram of a microfluidic chip module provided by an embodiment of the present application;
[0025] Figure 3 Schematic diagram of the structure of a snapshot hyperspectral camera provided by an embodiment of the present application;
[0026] Figure 4 Schematic diagram of the structure of a snapshot hyperspectral camera provided by another embodiment of the present application;
[0027] Figure 5 Schematic diagram of the structure of a mosaic filter provided by an embodiment of the present application;
[0028] Figure 6 Flowchart of a microfluidic polymerase chain reaction detection method provided by an embodiment of the present application;
[0029] Figure 7 Flow chart of the microfluidic polymerase chain reaction detection method provided by another embodiment of the present application.
[0030] Marking description:
[0031] 10. Microfluidic chip module; 11. Low-temperature annealing zone; 12. Medium-temperature extension zone; 13. High-temperature denaturation zone; 15. First inlet; 16. Second inlet; 17. Third inlet; 18. Outlet; A. Detection point
[0032] 21. Light source; 22. Filter element; 23. First converging element; 24. Illumination optical fiber; 25. Second converging element
[0033] 31. Acquisition optical fiber; 32. Third converging element
[0034] 4. Snapshot hyperspectral camera; 41. Mosaic filter; 42. Microlens array; 43. Image sensor; 44. First convex lens; 45. Second convex lens Specific implementation mode
[0035] To make the objectives, technical solutions and advantages of the present application clearer and more understandable, the present application will be further described in detail below in conjunction with specific implementation modes and with reference to the accompanying drawings.
[0036] It should be noted that unless otherwise defined, the technical terms or scientific terms used in the implementation modes of the present application should have the ordinary meaning understood by those of ordinary skill in the field to which the present application belongs. The "first", "second" and similar terms used in the implementation modes of the present application do not indicate any order, quantity or importance, but are only used to distinguish different components. The terms such as "including" or "comprising" mean that the elements or objects appearing before this word cover the elements or objects listed after this word and their equivalents, without excluding other elements or objects. The terms such as "connected" or "linked" are not limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect. The terms such as "upper", "lower", "left" and "right" are only used to represent relative positional relationships, and when the absolute position of the object being described changes, the relative positional relationship may also change accordingly.
[0037] As Figure 1-2 shown, the embodiment of the present application provides a microfluidic polymerase chain reaction detection device, which includes a microfluidic chip module 10, a temperature control module, an illumination module and an acquisition module.
[0038] Among them, the microfluidic chip module 10 is used for quantitatively injecting and flowing nucleic acid molecule samples, and the temperature control module is used to control the temperatures of different regions on the microfluidic chip module 10 and form a low-temperature annealing region 11, a medium-temperature extension region 12, and a high-temperature denaturation region 13 on the microfluidic chip module 10 to perform amplification processing on the nucleic acid molecule samples; among them, the medium-temperature extension region 12 includes multiple detection points A, and each detection point A corresponds to the amplification of different cycles of the nucleic acid molecule sample. The different regions refer to the low-temperature annealing region 11, the medium-temperature extension region 12, and the high-temperature denaturation region 13.
[0039] Specifically, the high-temperature denaturation region 13 is used for high-temperature denaturation of nucleic acid molecules, the low-temperature annealing region 11 is used for low-temperature annealing of nucleic acid molecules, and the medium-temperature extension region 12 is used for medium-temperature extension of nucleic acid molecules. In one embodiment, the temperature of the low-temperature annealing region 11 is 60 °C, the temperature of the medium-temperature extension region 12 is 77 °C, and the temperature of the high-temperature denaturation region 13 is 95 °C. In another embodiment, the temperatures of the low-temperature annealing region 11, the medium-temperature extension region 12, and the high-temperature denaturation region 13 can be set as needed, and are not specifically limited.
[0040] The illumination module is used to generate excitation light and irradiate the nucleic acid molecule samples in each detection point A to excite the nucleic acid molecule samples to generate fluorescence signals.
[0041] The acquisition module includes an acquisition component and a snapshot hyperspectral camera 4. The acquisition component is used to acquire fluorescence signals and converge the fluorescence signals to the snapshot hyperspectral camera 4, and the snapshot hyperspectral camera 4 is used to convert the fluorescence signals into image signals.
[0042] The microfluidic polymerase chain reaction detection device disclosed in this embodiment applies the snapshot hyperspectral camera 4 to microfluidic PCR detection. By using the 20 to thousands of spectral channels in the snapshot hyperspectral camera 4, the spectral curves of the detected multi-color mixed fluorescence signals can be drawn, and then the relative intensities of the monochromatic fluorescence signals can be obtained. It can realize multi-color, even 7-color and above microfluidic PCR detection, and has the advantages of miniaturization and low cost.
[0043] Furthermore, the temperature control module includes a temperature controller, a temperature sensor, a heating module, etc. By heating different regions and controlling them at the target temperature, and controlling the residence position of the nucleic acid sample through microfluidics, the denaturation, annealing, and extension of nucleic acid molecules are realized, that is, PCR amplification is realized.
[0044] Further, the microfluidic chip module 10 includes a first inlet 15, a second inlet 16, a third inlet 17, and an outlet 18. The nucleic acid molecule sample is injected from the first inlet 15; the second inlet 16 is used to inject a buffer solution to ensure the efficiency and accuracy of the reaction; the third inlet 17 is used to inject air or an isolation solution, and sample blocking is performed using air bubbles or an oily isolation reagent; the amplified nucleic acid molecules are discharged from the outlet 18.
[0045] Specifically, the detection point A includes detection points 1, 2, 3, and detection point M. Among them, detection point 1 corresponds to the first amplification of the nucleic acid molecule sample, detection point 2 corresponds to the second amplification of the nucleic acid molecule sample, and detection point M corresponds to the Mth amplification of the nucleic acid molecule sample.
[0046] Please continue to refer to Figure 2 As shown, in one embodiment, the illumination module includes a light source 21, a filter element 22, a first focusing element 23, and a plurality of illumination optical fibers 24. The illumination optical fibers 24 are arranged in one-to-one correspondence with the detection point A. The filter element 22 filters the light emitted by the light source 21 to obtain excitation light of a corresponding wavelength. The first focusing element 23 focuses the excitation light of the corresponding wavelength onto each illumination optical fiber 24, and the excitation light emitted from each illumination optical fiber 24 is incident on the corresponding detection point A. Among them, the filter element 22 is used to filter out light other than the excitation light. Optionally, the filter element 22 is a band-pass filter or a beam splitter prism, etc. Optionally, the light source 21 is an LED, a mercury lamp, a laser, a halogen lamp, a xenon lamp, etc. Optionally, the first focusing element 23 is a convex lens or a Fresnel lens, etc.
[0047] In one embodiment, the illumination module further includes a plurality of second focusing elements 25. The second focusing elements 25 are arranged in one-to-one correspondence with the illumination optical fibers 24. Each second focusing element 25 is located between the corresponding illumination optical fiber 24 and the detection point A. The second focusing element 25 is used to focus the excitation light emitted from the illumination optical fiber 24 onto the corresponding detection point A. Optionally, the second focusing element 25 is a convex lens or a Fresnel lens, etc.
[0048] In one embodiment, the acquisition component includes a plurality of acquisition optical fibers 31 and a third focusing element 32. Each acquisition optical fiber 31 acquires the fluorescence signal generated by the nucleic acid molecule sample in the corresponding detection point A. The third focusing element 32 is located between the acquisition optical fiber 31 and the snapshot hyperspectral camera 4. The third focusing element 32 focuses and couples the fluorescence signal into the snapshot hyperspectral camera 4. Optionally, the third focusing element 32 is a convex lens or a Fresnel lens, etc.
[0049] Refer to Figures 3-5 As shown, the snapshot hyperspectral camera 4 includes a mosaic filter 41, a microlens array 42, and an image sensor 43. Refer to Figure 3As shown, in one embodiment, the snapshot hyperspectral camera 4 includes two microlens arrays 42. The fluorescence signal sequentially passes through the mosaic filter 41 and then reaches the first microlens array 42 and the second microlens array 42 and converges on the image sensor 43. Refer to Figure 5 As shown, in another embodiment, the snapshot hyperspectral camera 4 includes a telescope optical system. The telescope optical system includes a first convex lens 44 and a second convex lens 45. After the fluorescence signal is collimated by passing through the first convex lens 44 and the second convex lens 45 in sequence, and then passes through the mosaic filter 41, it is converged by the microlens array 42 onto the image sensor 43.
[0050] Among them, the snapshot hyperspectral camera 4 can simultaneously image the spectral region of interest, such as the visible light band where most of the fluorescence signals are located. Therefore, when changing the fluorescent dye used, there is no need to change the filter.
[0051] Among them, the mosaic filter 41 has various implementation methods (absorption filter, interference filter, induced transmission filter (ITF), linear gradient filter, surface plasmon filter, metasurface structure filter). Their commonality is that at different positions in space, spectral information of different bands is transmitted, and then the signals at different positions in space are analyzed by an array-type optoelectronic sensor, so as to obtain the spectral information of the object in each spectral band.
[0052] In one embodiment, the snapshot hyperspectral camera 4 includes a 60-channel mosaic filter. The working principle is F-P cavity interference type. Because its optical density (OD) is low, the excitation light will pass through and form crosstalk to the fluorescence. Therefore, an additional band-pass filter needs to be added to filter out the excitation light and only allow the fluorescence signal to pass through.
[0053] Another embodiment of the present application provides a microfluidic polymerase chain reaction detection system, which includes a computer and the microfluidic polymerase chain reaction detection device in the above embodiment. The computer is connected to the snapshot hyperspectral camera 4, and the computer receives and records the image signal output by the snapshot hyperspectral camera 4.
[0054] Refer to Figure 6 As shown, another embodiment of the present application provides a microfluidic polymerase chain reaction detection method, which is applied to the microfluidic polymerase chain reaction detection device in the above embodiment. The method includes the following steps:
[0055] Step S10: Draw a multi-color mixed fluorescence spectrum curve according to the image signal output by the snapshot hyperspectral camera 4;
[0056] Step S20: Use an inversion algorithm to unmix the mixed fluorescence spectrum curve into a single-color fluorescence spectrum curve to obtain the relative intensity of each single-color fluorescence signal.
[0057] Reference Figure 7 As shown in Figure 7 , in one embodiment, step S10 of plotting a multi-color mixed fluorescence spectrum curve according to the image signal output by the snapshot hyperspectral camera 4 includes:
[0058] Step S11: Obtain the image signal intensity output by each spectral channel in the snapshot hyperspectral camera 4;
[0059] Step S12: Connect the image signal intensity output by the first spectral channel and the image signal intensity output by the last spectral channel, and fit to obtain a multi-color mixed fluorescence spectrum curve.
[0060] In one embodiment, step S20 of using an inversion algorithm to unmix the mixed fluorescence spectrum curve into a single-color fluorescence spectrum curve to obtain the relative intensity of each single-color fluorescence signal includes:
[0061] Construct the following formula:
[0062]
[0063] where N is the total number of spectral channels in the snapshot hyperspectral camera 4, D1, D2... D N is the intensity of the mixed fluorescence signal obtained by each spectral channel; J is the number of fluorescent dyes; the intensities of the fluorescence signals emitted by each fluorescent dye are I1, I2... I J ; where, when the fluorescence emitted by the first fluorescent dye is imaged separately using the snapshot hyperspectral camera 4, its intensities normalized in N spectral channels are F 11 , F 21 ,..., F N1 ; Connect F 11 and F N1 and fit to obtain the single-color fluorescence spectrum curve of the first fluorescent dye; when the fluorescence emitted by the Jth dye is imaged separately using the snapshot hyperspectral camera 4, its intensities normalized in N spectral channels are F 1J , F 2J ,..., F NJ .
[0064] Solve for I1 to I J , obtain the relative intensity of each single-color fluorescence signal, and obtain the nucleic acid molecule content corresponding to the dye target at the target detection point according to the relative intensity.
[0065] The microfluidic polymerase chain reaction detection method disclosed in this embodiment solves the problems in existing multi-color microfluidic PCR detection that each filter can only sample the peak region of the single-color fluorescence signal intensity and serious crosstalk occurs when the peak regions of two fluorescence signals overlap, and can achieve multi-color, even 7-color and above microfluidic PCR detection.
[0066] It should be noted that "a certain body" and "a certain part" can be part of the corresponding "component", that is, "a certain body" and "a certain part" are integrally formed with "other parts of the component"; or they can be an independent component separable from "other parts of the component", that is, "a certain body" and "a certain part" can be manufactured independently and then combined with "other parts of the component" to form a whole. The expression of "a certain body" and "a certain part" in this application is only one implementation manner for the convenience of reading, rather than a limitation on the protection scope of this application. As long as the above features are included and the functions are the same, it should be understood as an equivalent technical solution of this application.
[0067] It should be noted that some embodiments of this application have been described above. Other embodiments are within the scope of the appended claims. In some cases, the actions or steps recited in the claims can be performed in a different order than in the above embodiments and still achieve the desired result. Additionally, the processes depicted in the drawings do not necessarily require the specific order or sequential order shown to achieve the desired result.
[0068] Those of ordinary skill in the art should understand that: the discussion of any of the above embodiments is only exemplary and is not intended to imply that the scope of this application (including the claims) is limited to these examples; under the concept of this application, the technical features between the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations in different aspects of the embodiments of this application as described above, which are not provided in detail for the sake of brevity.
[0069] The embodiments of this application are intended to cover all such substitutions, modifications, and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the embodiments of this application shall be included within the protection scope of this application.
Claims
1. A microfluidic polymerase chain reaction detection device, characterized in that: include: A microfluidic chip module and a temperature control module, wherein the microfluidic chip module is used for quantitative injection and flow processing of nucleic acid molecule samples, and the temperature control module is used for controlling the temperature of different areas on the microfluidic chip module, and forming a low-temperature annealing area, a medium-temperature extension area and a high-temperature denaturation area on the microfluidic chip module to amplify the nucleic acid molecule samples; wherein the medium-temperature extension area includes a plurality of detection points, each of which corresponds to a different cycle of amplification of the nucleic acid molecule sample; An illumination module, used for generating excitation light and irradiating the excitation light to the nucleic acid molecule sample in each of the detection points, so as to excite the nucleic acid molecule sample to generate a fluorescent signal; The acquisition module includes an acquisition component and a snapshot hyperspectral camera, wherein the acquisition component is used to acquire the fluorescence signal and converge the fluorescence signal to the snapshot hyperspectral camera, and the snapshot hyperspectral camera is used to convert the fluorescence signal into an image signal.
2. The microfluidic polymerase chain reaction detection device according to claim 1, characterized in that: The lighting module comprises: A light source, a filter element, a first convergence element and a plurality of illumination optical fibers, wherein the illumination optical fibers are arranged in one-to-one correspondence with the detection points, the filter element filters the light emitted by the light source to obtain excitation light of a corresponding wavelength, the first convergence element converges the excitation light of the corresponding wavelength to each of the illumination optical fibers, and the excitation light emitted from each of the illumination optical fibers is incident on the corresponding detection point.
3. The microfluidic polymerase chain reaction detection device according to claim 2, characterized in that: The lighting module further comprises: A plurality of second converging elements are provided one-to-one with the illumination optical fibers, each of the second converging elements is located between the corresponding illumination optical fiber and the detection point, and the second converging element is used to converge the excitation light emitted from the illumination optical fiber to the corresponding detection point.
4. The microfluidic polymerase chain reaction detection device according to claim 3, characterized in that: The first converging element is a convex lens or a Fresnel lens.
5. The microfluidic polymerase chain reaction detection device according to claim 2, characterized in that: The filter element is a bandpass filter or a beam splitter prism.
6. The microfluidic polymerase chain reaction detection device according to claim 2, characterized in that: The light source is LED, mercury lamp, laser, halogen lamp or xenon lamp.
7. The microfluidic polymerase chain reaction detection device according to claim 2, characterized in that: The first converging element is a convex lens or a Fresnel lens.
8. The microfluidic polymerase chain reaction detection device according to claim 1, characterized in that: The acquisition component comprises: Multiple collection optical fibers and a third convergence element, each of the collection optical fibers collects the fluorescence signal generated by the nucleic acid molecule sample in the corresponding detection point, the third convergence element is located between the collection optical fiber and the snapshot hyperspectral camera, and the third convergence element converges the fluorescence signal and couples it into the snapshot hyperspectral camera.
9. The microfluidic polymerase chain reaction detection device according to claim 8, characterized in that: The third converging element is a convex lens or a Fresnel lens.
10. The microfluidic polymerase chain reaction detection device according to claim 1, characterized in that: The snapshot hyperspectral camera includes a mosaic filter, a microlens array and an image sensor.
11. A microfluidic polymerase chain reaction detection system, characterized in that: The invention comprises a computer and the microfluidic polymerase chain reaction detection device according to any one of claims 1 to 10, wherein the computer is connected to the snapshot hyperspectral camera, and the computer receives and records the image signal output by the snapshot hyperspectral camera.