A ratiometric up-conversion fluorescence detection device and method based on microfluidic chip

Abstract

The present invention provides a ratio-type up-conversion fluorescence detection device and method based on a microfluidic chip, and relates to the field of fluorescence detection. The ratio-type up-conversion fluorescence detection device based on a microfluidic chip comprises a device body, a paper-based microfluidic sampling module, an optical path module and a detection module, wherein two mounting slots are arranged inside the device body, the paper-based microfluidic sampling module comprises a chip sampling slider, a paper-based microfluidic chip is mounted on the sampling slider, and an array-distributed near-infrared laser diode is detachably mounted on the excitation optical path slider, and the excitation optical path direction emitted by the paper-based microfluidic chip and the near-infrared laser diode is 60°, which solves the problem that the existing up-conversion detection devices based on smart terminals all use external high-power lasers or semiconductor laser diodes combined with lenses as excitation light sources, the excitation spot covers a small area, and each test can only detect a single sample, and cannot achieve multi-functional, multi-sample rapid detection.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescence detection, and in particular to a ratiometric up-conversion fluorescence detection device and method based on a microfluidic chip. Background Art

[0002] Fluorescence sensors have long been widely used in the fields of chemistry, biology, and agriculture due to their advantages such as rapidity, sensitivity, non-destructiveness, and specificity. Different from traditional fluorescence sensors based on ultraviolet excitation such as organic dyes, semiconductor materials, and quantum dots, fluorescence sensors based on lanthanide rare earth ion-doped upconversion nanomaterials (referred to as upconversion materials) can convert near-infrared light into shorter wavelength emissions (visible light or ultraviolet light). This unique photophysical property effectively avoids problems such as susceptibility to spontaneous fluorescence interference from organisms, low signal-to-noise ratio and sensitivity, and co-excitation of energy donors caused by ultraviolet excitation. At the same time, rare earth upconversion materials also have the advantages of low toxicity, multiple emission peaks, narrow emission bands, and good stability, which make them unique in the field of detection.

[0003] At present, there are relatively few instruments and equipment specifically used for upconversion material detection. Experimenters mainly use fluorescence spectrometers close to infrared lasers to detect luminescent materials, which cannot meet the needs of on-site instant detection. Since smart terminals such as smart phones and tablets have portability, powerful application processing functions and camera functions, mobile detection systems based on smart mobile terminals have become a research hotspot in the field of analytical detection. However, due to the low conversion efficiency of upconversion materials, in order to ensure the output power density, the existing upconversion detection devices based on smart terminals all use external high-power lasers or semiconductor laser diodes combined with lenses as excitation light sources. The excitation spot covers a small area, and each test can only detect a single sample, which cannot achieve multi-functional and multi-sample rapid detection.

[0004] Although laser diode arrays can achieve higher output power and a larger coverage area, their poor uniformity will affect the accuracy and sensitivity of detection. By utilizing the characteristics of multiple emission peaks and narrow emission bands of upconversion materials combined with multi-camera mobile terminals, a ratiometric fluorescence detection device can be designed, which greatly reduces the impact of laser unevenness, external environment, sample concentration, etc., and improves detection accuracy and sensitivity. At the same time, using solar energy to achieve self-powered laser diode arrays can solve the problem of outdoor electricity use. Further combined with paper-based microfluidic chip technology, a new type of upconversion fluorescence detection system can be developed to achieve rapid detection of multiple functions or multiple samples and multiple targets such as sample preparation, reaction, separation, and enrichment. Summary of the invention

[0005] 1. Technical issues to be resolved

[0006] In view of the shortcomings of the prior art, the present invention provides a ratiometric up-conversion fluorescence detection device and method based on a microfluidic chip, which solves the problem that the existing up-conversion detection devices based on smart terminals all use external high-power lasers or semiconductor laser diodes combined with lenses as excitation light sources, the excitation spot covers a small area, and can only detect a single sample in each test, and cannot achieve multi-functional and multi-sample rapid detection.

[0007] (II) Technical solution

[0008] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0009] On the one hand, a ratiometric up-conversion fluorescence detection device based on a microfluidic chip is provided, comprising a device body, a paper-based microfluidic sampling module, an optical path module and a detection module, wherein two mounting grooves are arranged inside the device body, the paper-based microfluidic sampling module comprises a chip sampling slider slidably mounted in one of the mounting grooves, a paper-based microfluidic chip is mounted on the sampling slider, an end of the slider located outside one of the mounting grooves is provided with a light shielding cover slidably connected to one of the mounting grooves, the optical path module comprises an excitation optical path slider slidably mounted in the other mounting groove, and the excitation optical path slider can be The device is disassembled and installed with near-infrared laser diodes distributed in an array, the paper-based microfluidic chip and the excitation light path direction emitted by the near-infrared laser diode are 60°, a filter is installed on the top of the device body, a solar panel is installed on one side of the device body, the near-infrared laser diode and the solar panel are electrically connected through a connecting line, the detection module includes an intelligent terminal installed on the top of the device body, the intelligent terminal includes cameras respectively arranged directly above the filters and data detection software, and the data detection software is used to analyze the collected image signals and extract the mode of all pixel points to obtain the detection value.

[0010] Preferably, the paper-based microfluidic chip comprises a bottom plate detachably mounted above the chip injection slider, the bottom plate is provided with a cover plate automatically clamped by magnetic adsorption, and the cover plate is provided with a through hole.

[0011] Preferably, the through hole is in the shape of a round hole, a dumbbell-shaped hole, a branch-shaped hole, a flower-shaped hole, a five-pointed star-shaped hole, a Y-shaped hole, a bridge-type composite structure hole or an irregular hole.

[0012] Preferably, the through hole includes a sample addition area and a detection area.

[0013] Preferably, the laser wavelength of the near-infrared laser diodes distributed in an array is 980 nm or 808 nm.

[0014] Preferably, the number of the cameras is two or more.

[0015] Preferably, the data detection software is ImageJ, CIE Lab color space, photoshop, color extraction APP, color detector or self-developed APP program.

[0016] In another aspect, a detection method for the above-mentioned ratiometric up-conversion fluorescence detection device based on a microfluidic chip is provided, the detection method comprising:

[0017] Put the near-infrared laser diode array into the excitation light path slider, push the excitation light path slider into another of the mounting slots, connect the near-infrared laser diode and the solar panel via a connecting wire, and place the device body under sunlight;

[0018] Open the paper-based microfluidic chip, sandwich the filter paper between the bottom plate and the cover plate, draw the corresponding shape of the through hole on the filter paper along the through hole wall line with a marker pen, and drop the solution containing the upconversion material, the sample to be tested or the reaction solution in the sample addition area after the ink is completely dry;

[0019] Open the light shielding cover, pull the chip injection slider out of one of the mounting grooves, insert the paper-based microfluidic chip into the chip injection slider, push the chip injection slider into the mounting groove, irradiate the light emitted by the near-infrared laser diode on the sample detection area, and close the light shielding cover;

[0020] Select the filter according to the properties of the target object, place the smart terminal on the device housing, and use the camera of the smart terminal to shoot the color of the filter paper in real time through the filter;

[0021] The data detection software of the intelligent terminal is used to calculate the ratio of the mode of all pixels in the image detection area obtained by two or more cameras, obtain the signal strength, and obtain the detection result based on the relationship between the signal and the concentration of the detected substance.

[0022] (III) Beneficial effects

[0023] (1) The present invention provides a ratiometric up-conversion fluorescence detection device and method based on a microfluidic chip. The array-distributed near-infrared laser diodes can obtain a larger output power and a larger coverage area. The ratiometric design can greatly reduce the effects of laser unevenness, external environment, sample concentration, etc., which is conducive to fully stimulating the up-conversion material on the paper-based microfluidic chip and ensuring detection accuracy and sensitivity. At the same time, it adopts solar energy self-power supply to solve the problem of outdoor electricity use.

[0024] (2) The present invention provides a ratiometric upconversion fluorescence detection device and method based on a microfluidic chip. The paper-based microfluidic chip technology has the characteristics of cheap materials, simple production, easy recycling, diverse structures, low reagent consumption, environmental protection and degradability. The upconversion fluorescence detection device based on the microfluidic chip can realize multi-function (preparation, separation, enrichment, reaction, etc.), multi-sample, and rapid detection of the target object.

[0025] (3) The present invention provides a ratiometric up-conversion fluorescence detection device and method based on a microfluidic chip. The ratiometric up-conversion fluorescence detection device based on a microfluidic chip has a compact structure, a small size, and a low cost. Users only need to have a smart terminal that supports multiple cameras to use the detection device to perform self-detection. The device is easy to operate and easy to observe experimental results, which meets the needs of rapid on-site detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a schematic diagram of the structure of the present invention;

[0027] Figure 2 It is a schematic structural diagram of a ratiometric up-conversion fluorescence detection device based on a microfluidic chip of the present invention;

[0028] Figure 3 This is a schematic diagram of the chip sample injection slider structure of the present invention;

[0029] Figure 4 This is a schematic diagram of the structure of the circular hole paper-based microfluidic chip of the present invention;

[0030] Figure 5 This is a schematic diagram of the structure of the paper-based microfluidic chip with flower-shaped holes of the present invention;

[0031] Figure 6 This is a schematic diagram of the structure of the near-infrared laser diode array of the present invention;

[0032] Figure 7 This is a standard curve diagram for detecting target substances using the up-conversion material of the present invention.

[0033] Among them, 1. Paper-based microfluidic chip; 2. Chip sampling slider; 3. Light shielding cover; 4. Filter; 5. Smart terminal; 6. Solar panel; 7. Excitation light path slider; 8. Near-infrared laser diode; 9. Connecting wire; 10. Cover plate; 11. Through hole; 12. Bottom plate; 13. Sample loading area; 14. Detection area. DETAILED DESCRIPTION

[0034] The following will be combined with the accompanying drawings of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Embodiment 1

[0036] like Figure 1-6 As shown, an embodiment of the present invention provides a ratiometric up-conversion fluorescence detection device based on a microfluidic chip, comprising a device body, a paper-based microfluidic sampling module, an optical path module and a detection module. Two mounting grooves are arranged inside the device body. The paper-based microfluidic sampling module comprises a chip sampling slider 2 slidably mounted in one of the mounting grooves. A paper-based microfluidic chip 1 is mounted on the sampling slider 2. A light shielding cover 3 slidably connected to one of the mounting grooves is provided at the end of the slider outside one of the mounting grooves. The paper-based microfluidic chip 1 comprises a detachable mounting plate. A bottom plate 12 is mounted on the chip sample injection slider 2, and a cover plate 10 is provided on the bottom plate 12 and is clamped by magnetic force. A through hole 11 is provided on the cover plate 10. According to different requirements such as multi-function (preparation, separation, enrichment, reaction, etc.), multi-sample, multi-target rapid detection, etc., the shape of the through hole 11 is set to be a round hole, a dumbbell-shaped hole, a branch-shaped hole, a flower-shaped hole, a five-pointed star hole, a Y-shaped hole, a bridge-type composite structure hole or an irregular hole. The through hole 11 includes a sample loading area 13 and a detection area 14, wherein the through hole 11 of some shapes, the sample loading area The domain 13 and the detection area 14 are the entire through hole 11, for example, a circular through hole 11, the optical path module includes an excitation optical path slider 7 slidably installed in another mounting groove, and the excitation optical path slider 7 is detachably installed with near-infrared laser diodes 8 distributed in an array, and the size of the near-infrared laser diodes 8 distributed in an array is 980nm or 808nm, and the excitation optical path direction emitted by the paper-based microfluidic chip 1 and the near-infrared laser diode 8 is 60°, so that the reflected light of the excitation light is reflected in a direction of 30° vertically, which greatly reduces its interference with fluorescence, a filter 4 is installed on the top of the device body, and a solar panel 6 is installed on one side of the device body. The near-infrared laser diode 8 is electrically connected to the solar panel 6 through a connecting line 9, and the detection module includes an intelligent terminal 5 installed on the top of the device body, and the intelligent terminal 5 includes cameras and data detection software respectively arranged directly above the filter 4, and the number of cameras is two or more, and the data detection software is used to analyze the collected image signals and extract the mode of all pixel points to obtain the detection value, and the data detection software is ImageJ, CIE Lab color space, photoshop, color extraction APP, color detector or self-developed APP program.

[0037] Embodiment 2

[0038] The present invention also provides a detection method for the ratiometric up-conversion fluorescence detection device based on a microfluidic chip in the above embodiment, the detection method comprising:

[0039] Place the 808nm near-infrared laser diode 8 array into the excitation light path slider 7, push the slider into the device, connect the near-infrared laser diode 8 and the solar panel 6, and place the device under sunlight;

[0040] Select a paper-based microfluidic chip 1 with a circular through hole 11, such as Figure 3 As shown, the paper-based microfluidic chip 1 is opened, the filter paper is sandwiched between the cover plate 10 and the bottom plate 12, and a marker is used to draw a corresponding shape along the wall of the through hole 11. After the ink is completely dry, drop the ink containing :Blank sample of Er upconversion material, standard curve reagent and sample to be tested; due to the evaporation of the carrier liquid on the paper base, it plays a role of concentration and enrichment. If the sample concentration is too low, the sample can be added dropwise for enrichment multiple times;

[0041] Open the light shielding cover 3, pull the chip sample introduction slider 2 out of one of the installation grooves, insert the paper-based microfluidic chip 1 into the chip sample introduction slider 2, push the chip sample introduction slider 2 into the installation groove, irradiate the light emitted by the near-infrared laser diode 8 on the sample through hole 11, and close the light shielding cover 3;

[0042] Select 550nm and 650nm bandpass filters 4 respectively, place the smart terminal 5 on the device housing, and use the two cameras of the smart terminal 5 to shoot the color of the two filter papers in real time through the filters 4, so as to simultaneously shoot and obtain information;

[0043] The data detection software of the intelligent terminal 5 is used to calculate the ratio of the mode of all pixels in the image detection area 14 obtained by two or more cameras to obtain the signal strength. The signal strength of the up-conversion material when there is no target object is divided by the signal strength of the up-conversion material when there is a target object to obtain a standard curve for detecting the target object, such as Figure 7 As shown, the ratio of the signal intensity of the blank sample to the signal intensity of the sample to be tested is compared with the standard curve to obtain the concentration of the test sample.

[0044] Embodiment 3

[0045] Place the 980nm near-infrared laser diode 8 array into the excitation light path slider 7, push the excitation light path slider 7 into another of the mounting slots, connect the near-infrared laser diode 8 and the solar panel 6 via the connecting wire 9, and place the device body under sunlight;

[0046] Select a paper-based microfluidic chip 1 with flower-shaped holes 11, such as Figure 4As shown, the paper-based microfluidic chip 1 is opened, the filter paper is sandwiched between the bottom plate 12 and the cover plate 10, and the corresponding shape of the through hole 11 is drawn along the wall of the through hole 11 with a marker pen. After the ink is completely dry, the ink containing : Tm up-conversion material, sample to be tested, eluent and quencher; since the solvents contained in the up-conversion material, sample to be tested, eluent and quencher are slightly different and the flow rates are different, the sample to be tested is first separated by the eluent to remove impurities, then reacts with the quencher to make the quencher lose its quenching effect, and finally mixes with the up-conversion material to reach the detection area 14;

[0047] Open the light shielding cover 3, pull the chip sample introduction slider 2 out of one of the installation slots, insert the paper-based microfluidic chip 1 into the chip sample introduction slider 2, push the chip sample introduction slider 2 into the installation slot, irradiate the light emitted by the near-infrared laser diode 8 on the detection area 14, and close the light shielding cover 3;

[0048] Select 475nm and 650nm bandpass filters 4 respectively, place the smart terminal 5 on the device housing, and use the two cameras of the smart terminal 5 to shoot the color of the two filter papers in real time through the filters 4, so as to simultaneously shoot and obtain information;

[0049] The data detection software of the intelligent terminal 5 is used to calculate the ratio of the mode of all pixels in the image detection area 14 obtained by two or more cameras, obtain the signal intensity, and obtain the detection result according to the relationship between the signal and the concentration of the detection substance.

[0050] Embodiment 4

[0051] Place the 980nm near-infrared laser diode 8 array into the excitation light path slider 7, push the excitation light path slider 7 into another of the mounting slots, connect the near-infrared laser diode 8 and the solar panel 6 via the connecting wire 9, and place the device body under sunlight;

[0052] Select a paper-based microfluidic chip 1 having dendritic pores 11, such as Figure 5 As shown, the paper-based microfluidic chip 1 is opened, the filter paper is sandwiched between the bottom plate 12 and the cover plate 10, and the shape of the through hole 11 is drawn on the filter paper along the wall line of the through hole 11 with a marker pen. After the ink is completely dry, the ink containing : Tm and quencher for detecting copper ions, : Er and a quencher for detecting zinc ions, :Ho and quencher for detecting iron ions, : A solution of Eu / Tb and a quencher for detecting mercury ions; after the solution is completely dry, the sample to be tested is added to the sample adding area 13, and the sample to be tested flows to each detection area 14 and mixes with the up-conversion material and the quencher;

[0053] Open the light shielding cover 3, pull the chip sample introduction slider 2 out of one of the installation slots, insert the paper-based microfluidic chip 1 into the chip sample introduction slider 2, push the chip sample introduction slider 2 into the installation slot, irradiate the light emitted by the near-infrared laser diode 8 on the sample detection area 14, and close the light shielding cover 3;

[0054] Select 800nm ​​and 650nm bandpass filters 4 respectively, place the smart terminal 5 on the device housing, and use the two cameras of the smart terminal 5 to shoot the color of the two filter papers in real time through the filters 4, so as to simultaneously shoot and obtain information;

[0055] The data detection software of the intelligent terminal 5 is used to calculate the ratio of the mode of all pixel points in each detection area 14 of the image obtained by the two cameras to obtain the signal strength; based on the relationship between the signal and the concentration of the detection target, the results of the copper ion, zinc ion, iron ion and mercury ion content of the detection sample are obtained at the same time.

[0056] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

Claims

1. A ratiometric up-conversion fluorescence detection device based on a microfluidic chip, comprising a device body, a paper-based microfluidic injection module, an optical path module and a detection module, wherein two mounting slots are provided inside the device body, and characterized in that: The paper-based microfluidic sampling module comprises a chip sampling slider (2) slidably mounted in one of the mounting grooves, a paper-based microfluidic chip (1) being mounted on the sampling slider (2), an end of the slider located outside one of the mounting grooves being provided with a light shielding cover (3) slidably connected to one of the mounting grooves, the optical path module comprises an excitation optical path slider (7) slidably mounted in another of the mounting grooves, an array of near-infrared laser diodes (8) being detachably mounted on the excitation optical path slider (7), the paper-based microfluidic chip (1) and the near-infrared laser diodes (8) being detachably mounted on the excitation optical path slider (7). The excitation light path direction emitted by the diode (8) is 60°, a filter (4) is installed on the top of the device body, a solar panel (6) is installed on one side of the device body, the near-infrared laser diode (8) and the solar panel (6) are electrically connected via a connecting line (9), and the detection module comprises an intelligent terminal (5) installed on the top of the device body, the intelligent terminal (5) comprises cameras respectively arranged directly above the filter (4) and data detection software, and the data detection software is used to analyze the collected image signal and extract the mode of all pixel points to obtain the detection value; The paper-based microfluidic chip (1) comprises a base plate (12) detachably mounted above the chip sample injection slider (2), a cover plate (10) automatically clamped by magnetic adsorption is provided on the base plate (12), and a through hole (11) is provided on the cover plate (10).

2. A ratiometric up-conversion fluorescence detection device based on a microfluidic chip according to claim 1, characterized in that: The through hole (11) is in the shape of a round hole, a dumbbell-shaped hole, a tree-shaped hole, a flower-shaped hole, a five-pointed star-shaped hole, a Y-shaped hole, a bridge-type composite structure hole or an irregular hole.

3. The ratiometric up-conversion fluorescence detection device based on a microfluidic chip according to claim 1, characterized in that: The through hole (11) comprises a sample addition area (13) and a detection area (14).

4. The ratiometric up-conversion fluorescence detection device based on a microfluidic chip according to claim 1, characterized in that: The laser wavelength of the near-infrared laser diodes (8) distributed in an array is 980 nm or 808 nm.

5. The ratiometric up-conversion fluorescence detection device based on a microfluidic chip according to claim 1, characterized in that: The number of the cameras is two or more.

6. The ratiometric up-conversion fluorescence detection device based on a microfluidic chip according to claim 1, characterized in that: The data detection software is ImageJ, CIE Lab color space, photoshop, color extraction APP, color detector or self-developed APP program.

7. A detection method for the ratiometric up-conversion fluorescence detection device based on a microfluidic chip according to any one of claims 1 to 6, characterized in that: The test method includes: The near-infrared laser diode (8) array is placed in the excitation light path slider (7), the excitation light path slider (7) is pushed into another installation groove, the near-infrared laser diode (8) is connected to the solar panel (6) via a connecting line (9), and the device body is placed under sunlight; Open the paper-based microfluidic chip (1), sandwich the filter paper between the bottom plate (12) and the cover plate (10), draw the corresponding shape of the through hole (11) on the filter paper along the hole wall line of the through hole (11) with a marker pen, and after the ink is completely dry, drop a solution containing an upconversion material, a sample to be tested, or a reaction solution into the sample addition area (13); Open the light shielding cover (3), pull the chip sample introduction slider (2) out of one of the installation slots, insert the paper-based microfluidic chip (1) into the chip sample introduction slider (2), push the chip sample introduction slider (2) into the installation slot, irradiate the light emitted by the near-infrared laser diode (8) onto the sample detection area (14), and close the light shielding cover (3); A filter (4) is selected according to the properties of the target object, and the intelligent terminal (5) is installed on the device housing, and the camera of the intelligent terminal (5) is used to shoot the color of the filter paper in real time through the filter (4); The data detection software of the intelligent terminal (5) is used to calculate the ratio of the mode of all pixel points in the image detection area (14) obtained by two or more cameras, obtain the signal intensity, and obtain the detection result based on the relationship between the signal and the concentration of the detection substance.

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