WO2020119562A1

WO2020119562A1 - Droplet microfluidic chip for multicolor fluorescence synchronous detection

Info

Publication number: WO2020119562A1
Application number: PCT/CN2019/123120
Authority: WO
Inventors: 陈艳; 冯鸿涛
Original assignee: 中国科学院深圳先进技术研究院
Priority date: 2018-12-15
Filing date: 2019-12-04
Publication date: 2020-06-18
Also published as: CN111323399A

Abstract

Provided is a droplet microfluidic chip for multicolor fluorescence synchronous detection. The chip comprises a chip body (1), an excitation optical fiber (10), a detection optical fiber (20) and a reflection mirror (30), wherein an excitation optical fiber preformed groove (40), a detection optical fiber preformed groove (50) and a detection flowing channel (60) are provided in the chip body (1), the detection flowing channel (60) comprises a detection area (70), and the reflection mirror (30) is arranged close to the detection area (70); one end of the excitation optical fiber (10) inserted into the excitation optical fiber preformed groove (40) and one end of the detection optical fiber (20) inserted into the detection optical fiber preformed groove (50) converge at the detection area (70), the other end of the excitation optical fiber (10) is divided into a plurality of branch optical fibers such that same are respectively connected to a scattered light source and at least one excitation light source, and the other end of the detection optical fiber (20) is connected to a detection module (80); and a droplet flowing through the detection area (70) is irradiated by the excitation optical fiber (10), and a light signal generated by the droplet is reflected by the reflection mirror (30) and acquired by the detection optical fiber (20). The chip is small in spatial optical volume and high in integration level, and synchronous detection can be actually realized for various kinds of fluorescence.

Description

Droplet microfluidic chip for simultaneous detection of multicolor fluorescence

This application requires the priority of a Chinese patent application filed with the China Patent Office on December 15, 2018, with application number 201811540555.X. The content of the above-mentioned previous application is incorporated into this text by way of introduction.

Technical field

The invention relates to the technical field of biomedical application instruments, in particular to a droplet microfluidic chip for simultaneous detection of multi-color fluorescence.

Background technique

Digital PCR (Digital Polymerase Chain Reaction) technology is the third generation PCR technology after the first generation of ordinary PCR and the second generation of fluorescent quantitative PCR. The principle is to disperse the fluorescent quantitative PCR reaction system containing the nucleic acid molecule to be tested into tens of thousands of micro-volume units, each micro-volume contains no or at most one nucleic acid molecule to be tested. Each micro volume acts as an independent reaction unit. After the amplification is completed, the fluorescence signal of each micro volume unit is detected one by one. Only the micro volume unit containing the nucleic acid molecule to be tested can generate a fluorescent signal. The fluorescence signal is interpreted as 0, and the sample concentration is calculated based on the Poisson distribution of the signal count.

In the initial stage of digital PCR, a CCD (charge coupled device) photographing method is usually used to identify droplets and empty droplets carrying samples, but it requires continuous large-scale repeated photographing. In recent years, a spatial optical scheme has been commonly used, that is, using objective lens focusing to excite droplet fluorescence to detect fluorescence signals, which can realize the statistics of a large number of droplets in a short time, and is not limited by the photographing area. However, the volume of space optics is huge and the focus adjustment is complicated; in addition, another fluorescent dye is usually added as a negative signal for empty droplets without samples. When multi-color fluorescence signals need to be detected, the excitation system usually uses time-multiplexed optical paths, and the dimming is complicated, so that the two types of fluorescent signals cannot be detected in real time.

Summary of the invention

In view of this, the present invention provides a droplet microfluidic chip for simultaneous detection of multi-color fluorescence, which solves the problems of the huge volume of the optical space itself and the complicated focus control manipulation of the traditional spatial optical scheme. Really realize synchronous detection, and it is also possible to identify empty droplets without adding fluorescent marks to the empty droplets.

The invention provides a droplet microfluidic chip for synchronous detection of multi-color fluorescence, which includes a chip body, an excitation fiber, a detection fiber and a reflector. The chip body is provided with an excitation fiber reserve slot and a detection fiber reserve slot And a detection flow channel, the detection flow channel includes a detection area, the reflector is disposed near the detection area; the excitation fiber reserved slot is used to insert the excitation fiber, and the detection fiber reserved slot is used to insert The detection optical fiber, one end of the excitation optical fiber and one end of the detection optical fiber are converged in the detection area, and the other end of the excitation optical fiber is branched into a plurality of sub-fibers for respectively connecting a scattered light source and at least one excitation A light source, the other end of the detection fiber is connected to a detection module; the droplet flowing through the detection area is irradiated by the light from the excitation fiber, and the optical signal generated by the droplet is reflected by the mirror and detected by the reflector Optical fiber collection, when the droplet is an empty droplet, the optical signal is a scattered light signal, and when the droplet is a droplet that can generate fluorescence, the optical signal is a scattered light signal and at least two types of fluorescence The superimposed signal of the signal.

Wherein, the multi-color fluorescence is two kinds of fluorescence signals; the detection module includes a collimator mirror and a dichroic mirror arranged in sequence, the dichroic mirror is used to separate the two kinds of fluorescence signals; the detection module also It includes a first filter and a first detector which are sequentially arranged on the first light-emitting side of the dichroic mirror, and a second filter and the first detector which are sequentially arranged on the second light-emitting side of the dichroic mirror Two detectors; wherein, the dichroic mirror is also used to divide the scattered light signal to the first light exit side or the second light exit side.

Wherein, the droplet flowing through the detection area is located at the focal point of the mirror, the mirror is a curved mirror; the mirror and the detection fiber are on the same horizontal plane, and the mirror and the The detection optical fibers are perpendicular to the detection flow channel.

Wherein, the chip body includes a bottom chip and a top chip stacked on the bottom chip, the bottom chip is provided with a first gap and a second gap, and the top chip is provided with a suitable for the first gap A first groove matched, a second groove adapted to the second notch, and the detection flow path, when the bottom chip and the top chip are docked, the first notch is formed with the first groove The excitation optical fiber reserved groove for accommodating the excitation optical fiber, and the second notch and the second groove form the detection optical fiber reserved groove for accommodating the detection optical fiber.

Wherein, the central axis of the excitation optical fiber reserved groove, the detection optical fiber reserved groove and the detection flow channel are on the same horizontal plane.

Wherein, the detection flow channel includes a mixing inlet, and the top chip is provided with a first injection port, a regulating phase flow channel connected to the first injection port, a second injection port, and a second injection port The droplet flow channel, one end of the droplet flow channel and the adjustment phase flow channel all meet at the mixing inlet, and the adjustment phase interval in the adjustment phase flow channel at the mixing inlet is formed in the liquid Between the droplets in the drip channel to adjust the spacing between the droplets.

Wherein, the depth of the excitation optical fiber reserved groove and the detection optical fiber reserved groove are different.

Wherein, the numerical aperture, inner diameter and outer diameter of the excitation fiber are 0.1, 62.5 microns and 125 microns, respectively.

Wherein, the numerical aperture, inner diameter and outer diameter of the detection fiber are 0.38, 200 microns and 225 microns, respectively.

Wherein, the material of the reflector includes a low melting point metal alloy.

The droplet microfluidic chip provided by the invention integrates the divided excitation optical fiber, the detection optical fiber and the reflector, which has a small spatial optical volume and a high degree of integration, which solves the problem of the traditional spatial optical solution. The large volume of the space itself and the complicated focus control operation solve the accuracy of the sample concentration measurement due to the limitation of the photographing area; the droplet microfluidic chip can simultaneously detect the scattered light signal of the empty droplet and can generate The superimposed signal of the scattered light signal of the fluorescent droplet and the fluorescent signal; more importantly, it is suitable for the excitation and detection of single-excitation or multi-excitation multi-color fluorescence, and truly realizes the simultaneous detection of multi-color fluorescence without affecting many Signal response.

BRIEF DESCRIPTION

In order to clarify the technical solution and beneficial effects of the present invention more clearly, it will be described in detail below with reference to the drawings and specific embodiments.

FIG. 1 is a schematic structural diagram of a droplet microfluidic chip provided by an embodiment of the present invention.

FIG. 2 is a schematic structural diagram of the detection module connected to the detection optical fiber in FIG. 1.

3 is a schematic diagram of the side structure of the droplet microfluidic chip in FIG.

4 is a schematic diagram of the structure of the top chip in FIG.

detailed description

The technical solutions in the embodiments of the present invention will be described clearly and completely in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, but not all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

The embodiment of the present invention provides a droplet microfluidic chip, which is used for the simultaneous detection of multi-color fluorescence of droplets (especially PCR droplets).

As shown in FIG. 1, the droplet microfluidic chip includes a chip body 1, an excitation fiber 10, a detection fiber 20, and a reflector 30. The chip body 1 is provided with an excitation fiber reserve slot 40, a detection fiber reserve slot 50, and detection Flow channel 60. Among them, the excitation optical fiber reserved groove 40 is used for inserting/accommodating the excitation optical fiber 10, the detection optical fiber reserved groove 50 is used for inserting/accommodating the detection optical fiber 20, and the detection flow channel 60 is a flow path of droplets. In an embodiment of the present invention, the droplets may include empty droplets after PCR reaction, sample droplets that can generate fluorescence (especially multicolor fluorescence), and the like.

The detection flow path 60 includes a detection area 70, and the mirror 30 is disposed outside the detection flow path 60 and close to the detection area 70. One end of the excitation optical fiber 10 and one end of the detection optical fiber 20 converge in the detection area 70, and the other end of the excitation optical fiber 10 branches into a plurality of sub-fibers for connecting the scattered light source and at least one excitation light source, respectively, and the other end of the detection optical fiber 20 is connected Detection module 80. Here, the excitation optical fiber 10 is divided into multiple optical fibers, so that the emitted light is simultaneously mixed with scattered light and fluorescent excitation light, to avoid the difficulty of coupling multiple light sources in space optics, so as to irradiate droplets in the detection area 70 To generate an optical signal that can be detected by the detection module 80.

The droplets flowing through the detection area 70 are irradiated with light from the excitation fiber 10, and the optical signal generated by the droplets is reflected by the mirror 30 and collected by the detection fiber 20. When the droplet is an empty droplet, the light signal is scattered light Signal, when the droplet is a droplet capable of generating fluorescence, the optical signal is a superimposed signal of the scattered light signal and at least two fluorescent signals. Specifically, the droplet flowing through the detection area 70 is irradiated with light from the excitation fiber 10, and when the droplet is a non-fluorescent empty droplet, the scattered light signal generated by it is reflected by the mirror 30 and collected by the detection fiber 20 And transmitted to the detection module 80; when the droplet is a sample droplet that can generate fluorescence, the superimposed signal of the fluorescent signal and the scattered light signal generated by it is collected by the detection optical fiber 20 and transmitted to the detection module 80. The detection module 80 processes and counts the received optical signals, and distinguishes the scattered light signals and various types of multi-color fluorescent signals. The number of scattered light signals corresponds to the number of all droplets.

The droplet microfluidic chip provided by the present invention integrates the multi-divided excitation optical fiber 10 and the detection optical fiber 20 and the reflector 30, which has a small spatial optical volume and a high degree of integration, which solves the traditional space The optical space of the optical solution is bulky and the focus control is complicated, which further solves the accuracy of the sample concentration measurement due to the limitation of the photographing area; the droplet microfluidic chip can simultaneously detect the scattered light signal of the empty droplet , And the superimposed signal of the scattered light signal and the fluorescent signal of the droplets that can produce fluorescence; more importantly, it is suitable for the excitation and detection of single-excitation or multi-excitation multicolor fluorescence, and truly realizes the simultaneous detection of multicolor fluorescence. And does not affect the multi-signal response.

Further, the structure of the detection module 80 will be described below by taking multi-color fluorescence as two kinds of fluorescence signals as an example. As shown in FIG. 2, the detection module 80 includes a collimator mirror 81 and a dichroic mirror 82 arranged in sequence. The dichroic mirror 82 is used to separate the two fluorescent signals; the detection module 80 further includes a dichroic mirror arranged in sequence. 82, a first filter 83 and a first detector 84 on the first light-exiting side, and a second filter 85 and a second detector 86 that are sequentially disposed on the second light-emitting side of the dichroic mirror; The dichroic mirror 82 is also used to divide the scattered light signal to the first light emitting side or the second light emitting side (that is, the scattered light signal can be detected by the first detector 84 or by the second detector 86).

Obviously, the wavelengths allowed to pass by the first filter 83 and the second filter 85 are different to distinguish the two fluorescent signals. Based on the presence of the detection module 80, the fluorescence information of at least two wavelengths of the measured object can be obtained by one measurement, which simplifies the detection process and equipment. Based on the difference in the intensity of the scattered light signal and the first or second fluorescence signal, the two can still be distinguished by the detector on the same light exit side, so as to realize the counting statistics of each droplet and finally determine the sample concentration.

More specifically, taking the detection of single-excitation multicolor fluorescence as an example, the excitation fiber 10 is connected with a scattered light source with a wavelength of 510-530 nm; and a fluorescent excitation light source with a wavelength of 488 nm, which can excite droplets with samples to emit wavelengths between The first fluorescence at 510-530nm, and the second fluorescence at a wavelength between 560-580nm. After the light signal of the fluorescent droplets passes through the collimating mirror 81, the light beam is maintained in collimation, and after passing through the dichroic mirror 81 (the transmission cut-off wavelength is 530nm), the first fluorescent signal of 560-580nm is transmitted (Fall into the bandpass wavelength range of the first filter 83), reflect the scattered light of 510-530nm and the second fluorescent signal of 510-530nm (fall into the bandpass wavelength range of the second filter 85), so The first fluorescent signal can be detected by the first detector 84 placed behind the first filter 83, and the second fluorescent signal and the scattered light signal can be detected by the second detector 86 placed behind the second filter 85 To. Correspondingly, for the empty droplet, the optical signal collected by the detection fiber 20 is only the scattered light signal, which is detected by the second detector 86. In this way, the first fluorescence signal and the second fluorescence signal can independently respond as positive signals on the two detectors, and the scattered light of 510-530 nm after passing through the detection fiber 20 is weak, which is used as the second detector 86. The negative signal ensures the statistics of all droplets.

Optionally, the first detector 84 and the second detector 86 are photomultiplier tubes, which can convert the corresponding optical signals received into electrical signals and amplify them, and then can distinguish the yin and yang signals on the host computer connected to them.

Of course, as an extension of the present invention, the detection module 80 of the droplet microfluidic chip can also realize the simultaneous detection of more than three kinds of fluorescence, for example, two groups of fluorescence are divided by the dichroic mirror 81, of which The first group of fluorescence on the light exit side includes two kinds of fluorescence, and the second group of fluorescence on the first light exit side includes one kind of fluorescence. A second dichroic mirror can be placed after the first light exit side. In order to separate the two kinds of fluorescence in the first group of fluorescence.

Preferably, the droplets flowing through the detection area 70 are located at the focus of the mirror 30, and the mirror 30 is a curved mirror. In this way, the optical signal of the droplet irradiated by the excitation fiber 10 is reflected by the mirror 30 as much as possible. The reflector 30 and the detection fiber 20 are located on the same horizontal plane, and the reflector 30 and the detection fiber 20 are both perpendicular to the detection flow channel 60. In this way, the optical signal of the droplets reflected by the mirror 30 can be collected by the detection fiber 20 to the greatest extent, reducing signal loss.

Optionally, near the detection area 70, the angle between the excitation fiber 10 and the detection fiber 20 is 45°. In this way, the laser light source may not be reflected by the mirror 30 as much as possible to avoid interference with the fluorescent signal and the scattered light signal.

In this embodiment, the numerical aperture (NA) of the excitation fiber 10 is 0.1, and the inner diameter and outer diameter of the excitation fiber 10 are 62.5 microns and 125 microns, respectively. Among them, the inner diameter of 62.5 microns can meet the transmission of light intensity, NA is selected to 0.1, the emission angle is limited to a very small range, to avoid the excitation divergence angle is too large and the irradiation area is too large, thereby avoiding the adjacent droplets Signal crosstalk.

In this embodiment, the numerical aperture (NA) of the detection fiber 20 is 0.38, and the inner diameter and outer diameter of the detection fiber 20 are 200 μm and 225 μm, respectively. Among them, the inner diameter of 200 microns can ensure the maximum collection of fluorescent cross-sections emitted by the droplets and improve the fluorescence collection efficiency. NA 0.38 achieves the maximum emission angle collection of the scattered light reflected.

Referring to FIG. 3, in an embodiment of the present invention, the chip body 1 includes a bottom chip 11 and a top chip 12 stacked on the bottom chip 11. The bottom chip body 11 is provided with a first notch 111 on the surface facing the top chip 12. In the second notch 112 and the detection channel 60, the surface of the top chip 12 facing the bottom chip 11 is provided with a first groove 121 adapted to the first notch 111 and a second groove 122 adapted to the second notch 112. When the bottom chip 11 and the top chip 12 are docked, the first notch 111 and the first slot 121 form an excitation fiber reserved slot 40 for accommodating the excitation fiber 10, and the second notch 112 and the second slot 122 form a detection fiber for accommodating the detection fiber 20 Reserve slot 50. Wherein, the bottom chip 11 and the top chip 12 are connected by but not limited to using vacuum oxygen plasma for bonding and packaging. The material of the bottom chip 11 includes but is not limited to glass, and the material of the top chip 12 includes but is not limited to polydimethylsiloxane, glass and the like.

Optionally, the entrance end of the first slot 121 on the top chip 12 facing away from the detection area 70 is larger than its port close to the detection area 70. Correspondingly, the shape of the first notch 111 on the bottom chip 11 is the same. This can facilitate the insertion of the excitation fiber 10 from the docked excitation fiber reserved slot 40. For example, the inlet end of the first groove 121 on the top chip 12 facing away from the detection area 70 is in a single-sided dovetail shape, and the inlet end of the second groove 122 facing away from the detection area 70 is in a bilateral dovetail shape.

In an embodiment of this embodiment, the excitation fiber reserve groove 40 and the detection fiber reserve groove 50 are formed by a photolithography method. The depth of the excitation fiber reserve groove 40 and the detection fiber reserve groove 50 are different. In this way, the fixation of the excitation optical fiber 10 and the detection optical fiber 20 with different outer diameters can be satisfied.

In the present invention, the central axes of the excitation fiber reserve slot 40, the detection fiber reserve slot 50, and the detection flow channel 60 (L1 in FIG. 3 is the center axis) are on the same plane (L2 in FIG. 3 is the bottom chip 11. The dividing line of the top chip 12). Specifically, since the central axes of the excitation optical fiber reserved groove 40, the detection optical fiber reserved groove 50, and the detection flow channel 60 are on the same plane, the central axis of the excitation optical fiber 10, the detection optical fiber 20, and the detection flow channel 60 The center of the droplet in is located on the same plane, so that the light exciting the optical fiber 10 can be irradiated on the droplet in the largest area, and the detection fiber 20 can collect the optical signal of the droplet in the largest area, thereby improving the light collection efficiency To improve the sensitivity of detection.

Referring to FIG. 4, one end of the detection flow channel 60 has a mixing inlet 601, and the top chip 12 is provided with a first injection port 123, a regulating phase flow channel 124 connected to the first injection port 123, a second injection port 125, and a communication port The droplet flow path 126 of the second injection port 125, one end of the droplet flow path 126 and one end of the adjustment phase flow path 124 both meet at the mixing inlet 601, and the adjustment phase interval in the adjustment phase flow path 124 is formed at the mixing inlet 601 Between the droplets in the droplet flow channel 126 to adjust the spacing between the droplets. Specifically, the first injection port 123 is used to inject the adjustment phase into the adjustment phase flow channel 124 (for water-in-oil droplets, the adjustment phase is usually the oil phase), and the second injection port 125 is used to apply the droplet The droplet flow channel 126 is injected from it. Among them, the first injection port 123 and the second injection port 125 are through holes penetrating the top chip 12, and the droplet flow channel 123 and the adjustment phase flow channel 124 are provided on the surface of the top chip 12 facing the bottom chip 11. The droplets and the adjustment phase meet at the mixing inlet 601, and the adjustment phase interval is formed between the droplets, and the droplets are separated from each other at regular intervals, to avoid that the droplets are too close and cause the scattered light between the droplets to crosstalk and cannot Discrimination, which in turn improves the accuracy of the calculation results. Optionally, at the mixing inlet 601, the droplet flow path 126 and the adjustment phase flow path 124 are coaxial with the detection flow path 60.

Please continue to refer to FIG. 4. The top chip 12 is further provided with a third injection port 128 and a mirror accommodating channel 129 communicating with the third injection port 128. The mirror accommodating channel 129 is perpendicular to the detection flow channel 60, and the third injection port 128 is used for the injection of the material forming the mirror (such as a low melting point metal alloy), and the material forming the mirror is injected into the mirror from the third injection port 128 The channel 129 is received to form the reflector 30. Optionally, the mirror accommodating channel 129 is curved toward the port of the detection fiber 20 to form the mirror 30 with a curved surface. The final reflector is liquid or solid, preferably liquid.

The material of the reflector 30 is a low-melting metal alloy (for example, an alloy of indium, bismuth, and tin, or an alloy of gallium, indium, and tin), and the fluidity of the low-melting material is used to make the reflector 30 simple to manufacture and low in cost To avoid the problems of complicated production and high cost caused by the commonly used methods of magnetron sputtering coating and chemical redox deposition. The mirror accommodating channel 129 can be made by using micro-nano processing technology, which can realize the manufacture of the mirror 30 with different structures in the droplet microfluidic chip, and can realize the integration and complex design of tiny optical elements.

The above disclosure is only the preferred embodiments of the present invention, and of course it cannot be used to limit the scope of the present invention. Those of ordinary skill in the art can easily think of various equivalent modifications within the technical scope disclosed by the present invention Or replacement, these modifications or replacements should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

A droplet microfluidic chip for synchronous detection of multi-color fluorescence, which is characterized by comprising: a chip body, an excitation fiber, a detection fiber and a reflector, the chip body is provided with an excitation fiber reserve slot and a detection fiber reserve A groove and a detection flow channel, the detection flow channel includes a detection area, and the reflector is disposed near the detection area; the excitation fiber reserved slot is used to insert the excitation fiber, and the detection fiber reserved slot is used to Insert the detection optical fiber, one end of the excitation optical fiber and one end of the detection optical fiber converge in the detection area, and the other end of the excitation optical fiber branch into a plurality of branch fibers for connecting the scattered light source and at least one Excitation light source, the other end of the detection fiber is connected to the detection module; the droplets flowing through the detection area are illuminated by the light from the excitation fiber, and the optical signal generated by the droplets is reflected by the mirror and is reflected by the mirror Detection fiber collection, when the droplet is an empty droplet, the optical signal is a scattered light signal, and when the droplet is a droplet capable of generating fluorescence, the optical signal is a scattered light signal and at least two The superimposed signal of the fluorescent signal.
The droplet microfluidic chip according to claim 1, wherein the multi-color fluorescence is two kinds of fluorescence signals;

The detection module includes a collimator mirror and a dichroic mirror arranged in sequence, and the dichroic mirror is used to separate the two fluorescent signals; the detection module further includes a first one which is sequentially arranged in the dichroic mirror A first filter and a first detector on the light exit side, and a second filter and a second detector that are sequentially disposed on the second light exit side of the dichroic mirror; wherein the dichroic mirror also It is used to divide the scattered light signal to the first light emitting side or the second light emitting side.
The droplet microfluidic chip according to claim 1, wherein the droplet flowing through the detection area is located at the focal point of the mirror, the mirror is a curved mirror; the mirror and The central axis of the detection fiber is on the same horizontal plane, and the reflector and the detection fiber are both perpendicular to the detection channel.
The droplet microfluidic chip according to claim 1, wherein the chip body comprises a bottom chip and a top chip stacked on the bottom chip, the bottom chip is provided with a first notch and a second Notch, the top chip is provided with a first slot adapted to the first notch, a second slot adapted to the second notch, and the detection flow channel, the bottom chip and the top layer When the chip is docked, the first notch and the first groove form the excitation fiber reserved slot for accommodating the excitation fiber, and the second notch and the second slot form the Check the fiber reserved slot.
The droplet microfluidic chip according to claim 4, wherein the central axes of the excitation optical fiber reserved groove, the detection optical fiber reserved groove and the detection flow channel are on the same plane.
The droplet microfluidic chip according to claim 5, characterized in that the depth of the excitation optical fiber reserved groove and the detection optical fiber reserved groove are different.
The droplet microfluidic chip according to claim 4, wherein an inlet end of the first groove facing away from the detection area is larger than a port close to the detection area.
The droplet microfluidic chip according to claim 7, wherein the inlet end of the first groove facing away from the detection area has a single-sided dovetail shape, and the second groove faces away from the inlet end of the detecting area It has a bilateral dovetail shape.
The droplet microfluidic chip according to claim 4, wherein the detection flow channel includes a mixing inlet, and the top chip is provided with a first injection port and an adjustment phase connected to the first injection port A flow channel, a second injection port, and a droplet flow channel connected to the second injection port, the droplet flow channel and one end of the regulating phase flow channel all meet at the mixing inlet, and at the mixing inlet An adjustment phase interval in the adjustment phase flow channel is formed between the droplets in the droplet flow channel to adjust the spacing between the droplets.
The droplet microfluidic chip according to claim 4, wherein the top chip is further provided with a third injection port and a mirror accommodating channel communicating with the third injection port, wherein the The mirror accommodating channel is curved toward the port of the detection fiber.
The droplet microfluidic chip according to claim 1, wherein the angle between the excitation fiber and the detection fiber is 45° near the detection area.
The droplet microfluidic chip according to any one of claims 1-11, wherein the numerical aperture, inner diameter and outer diameter of the excitation fiber are 0.1, 62.5 microns and 125 microns, respectively.
The droplet microfluidic chip according to any one of claims 1 to 11, wherein the numerical aperture, inner diameter and outer diameter of the detection optical fiber are 0.38, 200 microns and 225 microns, respectively.
The droplet microfluidic chip according to any one of claims 1 to 11, wherein the material of the reflector includes a low-melting metal alloy.
The droplet microfluidic chip according to claim 14, wherein the material of the reflector is an alloy of indium, bismuth, and tin, or an alloy of gallium, indium, and tin.