CN111896516A - Microdroplet Dual Fluorescence Signal Detection Device - Google Patents

Abstract

The invention relates to a micro-droplet double fluorescence signal detection device, comprising: a light combining module, an objective lens, and a light processing module. The light processing module also includes a mounting housing configured with a light conducting channel including a single aperture corresponding to both the first photomultiplier tube and the second photomultiplier tube. The invention realizes that multiple detection parameters can be acquired in a single detection, and forms a single aperture confocal light processing structure. Save production costs.

Description

Microdroplet Dual Fluorescence Signal Detection Device

technical field

The invention relates to the technical field of microfluidic chips, in particular to a microdroplet double fluorescent signal detection device.

Background technique

Biochips have a wide range of applications in new drug development, disease diagnosis and gene expression analysis. Microfluidic chip technology is also becoming more and more mature, and has gradually become a hot spot of people's attention. There are various biological and chemical processes in the microfluidic detection chip, and the process is usually completed in the micron-scale flow channel space, which requires some devices that can detect the reaction process. At present, the detection methods can be divided into CCD scanning and laser confocal scanning. Compared with the laser confocal scanning system, the CCD scanning system has a simpler structure and faster detection speed, but the lateral resolution is low. It is necessary to increase the magnification of the imaging system, which will reduce the corresponding field of view (that is, the chip area for one measurement is small). , Since the block scanning actually makes the chip and the imaging system move relative to each other through mechanical motion, the mechanical positioning error will form the stitching error of the scanned image, so this method is not suitable for high-precision high-density biochip detection.

The biochip detection system constructed based on the principle of laser confocal scans the biochip point by point. Since the biochip is always in the focal plane, the spot size of the excitation light is very small and the lateral resolution is high. Due to the characteristics of high resolution and high sensitivity, laser confocal detection method can obtain clear digital fluorescence images and quantitative analysis results of fluorescently labeled antibodies on biochips, and can become a new detection method mainly used in high-density biochip scanning. However, when the two collection optical paths use different apertures, the excitation points of different lasers are not completely consistent, which may easily lead to inconsistent emission light efficiencies of the two paths, which in turn leads to different crosstalk correction parameters for each instrument. When two channels of PMT use the same small hole to shield stray light, the two channels of emission light efficiency increase or decrease proportionally to achieve the purpose of unifying the crosstalk correction parameters.

SUMMARY OF THE INVENTION

Therefore, the technical problem to be solved by the present invention is to provide a micro-droplet dual-fluorescence signal detection device, which can acquire the dual-fluorescence detection signals of micro-droplets through a light combining module and a light processing module, and form a single-hole confocal light In the processing architecture, the two detection optical paths use the same single small hole, so that the emission light efficiency of the two paths can be increased or decreased proportionally, saving production costs.

In order to solve the above problems, the present invention provides a microdroplet dual fluorescence signal detection device, comprising:

a light combining module for combining the first laser with the first wavelength and the second laser with the second wavelength into mixed excitation light;

The objective lens is located on the light conduction path of the mixed excitation light, and is used to confocal the mixed excitation light on the microdroplet to be detected, so as to excite and generate the first fluorescence corresponding to the first laser and corresponding to the the second fluorescence of the second laser;

The light processing module includes a first photomultiplier tube for acquiring first fluorescence, a second photomultiplier tube for acquiring second fluorescence, and a first dichroic for reflecting the mixed excitation light from the light combining module to the objective lens a mirror, and a second dichroic mirror that separates the first fluorescence from the second fluorescence;

The light processing module also includes a mounting housing configured with a light conducting channel including a single unit corresponding to both the first photomultiplier tube and the second photomultiplier tube. of small holes.

Preferably, the light processing module further comprises a convex lens between the first dichroic mirror and the second dichroic mirror, the convex lens is configured to focus the parallel light passing through the convex lens .

Preferably, the small hole is arranged between the convex lens and the second dichroic mirror, so that the light passing through the convex lens is focused on the small hole.

Preferably, the light processing module further includes a first color filter, the first color filter is located between the first photomultiplier tube and the second dichroic mirror, and/or the light The processing module further includes a second color filter between the second photomultiplier tube and the second dichroic mirror.

Preferably, the mounting housing includes a fixing piece, a first connecting piece, a second connecting piece, a third connecting piece, and a fourth connecting piece, and the fixing piece has a first light entrance and a first light exit, so The first light entrance is coaxial with the first light exit, and a small hole is arranged at the first light exit; the third connecting piece is connected to the first light exit of the fixing member, so the The third connector has a second light entrance, a second light exit, and a third light exit. The second light entrance is coaxial with the first light exit of the fixing member, and the second light exits. The exit is coaxial with the second light entrance, the third light exit is perpendicular to the second light entrance; the second connecting piece is connected to the first light entrance of the fixing piece, and the two are sandwiched therebetween. The convex lens is provided; the fourth connecting piece is connected to the second light exit of the third connecting piece, and the second dichroic mirror is sandwiched therebetween, and the fourth connecting piece is away from One end of the fixing member is provided with a first photomultiplier tube, and the first color filter is sandwiched between the fourth connecting member and the first photomultiplier tube along the light transmission path; the third connecting member A second photomultiplier tube is connected to the third light exit port, and the second color filter is sandwiched between the fourth connector and the second photomultiplier tube holder along the light transmission path; the first A connecting piece is connected to a side of the second connecting piece away from the fixing piece, and the first dichroic mirror is sandwiched therebetween.

Preferably, the connection between the first connection piece and the second connection piece, and/or the connection between the second connection piece and the fixing piece, and/or the first connection piece The connection between the third connection piece and the fourth connection piece, and/or the connection between the third connection piece and the fixing piece, and/or the connection between the first photomultiplier tube and the The connection between the fourth connection members, and/or the connection between the second photomultiplier tube and the third connection member is provided with a sealing damping member.

Preferably, the objective lens is movably connected to a side of the first connecting member away from the second connecting member.

Preferably, the light combining module includes a first laser, a second laser, a reflector and a third dichroic mirror, and the first laser emitted by the first laser is reflected by the reflector and is combined with the second laser. The second laser light emitted by the two lasers is combined in the third dichroic mirror to form the mixed excitation light.

Preferably, the light combining module further includes a fixing plate and a light combining cassette, the first laser, the second laser and the light combining cassette are fixedly connected to the fixing plate, the reflector and the third dichroic mirror It is arranged in the light-combining cassette, and the light-combining cassette has a mixed excitation light outlet.

Preferably, the first laser is a 532 nm laser; and/or the second laser is a 473 nm laser.

The invention provides a micro-droplet dual-fluorescence signal detection device, by combining a first laser and a second laser with different wavelengths to form the mixed excitation light, the micro-droplets to be detected are excited to form the first fluorescence and the second laser light. The second fluorescence is acquired by the first photomultiplier tube and the second photomultiplier tube, respectively, and then transmitted to the corresponding data processing equipment (such as a computer, etc.) for corresponding processing (such as counting), thereby realizing a single The detection can obtain multiple detection parameters, and form a single aperture confocal light processing structure. The two detection optical paths use a common aperture, so that the emission efficiency of the two paths can be increased or decreased proportionally, saving production costs.

Description of drawings

FIG. 1 is a schematic diagram of an exploded structure of a microdroplet dual fluorescence signal detection device according to an embodiment of the present invention;

2 is a schematic diagram of a light conduction path of a microdroplet dual fluorescence signal detection device according to an embodiment of the present invention;

FIG. 3 is a schematic diagram of an exploded structure of the optical processing module in FIG. 1;

FIG. 4 is a schematic diagram of an exploded structure of the light combining module in FIG. 1 .

The reference numbers are:

1. Light combining module; 11. First laser; 12. Second laser; 13. Fixing plate; 14. Light combining cassette; 141, Reflector; 142, Third dichroic mirror; processing module; 31, first photomultiplier tube; 32, second photomultiplier tube; 33, first dichroic mirror; 34, second dichroic mirror; 35, first color filter; 36, second filter Color chip; 371, fixing piece; 372, first connecting piece; 373, second connecting piece; 374, fourth connecting piece; 375, third connecting piece; 376, small hole; 38, convex lens; 391, shock-absorbing pad ; 392, sealing pad; 393, dense light pad; 100, microdroplets to be detected.

Detailed ways

Referring to FIG. 1 to FIG. 4 , according to an embodiment of the present invention, a microdroplet dual fluorescence signal detection device is provided, including: a light combining module 1 for combining a first laser with a first wavelength with a first laser with a second The second laser light of two wavelengths is synthesized into mixed excitation light; the objective lens 2 is located on the light conduction path of the mixed excitation light, and is used to confocal the mixed excitation light on the microdroplet 100 to be detected, so as to generate corresponding excitation light. For the first fluorescence of the first laser and the second fluorescence corresponding to the second laser; the light processing module 3 includes a first photomultiplier tube 31 for obtaining the first fluorescence, and a second photomultiplier for obtaining the second fluorescence The tube 32, the first dichroic mirror 33 for reflecting the mixed excitation light from the light combining module to the objective lens 2, and the second dichroic mirror 34 for separating the first fluorescence from the second fluorescence . In this technical solution, the mixed excitation light is formed by combining the first laser and the second laser with different wavelengths to excite the microdroplet 100 to be detected to form the first fluorescence and the second fluorescence, and pass the first and second fluorescence. A photomultiplier tube 31 and a second photomultiplier tube 32 are respectively acquired and then transmitted to the corresponding data processing equipment (such as a computer, etc.) for corresponding processing (such as counting), thereby realizing that a single detection can acquire multiple detection parameters , and a single aperture confocal light processing structure is formed. The two detection optical paths use a common aperture, so that the emission light efficiency of the two paths can be increased or decreased in equal proportions, saving production costs. It is worth mentioning that, since the detection device of the present application forms a single aperture confocal light processing structure, when the two detection optical paths use the same aperture to shield stray light, the emission light efficiencies of the two paths increase or decrease in equal proportions. To achieve the purpose of unifying the crosstalk correction parameters, at the same time, there are fewer structural parts, the debugging process is simpler, and the production efficiency is greatly improved.

Preferably, the light processing module 3 further includes a convex lens 38, the convex lens 38 is located between the first dichroic mirror 33 and the second dichroic mirror 34, so that the parallel light passing through it is focused to The small hole 376 makes the external interference light blocked by the small hole 376 to ensure the detection accuracy of the subsequent fluorescent signal.

Further, the light processing module 3 further includes a first color filter 35, the first color filter 35 is located between the first photomultiplier tube 31 and the second dichroic mirror 34, and/ Or, the light processing module 3 further includes a second color filter 36 , and the second color filter 36 is located between the second photomultiplier tube 32 and the second dichroic mirror 34 .

Specifically, the light processing module 3 further has an installation casing, the installation casing is configured with a light conduction channel, and the light conduction channel has corresponding to the first photomultiplier tube 31 and the second photomultiplier tube 32 aperture 376. More specifically, the mounting housing includes a fixing member 371 , a first connecting member 372 , a second connecting member 373 , a third connecting member 375 , and a fourth connecting member 374 , and the fixing member 371 has a first light entrance. , a first light exit, the first light entrance is coaxial with the first light exit, and a small hole 376 is arranged in the first light exit; the third connector 375 is connected to the At the first light exit of the fixing member 371 , the third connecting member 375 has a second light entrance, a second light exit, and a third light exit. A light exit port is coaxial, the second light exit port is coaxial with the second light entrance port, the third light exit port is perpendicular to the second light entrance port, and the second connecting member 373 is connected to the fixed The first light emitting port of the member 371, and the convex lens 38 is sandwiched therebetween; the fourth connecting member 374 is connected to the second light emitting port of the third connecting member 375, and the two are sandwiched therebetween The second dichroic mirror 34 is provided, the first photomultiplier tube 31 is provided at the end of the fourth connecting member 374 away from the fixing member 371 , and the fourth connecting member 374 is connected to the first photoelectric The first color filter 35 is sandwiched between the multiplier tubes 31 along the light transmission path; the third light outlet of the third connecting member 375 is connected with the second photomultiplier tube 32, and the fourth connecting member 374 is connected to the second photomultiplier tube 32. The second color filter 36 is sandwiched between the second photomultiplier tube fixing bases 32 along the light transmission path; one side, and the first dichroic mirror 33 is sandwiched between them. This technical solution provides a specific implementation of the light processing module 3, which is formed by piecing together relatively independent connectors and components. It can be understood that in order to ensure the light transmission channel Rationality, specifically, the design of the light conduction channel conforms to the light processing path shown in FIG. 2 of the present application, that is, although the first connector 372 , the second connector 373 , the Corresponding light conduction channels in the third connecting piece 375 and the fourth connecting piece 374 are specifically defined, but it is clear that the first connecting piece 372 , the second connecting piece 373 , the fixing piece 371 , and the third connecting piece 375. The fourth connector 374 is placed on the light conduction path in turn and meets the light conduction requirements shown in FIG. 2 . In order to ensure the light conduction requirements of FIG. 2 and the compactness of the overall structure of the light processing module 3 The first connecting piece 372 , the second connecting piece 373 , the third connecting piece 375 , and the fourth connecting piece 374 are all isosceles right triangles in shape, that is, they all have a 45° slope.

Further, in order to protect the optical lenses such as the convex lens 38 provided in the light processing module 3 from being damaged, and to ensure that it is not disturbed by external light, preferably, the first connecting member 372 is connected to the second connecting member 372. The connection between the connection pieces 373, and/or the connection between the second connection piece 373 and the fixing piece 371, and/or the third connection piece 375 and the fourth connection piece 374, and/or, the connection between the third connecting piece 375 and the fixing piece 371, and/or, the first photomultiplier tube 31 and the fourth connecting piece 374 A sealing damping member is provided at the connection between the second photomultiplier tube 32 and the third connecting member 375, and the sealing damping member may include, for example, a damping pad 391, one or more of the gasket 392 and the gasket 393.

The objective lens 2, as a component for focusing detection of the droplet 100 to be detected, is movably connected to the side of the first connecting member 372 away from the second connecting member 373, as a specific implementation manner. , it can use a 20x microscope objective lens. It can be understood that the movable connection here refers to that the objective lens 2 has the degree of freedom of rotation (around the Z axis) and translation (along the Z axis or the X axis). ) degrees of freedom to ensure that the inspector can flexibly adjust the focal point (focal plane) of the objective lens 2. It is further understandable that the aforementioned movement of the objective lens 2 can be controlled by a power module such as a screw motor.

As a specific implementation of the light combining module 1, preferably, the light combining module 1 includes a first laser 11, a second laser 12, a reflector 141 and a third dichroic mirror 142. The first laser light emitted by the laser 11 is reflected by the mirror 141 and then combined with the second laser light emitted by the second laser 12 at the third dichroic mirror 142 to form the mixed excitation light. For example, the first laser 11 is a 532 nm laser, which is a green laser at this time; and/or the second laser 12 is a 473 nm laser, which is a blue laser at this time. More specifically, the light combining module 1 further includes a fixing plate 13 and a light combining cassette 14 , and the first laser 11 , the second laser 12 and the light combining cassette 14 are fixedly connected to the fixing plate 13 . The reflecting mirror 141 and the third dichroic mirror 142 are disposed in the light-combining cassette 14 , and the light-combining cassette 14 has a mixed excitation light exit. At this point, it can be understood that the light combining module 1 is constructed as a whole, so that the structure of the microdroplet dual fluorescence signal detection device is further compact.

It can be easily understood by those skilled in the art that, on the premise of no conflict, the above advantageous manners can be freely combined and superimposed.

The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention shall be included in the protection scope of the present invention. Inside. The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the technical principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be It is regarded as the protection scope of the present invention.

Claims (10)

1. A microdroplet dual fluorescence signal detection device, characterized in that, the detection device comprises: a light combining module (1) for combining a first laser with a first wavelength and a second laser with a second wavelength into mixed excitation light; The objective lens (2) is located on the light conduction path of the mixed excitation light, and is used to confocal the mixed excitation light on the microdroplet (100) to be detected, so as to excite and generate the first laser light corresponding to the first laser light. a fluorescence and a second fluorescence corresponding to the second laser; A light processing module (3), comprising a first photomultiplier tube (31) for acquiring first fluorescence, a second photomultiplier tube (32) for acquiring second fluorescence, and used for reflecting the mixed excitation light from the light combining module to A first dichroic mirror (33) of the objective lens (2), and a second dichroic mirror (34) separating the first fluorescence from the second fluorescence, the light processing module (3) further comprises a mounting housing configured with a light conducting channel comprising a single unit corresponding to both the first photomultiplier tube (31) and the second photomultiplier tube (32) Small hole (376). 2. The detection device according to claim 1, characterized in that, the light processing module (3) further comprises a convex lens (38), and the convex lens (38) is located between the first dichroic mirror (33) and the Between the second dichroic mirrors (34), the convex lens (38) is configured to focus the parallel light passing through the convex lens. 3. The detection device according to claim 2, wherein the small hole (376) is provided between the convex lens (38) and the second dichroic mirror (34), The light of the convex lens (38) is focused on the small hole. 4. The detection device according to claim 2, characterized in that, the light processing module (3) further comprises a first color filter (35), and the first color filter (35) is located in the first color filter (35). between the photomultiplier tube (31) and the second dichroic mirror (34), and/or, the light processing module (3) further comprises a second color filter (36), the second color filter A sheet (36) is located between the second photomultiplier tube (32) and the second dichroic mirror (34). 5 . The detection device according to claim 4 , wherein the installation housing comprises a fixing member ( 371 ), a first connecting member ( 372 ), a second connecting member ( 373 ), and a third connecting member ( 375 ). 6 . ), a fourth connecting piece (374), the fixing piece (371) has a first light entrance and a first light exit, the first light entrance is coaxial with the first light exit, and is The first light exit port is provided with the small hole (376). It has a second light entrance, a second light exit and a third light exit, the second light entrance is coaxial with the first light exit of the fixing member (371), and the second light exit is The second light entrance is coaxial, the third light entrance is perpendicular to the second light entrance; the second connecting member (373) is connected to the first light entrance of the fixing member (371), and the two The convex lens (38) is sandwiched between them; the fourth connecting member (374) is connected to the second light exit of the third connecting member (375), and the first connecting member (374) is sandwiched therebetween. A dichroic mirror (34), a first photomultiplier tube (31) is provided at the end of the fourth connecting piece (374) facing away from the fixing piece (371), and the fourth connecting piece (374) is connected to the first photomultiplier tube (31). The first color filter (35) is sandwiched between the first photomultiplier tubes (31) along the light transmission path; the third light outlet of the third connector (375) is connected with a second photomultiplier tube (375). 32), and the second color filter (36) is sandwiched between the fourth connector (374) and the second photomultiplier tube holder (32) along the light transmission path; the first The connecting piece (372) is connected to the side of the second connecting piece (373) away from the fixing piece (371), and the first dichroic mirror (33) is sandwiched therebetween. 6. The detection device according to claim 5, characterized in that, the connection between the first connection member (372) and the second connection member (373), and/or the second connection The connection between the fixing member (373) and the fixing member (371), and/or the connection between the third connection member (375) and the fourth connection member (374), and/or , the connection between the third connecting piece (375) and the fixing piece (371), and/or, between the first photomultiplier tube (31) and the fourth connecting piece (374) and/or, a sealing damping member is provided at the connection between the second photomultiplier tube (32) and the third connecting member (375). 7 . The detection device according to claim 5 , wherein the objective lens ( 2 ) is movably connected to a side of the first connecting member ( 372 ) away from the second connecting member ( 373 ). 8 . 8 . The detection device according to claim 1 , wherein the light combining module ( 1 ) comprises a first laser ( 11 ), a second laser ( 12 ), and a reflector ( 141 ). 9 . and a third dichroic mirror (142), the first laser light emitted by the first laser (11) is reflected by the reflector (141) and the second laser light emitted by the second laser (12) The two lasers are combined into the mixed excitation light in the third dichroic mirror (142). 9. The detection device according to claim 8, characterized in that, the light combining module (1) further comprises a fixing plate (13), a light combining cassette (14), the first laser (11), the second laser (11), the second The laser (12) and the light-combining cassette (14) are fixedly connected to the fixing plate (13), and the reflector (141) and the third dichroic mirror (142) are arranged on the light-combining cassette (14) Among them, the light-combining cassette (14) has a mixed excitation light exit. 10. The detection device according to claim 8, wherein the first laser (11) is a 532nm laser; and/or the second laser (12) is a 473nm laser.

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