CN117554340A - Laser-induced fluorescence detection system and detection method - Google Patents

Laser-induced fluorescence detection system and detection method Download PDF

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Publication number
CN117554340A
CN117554340A CN202311329741.XA CN202311329741A CN117554340A CN 117554340 A CN117554340 A CN 117554340A CN 202311329741 A CN202311329741 A CN 202311329741A CN 117554340 A CN117554340 A CN 117554340A
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laser
dichroic mirror
light path
detection system
induced fluorescence
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王玮
王少峰
郭业昌
李沛玥
张盼
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Peking University
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Peking University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N2021/6463—Optics

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biomedical Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Dispersion Chemistry (AREA)
  • Hematology (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

The application relates to a laser-induced fluorescence detection system and a detection method in the technical field of analysis and detection, wherein the laser-induced fluorescence detection system comprises: a dichroic mirror formed with a laser incident light path, a laser reflecting light path, and a fluorescence receiving light path; the laser is arranged on a laser incident light path; the collecting objective lens is arranged on the laser reflection light path; the three-dimensional platform is arranged on the laser reflection light path, is positioned on one side of the collecting objective lens far away from the dichroic mirror, forms a microchip placing table on one side of the three-dimensional platform close to the collecting objective lens, and can adjust the space position of the microchip placing table; and the photoelectric detection module is arranged on the fluorescence receiving light path. The laser-induced fluorescence detection system can detect low-concentration proteins and other biochemical small molecules, has high sensitivity and high response speed, and is miniaturized so that the occupied space is small.

Description

Laser-induced fluorescence detection system and detection method
Technical Field
The application relates to the technical field of analysis and detection, in particular to a laser-induced fluorescence detection system and a detection method.
Background
The microfluidic chip technology integrates basic operation units of sample preparation, reaction, separation, detection and the like in biological, chemical and medical analysis processes onto a micron-scale chip, and automatically completes the whole analysis process. Since the advent of microfluidic chips, detector research has been a focus of attention; various biological and chemical processes in microfluidic chips are usually performed within a micron-scale channel geometry, but in actual biological sample detection, the content of many target components is very low (< nmol/L level), and the detection process is disturbed by other components, resulting in very low concentration components that are difficult to detect well.
Disclosure of Invention
In view of the problems existing in the background art, the application provides a laser-induced fluorescence detection system and a detection method, wherein the laser-induced fluorescence detection system can detect low-concentration proteins and other biochemical small molecules, and has high sensitivity and quick response speed.
According to one aspect of the present invention, there is provided a laser-induced fluorescence detection system comprising: a first dichroic mirror formed with a laser incident light path, a laser reflecting light path, and a fluorescence receiving light path; the laser is arranged on the laser incident light path; the collecting objective lens is arranged on the laser reflection light path; the three-dimensional platform is arranged on the laser reflection light path, is positioned on one side of the collecting objective lens far away from the first dichroic mirror, forms a microchip placing table on one side of the three-dimensional platform close to the collecting objective lens, and can adjust the space position of the microchip placing table; and the photoelectric detection module is arranged on the fluorescence receiving light path.
When the laser-induced fluorescence detection system in the technical scheme is used, a biological sample to be detected is firstly placed in a micro-channel of a micro-fluidic chip, the micro-fluidic chip is fixed on a micro-chip placing table, the space position of the micro-fluidic chip is adjusted by using a three-dimensional platform so as to move a detection point to a target detection position, then a laser is started, laser emitted by the laser irradiates a first dichroic mirror on a laser incidence light path, the laser is reflected to a collection objective lens by utilizing the high reflectivity of the first dichroic mirror to the laser with the wavelength lower than the cut-off, the laser is enabled to irradiate the detection point after passing through the collection objective lens on a laser reflection light path, the component in the biological sample generates fluorescence under the excitation action of the laser, then the fluorescence is collected by the collection objective lens, the fluorescence sequentially passes through the collection objective lens and the first dichroic mirror, the fluorescence irradiates a photoelectric detection module on a fluorescence receiving light path, the photoelectric detection module converts weak light signals into electric signals, and the concentration of the component and the component in the biological sample can be judged according to the electric signals, the detection of low-concentration proteins and other small molecules is realized, and the detection speed of the biological sample is very high in sensitivity and high in response speed is achieved.
In some embodiments of the invention, the laser-induced fluorescence detection system further comprises: and the ocular is arranged on the fluorescence receiving light path.
In some embodiments of the invention, the microchip rest is a transparent plate, and an illumination light source is arranged on one side of the transparent plate, which is close to the three-dimensional platform.
In some embodiments of the invention, the three-dimensional platform comprises: the X-axis sliding rail is connected with an X-axis sliding plate in a sliding manner; the Y-axis sliding rail is arranged on the X-axis sliding plate and is connected with the Y-axis sliding plate in a sliding manner; the stand, the stand is installed on the Y axle slide, stand one side is formed with the Z axle slide rail, Z axle slide rail sliding connection has the Z axle slide, the transparent plate with the illumination light source install in the Z axle slide is kept away from the one end of Y axle slide.
In some embodiments of the present invention, the photodetection module includes a short-pass filter, a first lens, a pinhole, a second lens, a focusing lens, and a photomultiplier tube sequentially disposed from a side closer to the first dichroic mirror to a side farther from the first dichroic mirror.
In some embodiments of the present invention, the photodetection module further includes a second dichroic mirror disposed on a side of the first dichroic mirror near the short-pass filter, and the fluorescence receiving light path is bent at 90 ° through the second dichroic mirror.
In some embodiments of the invention, the laser light incident optical path is at an angle of 45 ° to the first dichroic mirror.
In some embodiments of the invention, the laser is a semiconductor laser;
preferably, the semiconductor laser is a 488nm blue-violet semiconductor laser.
In some embodiments of the invention, a filter is disposed between the laser and the first dichroic mirror.
According to another aspect of the present invention, there is provided a detection method for detecting a component in a biological sample using the laser-induced fluorescence detection system described above, the detection method comprising: placing a biological sample to be detected in a micro-channel of a micro-fluidic chip, wherein a detection point is formed on the micro-channel, fixing the micro-fluidic chip on a micro-chip placing table, and adjusting the space position of the micro-fluidic chip by using a three-dimensional platform so as to move the detection point to a target detection position; the laser emitted by the laser is reflected by the first dichroic mirror and irradiates the detection point through the collecting objective to generate fluorescence, the fluorescence sequentially passes through the collecting objective and the first dichroic mirror and irradiates the photoelectric detection module, and the photoelectric detection module converts weak light signals into electric signals; and judging the concentration of the components in the biological sample according to the electric signals.
Drawings
Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the application. Also, like reference numerals are used to designate like parts throughout the figures. In the drawings:
FIG. 1 is a schematic diagram of the overall structure of a laser-induced fluorescence detection system according to an embodiment of the present application;
FIG. 2 is a schematic diagram of a structure embodying a photodetection module;
fig. 3 is a schematic diagram of the overall principle of the laser-induced fluorescence detection system according to the embodiment of the present application.
The reference numerals in the drawings are as follows:
1. a first dichroic mirror; 2. a laser; 3. a collection objective; 4. a three-dimensional platform; 5. a photoelectric detection module; 51. a short-pass filter; 52. a first lens; 53. a pinhole; 54. a second lens; 55. a focusing lens; 56. a photomultiplier tube; 57. a second dichroic mirror; 58. a total reflection mirror; 59. a total reflection mirror; 6. a microfluidic chip; 7. a light filter; 8. PMT drive circuit; 9. a printed circuit board; 10. a signal display table; 11. an eyepiece; 12. microchip placing table.
Detailed Description
It should be understood that the described embodiments are merely some, but not all, of the embodiments of the present application. All other embodiments, based on the embodiments herein, which would be apparent to one of ordinary skill in the art without making any inventive effort, are intended to be within the scope of the present application.
When the following description refers to the accompanying drawings, the same numbers in different drawings refer to the same or similar elements, unless otherwise indicated. The implementations described in the following exemplary examples are not representative of all implementations consistent with the present application. Rather, they are merely examples of apparatus and methods consistent with some aspects of the present application as detailed in the accompanying claims.
In the description of the present application, it should be understood that the terms "first," "second," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance. The specific meaning of the terms in this application will be understood by those of ordinary skill in the art in a specific context. Furthermore, in the description of the present application, unless otherwise indicated, "a plurality" means two or more. "and/or", describes an association relationship of an association object, and indicates that there may be three relationships, for example, a and/or B, and may indicate: a exists alone, A and B exist together, and B exists alone. The character "/" generally indicates that the context-dependent object is an "or" relationship.
Since the advent of microfluidic chips, detector research has been a focus of attention; various biological and chemical processes in the microfluidic chip are usually completed in a micro-scale channel geometry, so that the detector is required to have the characteristics of high sensitivity, high response speed, microminiaturization and the like.
The fluorescence detection method mainly utilizes the fluorescence generated by the detected substance when the ultraviolet light irradiates, and achieves the detection purpose by testing fluorescence response, so that the method can effectively eliminate the interference of other matrixes and has good response stability; the fluorescence detector has the characteristics of high sensitivity and good selectivity, and is widely applied to high performance liquid chromatography.
The fluorescence detector used at present depends on imported general fluorescence detection equipment, but the equipment usually consists of two groups of monochromators for exciting light and emitting light, a plurality of gratings, slits and lenses, and has the advantages of complex structure, high cost and larger occupied instrument space; the excitation light source is a key component of the fluorescence detection device, and the traditional excitation light source (such as a xenon lamp) has the problems of large volume, high energy consumption, short service life and the like.
Accordingly, embodiments of the present application disclose a laser-induced fluorescence detection system. As shown in fig. 1 to 3, the laser-induced fluorescence detection system includes a first dichroic mirror 1, a laser 2, a collection objective 3, a three-dimensional platform 4, and a photodetection module 5; wherein the first dichroic mirror 1 is formed with a laser light incident light path, a laser light reflecting light path, and a fluorescence receiving light path.
The laser 2 is arranged on a laser incidence light path, and the collecting objective 3 is arranged on a laser reflection light path; the laser light emitted from the laser 2 first irradiates the first dichroic mirror 1 on the laser light incident path, then reflects the laser light to the collection objective lens 3 by utilizing the high reflectivity of the first dichroic mirror 1 to the laser light lower than the cut-off wavelength, and makes the laser light pass through the collection objective lens 3 on the laser light reflected path by utilizing the condensing action of the collection objective lens 3.
The three-dimensional platform 4 is arranged on a laser reflection light path, the three-dimensional platform 4 is arranged on one side of the collecting objective 3, which is far away from the first dichroic mirror 1, a microchip placing table 12 is formed on one side of the three-dimensional platform 4, which is close to the collecting objective 3, the microchip placing table 12 is used for placing the microfluidic chip 6, the microfluidic chip 6 is detachably fixed on the microchip placing table 12, the microfluidic chip 6 is provided with a micro-channel, a detection point is formed on the micro-channel, the three-dimensional platform 4 can adjust the spatial position of the microchip placing table 12, namely, the three-dimensional platform 4 can adjust the three-dimensional spatial position of the detection point on the micro-channel of the microfluidic chip 6.
When detecting components in a biological sample, a known antibody is marked with fluorescein to prepare a fluorescent antibody, the fluorescent antibody can be excited to emit fluorescence, the fluorescent antibody is used as a probe to detect the corresponding components in the biological sample, the biological sample to be detected is placed in a micro-channel of a micro-fluidic chip 6, the micro-fluidic chip 6 is fixed on a micro-chip placing table 12, and then a three-dimensional platform 4 is used for adjusting the space position of the micro-fluidic chip 6 so as to move a detection point to a target detection position, so that laser irradiates the detection point after passing through a collecting objective lens 3 on a laser reflection light path.
The photoelectric detection module 5 is arranged on the fluorescence receiving light path; after the laser irradiates the detection point, the components in the biological sample generate fluorescence under the excitation action of the laser, then the fluorescence is collected by the same collecting objective 3, namely, after the fluorescence sequentially passes through the collecting objective 3 and the first dichroic mirror 1 (the high permeability of the fluorescence higher than the cut-off wavelength is utilized by the first dichroic mirror 1), the fluorescence irradiates the photoelectric detection module 5 in a fluorescence receiving light path, and then the photoelectric detection module 5 converts weak light signals into electric signals, and then the concentrations of the components in the biological sample and the components can be judged according to the electric signals, so that the detection of proteins and other biochemical small molecules with low concentration can be realized, the sensitivity and the response speed are very high, and the detection sensitivity can reach 10nM (nmol).
It can be understood that the laser has the characteristics of high energy, good directivity, easy focusing and the like, and can be used as an excitation light source in the microfluidic chip 6 to improve the detection sensitivity; the laser-induced fluorescence detection system only responds to molecules generating fluorescence, and can effectively eliminate interference of a matrix, so that detection of trace compounds is realized; for a certain compound, the fluorescence spectrum is fixed under a given condition, so that a basis is provided for sample characterization; when the given fluorescent substance concentration is low, the fluorescent intensity is in direct proportion to the fluorescent substance concentration, so that a basis is provided for quantitative detection, and the detection of the sample with low sample concentration and large matrix interference is realized under the capability of high-sensitivity and high-selectivity detection.
Further, the laser 2 may employ a semiconductor laser 2; preferably, the semiconductor laser 2 is a 488nm blue-violet semiconductor laser 2 (vincrist industry photoelectric), and the output power is 25mW; the laser 2 has the characteristics of miniaturization, high efficiency and low cost as an excitation light source, and the laser 2, the first dichroic mirror 1, the collecting objective 3 and the photoelectric detection module 5 can be arranged on a metal bracket to form a compact arrangement and fixed relative position, so that the laser-induced fluorescence detection system is miniaturized and space occupation is reduced, and the overall structure is simple and the cost is low.
In some embodiments of the present invention, as shown in fig. 2, the angle between the incident light path of the laser light and the first dichroic mirror 1 is 45 °; the laser light emitted from the laser 2 horizontally irradiates the first dichroic mirror 1, is reflected in a vertical direction by the first dichroic mirror 1, and finally is converged on a detection point of a micro channel of the micro fluidic chip 6.
In some embodiments of the present invention, as shown in fig. 2 and 3, a filter 7 is disposed between the laser 2 and the first dichroic mirror 1, and the filter 7 filters the laser light emitted from the laser 2, and selects the laser light with a desired radiation band, so as to further improve the analysis sensitivity.
It should be noted that, since the laser light emitted by the laser 2 has excellent monochromaticity and high excitation energy, so that stray light other than the required wavelength is less, the filter 7 between the laser 2 and the first dichroic mirror 1 can be removed, and the cost is reduced while ensuring the detection effect.
In some embodiments of the present invention, as shown in fig. 2, the photodetection module 5 includes a short-pass filter 51, a first lens 52, a pinhole 53, a second lens 54, a focusing lens 55, and a photomultiplier tube 56; the short-pass filter 51, the first lens 52, the pinhole 53, the second lens 54, the focusing lens 55, and the photomultiplier 56 are disposed in this order from the side close to the first dichroic mirror 1 to the side away from the first dichroic mirror 1.
The components in the biological sample generate fluorescence under the excitation action of laser, firstly, the fluorescence sequentially passes through the collecting objective lens 3 and the first dichroic mirror 1, then the fluorescence continuously passes through the short-pass filter 51, the first lens 52, the pinhole 53, the second lens 54 and the focusing lens 55 and irradiates the photomultiplier 56, the fluorescence is passed through the short-pass filter 51 by the light of a specific wave band (corresponding to the components to be detected), the light outside the pass band is cut off, the photomultiplier 56 is used as a detector, and weak light signals are converted into electric signals, so that the judgment of the components and the concentration of the components in the biological sample according to the electric signals is realized.
Further, the first lens 52 and the second lens 54 are identical bandpass filters, OD6FL508.5-10, FL508.5-10 from Thorlabs Inc. is 500 to 590nm in wavelength, 508.5nm in Center Wavelength (CWL) and 10nm in bandwidth (FWHM). The short-pass filter 51 is a <510nm short-pass filter that allows light having a wavelength less than the set wavelength 510nm to pass therethrough, and cuts off light having a wavelength greater than the set wavelength. The first dichroic mirror 1 is >495 long pass, i.e. light of a wavelength greater than 495nm can pass, less than 495nm reflected. The filter 7 is a band-pass filter, is an OD4 filter, has a central wavelength of 488.00 +/-2.0 nm and has a bandwidth of 10nm. Only 483.00-493nm wavelength is allowed to pass. Further, the photodetection module 5 further includes a second dichroic mirror 57, the second dichroic mirror 57 is disposed on a side of the first dichroic mirror 1 close to the short-pass filter 51, and the fluorescence receiving optical path is bent to 90 ° by the second dichroic mirror 57, that is, after the fluorescence passes through the first dichroic mirror 1, the second dichroic mirror 57 irradiates the second dichroic mirror 57 first, and then the second dichroic mirror 57 reflects the fluorescence to the short-pass filter 51, so as to improve the design flexibility of the laser induced fluorescence detection system, and make the structure compact, for example, avoid the occupation of large space and inconvenient use caused by the overall length of the laser induced fluorescence detection system being too long; the photo detection module 5 further comprises a total reflection mirror 59, the total reflection mirror 59 being arranged between the first dichroic mirror 1 and the collecting objective 3 for reflecting light from the first dichroic mirror 1 to the collecting objective 3 or for reflecting light from the collecting objective 3 to the first dichroic mirror 1.
Specifically, the second dichroic mirror 57 is short-pass for <512nm, i.e., light having a wavelength less than 512nm will travel straight through the second dichroic mirror 57, and light greater than 512nm will be reflected, leading to the short-pass filter 51.
The band of laser light is filtered to a central wavelength 488.00 + -2.0 nm by a filter 7 and the laser light with a bandwidth of 10nm is then passed through a first dichroic mirror 1, and is reflected by the first dichroic mirror 1 instead of passing through it because the laser light wavelength is now less than 495nm, and then excited to be irradiated on the sample through a collecting objective 3.
Alexa Fluor 488 fluorescein in the sample has an excitation wavelength of 488nm and a maximum emission wavelength of 519nm. The emitted fluorescence also contains background noise such as stray light and reflected excitation light, and therefore, filtering is required.
Fluorescence and noise are collected through the collecting objective lens 3, pass through the first dichroic mirror 1, and pass through the second dichroic mirror 57 instead of reflection because the wavelength is larger than 495nm, then pass through the short-pass filter 51, <510nm short-pass, the fluorescence at this time is limited to 495-510nm wave band, then pass through the first lens 52 and the second lens 54, the fluorescence passes through the light with the central wavelength of 508.5nm and the bandwidth of 10nm, and is further filtered to 503.5-510nm, and then enter the photomultiplier for detection; the pinhole 53 also functions to filter stray light.
It should be noted that the PMT driving circuit 8 of the photomultiplier tube 56 may be amplified and converted into a voltage signal by a circuit suggested by the photomultiplier tube 56 supplier; the direct current module can be used for supplying power to the operational amplifier; all components are arranged on one 38 x 78mm2 area of the printed circuit board 9 (PCB), which printed circuit board 9 can also accommodate signal and power connectors. The driving circuit of the laser 2 is also located on the printed circuit board 9.
In addition, the data acquisition of converting the weak light signals into the electric signals can be realized by a commercial multifunctional data acquisition card, and a USB hub is arranged in the system and used for accommodating a control circuit and the data acquisition card at the same time; further, the photomultiplier tube 56 may be directly connected to a signal display meter 10 (voltmeter), which displays the output signal value of the photomultiplier tube 56 for conversion into a concentration value.
In some embodiments of the present invention, as shown in fig. 1 and 3, the laser-induced fluorescence detection system further includes an eyepiece 11, where the eyepiece 11 is disposed on a fluorescence receiving optical path, specifically, the eyepiece 11 is located on a light-transmitting side of the second dichroic mirror 57, and specifically, reflects the light to the eyepiece 11 through a total reflection mirror 58; the microchip placing table 12 is a transparent plate, and an illumination light source is arranged on one side of the transparent plate, which is close to the three-dimensional platform 4; preferably, the illumination light source is a red LED, the LED is turned off after the alignment of the red LED is used, then signal acquisition is carried out, and the LED is turned on only when the alignment is carried out.
After the microfluidic chip 6 is fixed on the microchip placing table 12, an illumination light source can be turned on, the micro-channel of the microfluidic chip 6 is amplified by utilizing the illumination light source to form an image in bright field, the detection point of the micro-channel is found by matching an objective lens with an eyepiece 11, and meanwhile, the three-dimensional platform 4 is used for adjusting the space position of the microfluidic chip 6 until the detection point is aligned with the focusing point of laser, namely, the detection point moves to a target detection position, so that the system is compatible with the microfluidic chip 6 with different sizes and the microfluidic chip 6 designed by the micro-channel, more importantly, the necessity of filling fluorescent dye when the detection point is aligned is avoided, and the operation is greatly simplified.
In bright field imaging, a manual light barrier is arranged between the photomultiplier 56 and the focusing lens 55, and the acquisition signal is only turned on when the fluorescent signal is acquired, and is turned off at ordinary times.
In some embodiments of the present invention, three-dimensional platform 4 includes X-axis slide rails, Y-axis slide rails, and posts; wherein, the X-axis sliding rail is connected with an X-axis sliding plate in a sliding way; the Y-axis sliding rail is arranged on the X-axis sliding plate and is connected with the Y-axis sliding plate in a sliding way; the stand is installed on the Y-axis slide, and stand one side is formed with the Z-axis slide rail, and Z-axis slide rail sliding connection has the Z-axis slide, and transparent plate and illumination light source are installed in the one end that the Y-axis slide was kept away from to the Z-axis slide.
Through the movement of the X-axis sliding plate on the X-axis sliding rail and the Y-axis sliding plate on the Y-axis sliding rail, the position adjustment of the transparent plate and the illumination light source in the horizontal direction is realized, and through the movement of the Z-axis sliding plate on the Z-axis sliding rail, the position adjustment of the transparent plate and the illumination light source in the vertical direction is realized, and the spatial position of the microfluidic chip 6 is adjusted. The X-axis, Y-axis and Z-axis moving structures of the three-dimensional stage 4 are preferably precise translation stages, and the motion precision of each axis can reach 1nm.
Firstly, a chip to be detected is placed on a three-dimensional platform surface, and an illumination red light LED is turned on, and the chip is made of PDMS, so that the chip is transparent. Then the laser is turned on, under the darkroom condition, the chip is manually moved, so that light spots of blue laser irradiated on the chip are approximately close to the region to be detected, eyes observe the region through an eyepiece, XYZ axis movement of the three-dimensional platform is regulated, focus is aligned, and then the XY axis movement enables the laser light spots to move into the micro-channel. In the process, the picture observed from the ocular is red, the shape outline of the micro-channel and the laser light spot can be clearly seen, and the light spot is a blue light spot with the diameter of 20 um.
When the quick detection work is performed, the micro-fluidic chip 6 is detachably fixed on the micro-chip placing table 12, for example, the micro-fluidic chip 6 is fixed in a detachable manner by using a clamp, a tabletting clamp and the like, on one hand, the replacement work of the micro-fluidic chip 6 can be quickly completed after the detection of the last micro-fluidic chip 6 is completed, and on the other hand, the detection point of the micro-fluidic chip 6 is accurately adjusted to the optimal detection position by using the precise translation table, so that the analysis speed and the working efficiency of the micro-fluidic chip 6 are obviously improved.
Further, the housing of the three-dimensional platform 4, the housing of the laser 2, the first dichroic mirror 1, the collection objective 3 and the photo detection module 5 may be manufactured by 3D printing; one side of the microfluidic chip 6 is a probing point for manually placing the microfluidic chip 6 onto the microchip placing table 12.
The embodiment also provides a detection method, which uses the laser-induced fluorescence detection system to detect components in a biological sample, and comprises the following steps: placing a biological sample to be detected in a micro-channel of a micro-fluidic chip 6, wherein a detection point is formed on the micro-channel, fixing the micro-fluidic chip 6 on a micro-chip placing table 12, and adjusting the spatial position of the micro-fluidic chip 6 by using a three-dimensional platform 4 so as to move the detection point to a target detection position; the laser emitted by the laser 2 is reflected by the first dichroic mirror 1 and irradiates a detection point through the collecting objective 3 to generate fluorescence, the fluorescence sequentially passes through the collecting objective 3 and the first dichroic mirror 1 and irradiates the photoelectric detection module 5, and the photoelectric detection module 5 converts weak light signals into electric signals; and judging the concentration of the components in the biological sample according to the electric signals. For specific reference, the foregoing is omitted herein.
The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention. Therefore, the protection scope of the invention is subject to the protection scope of the claims.

Claims (10)

1. A laser-induced fluorescence detection system, comprising:
a first dichroic mirror formed with a laser incident light path, a laser reflecting light path, and a fluorescence receiving light path;
the laser is arranged on the laser incident light path;
the collecting objective lens is arranged on the laser reflection light path;
the three-dimensional platform is arranged on the laser reflection light path, is positioned on one side of the collecting objective lens far away from the first dichroic mirror, forms a microchip placing table on one side of the three-dimensional platform close to the collecting objective lens, and can adjust the space position of the microchip placing table;
and the photoelectric detection module is arranged on the fluorescence receiving light path.
2. The laser-induced fluorescence detection system of claim 1, further comprising:
and the ocular is arranged on the fluorescence receiving light path.
3. The laser-induced fluorescence detection system of claim 2, wherein the microchip placement stage is a transparent plate with an illumination source disposed on a side of the transparent plate adjacent to the three-dimensional platform.
4. The laser-induced fluorescence detection system of claim 3, wherein the three-dimensional platform comprises:
the X-axis sliding rail is connected with an X-axis sliding plate in a sliding manner;
the Y-axis sliding rail is arranged on the X-axis sliding plate and is connected with the Y-axis sliding plate in a sliding manner;
the stand, the stand is installed on the Y axle slide, stand one side is formed with the Z axle slide rail, Z axle slide rail sliding connection has the Z axle slide, the transparent plate with the illumination light source install in the Z axle slide is kept away from the one end of Y axle slide.
5. The laser-induced fluorescence detection system of claim 1, wherein the photodetection module comprises a short-pass filter, a first lens, a pinhole, a second lens, a focusing lens, and a photomultiplier tube sequentially disposed from a side closer to the first dichroic mirror to a side farther from the first dichroic mirror.
6. The laser induced fluorescence detection system of claim 5, wherein the photo detection module further comprises a second dichroic mirror, the second dichroic mirror is disposed on a side of the first dichroic mirror near the short-pass filter, and the fluorescence receiving light path is bent at 90 ° through the second dichroic mirror.
7. The laser-induced fluorescence detection system of claim 1, wherein the laser light incident optical path is at an angle of 45 ° to the first dichroic mirror.
8. The laser-induced fluorescence detection system of claim 1, wherein the laser is a semiconductor laser.
9. The laser-induced fluorescence detection system of claim 1, wherein a filter is disposed between the laser and the first dichroic mirror.
10. A method of detecting a component in a biological sample using the laser-induced fluorescence detection system of any one of claims 1-9, the method comprising:
placing a biological sample to be detected in a micro-channel of a micro-fluidic chip, wherein a detection point is formed on the micro-channel, fixing the micro-fluidic chip on a micro-chip placing table, and adjusting the space position of the micro-fluidic chip by using a three-dimensional platform so as to move the detection point to a target detection position;
the laser emitted by the laser is reflected by the first dichroic mirror and irradiates the detection point through the collecting objective to generate fluorescence, the fluorescence sequentially passes through the collecting objective and the first dichroic mirror and irradiates the photoelectric detection module, and the photoelectric detection module converts weak light signals into electric signals;
and judging the concentration of the components in the biological sample according to the electric signals.
CN202311329741.XA 2023-10-13 2023-10-13 Laser-induced fluorescence detection system and detection method Pending CN117554340A (en)

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CN202311329741.XA CN117554340A (en) 2023-10-13 2023-10-13 Laser-induced fluorescence detection system and detection method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202311329741.XA CN117554340A (en) 2023-10-13 2023-10-13 Laser-induced fluorescence detection system and detection method

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Publication Number Publication Date
CN117554340A true CN117554340A (en) 2024-02-13

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