CN112649411A - Multi-wave long-time resolution fluorescence measuring device - Google Patents
Multi-wave long-time resolution fluorescence measuring device Download PDFInfo
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- CN112649411A CN112649411A CN202011566752.6A CN202011566752A CN112649411A CN 112649411 A CN112649411 A CN 112649411A CN 202011566752 A CN202011566752 A CN 202011566752A CN 112649411 A CN112649411 A CN 112649411A
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- G—PHYSICS
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- 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
- G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
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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
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
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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
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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
- G01N2021/6417—Spectrofluorimetric devices
- G01N2021/6421—Measuring at two or more wavelengths
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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- Pathology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention discloses a multi-wavelength time-resolved fluorescence measuring device, which comprises five parts, namely an optical path inlet component, a measuring darkroom base component, a measuring darkroom upper seat plate component, a sample mirror component and a photon counting component, wherein the optical path inlet component is connected with the measuring darkroom base component through screws and is installed together; the sample mirror assembly is connected with the measurement darkroom base assembly through screws and is arranged together, the measurement darkroom is internally provided with the optical filter switching device, the optical filter switching device is driven by a rotating motor and driven by a synchronous belt to rotate, the optical filters with different wavelengths are switched, and then the photon counting assembly arranged above the upper seat plate assembly of the measurement darkroom collects two different optical signals so as to detect the concentration of the antigen. The device has compact structure, good light shading performance and accurate and reliable detection result.
Description
Technical Field
The invention belongs to the technical field of in-vitro diagnosis medical instruments, and particularly relates to a multi-wavelength time-resolved fluorescence measuring device.
Background
For more than half a century, the mainstream immunoassay technology is radio immunoassay, enzyme-linked immunoassay, chemiluminescence immunoassay and the like, wherein the radio immunoassay is used in a small amount at present due to the problem of radioactivity, the enzyme-linked immunoassay is gradually replaced due to long detection time, and the chemiluminescence immunoassay is the mainstream immunoassay technology at present. In the field of instant detection such as emergency treatment and the like, chemiluminescence is difficult to widely apply due to complex technology and high cost, and the demands promote instant detection type immunoassay technologies such as colloidal gold immunoassay, fluorescence chromatography immunoassay, colloidal turbidimetric immunoassay and the like. The chemiluminescence immunoassay technology is born in the seventies of the last century, the main technology adopted is magnetic particle chemiluminescence, the core hypothesis is that the separation of specific immune complexes and biological matrixes can be realized certainly through magnetic separation, but in clinical practical application, due to the diversity of clinical specimens, cases of magnetic separation failure cannot be avoided, such as 'jump value' phenomenon caused by nonspecific adsorption due to fibrin adhesion, false positive caused by heterophilic antibodies and the like. Half of the failures are due to magnetic cleaning failures for the instrument itself. Therefore, the cleaning-free immunoassay technology is a research hotspot for nearly two-thirty years, siemens, beckmann and other companies have related technology reserves, a series of high-flux immunoassay systems are provided by Beijing Koimei corporation of domestic enterprises based on the Siemens patent light-activated chemiluminescence technology, and the clinical application of cleaning-free detection is realized for the first time.
The time-resolved fluorescence resonance energy transfer immunoassay (TR-FRET) technology was originally developed by a supramolecular rare earth cryptand ether probe discovered by German Bolames (brahms) company based on Jean-Marie Lehn, Nobel chemical prize-winning France, and overcomes the influence of abnormal alignment accuracy of samples such as hemolysis, hyperlipidemia, jaundice and the like in homogeneous detection. Compared with other immunoassay technologies, the technology has many advantages, does not need a solid phase carrier and a solid phase probe, and has the advantages of good precision and accuracy, simple operation, easy automation and miniaturization and the like.
The core of the time-resolved fluorescence resonance energy transfer method is that two fluorescence molecules are called donor and acceptor respectively, wherein the donor can absorb exciting light and emit fluorescence, and the fluorescence can also be transmitted to the acceptor in a fluorescence resonance energy transfer mode and emitted by the acceptor. Specifically, a chelate label of a rare earth element having a cryptic structure is used as a fluorescence donor, and a short-lived fluorescent molecule having a good spectral overlap with the fluorescence donor is used as a fluorescence acceptor, and Fluorescence Resonance Energy Transfer (FRET) occurs between the donor and the acceptor (second fluorescent label) of the cryptic compound of a rare earth element. In fluorescence resonance energy transfer, the lifetime of the acceptor emitted fluorescence approaches that of the donor. Because the donor fluorescence decay period is long, the donor induces the acceptor to emit fluorescence for a long time, and the fluorescence generated after the acceptor is excited can last for a long time, so that the self-scattered fluorescence with short lifetime can be distinguished through time resolution, and the FRET signal can be easily distinguished from the background of the fluorescence with short lifetime.
The fluorescent donor and acceptor can be covalently linked to different partner molecules, e.g., protein dimers, complementary strands of DNA, antigens and antibodies, ligands and acceptors, and the like. Conventional FRET fluorescent compounds are susceptible to interference from background fluorescence of the sample (serum, plasma, buffers, proteins, chemicals and cell lysates). This background fluorescence has a very short lifetime (on the order of 10-9 seconds) and is easily removed by time-resolved methods. The time-resolved fluorescence resonance energy transfer (TR-FRET) technique combines the FRET technique and the time-resolved fluorescence measurement, and removes the extremely short-lived background fluorescence. After transient photoexcitation, the non-specific short-lived emission drops to zero after a delay of 50-150 microseconds. Whereas TR-FRET fluorophores emit long-lived fluorescence that participates in the FRET process. Thus, long-lived acceptor emission represents the energy transfer upon molecular binding.
Disclosure of Invention
The invention aims to provide a multi-wavelength time-resolved fluorescence measuring device, which is used for detecting the luminous intensity of luminous markers with different lengths and improving the detection rate of measurement.
The technical solution for realizing the purpose of the invention is as follows:
a multi-wavelength time-resolved fluorescence measuring device comprises a light path inlet component, a measuring darkroom base component, a measuring darkroom upper base plate component, a sample mirror component and a photon counting component;
the light path inlet component is connected with the measurement darkroom base component and is used as an incident light source;
a dichroic mirror is arranged in the measurement darkroom base component and used for reflecting light;
the sample mirror assembly is connected with the measurement darkroom base assembly and is used for collimating the reflection pipeline and emitting the collimated light into the analysis cup so as to generate light rays with different filter wavelengths, and the light rays are transmitted to the photon counting assembly through the dichroic mirror;
the upper seat plate assembly of the measurement darkroom is connected with the base assembly of the measurement darkroom to form a measurement darkroom main body; a light filter switching device and a position detection unit are arranged between the upper seat plate component of the measurement darkroom and the base component of the measurement darkroom, the light filter switching device rotates to complete the switching of different light filters so as to select the light rays with different wavelengths to pass through, and the light rays with the passing wavelengths are collected by the photon counting component after being collimated by the counting lens in the upper seat plate component of the measurement darkroom; the position detection unit is used for detecting the rotating positions of different optical filters.
Compared with the prior art, the invention has the following remarkable advantages:
the invention designs the optical filter switching device, can collect photon number signals with different wavelengths emitted by different luminous markers, switches different optical filters by driving the shading block to rotate through the rotating motor in one measurement, each optical filter can pass through light waves with different wavelengths, the rotating mode not only reduces the volume of the whole module, but also improves the measurement accuracy and the detection rate through reading with different wavelengths. The whole structure is simple and compact, the stability is good, the synchronous belt transmission efficiency is high, the detection is quick, and the synchronous belt transmission device can be transplanted to various equipment platforms as a core module.
Drawings
Fig. 1 is a schematic view of the overall structure of the present invention.
Fig. 2 is a schematic cross-sectional view of an optical device according to the present invention.
FIG. 3 is an exploded view of a measurement darkroom pedestal assembly in accordance with the present invention.
FIG. 4 is an exploded view of the seat plate assembly of the darkroom according to the present invention.
Fig. 5 is an exploded view of a sample mirror assembly of the present invention.
FIG. 6 is a schematic structural diagram of a photon counting assembly according to the present invention.
FIG. 7 is a schematic diagram of switching a filter according to the present invention.
Detailed Description
The invention is further described with reference to the following figures and embodiments.
Referring to fig. 1, the multi-wavelength time-resolved fluorescence measuring device of the present invention includes five parts, namely, an optical path component 1, a measurement darkroom base component 2, a measurement darkroom upper base component 3, a sample mirror component 4, and a photon counting component 5, wherein the optical path component 1 is connected to the measurement darkroom base component 2 by screws, the measurement darkroom upper base component 3 is connected to the measurement darkroom base component 2 by screws to form a measurement darkroom main body, the sample mirror component 4 is connected to the measurement darkroom base component 2 by screws to form a measurement darkroom main body, the photon counting component 5 is connected to fix the photon counter mounting plate 502 and the measurement darkroom upper base plate 301 together by screws, and an O-ring 303 is installed on a contact surface to ensure optical path light shading, so that the five parts form a multi-wavelength time-resolved fluorescence measuring device.
Referring to fig. 2, in the multi-wavelength time-resolved fluorescence measuring device of the present invention, the optical path component 1 is a light source composed of a xenon lamp base 104 and a xenon lamp socket 101, and is fixed on the light source mirror sleeve 105 through a xenon lamp gland 102, the fixing flange of the xenon lamp base 104 and the xenon lamp gland 102 are provided with a plurality of xenon lamp adjusting pads 103, and the distance from the xenon lamp light source to the light source lens 107 is adjusted by adjusting the number of the xenon lamp adjusting pads 103 in front of and behind the fixing flange of the xenon lamp base 101; the light source lens 107 is mounted in the lens cap 106, and is placed in the light source lens sleeve 105 together with the lens cap 106, then the light source spacer 108 is installed in the light source lens sleeve 105 to determine the mounting position of the light source lens 107, and then the light source filter 110 is installed, and the optical elements in the light source lens sleeve 105 are locked by the pressing ring 109 through screw-fit. The xenon lamp base 104 emits light, which is collimated by the light source lens 107 and filtered by the light source filter 110, and emits light with a fixed wavelength onto the reflecting surface of the dichroic mirror 203
Referring to fig. 3, the multi-wavelength time-resolved fluorescence measuring apparatus of the present invention, the measurement darkroom base assembly 2 is: the measurement darkroom base 201 is used as a main structure, the dichroic mirror 203 is arranged at a groove on the inclined plane of the measurement darkroom base 201, the dichroic mirror 203 is fixed in the groove on the inclined plane of the measurement darkroom base 201 through the connection of a light shielding block 204 through screws, the lower surface of the light shielding block 204 is provided with two slender grooves, and a cut O-shaped ring is arranged in the slender grooves, so that the dichroic mirror 203 can be conveniently fixed; the photoelectric switch seat 213 is connected and installed at a groove on the upper surface of the measurement darkroom base 201 through a screw, the photoelectric switch 214 is connected and fixed on the photoelectric switch seat 213 through a screw, and the photoelectric switch seat 213 is provided with a waist-shaped hole which can drive the photoelectric switch 214 to carry out fine adjustment; the cable waterproof connector 202 is matched with the measuring darkroom base 201 through threads and is installed on the side face of the measuring darkroom base 201, and the light shading performance of the photoelectric switch 214 when the cable leaves the measuring darkroom is guaranteed; the motor mounting plate 207 is installed on the side surface of the measurement darkroom base 201 through screw connection, the rotating motor 209 is installed on the motor mounting plate 207 through screw connection, and a motor mounting plate sealing gasket 206 is installed between the motor mounting plate and the motor mounting plate to ensure the light shielding property; the rotating motor 209 drives the synchronous pulley 208 to rotate, and drives the optical filter switching seat 310 to rotate through synchronous belt transmission; the motor cover 210 is connected with the motor mounting plate 207 and the side surface of the measurement darkroom base 201 through screws, and the motor cover sealing gasket 205 is arranged in the middle of the motor cover 210, so that a closed cavity is formed at one side of the rotating motor 209, which is provided with the synchronous belt pulley 208, and is only communicated with one side of the measurement darkroom, and the light shading performance of the measurement darkroom is ensured; the rear cover 211 is installed on the measurement darkroom base 201 through screw connection, and the middle part is provided with a rear cover sealing gasket 212 to ensure the light shading performance of the measurement darkroom.
Referring to fig. 4, the multi-wavelength time-resolved fluorescence measuring device of the present invention, the seat plate assembly 3 on the measurement darkroom is: taking the upper base plate 301 of the measurement darkroom as a main structure, the counting lens 305 is arranged in the lens cap 302, is arranged in a hole on the upper base plate 301 of the measurement darkroom together with the lens cap, and is locked by a pressing ring 304 through threaded connection; a photoelectric sensor retaining piece 311, a synchronous pulley 312 and a filter switching seat 310 are arranged on a filter wheel shaft 309 and fixed by a set screw, the filter wheel shaft 309 is arranged in a deep groove ball bearing 308 arranged in a hole of an upper seat plate 301 of a measurement darkroom and can rotate under the transmission of a synchronous belt, a pressing ring 307 locks the deep groove ball bearing 308 in the hole of the upper seat plate 301 of the measurement darkroom through threaded fit, and a shaft stopper 306 locks the inner rings of the filter wheel shaft 309 and the deep groove ball bearing 308 through screw connection; the optical filter A313 and the optical filter B are respectively arranged in two holes of the optical filter switching seat 310, are fixed by set screws, rotate along with the optical filter switching seat 310, and are matched with the photoelectric switch 214 through the photoelectric sensor retaining sheet 311 to determine the rotating positions of the two optical filters. After the filter switching seat 310 is in place, the corresponding filter a313 or filter B can be aligned with the counting lens 305.
Referring to fig. 5, the multi-wavelength time-resolved fluorescence measuring apparatus of the present invention, the sample mirror assembly 4 is: taking a sample mirror sleeve 401 as a main body, installing a sample lens 405 in a lens cap 403, installing the sample lens in the sample mirror sleeve 401 together with the lens cap, and distributing the number of sample mirror spacers 402 up and down at the installation position of the sample lens in the sample mirror sleeve 401 to adjust the distance from the sample lens 405 to an analysis cup; clamping ring 404 locks sample lens 405 and sample mirror spacer 402 in sample mirror sleeve 401 by a threaded fit.
Referring to fig. 6, in the multi-wavelength time-resolved fluorescence measuring apparatus of the present invention, the photon counting assembly 5 is: the photon counter 501 is fixed on the photon counter mounting plate 502, the photon counter mounting plate 502 is connected with the upper base plate 301 of the measurement darkroom through screws, and the O-shaped ring 303 is arranged in the middle to ensure the light shading performance of the light path.
Referring to fig. 1 to 7, light emitted from a xenon lamp base 104 is collimated by a light source lens 107 and filtered by a light source filter 110, and then emitted to a reflecting surface of a dichroic mirror 203, the reflected light is collimated by a sample lens 405 and emitted into an analyzing cup, the light excites a first luminescent marker to emit light with a wavelength capable of passing through a filter a313, the light excites another luminescent marker to emit light with a wavelength capable of passing through a filter B314, the light passes through the dichroic mirror 203 and propagates upwards, a rotating motor 209 drives a synchronous pulley 208 to rotate, a filter switching seat 310 is driven to rotate by synchronous belt transmission, the filter a313 and the filter B314 are switched, light with different wavelengths respectively passes through the filter a313 and the filter B314, and after being collimated by a counting lens 305, the light is collected by a photon counter 501, and then the antigen concentration is calculated by algorithm processing. In conclusion, the multi-wavelength time-resolved fluorescence measuring device has the advantages of compact structure, good light shading performance and accurate and reliable detection result, is used as a core module of in-vitro diagnostic equipment, can be transplanted to various equipment platforms, and is easy to popularize.
Claims (10)
1. A multi-wavelength time-resolved fluorescence measuring device is characterized by comprising an optical path entering component (1), a measuring darkroom base component (2), a measuring darkroom upper base plate component (3), a sample mirror component (4) and a photon counting component (5);
the light inlet path component (1) is connected with the measurement darkroom base component (2) and is used as an incident light source;
a dichroic mirror (203) is arranged in the measurement darkroom base component (2) and is used for reflecting light;
the sample mirror assembly (4) is connected with the measurement darkroom base assembly (2) and is used for collimating a reflection pipeline and emitting the collimated reflection pipeline into an analysis cup so as to generate light rays with different filter wavelengths, and the light rays are transmitted to the photon counting assembly (5) through the dichroic mirror (203);
the upper seat plate component (3) of the measurement darkroom is connected with the base component (2) of the measurement darkroom to form a measurement darkroom main body; a light filter switching device and a position detection unit are arranged between the upper seat plate component (3) of the measurement darkroom and the base component (2) of the measurement darkroom, the light filter switching device rotates to complete the switching of different light filters so as to select the light rays with different wavelengths to pass through, and the light rays with the passed wavelengths are collected by a photon counting component (5) after being collimated by a counting lens (305) in the upper seat plate component (3) of the measurement darkroom; the position detection unit is used for detecting the rotating positions of different optical filters.
2. The multiwavelength time-resolved fluorescence measuring device of claim 1, wherein the filter switching device comprises a measurement darkroom upper base plate (301), a filter switching base (310), a filter axle (309) photosensor flap (311), a synchronous pulley (312), and a rotating motor (209);
the counting lens 305 is fixed on a seat plate component (3) on a measurement darkroom; the optical filter switching seat (310) is rotationally connected with the upper seat plate (301) of the measurement darkroom through an optical filter wheel shaft (309); the optical filter switching seat (310) is internally provided with a plurality of optical filters capable of passing different wavelengths, a synchronous belt wheel (312) is arranged on an optical filter wheel shaft (309), the rotating motor (209) is connected with the synchronous belt wheel (312) through a synchronous belt, a photoelectric sensor blocking piece (311) is arranged at the lower end of the optical filter wheel shaft (309), a photoelectric switch (214) is arranged in the measurement darkroom main body, and the photoelectric switch (214) detects the position of the photoelectric sensor blocking piece (311) so as to detect the rotating positions of different optical filters.
3. The multiwavelength time-resolved fluorescence measurement device of claim 2, wherein the counter lens (305) is fixed in a hole on the base plate (301) on the measurement darkroom by a lens cap (302) and locked by a clamping ring.
4. The multi-wavelength time-resolved fluorescence measuring device according to claim 2, wherein the measuring darkroom upper seat plate (301) is provided with a deep groove ball bearing (308), and the deep groove ball bearing (308) is locked in a hole of the measuring darkroom upper seat plate (301) through a pressing ring; the filter wheel shaft (309) is connected with the inner ring of the deep groove ball bearing (308).
5. The multi-wavelength time-resolved fluorescence measuring device according to claim 1, wherein the light path component (1) comprises a xenon lamp head (104), a xenon lamp socket (101), a light source mirror sleeve (105), a light source lens (107) and a light source filter (110);
the xenon lamp head (104) is inserted into the xenon lamp holder (101) to form a xenon lamp light source; the light source mirror sleeve (105) is connected with a xenon lamp light source; the light source lens (107) and the light source filter (110) are arranged in the light source mirror sleeve (105).
6. The multi-wavelength time-resolved fluorescence measuring device according to claim 5, wherein the light source mirror sleeve (105) is connected with a xenon lamp light source through a xenon lamp gland (102), and the xenon lamp gland (102) is matched with the light source mirror sleeve (105) through threads;
the xenon lamp socket is characterized in that a circle of flange is arranged on the excircle of the xenon lamp socket (101), a plurality of xenon lamp adjusting pads (103) are arranged between the flange and the light source mirror sleeve (105), and the xenon lamp adjusting pads (103) and the light source mirror sleeve (105) are both sleeved on the excircle of the xenon lamp socket (101).
7. The multi-wavelength time-resolved fluorescence measuring device according to claim 5, wherein a light source spacer (108) is disposed between the light source lens (107) and the light source filter (110); and a pressing ring is connected in the light source lens sleeve (105) and used for axially positioning the light source filter (110).
8. The multi-wavelength time-resolved fluorescence measurement device of claim 1, wherein the measurement camera base assembly (2) comprises a measurement camera base (201); an inclined plane is arranged in the measurement darkroom base (201), the dichroic mirror (203) is installed at a groove on the inclined plane of the measurement darkroom base (201), and the dichroic mirror (203) is fixed in the groove on the inclined plane of the measurement darkroom base (201) through the shading block (204).
9. The multi-wavelength time-resolved fluorescence measuring device according to claim 1, wherein the sample mirror assembly (4) comprises a sample mirror housing (401), a sample lens (405), a lens cap (403), a sample mirror spacer (402);
the sample lens (405) is installed in the sample mirror sleeve (401) through a lens cap (403), sample mirror spacer sleeves (402) are arranged on the upper portion and the lower portion of the sample mirror sleeve (401), and the sample lens (405) is locked in the sample mirror sleeve (401) through a pressing ring.
10. The multi-wavelength time-resolved fluorescence measuring device according to claim 1, wherein the photon counting assembly (5) comprises a photon counter (501), a photon counter mounting plate (502); the photon counter mounting plate (502) is connected with the seat plate component (3) on the measurement darkroom and is sealed and light-proof, and the photon counter (501) is fixed on the photon counter mounting plate (502).
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| CN202011566752.6A CN112649411A (en) | 2020-12-25 | 2020-12-25 | Multi-wave long-time resolution fluorescence measuring device |
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| CN202011566752.6A CN112649411A (en) | 2020-12-25 | 2020-12-25 | Multi-wave long-time resolution fluorescence measuring device |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113694388A (en) * | 2021-07-19 | 2021-11-26 | 西安交通大学 | Photon treatment handle and optical filter rotation switching device thereof |
Citations (11)
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