CN113960009B - Capillary fluorometer with low background signal - Google Patents
Capillary fluorometer with low background signal Download PDFInfo
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- CN113960009B CN113960009B CN202111440339.XA CN202111440339A CN113960009B CN 113960009 B CN113960009 B CN 113960009B CN 202111440339 A CN202111440339 A CN 202111440339A CN 113960009 B CN113960009 B CN 113960009B
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Abstract
The invention provides a capillary fluorescent instrument with low background signal, which comprises a light source, a capillary, an optical filter and a photoelectric detector; the light source and the photoelectric detector are arranged on the end face of the capillary tube; the capillary tube is internally provided with a reflective film and/or a noise reduction film which are used for reducing stray excitation light and increasing fluorescent signals; the capillary tube can be of a Y-shaped bifurcation structure, so that a beam splitter is not required, and an object to be detected is not required to be placed at a focusing focus of excitation light. The fluorescence instrument has the characteristics of compact structure, high sensitivity and simple and convenient operation.
Description
Technical Field
The invention relates to a capillary fluorometer with low background signal, which can improve detection sensitivity by inhibiting the background signal.
Background
The fluorescence instrument can be used for detecting the content of trace substances (namely trace detection), the detection principle is that a high-energy light beam (namely excitation light) is utilized to excite the object to be detected, so that the object to be detected radiates characteristic fluorescence (the fluorescence wavelength is larger than the excitation light wavelength), and the components and the content of the trace substances in the object to be detected can be obtained through detecting the characteristic fluorescence. When laser light is used as excitation light, the fluorometer is referred to as a "Laser Induced Fluorometer (LIF)". Since excitation light is strong and fluorescence is weak, how to suppress background signals and background noise caused by leakage of excitation light becomes a key to improve detection sensitivity. And (3) injection: background noise comes from the background signal, with background noise generally increasing with background signal.
Currently, fluorometers typically employ capillaries as sample cells. The capillary tube provides a long optical path, thereby increasing excitation efficiency, enhancing fluorescence signals, and also has the advantage of low sample requirements (Optics and Lasers in Engineering,2021,139,106488). The existing capillary sample cell mainly has two types: transparent capillaries (e.g., quartz capillaries); opaque capillaries (e.g., metal capillaries). The following disadvantages exist respectively: for transparent capillary, the excitation light can penetrate the wall of the capillary (or be transmitted in the wall of the capillary), so that the excitation light leaks out of the capillary, and background noise is increased; for the metal capillary, the roughness of the inner wall of the metal tube is large, so that the inner wall can diffusely reflect excitation light and fluorescence, thereby causing fluorescence signal loss and excitation light leakage.
To sum up, in order to solve the above problems, a new structure of a fluorescence detector is sought, which is an innovative research machine of the present invention.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a capillary fluorometer with low background signals.
The technical scheme of the invention is as follows:
a capillary fluorescent instrument with low background signal is prepared as setting reflecting film and/or noise-reducing film in capillary tube and setting optical filters at two ends of capillary tube.
The reflecting film is used for reflecting excitation light and fluorescence, and is directly applied to the inner wall surface of the capillary tube or is applied to the inner wall surface of the capillary tube through the supporting film.
The reflective film is made of high-reflectivity materials (such as photonic crystals and metals), and the metals are preferably made of aluminum, silver, titanium, stainless steel, metal alloy and the like. The photonic crystal is a material with a refractive index which changes periodically, such as a dielectric film reflector and the like.
The thickness of the reflecting film is larger than 50nm.
The surface of the reflecting film is plated with a protective film (such as Al 2 O 3 ，SiO 2 ) To prevent damage (e.g., oxidation, or corrosion by solution) to the retroreflective sheeting.
The capillary tube is made of metal materialWhen the material is used (namely, the metal capillary tube), the inner wall of the material is polished and oxidized, and the material can also have the effects of a reflecting film and a protecting film. In this case, the metal capillary itself may be regarded as having a light reflecting film and a protective film. For example, after the inner wall of the aluminum tube is polished, the bright inner wall can play a role in reflecting light (as a reflecting film); further, the inner wall surface of the aluminum tube is oxidized to form Al 2 O 3 The layer can serve as a protective film, so that the polished and oxidized aluminum tube can be considered to have a light reflecting film and a protective film (i.e., the inner wall of the aluminum tube does not need to be coated with a light reflecting film and/or a protective film).
The noise reduction film is attached to the surface of the bracket and is arranged in the capillary tube and positioned at the light emitting end.
The noise reduction film is made of semiconductor material, such as GaN, inGaN, alGaN, tiO 2 、SnO 2 、ZnO、Ga 2 O 3 、WO 3 Etc. The semiconductor material has a band gap width (or forbidden band width) of an electron energy band between photon energy of excitation light and fluorescence, so that the semiconductor material can absorb stray excitation light and does not absorb fluorescence. Among them, tiO is preferable 2 The material (rutile structure) has a forbidden bandwidth of about 3.2eV, can absorb the excitation light with the wavelength of less than 387.5nm (namely photon energy is more than 3.2 eV), and does not absorb the fluorescence with the wavelength of more than 387.5nm (namely photon energy is less than 3.2 eV). And (3) injection: the data (e.g., forbidden band width and wavelength) are only for schematic illustration, and may deviate from the actual values (e.g., specific forbidden band width is related to lattice structure, grain size, and preparation mode, etc.).
The noise reduction film is preferably made of a semiconductor material with more indirect band gaps and/or crystal defects, so that the fluorescence radiated by the noise reduction film is reduced. And (3) injection: compared with a direct bandgap semiconductor, the indirect bandgap semiconductor has extremely low light emission efficiency; in addition, crystal defects also reduce the luminous efficiency, thereby reducing the background fluorescence signal.
The noise reduction film can be attached to the surface of the reflection film, namely, a double-layer composite structure of the reflection film and the noise reduction film is formed on the inner surface of the capillary tube. Wherein, the reflective film can restrict the excitation light and fluorescence to be transmitted in the capillary tube (the excitation light and the fluorescence cannot leak out of the tube); the noise reduction film can absorb stray excitation light and does not absorb fluorescence. Therefore, the double-layer composite structure has the triple functions of reducing stray excitation light, confining excitation light and confining fluorescence. And (3) injection: the reflecting film and the capillary tube can be made of the same material; the noise reduction film is arranged in parallel with the inner wall of the capillary tube, so that excitation light (non-stray light) transmitted in parallel along the capillary tube cannot enter the noise reduction film (i.e., cannot be absorbed by the noise reduction film).
The noise reduction film can be attached to the surface of the bracket and is arranged in the capillary tube. Wherein the shape of the bracket comprises a tubular shape, a columnar shape, a filiform shape, a lamellar shape and the like, and the material of the bracket is selected from substances with small fluorescence absorption (such as metal, quartz, metal oxide and the like). The sheet-shaped support can be folded into a polygonal or star-shaped tubular structure (namely, the noise reduction film presents a polygonal or star shape), so that the incidence times of stray excitation light and the noise reduction film are increased (namely, the absorption of the stray excitation light is increased), and the resistance of liquid flow is reduced (namely, the flow dead zone is reduced).
The noise reduction film is preferably the same metal material as the capillary (or light reflecting film). For example, the noise reduction film is TiO 2 The material and the corresponding capillary tube are made of Ti material, and the preparation process is as follows: oxidizing the Ti capillary tube to form a layer of TiO on its inner surface 2 Films (i.e. "Ti-TiO 2 "double layer composite structure"). The preparation method has simple process and stable structure (TiO) 2 The film and the Ti tube are of an integrated structure), and the TiO can be adjusted by adjusting the oxidation time and temperature 2 Thickness of the film.
The thickness of the noise reduction film is larger than 20nm.
The capillary is made of quartz glass, sapphire, aluminum, silver, titanium, tin, tungsten, iron, zinc, stainless steel, metal alloy and other materials, and preferably metal materials with high optical reflectivity (such as Ag and Al); the cross-sectional shape of the material can be round, rectangular or polygonal; in the length direction, the capillary tube may be straight, curved, bifurcated or spiral.
The capillary tube is of a Y-shaped bifurcation structure, one bifurcation is used for transmitting excitation light, the other bifurcation is used for transmitting fluorescence, and an object to be measured is placed outside a junction port. At this time, the analyte does not need to flow through the capillary (placed at the capillary port), so that it is more suitable for detecting solid analyte (or non-flowing liquid analyte). Compared with LIF of the traditional confocal structure, the Y-shaped branched structure capillary tube does not need to adopt a light splitting sheet, and an object to be detected does not need to be placed at a focusing focus of excitation light.
The Y-branch structure may also be used to test flowing samples, where one branch is used for flowing samples (i.e., a sample branch) and the other branch is used for transmitting excitation light (i.e., an excitation light branch). A transparent window is arranged at the joint of the two branches to prevent the sample from flowing into the excitation light branches; meanwhile, excitation light can enter the sample branch from the transparent window, so that the flowing sample is excited to generate fluorescence; the generated fluorescence is transmitted along the sample fork and received by the photodetector.
The Y-shaped bifurcation structure is not limited to two bifurcation, and may be two or more bifurcation. Wherein different branches are used for transmitting excitation light of different wavelengths.
The support film is preferably made of smooth-surface materials (such as plastics, quartz glass, sapphire and the like), so that the reflective film can improve the reflectivity of the inner wall of the capillary tube, and meanwhile, the support film can reduce the roughness of the inner wall, so that the generation of stray excitation light is reduced.
A light absorbing sheet or a reflecting mirror with holes is arranged between the end face of the capillary tube and the optical filter.
And a layer of noise reduction film is attached to the surface of the optical filter.
The filters were linear graded filters to test fluorescence spectra.
The end face of the capillary tube is provided with a light absorption sheet (or a reflecting mirror) with holes, so that excitation light can enter the capillary tube through the small holes, and the beam diameter of the excitation light is smaller than the inner diameter of the capillary tube, thereby effectively reducing the contact between the excitation light and the side wall of the capillary tube (namely reducing stray excitation light). The light absorbing sheet is used for absorbing excitation light, and the material of the light absorbing sheet is selected from noise reduction film materials, black materials (such as anodic aluminum oxide, inorganic nano materials, flannelette) and the like. Wherein the reflecting mirror is used for reflecting the excitation light.
The light source is used for emitting excitation light and is selected from a laser, an LED, plasma, a filament and the like.
The photoelectric detector is used for detecting fluorescence emitted by the object to be detected.
The object to be detected is arranged in the capillary or at the port of the capillary.
The stray excitation light means that when the excitation light is incident on the inner wall of the capillary, the rough surface of the inner wall of the capillary causes diffuse reflection (or scattering), thereby stray the propagation direction of the excitation light. Therefore, diffuse reflection increases the number of reflections and the reflection loss of the sidewall (the reflection loss increases with the number of reflections), thereby reducing the intensity of the excitation light and the fluorescent signal; in addition, stray excitation light can leak through the filter, thereby increasing the background signal. And (3) injection: the inner wall of the metal capillary is difficult to polish into a mirror surface, and thus the inner wall surface is rough.
The optical filter is positioned on the end face of the capillary tube. The filter at the outlet of the capillary tube is used for reflecting excitation light and transmitting fluorescence (namely a long-wave pass filter or a band-pass filter); the optical filter can be a linear gradient filter (linear gradient filter) to obtain a fluorescence spectrum; and a noise reduction film is attached to the surface of the optical filter and used for absorbing stray excitation light.
The invention has the technical effects that:
(1) The capillary tube adopted by the traditional fluorescent instrument is mainly a quartz capillary tube and a metal capillary tube, wherein only the metal capillary tube can play the dual functions of restraining excitation light and fluorescence. In contrast, the capillary tube of the invention can reduce stray excitation light in addition to confining excitation light and fluorescence due to the introduction of the noise reduction film and/or the reflection film, thereby more effectively inhibiting background signals and noise.
(2) The inner wall of the capillary tube can be attached with a reflective film layer or a supporting film-reflective film double-layer structure, so that the optical reflectivity of the inner wall of the capillary tube is improved, and meanwhile, the roughness and related diffuse reflection of the surface of the inner wall are reduced.
(3) The noise reduction film can be made into a polygonal or star-shaped tubular structure, thereby increasing the absorption of stray excitation light and reducing the resistance of liquid flow.
(4) The Y-shaped bifurcation structure capillary tube is characterized in that one bifurcation is used for transmitting excitation light, the other bifurcation is used for transmitting fluorescence, and the Y-shaped bifurcation structure capillary tube is more suitable for detecting solid objects to be detected and is simple and convenient to operate.
Drawings
FIG. 1 is a schematic diagram of a capillary fluorometer.
FIG. 2 is a schematic diagram of a Y-type bifurcation capillary fluorometer.
Fig. 3 is a schematic view of a capillary structure with a reflective film attached to the inner wall.
Fig. 4 is a schematic view of a capillary structure with a support film and a reflective film attached to the inner wall.
Fig. 5 is a schematic view of a capillary structure with a support film, a reflective film and a protective film attached to the inner wall.
Fig. 6 is a schematic view of a capillary structure with a noise reduction film attached to the inner wall.
Fig. 7 is a schematic view of a capillary structure with a reflective film and a noise reduction film attached to the inner wall.
Fig. 8 is a schematic view of a capillary structure with a holder and a noise reduction film placed inside.
Fig. 9 is a cross-sectional view of a triangular tubular stent disposed within a capillary tube.
Fig. 10 is a cross-sectional view of a star-shaped tubular stent placed in a capillary tube.
Fig. 11 is a cross-sectional view of a filter with a noise reduction film.
FIG. 12 is a schematic diagram of another Y-branch structure capillary fluorometer.
1. A light source; 2. excitation light; 3. a first filter (short-pass or band-pass); 4. a noise reduction film; 5. a capillary tube; 6. fluorescence; 7. a light absorbing sheet or mirror; 8. a second filter (long pass or band pass); 9. a lens; 10. a photodetector; 11. a first aperture; 12. a second aperture; 13. a reflective film; 14. a bracket; 15. an object to be measured; 16. a light shield; 17. a support film; 18. a protective film; 19. the excitation light diverges; 20. excitation light passes through the window; 21. an optical mirror; 22. and (5) a light absorbing sheet.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings and technical solutions.
The invention provides a fluorometer (fig. 1 and 2), which mainly comprises a light source 1, a capillary 5 and a photoelectric detector 10, wherein the light source 1 and the photoelectric detector 10 are arranged at two ends of the capillary 5. Wherein the inner diameter of the capillary 5 is 10 μm to 10cm.
The excitation light 2 emitted from the light source 1 enters the inlet of the capillary 5 (or one branched inlet of the Y-shaped capillary shown in fig. 2) and is transmitted along the axial direction of the capillary 5. When the excitation light 2 is transmitted in the capillary, the object 15 to be measured (or the object 15 to be measured outside the Y-shaped capillary port shown in fig. 2) inside the capillary is excited, so that the object 15 to be measured emits fluorescence 6. The fluorescence 6 is confined in the capillary 5, transmitted from the outlet of the capillary 5 (or the other branch outlet of the Y-shaped capillary shown in fig. 2), converged by the lens 9, and received by the photodetector 10.
The three ports of the Y-shaped capillary 5 (fig. 2) are respectively used for introducing excitation light 2, outputting fluorescence 6 and placing an object 15 to be measured, the excitation light 2 can be incident on the object 15 to be measured through one branch of the capillary, and the fluorescence 6 emitted by the object 15 to be measured can be transmitted to the photodetector 10 through the other branch of the capillary; thus, excitation light 2 and fluorescence light 6 are transmitted in different branches of the capillary tube, without a dichroic sheet separating the transmission paths of excitation light 2 and fluorescence light 6. Wherein a light shield 16 is used to prevent ambient light from entering the capillary tube 5. The Y-shaped capillary 5 may allow the analyte 15 to enter the capillary 5, and the fluorescence collection efficiency may be further improved.
The Y-capillary 5 may also take another configuration (fig. 12) that can be used to detect a flowing sample (liquid or gas sample). At this time, the Y-capillary has two branches: sample branch 5, excitation light branch 19. Wherein the sample flows within the bifurcation 5. Excitation light 2 is transmitted within the bifurcation 19 and enters the bifurcation 5 through the window 20 and is then reflected by the mirror 21, thereby becoming parallel transmitted along the axial direction of the bifurcation 5; thus, the excitation light 2 can be prevented from contacting the side walls of the bifurcation 5, thereby reducing the increase in background signal caused by the scattering of the side walls; meanwhile, the transmission directions of the excitation light 2 and the fluorescence 6 are opposite, so that the influence of the excitation light 2 on the fluorescence 6 (namely, the background signal is reduced) can be reduced to the greatest extent. Among them, the window 20 is preferably a short-wave pass filter (or a band-pass filter) that transmits the excitation light 2 and reflects the fluorescence light 6; the window 20 also serves to prevent the sample from flowing into the excitation light branches 19. Wherein the reflecting mirror 21, preferably a metal tube wall, is convex, and one side slope of the convex can reflect the excitation light 2. The light absorbing sheet 22 is located at one end of the sample branch 5, and is used for absorbing the excitation light 2, so as to avoid that the excitation light 2 is reflected back to the other end of the branch 5 (i.e. the receiving end of the fluorescence 6), thereby reducing the background signal. The light absorbing sheet 22 is preferably a black anodized aluminum sheet.
Note that: in the existing confocal LIF, the transmission light paths of excitation light and fluorescence are the same, so that a 45-degree beam splitting sheet is needed to separate the transmission light paths of the excitation light and the fluorescence; moreover, the object to be tested needs to be accurately placed at the focus of the excitation light, so that it is difficult to test the object to be tested (such as a solid object to be tested) with an unfixed position; in contrast, the Y-capillary does not need to be precisely placed.
Note that: the existing optical fiber LIF transmits excitation light and fluorescence through an optical fiber, and the collection efficiency of the optical fiber on the fluorescence is low due to the limited numerical aperture of the optical fiber, particularly, the collection efficiency of the fluorescence is further reduced by an object to be detected with a non-smooth surface; in contrast, since the side wall of the capillary has high reflectivity, the fluorescence collection efficiency of the capillary is far higher than that of the optical fiber, and the noise reduction film can be placed in the capillary to reduce the background noise (the noise reduction film is difficult to place in the optical fiber).
A short-wave pass filter (or band-pass filter) 3 is disposed between the light source 1 and the capillary 5 to filter out long-wave components in the excitation light 2. And (3) injection: since excitation light is usually not strictly monochromatic (the wavelength range of excitation light is broad), in order to avoid overlapping of excitation light wavelengths with fluorescence wavelengths (if the wavelengths overlap, excitation light and fluorescence cannot be separated, resulting in increased background noise), the long-wave portion of excitation light needs to be filtered out.
Between the light source 1 and the capillary 5, a light absorbing sheet (or mirror) 7 with holes 11 is provided for absorbing (or reflecting) excitation light. The excitation light 2 entering the interior of the capillary 5 through the aperture 11 is thus limited in its beam diameter by the aperture of the aperture 11. When the aperture of the small hole 11 is smaller than the inner diameter of the capillary 5, the diameter of the excitation light beam entering the capillary can be smaller than the inner diameter of the capillary, so that the excitation light 2 is effectively prevented from contacting with the side wall of the capillary, and stray excitation light caused by diffuse reflection of the side wall is reduced. And (3) injection: the filter is usually composed of a dielectric film, and the filtering effect of the filter is related to the incidence angle of the light beam (the filtering effect is the best in the case of normal incidence); since the incident angle of the stray excitation light is disordered, the optical filter 8 cannot effectively block the stray excitation light, thereby causing an increase in background signal.
The capillary 5 (fig. 3) may have a reflective film 13 coated on its inner wall to enhance the reflectivity of the excitation light 2 and the fluorescence 6.
The capillary 5 (fig. 4 and 5) may further have a support film 17 and a reflective film 13 attached to the inner wall thereof. The reflective film 13 is coated on the surface of the supporting film 17 to improve the reflectivity of the excitation light 2 and the fluorescence 6. Since the support film 17 can be made of a smooth-surfaced material, the roughness of the inner surface of the capillary tube and the related diffuse reflection can be reduced. In order to avoid oxidation and corrosion of the reflective film 13, a protective film 18 is coated on the surface thereof. And (3) injection: since it is difficult to polish the inner wall of the capillary 5, particularly, it is difficult to polish the metal capillary to a mirror surface (i.e., the inner wall is rough), there are light loss and light leakage due to diffuse reflection.
The inner wall of the capillary 5 (fig. 6) may be covered with a noise reduction film 4, so as to reduce diffuse reflection of the excitation light 2 by the inner wall of the capillary, and reduce the background signal.
The inner wall of the capillary tube 6 (fig. 7) can be covered with a double-layer composite structure of a reflective film 13 and a noise reduction film 4, wherein the noise reduction film 4 is plated on the surface of the reflective film 13. Wherein the reflective film 13 is used for reflecting the excitation light 2 and the fluorescence 6, so that the excitation light 2 and the fluorescence 6 are restrained to be transmitted in the capillary tube (cannot leak out of the tube); the noise reduction film 4 may absorb the excitation light 2 and not absorb the fluorescence 6. Therefore, the double-layer composite structure has the triple functions of absorbing excitation light, confining excitation light and confining fluorescence.
The noise reduction film 4 (figure 8) is coated on the surface of the bracket 14, wherein the noise reduction film 4 is TiO 2 The membrane and the bracket 14 are quartz columns. Then is plated with TiO 2 The quartz column 14 of the membrane 4 is placed inside the capillary 5, wherein the diameter and length of the quartz column are smaller than the inner diameter and length of the capillary 5, respectively.
The support 14 may be a quartz column, a quartz tube, a diaphragm, or the like. Wherein the membrane holders 14 (fig. 9 and 10) are in the shape of triangular tubes or star-shaped tubes (i.e. tubes with triangular or star-shaped cross-sections). After the surface of the triangular tube (or star-shaped tube) 14 is plated with the noise reduction film 4, it is placed in the capillary tube 5. At this time, the liquid to be measured can flow through the inside and the outside of the pipe simultaneously, so that the resistance and dead area to the fluid are reduced (the thinner the pipe wall is, the smaller the resistance and dead area to the flow are).
The light absorbing sheet (or mirror) 7 is disposed on the left or right side, preferably the right side, of the filter 3 (as shown in fig. 1). The light absorbing sheet (or reflecting mirror) 7 is selected from a light absorbing sheet, a reflecting mirror, or a combination of both. Wherein, the combination is a double-layer structure of overlapping the light absorbing sheet and the reflecting mirror.
The long-wave pass filter (or band-pass filter) 8 is disposed at the outlet end of the capillary 5. Since the wavelength of the excitation light 2 is smaller than the wavelength of the fluorescence 6, the filter 8 can transmit the fluorescence 6 and reflect the excitation light 2 (i.e., block the excitation light), thereby reducing the background signal. The surface of the filter 8 (fig. 11) facing the capillary tube may be further coated with a noise reduction film 4 for absorbing the excitation light 2, in particular, stray excitation light.
The light absorbing sheet 7 is disposed between the outlet of the capillary 5 and the optical filter 8, and is used for further reducing leakage of the excitation light 2. Since the excitation light 2 reflected by the filter 8 is likely to leak from the "port slit" (the slit between the capillary outlet and the filter 8). The light absorbing sheet 7 is thus introduced to absorb the excitation light leaking from the slit (the light absorbing sheet 7 is located in the slit), thereby preventing the excitation light 2 from leaking out of the port slit. The organic combination of the reflection and absorption functions (the combination of the optical filter 8 and the light absorbing sheet 7) can more effectively prevent the excitation light 2 from leaking out of the capillary 5, and further inhibit background noise. Similarly, the light absorbing sheet 7 can reduce leakage of excitation light from a port slit at the capillary inlet (the slit is located between the capillary inlet and the filter 3).
The middle of the light absorbing sheet 7 is provided with a second small hole 12. The aperture of the small hole is smaller than the outer diameter of the capillary, so that the excitation light leaked through the port gap is effectively absorbed; at the same time, the aperture allows fluorescence to pass through the aperture and filter 8 and be received by the photodetector 10.
The port slit is positioned at two ends of the capillary (the slit between the capillary port and the optical filter) and is used for circulating the sample to be measured, so that the slit cannot be eliminated. The width of the port slit is between 1 micron and 1 cm, preferably between 0.01 and 2 mm.
Example 1
As shown in fig. 1, the excitation light 2 emitted from the light source 1 passes through the short-wave pass filter 3 and the small hole 11 of the light absorbing sheet 7, then enters the capillary 5 from the inlet of the capillary 5, excites the object 15 to be measured in the capillary to generate fluorescence 6, and is finally absorbed by the light absorbing sheet 7 at the outlet end of the capillary and reflected back by the light absorbing sheet 8.
Since the stainless steel capillary 5 has the peek plastic support film 17 and the aluminum reflective film 13 (fig. 4) attached to the inner wall, the reflective film 13 can serve as a tube wall to reflect the fluorescence 6 and allow the fluorescence 6 to be transmitted inside the capillary 5. The fluorescence 6 passes through the light absorbing sheet small hole 12 and the optical filter 9, is converged by the lens 9 and enters the photoelectric detector 10.
Since the excitation light 2 emitted from the light source 1 has a certain beam divergence angle, the excitation light 2 diverges and enters the inner wall of the capillary tube when transmitted in the capillary tube even if the restriction of the aperture 11 (the beam diameter of the excitation light 5 at the inlet end is smaller than the inner diameter of the capillary tube) is imposed, thereby causing the generation of stray excitation light.
Compared with the inner wall of the stainless steel capillary 5, the surfaces of the supporting film 17 and the reflecting film 13 are smoother, so that stray excitation light caused by roughness of the inner wall can be effectively reduced, and background noise is reduced. Moreover, the smooth tube wall can reduce the transmission loss of the excitation light 2 and the fluorescence 6 in the tube caused by diffuse reflection, thereby enhancing the fluorescence signal.
Example 2
As shown in fig. 2, excitation light 2 emitted from a light source 1 enters an object 15 to be measured at the port of a Y-branched capillary 5 through one branch of the capillary 5. The fluorescence 6 generated by the excitation of the analyte is transmitted along the other branch of the Y-branch capillary 5 and received by the photodetector 10 at the other port of the capillary 5.
At this time, the fluorescence 6 and the excitation light 2 are transmitted along different capillary branches, so that a 45-degree beam splitter (reduction in the volume of the instrument) is not required; the object 15 to be tested does not need to enter the capillary, and is more suitable for testing solid objects to be tested; moreover, compared with the confocal LIF, the excitation light 2 is directly incident to the object 15 to be measured (i.e., the excitation light 2 does not need to be focused on the object 15 to be measured), so that the object 15 to be measured does not need to be placed on the focusing point of the excitation light 2 (no precise alignment is needed), and the operation is simple; in addition, compared with the optical fiber LIF, the inner wall of the quartz capillary 5 is provided with a silver reflecting film 13 (figure 3), the numerical aperture of the quartz capillary is larger, and fluorescence 6 can be collected more effectively.
The capillary 5 can be placed inside a triangular tubular quartz support 14. The preparation process of the bracket 14 is as follows: the outer surface of the quartz tube 14 with triangular cross section is plated with 200nm TiO 2 Film 4 (TiO) 2 The film acts as a noise reduction film). The TiO 2 The film absorbs excitation light 2 at a wavelength of 310nm and does not absorb fluorescence 6 at a wavelength of 500nm, so that stray excitation light can be absorbed, thereby reducing background signals.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention.
Claims (9)
1. The capillary tube fluorometer with low background signal is characterized by comprising a light source, a capillary tube and a photoelectric detector; a reflecting film and/or a noise reduction film are arranged in the capillary tube; the capillary tube is of a Y-shaped bifurcation structure and comprises two bifurcations, the bifurcations comprise a sample bifurcation and an excitation light bifurcation, an optical filter is arranged between the excitation light bifurcation and a light source, a photoelectric detector and a light absorbing sheet are respectively arranged at two ends of the sample bifurcation, the optical filter is arranged between the sample bifurcation and the photoelectric detector, the sample flows in the sample bifurcation, the excitation light is transmitted in the excitation light bifurcation, enters the sample bifurcation through a window and is reflected by a reflecting mirror, so that the excitation light is transmitted in parallel along the axial direction of the sample bifurcation, and the transmission direction of the excitation light and the fluorescence is opposite; the window is a short-wave pass filter which can transmit excitation light and reflect fluorescence, the reflector is of a convex structure, and an inclined plane at one side of the convex can reflect the excitation light; the light absorbing sheet is used for absorbing excitation light.
2. The capillary fluorometer of claim 1, wherein the reflective film is directly applied to the inner wall of the capillary tube or is applied to the inner wall of the capillary tube through a support film, and the reflective film is made of a high-reflectivity material and has a thickness of more than 50nm.
3. The capillary fluorometer of claim 2, wherein the reflective film is made of aluminum, silver, titanium, stainless steel, metal alloy or photonic crystal.
4. The capillary fluorometer of claim 3, wherein the reflective film is coated with a protective film.
5. The capillary fluorometer of any one of claims 1-4, wherein the noise reduction film is attached to the surface of the light reflection film, i.e., a double-layer composite structure of "light reflection film-noise reduction film" is formed on the inner surface of the capillary tube; or the noise reduction film is attached to the surface of the bracket and is arranged in the capillary tube and positioned at the light emitting end, and the noise reduction film is made of semiconductor material and has the thickness of more than 20nm.
6. The capillary fluorometer of claim 5, wherein the noise reduction film is made of a material that is
GaN、InGaN、AlGaN、TiO 2 、SnO 2 、ZnO、Ga 2 O 3 Or WO 3 。
7. The capillary tube fluorescence detector of claim 6, wherein a light absorbing sheet or a reflecting mirror with holes is arranged between the end face of the capillary tube and the optical filter.
8. The capillary fluorometer of claim 7, wherein a noise reduction film is attached to the surface of the filter.
9. The capillary fluorometer of claim 8, wherein the filter is a linear graded filter to test fluorescence spectra.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| JP2000019114A (en) * | 1998-07-03 | 2000-01-21 | Hitachi Ltd | Method and apparatus for detecting faint fluorescence |
| CN202057580U (en) * | 2011-04-23 | 2011-11-30 | 浙江大学 | Optical system for fluorescent detection of a photo-conductive relay (PCR) amplifier with quantitative property |
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| CN106399075A (en) * | 2016-08-26 | 2017-02-15 | 浙江科技学院 | Reflector based fluorescent quantitative PCR detection system |
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| CN113791059A (en) | 2021-12-14 |
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