CN211697502U

CN211697502U - Wavelength/angle modulation free conversion polarized light fluorescence imaging surface plasma resonance instrument

Info

Publication number: CN211697502U
Application number: CN201921623277.4U
Authority: CN
Inventors: 张洪艳; 马海涛; 张秀芹; 张美玲
Original assignee: Changchun Guoke Medical Technology Development Co ltd; Beijing Institute of Clothing Technology
Current assignee: Changchun Guoke Xilai Technology Co ltd; Beijing Institute Fashion Technology
Priority date: 2019-09-27
Filing date: 2019-09-27
Publication date: 2020-10-16
Anticipated expiration: 2029-09-27

Abstract

The utility model provides a wavelength/angle modulation free conversion polarisation fluorescence imaging surface plasma resonance appearance, including the positive fluorescence microscopic light path of polarisation, the three-dimensional objective table of sample, cage incident light path, cage reflection light path, electromechanical corner device, data processing system, syringe pump, the three-dimensional objective table of sample is integrated has right angle prism, the sample substrate and the micro-fluidic flow-through cell of multichannel of gold-plated membrane, and the inlet and the liquid outlet of the micro-fluidic flow-through cell of multichannel are passed through the pipeline and are connected with the liquid outlet and the inlet of syringe pump respectively. The utility model discloses can realize having the interact between anisotropic biological sampleThe real-time observation is carried out by polarized fluorescence imaging while the high-sensitivity quantitative and qualitative wavelength angle modulation free conversion surface plasmon resonance detection is carried out, and the refractive index change is as high as 1.0 × 10^‑5The detection sensitivity reaches 10^‑11g/ml, and the image resolution is up to 2 um.

Description

Wavelength/angle modulation free conversion polarized light fluorescence imaging surface plasma resonance instrument

Technical Field

The utility model relates to a multichannel detects biomolecule reaction technical field, more specifically relates to a fluorescence polarisation formation of light surface plasma resonance instrument based on the real-time observation of polarisation fluorescence microsystem and the free conversion surface plasma resonance multichannel micro-fluidic detection technology interact of a wavelength/angle modulation.

Background

In the life science research, the interaction between biomolecules is a basic life phenomenon, and is one of the major problems in modern life science research, and there are many conventional methods for studying the interaction between biomolecules, such as radioimmunoassay, enzyme-linked immunoassay, labeled tracer method, etc. However, since these methods involve different kinds and contents of cells and biomolecules and there is a complex interaction between various substances, it is difficult to accurately obtain related information transfer between biomolecules using these conventional research methods, and meanwhile, there is an urgent need for a method capable of rapidly identifying the interaction between biomolecules with accuracy and high throughput using the increasing number of new proteins and DNA sequence data. In particular, highly ordered molecular assemblies, such as chromosomes, cell walls, cytoplasm, protein chains, supramolecular self-assembly, and the like, are found in many bio-organic systems and chemical materials in biological systems, however, the organization structure and physiological function of these highly ordered molecular assemblies has not been well understood, let alone the information of species interactions in anisotropic systems. The polarization spectrum measurement can provide important information of a complex system containing anisotropic molecular assemblies, interaction information between light and the molecular assemblies can be described through a Mueller matrix, different polarization state differential spectra carry anisotropic distribution information in bipolar absorption, but quantitative kinetic information cannot be obtained, and the flux is limited. Therefore, in the early 90 s, the high-throughput chip detection technology developed along with the biochip technology is mainly divided into two types at present, one is a chemical method, such as an isotope labeling method, a fluorescence labeling method, an electrochemical method and the like, the most used is the fluorescence labeling method at present, a fluorescence laser confocal system is mainly adopted for carrying out high-throughput detection on the microarray biomolecule reaction, the sensitivity of the method is higher, but the sample needs to be subjected to early-stage treatment and is difficult to quantitatively detect, the sample to be detected is damaged and is easy to generate a light bleaching phenomenon; and the other is a physical method, such as a surface plasmon microscope, an atomic force microscope, a mass spectrometry, an ellipsometer and the like, wherein the ellipsometer is widely applied because the ellipsometer does not need to mark an object to be measured and does not cause any disturbance and damage to the activity of a biomolecule to be measured, and has the advantages of high real-time performance and sensitivity and the like. Extinction ellipsometers, photometric ellipsometers, infrared ellipsometers, imaging ellipsometers, generalized ellipsometers, and the like have been developed. The imaging ellipsometer is mainly used for detecting the interaction between the chip biomolecules, and can measure the thickness, the diameter and the three-dimensional appearance of the biomolecules; the scanning of the non-labeling real-time biochip, the measurement of the adsorption and desorption processes of various biomolecules and the kinetic research can observe the change condition in the interaction process between the molecules in real time, and obtain a plurality of pieces of information of the interaction between the biomolecules, which is difficult to provide by the traditional technology. However, since the research in this field is in the development stage, many scientific problems in practical applications are yet to be further explored, for example, imaging ellipsometers generally adopt polychromatic light or multi-wavelength laser light sources, and the mechanical structure is relatively complex; the CCD device is adopted, the polarization state of reflected light of a sample is interfered, strong background signals exist, the imaging speed is low, in-situ high-sensitivity online detection cannot be realized, the data processing is complex, the accuracy is not high enough, and the like; the accuracy measurement of different biochemical reactions of arrays on the same chip of a multi-element array is difficult to realize, most experimental data are obtained under inaccurate experimental design, and the accuracy and the convenience degree of detection are both deficient to a certain extent; specific adsorption cannot be avoided. The multi-channel technology can improve the detection flux to a certain extent and can also avoid the problems in the high-flux detection method. If the polarized light fluorescence microscopy technology is combined with the wavelength/angle free conversion surface plasma resonance technology, the polarized light fluorescence microscopy technology is utilized to obtain the imaging information of the interaction between anisotropic biological samples with high sensitivity, the wavelength/angle free conversion surface plasma resonance technology can expand the detection range of the samples, obtain the interaction kinetic information between the samples, and is assisted with the advantage of economic integration of the microfluidic technology, and the in-situ real-time, multi-channel, qualitative and quantitative multi-element sensitive detection of biological reactions is expected to be realized. At present, no commercial product integrating polarized fluorescence and wavelength/angle modulation surface plasmon resonance technology is available at home and abroad. Although the prior utility model discloses in have surface plasmon resonance device that application number 200710177653.7 was based on laser confocal formation of image and the surface plasmon resonance that application number 201110436241.7 polarization modulation laser confocal formation of image, the cost of confocal part is high, greatly increased anisotropic sample detection's cost, and the range of application receives the restriction of the single modulation mode of surface plasmon resonance also very limited.

SUMMERY OF THE UTILITY MODEL

In view of the above problem, the utility model aims at combining together polarization modulation fluorescence microscopic imaging technique and wavelength/angle modulation free conversion surface plasmon resonance, utilize the high sensitivity ability of wavelength/angle modulation free conversion surface plasmon resonance to acquire the kinetic information of intermolecular interaction, and utilize polarization modulation fluorescence microscopic imaging can realize the characteristics of anisotropic imaging detection, assist with micro-fluidic technique, in order to realize the multichannel to having the interaction between anisotropic biological sample, normal position is real-time, quantitative sensitive detection, thereby provide a wavelength/angle modulation free conversion polarization fluorescence imaging surface plasmon resonance device that can normal position qualitative quantitative detection can observe the biomolecular reaction in real time again.

The utility model discloses above-mentioned purpose is realized through following technical scheme:

the utility model provides a wavelength/angle modulation free conversion polarized light fluorescence imaging surface plasma resonance instrument, which comprises a polarized light positive fluorescence microscope system, a three-dimensional sample objective table, a cage type incident light path, a cage type reflection light path, an electric mechanical corner device, a data processing system and a multi-channel injection pump; the sample polarization upright fluorescence microscope system comprises an excitation light source, a micro-optical filtering block and a micro-objective which are sequentially arranged along the direction of an excitation light path, and a focusing lens, a first polarizer, a filtering block, an analyzer and a CMOS detector which are sequentially arranged along the direction of an emission light path, wherein the laser light path and the emission light path form an upright fluorescence microscope light path; the three-dimensional sample objective table is arranged at the objective table of the upright fluorescence microscopic light path and comprises a right-angle prism, a sample substrate and a multi-channel microfluidic flow cell, wherein the right-angle prism is coupled with the bottom of the sample substrate through refractive index matching liquid; the electromechanical corner device comprises two flat plates and two rotating platforms for fixing the two flat plates, the two rotating platforms are vertically, coaxially and symmetrically arranged, the two flat plates are respectively provided with a groove, and a cage-type incident light path and a cage-type reflection light path are respectively arranged in the two grooves; when the wavelength modulation surface plasma resonance detection is carried out, the cage type incident light path comprises an incident light source, a second polarizer and an incident focusing lens which are sequentially arranged along the direction of the incident light path; the cage type reflection light path comprises a reflection focusing lens and an optical fiber receiving head which are sequentially arranged along the direction of the reflection light path; the data processing system comprises a spectrometer, a data acquisition card and a computer, wherein the spectrometer is provided with a photomultiplier module, the data outlet end of the optical fiber receiving head is connected with the data inlet end of the spectrometer through a data line, the data outlet end of the spectrometer and the data outlet end of the CMOS detector are respectively connected with the data inlet end of the data acquisition card through data lines, the data outlet end of the data acquisition card is connected with one data inlet end of the computer through a data line, and the other data inlet end of the computer is connected with a controller of the multi-channel injection pump through a data line; the incident light source is a white light LED lamp; when angle modulation surface plasma resonance detection is carried out, the cage type incident light path comprises an incident light source, a second polarizer, a chopper and an incident focusing lens which are sequentially arranged along the direction of the incident light path; the cage type reflection light path comprises a reflection focusing lens, a polarization analyzer and a photoelectric detector which are sequentially arranged along the direction of the reflection light path; the data processing system comprises a phase-locked amplifier, a data acquisition card and a computer, the set frequency of a chopper is used as the reference frequency of the phase-locked amplifier, the data inlet end of the phase-locked amplifier is connected with the data outlet end of a photoelectric detector through a data line, the data outlet end of the phase-locked amplifier and the data outlet end of a CMOS detector are respectively connected with the data inlet end of the data acquisition card through the data line, the data outlet end of the data acquisition card is connected with one data inlet end of the computer through the data line, and the other data inlet end of the computer is connected with the controller of the multi-channel injection pump through the data line; wherein, the incident light source is a single-wavelength laser or an LED lamp.

In addition, the preferable structure is that the multi-channel microfluidic flow cell comprises a microfluidic plate and a fixed plate, and the microfluidic plate and the fixed plate are made of PMMA materials.

In addition, it is preferable that the excitation light source is an LED lamp or a mercury lamp.

Further, it is preferable that the single-wavelength laser is a He-Ne laser having a wavelength of 632.8nm when the incident light source is a single-wavelength laser, and the LED lamp has a wavelength of 632nm when the incident light source is an LED lamp.

In addition, the data acquisition card is preferably equipped with a BNC adapter.

In addition, the preferable structure is that the sample substrate is a glass substrate, a chromium film with the thickness of 2nm is plated on the surface of the glass substrate, and a gold film with the thickness of 10-60nm is plated on the chromium film.

Further, it is preferable that the right-angle prism has the same refractive index as the material of the sample substrate.

Further, it is preferable that the photodetector is a silicon photodiode or a CCD.

The technical effects of the utility model reside in that:

(1) the polarizer is added in the fluorescence microscopic light path, and the free conversion surface plasma resonance detection light path is modulated by combining the wavelength and the angle, so that the quantitative sensitive detection of the interaction between biological samples, particularly anisotropic biological samples, and the in-situ real-time fluorescence polarization microscopic imaging observation are realized, the application range of the detection equipment is widened, and the signal to noise ratio and the detection sensitivity are improved by combining with effective mechanical separation.

(3) Can realize multi-channel detection of solid-phase and liquid-phase anisotropic biological samples.

(3) And the microfluidic technology is combined, so that the simultaneous high-throughput detection of various reactions can be really realized.

(4) The device has the advantages of simple structure, low cost and easy operation, can be applied to a plurality of fields of chemistry, biology, medicine, materials, environment, safety and the like, and provides a new high-efficiency research means for the deep research of the scientific problems in the fields of nano science, material science, biochemistry and intersection.

Drawings

Other objects and results of the invention will be more apparent and readily appreciated by reference to the following description taken in conjunction with the accompanying drawings, and as the invention is more fully understood. In the drawings:

fig. 1 is a schematic structural diagram of a wavelength/angle modulation free-switching polarized fluorescence imaging surface plasmon resonance apparatus according to an embodiment of the present invention;

FIG. 2 is a diagram showing the results of the wavelength/angle modulation free-switching polarized fluorescence imaging surface plasmon resonance in detecting the interaction between different bioprotein molecules in four channels on the same sample substrate.

Wherein the reference numerals include: the device comprises a polarized light upright fluorescence microscope system 1, an excitation light source 101, a micro-optical filtering block 102, a micro objective 103, a focusing lens 104, a first polarizer 105, a color filtering block 106, an analyzer 107, a CMOS detector 108, a three-dimensional sample stage 2, a right-angle prism 201, a sample substrate 202, a multi-channel micro-fluidic flow cell 203, a cage-shaped incident light path 3, an incident light source 301, a second polarizer 302, a chopper 303, an incident focusing lens 304, a cage-shaped reflecting light path 4, a reflecting focusing lens 401, a polarization analyzer 402, an optical fiber receiving head 403, a photoelectric detector 403 ', a spectrometer 501, a lock-in amplifier 501', a data acquisition card 502, a computer 503 and a multi-channel injection pump 6.

Detailed Description

In the following description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of one or more embodiments. It may be evident, however, that such embodiment(s) may be practiced without these specific details. In other instances, well-known structures and devices are shown in block diagram form in order to facilitate describing one or more embodiments.

The utility model provides a wavelength/angle modulation free conversion polarisation fluorescence imaging surface plasma resonance appearance, introduce polarization modulation technique on traditional fluorescence microscopic imaging principle, realize that mutual action is real-time polarisation fluorescence imaging observation between anisotropic sample, through electromechanical corner device and the optical element in replacement cage incident light path and the cage reflection light path, realize wavelength/angle modulation free conversion surface plasma resonance and detect, it is in order to avoid non-specific adsorption to assist micro-fluidic and chemical means, realize multichannel normal position, real-time, it is quantitative, the high flux, the accurate detection of multicomponent reaction, can be applied to including the micromolecule, the protein molecule, the macromolecule, the nanometer monomer, the mutual action and the dynamics process realize normal position real-time between the anisotropic species between the cell, ration, in the qualitative sensitive detection, also make research field expand to nanometer from biological system simultaneously, The fields of information, materials and the like, thereby further enriching the understanding and understanding of people to the micro world and providing a new research method and means for revealing basic physicochemical problems in the research of life science, nano science, information science and material science. The utility model discloses an develop and to develop the many units anisotropic biochemical reaction detecting system of the independent intellectual property right of china, improve the international competitiveness of china in the aspect of the biological detection instrument, have huge application prospect and economic value.

Example one

Fig. 1 shows a structure of a wavelength/angle modulation free-conversion polarized fluorescence imaging surface plasmon resonance apparatus according to a first embodiment of the present invention.

As shown in fig. 1 and fig. 2 together, a wavelength/angle modulation free-conversion polarized fluorescence imaging surface plasmon resonance apparatus according to an embodiment of the present invention includes: the device comprises a polarized light upright fluorescence microscope system 1, a three-dimensional sample objective table 2, a cage type incident light path 3, a cage type reflection light path 4, an electromechanical corner device, a data processing system and a multi-channel injection pump 6; wherein, the polarized light positive fluorescence microscopic system 1 is used for carrying out polarized light microscopic imaging to the reaction process between the anisotropic biological samples on the three-dimensional sample objective table 2 and sending the polarized light microscopic imaging to the data processing system, the cage type incident light path 3 and the cage type reflection light path 4 are used for realizing the angle or wavelength modulation surface plasma resonance detection, and the electric mechanical corner device is used for realizing the rotation of the cage type incident light path 3 and the cage type reflection light path 4 in the vertical direction.

The polarized light upright fluorescence microscope system 1 comprises an excitation light source 101, a microscopic optical filtering block 102, a microscopic objective 103, a focusing lens 104, a first polarizer 105, a filtering block 106, an analyzer 107 and a CMOS detector 108 which are sequentially arranged along the direction of an excitation light path, wherein the focusing lens 104, the first polarizer 105, the filtering block 106, the analyzer 107 and the CMOS detector 108 are sequentially arranged along the direction of an emission light path, the laser light path and the emission light path form an upright fluorescence microscope light path, and the polarized fluorescence imaging function is realized by adding the first polarizer 105 and the analyzer 107 into the emission light path. The excitation light source 101 may be an LED lamp or a mercury lamp.

The three-dimensional sample stage 2 is arranged at a stage of the upright fluorescence microscopic light path and comprises a right-angle prism 201, a sample substrate 202 and a multi-channel microfluidic flow cell 203, wherein the right-angle prism 201 is coupled with the bottom of the sample substrate 202 through refractive index matching fluid, the multi-channel microfluidic flow cell 203 is arranged at the top of the sample substrate 202, and a liquid inlet and a liquid outlet of the multi-channel microfluidic flow cell 203 are respectively connected with a liquid outlet and a liquid inlet of a multi-channel injection pump 6 through pipelines.

The right angle prism 201 has dimensions of 20mm × 20mm × 28mm, but may have other dimensions. The sample substrate 202 has a size of 30mm × 25mm × 1mm or other sizes, the surface of the sample substrate 202 is plated with a reflective film, the inner layer of the reflective film is a chromium film with a thickness of 2nm, and the outer layer of the reflective film is a gold film with a thickness of 48 nm. The material of the right-angle prism 201 is the same as that of the sample substrate 202, both are made of K9 or lafn 9 glass, the refractive index of the material of the right-angle prism 201 is the same as that of the material of the sample substrate 202, and the refractive index of the refractive index matching fluid between the right-angle prism 201 and the sample substrate 202 is 1.52 or 1.70.

The peripheral dimension of the multi-channel microfluidic flow cell 203 is 35mm × 25mm × 1.5mm, the multi-channel microfluidic flow cell 203 comprises a microfluidic plate and a fixing plate, the microfluidic plate and the fixing plate are made of PMMA, the thickness of the microfluidic plate is 3mm, the number of channels of the multi-channel microfluidic flow cell 203 is 4-6, and the channel dimension is 25mm × 1mm × 1.5 mm.

The multi-channel injection pump 6 adopts an injection pump with a controller, the capacity of the multi-channel injection pump is 10 mu L-140 ML, and the flow rate of the multi-channel injection pump is 0.001 mu L/hr-147 ML/min.

The electromechanical corner device comprises two flat plates and two rotating platforms for fixing the two flat plates, the two flat plates are driven to rotate through the two rotating platforms, the two rotating platforms are vertically, coaxially and symmetrically arranged, the two flat plates are respectively provided with a groove, the cage-type incident light path 3 and the cage-type reflection light path 4 are respectively arranged in the two grooves, 0-90-degree adjustment can be realized in the vertical direction, and the rotating platforms adopt products of Japanese horse and company, the KS432-75 model and the rotating precision of the rotating platforms is 0.0012.

When the cage-type incident light path 3 and the cage-type reflection light path 4 carry out wavelength modulation surface plasmon resonance detection, the cage-type incident light path 3 comprises an incident light source 301, a second polarizer 302 and an incident focusing lens 304, and the incident light source 301 is a white light LED lamp; the cage-type reflection optical path 4 comprises a reflection focusing lens 401 and an optical fiber receiving head 403 which are sequentially arranged along the direction of the reflection optical path; the data processing system comprises a spectrometer 501 provided with a photomultiplier module, a data acquisition card 502 provided with a BNC adapter (BNC-2110), and a computer 503, wherein a data outlet end of the optical fiber receiving head 403 is connected with a data inlet end of the spectrometer 501 through a data line, a data outlet end of the spectrometer 501 and a data outlet end of the CMOS detector 108 are respectively connected with the data inlet end of the data acquisition card 502 through data lines, the data outlet end of the data acquisition card 502 is connected with one data inlet end of the computer 503 through a data line, and the other data inlet end of the computer 503 is connected with a controller of the multi-channel injection pump 6 through a data line. The wavelength modulation surface plasmon resonance detection can be realized through the matching of the optical fiber receiving head 403 and the spectrometer 501.

When the cage-type incident light path 3 and the cage-type reflected light path 4 perform angle modulation surface plasmon resonance detection, the cage-type incident light path 3 comprises an incident light source 301, a second polarizer 302, a chopper 303 (model is SR540 series products of Stanford research systems company, USA) and a focusing lens 304 which are sequentially arranged along the direction of the incident light path, and the incident light source 301 is a He-Ne laser with the wavelength of 632.8nm or an LED lamp with the wavelength of 632 nm; the cage-shaped reflection light path 4 comprises a reflection focusing lens 401, a polarization analyzer 402 (model is CVI Laser CPAD-10.0-425-; the data processing system comprises a data acquisition card 502 and a computer 503, wherein the data acquisition card 502 is provided with a phase-locked amplifier 501 ', a BNC adapter (BNC-2110) and the computer 503, the set frequency of a chopper 303 is used as the reference frequency of the phase-locked amplifier 501 ', the data inlet end of the phase-locked amplifier 501 ' is connected with the data outlet end of a photoelectric detector 403 ' through a data line, the data outlet end of the phase-locked amplifier 501 ' and the data outlet end of a CMOS detector 108 are respectively connected with the data inlet end of the data acquisition card 502 through a data line, the data outlet end of the data acquisition card 502 is connected with one data inlet end of the computer 503 through a data line, and the other data inlet end of the computer 503 is connected with the controller of the multi-channel injection pump. The angle modulation surface plasmon resonance detection can be realized through the cooperation of the photoelectric detector 403 'and the lock-in amplifier 501'.

The utility model discloses a change cage incident light path 3 and cage reflection light path 4 in partial component realize the free conversion of wavelength and angle modulation.

The data acquisition card 502 is the same as the data acquisition card in the patent granted under 200710177653.7 and the patent granted under 201110436241.7, and the computer 503 is the same as the data processing device in the patent granted under 200710177653.7 and the patent granted under 201110436241.7.

The Anti-immunoglobulin Anti-IgG is chemically modified on the gold film, an aqueous solution containing an anisotropic immunoglobulin IgG antibody molecule labeled with a fluorescent light is circulated in the multi-channel microfluidic flow cell 203, and the Anti-IgG and the IgG antibody molecule react when contacting, and the wavelength/angle modulation free-conversion polarized fluorescence imaging surface plasmon resonance instrument provided by the embodiment can be used for detecting a wavelength-modulated surface plasmon resonance signal and simultaneously obtaining a polarized fluorescence polarization image in the reaction process. The wavelength/angle modulation free-conversion polarized fluorescence imaging surface plasmon resonance provided by the embodiment can detect refractive index change of 2 × 10-5, detection sensitivity of 10-11g/ml and image resolution of 2 μm, as shown in fig. 2, and fig. 2a and 2b show a wavelength modulation surface plasmon resonance kinetic curve and a polarized fluorescence imaging result.

The above results show that: the utility model provides a wavelength/angle modulation free conversion polarisation fluorescence imaging surface plasma resonance appearance can realize the especially anisotropic polarisation fluorescence imaging of multichannel biomolecule reaction and surface plasma resonance dynamics process simultaneously and detect, can carry out ration and qualitative sensitive detection to the biomolecule reaction, and the non-specific absorption in the surface plasma resonance detection can be arranged to the fluorescence image for the testing result is more accurate.

Example two

The photodetector 403' is a photomultiplier tube, for example, model 9863/100B, the rest being the same as in the first embodiment. Compared with the first embodiment, the second embodiment provides a wavelength/angle modulation free-conversion polarized fluorescence imaging surface plasmon resonance instrument, which has improved sensitivity and detection accuracy by 2 orders of magnitude.

EXAMPLE III

The reflective film on the surface of the sample substrate 202 was made of an Au (45nm)/[ TiO2(20nm)/ITO (20nm) ]4/Au (20nm) composite film or an Au (25nm)/[ TiO2(20nm)/SiO2(20nm) ]2/Au (30nm) composite film, and the rest was the same as in example one. Compared with the first embodiment, the third embodiment provides a wavelength/angle modulation free-conversion polarized fluorescence imaging surface plasmon resonance instrument, the sensitivity and the detection accuracy of which can be improved by 1-2 orders of magnitude.

Example four

The reflective film on the surface of the sample substrate 202 is, for example, a gold nano-array structure prepared by a nano-etching method, and the rest is the same as in the first embodiment. Compared with the first embodiment, the sensitivity and the detection precision of the wavelength/angle modulation free-conversion polarized fluorescence imaging surface plasmon resonance instrument provided by the fourth embodiment can be improved by 1-2 orders of magnitude.

EXAMPLE five

The reflective film on the surface of the sample substrate 202 was of Au (45nm)/[ TiO2(20nm)/ITO (20nm) ]4/Au (20nm) nanoarray structure or Au (25nm)/[ TiO2(20nm)/SiO2(20nm) ]2/Au (30nm) nanoarray structure, and the rest was the same as in example one. Compared with the first embodiment, the sensitivity and the detection precision of the wavelength/angle modulation free-conversion polarized fluorescence imaging surface plasmon resonance instrument provided by the fifth embodiment can be improved by 1-3 orders of magnitude.

EXAMPLE six

By adopting the structure of the first embodiment and a chemical modification method, the antibody of the core protein p24 of the AIDS virus type 1 (HIV-1) is modified on the surface of the sample substrate 202, and the antigen of the core protein p24 of the AIDS virus type 1 (HIV-1) with different concentrations flows in the multichannel constant-temperature flow cell 203, and the kinetics analysis of the diagnosis and detection of the AIDS virus type 1 can be simultaneously carried out by adopting the structure of the first embodiment and the chemical modification method.

EXAMPLE seven

By adopting the structure of the first embodiment and a chemical modification method, the surface of the sample substrate 202 is modified with a fluorescent probe capable of identifying mercury ions, biological cell proteins polluted by the mercury ions flow in the multi-channel constant-temperature flow cell 203, and other steps are the same as those of the first embodiment, and the mercury ions in a living body can be detected.

Example eight

By adopting the structure of the first embodiment and a chemical modification method, the surface of the sample substrate 202 is modified with a fluorescent probe capable of identifying zinc ions, biological cell protein polluted by zinc ions flows in the multi-channel constant-temperature flow cell 203, and other steps are the same as those of the first embodiment, so that zinc ions in a living body can be detected.

Example nine

By adopting the structure of the first embodiment and a chemical modification method, the surface of the sample substrate 202 is modified with a fluorescent probe capable of identifying magnesium ions, biological cell protein polluted by magnesium ions flows in the multi-channel constant-temperature flow cell 203, and other steps are the same as those of the first embodiment, so that magnesium ions in a living body can be detected.

Example ten

By adopting the structure of the first embodiment and the chemical modification method, the surface of the sample substrate 202 is modified with the fluorescent probe capable of identifying the fluorine ions, biological cell protein polluted by the fluorine ions flows in the multi-channel constant-temperature flow cell 203, and the fluorine ions in the organism can be detected as in the first embodiment.

EXAMPLE eleven

By adopting the structure of the first embodiment and the chemical modification method, the fluorescent probes capable of simultaneously identifying the mercury ions, the zinc ions, the magnesium ions, the fluorine ions and the cadmium ions are modified in different channels on the surface of the sample substrate 202, biological cell proteins polluted by the mercury ions, the zinc ions, the magnesium ions, the fluorine ions and the cadmium ions flow in the multi-channel constant-temperature flow cell 203, and the other steps are the same as those in the first embodiment, so that the mercury ions, the zinc ions, the magnesium ions, the fluorine ions and the cadmium ions in a living body can be simultaneously detected.

Example twelve

The tumor markers such as alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), sugar chain antigen 19-19(CA199), sugar chain antigen 24-2(CA242) and the like are modified to different channels on the surface of the same sample substrate 202, and the other steps are the same as the first embodiment, so that the real-time quantitative detection of various tumor markers can be realized simultaneously.

The above description is only for the specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto, and any person skilled in the art can easily think of the changes or substitutions within the technical scope of the present invention, and all should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. A wavelength/angle modulation free conversion polarized light fluorescence imaging surface plasma resonance instrument comprises a multi-channel injection pump (6) and an electromechanical corner device, and is characterized by further comprising a polarized light positive fluorescence microscope system (1), a three-dimensional sample stage (2), a cage type incident light path (3), a cage type reflection light path (4) and a data processing system; wherein,

the sample polarization upright fluorescence microscope system (1) comprises an excitation light source (101), a micro-optical color filtering block (102), a micro objective (103) and a focusing lens (104), a first polarizer (105), a color filtering block (106), an analyzer (107) and a CMOS detector (108) which are sequentially arranged along the direction of an excitation light path, wherein the focusing lens (104), the first polarizer (105), the color filtering block, the analyzer (107) and the CMOS detector (108) are sequentially arranged along the direction of an emission light path, and the excitation light path and the emission light path form an upright fluorescence microscope path;

the three-dimensional sample stage (2) is arranged at the stage of the upright fluorescence microscopic light path and comprises a right-angle prism (201), a sample substrate (202) and a multi-channel microfluidic flow cell (203), the right-angle prism (201) is coupled with the bottom of the sample substrate (202) through a refractive index matching fluid, the multi-channel microfluidic flow cell (203) is arranged at the top of the sample substrate (202), and a liquid inlet and a liquid outlet of the multi-channel microfluidic flow cell (203) are respectively connected with a liquid outlet and a liquid inlet of the injection pump (6) through pipelines;

the electromechanical corner device comprises two flat plates and two rotating platforms for fixing the two flat plates, the two rotating platforms are vertically, coaxially and symmetrically arranged, the two flat plates are respectively provided with a groove, and the cage-type incident light path (3) and the cage-type reflecting light path (4) are respectively arranged in the two grooves;

the cage-type incident light path (3) comprises an incident light source (301), a second polarizer (302) and an incident focusing lens (304) which are sequentially arranged along the direction of the incident light path; the cage type reflection light path (4) comprises a reflection focusing lens (401) and an optical fiber receiving head (403) which are sequentially arranged along the direction of the reflection light path; the data processing system comprises a spectrometer (501) provided with a photomultiplier module, a data acquisition card (502) and a computer (503), wherein the data outlet end of the optical fiber receiving head (403) is connected with the data inlet end of the spectrometer (501) through a data line, the data outlet end of the spectrometer (501) and the data outlet end of the CMOS detector (108) are respectively connected with the data inlet end of the data acquisition card (502) through a data line, the data outlet end of the data acquisition card (502) is connected with one data inlet end of the computer (503) through a data line, and the other data inlet end of the computer (503) is connected with the controller of the multi-channel injection pump (6) through a data line; wherein the incident light source (301) is a white LED lamp; or,

the cage-type incident light path (3) comprises an incident light source (301), a second polarizer (302), a chopper (303) and an incident focusing lens (304) which are sequentially arranged along the direction of the incident light path; the cage type reflection light path (4) comprises a reflection focusing lens (401), a polarization analyzer (402) and a photoelectric detector (403') which are sequentially arranged along the direction of the reflection light path; the data processing system comprises a phase-locked amplifier (501 '), a data acquisition card (502) and a computer (503), the set frequency of the chopper (303) is used as the reference frequency of the phase-locked amplifier (501 '), the data inlet end of the phase-locked amplifier (501 ') is connected with the data outlet end of the photoelectric detector (403 ') through a data line, the data outlet end of the phase-locked amplifier (501 ') and the data outlet end of the CMOS detector (108) are respectively connected with the data inlet end of the data acquisition card (502) through data lines, the data outlet end of the data acquisition card (502) is connected with one data inlet end of the computer (503) through a data line, and the other data inlet end of the computer (503) is connected with the controller of the multi-channel injection pump (6) through a data line; wherein the incident light source (301) is a single wavelength laser or an LED lamp.

2. The wavelength/angle modulation free-switching polarized fluorescence imaging surface plasmon resonance apparatus of claim 1, wherein the multi-channel microfluidic flow cell (203) comprises a microfluidic plate and a fixed plate, and the microfluidic plate and the fixed plate are both made of PMMA.

3. The wavelength/angle modulation free-switching polarized fluorescence imaging surface plasmon resonance apparatus of claim 2, wherein: the excitation light source (101) is an LED lamp or a mercury lamp.

4. The wavelength/angle-modulated free-switching polarized fluorescence imaging surface plasmon resonance instrument of claim 3, characterized in that when the incident light source (301) is a single wavelength laser, the single wavelength laser is a He-Ne laser with a wavelength of 632.8nm, and when the incident light source (301) is an LED lamp, the LED lamp has a wavelength of 632 nm.

5. The wavelength/angle modulation free-switching polarized fluorescence imaging surface plasmon resonance instrument of claim 1, wherein said data acquisition card (502) is equipped with a BNC adapter.

6. The wavelength/angle-modulated free-switching polarized fluorescence imaging surface plasmon resonance apparatus of claim 1, wherein said sample substrate (202) is a glass substrate, a 2nm thick chromium film is coated on the surface of said glass substrate, and a 10-60nm thick gold film is coated on said chromium film.

7. The wavelength/angle-modulation free-switching polarized fluorescence imaging surface plasmon resonance apparatus of claim 1, wherein the right angle prism (201) and the material of the sample substrate (202) have the same refractive index.

8. The wavelength/angle modulation free-switching polarized fluorescence imaging surface plasmon resonance instrument of claim 1, wherein the photodetector (403') is a silicon photodiode or a CCD.