CN110806401A - Wavelength/angle modulation free conversion polarized light fluorescence imaging surface plasma resonance instrument - Google Patents
Wavelength/angle modulation free conversion polarized light fluorescence imaging surface plasma resonance instrument Download PDFInfo
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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
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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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
- G01N21/6445—Measuring fluorescence polarisation
Abstract
The invention provides a wavelength/angle modulation free conversion polarized light fluorescence imaging surface plasma resonance instrument, which comprises a polarized light positive fluorescence microscopic light path, a sample three-dimensional objective table, a cage type incident light path, a cage type reflection light path, an electric mechanical corner device, a data processing system and an injection pump, wherein the sample three-dimensional objective table is integrated with a right-angle prism, a sample substrate coated with a gold film and a multi-channel microfluidic flow cell, and a liquid inlet and a liquid outlet of the multi-channel microfluidic flow cell are respectively connected with a liquid outlet and a liquid inlet of the injection pump through pipelines. The invention can realize the high-sensitivity quantitative and qualitative wavelength angle modulation free conversion surface plasma resonance detection of the interaction between anisotropic biological samples and simultaneously realize real-time observation through polarized light fluorescence imaging; the refractive index variation is up to 1.0 × 10‑5The detection sensitivity reaches 10‑11g/ml, and the image resolution is up to 2 um. The invention gives consideration to all levels of standing balance training modes and meets the requirement of standing balance and stability training of each stage of a patient group.
Description
Technical Field
The invention relates to the technical field of multi-channel detection of biomolecular reaction, in particular to a fluorescence polarization imaging surface plasma resonance instrument based on interaction of real-time observation of a polarization fluorescence microscope system and a wavelength/angle modulation free conversion surface plasma resonance multi-channel micro-fluidic detection technology.
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 invention has a surface plasma resonance device with the application number of 200710177653.7 based on laser confocal imaging and a surface plasma resonance device with the application number of 201110436241.7 polarization modulation laser confocal imaging, the manufacturing cost of a confocal part is high, the cost of anisotropic sample detection is greatly increased, and the application range is limited by the single modulation mode of the surface plasma resonance.
Disclosure of Invention
In view of the above problems, the present invention aims to combine the polarization modulation fluorescence microscopic imaging technology with the wavelength/angle modulation free conversion surface plasmon resonance, utilize the high sensitivity of the wavelength/angle modulation free conversion surface plasmon resonance to obtain the kinetic information of the intermolecular interaction, and utilize the polarization modulation fluorescence microscopic imaging to realize the anisotropic imaging detection, and the microfluidic technology is assisted to realize the multi-channel, in-situ, real-time, quantitative and sensitive detection of the interaction between anisotropic biological samples, thereby providing a wavelength/angle modulation free conversion polarized light fluorescence imaging surface plasmon resonance apparatus which can perform both the in-situ quantitative detection and the real-time observation of the biomolecular reaction.
The above purpose of the invention is realized by the following technical scheme:
the invention 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, wherein the cage type incident light path is arranged on the polarized light positive fluorescence microscope system; the sample polarization upright fluorescence microscope system comprises an excitation light source, a microscopic optical filtering block and a microscopic objective which are sequentially arranged along the direction of an excitation light path, and a focusing lens, a 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 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 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 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.
In addition, it is preferable that the photodetector is a silicon photodiode or a CCD.
The invention has the technical effects 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 present invention will become more apparent and more readily appreciated as the same becomes better understood by reference to the following description taken in conjunction with the accompanying drawings. In the drawings:
FIG. 1 is a schematic 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 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-type incident light path 3, an incident light source 301, a polarizer 302, a chopper 303, an incident focusing lens 304, a cage-type reflected light path 4, a reflected 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 invention provides a wavelength/angle modulation free conversion polarized light fluorescence imaging surface plasma resonance instrument, which introduces a polarization modulation technology on the traditional fluorescence microscopic imaging principle to realize the real-time polarized light fluorescence imaging observation of the interaction between anisotropic samples, realizes the wavelength/angle modulation free conversion surface plasma resonance detection by an electromechanical corner device and replacing optical elements in a cage-type incident light path and a cage-type reflection light path, is assisted by micro-fluidic and chemical means to avoid non-specific adsorption, realizes the accurate detection of multichannel in-situ, real-time, quantitative, qualitative, high-flux and multi-element reaction, can be applied to the realization of in-situ real-time, quantitative and qualitative sensitive detection of the interaction between anisotropic species and the kinetic process between micromolecules, protein molecules, macromolecules, nano monomers and cells, and simultaneously expands the research field from a biological system to nano, 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 development of the invention can develop a multivariate anisotropic biochemical reaction detection system of the independent intellectual property rights of China, improve the international competitiveness of China in the aspect of biological detection instruments and have huge application prospect and economic value.
Example one
Fig. 1 shows a structure of a wavelength/angle modulation free-switching polarized fluorescence imaging surface plasmon resonance apparatus according to an embodiment of the present invention.
As shown in fig. 1 and fig. 2 together, a wavelength/angle modulation free-switching 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 and a microscopic objective 103 which are sequentially arranged along the direction of an excitation light path, and a focusing lens 104, a polarizer 105, a filtering block 106, an analyzer 107 and a CMOS detector 108 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, and the polarizer 105 and the analyzer 107 are added into the emission light path to realize the polarized light fluorescence imaging function. 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 20 mm × 20 mm × 28 mm, but may have other dimensions. The sample substrate 202 has a size of 30 mm × 25mm × 1 mm 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 35 mm × 25mm × 1.5 mm, 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 × 1 mm × 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 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 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 invention realizes free conversion of wavelength and angle modulation by replacing part of elements in the cage type incident light path 3 and the cage type reflection light path 4.
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 the refractive index change of 2 multiplied by 10-5The detection sensitivity is as high as 10-11g/ml, high image resolutionUp to 2 μm, as shown in fig. 2, fig. 2a and 2b show wavelength modulated surface plasmon resonance kinetic curves and polarized fluorescence imaging results.
The above results show that: the wavelength/angle modulation free conversion polarized light fluorescence imaging surface plasma resonance instrument provided by the invention can simultaneously realize multi-channel biomolecular reaction, particularly anisotropic polarized light fluorescence imaging and surface plasma resonance dynamic process detection, can carry out quantitative and qualitative sensitive detection on the biomolecular reaction, and can eliminate non-specific adsorption in the surface plasma resonance detection by a fluorescence image, so that the detection 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 sensitivity and the detection precision of the wavelength/angle modulation free-conversion polarized fluorescence imaging surface plasmon resonance instrument provided by the second embodiment can be improved 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 (CA 199), sugar chain antigen 24-2 (CA 242) 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 scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (8)
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 content of the first and second substances,
the sample polarization upright fluorescence microscope system (1) comprises an excitation light source (101), a microscopic optical color filtering block (102) and a microscopic objective (103) which are sequentially arranged along the direction of an excitation light path, and a focusing lens (104), a polarizer (105), a color filtering block (106), an analyzer (107) and a CMOS detector (108) 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 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;
when the wavelength modulation surface plasma resonance detection is carried out, the cage-shaped incident light path (3) comprises an incident light source (301), a 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;
when angle modulation surface plasma resonance detection is carried out, the cage-shaped incident light path (3) comprises an incident light source (301), a polarizer (302), a chopper (303) and a 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 3, 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 wavelength 632.8nm, and when the incident light source (301) is an LED lamp, the LED lamp has wavelength 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-modulating 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.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112268885A (en) * | 2020-10-22 | 2021-01-26 | 清华大学 | Semiconductor nanowire photoluminescence characteristic in-situ characterization system for scanning electron microscope |
| CN113109297A (en) * | 2021-03-04 | 2021-07-13 | 北京化工大学 | Small multifunctional biochemical analyzer based on total internal reflection resonance principle |
| CN114994346A (en) * | 2022-08-04 | 2022-09-02 | 天津长和生物技术有限公司 | Cell detection device and detection method thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112268885A (en) * | 2020-10-22 | 2021-01-26 | 清华大学 | Semiconductor nanowire photoluminescence characteristic in-situ characterization system for scanning electron microscope |
| CN112268885B (en) * | 2020-10-22 | 2021-07-09 | 清华大学 | Semiconductor nanowire photoluminescence characteristic in-situ characterization system for scanning electron microscope |
| CN113109297A (en) * | 2021-03-04 | 2021-07-13 | 北京化工大学 | Small multifunctional biochemical analyzer based on total internal reflection resonance principle |
| CN113109297B (en) * | 2021-03-04 | 2023-04-28 | 北京化工大学 | Small-sized multifunctional biochemical analyzer based on total internal reflection resonance principle |
| CN114994346A (en) * | 2022-08-04 | 2022-09-02 | 天津长和生物技术有限公司 | Cell detection device and detection method thereof |
| CN114994346B (en) * | 2022-08-04 | 2022-10-04 | 天津长和生物技术有限公司 | Cell detection device and detection method thereof |
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