CN110806401B - Wavelength/angle modulation free conversion polarization fluorescence imaging surface plasmon resonance instrument - Google Patents
Wavelength/angle modulation free conversion polarization fluorescence imaging surface plasmon resonance instrument Download PDFInfo
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Abstract
本发明提供一种波长/角度调制自由转换偏光荧光成像表面等离子共振仪，包括偏光正置荧光显微光路、样品三维载物台、笼式入射光路、笼式反射光路、电动机械转角装置、数据处理系统、注射泵，样品三维载物台集成有直角棱镜、镀金膜的样品基片和多通道微流控流通池，多通道微流控流通池的进液口和出液口通过管路分别与注射泵的出液口和进液口连接。本发明能够实现对具有各向异性生物样品间的相互作用进行高灵敏定量和定性波长角度调制自由转换表面等离子共振检测的同时通过偏光荧光成像实时观察；折射率变化高达1.0×10‑5，检测灵敏度达10‑11 g/ml，图像分辨率高达2 um。本发明兼顾各级站立平衡训练模式，满足患者群各阶段站立平衡稳定训练需求。
The present invention provides a wavelength/angle modulation free conversion polarized light fluorescence imaging surface plasma resonance instrument, including a polarized upright fluorescence microscope optical path, a sample three-dimensional stage, a cage-type incident optical path, a cage-type reflected optical path, an electric mechanical corner device, a data processing system, and a syringe pump. The sample three-dimensional stage is integrated with a right-angle prism, a sample substrate with a gold-plated film, and a multi-channel microfluidic flow cell. The inlet and outlet of the multi-channel microfluidic flow cell are respectively connected to the outlet and inlet of the syringe pump through a pipeline. The present invention can realize highly sensitive quantitative and qualitative wavelength angle modulation free conversion surface plasma resonance detection of the interaction between anisotropic biological samples while real-time observation through polarized fluorescence imaging; the refractive index change is up to 1.0×10 ‑5 , the detection sensitivity is up to 10 ‑11 g/ml, and the image resolution is up to 2 um. The present invention takes into account the standing balance training mode at all levels and meets the standing balance stability training needs of the patient group at all stages.
Description
Technical Field
The invention relates to the technical field of multichannel detection biomolecular reaction, in particular to a fluorescence polarized imaging surface plasma resonance instrument based on interaction of real-time observation of a polarized fluorescence microscopy system and a wavelength/angle modulation free conversion surface plasma resonance multichannel microfluidic detection technology.
Background
In life science research, interactions between biomolecules are a basic life phenomenon, and are one of the major problems in modern life science research, and various traditional methods for researching interactions between biomolecules are available, such as radioimmunoassay, enzyme-linked immunoassay, labeling and tracing methods. However, since these methods involve different kinds and amounts of cells and biomolecules and there are complex interactions between various substances, it is difficult to accurately obtain the related transfer information between biomolecules using these conventional research methods, and there is an urgent need for a method capable of accurately and rapidly identifying interactions between biomolecules with high throughput using increasingly new protein and DNA sequence data. In particular, highly ordered molecular assemblies such as chromosomes, cell walls, cytoplasms, protein chains, and supramolecular self-assembly are found in many bio-organic systems and chemical materials in biological systems, however, it has not been clear that the tissue structure and physiological function of these highly ordered molecular assemblies, let alone the information of species interactions in anisotropic systems. Polarization spectrometry 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, and differential spectra of different polarization states 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-flux chip detection technology developed along with the biochip technology is mainly divided into two types, namely a chemical method, such as a position element marking method, a fluorescent marking method, an electrochemical method and the like, and the most used fluorescent marking method is the fluorescent marking method at present, and a fluorescent laser confocal system is mainly adopted for high-flux detection of the micro-array biomolecule reaction, so that the method has higher sensitivity, but the sample needs to be subjected to pretreatment, the quantitative detection is difficult, the sample to be detected is damaged, and the phenomenon of light floating easily occurs; and the second 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 detected, does not cause any disturbance and damage to the activity of biological molecules to be detected, and has the advantages of real time, higher sensitivity and the like. Extinction ellipsometers, photometric ellipsometers, infrared ellipsometers, imaging ellipsometers, generalized ellipsometers, and the like have appeared. The imaging ellipsometer is mainly used for detecting the interaction between the chip biomolecules, and can be used for measuring the thickness, the diameter and the three-dimensional morphology of the biomolecules; the scanning of the non-marked real-time biochip, the measurement of adsorption and desorption processes of various biomolecules and the dynamic research can be used for observing the change condition of the interaction process among the molecules in real time, so as to obtain the information of the interaction among the biomolecules which is difficult to be provided by the traditional technology. However, as the research in the field is in the development stage, many scientific problems in practical application remain to be further explored, such as an imaging ellipsometer generally adopts a polychromatic light or multi-wavelength laser source, and the mechanical structure is relatively complex; the CCD device is adopted, so that the polarization state of reflected light of a sample is disturbed, a strong background signal exists, 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 precision measurement of different biochemical reactions on the array on the same chip of the multi-element array is difficult to realize, most experimental data are obtained under the inaccurate experimental design, and the precision and the convenience degree of detection are deficient to a certain extent; specific adsorption and the like cannot be avoided. The multichannel technology can improve the detection flux to a certain extent and can also avoid the problems existing in the high-flux detection method. If the polarized fluorescence microscopy technology is combined with the wavelength/angle free conversion surface plasma resonance technology, the polarized fluorescence microscopy technology is utilized to obtain the interaction imaging information 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 the advantage of economic integration of the microfluidic technology is assisted, so that the in-situ real-time, multichannel, qualitative and quantitative multiplex sensitive detection of biological reactions is hopefully realized. At present, no commercial product integrating polarized fluorescence and wavelength/angle modulation surface plasma resonance technology exists at home and abroad. Although the prior invention has a surface plasma resonance device with the application number 200710177653.7 based on laser confocal imaging and surface plasma resonance with the application number 201110436241.7 polarization modulation laser confocal imaging, the cost of the confocal part is high, the cost of anisotropic sample detection is greatly increased, and the application range is limited by a 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 microscopy imaging technology with the wavelength/angle modulation free conversion surface plasmon resonance, obtain the kinetic information of the intermolecular interaction by utilizing the high sensitivity performance of the wavelength/angle modulation free conversion surface plasmon resonance, and realize the anisotropic imaging detection by utilizing the characteristic of the polarization modulation fluorescence microscopy imaging, and assist the microfluidic technology to realize the multichannel, in-situ real-time and quantitative sensitive detection of the interactions between anisotropic biological samples, thereby providing a wavelength/angle modulation free conversion fluorescence imaging surface plasmon resonance device capable of in-situ qualitative and quantitative detection and real-time observation of the biomolecular reaction.
The above object of the present invention is achieved by the following technical scheme:
The invention provides a wavelength/angle modulation free-conversion polarized light fluorescence imaging surface plasma resonator, which comprises a polarized light forward fluorescence microscope system, a three-dimensional sample object stage, a cage-type incident light path, a cage-type reflecting light path, an electromechanical corner device, a data processing system and a multichannel injection pump; the sample polarization forward fluorescence microscopy system comprises an excitation light source, a micro optical color filter block, a micro objective lens, a focusing lens, a first polarizer, a color filter block, an analyzer and a CMOS detector, wherein the excitation light source, the micro optical color filter block and the micro objective lens are sequentially arranged along the direction of an excitation light path, and the focusing lens, the first polarizer, the color filter block, the analyzer and the CMOS detector are sequentially arranged along the direction of an emission light path; the three-dimensional sample stage is arranged at the stage of the forward fluorescence microscopic light path and comprises a rectangular prism, a sample substrate and a multichannel microfluidic flow cell, wherein the rectangular prism is coupled with the bottom of the sample substrate through refractive index matching liquid, the multichannel microfluidic flow cell is arranged at the top of the sample substrate, and a liquid inlet and a liquid outlet of the multichannel microfluidic flow cell are respectively connected with a liquid outlet and a liquid inlet of the injection pump through pipelines; the electromechanical corner device comprises two flat plates and two rotating platforms for fixing the two flat plates, wherein 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 reflecting 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 spectrum instrument provided with a photomultiplier module, a data acquisition card and a computer, wherein the data outlet end of the optical fiber receiving head is connected with the data inlet end of the spectrum instrument through a data line, the data outlet end of the spectrum instrument and the data outlet end of the CMOS detector are respectively connected with the data inlet end of the data acquisition card through a data line, 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 the controller of the multichannel injection pump through a data line; the incident light source is a white light LED lamp; when the 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 lock-in amplifier, a data acquisition card and a computer, wherein the set frequency of the chopper is used as the reference frequency of the lock-in amplifier, the data inlet end of the lock-in amplifier is connected with the data outlet end of the photoelectric detector through a data line, the data outlet end of the lock-in amplifier 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 the controller of the multichannel injection pump through a data line; the incident light source is a single-wavelength laser or an LED lamp.
In addition, the multichannel microfluidic flow cell preferably comprises a microfluidic plate and a fixing plate, wherein the microfluidic plate and the fixing plate are made of PMMA.
In addition, the excitation light source is preferably 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.
Furthermore, the preferred construction is that the data acquisition card is equipped with a BNC adapter.
In addition, 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 and the sample substrate are made of the same material having the same refractive index.
Furthermore, the preferred construction is 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 wavelength angle modulation free conversion surface plasma resonance detection light path is combined to be motionless, so that interaction quantitative sensitive detection and in-situ real-time fluorescence polarized light microscopic imaging observation of biological samples, particularly anisotropic biological samples, are realized, the application range of detection equipment is widened, and the signal to noise ratio and the detection sensitivity are improved by combining effective mechanical decoupling.
(3) The multi-channel detection of the solid-phase and liquid-phase anisotropic biological samples can be realized.
(3) The microfluidic technology is combined, so that multiple reactions can be truly realized, and high-flux detection can be realized at the same time.
(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 scientific problems in the fields of nano science, material science, biochemistry and intersection.
Drawings
Other objects and attainments together with a more complete understanding of the invention will become apparent and appreciated by referring 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-conversion polarized fluorescence imaging surface plasmon resonance apparatus according to an embodiment of the present invention;
FIG. 2 is a graph showing the results of wavelength/angle modulation free-switching polarized fluorescence imaging Surface Plasmon Resonance (SPR) detection of interactions between different biological protein molecules in four channels on the same sample substrate.
Wherein reference numerals include: the system comprises a polarized light forward fluorescence microscope system 1, an excitation light source 101, a micro optical color filter block 102, a micro objective lens 103, a focusing lens 104, a first polarizer 105, a color filter block 106, an analyzer 107, a CMOS detector 108, a three-dimensional sample stage 2, a rectangular prism 201, a sample substrate 202, a multichannel microfluidic flow cell 203, a cage-type incident light path 3, an incident light source 301, a second polarizer 302, a chopper 303, an incident focusing lens 304, a cage-type reflected light path 4, a reflection 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 multichannel 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 in 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 an optical element in a displacement cage-type incident light path and a cage-type reflection light path, is assisted with micro-fluidic and chemical means to avoid nonspecific adsorption, realizes the precise detection of multi-channel in-situ, real-time, quantitative, qualitative, high-flux and multi-element reactions, can be applied to the in-situ real-time, quantitative and qualitative sensitive detection of the interaction and dynamic process among anisotropic species including small molecules, protein molecules, macromolecules, nanometer monomers and cells, and simultaneously expands the research field from a biological system to the fields of nanometer, information, materials and the like, thereby further enriching the understanding and understanding of people on the microcosmic world, and providing new research methods and means for revealing basic physical and chemical problems in life science, nanometer science, information science and material science research. The development of the invention can develop a multi-element anisotropic biochemical reaction detection system of independent intellectual property rights in China, improves international competitiveness of China in terms of biological detection instruments, and has great application prospect and economic value.
Example 1
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 light fluorescent imaging surface plasmon resonance apparatus according to a first embodiment of the present invention includes: the device comprises a polarized light orthotopic fluorescence microscope system 1, a three-dimensional sample object stage 2, a cage-type incident light path 3, a cage-type reflecting light path 4, an electromechanical corner device, a data processing system and a multichannel injection pump 6; the polarized light forward fluorescence microscopy system 1 is used for carrying out polarized light microscopic imaging on the reaction process between anisotropic biological samples on the three-dimensional sample object stage 2 and sending the polarized light microscopic imaging to the data processing system, the cage-type incident light path 3 and the cage-type reflecting light path 4 are used for realizing angle or wavelength modulation surface plasma resonance detection, and the electromechanical corner device is used for realizing rotation of the cage-type incident light path 3 and the cage-type reflecting light path 4 in the vertical direction.
The polarized light forward fluorescence microscope system 1 comprises an excitation light source 101, a micro-optical color filter block 102, a micro-objective 103, a focusing lens 104, a first polarizer 105, a color filter block 106, an analyzer 107 and a CMOS detector 108 which are sequentially arranged along the direction of an excitation light path, wherein the laser light path and the emission light path form a forward fluorescence microscopic light path, and a polarized light 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 the stage of the forward fluorescence microscopic light path and comprises a rectangular prism 201, a sample substrate 202 and a multichannel microfluidic flow cell 203, wherein the rectangular prism 201 is coupled with the bottom of the sample substrate 202 through refractive index matching liquid, the multichannel microfluidic flow cell 203 is arranged at the top of the sample substrate 202, and a liquid inlet and a liquid outlet of the multichannel microfluidic flow cell 203 are respectively connected with a liquid outlet and a liquid inlet of the multichannel injection pump 6 through pipelines.
The rectangular prism 201 has dimensions of 20 mm x 20 mm x 28 mm, but may have other dimensions. The sample substrate 202 has dimensions of 30 mm ×25 mm ×1mm or other dimensions, the surface of the sample substrate 202 is coated with a reflective film, the inner layer of the reflective film is a 2nm thick chromium film, and the outer layer of the reflective film is a 48 nm thick gold film. The right-angle prism 201 and the sample substrate 202 are made of K9 or LaSFN9 glass, and the refractive indexes of the right-angle prism 201 and the sample substrate 202 are the same, and the refractive index of the refractive index matching liquid between the right-angle prism 201 and the sample substrate 202 is 1.52 or 1.70.
The peripheral size of the multichannel microfluidic flow cell 203 is 35 mm ×25mm ×1.5 mm, the multichannel 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 multichannel microfluidic flow cell 203 is 4-6, and the channel size is 25mm×1mm ×1.5 mm.
The multichannel syringe pump 6 employs a syringe pump with a controller, and its capacity is exemplified by 10. Mu.L-140 ML, and its flow rate 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 and coaxially symmetrically arranged, the two flat plates are respectively provided with grooves, a cage-type incident light path 3 and a cage-type reflecting light path 4 are respectively arranged in the two grooves, 0-90-degree adjustment can be achieved in the vertical direction, the rotating platforms adopt KS432-75 products manufactured by Japanese jun and company, and the rotating precision is 0.0012 degrees.
When the cage-type incident light path 3 and the cage-type reflecting light path 4 carry out wavelength modulation surface plasma 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 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 wire, 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 wire, the data outlet end of the data acquisition card 502 is connected with one data inlet end of the computer 503 through a data wire, and the other data inlet end of the computer 503 is connected with the controller of the multichannel injection pump 6 through a data wire. Wavelength modulated surface plasmon resonance detection can be achieved by the cooperation of the fiber receiving head 403 with the spectroscopic instrument 501.
When the cage-type incident light path 3 and the cage-type reflecting light path 4 perform angle modulation surface plasma 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 product of Stuft research systems Co., USA) and an incident focusing lens 304 which are sequentially arranged along the direction of the incident light path, wherein 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-type reflection light path 4 comprises a reflection focusing lens 401, a polarization analyzer 402 (model number CVI LASER CPAD-10.0-425-675) and a photoelectric detector 403' which are sequentially arranged along the direction of the reflection light path; the data processing system comprises a data acquisition card 502 provided with a lock-in amplifier 501 ', a BNC adapter (BNC-2110) and a computer 503, wherein the set frequency of the chopper 303 is used as the reference frequency of the lock-in amplifier 501 ', the data inlet end of the lock-in amplifier 501 ' is connected with the data outlet end of the photoelectric detector 403 ' through a data line, the data outlet end of the lock-in 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. The angle modulation surface plasmon resonance detection can be realized by the cooperation of the photodetector 403 'and the lock-in amplifier 501'.
The invention realizes the free conversion of wavelength and angle modulation by replacing part of elements in the cage-type incident light path 3 and the cage-type reflecting light path 4.
The data acquisition card 502 is the same as the data acquisition card in 200710177653.7 and 201110436241.7, and the computer 503 is the same as the data processing device in 200710177653.7 and 201110436241.7.
The gold film is provided with Anti-immunoglobulin Anti-IgG modified by a chemical method, the multichannel microfluidic flow cell 203 is flowed with an aqueous solution containing fluorescent labeled anisotropic immunoglobulin IgG antibody molecules, the Anti-IgG reacts when contacting with the IgG antibody molecules, the wavelength/angle modulation free conversion polarized fluorescence imaging surface plasmon resonance instrument provided by the embodiment can detect the surface plasmon resonance signal modulated by the wavelength, and polarized fluorescence polarization images in the reaction process are obtained. The refractive index change that can be detected by the wavelength/angle modulation free-conversion polarized fluorescence imaging surface plasmon resonance apparatus provided in the first embodiment can reach 2×10 -5, the detection sensitivity can reach 10 -11 g/ml, the image resolution can reach 2 μm, as shown in fig. 2, and fig. 2a and 2b show the wavelength modulation surface plasmon resonance dynamics curve and polarized fluorescence imaging result.
The above results indicate that: the wavelength/angle modulation free-conversion polarized light fluorescence imaging surface plasmon resonance instrument provided by the invention can simultaneously realize multichannel biomolecular reaction, especially anisotropic polarized light fluorescence imaging and surface plasmon resonance dynamic process detection, can perform quantitative and qualitative sensitive detection on biomolecular reaction, and can discharge nonspecific adsorption in surface plasmon resonance detection by fluorescent images, so that the detection result is more accurate.
Example two
The photodetector 403' employs, for example, a photomultiplier tube model 9863/100B, otherwise identical to that of the first embodiment. Compared with the first embodiment, the sensitivity and the detection precision of the wavelength/angle modulation free-conversion polarized light fluorescence imaging surface plasma resonator 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 an Au (45 nm)/[ TiO2 (20 nm)/ITO (20 nm) ]4/Au (20 nm) complex film or an Au (25 nm)/[ TiO2 (20 nm)/SiO 2 (20 nm) ]2/Au (30 nm) complex film, and the other 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 light fluorescence imaging surface plasma resonator provided by the third embodiment can be improved by 1-2 orders of magnitude.
Example IV
The reflective film on the surface of the sample substrate 202 adopts a gold nano array structure prepared by nano etching, for example, and the other steps are the same as those in the first embodiment. Compared with the first embodiment, the sensitivity and the detection precision of the wavelength/angle modulation free-conversion polarized light fluorescence imaging surface plasma resonator 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 (45 nm)/[ TiO2 (20 nm)/ITO (20 nm) ]4/Au (20 nm) nanoarray structure or Au (25 nm)/[ TiO2 (20 nm)/SiO 2 (20 nm) ]2/Au (30 nm) nanoarray structure, and the other 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 light fluorescence imaging surface plasma resonator 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 adopting a chemical modification method, antibodies of HIV 1 type (HIV-1) core protein p24 are modified on the surface of a sample substrate 202, antigens of HIV 1 type (HIV-1) core protein p24 with different concentrations flow into a multichannel constant temperature flow cell 203, and other dynamic analysis of various concentration diagnostic tests can be carried out on HIV 1 type simultaneously.
Example seven
The structure of the first embodiment is adopted, a fluorescent probe capable of identifying mercury ions is modified on the surface of the sample substrate 202 by adopting a chemical modification method, and biological cell proteins polluted by the mercury ions flow into the multichannel constant temperature flow cell 203, and other methods are the same as the first embodiment, so that the mercury ions in the living body can be detected.
Example eight
The structure of the first embodiment is adopted, a fluorescent probe capable of identifying zinc ions is modified on the surface of the sample substrate 202 by adopting a chemical modification method, biological cell proteins polluted by zinc ions flow in the multichannel constant temperature flow cell 203, and other methods are the same as the first embodiment, so that zinc ions in a living body can be detected.
Example nine
The structure of the first embodiment is adopted, a fluorescent probe capable of recognizing magnesium ions is modified on the surface of the sample substrate 202 by a chemical modification method, biological cell proteins polluted by magnesium ions flow in the multichannel constant temperature flow cell 203, and other materials are the same as the first embodiment, so that magnesium ions in a living body can be detected.
Examples ten
The structure of the first embodiment is adopted, a fluorescent probe capable of identifying fluorine ions is modified on the surface of the sample substrate 202 by a chemical modification method, and biological cell proteins polluted by the fluorine ions flow in the multichannel constant temperature flow cell 203, and other biological cell proteins can be detected by the fluorine ions in the living body in the same way as the first embodiment.
Example eleven
By adopting the structure of the first embodiment, fluorescent probes capable of identifying mercury ions, zinc ions, magnesium ions, fluorine ions and cadmium ions simultaneously are modified in different channels on the surface of the sample substrate 202 by adopting a chemical modification method, and biological cell proteins polluted by the mercury ions, the zinc ions, the magnesium ions, the fluorine ions and the cadmium ions flow in the multichannel constant temperature flow cell 203.
Example twelve
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 channels are the same as the first embodiment, so that real-time quantitative detection of various tumor markers can be realized simultaneously.
The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are 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 (6)
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