CN109490202B - Immune turbidimetric protein analyzer based on optical microfluidic laser - Google Patents
Immune turbidimetric protein analyzer based on optical microfluidic laser 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
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/39—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using tunable lasers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
- G01N2021/0106—General arrangement of respective parts
- G01N2021/0112—Apparatus in one mechanical, optical or electronic block
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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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/39—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using tunable lasers
- G01N2021/391—Intracavity sample
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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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/39—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using tunable lasers
- G01N2021/392—Measuring reradiation, e.g. fluorescence, backscatter
- G01N2021/393—Measuring reradiation, e.g. fluorescence, backscatter and using a spectral variation of the interaction of the laser beam and the sample
Abstract
The invention discloses an immune turbidimetric protein analyzer based on optical microfluidic laser, and belongs to the technical field of sensors. The analyzer comprises a pumping light source module, a sample detection module and a signal collection and processing module. The pulse pump light is output by the optical parametric oscillator in the pump light source module, converged by the converging lens and then incident into the Fabry-Perot resonant cavity through the reflector. The upper reflector and the lower reflector in the sample detection module are arranged in parallel to form a Fabry-Perot resonant cavity, and when pulse pumping light enters the Fabry-Perot resonant cavity, dye molecules are subjected to stimulated radiation to generate laser signals to be output. And the signal collection processing module processes the laser signal and outputs a processing result to the computer. The invention combines the optical microfluidic laser technology with the immunoturbidimetry, utilizes the optical microfluidic laser to reflect for many times in the Fabry-Perot resonant cavity, increases the action times between the laser and the compound, and improves the sensitivity of protein detection; the amount of the sample to be detected is small, and the content of various immunoglobulins can be determined.
Description
Technical Field
The invention belongs to the technical field of sensors, and particularly relates to an immune turbidimetric protein analyzer based on optical microfluidic laser.
Technical Field
Immunoturbidimetry has been widely used in the fields of biomedicine, food hygiene, environmental monitoring, and the like. The traditional immunoturbidimetric immunoassay method mainly utilizes the specific binding of antigen and antibody to form a compound with larger particle size under the action of strong electrolyte. In the case of an excess of antibody, the amount of complex formation is directly proportional to the concentration of antigen. Light interacts with the complex, scattering, absorption and reflection occur, and the intensity of transmitted or scattered light is proportional to the concentration of the antigen to be measured. The method has the advantages of short time consumption, no washing, homogeneous reaction and the like, but the application range of the method is limited by the defects of low sensitivity, large sample consumption and the like.
Optical microfluidics is the integration of optical technology (sensing, manipulation, imaging) and microfluidic systems. The microfluidic channel is arranged between the two reflectors to form a Fabry-Perot cavity. The laser dye in the channel generates stimulated radiation under the action of the pump light. The excited radiation photons are reflected back and forth between the two mirrors and are continuously enhanced, and finally laser output is formed. The compound formed by the sample to be detected is mixed with the dye, and the concentration of the compound generates different losses to the laser in the laser cavity, so that the intensity of the output laser is influenced. Therefore, this detection method has high sensitivity. The traditional immune protein analyzer mostly adopts a mode that detection light penetrates a cuvette once to detect, and the dosage of samples and reagents is large. Compared with the traditional method, the invention provides a novel immune turbidimetric protein analyzer by utilizing the high sensitivity characteristic of Fabry-Perot cavity optical microfluidic laser, and the detection performance of the traditional immune turbidimetric method can be obviously improved by utilizing the multiple reflection of the microfluidic laser in the cavity. Meanwhile, the measurement of different protein concentrations can be realized by changing the concentration, solvent or variety of the dye.
Disclosure of Invention
The invention designs and manufactures an immune turbidimetric protein analyzer based on optical microfluidic laser aiming at the defects mentioned in the background technology. The protein analyzer has the characteristics of simple structure, high measurement precision and the like.
An immune turbidimetric protein analyzer based on optical microfluidic laser comprises a pump light source module 1, a sample detection module 2 and a signal collection and processing module 3.
The pumping light source module includes: the pulse pump light source comprises an optical parametric oscillator (1), a converging lens (5) and a reflector (6), wherein the optical parametric oscillator (1) outputs pulse pump light, the pulse pump light is converged by the converging lens (5), and then enters the Fabry-Perot resonant cavity at an angle of 13-17 degrees with the normal direction of an upper reflector (8) through the reflector (6).
The sample detection module includes: the Fabry-Perot laser comprises a micro-flow channel (7), an upper reflector (8), a supporting structure (9) and a lower reflector (10), wherein the upper reflector (8) and the lower reflector (10) are arranged in parallel to form a Fabry-Perot resonant cavity and provide optical feedback for micro-flow laser, and the micro-flow channel (7) and the supporting structure (9) are arranged in the Fabry-Perot resonant cavity; after the pulse pump light is emitted into the Fabry-Perot resonant cavity, dye molecules absorb pulse pump light energy and jump from a ground state to an excited state, when a large number of dye molecules are in the excited state, population inversion can occur, the dye molecules are subjected to stimulated radiation, and laser signals are output.
The signal collection processing module comprises: the device comprises a collecting element (11), an optical fiber (12), a spectrometer (13) and a computer (14), wherein the collecting element (11) couples the collected laser signals into the optical fiber (12), the laser signals are transmitted to the spectrometer (13) along the optical fiber (12), the spectrometer (13) processes the laser signals, and the processing result is output to the computer (14).
Furthermore, the pump light source module also comprises an aperture diaphragm (2), an adjustable attenuation sheet (3) and an energy probe (4). The pulse pump light output by the optical parametric oscillator (1) passes through the small-hole diaphragm (2), is adjusted by the adjustable attenuation sheet (3), passes through the energy probe (4), and is converged by the converging lens (5). The small aperture diaphragm (2) can carry out spatial filtering and beam shaping on the pulse pump light. The adjustable attenuation sheet (3) can adjust the energy of the pulse pump light. The energy probe (4) can monitor the energy of the pulse pump light in real time.
Furthermore, the reflectivity of the upper reflector and the reflectivity of the lower reflector are both higher than 90%, a low-cost metal film or a dielectric film with excellent performance can be adopted, the reflectivity is improved, and the detection sensitivity is further improved.
Further, the tunable range of the laser wavelength of the optical parametric oscillator is 189-2500 nm. The pulse laser with different wavelengths can be selected for pumping according to the types of the dyes, and the common pulse laser wavelength can be 355nm, 532nm, 635nm, 808nm, 1064nm and the like.
Further, the microfluidic channel (7) is composed of a square quartz capillary tube and a hose, wherein the square quartz capillary tube has an outer diameter of 1.4mm × 1.4mm and an inner diameter of 1.1mm × 1.1 mm. The square quartz capillary tube and the plastic hose are connected by ultraviolet glue curing. The microflow channel can adopt the material that the wall thickness is thinner, the transmissivity is higher, further reduces the loss in the Fabry-Perot resonant cavity, and then improves the detectivity. The supporting structure (9) can adopt a square capillary tube, a metal block and the like with the same size as the microfluidic channel (7) so as to enhance the stability of the Fabry-Perot resonant cavity.
The invention combines the optical microfluidic laser technology with the immunoturbidimetry. On the one hand, antigen-antibody can be specifically combined under specific conditions, and a complex with larger particle size is formed under the action of strong electrolyte. When the antibody is in excess, the concentration of complex formed is directly proportional to the antigen concentration. On the other hand, the optical microfluidic laser technology generally adopts a pulse pump light source as a pump light source, and the dye is introduced into the resonant cavity as a gain medium to realize laser output, and laser signal output with different wavelengths can be realized by changing the wavelength of the pump light, the type of the dye and the size of the Fabry-Perot resonant cavity. The complex formed by the antigen and the antibody and the dye are mixed uniformly and are introduced into the Fabry-Perot resonant cavity together, and the generated micro-flow laser can be reflected for many times in the cavity to enhance the interaction between the light and the complex. When the particle size of the compound is far smaller than the wavelength of the microfluidic laser, Rayleigh scattering is generated, and the intensity of the microfluidic laser is obviously reduced. And the intensity of the micro-flow laser is reduced in proportion to the concentration of the compound, so that the sensing of the concentration of the protein to be detected can be realized by detecting the intensity of the micro-flow laser.
The invention has the beneficial effects that:
1. the invention utilizes the multiple reflection of the microfluidic laser in the Fabry-Perot resonant cavity, increases the action times between the laser and the compound, and can improve the sensitivity of protein detection.
2. The immunoglobulin G concentration measuring method provided by the invention has the advantages of high sensitivity, simple operation and less required sample amount, and can be used for measuring the content of various immunoglobulins.
3. The immunoglobulin G concentration measuring method provided by the invention can change the types of dyes and the wavelengths of pulse pump light, thereby realizing laser output with different wavelengths, and different dyes and pulse pump light are selected according to the particle size of a compound formed by the protein to be detected, thereby realizing analysis of the concentrations of different types of proteins.
Drawings
FIG. 1 is a schematic structural diagram of an optical microfluidic laser immunoturbidimetric protein analyzer provided by the present invention
FIG. 2 is a cross-sectional view of the optical microfluidic laser immunoturbidimetric protein analyzer provided by the present invention
FIG. 3 is a schematic diagram of antigen-antibody reaction
FIG. 4 is a graph showing the relationship between the laser intensity and the protein concentration of the optical microfluidic laser immunoturbidimetric protein analyzer of the present invention
Detailed description of the invention
A manufacturing and using method of an immune turbidimetric protein analyzer based on optical microfluidic laser specifically comprises the following steps:
the immune turbidimetric protein analyzer of the optical microfluidic laser comprises a pump light source module 1, a sample detection module 2 and a signal collection and processing module 3. The pumping light source module includes: the pulse laser comprises a 532nm pulse laser (1), a converging lens (5) and a reflector (6), wherein the 532nm pulse laser (1) outputs pulse pump light, the pulse pump light is subjected to spatial filtering and beam shaping by using an aperture diaphragm (2), the energy of the pulse pump light is adjusted by an adjustable attenuation plate (3), the energy of the pulse pump light is monitored in real time by an energy probe (4), then the pulse pump light is converged by the converging lens (5), and then the pulse pump light enters a Fabry-Perot resonant cavity at an angle of 15 degrees with the normal direction of an upper reflector (8) through the reflector (6); at the same time, the collecting element (11) is positionally adjusted and fixed, and then connected to the spectrometer (13) by means of an optical fiber (12), so that the output signal of the spectrometer is transmitted to the computer (14).
The sample detection module includes: the reflection ratio is 91.5% of the upper reflector (8), 99.5% of the lower reflector (10), and the material of the reflecting film is dielectric film. The micro-flow channel (7) comprises a square quartz capillary tube and a round plastic hose, wherein the two sides of the square quartz capillary tube are communicated with each other, the two ends of the square capillary tube are connected with the round plastic hose, and ultraviolet glue is used for curing and filling the joint. The microfluidic channel (7) and the supporting structure (9) are placed between the two reflectors together to form a Fabry-Perot resonant cavity. And meanwhile, the position of the Fabry-Perot resonant cavity is adjusted, so that the convergent light beam just enters the Fabry-Perot resonant cavity.
Diluting the standard substance according to a certain concentration gradient, mixing 10 μ L of diluted sample with 1125 μ L of R1 reagent (Elizan, immunoglobulin G detection kit), placing in a water bath at 37 deg.C, and incubating at constant temperature for 5 min. Then, 375. mu.L of R2 reagent (Elizan, immunoglobulin G detection kit) was added to the mixed solution and mixed well, and the mixture was placed in a water bath at 37 ℃ and incubated at constant temperature for 5 min. And putting 900 mu L of the solution after the reaction into another centrifuge tube, adding 100 mu L of 1.2mM rhodamine B aqueous solution, and uniformly mixing.
The mixed solution was passed into the microfluidic channel (7) as shown in fig. 2. And turning on the pulse pumping light source, and recording the laser intensities corresponding to the standard substances with different concentrations. And in each measurement, the indication of the energy probe (4) is observed, and the adjustable attenuation sheet (3) is adjusted to ensure that the energy of each pumping is consistent. And drawing a standard curve by using the standard substances with different concentrations and the corresponding laser intensity.
And repeating the measurement steps for the sample to be measured, and comparing the measured laser intensity with the standard curve to obtain the specific concentration of the sample.
As shown in fig. 3, the antigen-antibody specifically binds to each other, and forms a complex having a large particle size under the action of strong electrolyte after a lapse of time. A Fabry-Perot cavity can be formed by arranging the micro-flow channel (7) between the two reflectors. The laser dye in the channel generates stimulated radiation under the action of the pump light. The stimulated radiation photons are reflected back and forth between the two mirrors and are continuously enhanced, and finally laser signal output is formed. The compound formed by the sample to be detected is mixed with the dye, and the compound concentration generates different losses to the laser in the laser cavity, so that the intensity of the output laser signal is influenced. When the pumping energy is fixed at 35.37uJ/mm2The relationship curve of the concentration of the standard substance and the laser intensity calibrated by the experiment is shown in figure 4.
Corresponding dyes and pump wavelengths may be selected for different proteins to be detected. For example, the IgM kit manufactured by Siemens, the particle size of the formed complex is about 800nm, so that the dye can be changed into HITC, and the pumping wavelength is 600 nm. Thus, detection of IgM concentration can also be accomplished on this device.
Claims (5)
1. An immune turbidimetric protein analyzer based on optical microfluidic laser comprises a pump light source module, a sample detection module and a signal collection and processing module;
the pumping light source module includes: the pulse pump light output by the optical parametric oscillator (1) is subjected to spatial filtering and beam shaping through the small aperture diaphragm (2), then is subjected to energy adjustment through the adjustable attenuation plate (3), is subjected to real-time monitoring through the energy probe (4), is converged through the converging lens (5), and is incident into a Fabry-Perot resonant cavity at an angle of 13-17 degrees with the normal direction of the upper reflector (8) through the reflector (6); the sample detection module includes: the Fabry-Perot laser comprises a micro-flow channel (7), an upper reflector (8), a supporting structure (9) and a lower reflector (10), wherein the upper reflector (8) and the lower reflector (10) are arranged in parallel to form a Fabry-Perot resonant cavity and provide optical feedback for micro-flow laser, and the micro-flow channel (7) and the supporting structure (9) are arranged in the Fabry-Perot resonant cavity; after the pulse pump light is incident into the Fabry-Perot resonant cavity, dye molecules absorb the energy of the pulse pump light, the dye molecules are transited from a ground state to an excited state, the population inversion occurs, the dye molecules are subjected to excited radiation, and laser signals are generated and output;
the signal collection processing module comprises: the device comprises a collecting element (11), an optical fiber (12), a spectrometer (13) and a computer (14), wherein the collecting element (11) couples collected laser signals into the optical fiber (12), the laser signals are transmitted to the spectrometer (13) along the optical fiber (12), the spectrometer (13) processes the laser signals and outputs the processing results to the computer (14);
the detection method of the immune turbidimetric protein analyzer for the sample to be detected comprises the following steps: diluting the standard substance according to concentration gradient, and taking 10
With 1125
R1 reagentMixing, placing in 37 deg.C water bath, and incubating at constant temperature for 5 min; then added 375 again
The R2 reagent is evenly mixed and placed in a water bath kettle at 37 ℃, and the mixture is heated and incubated for 5min at constant temperature; get 900
Putting the solution after the reaction into a centrifuge tube, and adding 100 percent
Uniformly mixing 1.2mM of rhodamine B water solution; introducing the mixed solution into a microfluidic channel (7), turning on a pulse pumping light source, and recording laser intensities corresponding to standard substances with different concentrations; during each measurement, the reading of the energy probe (4) is observed, and the adjustable attenuation sheet (3) is adjusted to ensure that the energy of each pumping is consistent; drawing a standard curve by using the standard substances with different concentrations and the corresponding laser intensity; and comparing the measured laser intensity with the standard curve for the sample to be measured to obtain the specific concentration of the sample.
2. The optical microfluidic laser-based immunoturbidimetric protein analyzer of claim 1, wherein: the reflectivity of the upper reflector and the reflectivity of the lower reflector are both higher than 90%, and metal films or dielectric films are adopted.
3. The optical microfluidic laser-based immunoturbidimetric protein analyzer of claim 1, wherein: the adjustable range of the laser wavelength of the optical parametric oscillator is 189-2500 nm.
4. The optical microfluidic laser-based immunoturbidimetric protein analyzer of claim 1, wherein: the micro-flow channel (7) is composed of a square quartz capillary tube and a hose, wherein the two ends of the square quartz capillary tube are solidified and communicated by ultraviolet glue.
5. The optical microfluidic laser-based immunoturbidimetric protein analyzer of claim 1, wherein: the supporting structure (9) is a square capillary or a metal block with the same size as the microfluidic channel (7).
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| CN106404729A (en) * | 2016-08-26 | 2017-02-15 | 电子科技大学 | Manufacturing and application method of ion sensor based on optofluidic bio-laser |
| CN107589275A (en) * | 2017-08-02 | 2018-01-16 | 电子科技大学 | A kind of flow-velocity sensing method and device based on light miniflow dye laser |
| CN108398388A (en) * | 2018-01-16 | 2018-08-14 | 电子科技大学 | A kind of integrated array colorimetric analysis device based on one-dimension optical-fiber micro-flow laser |
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| CN106404729A (en) * | 2016-08-26 | 2017-02-15 | 电子科技大学 | Manufacturing and application method of ion sensor based on optofluidic bio-laser |
| CN107589275A (en) * | 2017-08-02 | 2018-01-16 | 电子科技大学 | A kind of flow-velocity sensing method and device based on light miniflow dye laser |
| CN108398388A (en) * | 2018-01-16 | 2018-08-14 | 电子科技大学 | A kind of integrated array colorimetric analysis device based on one-dimension optical-fiber micro-flow laser |
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