CN111443213A - Multivariable reaction kinetics real-time detector device and detection method - Google Patents

Multivariable reaction kinetics real-time detector device and detection method Download PDF

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Publication number
CN111443213A
CN111443213A CN202010274339.6A CN202010274339A CN111443213A CN 111443213 A CN111443213 A CN 111443213A CN 202010274339 A CN202010274339 A CN 202010274339A CN 111443213 A CN111443213 A CN 111443213A
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reaction
test tube
light source
temperature
light guide
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CN111443213B (en
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吴景洪
苏星
马骏
张代化
苏成宽
苏卫
吴开原
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Xingyuanzhi Zhuhai Biological Technology Co ltd
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Xingyuanzhi Zhuhai Biological Technology Co ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00584—Control arrangements for automatic analysers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/48—Automatic or computerized control
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N2035/00346—Heating or cooling arrangements
    • G01N2035/00356—Holding samples at elevated temperature (incubation)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P90/00—Enabling technologies with a potential contribution to greenhouse gas [GHG] emissions mitigation
    • Y02P90/02—Total factory control, e.g. smart factories, flexible manufacturing systems [FMS] or integrated manufacturing systems [IMS]

Abstract

The invention provides a multivariable reaction kinetics real-time detector device and a detection method, wherein the detector device comprises a detector and a computer; wherein the detector is in communicative connection with the computer. The detector comprises a shell, a main control circuit board and a detection structure; the main control circuit board and the detection structure are both arranged in the shell, and the detection structure is in communication connection with the main control circuit board. The multivariable reaction kinetics real-time detector device and the detection method can read optical signals related to temperature, time and space variables in a test tube in real time, display the result in real time through a computer, obtain the reaction result more quickly, know the reaction process in real time and effectively improve the analysis efficiency.

Description

Multivariable reaction kinetics real-time detector device and detection method
Technical Field
The invention relates to the technical field of biochemical reaction detection equipment, in particular to a multivariable reaction kinetics real-time detector device and a detection method.
Background
The reaction kinetic analysis is a method for analyzing the characteristics and the quantity of a reaction substrate or a catalyst by measuring the reaction speed by means of the relation between the speed of a chemical reaction and the concentration of the reaction substrate or the relation between the speed of the chemical reaction and the concentration of the catalyst for accelerating the reaction, including the concentration of biological enzymes. The reaction kinetics are directly related to temperature. Reaction kinetics monitoring is important for chemical and biochemical (biochemical) reaction characterization. Chemical and biochemical reactions typically involve catalysts or biological enzymes that affect the rate and yield of reaction products, which are critical for drug development and medical diagnostics, public health and biological testing, and reaction kinetics also affect the efficacy of drugs.
Kinetic analysis can be accomplished by monitoring directly or indirectly generated optical signals, however, existing detection instruments typically use separate thermodynamic and optical components and are therefore bulky and expensive; and the optical signal needs to be read step by step, and the result is displayed after the reaction is finished. Therefore, the existing apparatus cannot read the optical signal in real time or continuously and display the result in real time under the condition of different variables, so that the time is long and the analysis efficiency is low.
Disclosure of Invention
The invention provides a multivariable reaction kinetics real-time detector device and a detection method, which can read optical signals in real time, display results in real time and improve analysis efficiency.
The technical scheme adopted by the invention is as follows: a multivariate reaction kinetics real-time detector device, comprising: the detector is in communication connection with the computer. The detector comprises a shell, a main control circuit board and a detection structure; the main control circuit board and the detection structure are arranged in the shell, and the detection structure is in communication connection with the main control circuit board; a mounting seat is mounted in the shell, and a test tube mounting hole is formed in the mounting seat and used for mounting a reaction test tube; the detection structure is arranged on the periphery of the test tube mounting hole;
the detection structure comprises an excitation light source, a temperature control assembly and a photoelectric detection assembly.
The excitation light source is arranged above or below the test tube mounting hole and faces the test tube mounting hole; and a first light source optical filter is arranged between the test tube mounting hole and the excitation light source. The temperature control assembly comprises an upper heater and a lower heater, the upper heater is installed at the upper end of the test tube mounting hole, the lower heater is installed at the lower end of the test tube mounting hole, and the upper heater and the lower heater are all surrounded on the periphery of the reaction test tube.
The photoelectric detection component comprises a photoelectric detector, a solid light guide and a second light source optical filter. The photoelectric detector, the solid light guide and the second light source optical filter are all arranged on the same side of the test tube mounting hole; the solid light guide is positioned between the cuvette mounting hole and the photodetector; the second source filter is positioned between the solid light guide and the photodetector.
The lighting time of the excitation light source is adjusted between 1 millisecond and 1000 milliseconds, the interval period of lighting the excitation light source every time is between 1 second and 5 minutes, and the collection of optical signals by the photoelectric detection assembly is completed in the lighting process of the excitation light source; the first light source filter and the second light source filter limit part of light with wavelengths to pass through;
the master control circuit board comprises an MCU module, a light temperature control module and a light signal amplification module, wherein the light signal amplification module is in communication connection with the MCU module, and the MCU module is in communication connection with the light temperature control module; the excitation light source and the temperature control assembly are both in communication connection with the light temperature control module, and the photoelectric detection assembly is in communication connection with the light signal amplification module; the MCU module can be expanded into a computer module, and partially or completely replaces the computer function;
the computer can be an MCU module with expanded functions and comprises a user interface, and a user can input variable parameters through the user interface and can monitor optical signals determined by temperature, time or spatial positions in the reaction test tube in real time; the user interface sets the working temperature of the upper heater and the lower heater, the light emitting time and period of the excitation light source, and selectively turns on the photoelectric detector to realize the collection of optical signals of a certain wave band;
after the detector starts to work, the user interface can display information corresponding to the temperature and the time fed back in real time; and simultaneously displaying the information corresponding to the intensity and time of the optical signal fed back in real time.
Further, go up the heater with the heater is adjustable down the temperature of reaction test tube heats the reaction test tube, makes reaction test tube interior liquid heat to 100 ℃ or cool down to the room temperature to promote or prevent liquid convection through the difference in temperature.
Further, the photodetector monitors a chemical reaction or a biochemical reaction of the liquid in the reaction cuvette with changes in temperature and time by an optical signal.
Further, the detector apparatus determines the amount, structural characteristics or biochemical activity of the target molecule in the sample by real-time monitoring of reaction kinetics.
Further, the photodetection assembly comprises two photodetectors, two light source filters and two solid light guides. One the photoelectric detector, one the light source light filter and one the solid light guide are one group, divide into two sets, set up in the both sides of test tube mounting hole relatively, and two the solid light guide is located go up the heater with between the heater.
Furthermore, the photoelectric detection components comprise two groups of upper photoelectric detection components and two groups of lower photoelectric detection components; the two groups of upper photoelectric detection assemblies are oppositely arranged on two sides of the upper end of the test tube mounting hole, and the two groups of lower photoelectric detection assemblies are oppositely arranged on two sides of the lower end of the test tube mounting hole; each set of the upper photoelectric detection components comprises an upper photoelectric detector, an upper solid light guide and a second light source filter; the upper solid light guide is located below the upper heater, and the upper solid light guide is located between the cuvette mounting hole and the upper photodetector, and the second light source filter is located between the upper solid light guide and the upper photodetector.
Each group of the lower photoelectric detection components comprises a lower photoelectric detector, a third light source optical filter and a lower solid light guide; the lower solid light guide is positioned above the lower heater, the lower solid light guide is positioned between the test tube mounting hole and the lower photoelectric detector, and the third light source optical filter is positioned between the lower solid light guide and the lower photoelectric detector.
Further, the photoelectric detection components comprise a group of upper photoelectric detection components and a group of lower photoelectric detection components; the upper photoelectric detection assembly and the lower photoelectric detection assembly are arranged on the same side of the test tube mounting hole;
or the photoelectric detection components comprise a group of upper photoelectric detection components and a group of lower photoelectric detection components; the upper photoelectric detection assembly and the lower photoelectric detection assembly are respectively arranged on two sides of the test tube mounting hole;
the upper photoelectric detection assembly comprises an upper photoelectric detector, an upper solid light guide and a second light source optical filter; the upper solid light guide is positioned below the upper heater, and the upper solid light guide is positioned between the cuvette mounting aperture and the upper photodetector, and the second light source filter is positioned between the upper solid light guide and the upper photodetector;
the lower photoelectric detection assembly comprises a lower photoelectric detector, a third light source optical filter and a lower solid light guide; the lower solid light guide is positioned above the lower heater, the lower solid light guide is positioned between the test tube mounting hole and the lower photoelectric detector, and the third light source optical filter is positioned between the lower solid light guide and the lower photoelectric detector.
Furthermore, the photoelectric detection components comprise two groups of upper photoelectric detection components and one group of lower photoelectric detection components; the two groups of upper photoelectric detection assemblies are oppositely arranged on two sides of the upper end of the test tube mounting hole, and the lower photoelectric detection assembly is arranged on one side of the lower end of the test tube mounting hole;
or the photoelectric detection components comprise a group of upper photoelectric detection components and two groups of lower photoelectric detection components; the upper photoelectric detection assemblies are arranged on one side of the upper end of the test tube mounting hole, and the two groups of lower photoelectric detection assemblies are oppositely arranged on two sides of the lower end of the test tube mounting hole;
the upper photoelectric detection assembly comprises an upper photoelectric detector, an upper solid light guide and a second light source optical filter; the upper solid light guide is positioned below the upper heater, and the upper solid light guide is positioned between the cuvette mounting aperture and the upper photodetector, and the second light source filter is positioned between the upper solid light guide and the upper photodetector;
the lower photoelectric detection assembly comprises a lower photoelectric detector, a third light source optical filter and a lower solid light guide; the lower solid light guide is positioned above the lower heater, the lower solid light guide is positioned between the test tube mounting hole and the lower photoelectric detector, and the third light source optical filter is positioned between the lower solid light guide and the lower photoelectric detector.
Further, the test tube mounting hole is inclined at an angle ranging from 5 ° to 45 ° when in use.
Further, the lower heater comprises a lower heating plate and a lower heating ring arranged on the lower heating plate, and the lower heating ring is in thermal expansion reversible locking contact with the reaction test tube.
Further, the solid light guide can be coated with a filter film and can play a role in full transmission or partial transmission or selective transmission on incident light, and the solid light guide is cylindrical or square.
Further, the first light source optical filter, the second light source optical filter and the third light source optical filter are made of inorganic glass, organic glass or quartz, and the surfaces of the first light source optical filter, the second light source optical filter and the third light source optical filter can be coated with optical filter films, and have the performance of low pass, high pass or band pass.
Further, the detector device is used for performing enzyme-catalyzed reaction kinetic analysis, molecular conformation and configuration analysis by detecting chemiluminescence, electrochemiluminescence, microsomal luminescence and fluorescence.
Further, the computer is mounted inside or outside the housing.
Furthermore, the distance between the main control circuit board and the mounting seat is less than or equal to 100 mm.
The invention also provides the following technical scheme: a detection method using the detector device comprises the following steps:
adding a reaction solution into a reaction test tube, placing the closed reaction test tube into a test tube mounting hole, and heating the reaction solution in the reaction test tube by an upper heater and a lower heater to ensure that the temperature difference between the upper part and the lower part of the reaction solution in the reaction test tube is more than or equal to 5 ℃ so as to circulate, mix or stand the solution;
starting a computer real-time monitoring program, and recording and outputting a reaction result in real time;
and carrying out medical diagnosis or biological sample detection or drawing a regional distribution map of related information according to the reaction result.
Further, the method comprises the following steps:
firstly, adding a reaction solution into a reaction test tube, wherein the reaction solution contains a mixed nucleic acid sample, a chemical reagent for nucleic acid amplification and a nucleic acid fluorescent dye or a fluorescent labeling molecular probe, and when the nucleic acid sample is RNA, transcribing the RNA into complementary DNA; then, carrying out nucleic acid sample amplification by using a constant-temperature amplification or variable-temperature amplification enzymatic method, and controlling the temperature of the liquid to circulate between 45 ℃ and 98 ℃ in the variable-temperature nucleic acid amplification process; when the photodetection assembly detects the signal of the nucleic acid fluorochrome or fluorescently labeled molecular probe, the intensity of the fluorescent signal generated by the amplification reaction is proportional to the amount of amplified DNA product.
Further, the method comprises the following steps:
when the temperature of the solution at the bottom of the reaction test tube is reduced to be lower than the nucleic acid dissolution temperature point after the nucleic acid amplification reaction is finished, most DNA molecules are combined into double-stranded molecules and combined with the nucleic acid fluorescent dye to release stronger fluorescence, the upper heater and the lower heater are controlled to start heating at a slow speed, and time and temperature variables are recorded through a computer; when the temperature variable is adjusted, the change of the optical signal is recorded in real time through the lower photoelectric detector or the upper photoelectric detector, and the relation between the temperature and the fluorescence signal is displayed through a computer; as the temperature is increased, the double-stranded DNA molecules are dissolved into single-stranded molecules, so that the fluorescence quantity is reduced, and the dissolution point or the dissolution curve of the amplified double-stranded DNA molecules is measured by data processing of a computer.
Further, the method comprises the following steps:
after the nucleic acid amplification reaction is finished, keeping the temperature of the solution at the bottom of the reaction test tube at 90-98 ℃, raising the temperature of the liquid at the upper end to be not more than 95 ℃ by an upper heater, dissolving most double-stranded DNA molecules into single-stranded DNA molecules, controlling the upper heater and the lower heater to start to reduce the temperature at a slow speed, and recording time and temperature variables by a computer; when the temperature variable is adjusted, the change of the optical signal is recorded in real time through the lower photoelectric detector or the upper photoelectric detector, and the relation between the temperature and the fluorescence signal is displayed through a computer; as the temperature is lowered, the single-stranded DNA molecules are combined into double-stranded molecules, so that the fluorescence quantity is increased, and the dissolution point or the dissolution curve of the amplified DNA molecules is measured through data processing by a computer.
Compared with the prior art, the multivariate reaction kinetics real-time detector device and the detection method of the invention monitor multivariate (such as temperature, time and space parts) related optical signals in real time by arranging the photoelectric detection component, the temperature control component and the excitation light source on the detector to be in communication connection with the main control circuit board and connecting the detector with the computer in communication, such as: controlling the temperature of different positions of the reaction test tube, and detecting optical signals of relative positions changing along with time; controlling the temperature of different positions of the reaction test tube, and detecting optical signals of relative positions changing along with the temperature; controlling the temperature gradient of the liquid in the reaction tube, and detecting an optical signal of which the relative position changes along with the temperature and the time; therefore, the detector device can read optical signals in real time, display results in real time, obtain reaction results more quickly and know the reaction process in real time. And the results can be uploaded to the cloud, and the data can be remotely monitored and analyzed and used to generate a regional profile of information (e.g., by monitoring (immune response or nucleic acid amplification) an infectious disease, profiling an infectious disease and thereby predicting a trend).
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention without limiting the invention. In the drawings, there is shown in the drawings,
FIG. 1: the invention discloses a three-dimensional view of a multivariable reaction kinetics real-time detector device;
FIG. 2: the invention discloses a perspective view of a first detection structure embodiment;
FIG. 3: the invention detects the sectional view of the structural embodiment one;
FIG. 4: another perspective view of the first embodiment of the detection structure of the present invention;
FIG. 5: the invention discloses a block diagram of a multivariable reaction kinetics real-time detector device;
FIG. 6: the invention detects the perspective view of the structural embodiment two;
FIG. 7: the invention detects the perspective view of the third structural embodiment;
FIG. 8: the invention detects the perspective view of the structural embodiment IV;
FIG. 9: a perspective view of a fifth embodiment of the detection structure of the present invention;
FIG. 10: the invention detects the perspective view of the structural embodiment six;
FIG. 11: the invention detects the perspective view of the structure embodiment seventh;
FIG. 12: a schematic diagram of a nucleic acid detection process;
FIG. 13: a plot of the fluorescence signal of the nucleic acid detection process;
FIG. 14: temperature change profiles of the upper heater and the lower heater.
Detailed Description
The following detailed description of embodiments of the invention refers to the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.
Example one
As shown in FIG. 1, the multivariate reaction kinetics real-time detector device of the present invention comprises a base 1, a detector 2 and a computer; wherein, the detector 2 is arranged on the base 1, and the detector 2 is connected with the computer through a USB connection line or wireless (such as Bluetooth, WIFI, 5G) communication, or the detector 2 and the computer are integrated into a whole; and sending a control command in real time through a computer to control the detector 2 to work.
The detector 2 comprises a shell 201, a main control circuit board and a detection structure; the shell 201 is rotatably mounted on the base 1, the main control circuit board and the detection structure are mounted in the shell 201, and the detection structure is in communication connection with the main control circuit board. In addition, the housing 201 is further provided with a ventilation and heat dissipation hole 202, so that the phenomenon that the temperature of the detector 2 is too high after long-time work is effectively avoided.
The base 1 includes a base plate 101 and a rotating plate 102, wherein one end of the rotating plate 102 is rotatably mounted to one end of the base plate 101 through a rotating shaft 3 and is locked by an angle locking nut 4. The housing 201 is mounted on the rotating plate 102, a mounting seat 5 is mounted in the housing 201, and a plurality of test tube mounting holes 6 are formed in the mounting seat 5 and used for mounting reaction test tubes 7; each of the detecting structures is mounted to the outer periphery of each of the test tube mounting holes 6, respectively. Further, the position that shell 201 corresponds a plurality of test tube mounting holes 6 is equipped with rotatory lid 203, is convenient for open shell 201, exposes a plurality of test tube mounting holes 6, easy to assemble reaction test tube 7.
Through installing detector 2 in rotor plate 102, when the inclination of reaction test tube 7 needs to be adjusted, can be through loosening angle lock nut 4, rotate rotor plate 102 around rotation axis 3 to required angle, then again locking angle lock nut 4 can for the solution in the reaction test tube 7 realizes more effective thermal convection. Wherein, the reaction test tube 7 is a straight test tube, when in use, the inclination angle of the test tube mounting hole 6 can be adjusted by adjusting the angle of the rotating plate 102, and then the inclination angle of the reaction test tube 7 is adjusted, the angle adjustment range is 5-45 degrees, and the ratio of the length to the diameter of the reaction test tube 7 is more than or equal to 10.
As shown in fig. 1 and fig. 2, the detection structure includes an excitation light source 8, a temperature control component, and a photodetection component; wherein, excitation light source 8 installs the below that the test tube mounting hole 6 faces test tube mounting hole 6, and still installs first light source light filter 9 between test tube mounting hole 6 and the excitation light source 8. When the reaction cuvette 7 is loaded in the cuvette mounting hole 6, the first light source filter 9 is positioned below the bottom of the reaction cuvette 7. As shown in fig. 4, it is understood that the excitation light source 8 and the first light source filter 9 may also be installed above the cuvette mounting hole 6 and opposite to the cuvette mounting hole 6, specifically, on the inner wall surface of the rotary cap 203, as shown in fig. 4, but not limited thereto.
The lighting time of the excitation light source 8 is adjusted between 1 millisecond and 1000 milliseconds, the lighting interval period of each time of the excitation light source 8 is between 1 second and 5 minutes, the lighting interval period can be adjusted according to different reaction substances, and the collection of optical signals by the photoelectric detection assembly is completed in the lighting process of the excitation light source 8.
As shown in fig. 1 to 3, the temperature control assembly includes an upper heater 10 and a lower heater 11, the upper heater 10 is mounted at the upper end of the test tube mounting hole 6, and the lower heater 11 is mounted at the lower end of the test tube mounting hole 6; when the upper reaction cuvette 7 is loaded in the cuvette mounting hole 6, the upper heater 10 and the lower heater 11 are both wound around the circumference of the reaction cuvette 7. The method specifically comprises the following steps: the upper heater 10 includes an upper heating plate and an upper heating ring mounted on the upper heating plate; the lower heater 11 includes a lower heating plate and a lower heating ring mounted on the lower heating plate, and the reaction cuvette 7 is reversibly locked in the lower heating ring by thermal expansion. Further, thermistors are mounted on the upper heater 10 and the lower heater 11; the control of the temperature of the solution in the reaction cuvette 7 by the temperature control assembly comprises: heating, keeping constant temperature, and cooling to form temperature gradient to make reaction solution generate or stop thermal convection.
The computer includes a user interface and associated software through which a user inputs variable parameters to monitor in real time optical signals that are dependent on temperature, time or spatial position in the reaction cuvette 7.
The method specifically comprises the following steps: the user interface sets the working temperature of the upper heater 10 and the lower heater 11, the light emitting time and period of the excitation light source 8, and selectively turns on one of the photodetectors to realize the collection of the optical signals of a certain waveband. After the detector is started to work, the user interface can display the corresponding curve of the temperature and the time fed back in real time; and simultaneously displaying a curve corresponding to the intensity and the time of the optical signal fed back in real time. By comparing the intensity of the optical signal, the amount of the product corresponding to the reactant can be analyzed.
The continuous temperature change of the solution in the reaction cuvette 7 may be achieved in various ways, and one of the upper heater 10 and the lower heater 11 is changed to be transferred to the reaction cuvette 7, thereby causing the temperature of the liquid in the reaction cuvette 7 to be changed. Another way is to convect the liquid by adjusting the temperature difference between the upper and lower parts of the reaction tube 7. Even though the upper heater 10 and the lower heater 11 maintain the temperature of the respective portions constant, molecules in the liquid may undergo different temperature changes during convection. Therefore, a continuous temperature change refers to a temperature change of the liquid sample in the effective reaction cuvette 7. The photoelectric detection subassembly includes two sets of photoelectric detection subassemblies and two sets of photoelectric detection subassemblies down, and wherein, two sets of photoelectric detection subassemblies set up the upper end both sides at test tube mounting hole 6 relatively on two sets of photoelectric detection subassemblies, and two sets of lower photoelectric detection subassemblies set up the lower extreme both sides at test tube mounting hole 6 relatively.
The method specifically comprises the following steps: each group of upper photoelectric detection components comprises an upper photoelectric detector 12, an upper solid light guide 13 and a second light source filter 17, wherein the upper solid light guide 13 is positioned below the upper heater 10, the upper solid light guide 13 is positioned between the test tube mounting hole 6 and the upper photoelectric detector 12 and is used for transmitting a fluorescence signal from the reaction test tube 7 to the upper photoelectric detector 12, and then the upper photoelectric detector 12 outputs a voltage or current analog signal corresponding to the intensity of the fluorescence signal to the main control circuit board. A second light source filter 17 is located between the upper solid light guide 13 and the upper photodetector 12.
Each set of lower photo-detection assemblies comprises a lower photo-detector 14, a third light source filter 18 and a lower solid light guide 15, the lower photo-detector 14 being located above the lower heater 11, and the lower solid light guide 15 being located between the cuvette mounting hole 6 and the lower photo-detector 14 for conducting a source of light from the reaction cuvette 7 to the lower photo-detector 14. Further, a third source filter 18 is located between the lower solid light guide 15 and the lower photodetector 14. In addition, the vertical distance between any one upper photodetector 12 and any one lower photodetector 14 is greater than or equal to 5 mm.
The upper and lower solid light guides 13, 15 are capable of full or partial or selective transmission of incident light, and the shape may be different shapes, such as: cylindrical, square, etc.; the material can be inorganic glass, organic glass, quartz and the like, and the surface can be coated with a light filtering film. The first light source filter 9, the second light source filter 17 and the third light source filter 18 limit the light with partial wavelengths to pass through, including low pass, high pass and band pass. The first light source optical filter 9, the second light source optical filter 17 and the third light source optical filter 18 play a role in distinguishing different fluorescence wavelengths; the wavelength of the emitted fluorescence spectrum is different according to the reaction substance in the reaction tube 7, and filters with different wave bands can be used to distinguish one or more reaction substances according to the needs of the reaction substance.
In this embodiment, a row of test tube mounting holes 6 is formed in the mounting seat 5, a row of four or eight test tube mounting holes 6 is formed in the mounting seat, and a detection structure is mounted corresponding to each test tube mounting hole 6; it is understood that in other embodiments, the mounting seat 5 may be provided with a test tube mounting hole 6 and a detection structure mounted on the periphery of the test tube mounting hole 6; or the multiple rows of test tube mounting holes 6 are arranged in an array, and a detection structure is mounted corresponding to each test tube mounting hole 6, which is not limited to this.
As shown in fig. 2 and 5, the main control circuit board includes an MCU module, a light temperature control module, and a light signal amplification module; the light signal amplification module is in communication connection with the MCU module, and the MCU module is in communication connection with the light temperature control module. The excitation light source 8 and the temperature control component are both in communication connection with the light temperature control module, and the photoelectric detection component is in communication connection with the light signal amplification module.
The optical signal within a specific wavelength range is acquired in less than one second through the upper solid light guide 13, the lower solid light guide 15, the second light source optical filter 17 and the third light source optical filter 18, and the optical signal is amplified through the optical signal amplification module, so that the detection instrument can realize real-time detection.
Example two
As shown in fig. 1 and fig. 6, the same structure of this embodiment as that of the first embodiment is not repeated herein, and the differences are only: the photodetection assembly consists of only one photodetector 19, one light source filter 20, and one solid light guide 21, and is located at one side of the test tube mounting hole 6, and the solid light guide 21 is located between the upper heater 10 and the lower heater 11.
Example three:
as shown in fig. 1 and fig. 7, the same structure of this embodiment as that of the first embodiment is not repeated herein, and the differences are only: the photoelectric detection component comprises two photoelectric detectors 19, two light source filters 20 and two solid light guides 21; one photodetector 19, one light source filter 20, and one solid light guide 21 are grouped into two groups, oppositely disposed on both sides of the cuvette mounting hole 6, and the two solid light guides 21 are located between the upper heater 10 and the lower heater 11.
Example four
As shown in fig. 1 and fig. 8, the same structure of this embodiment as that of the first embodiment is not repeated herein, and the differences are only: the photoelectric detection assembly comprises a group of upper photoelectric detection assemblies and a group of lower photoelectric detection assemblies; wherein, go up the photoelectric detection subassembly and all set up in the same one side of test tube mounting hole 6 with lower photoelectric detection subassembly.
EXAMPLE five
As shown in fig. 1 and fig. 9, the same structure of this embodiment and the embodiment is not repeated herein, and the difference is only: the photoelectric detection assembly comprises a group of upper photoelectric detection assemblies and a group of lower photoelectric detection assemblies; wherein, go up photoelectric detection subassembly and lower photoelectric detection subassembly and set up respectively in the both sides of test tube mounting hole 6.
Example six:
as shown in fig. 1 and fig. 10, the same structure of this embodiment as that of the first embodiment is not repeated herein, and the differences are only: the photoelectric detection components comprise two groups of upper photoelectric detection components and one group of lower photoelectric detection components; wherein, two sets of upper photoelectric detection subassemblies set up the upper end both sides at test tube mounting hole 6 relatively, and lower photoelectric detection subassembly sets up one of them side at test tube mounting hole 6 lower extreme.
Example seven:
as shown in fig. 1 and fig. 11, the same structure of this embodiment as that of the first embodiment is not repeated herein, and the differences are only: the photoelectric detection components comprise a group of upper photoelectric detection components and two groups of lower photoelectric detection components; wherein, go up photoelectric detection subassembly setting in one of them side of 6 upper ends of test tube mounting hole, two sets of lower photoelectric detection subassemblies set up the lower extreme both sides at test tube mounting hole 6 relatively.
As shown in fig. 1 and 11, one function of the multiple sets of photodetecting elements in various embodiments is to measure optical signals at different spatial locations; another effect is to measure optical signals of different frequencies (wavelengths), spectra, or a combination of both. Therefore, multiple detections can be realized, and the detection efficiency or the detection accuracy can be improved.
The working principle of the multivariable reaction kinetics real-time detector device and the detection method is as follows:
loading a solution to be detected (the solution is provided with fluorescent molecules, added with the fluorescent molecules or can generate the fluorescent molecules or precursors of luminescent molecules or substrate molecules in the reaction process) into a reaction test tube 7, plugging a plug 16, and then installing the reaction test tube 7 into a test tube installation hole 6; the mounting 5 is tilted, if necessary to adjust the reaction tube 7 to a tilt of 5 ° -45 ° (note: the tilt angle is useful only for thermal convection); starting a real-time monitoring program, sending a control instruction through a computer, controlling the detector 2 to start working, heating the solution in the reaction test tube 7 through the upper heater 10 and the lower heater 11, when the temperature is continuously increased, thermally expanding the reaction test tube 7, realizing reversible locking with the lower heater 11, and conducting heat to the reaction test tube 7 through the upper heater 10 and the lower heater 11; when the solution in the reaction test tube 7 generates a temperature gradient, the solution realizes more effective heat convection;
meanwhile, the MCU module controls the output power of the upper heater 10 and the lower heater 11 in real time according to the instruction, thereby achieving the purpose of controlling the working temperature of the upper heater and the lower heater and conducting the corresponding temperature to the reaction test tube 7; the temperatures of the upper heater 10 and the lower heater 11 can be adjusted to different temperatures according to the reaction requirements, and the actual temperatures are simultaneously fed back to the computer through corresponding thermistors;
when the detector 2 receives a light-emitting instruction from the computer, the excitation light source 8 is lighted according to the instruction to irradiate the solution in the reaction tube 7, the solution in the reaction tube 7 emits fluorescence after being irradiated by the excitation light source 8, and the fluorescence is transmitted to the upper photodetector 12 and the lower photodetector 14 through the corresponding upper solid light guide 13 and the lower solid light guide 15 respectively; when the upper photoelectric detector 12 and the lower photoelectric detector 14 detect the fluorescent signal emitted by the solution in the reaction test tube 7, the analog signal of voltage or current corresponding to the fluorescent signal is directly output, the analog signal is converted into an amplified electric signal through the optical signal amplification module, the amplified electric signal corresponds to the fluorescent signal with corresponding intensity, and the fluorescent signal is fed back to the computer through the MCU module in real time, so that the optical signal is read in real time, and the result is displayed in real time.
Wherein, the computer is provided with a temperature sensor and a light source 8, which can select different temperatures and irradiation times of the excitation light source according to different reaction solutions, and feed back the solution to react in real time at different spatial parts; the lighting time of the excitation light source 8 is adjusted between 1 millisecond and 1000 milliseconds; the interval period of each lighting of the excitation light source is between 1 second and 5 minutes, and the lighting interval period can be adjusted according to different reaction substances; the collection of the optical signal by the photoelectric detection component is completed in the process after the excitation light source 8 is lightened.
The result obtained by monitoring the multivariate reaction kinetics real-time detector device and the detection method can be uploaded to the cloud end through a computer, and the data can be remotely monitored and analyzed and can be used for making an information regional distribution map (for example, by monitoring an infectious disease (immune reaction or nucleic acid amplification), drawing an infectious disease distribution map and predicting the development trend of the infectious disease distribution map).
Referring to FIG. 12, the process of detecting nucleic acid amplification is as follows:
loading a reaction tube 7 with a reaction solution containing a mixed nucleic acid sample, a chemical reagent for nucleic acid amplification and a nucleic acid fluorescent dye (intercalling dye) or a fluorescent labeled molecular probe (fluorescent dye labeled molecular probe), wherein the nucleic acid sample can be RNA or DNA of animal and plant cells, bacteria, fungi or viruses, when the nucleic acid sample is an RNA sample, the RNA is transcribed into complementary DNA, namely cDNA (cDNA and DNA can be amplified in the tube to achieve the purpose of detection), performing nucleic acid amplification by an enzymatic method, including isothermal amplification, such as RPA (recombinant amplification), L AMP (L oop-mediated isothermal amplification), and variable temperature amplification, such as PCR (polymerase chain reaction in reaction), wherein the temperature of the liquid needs to be controlled so that the liquid is circulated between 45 ℃ and 98 ℃;
when the photoelectric detection component detects that the solution has a fluorescence signal released by the nucleic acid fluorescent dye or the fluorescence labeling molecular probe, the intensity of the fluorescence signal generated by the amplification reaction is proportional to the quantity of the amplified DNA product (the process is a reaction kinetic process which is determined by variable factors such as temperature, time and the like, and the initial copy number of the target nucleic acid in the original sample can be presumed through reaction kinetic data, which has important significance in the medical diagnosis, public health and biological detection industries);
determining a dissolution curve, and when the amplification reaction is finished, reducing the temperature of the solution at the bottom of the reaction test tube 7 to about 45 ℃, and then combining DNA molecules into double-stranded molecules, combining the double-stranded molecules with the nucleic acid fluorescent dye and then releasing strong fluorescence; in order to determine the dissolution curve, the upper heater 10 and the lower heater 11 start to be heated at a slow speed, and the time and temperature variables are recorded by the computer, while the temperature variables are adjusted, the changes of the optical signals are recorded in real time by the lower or upper photodetector 14, and the relationship between the temperature and the fluorescence signals is displayed by the computer; with the increase of temperature, double-stranded DNA molecules can be dissolved into single-stranded molecules, so that the fluorescence quantity is reduced (the process is related to the length of a DNA molecular chain, namely the DNA molecule with a certain length has a characteristic dissolving temperature); the computer is used to process data to obtain the dissolution point of amplified DNA molecule, and the determination of dissolution curve is one method of confirming the accuracy of DNA amplification.
Another method for determining the melting curve is to maintain the temperature of the solution at the bottom of the reaction tube at a high temperature (90 ℃ to 98 ℃) and to raise the temperature of the upper end liquid to not more than 95 ℃ by an upper heater after the nucleic acid amplification reaction is completed, at which time most of the double-stranded DNA molecules are dissolved into single-stranded DNA molecules; then controlling the upper heater and the lower heater to start to reduce the temperature at a slow speed, and recording time and temperature variables through a computer; when the temperature variable is adjusted, the change of the optical signal is recorded in real time through the lower photoelectric detector or the upper photoelectric detector, and the relation between the temperature and the fluorescence signal is displayed through a computer; as the temperature is lowered, the single-stranded DNA molecules are combined into double-stranded molecules, so that the fluorescence quantity is increased, and the dissolution point or the dissolution curve of the amplified DNA molecules is measured through data processing by a computer.
Referring to fig. 13 and 14, fig. 13 is a graph of 8 fluorescence signal curves (V1, V2, V3, V4, V5, V6, V7, V8) collected in real time; the ordinate is the signal intensity and the abscissa is the time (minutes). Reading-time signals every 10 seconds, fig. 14 is a temperature change graph, T1 is the upper heater temperature, T2 is the lower heater temperature change graph, the ordinate is the temperature parameter, the relative high value is the relative low temperature, such as 60 ℃ for duration 350 and 95 ℃ for duration 185; the horizontal axis is time (minutes).
The data shown is a nucleic acid amplification experiment used for proof of principle. The reaction tube contains reaction liquid, which contains respiratory polynuclear virus (RSV) RNA to be detected, primer required for nucleic acid amplification, reverse transcriptase, DNA polymerase, buffer solution, nucleic acid fluorescent dye and the like. First the RNA is reverse transcribed to cDNA (first seven minutes) and then DNA amplification is performed in the same tube. As shown in FIG. 13, PCR-like amplification was achieved by controlling the temperature gradient in the reaction tube, allowing the reaction solution to undergo convection in the reaction tube, and subjecting the nucleic acid to temperature cycling. In real-time monitoring of 4 reaction tubes simultaneously with the apparatus of the present invention, reaction status information related to 520nm wavelength (V1, V2, V3, V4) and 530nm wavelength (V5, V6, V7, V8) was read and displayed from each reaction tube at a time point. The corresponding curves for the reaction tubes are: reaction tube one (V1, V2), reaction tube two (V2, V6), reaction tube three (V3, V7), and reaction tube four (V4, V8). As can be seen from FIG. 13, the reaction tube emits non-specific fluorescence (first few minutes) when the liquid temperature is low. The non-specific fluorescence signal decreases when the temperature increases (ten to twenty-five minutes), and the specific fluorescence signal starts to increase when the DNA is amplified to a certain amount (twenty-five minutes later). By similar procedures, the identity (source, genetic structure, number of target molecules, etc.) of the target molecule in the original sample can be determined.
The multivariable reaction kinetics real-time detector device is suitable for measuring reaction kinetics taking light as a signal, and comprises the following components:
① chemiluminescence, electrochemiluminescence, microsomal luminescence, fluorescence, etc. in immunoassays;
chemiluminescence immunoassay (C L IA) is a common method in medical diagnosis, which combines chemiluminescence assay technology with high sensitivity and high specificity immunoreaction, is a detection and analysis technology for various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins, drugs and the like, and is a new immunoassay technology developed after radiochemical immunoassay, enzyme immunoassay, fluorescence immunoassay and time-resolved fluorescence immunoassay.
The chemiluminescence immunoassay comprises two parts, namely an immunoreaction system and a chemiluminescence system, wherein the chemiluminescence system utilizes a chemiluminescent substance (luminescent substrate) to form an excited intermediate through catalysis of a catalyst (enzyme) and oxidation of an oxidant, the excited intermediate simultaneously emits photons when returning to a stable ground state, and a luminescence signal measuring instrument can measure the yield of photon.
a. The change of the luminous kinetics is observed in real time under the control of different variables (temperature and time); the luminous rate is influenced by the temperature, the liquid can be continuously mixed by the temperature gradient, the luminous reaction is more uniform, the indexes such as the amount of the antigen, the quality of the luminous substrate, the stability of the luminous substrate and the like can be determined according to the luminous rate, and the dynamic method is more accurate than the end point luminous quantity detection.
b. Detecting the affinity between the antigen and the antibody and the stability of the combination by a fluorescence resonance energy transfer method; fluorescence resonance energy transfer refers to when two fluorescent chromophore groups are close enough, the donor molecule is excited to a higher electron energy state after absorbing a photon with a certain frequency, and energy transfer to an adjacent acceptor molecule is realized through dipole interaction before the electron returns to the ground state (i.e. energy resonance transfer occurs). Antibodies are the core reagents of immune reactions, and the specificity and the affinity of the antibodies determine the specificity and the sensitivity of the immune reactions. The conventional immune reaction is a sandwich method, i.e., the capture antibody and the detection antibody are used to recognize the same antigen. When the capture antibody and the detection antibody are labeled with fluorescein capable of fluorescence energy transfer, respectively, the affinity of the antigen and the antibody can be estimated by detecting the kinetics of fluorescence resonance energy transfer (the relationship between the antibody concentration and the light emission rate).
② enzymatic reaction kinetics analysis including but not limited to nucleic acid detection (isothermal or thermal cycling).
③ molecular conformation and configuration analysis, including but not limited to DNA melting curves.
④ tests performed according to the above principles include medical diagnostics, public health, environmental monitoring, food quality, industrial and agricultural production, commodity inspection, beauty, etc.
In conclusion, the multivariable reaction kinetics real-time detector device and the detection method have the following advantages:
1. by arranging a photoelectric detection component, a temperature control component and an excitation light source 8 on the detector 2 to be in communication connection with a main control circuit board, and connecting the detector 2 with a computer to be in communication connection, multivariate (such as temperature, time and space part) related optical signals are monitored in real time, such as: controlling the temperature of different positions of the reaction test tube 7, and detecting optical signals of relative positions changing along with time; controlling the temperature of different positions of the reaction test tube 7, and detecting optical signals of relative positions changing along with the temperature; controlling the temperature gradient of the liquid in the reaction test tube 7, and detecting the optical signal of which the relative position changes along with the temperature and the time; therefore, the detector device can read the optical signal in real time, display the result in real time, obtain the reaction result more quickly and know the reaction process in real time, and an important tool is provided for scientific research, drug development, medical diagnosis, public health and biological detection.
2. By providing the upper heater 10 and the lower heater 11, the reaction states in different regions (spatial portions) in the same reaction cuvette 7 can be measured at the same time.
3. By arranging the upper photoelectric detector 12 and the lower photoelectric detector 14, the photoelectric detection assembly can collect optical signals at a plurality of different positions of the reaction test tube 7, so that reaction data can be collected more effectively; realizing multiple detection;
4. through setting up solid light guide 13 and lower solid light guide 15 for the structure of photoelectric detection subassembly is more nimble, can increase photon collection volume moreover, thereby improves sensitivity and realizes real-time reaction state and shows.
5. Through installing detector 2 rotation in base 1, realize adjustable reaction tube 7's angle of inclination for the solution in the reaction tube 7 realizes more orderly thermal convection.
6. Simple structure, lower heater 11 realizes effective heat-conduction through the locking of the thermal energy mode reversibility of reaction test tube 7, and integrated optical-mechanical-electrical components is in an organic whole simultaneously, conveniently carries, and can be in the field work.
The related terms related to the present invention are defined as follows:
1. reaction kinetics: the reaction kinetics is a branch discipline of chemical reaction engineering for researching the influence of various physical and chemical factors (such as temperature, pressure, concentration, media in a reaction system, catalyst, flow field and temperature field distribution, residence time distribution and the like) on the reaction rate and corresponding reaction mechanism, mathematical expressions and the like. The reaction kinetics in the present invention refer to the course and the results of the reaction in relation to the above principles.
2. Real-time monitoring, including reading optical signals in real time, and displaying results in real time: the continuously changing reaction state is monitored during a longer (more than ten minutes) reaction. The monitoring can be continuous or intermittent, and the time interval of the intermittent monitoring is not more than 5 minutes.
3. A computer: the computer is commonly called as a computer, is a modern electronic computing machine for high-speed computation, can perform numerical computation and logic computation, and also has a memory function; the intelligent electronic device can be operated according to a program, and can automatically process mass data at a high speed. The system is composed of a hardware system and a software system. The computer in the invention comprises a display interface and a related software program.
4. Communication connection: a connection mode, through the transmission interaction of signals, communication is formed between connected devices; the communication connection comprises a wired connection and a wireless connection; the change of the wired connection level comprises a change of an analog level or a change of a digital level; wireless connections include radio waves, magnetic fields, light propagation, and the like.
5. A circuit board: the circuit board has the name: ceramic circuit boards, alumina ceramic circuit boards, aluminum nitride ceramic circuit boards, PCB boards, aluminum substrates, high-frequency boards, thick copper boards, impedance boards, PCBs, ultra-thin circuit boards, printed (copper etching technology) circuit boards, and the like; the circuit board may be referred to as a printed wiring board or a printed circuit board.
6. A reaction tube; the light-permeable reaction vessel can be made of glass, quartz, organic glass (plastic) and the like.
7. The excitation light source can be visible light such as laser, L ED light, incandescent light and the like, or invisible light such as ultraviolet light, infrared light and the like.
8. Temperature control: the heating power of the heating part is changed, and the forms of hot air, infrared, heat conduction, heat radiation and the like are adopted.
The MCU module: a circuit board containing a microcontroller integrated circuit and bearing communication with the computer and peripheral modules; can be expanded to a computing module, and partially or completely replaces the computer function.
10. The light temperature control module: and the circuit board and the device receive the control instruction and control the temperature and the light emission of the detection structure.
11. An optical signal amplification module; the optical signal amplifying circuit board and the device amplify the analog electric signal output by the photoelectric detector to the electric signal which can be identified by the MCU module.
12. Heating plate: the circuit board provided with the heating part (heating ring) is convenient for installing components of the circuit and plays a role in heat conduction.
13. Heating a ring: a heat conducting part, which can rapidly conduct heat to the reaction test tube and the heat sensor (thermistor).
14. A photoelectric detector: a semiconductor component or a packaged device of integrated semiconductor components that receives photons and then outputs an analog electrical signal.
15. An optical filter: and limiting the light of partial wavelength to pass through, including low-pass, high-pass and band-pass. Is made of inorganic glass, organic glass or quartz, etc.
16. Solid light guide: the light guide solid material can play a role in full transmission or partial transmission or selective transmission on incident light; can be in different shapes, such as column shape, square shape, etc.; can be made of inorganic glass, organic glass, quartz and the like; the surface can be coated with a filter film, including low-pass, high-pass and band-pass.
17. A user interface: the User Interface (UI) is a medium for interaction and information exchange between the system and the User, and it realizes the conversion between the internal form of information and the form acceptable to human. The user interface is designed to interact and communicate with the related software between the user and the hardware, so that the user can conveniently and efficiently operate the hardware to achieve bidirectional interaction and complete the work expected to be completed by the hardware. User interfaces are widely defined, including human-computer interaction and graphical user interfaces, and exist in the field of human and mechanical information communication.
18. Biochemical reaction: chemical reactions involving biomolecules, typically reactions catalyzed by biological enzymes.
19. Target molecule: the molecular object to be detected can be pathogen, protein molecule, nucleic acid molecule, glycoprotein molecule, carbohydrate or lipid molecule, organic molecule and inorganic molecule.
20. Chemiluminescence: chemiluminescence is a light radiation phenomenon accompanying substances in the process of carrying out chemical reactions and can be divided into direct luminescence and indirect luminescence.
21. Electrochemiluminescence (EC L) refers to a luminescence phenomenon that is generated by directly subjecting a luminescent material on the surface of an electrode to oxidation-reduction reaction by using energy supplied from the electrode to form an excited state and returning the excited state to a ground state
22. Fluorescence: refers to a photoluminescence cold light phenomenon. When a certain normal temperature substance is irradiated by incident light with a certain wavelength, the substance enters an excited state after absorbing light energy, and immediately excites and emits emergent light (the wavelength is usually in a visible light band) which is longer than the wavelength of the incident light.
23. Molecular conformation: the molecules present different steric images by changing the relative position of their atoms or groups of atoms in space due to the rotation of single bonds.
24. Molecular configuration: the spatial configuration of a molecule refers to the geometry of the spatial distribution of various groups or atoms in the molecule.
25. Medical diagnosis: products and services for judging diseases or body functions are obtained by detecting human samples (blood, body fluid, tissues and the like) to obtain clinical diagnosis information.
26. Biological detection: is a detection means and method for biochemical analysis using biomolecules derived from an organism. Biological detection relates to field detection and on-line monitoring in the fields of medical diagnosis, clinical examination, disease prevention and control, agriculture, animal husbandry, aquaculture, food safety guarantee and the like, and even separation and analysis of components.
27. Nucleic acid sample: it refers to ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), and may be artificially synthesized or derived from natural animal, plant, and microorganism (including virus).
28. Nucleic acid amplification: the process of specific or specific repeated replication of nucleic acid target molecules to be detected in test tubes is carried out by the action of enzymes.
29. Nucleic acid fluorescent dye: a fluorescent dye specific for nucleic acids. When the fluorescent dye is combined with nucleic acid molecules, the fluorescence effect can be greatly improved, and when a fixed amount of nucleic acid fluorescent dye is contained in a test tube, the total fluorescence amount is in direct proportion to the amount of the nucleic acid.
30. Fluorescence labeling of molecular probes: and the specific fluorescent probe is an oligonucleotide, and two ends of the specific fluorescent probe are respectively marked with a reporter fluorescent group and a quenching fluorescent group. When the probe is complete, the fluorescent signal emitted by the reporter group is absorbed by the quenching group; when nucleic acid amplification is carried out (such as PCR amplification), the probe is subjected to enzyme digestion degradation by the exonuclease activity of polymerase, so that the report fluorescent group and the quenching fluorescent group are separated, a fluorescence monitoring system can receive a fluorescence signal, namely, a free fluorescent molecule is formed when one DNA strand is amplified, and the complete synchronization of the accumulation of the fluorescence signal and the formation of an amplification product is realized.
31. Dissolution point: also called DNA denaturation temperature point, refers to the hydrogen bond breakage of double-helix base pairs of nucleic acid, the stacking force between bases is destroyed, 50% of double-strand becomes single-strand, the natural conformation and properties of nucleic acid are changed, but the change of primary structure is not related.
32. Dissolution curve: a graphical representation of the change in fluorescence signal associated with denaturation (lysis) of DNA with changes in temperature allows the determination of the DNA lysis point.
33. Multiplex detection, a method and a process for detecting two or more target molecules in the same reaction system. Different target molecules have different molecular characteristics from one target molecule to another.
34. Curves, information, data in the present invention, these terms all represent results obtained using the device of the present invention, and are interchangeable.
35. Liquid in the present invention, the concept of liquid includes an aqueous solution-based liquid or an organic solution-based liquid.
Any combination of the various embodiments of the present invention should be considered as disclosed in the present invention, unless the inventive concept is contrary to the present invention; within the scope of the technical idea of the invention, any combination of various simple modifications and different embodiments of the technical solution without departing from the inventive idea of the present invention shall fall within the protection scope of the present invention.

Claims (19)

1. A multivariate reaction kinetics real-time detector device, comprising: the detector is in communication connection with the computer; the detector comprises a shell, a main control circuit board and a detection structure; the main control circuit board and the detection structure are arranged in the shell, and the detection structure is in communication connection with the main control circuit board; a mounting seat is mounted in the shell, and a test tube mounting hole is formed in the mounting seat and used for mounting a reaction test tube; the detection structure is arranged on the periphery of the test tube mounting hole;
the detection structure comprises an excitation light source, a temperature control assembly and a photoelectric detection assembly; the excitation light source is arranged above or below the test tube mounting hole and faces the test tube mounting hole, a first light source optical filter is arranged between the test tube mounting hole and the excitation light source, the temperature control assembly comprises an upper heater and a lower heater, the upper heater is arranged at the upper end of the test tube mounting hole, the lower heater is arranged at the lower end of the test tube mounting hole, and the upper heater and the lower heater are both surrounded on the periphery of the reaction test tube;
the photoelectric detection assembly comprises a photoelectric detector, a solid light guide and a second light source optical filter, and the photoelectric detector, the solid light guide and the second light source optical filter are all arranged on the same side of the test tube mounting hole; the solid light guide is positioned between the test tube mounting hole and the photoelectric detector, and the second light source optical filter is positioned between the solid light guide and the photoelectric detector; the lighting time of the excitation light source is adjusted between 1 millisecond and 1000 milliseconds, the interval period of lighting the excitation light source every time is between 1 second and 5 minutes, and the collection of optical signals by the photoelectric detection assembly is completed in the lighting process of the excitation light source; the first light source filter and the second light source filter limit part of light with wavelengths to pass through;
the master control circuit board comprises an MCU module, a light temperature control module and a light signal amplification module; the optical signal amplification module is in communication connection with the MCU module, and the MCU module is in communication connection with the light temperature control module; the excitation light source and the temperature control assembly are both in communication connection with the light temperature control module, and the photoelectric detection assembly is in communication connection with the light signal amplification module;
the computer comprises a user interface, and a user inputs variable parameters through the user interface, so that optical signals determined by temperature, time or spatial positions in the reaction test tube can be monitored in real time; the user interface sets the working temperature of the upper heater and the lower heater, the light emitting time and period of the excitation light source, and selectively turns on the photoelectric detector to realize the collection of optical signals of a certain wave band; after the detector starts to work, the user interface can display information corresponding to the temperature and the time fed back in real time; and simultaneously displaying the information corresponding to the intensity and time of the optical signal fed back in real time.
2. The multivariate reaction kinetics real-time detector device of claim 1, wherein: go up the heater with the heater is adjustable down the temperature of reaction test tube heats the reaction test tube makes reaction test tube internal liquid heats up to 100 ℃ or cools down to the room temperature to promote or prevent liquid convection through the difference in temperature.
3. The multivariate reaction kinetics real-time detector device of claim 1, wherein: the photodetector monitors a chemical reaction or a biochemical reaction of the liquid in the reaction cuvette as a function of temperature and time by means of an optical signal.
4. The multivariate reaction kinetics real-time detector device of claim 3, wherein: the detector apparatus determines the amount, structural characteristics or biochemical activity of a target molecule in a sample by real-time monitoring of reaction kinetics.
5. The multivariate reaction kinetics real-time detector device of claim 1, wherein: the photoelectric detection component comprises two photoelectric detectors, two light source optical filters and two solid light guides, wherein one of the photoelectric detectors, one of the light source optical filters and one of the solid light guides are in one group and divided into two groups, the two groups are oppositely arranged on two sides of the test tube mounting hole, and the two groups of the solid light guides are positioned between the upper heater and the lower heater.
6. The multivariate reaction kinetics real-time detector device of claim 1, wherein: the photoelectric detection components comprise two groups of upper photoelectric detection components and two groups of lower photoelectric detection components; the two groups of upper photoelectric detection assemblies are oppositely arranged on two sides of the upper end of the test tube mounting hole, and the two groups of lower photoelectric detection assemblies are oppositely arranged on two sides of the lower end of the test tube mounting hole; each set of the upper photodetection components comprises an upper photodetector, an upper solid light guide, a second light source filter, the upper solid light guide being located below the upper heater, and the upper solid light guide being located between the cuvette mounting aperture and the upper photodetector, the second light source filter being located between the upper solid light guide and the upper photodetector;
each group of the lower photoelectric detection assembly comprises a lower photoelectric detector, a third light source optical filter and a lower solid light guide, the lower solid light guide is positioned above the lower heater, the lower solid light guide is positioned between the test tube mounting hole and the lower photoelectric detector, and the third light source optical filter is positioned between the lower solid light guide and the lower photoelectric detector.
7. The multivariate reaction kinetics real-time detector device of claim 1, wherein: the photoelectric detection assembly comprises a group of upper photoelectric detection assemblies and a group of lower photoelectric detection assemblies; the upper photoelectric detection assembly and the lower photoelectric detection assembly are arranged on the same side of the test tube mounting hole;
or the photoelectric detection components comprise a group of upper photoelectric detection components and a group of lower photoelectric detection components; the upper photoelectric detection assembly and the lower photoelectric detection assembly are respectively arranged on two sides of the test tube mounting hole;
the upper photodetection assembly comprises an upper photodetector, an upper solid light guide, a second light source filter, the upper solid light guide being located below the upper heater, and the upper solid light guide being located between the cuvette mounting aperture and the upper photodetector, the second light source filter being located between the upper solid light guide and the upper photodetector;
the lower photoelectric detection assembly comprises a lower photoelectric detector, a third light source optical filter and a lower solid light guide, the lower solid light guide is located above the lower heater, the lower solid light guide is located between the test tube mounting hole and the lower photoelectric detector, and the third light source optical filter is located between the lower solid light guide and the lower photoelectric detector.
8. The multivariate reaction kinetics real-time detector device of claim 1, wherein: the photoelectric detection components comprise two groups of upper photoelectric detection components and one group of lower photoelectric detection components; the two groups of upper photoelectric detection assemblies are oppositely arranged on two sides of the upper end of the test tube mounting hole, and the lower photoelectric detection assembly is arranged on one side of the lower end of the test tube mounting hole;
or the photoelectric detection components comprise a group of upper photoelectric detection components and two groups of lower photoelectric detection components; the upper photoelectric detection assemblies are arranged on one side of the upper end of the test tube mounting hole, and the two groups of lower photoelectric detection assemblies are oppositely arranged on two sides of the lower end of the test tube mounting hole;
the upper photodetection assembly comprises an upper photodetector, an upper solid light guide, a second light source filter, the upper solid light guide being located below the upper heater, and the upper solid light guide being located between the cuvette mounting aperture and the upper photodetector, the second light source filter being located between the upper solid light guide and the upper photodetector;
the lower photoelectric detection assembly comprises a lower photoelectric detector, a third light source optical filter and a lower solid light guide, the lower solid light guide is located above the lower heater, the lower solid light guide is located between the test tube mounting hole and the lower photoelectric detector, and the third light source optical filter is located between the lower solid light guide and the lower photoelectric detector.
9. The multivariate reaction kinetics real-time detector device of claim 1, wherein: when in use, the test tube mounting hole is inclined at an angle ranging from 5 degrees to 45 degrees.
10. The multivariate reaction kinetics real-time detector device of claim 1, wherein: the lower heater comprises a lower heating plate and a lower heating ring arranged on the lower heating plate, and the lower heating ring is in thermal expansion reversible locking contact with the reaction test tube.
11. The multivariate reaction kinetics real-time detector device of claim 1, wherein: the solid light guide can be coated with a filter film and can play a role in full transmission or partial transmission or selective transmission on incident light, and the solid light guide is cylindrical or square.
12. The multivariate reaction kinetics real-time detector device of claim 1, wherein: the first light source optical filter, the second light source optical filter and the third light source optical filter are made of inorganic glass, organic glass or quartz, and the surfaces of the first light source optical filter, the second light source optical filter and the third light source optical filter can be coated with optical filter films, and the optical filter films have low-pass, high-pass or band-pass performance.
13. The multivariate reaction kinetics real-time detector device according to any one of claims 1 to 12, wherein: the detector device is used for enzyme catalysis reaction kinetic analysis and molecular conformation and configuration analysis by detecting chemiluminescence, electrochemiluminescence, microsome luminescence and fluorescence.
14. The multivariate reaction kinetics real-time detector device of claim 1, wherein: the computer is mounted inside or outside the housing.
15. The multivariate reaction kinetics real-time detector device of claim 1, wherein: the distance between the main control circuit board and the mounting seat is less than or equal to 100 mm.
16. A method of testing using the meter device of any of claims 6 to 8, comprising the steps of:
adding a reaction solution into a reaction test tube, placing the closed reaction test tube into a test tube mounting hole, and heating the reaction solution in the reaction test tube by an upper heater and a lower heater to ensure that the temperature difference between the upper part and the lower part of the reaction solution in the reaction test tube is more than or equal to 5 ℃ so as to circulate, mix or stand the solution;
starting a computer real-time monitoring program, and recording and outputting a reaction result in real time;
and carrying out medical diagnosis or biological sample detection or drawing a regional distribution map of related information according to the reaction result.
17. The detection method according to claim 16, comprising the steps of:
firstly, adding a reaction solution into a reaction test tube, wherein the reaction solution contains a mixed nucleic acid sample, a chemical reagent for nucleic acid amplification and a nucleic acid fluorescent dye or a fluorescent labeling molecular probe, and when the nucleic acid sample is RNA, transcribing the RNA into complementary DNA;
then, carrying out nucleic acid sample amplification by using a constant-temperature amplification or variable-temperature amplification enzymatic method, and controlling the temperature of the liquid to circulate between 45 ℃ and 98 ℃ in the variable-temperature nucleic acid amplification process; when the photodetection assembly detects the signal of the nucleic acid fluorochrome or fluorescently labeled molecular probe, the intensity of the fluorescent signal generated by the amplification reaction is proportional to the amount of amplified DNA product.
18. The detection method of claim 17, comprising the steps of:
when the temperature of the solution at the bottom of the reaction test tube is reduced to be lower than the nucleic acid dissolution temperature point after the nucleic acid amplification reaction is finished, most DNA molecules are combined into double-stranded molecules and combined with the nucleic acid fluorescent dye to release stronger fluorescence, the upper heater and the lower heater are controlled to start heating at a slow speed, and time and temperature variables are recorded through a computer; when the temperature variable is adjusted, the change of the optical signal is recorded in real time through the lower photoelectric detector or the upper photoelectric detector, and the relation between the temperature and the fluorescence signal is displayed through a computer; as the temperature is increased, the double-stranded DNA molecules are dissolved into single-stranded molecules, so that the fluorescence quantity is reduced, and the dissolution point or the dissolution curve of the amplified double-stranded DNA molecules is measured by data processing of a computer.
19. The detection method of claim 17, comprising the steps of:
after the nucleic acid amplification reaction is finished, keeping the temperature of the solution at the bottom of the reaction test tube at 90-98 ℃, raising the temperature of the liquid at the upper end to be not more than 95 ℃ by an upper heater, dissolving most double-stranded DNA molecules into single-stranded DNA molecules, controlling the upper heater and the lower heater to start to reduce the temperature at a slow speed, and recording time and temperature variables by a computer; when the temperature variable is adjusted, the change of the optical signal is recorded in real time through the lower photoelectric detector or the upper photoelectric detector, and the relation between the temperature and the fluorescence signal is displayed through a computer; as the temperature is lowered, the single-stranded DNA molecules are combined into double-stranded molecules, so that the fluorescence quantity is increased, and the dissolution point or the dissolution curve of the amplified DNA molecules is measured through data processing by a computer.
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