CN111443213B - Multi-variable reaction dynamics real-time detector device and detection method - Google Patents

Multi-variable reaction dynamics real-time detector device and detection method Download PDF

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Publication number
CN111443213B
CN111443213B CN202010274339.6A CN202010274339A CN111443213B CN 111443213 B CN111443213 B CN 111443213B CN 202010274339 A CN202010274339 A CN 202010274339A CN 111443213 B CN111443213 B CN 111443213B
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reaction
test tube
temperature
light source
detector
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CN111443213A (en
Inventor
吴景洪
苏星
马骏
张代化
苏成宽
苏卫
吴开原
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Zhuhai Astrobio Biotechnology Co ltd
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Zhuhai Astrobio Biotechnology 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 communicatively coupled to 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 dynamics real-time detector device and the detection method can read optical signals related to temperature, time and space variables in the test tube in real time, display results in real time through a computer, obtain reaction results more quickly, know the reaction process in real time and effectively improve analysis efficiency.

Description

Multi-variable reaction dynamics 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 dynamics real-time detector device and a detection method.
Background
The reaction kinetics analysis is a method of analyzing the characteristics of a reaction substrate or a catalyst and the amount thereof by measuring the reaction rate by means of the relationship between the rate of a chemical reaction and the concentration of the reaction substrate or the relationship between the reaction acceleration catalyst including the concentration of a biological enzyme. The reaction kinetics has a direct relationship with temperature. Reaction kinetics monitoring is important for chemical reactions 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 to drug development and medical diagnostics, public health and biological detection, and reaction kinetics can also affect the efficacy of the drug.
Kinetic analysis can be accomplished by monitoring the directly or indirectly generated optical signals, however, existing detection instruments typically use discrete thermodynamic and optical components and are therefore bulky and expensive; and the optical signals need to be read step by step, and finally the result is displayed after the reaction is completed. Therefore, the existing instrument cannot read the optical signal in real time or continuously under the condition of different variables and display the result in real time, so that the time required for the existing instrument is long and the analysis efficiency is low.
Disclosure of Invention
The invention provides a multivariable reaction dynamics 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 invention adopts the technical scheme that: a multivariable reaction kinetics real-time detector device, comprising: the detector and computer, the detector is connected with the computer communication. 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 arranged 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 component and a photoelectric detection component.
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 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, wherein the upper heater is installed at the upper end of the test tube installation hole, the lower heater is installed at the lower end of the test tube installation hole, and the upper heater and the lower heater are all enclosed at the periphery of the reaction test tube.
The photoelectric detection assembly comprises a photoelectric detector, a solid light guide and a second light source filter. The photoelectric detector, the solid light guide and the second light source 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; the second light source filter is located 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 each lighting of the excitation light source is between 1 second and 5 minutes, and the acquisition of the light signal by the photoelectric detection component is completed in the lighting process of the excitation light source; the first light source filter and the second light source filter limit the light with partial wavelength to pass through;
The main control circuit board comprises an MCU module, an optical temperature control module and an optical signal amplification module, wherein the optical signal amplification module is in communication connection with the MCU module, and the MCU module is in communication connection with the optical temperature control module; the excitation light source and the temperature control component are both in communication connection with the optical temperature control module, and the photoelectric detection component is in communication connection with the optical signal amplification module; the MCU module can be expanded into a computer module to partially or completely replace the computer function;
the computer can be a MCU module with expanded functions, and comprises a user interface, wherein a user inputs variable parameters through the user interface, and can monitor optical signals which are determined by temperature, time or space position in the reaction test tube in real time; the user interface sets the working temperatures of the upper heater and the lower heater, the time and the period of the light emission of the excitation light source, and selectively opens the photoelectric detector to collect the optical signals of a certain wave band;
after the detector starts to work, the user interface can display information corresponding to the temperature and time fed back in real time; and simultaneously displaying information corresponding to the optical signal intensity and time fed back in real time.
Further, the upper heater and the lower heater can adjust the temperature of the reaction tube, heat the reaction tube, enable the liquid in the reaction tube to be heated to 100 ℃ or cooled to room temperature, and promote or prevent liquid convection through temperature difference.
Further, the photodetector monitors a chemical reaction or a biochemical reaction of the liquid in the reaction cuvette with temperature and time by means of an optical signal.
Further, the detector device monitors in real time by reaction kinetics to determine the amount, structural characteristics or biochemical activity of the target molecule in the sample.
Further, the photodetection assembly comprises two photodetectors, two light source filters, and two solid light guides. One photoelectric detector, one light source filter and one solid light guide are divided into two groups, are oppositely arranged on two sides of the test tube mounting hole, and are positioned between the upper heater and the lower heater.
Further, the photoelectric detection assemblies comprise two groups of upper photoelectric detection assemblies and two groups of lower photoelectric detection assemblies; the two groups of upper photoelectric detection assemblies are oppositely arranged at two sides of the upper end of the test tube mounting hole, and the two groups of lower photoelectric detection assemblies are oppositely arranged at two sides of the lower end of the test tube mounting hole; each group of 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 positioned below the upper heater, the upper solid light guide is positioned between the test tube mounting hole and the upper photoelectric detector, and the second light source filter is positioned between the upper solid light guide and the upper photoelectric detector.
Each group of lower photoelectric detection components comprises a lower photoelectric detector, a third light source 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 cuvette mounting hole and the lower photodetector, and the third light source filter is located between the lower solid light guide and the lower photodetector.
Further, the photoelectric detection assembly comprises an upper photoelectric detection assembly and a lower photoelectric detection assembly; 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 assembly comprises an upper photoelectric detection assembly and a lower photoelectric detection assembly; the upper photoelectric detection assembly and the lower photoelectric detection assembly are respectively arranged at 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 filter; the upper solid light guide is positioned below the upper heater, the upper solid light guide is positioned between the test tube mounting hole and the upper photoelectric detector, and the second light source filter is positioned between the upper solid light guide and the upper photoelectric detector;
The lower photoelectric detection assembly comprises a lower photoelectric detector, a third light source 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 cuvette mounting hole and the lower photodetector, and the third light source filter is located between the lower solid light guide and the lower photodetector.
Further, the photoelectric detection assembly comprises two groups of upper photoelectric detection assemblies and one group of lower photoelectric detection assemblies; the two groups of upper photoelectric detection assemblies are oppositely arranged at two sides of the upper end of the test tube mounting hole, and the lower photoelectric detection assemblies are arranged at one side of the lower end of the test tube mounting hole;
or the photoelectric detection assembly comprises an upper photoelectric detection assembly and two lower photoelectric detection assemblies; 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 filter; the upper solid light guide is positioned below the upper heater, the upper solid light guide is positioned between the test tube mounting hole and the upper photoelectric detector, and the second light source filter is positioned between the upper solid light guide and the upper photoelectric detector;
The lower photoelectric detection assembly comprises a lower photoelectric detector, a third light source 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 cuvette mounting hole and the lower photodetector, and the third light source filter is located between the lower solid light guide and the lower photodetector.
Further, the tube mounting aperture is inclined at an angle in the range of 5 ° to 45 ° 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, can play a role of total transmission or partial transmission or selective transmission on incident light, and is cylindrical or square.
Further, the first light source filter, the second light source filter and the third light source filter are inorganic glass, organic glass or quartz, and the surfaces of the first light source filter, the second light source filter and the third light source filter can be coated with filter films, including performances 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 the housing or outside the housing.
Further, the distance between the main control circuit board and the mounting seat is less than or equal to 100mm.
The invention also provides the following technical scheme: a detection method using the detector device, 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, heating the reaction solution in the reaction test tube by an upper heater and a lower heater, and enabling the temperature difference between the upper temperature and the lower temperature of the reaction solution in the reaction test tube to be more than or equal to 5 ℃ so as to enable the solution to circulate, mix or stand still;
starting a computer real-time monitoring program, 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 the 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, firstly transcribing the RNA into complementary DNA; then amplifying the nucleic acid sample by using an enzymatic method of isothermal amplification or variable temperature amplification, wherein in the variable temperature nucleic acid amplification process, the temperature circulation of the liquid between 45 ℃ and 98 ℃ is controlled; when the photodetection assembly detects the signal of the nucleic acid fluorescent dye or the fluorescent 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 tube is reduced below the nucleic acid dissolution temperature point after the nucleic acid amplification reaction is finished, most DNA molecules are combined into double-stranded molecules and are combined with nucleic acid fluorescent dye to emit stronger fluorescence, the upper heater and the lower heater are controlled to start heating at a low speed, and time and temperature variables are recorded through a computer; when the temperature variable is regulated, the change of the optical signal is recorded in real time through a lower photoelectric detector or an upper photoelectric detector, and the relation between the temperature and the fluorescent signal is displayed through a computer; with increasing temperature, the double-stranded DNA molecules are dissolved into single-stranded molecules, so that the fluorescence amount is reduced, and the dissolution point or dissolution curve of the amplified double-stranded DNA molecules is measured by performing data processing on a computer.
Further, the method comprises the following steps:
when the nucleic acid amplification reaction is completed, maintaining the temperature of the solution at the bottom of the reaction tube at 90-98 ℃ and raising the temperature of the upper liquid to not more than 95 ℃ by an upper heater, at this time, most of the double-stranded DNA molecules are dissolved into single-stranded DNA molecules, then controlling the upper heater and a lower heater to start to slowly cool down, and recording time and temperature variables by a computer; when the temperature variable is regulated, the change of the optical signal is recorded in real time through a lower photoelectric detector or an upper photoelectric detector, and the relation between the temperature and the fluorescent signal is displayed through a computer; as the temperature decreases, single-stranded DNA molecules will bind to double-stranded molecules, causing an increase in fluorescence, and the computer is used to perform data processing to determine the dissolution point or dissolution profile of the amplified DNA molecules.
Compared with the prior art, the multivariable reaction kinetics real-time detector device and the detection method of the invention are characterized in that the photoelectric detection component, the temperature control component and the excitation light source are arranged on the detector and are in communication connection with the main control circuit board, the detector is in communication connection with the computer, and the related optical signals of the multivariable (such as temperature, time and space part) are monitored in real time, such as: controlling the temperature of different positions of the reaction test tube, and detecting optical signals of the relative position changing along with time; controlling the temperature of different positions of the reaction test tube, and detecting optical signals of the relative positions along with the temperature change; controlling the temperature gradient of the liquid in the reaction test tube, and detecting an optical signal of the relative position along with the change of temperature and 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 the results can be uploaded to the cloud, the data can be monitored and analyzed remotely, and can be used to generate a regional distribution map of information (e.g., by monitoring for infectious disease (immune response or nucleic acid amplification), infectious disease distribution map and thus predicting its trend).
Drawings
The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate the invention and together with the description serve to explain the invention. In the drawings of which there are shown,
fig. 1: the invention relates to a perspective view of a multivariable reaction dynamics real-time detector device;
fig. 2: a perspective view of a first embodiment of the detection structure of the present invention;
fig. 3: a cross-sectional view of a first embodiment of the detection structure of the present invention;
fig. 4: another perspective view of the first embodiment of the detection structure of the present invention;
fig. 5: a block diagram of the multivariable reaction kinetics real-time detector device of the invention;
fig. 6: a perspective view of a second embodiment of the detection structure of the present invention;
fig. 7: a perspective view of a third embodiment of the detection structure of the present invention;
fig. 8: a perspective view of a fourth embodiment of the detection structure of the present invention;
fig. 9: a perspective view of a fifth embodiment of the detection structure of the present invention;
fig. 10: a perspective view of a sixth embodiment of the detection structure of the present invention;
fig. 11: a perspective view of a seventh embodiment of the detection structure of the present invention;
fig. 12: schematic diagram of nucleic acid detection process;
fig. 13: a plot of fluorescent signal from a nucleic acid detection process;
fig. 14: temperature change profiles of the upper and lower heaters.
Detailed Description
The following describes specific embodiments of the present invention in detail with reference to the drawings. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.
Example 1
As shown in fig. 1, the multivariable reaction kinetics real-time detector device of the present invention comprises a base 1, a detector 2, and a computer; the detector 2 is arranged on the base 1, and the detector 2 and the computer are connected through USB connection or wireless (such as Bluetooth, WIFI and 5G) communication, or the detector 2 and the computer are integrated into a whole; the detector 2 is controlled to work by sending a control command in real time through a computer.
The detector 2 comprises a shell 201, a main control circuit board and a detection structure; the shell 201 is rotatably installed on the base 1, the main control circuit board and the detection structure are installed 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 ventilation and heat dissipation holes 202, so that the phenomenon that the temperature of the detector 2 is too high after long-time working is effectively avoided.
The base 1 includes a bottom plate 101 and a rotating plate 102, wherein one end of the rotating plate 102 is rotatably mounted at one end of the bottom plate 101 through a rotating shaft 3, and is locked through an angle lock nut 4. The shell 201 is arranged on the rotating plate 102, the mounting seat 5 is arranged in the shell 201, and a plurality of test tube mounting holes 6 are formed in the mounting seat 5 and used for mounting the reaction test tubes 7; each of the detecting structures is mounted on the outer periphery of each test tube mounting hole 6, respectively. Further, the position of the housing 201 corresponding to the plurality of test tube mounting holes 6 is provided with a rotary cover 203, so that the housing 201 is conveniently opened, the plurality of test tube mounting holes 6 are exposed, and the reaction test tube 7 is conveniently mounted.
Through installing detector 2 in rotor plate 102, when the inclination of needs regulation reaction tube 7, can be through loosening angle lock nut 4, with rotor plate 102 around the rotation axis 3 rotation to the angle of needs, then lock angle lock nut 4 again can for solution in the reaction tube 7 realizes more effective heat convection. Wherein, the reaction test tube 7 is straight test tube, when using, can adjust the inclination of test tube mounting hole 6 through the angle of adjusting rotor plate 102, and then adjust the angle of reaction test tube 7 slope, angle adjustment's scope 5-45, and the ratio of reaction test tube 7's length and diameter is more than or equal to 10.
As shown in fig. 1 and 2, the detection structure includes an excitation light source 8, a temperature control component, and a photoelectric detection component; wherein, excitation light source 8 is installed in test tube mounting hole 6 below and is faced 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 mounted 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 can be understood that the excitation light source 8 and the first light source filter 9 may also be mounted above the test tube mounting hole 6 opposite to the test tube mounting hole 6, specifically, mounted on the inner wall surface of the rotary cover 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 the excitation light source 8 is between 1 second and 5 minutes each time, the lighting interval period can be adjusted according to different reaction substances, and the light signal acquisition of the photoelectric detection component 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 installed at the upper end of the test tube installation hole 6, and the lower heater 11 is installed at the lower end of the test tube installation hole 6; when the reaction cuvette 7 is mounted in the cuvette mounting hole 6, the upper heater 10 and the lower heater 11 are both enclosed around the outer circumference of the reaction cuvette 7. The method 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 comprises a lower heating plate and a lower heating ring mounted on the lower heating plate, in which the reaction tube 7 is reversibly locked by thermal expansion. Further, the upper heater 10 and the lower heater 11 are each mounted with a thermistor; the temperature control of the solution in the reaction test tube 7 is realized through the temperature control component, and the temperature control device comprises: heating, keeping constant temperature, cooling to form a temperature gradient, so that the reaction liquid generates or stops heat convection.
The computer comprises a user interface and associated software by which the user inputs variable parameters for monitoring in real time the optical signals dependent on temperature, time or spatial position in the reaction cuvette 7.
The method comprises the following steps: the user interface sets the working temperature of the upper heater 10 and the lower heater 11, the time and the period of the light emission of the excitation light source 8, and selectively turns on one of the photodetectors to collect the light signal of a certain wave band. After the detector is started to work, the user interface can display a temperature and time corresponding curve fed back in real time; and simultaneously displaying the curve corresponding to the optical signal intensity and time fed back in real time. By comparing the intensity of the optical signal, the number of products corresponding to the reactants can be analyzed.
The continuous temperature change of the solution in the reaction tube 7 can be achieved in various ways, firstly the upper heater 10 and the lower heater 11 are changed by themselves and conducted to the reaction tube 7, so that the temperature of the liquid in the reaction tube 7 is changed. Another way is to make the liquid convect 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 corresponding portions constant, the molecules in the liquid may undergo different temperature changes during convection. Therefore, continuous temperature change refers to the temperature change of the liquid sample in the reaction tube 7 that is effective. The photoelectric detection components comprise two groups of upper photoelectric detection components and two groups of lower photoelectric detection components, wherein the two groups of upper photoelectric detection components are oppositely arranged on two sides of the upper end of the test tube mounting hole 6, and the two groups of lower photoelectric detection components are oppositely arranged on two sides of the lower end of the test tube mounting hole 6.
The method 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, and 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 fluorescent signals from the reaction test tube 7 to the upper photoelectric detector 12 and outputting voltage or current analog signals corresponding to the intensity of the fluorescent signals to the main control circuit board through the upper photoelectric detector 12. 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 the light signal source from the reaction cuvette 7 to the lower photo-detector 14. Further, a third light 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 not less than 5mm.
The upper solid light guide 13 and the lower solid light guide 15 can be fully or partially or selectively transmissive to the incident light, and the shape can be different, such as: cylindrical, square, etc.; the material can be inorganic glass, organic glass, quartz and other materials, and the surface can be coated with a filter film. The first, second and third light source filters 9, 17, 18 restrict the passage of light of a part of wavelengths, including low pass, high pass, band pass. The first light source filter 9, the second light source filter 17 and the third light source filter 18 play a role in distinguishing different fluorescence wavelengths; the wavelength of the emitted fluorescence spectrum is different according to the different reaction substances in the reaction tube 7, and filters with different wave bands can be used for distinguishing one or more reaction substances according to the requirements of the reaction substances.
In this embodiment, a row of test tube mounting holes 6 are formed in the mounting seat 5, and a row of four or eight test tube mounting holes 6 are formed in the row, and a detection structure is mounted corresponding to each test tube mounting hole 6; it will be appreciated that in other embodiments, the mounting base 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 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 thereto.
As shown in fig. 2 and 5, the main control circuit board comprises an MCU module, an optical temperature control module and an optical signal amplifying module; the optical signal amplifying module is in communication connection with the MCU module, and the MCU module is in communication connection with the optical temperature control module. The excitation light source 8 and the temperature control component are both in communication connection with the optical temperature control module, and the photoelectric detection component is in communication connection with the optical signal amplification module.
The optical signals in a specific wavelength range are acquired in less than one second through the upper solid light guide 13, the lower solid light guide 15, the second light source filter 17 and the third light source filter 18, and the optical signals are amplified through the optical signal amplifying module, so that the detection instrument can realize real-time detection.
Example two
As shown in fig. 1 and 6, the same structure as that of the first embodiment of the present embodiment is not described herein, and the difference is that: the photodetection assembly consists of only one photodetector 19, one light source filter 20 and one solid light guide 21, which is located at one side of the cuvette mounting hole 6, and the solid light guide 21 is located between the upper heater 10 and the lower heater 11.
Embodiment III:
as shown in fig. 1 and 7, the same structure as that of the first embodiment of the present embodiment is not described herein, and the difference is that: the photoelectric detection assembly 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, which are oppositely disposed at 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 IV
As shown in fig. 1 and 8, the same structure as that of the first embodiment of the present embodiment is not described herein, and the difference is that: 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 all set up in test tube mounting hole 6 same one side.
Example five
As shown in fig. 1 and 9, the same structure as that of the embodiment is not described herein, and the difference is that: 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 10, the same structure as that of the first embodiment of the present embodiment is not described herein, and the difference is that: the photoelectric detection assembly comprises two groups of upper photoelectric detection assemblies and one group of lower photoelectric detection assemblies; wherein, two sets of upper photoelectric detection components set up in the upper end both sides of test tube mounting hole 6 relatively, and lower photoelectric detection component sets up in one of them one side of test tube mounting hole 6 lower extreme.
Embodiment seven:
as shown in fig. 1 and 11, the same structure as that of the first embodiment of the present embodiment is not described herein, and the difference is that: the photoelectric detection assembly comprises a group of upper photoelectric detection assemblies and two groups of lower photoelectric detection assemblies; wherein, go up photoelectric detection subassembly setting and be in one of them one side of test tube mounting hole 6 upper end, two sets of lower photoelectric detection subassemblies set up in test tube mounting hole 6's lower extreme both sides relatively.
As shown in fig. 1 and 11, one function of employing multiple sets of photodetection elements in various embodiments is to determine optical signals at different spatial locations; another function is to determine optical signals at different frequencies (wavelengths), spectra, or a combination of both. Therefore, multiple detection can be realized, and the detection efficiency or the detection accuracy can be improved.
The working principle of the multivariable reaction dynamics real-time detector device and the detection method of the invention is as follows:
filling a solution to be tested (the solution is provided with fluorescent molecules, is added with fluorescent molecules or is capable of generating fluorescent molecules or precursors or substrate molecules of the fluorescent molecules in the reaction process) into a reaction test tube 7, plugging a plug 16, and then installing the reaction test tube 7 in a test tube installation hole 6; tilting the mounting 5 by 5 ° -45 ° (note: tilt angle is only useful when heat convection is performed) if necessary adjusting the reaction tube 7; starting a real-time monitoring program, sending a control instruction through a computer to control the detector 2 to start working, heating a solution in the reaction test tube 7 through the upper heater 10 and the lower heater 11, and when the temperature is continuously increased, performing thermal expansion on 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 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 controlling the working temperatures of the upper heater and the lower heater and transmitting the corresponding temperatures to the reaction test tube 7; wherein, the temperature of the upper heater 10 and the lower heater 11 can be adjusted to different temperatures according to the reaction requirement, and the actual temperature is simultaneously fed back to the computer through the corresponding thermistor;
when the detector 2 receives a light-emitting instruction from a computer, the excitation light source 8 is lightened according to the instruction to irradiate the solution in the reaction test tube 7, the solution in the reaction test tube 7 is irradiated by the excitation light source 8 to emit fluorescence, and the fluorescence is respectively transmitted to the upper photoelectric detector 12 and the lower photoelectric detector 14 through the corresponding upper solid light guide 13 and the lower solid light guide 15; when the upper photoelectric detector 12 and the lower photoelectric detector 14 detect fluorescent signals sent by the solution in the reaction test tube 7, voltage or current analog signals corresponding to the fluorescent signals are directly output, the analog signals are converted into amplified electric signals through the optical signal amplification module, the amplified electric signals correspond to the fluorescent signals with corresponding intensity, and the amplified electric signals are fed back to the computer through the MCU module in real time, so that the optical signals are read in real time, and the results are displayed in real time.
Wherein, the computer can select different temperatures and irradiation time of the excitation light source 8 according to different reaction solutions, and feed back the conditions of the solution for reaction in different space positions in real time; 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 acquisition of the optical signal by the photoelectric detection component is completed in the process after the excitation light source 8 is lightened.
The results obtained by monitoring the multi-variable reaction dynamics real-time detector device and the detection method can be uploaded to the cloud 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.
Referring to FIG. 12, the process of detecting nucleic acid amplification is as follows:
filling a reaction solution containing a mixed nucleic acid sample, a chemical reagent for nucleic acid amplification and a nucleic acid fluorescent dye (intercalating dye) or a fluorescent-labeled molecular probe (fluorescent dye labeled molecular probe) into a reaction tube 7; wherein the nucleic acid sample may be taken from RNA or DNA of an animal or plant cell, a bacterium, a fungus or a virus; when the nucleic acid sample is an RNA sample, the RNA is transcribed into complementary DNA, i.e.: cDNA (cDNA and DNA can be amplified in a test tube to achieve the aim of detection); nucleic acid amplification using enzymatic methods, including isothermal amplification, such as RPA (Recombinase polymerase amplification), LAMP (Loop-mediated isothermal amplification), and variable temperature amplification, such as PCR (Polymerase chain reaction); in the variable temperature nucleic acid amplification process, the temperature of the liquid needs to be controlled, so that the liquid is subjected to temperature circulation between 45 ℃ and 98 ℃;
When the photoelectric detection component detects that the solution generates fluorescent signals released by nucleic acid fluorescent dye or fluorescent labeling molecular probes, the intensity of the fluorescent signals generated by the amplification reaction is proportional to the amount of amplified DNA products (the process is a reaction kinetics process which is determined by variable factors such as temperature, time and the like, and the initial copy number of target nucleic acid in a raw sample can be estimated through reaction kinetics data, so that the method has great significance in medical diagnosis, public health and biological detection industries);
determining a dissolution curve, and reducing the temperature of a solution at the bottom of the reaction test tube 7 to about 45 ℃ after the amplification reaction is finished, wherein DNA molecules are combined into double-stranded molecules, combined with nucleic acid fluorescent dye and then emit stronger fluorescence; to determine the dissolution profile, the upper heater 10 and the lower heater 11 start to heat at a slow speed, and time and temperature variables are recorded by a computer, and when the temperature variables are adjusted, changes of optical signals are recorded in real time by the lower or upper photodetectors 14, and the relationship between the temperature and fluorescent signals is displayed by the computer; with increasing temperature, the double-stranded DNA molecules are dissolved into single-stranded molecules, leading to a decrease in fluorescence (this process is related to the length of the DNA molecule chain, i.e. the dissolution temperature characteristic of a DNA molecule of a certain length); the data processing is carried out by a computer to obtain the dissolution point of the amplified DNA molecule, and the dissolution curve is one method for confirming the accuracy of DNA amplification because the dissolution point or the dissolution curve is related to the length of the molecule chain.
Another method for determining the dissolution profile is to maintain the temperature of the solution at the bottom of the reaction tube at a high temperature (90℃to 98 ℃) and raise the temperature of the upper liquid to not more than 95℃by an upper heater after the completion of the nucleic acid amplification reaction, 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 cool down at a low speed, and recording time and temperature variables through a computer; when the temperature variable is regulated, the change of the optical signal is recorded in real time through a lower photoelectric detector or an upper photoelectric detector, and the relation between the temperature and the fluorescent signal is displayed through a computer; as the temperature decreases, single-stranded DNA molecules will bind to double-stranded molecules, causing an increase in fluorescence, and the computer is used to perform data processing to determine the dissolution point or dissolution profile of the amplified DNA molecules.
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 signal intensity and the abscissa is time (minutes). Every 10 seconds, the signal is read-times, 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 relatively high value is the relatively low temperature, such as the number of steps 350 is 60 ℃, and the number of steps 185 is 95 ℃; the horizontal axis is time (minutes).
The data shown is a nucleic acid amplification experiment for principle verification. The reaction tube contains a reaction solution containing respiratory Polynucleosis virus (RSV) RNA to be tested, primers required for nucleic acid amplification, reverse transcriptase and DNA polymerase, buffer solution and nucleic acid fluorescent dye, etc. RNA was first reverse transcribed into cDNA (first seven minutes) and then DNA amplified using the same tube. As shown in FIG. 13, by controlling the temperature gradient of the reaction tube, the reaction solution is convected in the reaction tube, and the nucleic acid is subjected to temperature cycling, thereby realizing PCR-like amplification. In the simultaneous monitoring of 4 reaction cuvettes with the device according to the invention, the reaction status information associated with 520nm wavelengths (V1, V2, V3, V4) and 530nm wavelengths (V5, V6, V7, V8) is read from each reaction cuvette at a time point and displayed. The corresponding curves for the reaction tubes are: reaction tube one (V1, V2), reaction tube two (V2, V6), reaction tube three (V3, V7), reaction tube four (V4, V8). As can be seen from FIG. 13, when the liquid temperature is low, the reaction tube emits nonspecific fluorescence (first few minutes). The nonspecific fluorescent signal decreases (ten to twenty-five minutes) when the temperature increases, and the specific fluorescent signal begins to increase (twenty-five minutes later) when the DNA is amplified to a certain amount. By similar operations, the characteristics (source, genetic structure, number of target molecules, etc.) of the target molecules in the original sample can be detected.
The multivariable reaction kinetics real-time detector device of the present invention is suitable for measuring the reaction kinetics with light as a signal, and comprises:
(1) chemiluminescence, electrochemiluminescence, microsomal luminescence, fluorescence, etc. in immunoassays;
chemiluminescent immunoassay (chemiluminescence immunoassay, CLIA) is a common method in medical diagnosis, which combines a chemiluminescent assay technology with high sensitivity with a high specificity immunoreaction, is used for detection and analysis of various antigens, hapten, antibody, hormone, enzyme, fatty acid, vitamin, medicine and the like, and is a new immunoassay developed after metabolic immunoassay, enzyme immunoassay, fluorescent immunoassay and time-resolved fluorescent immunoassay.
The chemiluminescent immunoassay comprises two parts, namely an immunoreaction system and a chemiluminescent system, wherein the chemiluminescent system is formed into an excited intermediate by catalyzing a chemiluminescent substance (luminescent substrate) through a catalyst (enzyme) and oxidizing an oxidant, photons are emitted simultaneously when the excited intermediate returns to a stable ground state, and the light quantum yield can be measured by using a luminescent signal measuring instrument. The immunoreaction system is to directly or indirectly mark the substances (markers or enzymes) related to the luminous system on the antigen or antibody, and the enzyme quantity in the luminous system is directly proportional to the antigen quantity after immunoreaction and washing, so that when the enzyme acts on the luminous substrate, the luminous quantity is also directly proportional to the antigen quantity. Other luminescent immunoassay techniques also include microsomal luminescent immunoassay (microparticle luminescence enzyme immunoassay, MLEIA), electrochemiluminescent immunoassay (electrochemiluminescence immunoassay, ECLIA). Other methods may not require an excitation light source other than a fluorescence immunoassay method. Specific applications include, but are not limited to, the following:
a. Under the control of different variables (temperature and time), the change of luminescence dynamics is observed in real time; the luminous rate is influenced by temperature, the temperature gradient can enable the liquid to be continuously mixed, the luminous reaction is more uniform, the quantity of the antigen, the quality of the luminous substrate, the stability of the luminous substrate and other indexes can be determined according to the luminous rate, and the kinetic method is more accurate than the detection of the endpoint luminous quantity.
b. The fluorescent resonance energy transfer method detects the affinity and the binding stability between the antigen and the antibody; fluorescence resonance energy transfer refers to the fact that when two fluorescent chromophores are close enough, when a donor molecule absorbs photons with a certain frequency, the two fluorescent chromophores are excited to a higher electron energy state, and before the electrons return to a ground state, energy transfer to an adjacent acceptor molecule is achieved through dipole interaction (namely energy resonance transfer occurs). Antibodies are the core reagents for immune reactions, and their specificity and affinity determine immune reaction specificity and sensitivity. The conventional immune response is a sandwich method, i.e., the capture and detection antibodies are used to recognize the same antigen. When the capture antibody and the detection antibody are labeled with fluorescein, respectively, which can perform fluorescence energy transfer, the affinity of the antigen-antibody can be estimated by detecting the kinetics of fluorescence resonance energy transfer (relationship between the antibody concentration and the luminescence rate).
(2) Enzymatic reaction kinetic analysis including, but not limited to, nucleic acid detection (isothermal or thermocycling).
(3) Molecular conformation and configuration analysis, including but not limited to DNA melting curves.
(4) Tests performed according to the principles described above include medical diagnostics, public health, environmental monitoring, food quality, industrial and agricultural production, commodity inspection, beauty treatment, and the like.
In summary, the multivariable reaction kinetics real-time detector device and the detection method have the following advantages:
1. through setting up photoelectric detection subassembly, control by temperature change subassembly and excitation light source 8 and main control circuit board communication connection at detector 2 to with detector 2 and computer communication connection, the relevant light signal of real-time supervision multivariable (e.g. temperature, time, space position), if: controlling the temperature of different positions of the reaction test tube 7, and detecting optical signals of the relative position changing along with time; controlling the temperature of different positions of the reaction test tube 7, and detecting optical signals of the relative positions along with the temperature change; controlling the temperature gradient of the liquid in the reaction test tube 7, and detecting an optical signal of the relative position changing along with the temperature and 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 provides an important tool for scientific research, medicine development, medical diagnosis, public health and biological detection.
2. By providing the upper heater 10 and the lower heater 11, it is possible to measure the reaction states of different regions (space portions) in the same reaction tube 7 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, and reaction data can be collected more effectively; multiple detection is realized;
4. by arranging the upper solid light guide 13 and the lower solid light guide 15, the structure of the photoelectric detection assembly is more flexible, and the photon collection amount can be increased, so that the sensitivity is improved and the real-time reaction state display is realized.
5. By rotatably mounting the detector 2 to the base 1, an adjustable tilt angle of the reaction cuvette 7 is achieved, so that more orderly thermal convection of the solution in the reaction cuvette 7 is achieved.
6. The structure is simple, the lower heater 11 is reversibly locked in a thermal expansion mode through the reaction test tube 7, effective heat conduction is realized, and meanwhile, the optical-electromechanical device is integrated into a whole, so that the portable electric heating device is convenient to carry and can work outdoors.
The related nouns related to the invention are defined as follows:
1. reaction kinetics: reaction kinetics is a branch discipline of chemical reaction engineering that studies the effects of various physical and chemical factors (e.g., temperature, pressure, concentration, medium in the reaction system, catalyst, flow field and temperature field distribution, residence time distribution, etc.) on reaction rates, and corresponding reaction mechanisms and mathematical expressions, etc. The reaction kinetics in the present invention refer to the course of the reaction and the results associated with the above-described principles.
2. Real-time monitoring, including reading the optical signal in real time, and real-time display result: the state of the reaction was monitored over a longer period (more than ten minutes). The monitoring may be continuous or intermittent, with the intermittent monitoring being for a time period of no more than 5 minutes.
3. And (3) a computer: a computer (commonly called as a computer) is a modern electronic computing machine for high-speed computing, can perform numerical computation and logic computation, and also has a memory function; the intelligent electronic device is modern intelligent electronic equipment which can automatically and rapidly process mass data according to program operation. Consists of a hardware system and a software system. In the present invention, the computer includes a display interface and associated software programs.
4. Communication connection: a connection mode, wherein communication is formed between connected devices through signal transmission interaction; the communication connection comprises a wired connection and a wireless connection; the change of the wired connection level includes a change of an analog level or a change of a digital level; wireless connections include radio waves, magnetic fields, optical propagation, and the like.
5. And (3) a circuit board: the names of the circuit boards are: ceramic circuit boards, alumina ceramic circuit boards, aluminum nitride ceramic circuit boards, PCB boards, aluminum substrates, high frequency boards, thick copper plates, 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 may be made of glass, quartz, organic glass (plastic), or the like.
7. Excitation light source: the light source for exciting fluorescence may be visible light such as laser light, LED light, incandescent light, etc., or invisible light such as ultraviolet light, infrared light, etc.
8. And (3) temperature control: by changing the heating power of the heating component and through the forms of hot air, infrared, heat conduction, heat radiation and the like.
MCU module: a circuit board containing a microcontroller integrated circuit and carrying out communication with the computer and peripheral modules; and can be extended to computing modules to replace computer functions in part or in whole.
10. And the light temperature control module is used for: and receiving a control instruction, and controlling the circuit board and the device for detecting the temperature of the structure and emitting light.
11. An optical signal amplifying 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. And (3) heating a plate: the circuit board provided with the heating component (heating ring) is convenient for installing components of a circuit and plays a role in heat conduction.
13. A heating ring: and a heat conduction member capable of rapidly conducting heat to the reaction tube and the thermal sensor (thermistor).
14. Photo detector: semiconductor components or packages incorporating semiconductor components that receive photons and then output analog electrical signals.
15. An optical filter: limiting the passage of light at a portion of the wavelength includes low pass, high pass, band pass. Inorganic glass, organic glass, quartz, etc.
16. Solid light guide: the light-guiding solid material can play a role in total transmission or partial transmission or selective transmission of incident light; may be of different shapes, such as cylindrical, square, etc.; can be inorganic glass, organic glass, quartz and other materials; the surface can be coated with a filter film comprising low pass, high pass and band pass.
17. User interface: a User Interface (UI) is a medium for interaction and information exchange between a system and a User, and it converts an internal form of information into a human acceptable form. The user interface is designed to interact with the related software between the user and the hardware, so that the user can conveniently and effectively operate the hardware to achieve bidirectional interaction, and the work expected to be completed by the hardware is completed. User interfaces are widely defined, including human-machine interaction and graphical user interfaces, and exist in all fields involving human and machine information exchange.
18. Biochemical reaction: chemical reactions involving biological molecules are typically catalyzed by biological enzymes.
19. Target molecule: the molecular target to be detected can be pathogen, protein molecule, nucleic acid molecule, glycoprotein molecule, saccharide or lipid molecule, organic molecule and inorganic molecule.
20. Chemiluminescence: chemiluminescence is a phenomenon of light radiation that accompanies a substance in performing a chemical reaction, and can be classified into direct luminescence and indirect luminescence.
21. Electrochemiluminescence: electrochemiluminescence (ECL) refers to the phenomenon of luminescence generated by directly oxidizing and reducing an electrode surface illuminant by using energy supplied by an electrode to form an excited state, and returning the excited state to a ground state
22. Fluorescence: refers to a photoluminescence cold luminescence phenomenon. When a certain normal temperature substance is irradiated with incident light of a certain wavelength, the light energy is absorbed and then enters an excited state, and immediately de-excited and emits outgoing light (generally, the wavelength is in the visible light band) longer than the wavelength of the incident light.
23. Molecular conformation: the molecule changes its relative position in space of atoms or groups of atoms by single bond rotation to present different steric images.
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: clinical diagnosis information is obtained by detecting human body samples (blood, body fluid, tissue, etc.), and products and services of diseases or body functions are further judged.
26. Biological detection: is a detection means and method for biochemical analysis using biomolecules derived from organisms. Biological detection relates to on-site detection, on-line monitoring and even component separation and analysis in the fields of medical diagnosis, clinical examination, disease prevention and control, agriculture, animal husbandry, aquaculture, food safety guarantee and the like.
27. Nucleic acid sample: ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) can be artificially synthesized or derived from animals, plants and microorganisms (including viruses) in nature.
28. Nucleic acid amplification: the process of specific or specific-specific repeated replication of the nucleic acid target molecules to be detected is carried out in a test tube by the action of an enzyme.
29. Nucleic acid fluorescent dye: fluorescent dyes specific for nucleic acids. When combined with nucleic acid molecules, the fluorescence effect can be greatly improved, and when the fixed amount of nucleic acid fluorescent dye is arranged in the test tube, the total fluorescence amount is proportional to the nucleic acid amount.
30. Fluorescent-labeled molecular probes: a specific fluorescent probe, which is an oligonucleotide, and a reporter fluorophore and a quencher fluorophore are labeled at each end. When the probe is complete, the fluorescent signal emitted by the reporter group is absorbed by the quencher group; when nucleic acid amplification (such as PCR amplification) is carried out, the exonuclease activity of the polymerase carries out enzyme digestion degradation on the probe to separate the report fluorescent group from the quenching fluorescent group, so that a fluorescence signal can be received by a fluorescence monitoring system, namely, a free fluorescent molecule is formed for each amplified DNA chain, and the accumulation of the fluorescence signal and the formation of amplified products are completely synchronous.
31. Dissolution point: also called DNA denaturation temperature point, refers to the breaking of hydrogen bonds of double helix base pairs of nucleic acid, the break of stacking force between bases, and the change of 50% double strand into single strand, which changes the natural conformation and property of nucleic acid, but not the primary structure.
32. Dissolution profile: a graphical method of the change in the associated fluorescence signal during denaturation (dissolution) of DNA with temperature changes, by which the DNA dissolution point can be determined.
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.
34. Curves, information, data, in this disclosure, all refer to results obtained using the apparatus of the present invention, which are interchangeable.
35. Liquid the concept of liquid in the present invention includes aqueous solution based liquids or organic solution based liquids.
Any combination of the various embodiments of the invention should be considered as being within the scope of the present disclosure, as long as the inventive concept is not violated; within the scope of the technical idea of the invention, any combination of various simple modifications and different embodiments of the technical proposal without departing from the inventive idea of the invention should be within the scope of the invention.

Claims (15)

1. A multivariable reaction kinetics real-time detector device, comprising: a detector and a computer, the detector being communicatively connected to 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 arranged 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 component and a photoelectric detection component; the excitation light source is arranged above or below the test tube mounting hole, a first light source 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 enclosed at the periphery of the reaction test tube;
The photoelectric detection assembly comprises a photoelectric detector, a solid light guide and a second light source filter, and the photoelectric detector, the solid light guide and the second light source 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 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 each lighting of the excitation light source is between 1 second and 5 minutes, and the acquisition of the light signal by the photoelectric detection component is completed in the lighting process of the excitation light source; the first light source filter and the second light source filter limit the light with partial wavelength to pass through;
the main control circuit board comprises an MCU module, an optical temperature control module and an optical 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 optical temperature control module; the excitation light source and the temperature control component are both in communication connection with the optical temperature control module, and the photoelectric detection component is in communication connection with the optical signal amplification module;
The computer comprises a user interface through which a user inputs variable parameters, and can monitor optical signals which are determined by temperature, time or space position in the reaction test tube in real time; the user interface sets the working temperatures of the upper heater and the lower heater, the time and the period of the light emission of the excitation light source, and selectively opens the photoelectric detector to collect the optical signals of a certain wave band; after the detector starts to work, the user interface can display information corresponding to the temperature and time fed back in real time; simultaneously displaying information corresponding to the optical signal intensity and time fed back in real time;
the photoelectric detection assemblies comprise two groups of upper photoelectric detection assemblies and two groups of lower photoelectric detection assemblies, wherein the two groups of upper photoelectric detection assemblies are oppositely arranged at two sides of the upper end of the test tube mounting hole, and the two groups of lower photoelectric detection assemblies are oppositely arranged at two sides of the lower end of the test tube mounting hole; each group of upper photoelectric detection assemblies comprises an upper photoelectric detector, an upper solid light guide and a second light source filter, wherein the upper solid light guide is positioned below the upper heater, the upper solid light guide is positioned between the test tube mounting hole and the upper photoelectric detector, and the second light source filter is positioned between the upper solid light guide and the upper photoelectric detector;
Each group of lower photoelectric detection assemblies comprises a lower photoelectric detector, a third light source filter and a lower solid light guide, wherein 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 filter is positioned between the lower solid light guide and the lower photoelectric detector;
or the photoelectric detection assembly comprises an upper photoelectric detection assembly and a lower photoelectric detection assembly; 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 assembly comprises an upper photoelectric detection assembly and a lower photoelectric detection assembly; the upper photoelectric detection assembly and the lower photoelectric detection assembly are respectively arranged at two sides of the test tube mounting hole;
or the photoelectric detection assembly comprises two groups of upper photoelectric detection assemblies and one group of lower photoelectric detection assemblies; the two groups of upper photoelectric detection assemblies are oppositely arranged at two sides of the upper end of the test tube mounting hole, and the lower photoelectric detection assemblies are arranged at one side of the lower end of the test tube mounting hole;
or the photoelectric detection assembly comprises an upper photoelectric detection assembly and two lower photoelectric detection assemblies; 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.
2. The multi-variable reaction kinetics real-time detector apparatus as claimed in claim 1 wherein: the upper heater and the lower heater can adjust the temperature of the reaction test tube, heat the reaction test tube, enable the liquid in the reaction test tube to be heated to 100 ℃ or cooled to room temperature, and promote or prevent liquid convection through temperature difference.
3. The multi-variable reaction kinetics real-time detector apparatus as claimed in claim 1 wherein: the photodetector monitors the chemical or biochemical reaction of the liquid in the reaction cuvette with temperature and time by means of an optical signal.
4. The multi-variable reaction kinetics real-time detector apparatus as claimed in claim 3 wherein: the detector device monitors in real time through reaction kinetics to determine the amount, structural characteristics or biochemical activity of the target molecules in the sample.
5. The multi-variable reaction kinetics real-time detector apparatus as claimed in claim 1 wherein: the tube mounting hole is inclined at an angle in the range of 5-45 deg. when in use.
6. The multi-variable reaction kinetics real-time detector apparatus as claimed in 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.
7. The multi-variable reaction kinetics real-time detector apparatus as claimed in claim 1 wherein: the solid light guide is coated with a filter film, can play a role in total transmission or partial transmission or selective transmission of incident light, and is cylindrical or square.
8. The multi-variable reaction kinetics real-time detector apparatus as claimed in claim 1 wherein: the first light source filter, the second light source filter and the third light source filter are inorganic glass, organic glass or quartz, and the surfaces of the first light source filter, the second light source filter and the third light source filter are coated with filter films, and the filter films have low-pass, high-pass or band-pass performances.
9. The multi-variable reaction kinetics real-time detector apparatus as claimed in any one of claims 1 to 8 wherein: 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.
10. The multi-variable reaction kinetics real-time detector apparatus as claimed in claim 1 wherein: the computer is installed inside the housing or outside the housing.
11. The multi-variable reaction kinetics real-time detector apparatus as claimed in claim 1 wherein: the distance between the main control circuit board and the mounting seat is less than or equal to 100mm.
12. A detection method, characterized in that the detector device according to claim 1 is used, 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, heating the reaction solution in the reaction test tube by an upper heater and a lower heater, and enabling the temperature difference between the upper temperature and the lower temperature of the reaction solution in the reaction test tube to be more than or equal to 5 ℃ so as to enable the solution to circulate, mix or stand still;
starting a computer real-time monitoring program, 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 the related information according to the reaction result.
13. The method of detecting as claimed in claim 12, 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, firstly transcribing the RNA into complementary DNA;
Then amplifying the nucleic acid sample by using an enzymatic method of isothermal amplification or variable temperature amplification, wherein in the variable temperature nucleic acid amplification process, the temperature circulation of the liquid between 45 ℃ and 98 ℃ is controlled; when the photodetection assembly detects the signal of the nucleic acid fluorescent dye or the fluorescent labeled molecular probe, the intensity of the fluorescent signal generated by the amplification reaction is proportional to the amount of amplified DNA product.
14. The method of detecting as claimed in claim 13, comprising the steps of:
when the temperature of the solution at the bottom of the reaction tube is reduced below the nucleic acid dissolution temperature point after the nucleic acid amplification reaction is finished, most DNA molecules are combined into double-stranded molecules and are combined with nucleic acid fluorescent dye to emit stronger fluorescence, the upper heater and the lower heater are controlled to start heating at a low speed, and time and temperature variables are recorded through a computer; when the temperature variable is regulated, the change of the optical signal is recorded in real time through a lower photoelectric detector or an upper photoelectric detector, and the relation between the temperature and the fluorescent signal is displayed through a computer; with increasing temperature, the double-stranded DNA molecules are dissolved into single-stranded molecules, so that the fluorescence amount is reduced, and the dissolution point or dissolution curve of the amplified double-stranded DNA molecules is measured by performing data processing on a computer.
15. The method of detecting as claimed in claim 13, comprising the steps of: when the nucleic acid amplification reaction is completed, maintaining the temperature of the solution at the bottom of the reaction tube at 90-98 ℃ and raising the temperature of the upper liquid to not more than 95 ℃ by an upper heater, at this time, most of the double-stranded DNA molecules are dissolved into single-stranded DNA molecules, then controlling the upper heater and a lower heater to start to slowly cool down, and recording time and temperature variables by a computer; when the temperature variable is regulated, the change of the optical signal is recorded in real time through a lower photoelectric detector or an upper photoelectric detector, and the relation between the temperature and the fluorescent signal is displayed through a computer; as the temperature decreases, single-stranded DNA molecules will bind to double-stranded molecules, causing an increase in fluorescence, and the computer is used to perform data processing to determine the dissolution point or dissolution profile of the amplified DNA molecules.
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