Multifunctional automatic analysis system and method for intracellular and extracellular key biochemical parameters

Abstract

The invention discloses a multifunctional automatic analysis system and method for intracellular and extracellular key biochemical parameters, which comprises a sample pretreatment system, a cell disruption system, a solid-liquid separation system, an auxiliary liquid storage system, a sample dilution system, a chemical analysis system and an enzyme electrode analysis system, the sample analysis system is used for analyzing a sample from the sample pretreatment system, the cell disruption system or the solid-liquid separation system; the rotatable sampling needle mechanical moving device is used for sucking a sample and sending the sample to the chemical analysis system and the enzyme electrode analysis system through a sampling needle for related determination; and the instrument control and data integration system is used for controlling each system and recording related data. Detection of biomass, key biochemical parameters inside and outside cells and enzyme activity of key metabolic enzymes in the biological manufacturing process can be realized. The problems that a traditional detection means is poor in timeliness, complex in operation, prone to result interference and the like are solved, and great large-scale application value is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biological manufacturing and analysis and detection, and in particular relates to a multifunctional automatic analysis system and method for key biochemical parameters inside and outside cells. Background Art

[0002] The statements in this section merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] Biomanufacturing is a new industrial model that uses microbial cells or enzyme proteins as catalysts to synthesize chemicals or convert biomass into energy chemicals and materials. It is different from the petrochemical industry. It has the characteristics of low carbon emissions, recycling and green cleaning, which helps to promote environmental protection and sustainable industrial development. At present, the field of biomanufacturing has developed rapidly, especially the development of disciplines such as synthetic biology, which has enabled biomanufacturing to develop rapidly towards practical application and industrialization. At the same time, digitalization and intelligence have put forward new requirements for the future development of biomanufacturing.

[0004] The biomanufacturing process mainly relies on the transformation of living cells and is a complex system. The key parameters of the production process are nonlinear, time-varying, irreversible and hysteresis. At present, online detection and control have been realized for physicochemical parameters such as temperature, pH, dissolved oxygen, rotation speed, flow rate, etc. However, for many key factors in the biomanufacturing process, especially the detection of raw materials (substrates), products and intermediate metabolites in the fermentation process, the current main detection technology is chromatography, mass spectrometry and other offline detection technologies. Although a variety of key process parameters such as glucose, glycerol, lactic acid, glutamic acid, pyruvic acid and other concentrations have been developed, most of them are offline detection and have poor timeliness. At the same time, the detection of the enzyme activity of key enzymes in the cell metabolism process, the concentration of key intracellular substrates and metabolites is blank, and it is impossible to establish effective intracellular and extracellular data coupling, thus limiting the data source of biomanufacturing.

[0005] Data such as key biochemical parameters inside and outside cells and the activity of key enzymes are of great significance for the real-time regulation of the biomanufacturing process. Current detection methods are relatively limited and cannot achieve real-time synchronization of key data inside and outside cells, resulting in delays in the regulation of the biomanufacturing process and increased uncertainty in production and experimental results. Summary of the invention

[0006] In order to overcome the deficiencies of the above-mentioned prior art, the present invention provides a multifunctional automatic analysis system and method for key biochemical parameters inside and outside cells, which can realize the detection of biomass, key biochemical parameters inside and outside cells, and key metabolic enzyme activities in the biomanufacturing process. It overcomes the problems of poor timeliness, complex operation, and susceptibility to interference of traditional detection methods, and has great value for large-scale application.

[0007] To achieve the above objectives, one or more embodiments of the present invention provide the following technical solutions:

[0008] In a first aspect, the present invention provides a multifunctional automatic analysis system for key biochemical parameters inside and outside cells, comprising:

[0009] Sample pretreatment system, used for sampling and measurement;

[0010] A cell disruption system, used for disrupting cells of the sample from the sample pretreatment system;

[0011] A solid-liquid separation system, used to separate the solid and liquid of the sample using a centrifuge;

[0012] Auxiliary liquid storage system, used to store a variety of test reagents and provide refrigeration function;

[0013] Sample dilution system, used to dilute samples to different degrees;

[0014] A chemical analysis system, used for chemical analysis of samples from a sample pretreatment system, a cell disruption system or a solid-liquid separation system;

[0015] The enzyme electrode analysis system is specifically an enzyme reaction device composed of an enzyme membrane and an electrode, which is used to analyze samples from a sample pretreatment system, a cell disruption system or a solid-liquid separation system;

[0016] A rotatable sampling needle mechanical moving device is used to absorb samples from the sample pretreatment system, cell disruption system, solid-liquid separation system, auxiliary liquid storage system and sample dilution system, and send them to the chemical analysis system and enzyme electrode analysis system through the sampling needle for relevant determination;

[0017] The instrument control and data integration system is used to control the sample pretreatment system, cell disruption system, solid-liquid separation system, auxiliary liquid storage system, sample dilution system, chemical analysis system, enzyme electrode analysis system and rotatable sampling needle mechanical moving device, and record relevant data.

[0018] A further technical solution is that the sample pretreatment system includes online real-time sampling and measurement and offline sampling and measurement. The online real-time sampling and measurement is realized by an online sample processing system, which includes a sampling device and a sample storage pool. The sampling device is specifically a sampling tube with a straight plug type. The sampling tube is connected to the sample storage pool through a hose. A peristaltic pump is provided between the sample storage pool and the sampling tube, and the liquid in the reactor is pumped into the sample storage pool through the peristaltic pump.

[0019] According to a further technical solution, the cell disruption system includes a sample pool, a vibration module and a control module. The vibration module includes a vibration motor and an ultrasonic probe. A plurality of cooling metals are arranged on the circumference of the sample pool chamber. The ultrasonic probe is arranged at the bottom of the chamber. The bottom of the ultrasonic probe is connected to the vibration motor, and the vibration motor is electrically connected to the control module.

[0020] A further technical solution is that the solid-liquid separation system is implemented by a centrifuge, which includes a cold storage box, wherein an annular centrifuge tube groove is arranged inside the cold storage box, a plurality of centrifuge tubes are placed on the annular centrifuge tube groove, a centrifuge shaft is arranged in the center of the annular centrifuge tube groove, and the bottom of the centrifuge shaft is connected to a motor.

[0021] A further technical solution is that the chemical analysis system includes a light source, a lens, a monochromator, a reaction cell, a detector, a signal amplifier and a display. The light emitted by the light source is imaged on the entrance slit of the monochromator through the lens. The monochromator decomposes the light emitted by the light source into monochromatic light and irradiates the light onto the sample solution in the reaction cell. The detector responds to the light passing through the reaction cell through a photoelectric conversion element, converts the optical signal into an electrical signal and outputs it to the signal amplifier. The signal amplifier amplifies the electrical signal and outputs it to the display to show the measurement result.

[0022] According to a further technical solution, the enzyme electrode analysis system is specifically an enzyme reaction device composed of an enzyme membrane and an electrode, with an injection needle sensor on the top, multiple enzyme electrodes inserted on the side wall, a magnetic stirring motor at the bottom, and a magnetic rotor inside.

[0023] A further technical solution is that the mechanical moving device of the rotatable sampling needle includes a slide rail, a positioning rail is provided on the slide rail, a mobile sampling base is movably provided on the positioning rail, a volume controller is connected to one side wall of the mobile sampling base, a rotatable mechanical axis is provided on the top of the mobile sampling base, the rotatable mechanical axis is connected to one end of a transverse rotatable mechanical arm, the other end of the transverse rotatable mechanical arm is connected to a vertical rotatable mechanical arm, and a sampling needle is connected to the end of the vertical rotatable mechanical arm.

[0024] In a second aspect, the present invention provides a multifunctional automatic analysis method for key biochemical parameters inside and outside cells, comprising:

[0025] Select the required measurement parameters before measuring;

[0026] Start the sample pretreatment system to take samples, and pump the samples into the sample temporary storage pool;

[0027] The rotatable sampling needle mechanical moving device takes samples from the sample temporary storage pool according to the command, and sends the samples to the sample dilution system, solid-liquid separation system, cell disruption system, or directly to the chemical analysis system, or directly to the enzyme electrode analysis system according to the measurement requirements of the measurement parameters;

[0028] The rotatable sampling needle mechanical moving device takes liquid from the auxiliary liquid storage system according to the command to assist in sample dilution, solid-liquid separation, cell disruption and chemical analysis or enzyme electrode analysis;

[0029] The samples are finally sent to the chemical analysis system or enzyme electrode analysis system for final measurement to obtain the measurement results.

[0030] In a third aspect, the present invention provides a computer-readable storage medium having a computer program stored thereon, which, when executed by a processor, implements the steps of a multifunctional automatic analysis method for key intracellular and extracellular biochemical parameters as described in the second aspect.

[0031] In a fourth aspect, the present invention provides a computer device comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein when the processor executes the program, the steps of a multifunctional automatic analysis method for key intracellular and extracellular biochemical parameters as described in the second aspect are implemented.

[0032] One or more of the above technical solutions have the following beneficial effects:

[0033] The present invention combines the data chimneys that have emerged in current intelligent biomanufacturing to propose an automatic analysis system that can simultaneously measure multiple parameters in the biomanufacturing process, which can realize the automatic measurement of complex parameters, greatly improve the sampling frequency, accuracy and data volume, and form a database for specific products.

[0034] It is particularly pointed out that the present invention is different from other analysis and detection systems in that the present invention can measure the key biochemical parameters in the fermentation broth, whole cell wall breaking liquid, and cell wall breaking liquid after washing, as well as the key enzyme activities in the main metabolic pathways in the cells at one time, and couple the data inside and outside the cells as the basis for the next step of database construction.

[0035] The parameters measured by the present invention not only include traditional biochemical parameters in conventional fermentation broth, but also add a fragmentation system, so that various parameters in cells and key enzyme activities in metabolic pathways can be measured promptly and quickly, further enriching the source and effectiveness of data.

[0036] The present invention realizes the collection and recording of intracellular and extracellular data through the integration of various systems, providing a detailed data basis for intelligent biomanufacturing. At the same time, the automated analysis system provided by the present invention can provide a data basis for the automated control of future biomanufacturing processes. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0038] Figure 1 It is a module structure diagram of the multifunctional automatic analysis system according to an embodiment of the present invention;

[0039] Figure 2 2 is a schematic diagram of the structure of an online sample processing system of a sample preprocessing system according to an embodiment of the present invention;

[0040] Figure 3 2 is a schematic diagram of the structure of an off-line sample processing system of a sample preprocessing system according to an embodiment of the present invention;

[0041] Figure 4 is a schematic structural diagram of a cell disruption system according to an embodiment of the present invention;

[0042] Figure 5 is a schematic structural diagram of a solid-liquid separation system according to an embodiment of the present invention;

[0043] Figure 6 is a schematic structural diagram of an auxiliary liquid storage system according to an embodiment of the present invention;

[0044] Figure 7 is a schematic structural diagram of a sample dilution system according to an embodiment of the present invention;

[0045] Figure 8 is a schematic diagram of the structure of a chemical analysis system according to an embodiment of the present invention;

[0046] Fig. 9 is a schematic structural diagram of an enzyme electrode analysis system according to an embodiment of the present invention;

[0047] Fig.10 It is a structural diagram of a mechanical moving device for a rotatable sampling needle according to an embodiment of the present invention.

[0048] Among them, 101-channel 1 sampling tube, 102-channel 2 sampling tube, 103-channel 1 compression valve, 104-channel 2 compression valve, 105-peristaltic pump, 106-sample temporary storage pool, 107-first magnetic rotor, 108-pH electrode, 109-waste liquid pipe, 110-refrigeration pool, 111-sampling needle sensor, 112-first liquid level sensing device, 113-first magnetic stirrer, 201-sample storage pool, 202-circular sample pool, 203-second magnetic rotor, 204-second liquid level sensing device, 205-second magnetic stirrer, 301-soundproof chamber, 302-wall breaking pool, 303-cooling metal, 304-ultrasonic probe, 305-first temperature probe, 306-liquid level probe, 307-first motor, 308-first base, 401-refrigerated box, 402-centrifuge tube, 403-centrifuge tube tank, 404-centrifuge tank positioner, 405-centrifuge shaft, 4 06-second temperature probe, 407-second motor, 408-second base, 501-reagent storage position, 502-third temperature probe, 503-refrigeration chamber, 601-dilution chamber, 602-third magnetic rotor, 603-secondary dilution pool, 604-primary dilution pool, 605-third magnetic stirrer, 701-light source, 702-lens, 703-monochromator, 704-reaction pool, 705-detector, 706-signal amplifier , 707-display, 801-injection needle sensor, 802-enzyme electrode, 803-fourth magnetic rotor, 804-magnetic stirring motor, 901-slide rail, 902-mobile sampling base, 903-rotatable mechanical axis, 904-sampling needle, 905-lateral rotatable mechanical arm, 906-volume controller, 907-connecting tube, 908-rotator, 909-positioning track, 910-positioner, 911-vertical rotatable mechanical arm. DETAILED DESCRIPTION

[0049] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0050] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0051] In the absence of conflict, the embodiments of the present invention and the features of the embodiments may be combined with each other.

[0052] Embodiment 1

[0053] like Figure 1 As shown, this embodiment discloses a multifunctional automatic analysis system for key biochemical parameters inside and outside cells, including a sample pretreatment system for sampling and measurement;

[0054] Sample pretreatment system, used for sampling and measurement;

[0055] A cell disruption system, used for disrupting cells of the sample from the sample pretreatment system;

[0056] A solid-liquid separation system, used to separate the solid and liquid of the sample using a centrifuge;

[0057] Auxiliary liquid storage system, used to store a variety of test reagents and provide refrigeration function;

[0058] Sample dilution system, used to dilute samples to different degrees;

[0059] A chemical analysis system, used for chemical analysis of samples from a sample pretreatment system, a cell disruption system or a solid-liquid separation system;

[0060] The enzyme electrode analysis system is specifically an enzyme reaction device composed of an enzyme membrane and an electrode, which is used to analyze samples from a sample pretreatment system, a cell disruption system or a solid-liquid separation system;

[0061] A rotatable sampling needle mechanical moving device is used to absorb samples from the sample pretreatment system, cell disruption system, solid-liquid separation system, auxiliary liquid storage system and sample dilution system, and send them to the chemical analysis system and enzyme electrode analysis system through the sampling needle for relevant determination;

[0062] The instrument control and data integration system is used to control the sample pretreatment system, cell disruption system, solid-liquid separation system, auxiliary liquid storage system, sample dilution system, chemical analysis system, enzyme electrode analysis system and rotatable sampling needle mechanical moving device, and record relevant data.

[0063] It should be noted that the sample pretreatment system, cell disruption system, solid-liquid separation system, auxiliary liquid storage system, sample dilution system, chemical analysis system, and enzyme electrode analysis system in the multifunctional automatic analysis system are independent of each other. There is no clear sequential relationship between the above systems. Each individual system may be used or not used in each measurement and analysis. It needs to be selected according to different measurement parameters. Different systems are called up by the instrument control and data integration system. The operation of each system will be implemented in the instrument control and data integration system.

[0064] In this embodiment, if Figure 1As shown, the multifunctional automatic analysis system specifically includes: (I) sample pretreatment system, (II) cell disruption system, (III) solid-liquid separation system, (IV) auxiliary liquid storage system, (V) sample dilution system, (VI) chemical analysis system, (VII) enzyme electrode analysis system, (VIII) rotatable sampling needle mechanical moving device and (IX) instrument control and data integration system.

[0065] (I) The sample pretreatment system is used for sampling and measurement, including online real-time sampling and measurement and offline sampling and measurement, which can be selected according to actual needs.

[0066] 1) Online real-time sampling and measurement

[0067] like Figure 2 As shown, the online real-time sampling and determination method is selected to be matched with an online sample processing system, including a sampling device, a sample temporary storage pool, a cleaning system, a sample delivery system (completed by a Ⅷ rotatable sampling needle mechanical moving device) and a sampling control system.

[0068] The sampling device is configured according to the number of reactors. The sampling device is specifically a stainless steel sampling tube and a sheath with a straight-insertion type on the outside. The sampling tube is connected to a silicone tube or a hose made of other materials. The sampling device is connected to the sample temporary storage tank through a hose. A peristaltic pump 105 is provided on the hose between the sampling device and the sample temporary storage tank for conveying liquid. One end of the peristaltic pump 105 is respectively connected to multiple channel sampling tubes and a waste liquid pipe 109, and the other end is connected to the sample temporary storage tank 106. The sample temporary storage tank 106 is arranged in a cold storage tank 110.

[0069] In some embodiments, the length of the stainless steel sampling tube can be adjusted according to the size of the actual biological reaction, and the sampling aperture size of the sampling tube can be adjusted according to the actual fermentation product. The length of the stainless steel sampling tube is 100mm-350mm, preferably 120mm-200mm, and more preferably 150mm-160mm. The thickness of the sampling hole is 0.5mm-10mm, preferably 1mm-5mm, and more preferably 1.5mm-2mm.

[0070] Furthermore, Figure 2Two channel sampling tubes are arranged in the system, namely, one end of the peristaltic pump is connected to the channel 1 sampling tube 101 and the channel 2 sampling tube 102 respectively, and a channel 1 pressure tube valve 103 is arranged between the peristaltic pump and the channel 1 sampling tube, and a channel 2 pressure tube valve 104 is arranged between the peristaltic pump and the channel 2 sampling tube. Peristaltic pump (positive and negative rotation): it is opened when sampling, and is used to pump the liquid in the fermentation tank or reactor into the sample temporary storage pool, and is used in conjunction with 103 or 104, and is controlled by the analysis and control system; channel 1 sampling tube: it is used to sample from the fermentation tank or other reaction, channel 2 sampling tube: it is used to sample from the fermentation tank or other reaction; channel 1 pressure tube valve: it is in a closed state when not sampling, and is opened when sampling, and is controlled by the analysis and control system; channel 2 pressure tube valve: it is in a closed state when not sampling, and is opened when sampling, and is controlled by the analysis and control system; waste liquid pipe: it is used to discharge the sample when the sample analysis is completed, and is used to discharge the cleaning waste liquid when the sample pool is cleaned; cold storage pool: it provides temperature maintenance for sample storage, and the temperature is adjustable.

[0071] like Figure 2 As shown in the side view of the sample storage tank 106, a sampling needle sensor 111 is provided on the top of the sample storage tank 106, one side is connected to the sampling device through a hose, a first liquid level sensing device 112 is provided at a set position on the side, a first magnetic stirrer 113 is provided at the bottom, a first magnetic rotor 107 is placed inside, and a pH electrode 108 is provided on the side. The sampling needle sensor: used to sense the entry and exit of the sampling needle, and is connected to the analysis control system to transmit signals; the liquid level sensing device: used to detect whether there is liquid in the sample tank and whether the liquid level of the sample meets the analysis requirements; the magnetic stirrer (also called a magnetic stirring device): provides magnetic force for the first magnetic rotor 107 to achieve magnetic stirring; the first magnetic rotor (magnetic stirring bar): used to mix the sample, mix the liquid, and keep the sample in a uniform state, and the specific number matches the sampling channel and the sampling device; the pH electrode: used to measure the pH value of the sample, connected to the instrument control and data integration system, transmit the pH value to the instrument control and data integration system, and measure and record the data according to the system instructions.

[0072] Furthermore, the sample storage pool is used to store samples that have not been analyzed, and the temperature is adjustable. Preferably, the sample storage pool has a low temperature storage function, and the temperature control range is 0-80°C, and the more preferred temperature control range is 4-8°C.

[0073] The cleaning system is specifically a cleaning fluid connected to the sample storage tank.

[0074] The sample delivery system is implemented by (VIII) a rotatable sampling needle mechanical moving device, which is synchronized with the instrument control and data integration system and is controlled by the instrument control and data integration system. Specifically, it is a movable mechanical arm and a sampling needle, which is implemented by the VIII rotatable sampling needle mechanical moving device. The sampling needle can absorb liquids from the sample pretreatment system, solid-liquid separation system, cell disruption system, chemical analysis system, auxiliary liquid storage system, dilution system and enzyme electrode analysis system, and can also deliver samples to the above-mentioned different systems. The sampling needle can take out the corresponding liquids from different sample storage pools and deliver them to the reaction pool of the chemical analysis system or the enzyme electrode analysis system. Specifically, it can be a horizontally and vertically movable mechanical arm, or a rotatably movable sampling needle. The preferred sampling volume range is 2μL-10mL, and the more preferred sampling volume is 10-3000μL.

[0075] The sampling control system is connected to the instrument control and data integration system, and can control the sampling operation of 1-8 bioreactors. After receiving the sampling command from the instrument control and data integration system, the peristaltic pump 105 channel is opened to take the liquid in the reactor out of the bioreactor, and the other channels are closed. The sampling channels are 1-8 channels, preferably 1-5 channels, and more preferably 2-3 channels.

[0076] The specific process of the online real-time sampling and measurement method is as follows: the instrument control and data integration system sends a sampling command to the sample pretreatment system, and the sample pretreatment system starts the peristaltic pump after receiving the command. The peristaltic pump pumps the liquid in the reactor into the sample storage tank, and the sample is mixed in the sample storage tank by a magnetic rotor, and the pH value of the sample is measured by the internal pH electrode and transmitted to the instrument control and data integration system; then the instrument control and data integration system sends an instruction to the rotatable sampling needle mechanical moving device, the moving device receives the instruction and obtains the sample in the sample storage tank through the sampling needle, and sends the obtained sample to the cell disruption system or the solid-liquid separation system or the sample dilution system or the chemical analysis system or the enzyme electrode analysis system.

[0077] The specific process of online real-time sampling and measurement is as follows:

[0078] First, the user sets the relevant sampling parameters (sampling interval, number of repeated sampling, sampling cycle, etc.) and measurement parameters (preset parameters in the system: cell concentration, fermentation broth pH, ​​intracellular pH, fermentation broth glucose concentration, intracellular glucose concentration, fermentation broth lactate concentration, intracellular lactate concentration, intracellular ATP concentration, intracellular succinate dehydrogenase activity, intracellular malate dehydrogenase activity, intracellular oxidase activity, etc.) in the instrument control and data integration system. The user can also customize the parameters according to the properties of the instrument itself and the relevant measurement principles. For example, by measuring the absorbance change of NAD(H) at 450nm, the redox state and energy metabolism in the cell can be determined by the ratio of NAD and NADH. At the same time, according to the change of NAD(H) concentration, the activity of phosphofructokinase and alcohol dehydrogenase can also be determined.

[0079] Then, the sampling device of the online sampling sample processing system of the sample pretreatment system starts sampling after receiving the command, and transports the sample to the sample temporary storage tank according to the sampling parameters. At this time, the pH value of the sample can be measured, and the pH value measurement result is transmitted to the instrument control and data integration system;

[0080] Next, according to the test requirements, the mechanical moving device of the rotatable sampling needle can absorb the sample and enter the chemical analysis system or the dilution system to dilute the sample. The absorbance value of the bacterial solution at 600nm can be measured, and the dilution multiple is calculated after reading the data. The calculated data is the bacterial volume parameter of the fermentation liquid; at the same time, the mechanical moving device of the rotatable sampling needle can absorb the sample to the solid-liquid separation system to separate the sample bacteria, and then send the supernatant after solid-liquid separation to the enzyme electrode analysis system to measure parameters such as glucose concentration, lactic acid concentration, and glutamate concentration of the fermentation liquid. If necessary, dilution is performed according to the measurement results, and the measurement value and the dilution multiple are combined and calculated to obtain the measurement result, and the measurement result is transmitted to the instrument control and data integration system system; the rotatable sampling needle mechanical moving device can absorb samples into the solid-liquid separation system, and the distilled water or buffer solution absorbed by the auxiliary liquid storage system is combined to perform repeated solid-liquid separation on the fermentation liquid to achieve the purpose of bacterial washing, and then the washed bacteria and buffer solution or distilled water suspension are sent to the cell disruption system. After the cells are disrupted, the cell disruption liquid is respectively sent to the chemical analysis system and the enzyme electrode analysis system through the rotating sampling needle mechanical moving device, wherein the enzyme electrode analysis system is used to determine the intracellular glucose concentration, intracellular lactic acid concentration, intracellular glutamate concentration, etc., and the chemical analysis system is combined with different auxiliary liquids stored in the auxiliary liquid storage system to determine the intracellular succinate dehydrogenase activity, intracellular malate dehydrogenase activity, intracellular oxidase activity, etc.

[0081] Finally, all operation processes and measurement results are transmitted to the instrument control and data integration system.

[0082] 2) Offline sampling and measurement

[0083] Offline sampling and determination requires an offline sample processing system, which includes a sample bottle storage pool and a sample delivery system. The offline sample processing system is connected to the overall analysis control system and is controlled by the overall control system.

[0084] The sample bottle storage pool is specifically a grooved sample pool that can hold multiple circular sample bottles. Each circular groove can be used as a sample pool or can hold a cylindrical sample bottle. The sample bottle storage pool can be equipped with different numbers of sample bottles according to actual needs. According to actual needs, 12-48 sample bottles can be placed, and preferably 20 sample bottles can be placed. The sample storage pool has a temperature control function, and the specific temperature control range is 0-100°C, the preferred temperature control range is 4-30°C, and the more preferred temperature control range is 4-25°C. The bottom of the sample storage pool is equipped with a magnetic stirring function, and each sample bottle bottom can achieve stirring at different stirring rates. The offline sample processing system is not equipped with a pH electrode.

[0085] like Figure 3 As shown, a plurality of circular sample pools 202 are provided in the sample storage pool 201, and sample tubes can be placed in the circular sample pools 202. Sample storage pool: used to store sample tubes to be analyzed; Circular sample pool: used to store a single sample tube, which can be adjusted according to the actual size, and the number of samples stored and the storage volume can be customized, ranging from 0.2mL to 50mL.

[0086] like Figure 3 As shown in the side view of the circular sample pool, a second liquid level sensing device 204 is provided at a setting position on the side of the circular sample pool 202, a second magnetic stirrer 205 is provided at the bottom, and a second magnetic rotor 203 is provided inside. The second liquid level sensing device is used to detect whether there is liquid in the sample pool and whether the liquid level of the sample meets the analysis requirements; the second magnetic stirrer provides magnetic force for the magnetic stirring rotor to achieve magnetic stirring; the second magnetic rotor is used to stir the sample to keep the sample uniform.

[0087] The sample delivery system in the offline sample processing system is the same as that in the online sample processing system. The sample delivery system is synchronized with the instrument control and data integration system and is controlled by the instrument control and data integration system. Specifically, it is a movable mechanical arm and a sampling needle, which is realized by a rotatable sampling needle mechanical moving device VIII. The sampling needle can take out corresponding liquids from different sample bottles and send them to the reaction pool of the analysis system. Specifically, it can be a horizontally and vertically movable mechanical arm, or a rotatably movable sampling needle. The preferred sampling volume range is 2μL-10mL, and the more preferred sampling volume is 10-5000μL.

[0088] The specific process of offline sampling and determination is as follows:

[0089] First, the user sets the relevant sampling parameters (sampling interval, number of repeated sampling, sampling cycle, etc.) and measurement parameters (preset parameters in the system: cell concentration, fermentation broth pH, ​​intracellular pH, fermentation broth glucose concentration, intracellular glucose concentration, fermentation broth lactate concentration, intracellular lactate concentration, intracellular ATP concentration, intracellular succinate dehydrogenase activity, intracellular malate dehydrogenase activity, intracellular oxidase activity, etc.) in the instrument control and data integration system. The user can also customize the parameters according to the properties of the instrument itself and the relevant measurement principles. For example, by measuring the absorbance change of NAD(H) at 450nm, the redox state and energy metabolism in the cell can be determined by the ratio of NAD and NADH. At the same time, according to the change of NAD(H) concentration, the activity of phosphofructokinase and alcohol dehydrogenase can also be determined.

[0090] Then, the sampling device of the off-line sample processing system of the sample preprocessing system starts the sample testing program after receiving the command, and first starts the sample mixing mode according to the sampling parameters, and then accepts the sample suction and transmission of the rotatable sampling needle mechanical moving device;

[0091] Next, we enter the sample testing phase, which is similar to the specific process of the online real-time sampling and measurement method;

[0092] Finally, all the measurement result data are transmitted to the instrument control and data integration system, and the relevant batch number, tube number, etc. are recorded to establish a test result database.

[0093] The instrument control and data integration system sends instructions to the mechanical moving device of the rotatable sampling needle. The moving device receives the instructions and obtains samples in the circular sample pool of the sample storage pool through the sampling needle, and sends the obtained samples to the dilution system, solid-liquid separation system, cell disruption system, chemical analysis system or enzyme electrode analysis system.

[0094] (II) A cell disruption system, which specifically disrupts the samples from the sample pretreatment system and provides the broken samples for subsequent analysis. The cell disruption system is connected to the sample sampling system through the robotic arm of the sample delivery system, and the sample from the sample pool is injected into the sample pool of the cell disruption system through the sampling needle. The cell disruption method used in the present invention is ultrasonic cell disruption. Specifically, the cell disruption system is divided into three parts: a sample pool, a vibration module, and a control module, wherein the vibration system mainly converts electrical energy into mechanical energy.

[0095] The sample pool of the cell disruption system is a temperature-controllable metal bath, with a specific temperature control range of 0-100°C, a preferred control range of 0-25°C, and a more preferred temperature control range of 0-4°C. The sample pool is connected to the vibration module in a mechanical seal, and the ultrasonic probe of the vibration module protrudes from the bottom of the sample pool. The probe size matches the sample pool size, the sample pool size is 0.2-10mL, the preferred sample pool size is 0.5-5mL, and the more preferred sample pool size is 1-2mL.

[0096] like Figure 4 As shown, the wall-breaking pool (sample pool) 302 is placed in the soundproof chamber 301, and a liquid level probe 306 is set at a position on one side of the wall-breaking pool 302, and a first temperature probe 305 is set at a position on the other side. Soundproof chamber: isolates the sound caused by ultrasound and plays a buffering role; wall-breaking pool (metal bath cooling): used to cool the sample during the wall-breaking process, and the metal bath can be achieved by protruding metal, and the specific shape includes but is not limited to threads, fans, barbs, etc., and the sample is cooled by cooling the metal; liquid level probe: used to measure the liquid level of the sample, prompt the sample amount, and alarm when the sample amount is insufficient; first temperature probe: used to measure the temperature change of the sample during the ultrasound process, and can have a temperature alarm function. When the temperature is higher than a certain set temperature, it prompts over-temperature, stops ultrasound according to the program setting, or prompts that the sample is invalid.

[0097] Furthermore, a plurality of cooling metals 303 are arranged on the circumference of the chamber of the cell-breaking pool, an ultrasonic probe 304 is arranged at the bottom of the chamber, the bottom of the ultrasonic probe 304 is connected to the first motor 307, and the bottom of the motor is fixedly arranged on the first base 308. Cooling metal: The temperature of the protruding metal can be reduced to below 0 degrees Celsius by cooling the system, thereby realizing cooling of the sample in the ultrasonic process; Ultrasonic probe: It is used to break the bacteria in the sample, and the specific power, time, interval, etc. are controlled by the analysis and control system.

[0098] The vibration module mainly includes a vibration motor and an ultrasonic probe. The ultrasonic probe protrudes from the bottom of the sample pool. Parameters such as vibration frequency and time are controlled by the control module of the cell disruption system. The control module is an important part of the system and mainly includes a power controller, a vibration frequency controller, a temperature control, etc.

[0099] The control module achieves fine crushing of samples by precisely adjusting various parameters, while ensuring that the integrity and quality of the samples are not damaged. For example, the power controller can flexibly adjust the output power; the frequency controller can accurately control the vibration frequency of the ultrasound to meet the specific crushing requirements of different samples; the temperature controller effectively monitors and adjusts the sample temperature during the crushing process, maintaining the sample in a suitable temperature environment for processing.

[0100] The specific process of instruction transmission and sample processing in the cell disruption system is as follows:

[0101] The cell disruption system mainly disrupts the samples from the sample pretreatment system, dilution system or solid-liquid separation system. According to different test requirements and sequences, the instrument control and data integration system issues a command, and then the rotatable sampling needle mechanical moving device sends the sample from the sample temporary storage pool, dilution pool or solid-liquid separation system into the cell disruption system. The cell disruption system turns on temperature control, vibration electrodes and probes according to pre-set parameters, and can achieve fixed-time and fixed-frequency vibration according to different test requirements, or interval time and variable-frequency vibration, and finally achieves fine disruption of the sample, and transmits the cell disruption temperature change, time, frequency, interval and other related parameters back to the instrument control and data integration system. After the cell wall is broken, the rotatable sampling needle mechanical moving device sends the broken sample to the solid-liquid separation system, chemical analysis system or enzyme electrode analysis system.

[0102] (III) Solid-liquid separation system

[0103] The solid-liquid separation of the present invention is mainly achieved by centrifugation, specifically a centrifuge with adjustable speed and controllable temperature. The centrifuge carried by this multifunctional automatic analysis system can achieve the following functions: the speed is adjustable from 400 to 12000 rpm; the temperature control range is adjustable from 0 to 50 ° C; the centrifuge tube size is optional from 0.5 mL to 25 mL; the number of centrifuge tubes is optional from 2 to 10, which can be customized according to needs; at the same time, the centrifuge has a positioning function. After each centrifugation, the centrifuge tube automatically returns to the set fixed position, and the centrifuge tube is in a vertical state.

[0104] like Figure 5 As shown, the centrifuge includes a refrigerator 401, and an annular centrifuge tube slot 403 is arranged inside the refrigerator 401, and a plurality of centrifuge tubes 402 are placed on the annular centrifuge tube slot 403. Refrigerator (with refrigeration): provides a constant temperature environment for the solid-liquid separation system, and the temperature range is adjustable from 0 to 50°C; Centrifuge tube slot: used to place centrifuge tubes of different specifications, and different centrifuge tube slots can be replaced according to the size of the centrifuge tube. The number of centrifuge tubes is 2-10, which can be customized according to needs; At the same time, the centrifuge has a positioning function. After each centrifugation, the centrifuge tube automatically returns to the set fixed position, and the centrifuge tube is in a vertical state; Centrifuge tube: used to place samples for centrifugation, and the specific specifications can be adjusted according to actual needs. The size of the centrifuge tube is optional from 0.5mL to 25mL.

[0105] Furthermore, a centrifuge slot positioner 404 is fixedly provided on one side of the cold storage box 401, and a second temperature probe 406 is provided at a set position on the other side. The centrifuge slot positioner is used to initialize the position of the centrifuge tube slot, which is convenient for the injection needle and the sampling needle to absorb the sample; the second temperature probe is used to monitor the temperature of the solid-liquid separation system.

[0106] Furthermore, a centrifugal shaft 405 is provided at the center of the annular centrifugal tube slot 403, and the bottom of the centrifugal shaft 405 is connected to the second motor 407, and the second motor 407 is fixedly arranged on the second base 408. Centrifugal shaft: a centrifugal component, connecting the centrifugal tube slot box centrifuge; the second motor: used to provide power for the centrifugal component; the second base: used to stabilize and fix the centrifugal system.

[0107] The specific process of instruction transmission and sample processing in the solid-liquid separation system is as follows:

[0108] The solid-liquid separation system mainly performs solid-liquid separation on samples from the sample pretreatment system, dilution system or cell disruption system. According to different test requirements and sequences, the instrument control and data integration system issues a command, and then the rotatable sampling needle mechanical moving device sends the sample from the sample temporary storage pool, dilution pool or cell disruption system into the solid-liquid separation system. The solid-liquid separation system turns on temperature control, speed control, centrifugation time, etc. according to the pre-set parameters to perform solid-liquid separation on the samples. Finally, the solid-liquid separation of the sample is achieved, and the temperature change, time, speed and other related parameters of the solid-liquid separation process are transmitted back to the instrument control and data integration system. After the solid-liquid separation is completed, the rotatable sampling needle mechanical moving device sends the broken sample to the cell disruption system, chemical analysis system or enzyme electrode analysis system.

[0109] (IV) Auxiliary liquid storage system

[0110] The auxiliary liquid storage system can hold reagent bottles of different sizes, which store various buffers, washing solutions, diluents, standard solutions, enzyme solutions and other liquids that can be used for bacterial washing, dilution, sample determination, and sample reaction. The auxiliary liquid storage system has a refrigeration function, and the temperature control range is 0-15°C, the preferred temperature control range is 4-10°C, and the more preferred temperature control range is 4-6°C. The auxiliary liquid storage system determines the position according to the size of the storage bottle, and different positions have different numbers. Preferably, 10 kinds of auxiliary liquids can be placed, and the position numbers are R1-R10; more preferably, 20 kinds of auxiliary liquids can be placed, and the position numbers are R1-R20. Select the reagent number and volume corresponding to different positions under different measurement parameters.

[0111] like Figure 6 As shown, the auxiliary liquid storage system includes a refrigeration bin 503, in which a plurality of reagent storage positions 501 are arranged, and a third temperature probe 502 is also arranged inside the refrigeration bin 503. Reagent storage positions (R1-R12): used to store different reagents, enzyme solutions, buffer solutions, etc., and the corresponding names and uses can be re-entered in the analysis system according to the actual storage positions and actual names; the third temperature probe: used to sense the temperature of the storage system; the refrigeration bin: controls the temperature of the storage system within a certain range.

[0112] (V) Sample dilution system

[0113] The sample dilution system mainly consists of two parts. The first part is the primary dilution with a dilution range of 1-20 times, and the second part is the secondary dilution with a dilution range of 20-400 times. The liquid used for dilution includes but is not limited to distilled water, tap water, phosphate buffer of different pH, citrate buffer, etc. Different liquids can be used according to the specific reaction. The dilution of the sample is mainly completed by the movable robotic arm sampling needle of the sample delivery system. The primary dilution can be completed in the following way: a portion of the sample is drawn by the robotic arm sampling needle, and then 0-19 portions of the dilution liquid are drawn to complete a 1-20 times dilution. The secondary dilution is to draw a portion of the primary dilution liquid after the primary dilution is completed, and then 0-19 portions of the dilution liquid are drawn to complete the secondary dilution. The final maximum dilution multiple can reach 400 times.

[0114] like Figure 7 As shown, the sample dilution system includes a dilution chamber 601, in which a primary dilution pool 604 and a secondary dilution pool 603 are arranged, and a third magnetic stirrer 605 is respectively arranged at the bottom of the primary dilution pool 604 and the secondary dilution pool 603, and a third magnetic rotor 602 is respectively arranged inside the primary dilution pool 604 and the secondary dilution pool 603. Dilution chamber: used to place the dilution pool required for dilution; primary dilution pool: primary dilution of the sample is performed, and the primary dilution is achieved by the robotic arm sampling needle in combination with buffer or distilled water, etc., and the dilution multiple of the primary dilution is generally 2-25 times; secondary dilution pool: secondary dilution of the sample is performed, and the secondary dilution is achieved by the robotic arm sampling needle in combination with buffer or distilled water, etc., and the sample of the secondary dilution comes from the primary dilution pool, and the dilution multiple of the secondary dilution is generally 10-500 times; third magnetic stirrer: provides power for the magnetic rotor; third magnetic rotor: used for mixing the sample.

[0115] (VI) Chemical Analysis System

[0116] The chemical analysis system mainly includes a reaction cell, an adjustable light source, a monochromator, a detector, etc. The nanometer number of the adjustable light source can be adjusted according to different measurement requirements, and the adjustment range is 300-900nm. The absorbance value and transmittance at different nanometers can be measured to calculate the concentration of the corresponding substance or parameters such as enzyme activity. Specifically for the present invention, the chemical analysis system can measure parameters including but not limited to the following: OD value of the bacterial volume in the fermentation broth, urea, protein concentration, succinate dehydrogenase activity, malate dehydrogenase activity, lipase activity, etc.

[0117] like Figure 8As shown, the chemical analysis system includes a light source 701, a lens 702, a monochromator 703, a reaction cell (sample cell) 704, a detector 705, a signal amplifier 706, and a display 707. The light emitted by the light source is imaged on the incident slit of the monochromator through the lens, and the monochromator decomposes the light emitted by the light source into monochromatic light and irradiates the sample solution in the reaction cell; the detector responds to the light passing through the reaction cell through the photoelectric conversion element, converts the optical signal into an electrical signal and outputs it to the signal amplifier, and the signal amplifier amplifies the electrical signal and outputs it to the display to display the measurement result.

[0118] Furthermore, the light source: provides light source for the chemical analysis system, and can realize the emission of ultraviolet light and visible light of different nanometers through filters, monochromators, etc., with a wavelength range of 190-2500nm; lens: images the light emitted by the light source on the incident slit of the monochromator and evenly illuminates the slit; monochromator: emits continuous radiation light through the light source, which is decomposed into monochromatic light by the monochromator and then irradiated onto the sample solution; sample cell (absorption cell): stores the samples to be analyzed; detector: responds to the light passing through the cuvette through the photoelectric conversion element, and converts the optical signal into an electrical signal for output; signal amplifier: after converting the optical signal into an electrical signal, it plays the role of amplifying these signals; display: used to present the measurement results.

[0119] The processing and transmission process of instructions, samples, and test result data in the chemical analysis system is as follows:

[0120] The chemical analysis system mainly uses spectrophotometry to determine the absorbance of samples at different wavelengths, and records or calculates the parameters of the samples based on the absorbance. According to different test requirements, the instrument control and data integration system issues a command, and then the rotatable sampling needle mechanical moving device sends the sample from the sample storage pool, dilution pool, cell disruption system or solid-liquid separation system into the reaction pool of the chemical analysis system. The chemical analysis system sets the wavelength and measurement frequency according to the preset parameters, and then records the measurement results after the test is completed, and transmits the measurement results to the instrument control and data integration system. After the test is completed, the rotatable sampling needle mechanical moving device takes the sample away and discharges it.

[0121] (VII) Enzyme Electrode Analysis System

[0122] The enzyme electrode analysis system is specifically an enzyme reaction device composed of an enzyme membrane and an electrode. It is mainly used to measure various parameters in the supernatant after direct centrifugation of the fermentation liquid, various parameters in the liquid after centrifugation of the fermentation liquid bacteria, and various biochemical parameters in the supernatant after the bacteria are washed and broken. The parameters that can be measured include but are not limited to glucose, glutamic acid, lactic acid, ethanol, glycerol, pyruvic acid, methanol, glutamine, formic acid, bilirubin, etc. The specific parameters can be determined by the selected enzyme membrane. The electrodes used in the present invention are the same, and the specific measurement parameters are determined according to the different enzyme membranes used. Glucose enzyme membranes are used to measure the glucose concentration in various liquids; glutamate enzyme membranes are used for glutamate concentrations in various liquids. In general, the enzyme electrode analysis system provided by the present invention includes two electrodes or three electrodes, and 2 or 3 parameters are measured at the same time. The enzyme electrode bioanalysis system provided by the present invention is mainly to illustrate the parameters that need to be measured. In the specific analysis system, other measurement methods that can achieve the measurement of various parameters mentioned in the present invention can also be selected.

[0123] like Fig. 9 As shown, the enzyme reaction device is provided with a sampling needle sensor 801 on the top, a plurality of enzyme electrodes 802 are interspersed on the side wall, a magnetic stirring motor 804 is provided at the bottom, and a fourth magnetic rotor 803 is provided inside. The sampling needle sensor: senses the entry and exit of the sampling needle, and calculates the reaction time of the enzyme membrane and the sample through the entry and exit time of the sampling needle; the enzyme electrode: specifically an electrode based on oxidase, including three parts: a counter electrode, a reference electrode and a working electrode; the fourth magnetic rotor: used for mixing the sample; the magnetic stirring motor: provides power for the stirring rotor.

[0124] The processing and transmission process of instructions, samples, and test result data in the enzyme electrode analysis system is as follows:

[0125] The enzyme electrode analysis system mainly uses the enzyme electrode method to determine the concentration of compounds in the sample. The core working principle is that the enzyme catalyzes the conversion of substrates into products, which are detected by the converter and converted into quantifiable signal outputs to achieve the purpose of detecting the concentration of the substrate being tested. According to different test requirements, the instrument control and data integration system issues a command, and then the rotatable sampling needle mechanical moving device sends the sample from the sample storage pool, dilution pool, cell disruption system or solid-liquid separation system into the reaction pool of the enzyme electrode analysis system. The enzyme electrode analysis system sets the injection volume, reaction time and other parameters according to the preset parameters, and then records the measurement results after the test is completed, and transmits the measurement results to the instrument control and data integration system. After the test is completed, the sample is taken away by the rotatable sampling needle mechanical moving device and then discharged.

[0126] (VIII) Rotatable sampling needle mechanical moving device

[0127] The rotatable sampling needle mechanical moving device can absorb samples and reagents from (I) sample pretreatment system, (II) cell disruption system, (III) solid-liquid separation system, (VI) auxiliary liquid storage system, and (V) sample dilution system, and send them to (IV) chemical analysis system and (VII) enzyme electrode analysis system through the sampling needle for relevant determination. The specific sampling volume, number of times, interval, position and other information are controlled by (IX) instrument control and data integration system. The mechanical moving device can realize sample transmission, dilution, cleaning and discharge between different systems.

[0128] like Fig.10 As shown, the mechanical moving device of the rotatable sampling needle includes a slide rail 901, a positioning rail 909 is arranged on the slide rail 901, a mobile sampling base 902 is movably arranged on the positioning rail 909, a positioner 910 is arranged on the bottom surface of the mobile sampling base 902 close to the positioning rail 909, a volume controller 906 is connected to one side wall of the mobile sampling base 902 through a connecting tube 907, a rotator 908 is arranged on the top of the mobile sampling base 902, a rotatable mechanical shaft 903 is arranged on the top of the rotatable mechanical shaft 903, a rotator 908 is arranged on the top of the rotatable mechanical shaft 903, the rotator 908 is connected to one end of a horizontally rotatable mechanical arm 905, the other end of the horizontally rotatable mechanical arm 905 is connected to a vertically rotatable mechanical arm 911, and a sampling needle (or sample delivery needle) 904 is connected to the end of the vertically rotatable mechanical arm 911.

[0129] The mobile sampling base is equipped with a single-chip microcomputer MCU, which is connected to the instrument control and data integration system and receives its commands. The positions of each system and the positions where liquid transfer and liquid delivery are required are fixed. The positions of each system and the positions where liquid transfer and liquid delivery are required are preset in the single-chip microcomputer. These positions are defined based on the coordinate system of the positioner and the positioning track.

[0130] The single-chip microcomputer is respectively connected to the positioner, volume controller, and rotator control. The positioner captures the position information in real time and feeds it back to the single-chip microcomputer. The single-chip microcomputer determines that the moving device can accurately reach the preset position in the command based on the feedback of the positioner; the single-chip microcomputer sends a control signal (the signal contains the target volume of liquid to be sucked) to the volume controller, and the volume controller monitors the change of liquid volume to ensure that the sucked liquid volume is consistent with the target volume; the single-chip microcomputer sends a control signal to the rotator (the signal contains the angle to be selected) according to the preset position of pipetting and liquid delivery in each system, and the rotator responds to the control signal and rotates the mechanical arm to the corresponding position to achieve liquid absorption. It should be noted that the movement, mechanical arm rotation, target position pipetting, and target position liquid delivery functions in the rotatable sampling needle mechanical moving device are all implemented using well-known mechanical control methods, and the specific control content and working principle will not be repeated here.

[0131] The overall working process of the mobile device is:

[0132] The mechanical moving device of the rotatable sampling needle mainly realizes the operation of transferring or injecting samples, reagents and other liquids by sliding or rotating the mechanical arm horizontally. The moving device is controlled by the instrument control and data integration system. Before the entire multifunctional automatic analysis system is used, the initial position of the injection needle of each system needs to be preset and marked. After receiving the command from the instrument control and data integration system, the mechanical device moves and rotates according to the position mark in the control command, thereby achieving the purpose of transferring and delivering liquids.

[0133] According to different test requirements, the instrument control and data integration system issues a command, and the rotatable sampling needle mechanical moving device first determines the position where liquid transfer or liquid delivery is required, and then determines the volume and number of liquid transfer and delivery according to the test requirements, and removes the sample from the pretreatment system, cell disruption system, solid-liquid separation system, chemical analysis system, enzyme electrode analysis system, auxiliary liquid storage system or sample dilution system through lateral and rotational movement, and transports it to the cell disruption system, sample dilution system, solid-liquid separation system, chemical analysis system or enzyme electrode analysis system. After the liquid transfer is completed, the mechanical movement trajectory, liquid transfer volume, and liquid transfer times are recorded, and the data is transmitted to the instrument control and data integration system in real time.

[0134] (IX) Instrument control and data integration system

[0135] The instrument control and data integration system is connected to the above 7 systems, that is, each system is controlled by the instrument control and data integration system, and the instrument control and data integration system records the relevant operations and measurement results, etc., specifically:

[0136] (1) The instrument control and data integration system is connected to the sample pretreatment system and can control the sample pretreatment system. If an online sample processing system is selected, the sampling channel, channel name, sampling order, sampling time, sampling frequency, and repeated sampling times are controlled, and the data measured by the pH electrode are recorded at the same time, and recorded under the corresponding batch in the data integration system. Preferably, the sampling method, the length and aperture of the sampling tube, and other information can be recorded according to different online sampling methods, so as to form a sampling data record. More preferably, the online analysis control system controls the sampling volume, the liquid level of the sample pool, and the temperature of the sample storage pool.

[0137] If an off-line sample pretreatment system is used, the sample serial number, sampling time, number of measurements, etc. are controlled. The preferred off-line analysis system controls the storage tank temperature, sampling volume, etc. of the sample bottles, and records them in the corresponding batch data table.

[0138] (2) The instrument control and data integration system is connected to the cell disruption system and can control the cell disruption system. The specific controlled parameters mainly include sample cell disruption volume, cell disruption frequency, cell disruption power, and the temperature of the cell disruption pool.

[0139] (3) The instrument control and data integration system is connected to the solid-liquid separation system and can control the solid-liquid separation system. The specific control parameters mainly include centrifugal speed, the size of the centrifugal rotor, and the centrifugal temperature. The preferred analysis control system can record the position of each centrifuge tube in detail and return to the initial state after the centrifugation. At the same time, when performing the initialization setting, the model of the centrifuge tube needs to be reset in the system according to the size of the sample.

[0140] (4) The instrument control and data integration system is connected to the auxiliary liquid storage system and can control the auxiliary liquid storage system, specifically, selecting different test reagents according to different test items and registering the names and positions of the reagents in sequence. The analysis control system can also control the sampling needle to absorb reagents at different positions and different volumes of reagents according to different selected test parameters, and absorb reagents at corresponding positions in sequence according to the reaction sequence.

[0141] (5) The instrument control and data integration system is connected to the sample dilution system and can control the sample dilution system. The specific control parameters include the volume of the sample to be drawn, the volume of the diluent, the dilution factor and other parameters. The dilution factor can be selected as a fixed dilution factor or a step-by-step dilution method from low to high. If the range of the sample measurement exceeds the range after the previous dilution, the next dilution is expanded by 10 times. If the measurement requirements can be met, the dilution is stopped. If not, the dilution factor is continued to be increased or decreased. The appropriate dilution factor is determined through repeated dilution.

[0142] (6) The instrument control and data integration system is connected to the chemical analysis system and can control the chemical analysis system. The specific controlled parameters include the nanometer number of the light source, the data recording frequency, etc. It is also possible to set the absorbance value measurement at different time intervals according to the needs of the measurement method. Before the measurement, it is necessary to register the name of the parameter to be measured in the analysis control system and select the required reagent number and position.

[0143] (7) The instrument control and data integration system is connected to the enzyme electrode analysis system and can control the enzyme electrode analysis system. The controlled parameters include but are not limited to parameter name, measurement times, etc. Before the measurement, it is necessary to register the name of the parameter to be measured in the analysis control system and select the required reagent number and position.

[0144] Data integration in the instrument control and data integration system mainly includes the following functions: first, record product information, sample batch name, measurement date and other information. If it is online sampling and measurement, it can also record the sampling interval information since the first sampling; secondly, the instrument control and data integration system also records but is not limited to the following information from the beginning of the system: sample number, name, sampling method, sampling volume, dilution factor, bacterial concentration, sample pH value, wall breaking power, temperature change during wall breaking, centrifugal speed and other information; finally, record the measured values ​​according to the different parameters measured by the chemical analysis system and the enzyme electrode analysis system, and form a table. The instrument control and data integration system can present the data in the form of a table or a curve. It has a data output port, including but not limited to USB data output.

[0145] Here are some specific examples:

[0146] Example 1

[0147] In the succinic acid production process, the cell concentration, fermentation liquid pH, fermentation liquid glucose, fermentation liquid lactic acid and intracellular glucose, intracellular lactic acid, intracellular pyruvic acid, intracellular succinate dehydrogenase activity, intracellular ATP concentration and NAD (H) concentration based on metabolism in the fermentation liquid are measured. The bacterial strain used in the present embodiment is Corynebacterium glutamicum, which first passes through the aerobic culture stage, and then carries out anaerobic fermentation to produce succinic acid. The culture medium formula comprises glucose, ammonium salt and organic nitrogen source, and the fermentation process carries out process regulation to oxygen supply and pH. Note that the present embodiment is intended to illustrate the implementation and determination of the key biochemical parameters inside and outside the cell during the fermentation process, and the fermentation control process is no longer described in detail.

[0148] In this embodiment, an online sample pretreatment system is selected, and samples are taken every 240 minutes to measure relevant parameters.

[0149] To measure the above parameters, the required reagents, buffer solutions, etc. are placed in the auxiliary liquid storage system. The embodiments provided in the present invention are only for illustrative purposes. Users can set and modify different test parameters based on different test kits, reagents and methods according to different test requirements.

[0150] The auxiliary reagents involved in the present invention are specifically:

[0151] Distilled water;

[0152] Phosphate buffer (pH 6.5-7);

[0153] Phenazine dimethyl sulfate (PMS): 0.7 g / L;

[0154] 2,6-Dichlorophenol indophenol (DCPIP): 0.07 g / L;

[0155] ATP detection reagents A, B, C, and D are all commercial reagents in the kit and were purchased from MacLean;

[0156] ATP standard solution, a commercial reagent in the kit, was purchased from MacLean;

[0157] NAD / NADH assay buffer reagent;

[0158] Ethanol solution;

[0159] Alcohol dehydrogenase;

[0160] CCK-8 solution;

[0161] NADH standards; all are commercial reagents of the colorimetric detection kit for the enzyme activity of coenzyme I NAD(H), purchased from Beijing Pulilai Gene Technology Co., Ltd.

[0162] The pH value is directly measured by the sample storage tank of the pretreatment system;

[0163] The cell concentration of the fermentation broth is determined by a chemical analysis system. Specifically, after the sample is taken to the temporary storage pool of the pretreatment system, the instrument control and data integration system issues a command, and the rotatable sampling needle mechanical moving device moves the sample into the sample pool of the chemical analysis system to measure the absorbance value at 600nm. When the measurement result is greater than 1.5, it is determined to enter the dilution stage, and the rotatable sampling needle mechanical moving device moves the sample into the dilution system for dilution. The dilution multiple is increased step by step according to different OD values. The diluted liquid is moved by the rotatable sampling needle mechanical moving device into the sample pool of the chemical analysis system for measurement until the OD value is within the range of 0.2-1.5, and the final cell concentration is recorded according to the dilution multiple and the measurement result.

[0164] The concentration of glucose and lactic acid in the fermentation broth is determined by the enzyme electrode analysis system, specifically: after the sample is taken to the temporary storage pool of the pretreatment system, the instrument control and data integration system issues an instruction, firstly, the solid-liquid separation of cells and fermentation broth is carried out, and the sample is moved into the solid-liquid separation system by the rotatable sampling needle mechanical moving device to achieve the separation of cells and fermentation broth, and then the supernatant after solid-liquid separation can be directly sampled and analyzed by the sampling needle, and the sample is injected into the enzyme electrode analysis system and the result is determined by the enzyme electrode analysis system. Similarly, when the concentration of glucose and lactic acid exceeds the measurement range (0.0-1g / L) of the enzyme electrode analysis system, the sample enters the dilution link, and the rotatable sampling needle mechanical moving device realizes the transfer of samples and distilled water to achieve the dilution of the sample, and the dilution multiple is gradually increased according to different measurement result values. The diluted liquid is moved into the sample pool of the enzyme electrode analysis system by the rotatable sampling needle mechanical moving device for measurement until the measurement value is within the range of 0.0-1g / L, and the final glucose and lactic acid concentrations are recorded according to the dilution multiple and the measurement result. The results recorded at this time are the glucose and lactate concentrations in the cells.

[0165] The intracellular glucose concentration, intracellular lactate concentration, and intracellular pyruvate concentration are determined by the enzyme electrode analysis system. Specifically, after the sample is taken to the temporary storage pool of the pretreatment system, the instrument control and data integration system issues instructions to first perform solid-liquid separation of cells and fermentation broth, then take away the supernatant through the rotatable sampling needle mechanical moving device, and re-absorb distilled water or buffer into the solid-liquid separation system. After repeated blowing, solid-liquid separation is performed here, and the bacteria are washed after more than 3 operations. Then the mixed liquid is blown again and moved into the cell disruption system by the sampling needle to disrupt the cells. After cell disruption, the sample is divided into two parts. Part of the liquid enters the solid-liquid separation system again to determine the intracellular glucose concentration, intracellular lactate concentration, and intracellular pyruvate concentration; part of the liquid directly enters the chemical analysis system to determine the intracellular succinate dehydrogenase activity, intracellular ATP concentration, and metabolic-based NAD (H) concentration. After the liquid after cell crushing is separated into solid and liquid, the sample is injected into the enzyme electrode analysis system by the rotatable sampling needle mechanical moving device, and the result is measured by the enzyme electrode analysis system. Similarly, when the concentration of glucose, lactic acid, and pyruvic acid exceeds the measurement range (0.0-1g / L) of the enzyme electrode analysis system, the sample enters the dilution link, and the sample is transferred and distilled water is transferred by the rotatable sampling needle mechanical moving device to achieve the dilution of the sample. The dilution multiple is increased step by step according to different measurement result values. The diluted liquid is moved into the sample pool of the enzyme electrode analysis system by the rotatable sampling needle mechanical moving device for measurement until the measured value is within the range of 0.0-1g / L, and the final glucose and lactic acid concentrations are recorded according to the dilution multiple and the measurement result. The result recorded at this time is the intracellular glucose, lactic acid, and pyruvic acid concentrations.

[0166] The intracellular succinate dehydrogenase enzyme is measured by a chemical analysis system. The washed bacterial cells are subjected to a cell disruption system, and the sample is divided into two parts, one of which enters a solid-liquid separation system to measure the intracellular glucose concentration, intracellular lactic acid concentration, and intracellular pyruvic acid concentration. The remaining liquid directly enters a chemical analysis system to measure the intracellular succinate dehydrogenase enzyme activity, intracellular ATP concentration, and NAD (H) concentration based on metabolism. Specifically, after the instrument control and data integration system issues an instruction, a rotatable sampling needle mechanical moving device adds phosphate buffer, phenazine dimethyl sulfate (PMS), and 2,6-dichlorophenol indophenol (DCPIP) required for the determination of succinate dehydrogenase to the sample pool of the chemical analysis system, and then records the initial absorbance value at 600nm. Then, the rotatable sampling needle mechanical moving device injects the broken sample into the sample pool of the chemical analysis system, and starts the timing function at the same time. After the 1min or 2min timing is over, the absorbance value at 540nm is measured again. At the same time, the operation time, absorbance value change and other data are recorded and synchronously transmitted to the instrument control and data integration system, which calculates the intracellular succinate dehydrogenase concentration based on the measurement results.

[0167] The intracellular ATP concentration is determined by the chemical analysis system. The washed bacteria are passed through the cell disruption system, and the sample is divided into two parts. One part enters the solid-liquid separation system to determine the intracellular glucose concentration, intracellular lactate concentration, and intracellular pyruvate concentration. The remaining part of the liquid directly enters the chemical analysis system to determine the intracellular succinate dehydrogenase activity, intracellular ATP concentration, and metabolic NAD (H) concentration. Specifically: After the instrument control and data integration system issues an instruction, the rotatable sampling needle mechanical moving device moves the ATP detection working solution C and D required for the intracellular ATP concentration determination, and then adds the cell disruption sample, adds the sample pool added to the chemical analysis system respectively, and then adds a fixed volume of cell disruption solution according to the instructions of the kit. After incubation at room temperature for 10s, the measurement is immediately carried out. If the ATP concentration in the test sample is too high, it can be diluted with ATP detection working solution A and B before detection. After the test is completed, the ATP concentration in the cell disruption solution is calculated, and the detection concentration range has a good linear relationship in the 1nM-5,000nM system. If the concentration is too high, it will enter the dilution stage. At the same time, the operation time, absorbance value changes and other data are recorded and synchronously transmitted to the instrument control and data integration system, which calculates the intracellular ATP concentration based on the measurement results.

[0168] The intracellular NAD(H) concentration based on metabolism is determined by the chemical analysis system. The washed bacteria are passed through the cell disruption system, and the sample is divided into two parts, one of which enters the solid-liquid separation system to determine the intracellular glucose concentration, intracellular lactate concentration, and intracellular pyruvate concentration. The remaining liquid directly enters the chemical analysis system to determine the intracellular succinate dehydrogenase activity, intracellular ATP concentration, and intracellular NAD(H) concentration based on metabolism. Specifically, after the instrument control and data integration system issues an instruction, the rotatable sampling needle mechanical moving device adds the detection reagent buffer, CCK-8 solution, and the sample after cell disruption required for the determination of the intracellular NAD(H) concentration to the sample pool of the chemical analysis system in turn, mixes at room temperature for 10s, reacts for 30min, and then determines the absorbance value at 450nm. At the same time, the time of the operation process, the change in absorbance value and other data are recorded, and synchronously transmitted to the instrument control and data integration system, and the instrument control and data integration system calculates the intracellular NAD(H) concentration based on the measurement results.

[0169] Embodiment 2

[0170] This embodiment discloses a multifunctional automatic analysis method for key biochemical parameters inside and outside cells, comprising:

[0171] Select the required measurement parameters before measuring;

[0172] Start the sample pretreatment system to take samples, and pump the samples into the sample temporary storage pool;

[0173] The rotatable sampling needle mechanical moving device takes samples from the sample temporary storage pool according to the command, and sends the samples to the sample dilution system, solid-liquid separation system, cell disruption system, or directly to the chemical analysis system, or directly to the enzyme electrode analysis system according to the measurement requirements of the measurement parameters;

[0174] The rotatable sampling needle mechanical moving device takes liquid from the auxiliary liquid storage system according to the command to assist in sample dilution, solid-liquid separation, cell disruption and chemical analysis or enzyme electrode analysis;

[0175] The samples are finally sent to the chemical analysis system or enzyme electrode analysis system for final measurement to obtain the measurement results.

[0176] In this embodiment, the required measurement parameters are first selected before the measurement, such as: cell concentration in the fermentation broth, fermentation broth pH, ​​fermentation broth glucose, fermentation broth lactate and intracellular glucose, intracellular lactate, intracellular pyruvate, intracellular succinate dehydrogenase activity, intracellular ATP concentration and metabolism-based NAD (H) concentration.

[0177] Note: The multifunctional automatic analysis system involved in the present invention is carried out according to the difference of each test parameter and the difference of sample concentration, and there is no fixed liquid movement order. For example, a detailed measurement parameter can be preset here, such as each sample is measured for biomass, pH value, glucose (intracellular, fermentation broth), lactic acid (intracellular, fermentation broth), intracellular succinate dehydrogenase activity, and intracellular phosphofructokinase activity. If the cell concentration is measured, the sample is diluted by the sample dilution system after the sample pretreatment system, and then the absorbance value at 600nm is measured by the chemical analysis system; if the enzyme activity of succinate dehydrogenase needs to be measured, a sample pretreatment system, a cell disruption system, a solid-liquid separation system, and a chemical analysis system are required; if the fermentation broth (extracellular) glucose concentration is measured, a pretreatment system, a solid-liquid separation system, a sample dilution system, and an enzyme electrode analysis system are required; if the (intracellular) glucose concentration is measured, a pretreatment system, a solid-liquid separation system, a sample dilution system, a cell disruption system, and an enzyme electrode analysis system are required.

[0178] Likewise, researchers can customize the measurement parameters based on mature detection methods or their own customized experimental methods in combination with the multifunctional automatic analysis system provided by the present invention.

[0179] Embodiment 3

[0180] The purpose of this embodiment is to provide a computing device, including a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the processor implements the steps of the method of Embodiment 2 when executing the program.

[0181] Embodiment 4

[0182] The purpose of this embodiment is to provide a computer-readable storage medium, a computer-readable storage medium on which a computer program is stored, and when the program is executed by a processor, the steps of the method of embodiment 2 are performed.

[0183] The steps involved in the apparatus of the above embodiments 3 and 4 correspond to the method embodiment 1, and the specific implementation method can refer to the relevant description part of embodiment 1. The term "computer-readable storage medium" should be understood as a single medium or multiple media including one or more instruction sets; it should also be understood to include any medium that can store, encode or carry an instruction set for execution by a processor and enable the processor to execute any method in the present invention.

[0184] Those skilled in the art should understand that the modules or steps of the present invention described above can be implemented by a general-purpose computer device, or alternatively, they can be implemented by a program code executable by a computing device, so that they can be stored in a storage device and executed by the computing device, or they can be made into individual integrated circuit modules, or multiple modules or steps therein can be made into a single integrated circuit module for implementation. The present invention is not limited to any specific combination of hardware and software.

[0185] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0186] Although the above describes the specific implementation mode of the present invention in conjunction with the accompanying drawings, it is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art on the basis of the technical solution of the present invention without creative work are still within the scope of protection of the present invention.

Claims

1. A multifunctional automatic analysis system for key biochemical parameters inside and outside cells, characterized in that: include: Sample pretreatment system, used for sampling and measurement; A cell disruption system, used for disrupting cells of the sample from the sample pretreatment system; A solid-liquid separation system, used to separate the solid and liquid of the sample using a centrifuge; Auxiliary liquid storage system, used to store a variety of test reagents and provide refrigeration function; Sample dilution system, used to dilute samples to different degrees; A chemical analysis system, used for chemical analysis of samples from a sample pretreatment system, a cell disruption system or a solid-liquid separation system; The enzyme electrode analysis system is specifically an enzyme reaction device composed of an enzyme membrane and an electrode, which is used to analyze samples from a sample pretreatment system, a cell disruption system or a solid-liquid separation system; A rotatable sampling needle mechanical moving device is used to absorb samples from the sample pretreatment system, cell disruption system, solid-liquid separation system, auxiliary liquid storage system and sample dilution system, and send them to the chemical analysis system and enzyme electrode analysis system through the sampling needle for relevant determination; The instrument control and data integration system is used to control the sample pretreatment system, cell disruption system, solid-liquid separation system, auxiliary liquid storage system, sample dilution system, chemical analysis system, enzyme electrode analysis system and rotatable sampling needle mechanical moving device, and record relevant data.

2. A multifunctional automatic analysis system for key intracellular and extracellular biochemical parameters as claimed in claim 1, characterized in that: The sample pretreatment system includes online real-time sampling and measurement and offline sampling and measurement. The online real-time sampling and measurement is realized by an online sample processing system, which includes a sampling device and a sample storage pool. The sampling device is specifically a sampling tube with a straight plug-in type. The sampling tube is connected to the sample storage pool through a hose. A peristaltic pump is provided between the sample storage pool and the sampling tube, and the liquid in the reactor is pumped into the sample storage pool through the peristaltic pump.

3. A multifunctional automatic analysis system for key intracellular and extracellular biochemical parameters as claimed in claim 1, characterized in that: The cell disruption system includes a sample pool, a vibration module and a control module. The vibration module includes a vibration motor and an ultrasonic probe. A plurality of cooling metals are arranged on the circumference of the sample pool chamber. The ultrasonic probe is arranged at the bottom of the chamber. The bottom of the ultrasonic probe is connected to the vibration motor, and the vibration motor is electrically connected to the control module.

4. A multifunctional automatic analysis system for key intracellular and extracellular biochemical parameters as claimed in claim 1, characterized in that: The solid-liquid separation system is implemented by a centrifuge, which includes a refrigerator. An annular centrifuge tube groove is arranged inside the refrigerator. A plurality of centrifuge tubes are placed on the annular centrifuge tube groove. A centrifuge shaft is arranged at the center of the annular centrifuge tube groove. The bottom of the centrifuge shaft is connected to a motor.

5. A multifunctional automatic analysis system for key intracellular and extracellular biochemical parameters as claimed in claim 1, characterized in that: The chemical analysis system includes a light source, a lens, a monochromator, a reaction cell, a detector, a signal amplifier and a display. The light emitted by the light source is imaged on the incident slit of the monochromator through the lens. The monochromator decomposes the light emitted by the light source into monochromatic light and irradiates the light onto the sample solution in the reaction cell. The detector responds to the light passing through the reaction cell through a photoelectric conversion element, converts the optical signal into an electrical signal and outputs it to the signal amplifier. The signal amplifier amplifies the electrical signal and outputs it to the display to display the measurement result.

6. A multifunctional automatic analysis system for key intracellular and extracellular biochemical parameters as claimed in claim 1, characterized in that: The enzyme electrode analysis system is specifically an enzyme reaction device composed of an enzyme membrane and an electrode, with an injection needle sensor on the top, multiple enzyme electrodes inserted on the side wall, a magnetic stirring motor at the bottom, and a magnetic rotor inside.

7. A multifunctional automatic analysis system for key intracellular and extracellular biochemical parameters as claimed in claim 1, characterized in that: The mechanical moving device of the rotatable sampling needle includes a slide rail, a positioning rail is arranged on the slide rail, a mobile sampling base is movably arranged on the positioning rail, a volume controller is connected to a side wall of the mobile sampling base, a rotatable mechanical axis is arranged on the top of the mobile sampling base, the rotatable mechanical axis is connected to one end of a transverse rotatable mechanical arm, the other end of the transverse rotatable mechanical arm is connected to a vertical rotatable mechanical arm, and a sampling needle is connected to the end of the vertical rotatable mechanical arm.

8. A multifunctional automatic analysis method for key biochemical parameters inside and outside cells, characterized in that: include: Select the required measurement parameters before measuring; Start the sample pretreatment system to take samples, and pump the samples into the sample temporary storage pool; The rotatable sampling needle mechanical moving device takes samples from the sample temporary storage pool according to the command, and sends the samples to the sample dilution system, solid-liquid separation system, cell disruption system, or directly to the chemical analysis system, or directly to the enzyme electrode analysis system according to the measurement requirements of the measurement parameters; The rotatable sampling needle mechanical moving device takes liquid from the auxiliary liquid storage system according to the command to assist in sample dilution, solid-liquid separation, cell disruption and chemical analysis or enzyme electrode analysis; The samples are finally sent to the chemical analysis system or enzyme electrode analysis system for final measurement to obtain the measurement results.

9. A computer-readable storage medium having a computer program stored thereon, characterized in that: When the program is executed by a processor, the steps of a multifunctional automatic analysis method for key intracellular and extracellular biochemical parameters as described in claim 8 are implemented.

10. A computer device comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, characterized in that: When the processor executes the program, the steps of the multifunctional automatic analysis method for key intracellular and extracellular biochemical parameters as described in claim 8 are implemented.

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