Liquid handling method and analysis device

Abstract

The application provides a liquid processing method and an analysis device. The method comprises: performing a plurality of first pipetting operations on the liquid in the reaction container; wherein the first pipetting operation comprises liquid injection operation and liquid suction operation; performing a second pipetting operation on the liquid in the reaction container; wherein the second pipetting operation comprises liquid injection operation and liquid suction operation, and the injection speed of the second pipetting operation is less than that of the first pipetting operation. Through the first pipetting operation and the second pipetting operation, the residual liquid in the first pipetting operation can be washed into the reaction container, and by controlling the injection speed of the second pipetting operation to be less than that of the first pipetting operation, the adsorption of the magnetic beads can be reduced, and the accuracy of the nucleic acid detection result can be improved.

Description

Technical Field

[0001] This invention relates to the field of medical technology, and in particular to a liquid processing method and analysis apparatus. Background Technology

[0002] Molecular detection technologies have been widely applied in various fields, including infectious disease diagnosis, epidemiological investigation, food hygiene inspection, early diagnosis of tumors and genetic diseases, blood screening, prenatal screening, and pharmacokinetics. It is one of the fastest-growing clinical testing technologies. Polymerase chain reaction (PCR), a molecular biology technique used to amplify specific DNA (Deoxyribonucleic acid) fragments, is widely used in molecular biological detection and analysis due to its high specificity, high sensitivity, simplicity, and speed. However, the cumbersome sample extraction process is a significant barrier limiting the further clinical application of PCR technology.

[0003] Currently, in automated analytical equipment, nucleic acid extraction often involves immobilizing the analyte with a reaction solution. This mixture is then brought into contact with a carrier, immobilizing the analyte on the carrier for detection. The carrier can be magnetic beads. To ensure thorough mixing of the analyte, reaction solution, and magnetic beads, a repeated aspiration method is typically used. The analyte, reaction solution, and magnetic beads are drawn into a pipette and then dispensed into a test tube. The aspiration pump reciprocates mechanically, achieving uniform mixing and immobilizing the analyte on the magnetic beads. This method is called pipetting (see [link to pipetting method]). Figure 1 The diagram shows a first schematic of a pipetting and mixing method, in which 800 μL of liquid is drawn into the pipette. During the dispensing process, some magnetic beads and liquid may remain at the pipette opening (see...). Figure 2 (A second schematic diagram of a mixing method shown) When the liquid in the pipette is refilled into the test tube, some liquid, still containing the magnetic bead, will remain at the opening because... Figure 2 Under these conditions, when the pipette is suspended in the air, the external temperature of its location is lower than the temperature of the liquid inside the test tube, and the liquid inside the pipette moves upward along the inner wall of the pipette.

[0004] See Figure 3 The third schematic diagram of a mixing method illustrates that when the pipette draws in liquid again, the liquid drawn in pushes upwards along with some gas and liquid residue from the pipette opening. The mechanical reciprocating mixing operation of the suction pump cannot remove the magnetic beads adsorbed on the inner wall of the pipette. Because the magnetic beads are partially adsorbed, the concentration of the analyte may decrease, or magnetic beads containing the analyte may be left on the inner wall of the pipette, affecting the detection results. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a liquid processing method and an analytical apparatus to reduce the adsorption of magnetic beads and improve the accuracy of nucleic acid detection results.

[0006] In a first aspect, embodiments of the present invention provide a liquid processing method for processing liquid within a reaction vessel. The method includes: performing multiple first pipetting operations on the liquid within the reaction vessel; wherein the first pipetting operation includes a liquid injection operation and a liquid aspiration operation; performing a second pipetting operation on the liquid within the reaction vessel; wherein the second pipetting operation includes a liquid injection operation and a liquid aspiration operation, and the injection rate of the second pipetting operation is less than the injection rate of the first pipetting operation.

[0007] In optional embodiments of this application, before performing multiple first pipetting operations on the liquid in the reaction vessel, the method further includes: raising the temperature of the reaction vessel region from a first temperature to a second temperature; wherein the second temperature is greater than the first temperature, the first temperature is not less than 25°C, the first temperature is not greater than 37°C, the second temperature is not less than 25°C, and the second temperature is not greater than 60°C; before performing a second pipetting operation on the liquid in the reaction vessel, the method further includes: lowering the temperature of the reaction vessel region from the second temperature to the first temperature.

[0008] In an optional embodiment of this application, the step of performing multiple first pipetting operations on the liquid in the reaction vessel includes: determining whether the liquid volume in the reaction vessel is greater than a preset threshold; if so, controlling the sampling mechanism to move continuously in the vertical direction; if not, controlling the sampling mechanism to remain stationary in the vertical direction.

[0009] In an optional embodiment of this application, the ratio of the injection rate of the second pipetting operation to the injection rate of the first pipetting operation is not greater than 0.75; the ratio of the injection volume of the second pipetting operation to the injection volume of the first pipetting operation is related to the ratio of the second temperature to the first temperature.

[0010] In an optional embodiment of this application, the ratio of the injection rate of the second pipetting operation to the injection rate of the first pipetting operation is not greater than 0.5, and the ratio of the injection volume of the second pipetting operation to the injection volume of the first pipetting operation is not less than 1.07.

[0011] In an optional embodiment of this application, the first temperature is 37°C and the second temperature is 60°C.

[0012] In optional embodiments of this application, after the step of lowering the temperature of the reaction vessel region from the second temperature to the first temperature, the method further includes: raising the magnet disposed on the side of the reaction vessel from the initial position to the first position; before the step of performing the second liquid transfer operation on the liquid in the reaction vessel, the method further includes: raising the magnet from the first position to the second position; after the step of performing the second liquid transfer operation on the liquid in the reaction vessel, the method further includes: lowering the magnet from the second position to the first position.

[0013] In an optional embodiment of this application, the process applied to nucleic acid extraction includes: a lysis process; and a step of raising the temperature of the reaction vessel region from a first temperature to a second temperature, including: maintaining the temperature of the reaction vessel region at the first temperature during the lysis process; mixing the lysis agent with the sample within the reaction vessel; and raising the temperature of the reaction vessel region from the first temperature to the second temperature.

[0014] In optional embodiments of this application, the process applied to nucleic acid extraction further includes: an elution process; and a step of raising the temperature of the reaction vessel region from a first temperature to a second temperature, including: maintaining the temperature of the reaction vessel region at the first temperature during the elution process; mixing the eluent with magnetic beads within the reaction vessel; and raising the temperature of the reaction vessel region from the first temperature to the second temperature.

[0015] Secondly, embodiments of the present invention also provide an analytical apparatus for performing the above-described liquid processing method.

[0016] The embodiments of the present invention bring the following beneficial effects:

[0017] The present invention provides a liquid processing method and analysis device. Through a first pipetting operation and a second pipetting operation, the liquid remaining during the first pipetting operation can be washed into the reaction vessel. By controlling the injection rate of the second pipetting operation to be less than that of the first pipetting operation, the adsorption of magnetic beads can be reduced, thereby improving the accuracy of nucleic acid detection results.

[0018] Other features and advantages of this disclosure will be set forth in the following description, or some features and advantages may be inferred from the description or determined without doubt, or may be learned by practicing the techniques described above.

[0019] To make the above-mentioned objects, features and advantages of this disclosure more apparent and understandable, preferred embodiments are described below in detail with reference to the accompanying drawings. Attached Figure Description

[0020] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 This is a first schematic diagram of a blowing and mixing method provided in an embodiment of the present invention;

[0022] Figure 2 This is a second schematic diagram of a blowing and mixing method provided in an embodiment of the present invention;

[0023] Figure 3 A third schematic diagram of a blowing and mixing method provided in an embodiment of the present invention;

[0024] Figure 4 A flowchart of a liquid treatment method provided in an embodiment of the present invention;

[0025] Figure 5 This is a first three-dimensional schematic diagram of a nucleic acid detection system provided in an embodiment of the present invention;

[0026] Figure 6 This is a second three-dimensional schematic diagram of a nucleic acid detection system provided in an embodiment of the present invention;

[0027] Figure 7 This is a third perspective view of a nucleic acid detection system provided in an embodiment of the present invention;

[0028] Figure 8 This is a fourth three-dimensional schematic diagram of a nucleic acid detection system provided in an embodiment of the present invention;

[0029] Figure 9 A flowchart of another liquid treatment method provided in an embodiment of the present invention;

[0030] Figure 10 This is a schematic diagram illustrating the movement of a tip header according to an embodiment of the present invention;

[0031] Figure 11 A schematic diagram illustrating magnetic bead adsorption according to an embodiment of the present invention;

[0032] Figure 12 A schematic diagram of a pyrolysis process provided in an embodiment of the present invention;

[0033] Figure 13 A schematic diagram illustrating the movement of a magnet, provided as an embodiment of the present invention;

[0034] Figure 14This is a schematic diagram illustrating another tip head movement provided in an embodiment of the present invention;

[0035] Figure 15 A schematic diagram of an elution process provided in an embodiment of the present invention;

[0036] Figure 16 This is a schematic diagram of the structure of an electronic device provided in an embodiment of the present invention. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] Currently, molecular detection technologies are widely used in various fields such as infectious disease diagnosis, epidemiological investigation, food hygiene inspection, early diagnosis of tumors and genetic diseases, blood screening, prenatal screening, and pharmacokinetics. It is one of the fastest-growing clinical testing technologies. Polymerase chain reaction (PCR), a molecular biology technique used to amplify specific DNA fragments, is widely used in molecular biological detection and analysis due to its high specificity, high sensitivity, simplicity, and speed. However, the cumbersome sample extraction process is a significant barrier limiting the further clinical application of PCR technology.

[0039] Currently, in automated analytical equipment, nucleic acid extraction often involves immobilizing the analyte with a reaction solution. This mixture is then brought into contact with a carrier, immobilizing the analyte on the carrier for detection. The carrier can be magnetic beads. To ensure thorough mixing of the analyte, reaction solution, and magnetic beads, a repeated aspiration method is typically used. The analyte, reaction solution, and magnetic beads are drawn into a pipette and then dispensed into the reaction container. The aspiration pump reciprocates mechanically, achieving uniform mixing and immobilizing the analyte on the magnetic beads. This method is called pipetting (see [link to relevant documentation]). Figure 1 The diagram shows a first schematic of a pipetting and mixing method, in which 800 μL of liquid is drawn into the pipette. During the dispensing process, some magnetic beads and liquid may remain at the pipette opening (see...). Figure 2 (A second schematic diagram of a mixing method shown) When the liquid in the pipette is refilled into the reaction vessel, some liquid, still containing the magnetic beads, will remain at the opening because... Figure 2 Under these conditions, when the pipette is suspended in the air, the external temperature of its location is lower than the temperature of the liquid inside the reaction vessel, and the liquid inside the pipette moves upward along the inner wall of the pipette.

[0040] See Figure 3 The third schematic diagram of a mixing method illustrates that when the pipette draws in liquid again, the liquid drawn in pushes upwards along with some gas and liquid residue from the pipette opening. The mechanical reciprocating mixing operation of the suction pump cannot remove the magnetic beads adsorbed on the inner wall of the pipette. Because the magnetic beads are partially adsorbed, the concentration of the analyte may decrease, or magnetic beads containing the analyte may be left on the inner wall of the pipette, affecting the detection results.

[0041] Based on this, the liquid processing method and analysis device provided in this embodiment of the invention can reduce the adsorption of magnetic beads and improve the accuracy of nucleic acid detection results.

[0042] To facilitate understanding of this embodiment, a liquid treatment method disclosed in this embodiment of the invention will first be described in detail. This method is used to treat liquids in a reaction vessel.

[0043] A reaction vessel is a container used for mixing liquids by blowing and stirring, including but not limited to the following containers: test tubes, amplification tubes, extraction tubes, centrifuge tubes, eight-tube sets, reaction cups, etc.

[0044] Example 1:

[0045] This invention provides a liquid treatment method, see [link to relevant documentation]. Figure 4 The flowchart shown illustrates a liquid treatment method, which includes the following steps:

[0046] Step S402: Perform multiple first pipetting operations on the liquid in the reaction vessel; wherein the first pipetting operation includes liquid injection operation and liquid aspiration operation.

[0047] The method provided in this embodiment can be applied to the analysis device of a nucleic acid detection system. The analysis device includes an incubation module and a pipetting module. The analysis device in this embodiment may include a pipetting module, an incubation module, and a control module; the control module includes a pipetting control system for controlling the pipetting module and an incubation control system for controlling the incubation module.

[0048] The method provided in this embodiment can be applied to the process of nucleic acid extraction, wherein the nucleic acid extraction process includes at least a lysis process, a washing process, and an elution process in sequence. This embodiment generally takes the lysis process and the elution process as examples.

[0049] When the analytical device uses the pipetting module for extraction, the pipetting control system controls the pipetting module to perform two pipetting operations on the liquid in the incubation module, namely the first pipetting operation and the second pipetting operation.

[0050] The pipetting module can perform multiple first-stage pipetting operations on the liquid in a reaction vessel, which can be a test tube. Each first-stage pipetting operation includes a liquid dispensing operation and a liquid aspiration operation. During these multiple first-stage pipetting operations, the pipetting module can keep the pipette stationary in a vertical direction, or it can repeatedly move the pipette up and down while keeping the pipette opening below the mineral oil surface. The dispensing and aspiration rates of the liquid in the reaction vessel can be constant or varied. The volume of liquid dispensed and aspirated by the pipetting module in the reaction vessel can also be constant or varied.

[0051] Step S404: Perform a second pipetting operation on the liquid in the reaction vessel; wherein the second pipetting operation includes a liquid injection operation and a liquid aspiration operation, and the injection rate of the second pipetting operation is less than the injection rate of the first pipetting operation.

[0052] After the reaction in the reaction vessel is complete, a second pipetting operation can be performed on the liquid inside the reaction vessel. The purpose of the second pipetting operation is to wash away any residual liquid from the first pipetting operation and bring it into the reaction vessel. Therefore, the dispensing rate of the second pipetting operation can be set to be lower than that of the first pipetting operation; specifically, the dispensing rate of the second pipetting operation can be set to be lower than that of the last first pipetting operation. During this process, any magnetic beads remaining in the pipette are carried into the liquid in the reaction vessel by the liquid. Under the action of the magnet, the magnetic beads are attracted to the inner wall of the reaction vessel. Through this operation, the number of residual magnetic beads in the pipette can be reduced, avoiding the inhibition of nucleic acid detection by the magnetic beads without subsequently replacing the pipette.

[0053] The present invention provides a liquid processing method that, through a first pipetting operation and a second pipetting operation, can wash the liquid remaining from the first pipetting operation into the reaction vessel. By controlling the dispensing speed of the second pipetting operation to be less than that of the first pipetting operation, the adsorption of magnetic beads can be reduced, thereby improving the accuracy of nucleic acid detection results.

[0054] Example 2:

[0055] This embodiment provides another liquid treatment method, which is implemented based on the above embodiment. See [link to embodiment]. Figure 5 The first three-dimensional schematic diagram of a nucleic acid detection system is shown below. Figure 6 The diagram shown is a second three-dimensional representation of a nucleic acid detection system. Figure 7 The diagram shows a third-dimensional representation of a nucleic acid detection system. Figure 8 The diagram shows a fourth three-dimensional representation of a nucleic acid detection system.

[0056] like Figures 5-8As shown, the nucleic acid detection system includes a nucleic acid extraction device and a nucleic acid detection device 8. The nucleic acid extraction device includes a frame, a pipetting module, an incubation magnetic adsorption module, and a sample injection module 1. The sample injection module 1 contains a reagent kit 3, which includes nucleic acid extraction reagents, test tubes, nucleic acid detection reagents, and a tip 61. The nucleic acid extraction reagents include a lysis solvent, mineral oil, washing solution, and elution solution. The lysis solvent contains magnetic microparticles that can adsorb the target analyte.

[0057] The pipetting module includes a pipetting track 62 fixed on a frame and a pipetting mechanism 6 that moves along the pipetting track 62. A pipetting element 61 can be installed or removed from the pipetting mechanism 6. The pipetting element referred to in this invention can be a tip or other sampling structure used for liquid aspiration and dispensing.

[0058] The incubation magnetic module includes an incubation module and a magnetic module. The incubation magnetic module includes a base plate, a fixing plate 111 fixed to one side of the base plate, a fan 11 fixedly mounted on the fixing plate 111, a motor mounting bracket 151 fixed to the base plate and located behind the fixing plate 111, a lead screw motor 15 fixed to the motor mounting bracket 151, a left support 161 and a right support 162 fixed to the base plate and located on both sides of the motor mounting bracket 151, a guide rod 163 fixed to the left support 161 and the right support 162, a linear bearing 164 slidably connected to the guide rod 163, and a central fixed connection. The components include a magnet fixing mechanism fixed to the output shaft of the lead screw motor 15 and both ends fixed to the linear bearings 164, two magnets 131 installed in the magnet fixing mechanism, a light shield 19 fixed below the magnet fixing mechanism, an incubation chamber 12 fixed on the left support 161 and the right support 162, a heating film 17 fixedly connected to one side of the incubation chamber 12, a temperature sensor 14 installed in the incubation chamber 12 to detect the temperature around the test tube, a circuit board 18 fixed to one side of the incubation chamber 12, and a photoelectric sensor 181 fixed below the circuit board that can cooperate with the light shield 19.

[0059] The incubation module includes a heating membrane 17, a temperature sensor 14 for sensing the temperature of the test tube periphery, and a fan 11 for heat dissipation. The incubation module is used to heat the liquid in the reaction vessel placed in the heating module, which can speed up the extraction of nucleic acids from the sample.

[0060] The magnetic attraction module includes a lifting mechanism and a magnet 131 mounted on the lifting mechanism. The magnetic attraction module is used to attract magnetic particles to the side of the test tube, allowing the magnetic particles to move vertically along with the magnet, and also to separate the liquid in the test tube from the magnetic particles. The magnet can move between an initial position and a maximum position. The initial position of the magnet is H0, and it moves to position H1 as needed to attract magnetic beads from the bottom of the reaction vessel to position H1. The magnet can move up to position H2, which can be used to attract magnetic beads inside the reaction vessel to position H2.

[0061] Based on the above description, see Figure 9 The flowchart shown represents another liquid treatment method, which includes the following steps:

[0062] Step S902: Raise the temperature of the reaction vessel region from a first temperature to a second temperature; wherein the second temperature is greater than the first temperature, the first temperature is not less than 25°C, the first temperature is not greater than 37°C, the second temperature is not less than 25°C, and the second temperature is not greater than 60°C.

[0063] The purpose of raising the temperature of the reaction vessel area from the first temperature to the second temperature is to accelerate the reaction rate of the liquid within the reaction vessel, such as the lysis rate or the elution rate. The nucleic acid extraction process in this embodiment can sequentially include a lysis process, a washing process, and an elution process. This embodiment will describe the process according to the order of nucleic acid extraction.

[0064] (1) Pyrolysis process: During pyrolysis, the temperature of the reaction vessel area is maintained at the first temperature; the pyrolysis agent is mixed with the sample in the reaction vessel; the temperature of the reaction vessel area is raised from the first temperature to the second temperature.

[0065] The sample to be tested is placed into reagent kit 3, and the pipetting mechanism 6 presses the tip 61 onto the pipetting mechanism 6. The incubation chamber 12 maintains the temperature of the reaction container area at approximately 37°C. The pipetting mechanism 6 mixes the nucleic acid extraction reagent and the sample in the reaction container. In this embodiment, the pipetting mechanism 6 uses a 1000 μL tip to mix 500 μL of lysis reagent with 500 μL of sample. The volumes of lysis reagent and sample can be replaced according to actual needs.

[0066] After mixing, mineral oil can be added to the surface of the lysis reagent and sample. The tip 61 of the pipetting mechanism 6 transfers the mineral oil from kit 3 to the surface of the lysis reagent and sample mixture. The added mineral oil acts as a seal, preventing the diffusion of aerosols generated during nucleic acid extraction.

[0067] Preferably, t1 = 37°C and t2 = 60°C can be determined, where t1 is the first temperature and t2 is the second temperature. The incubation module can raise the temperature from the first temperature of 37°C to the second temperature of 60°C to accelerate the pyrolysis rate, that is, to accelerate the rate at which the target analyte binds to the magnetic beads in the reaction vessel.

[0068] Step S904: Perform multiple first pipetting operations on the reaction vessel; wherein the first pipetting operation includes liquid dispensing operation and liquid aspiration operation.

[0069] In this embodiment, the sampling mechanism is the tip. The pipetting module inserts the tip 61 into the liquid in the reaction container, and keeps the opening of the tip 61 below the mineral oil. The pipetting module uses the tip to repeatedly inject and draw liquid from the reaction container for 2 minutes.

[0070] By repeatedly injecting and drawing liquid into the reaction vessel over 2 minutes, the magnetic beads remain in a disordered state within the liquid, facilitating the extraction of the target analyte. During this process, the pipetting module can either keep the tip 61 vertically stationary or move it up and down repeatedly while maintaining the tip opening below the mineral oil surface. The injection and drawing rates of the liquid into the reaction vessel can be constant or variable. The volume of liquid injected and drawn into the reaction vessel can also be constant or variable. The final injection rate is 'v', and the final volume is 'a'. The duration of this process is variable.

[0071] During the 2-minute process, the pipetting module repeatedly moved the tip up and down while keeping the tip opening below the mineral oil surface. The pipetting module maintained a constant injection and aspiration rate (v) and a constant volume (a) of liquid (a = 800 μL) within the reaction vessel. The internal liquid temperature was 60°C, and the external temperature was room temperature. After the final injection, the pipetting module completely lifted the tip out of the liquid, at which point a magnetic bead would be adsorbed inside the tip's sampling mechanism.

[0072] See Figure 10 The diagram illustrates a tip-tip movement process. First, a 1000 μL syringe tip is used to mix the liquid by blowing it onto the liquid surface. The mixing volume is 800 μL, the mixing temperature is 60°C, and the mixing speed is 380.9 μL / s. After mixing, the temperature inside the syringe is 28°C. Due to the pressure change inside the syringe, the mixture at the tip is drawn back into the syringe. Therefore, the magnetic beads remaining in the tip-tip sampling mechanism move upwards under the influence of room temperature and pressure.

[0073] Step S906: Reduce the temperature of the reaction vessel region from the second temperature to the first temperature.

[0074] After the reaction in the reaction vessel is complete, the incubation module can lower the temperature of the reaction vessel area from the second temperature to the first temperature. This cooling can be achieved using a fan. For example, activating fan 11 lowers the temperature inside the reaction vessel from 60°C to 37°C.

[0075] After the temperature in the reaction vessel area is reduced from the second temperature to the first temperature, the magnet located on the side of the reaction vessel can be raised from its initial position to the first position H1. For example, during the cooling process, the lifting mechanism in the magnetic attraction module first raises the magnet 131 from the initial position H0 to the first position H1. At this time, the magnetic beads will be concentrated and attracted to the side wall of the reaction vessel at a height of H1 under the magnetic force of the magnet.

[0076] Before performing the second pipetting operation on the reaction vessel, the magnet can be raised from the first position H1 to the second position H2. See also Figure 11 The diagram shows a magnetic bead adsorption process. The magnet attracts the magnetic beads to both sides of the reaction vessel wall. The lifting mechanism in the magnetic adsorption module then raises the magnet from the first position H1 to the second position H2. At this time, the magnetic beads will be concentrated and adsorbed on the side wall of the reaction vessel at the height of H2 under the magnetic force of the magnet, and the magnetic beads will detach from the liquid in the reaction vessel.

[0077] Step S908: Perform a second pipetting operation on the reaction vessel; wherein the second pipetting operation includes a liquid dispensing operation and a liquid aspiration operation, and the dispensing rate of the second pipetting operation is less than the dispensing rate of the first pipetting operation.

[0078] At this time, the temperature inside the reaction vessel is maintained at 37°C. The pipetting module uses the tip to perform one suction and one injection of liquid in the reaction vessel. During this process, the speed at which the pipetting module injects and suctions liquid in the reaction vessel is constant, and the volume of liquid injected and suctioned by the pipetting module in the reaction vessel is constant.

[0079] In this embodiment, the ratio of the injection rate of the second pipetting operation to the injection rate of the first pipetting operation is not greater than 0.75, and the ratio of the injection volume of the second pipetting operation to the injection volume of the first pipetting operation is related to the ratio of the second temperature to the first temperature.

[0080] For example, the injection rate and volume of the second pipetting operation can be determined by the following formulas: v1 / v≤0.75; a1 / a≥(273.15+t1) / (273.15+t2); where v is the injection rate of the last first pipetting operation, a is the volume of the last first pipetting operation, v1 is the injection rate of the second pipetting operation, and a1 is the volume of the second pipetting operation.

[0081] Preferably, the ratio of the injection rate of the second pipetting operation to the injection rate of the first pipetting operation is not greater than 0.5, i.e., v1 / v ≤ 0.5; the ratio of the injection volume of the second pipetting operation to the injection volume of the first pipetting operation is not less than 1.07, i.e., a1 / a ≥ 1.07. Optimally, v1 / v = 0.5, a1 / a = 1.07.

[0082] During this process, the magnetic beads remaining inside the tip are carried into the liquid in the reaction vessel by the liquid. Under the action of the magnet, the magnetic beads are adsorbed and transferred to the inner wall of the reaction vessel.

[0083] After the second pipetting operation on the reaction vessel, the magnet can be lowered from the second position H2 to the first position H1. For example, the magnet is lowered from the second position H2 to the first position H1 by the lifting mechanism, moving the magnetic bead back to the bottom of the reaction vessel. The pipetting module then detaches the used tip. The pyrolysis operation is now complete.

[0084] See Figure 12 The diagram illustrates a pyrolysis process. During repeated injection and aspiration of liquid by the pipetting module, the vertical movement of the tip can be selected based on the liquid volume in the reaction vessel. When the liquid volume is less than or equal to a threshold, the tip does not need to move vertically. When the liquid volume exceeds the threshold, the tip needs to move vertically. For example, it determines whether the liquid volume in the reaction vessel is greater than a preset threshold; if so, it controls the sampling mechanism to move continuously in the vertical direction; if not, it controls the sampling mechanism to remain stationary in the vertical direction.

[0085] (2) Washing process: Add 500 μL of washing reagent to the reaction vessel. Use the tip of the pipetting module to repeatedly aspirate to ensure the washing reagent and magnetic beads react fully for 60 seconds at 37°C. This further washes away impurities on the surface of the magnetic beads, resulting in higher purity nucleic acid. After mixing, since no heating is applied during the process, pressure changes are not considered when cleaning the tip. Move the magnet to the bottom of the reaction vessel, adsorbing the magnetic beads onto the vessel wall where the magnet is located. Then, move the magnet to position H2 to adsorb all the magnetic beads together. After approximately 30 seconds, move the tip to the bottom of the reaction vessel to remove the waste liquid containing impurities. Move the magnet down to position H1 to pull the magnetic beads to the bottom of the tube, completing the first wash. In practical applications, to obtain high-purity nucleic acid, a second wash is often performed. The process of the second wash is the same as the first wash and will not be repeated. The tip can be replaced during the washing and elution process, or it can remain unchanged.

[0086] (3) Elution process: During the elution process, the temperature of the reaction vessel area is maintained at the first temperature; the eluent and magnetic beads are mixed in the reaction vessel; the temperature of the reaction vessel area is raised from the first temperature to the second temperature.

[0087] See Figure 13The diagram illustrates the movement of a magnet. After the needle is cleaned, a lifting mechanism lowers the magnet from position H1 to H0. During the washing process, a new tip 61 is pressed and installed onto the pipetting mechanism 6. In this embodiment, the tip is 200 μL. The incubation module maintains the temperature of the reaction vessel area at approximately 37°C. The pipetting mechanism 6 mixes the eluent with the magnetic beads within the reaction vessel. In this embodiment, the pipetting mechanism 6 uses a 200 μL tip to mix 60 μL of eluent with the magnetic beads, but the volume of eluent can be adjusted according to actual conditions. The incubation module raises the temperature from 37°C to 60°C to accelerate the detachment of the target analyte from the magnetic beads.

[0088] The pipetting module inserts the tip into the liquid in the reaction vessel, keeping the tip opening below the liquid surface and maintaining a vertical position. The module repeatedly injects and aspirates liquid from the reaction vessel using the tip for 1 minute. This continuous injection and aspiration keeps the magnetic beads in a disordered state within the liquid, facilitating the elution of the target analyte. The injection and aspiration rates can be constant or varied. The volume of liquid injected and aspirated can also be constant or varied. The final injection rate is v2, and the final volume is a2. The duration of this process is variable.

[0089] During the 1-minute process in this embodiment, the liquid injection and aspiration rates of the pipetting module in the reaction vessel are constant, both v2, and the liquid injection and aspiration volumes are constant, both a2, where a = 48 μL. The internal temperature of the liquid is 60°C, and the external temperature is room temperature. After the last liquid injection, the pipetting module raises the tip completely out of the liquid, at which point a magnetic bead will be adsorbed inside the tip.

[0090] Fan 11 is activated, causing the temperature inside the reaction vessel to drop from 60°C to 37°C. During the cooling process, the lifting mechanism 132 in the magnetic attraction module first raises the magnet 131 from its initial position H0 to H1. At this point, the magnetic beads are concentrated and attracted to the side wall of the reaction vessel at height H1 under the magnetic force of the magnet. Then, the lifting mechanism 132 in the magnetic attraction module raises the magnet 131 from H1 to H2. At this point, the magnetic beads are concentrated and attracted to the side wall of the reaction vessel at height H2 under the magnetic force of the magnet 131, and the magnetic beads detach from the liquid inside the reaction vessel.

[0091] See Figure 14The diagram shows another type of tip movement, in which the tip follows the liquid surface to aspirate and expel twice, with a liquid volume of 856 μL, an aspiration and expulsion temperature of 37°C, and an aspiration and expulsion rate of 190 μL / s. The needle tip mixture is rinsed into the reaction vessel, and after the needle is cleaned, the liquid is aspirated, leaving magnetic beads with adsorbed nucleic acids in the tube.

[0092] At this point, the temperature inside the reaction vessel is maintained at 37°C. The pipetting module uses the tip to perform one aspiration and dispensing operation on the liquid inside the reaction vessel. During this process, the dispensing and aspiration rates of the liquid are constant, v3, where v3 = 0.5v2; the volume of liquid dispensed and aspirated is also constant, a3, where a3 = 1.07a2. During this process, the magnetic beads remaining in the tip are carried into the liquid in the reaction vessel and, under the influence of the magnet, are attracted to the inner wall of the reaction vessel. This operation reduces the number of magnetic beads remaining in the tip, preventing their inhibition of nucleic acid detection if the tip is not replaced subsequently.

[0093] Under the action of the lifting mechanism, magnet 131 descends from the second position H2 to the first position H1, moving the magnetic bead back to the bottom of the reaction vessel. Then, under the action of the lifting mechanism, the magnet descends from position H1 to position H0. At this point, the elution operation is complete.

[0094] See Figure 15 The diagram illustrates an elution process where, during repeated dispensing and aspiration of liquid in the pipetting module, the tip can be positioned vertically based on the liquid volume in the reaction vessel. When the liquid volume is less than or equal to a threshold, the tip will not move vertically. When the liquid volume exceeds the threshold, the tip will move vertically.

[0095] This embodiment also provides a method for calculating the magnetic bead retrieval rate, for example: determining the initial magnetic bead mass of the reaction vessel; injecting the pyrolysis agent into the reaction vessel after pyrolysis and after elution respectively, performing a freeze-drying operation on the liquid in the reaction vessel, and obtaining the remaining magnetic bead mass after the pyrolysis process and the remaining magnetic bead mass after the elution process respectively; calculating the magnetic bead retrieval rate after pyrolysis based on the initial magnetic bead mass and the remaining magnetic bead mass after the pyrolysis process; calculating the magnetic bead retrieval rate after elution based on the initial magnetic bead mass and the remaining magnetic bead mass after the elution process.

[0096] Multiple 500 μL lysis reagents were injected into several reaction vessels. One reaction vessel was then used for lyophilization of the reagent. Only magnetic beads and a very small amount of reagent residue remained in the reaction vessel; the reagent weight was much smaller than the magnetic bead weight and therefore negligible. The mass of the magnetic beads in each reaction vessel was determined to be m. Using the lysis and elution method described above, the magnetic beads after lysis and elution were weighed. During this process, the mass of the analyte on the magnetic beads during lysis was much smaller than the mass of the magnetic beads and therefore negligible. The mass of the lysed magnetic beads was determined to be m1, and the mass of the eluted magnetic beads was determined to be m2. The values ​​of m1 / m and m2 / m were calculated to obtain the recovery rates of the lysed and eluted magnetic beads, respectively.

[0097] Furthermore, this embodiment also provides a test method for lysis conditions. Multiple reaction vessels injected with 500 μL of lysis reagent were subjected to the lysis process described in Table 1 below, and the magnetic bead retrieval rate was calculated after lysis. The sample types and volumes used were identical. The results showed that, under the conventional conditions of v1 / v = 1 and a1 / a = 1, the magnetic bead retrieval rate after lysis was 85%, which was not high enough. To achieve a 90% retrieval rate, v1 / v = 0.75 and a1 / a = 1.03. With v1 / v = 0.125-0.5 and a1 / a = 1.07-1.187, the magnetic bead retrieval rate after lysis reached 98%, achieving an unexpected technical effect. Observations during the experiment indicated that the repeated injection and aspiration of liquid from the reaction vessel by the pipetting module during the lysis process carried away some magnetic beads, resulting in a 2% magnetic bead loss. Considering the time required, the slower the second pipetting speed and the larger the volume of liquid pipetted, the longer the time required. Therefore, v1 / v = 0.5 and a1 / a = 1.07 are the optimal conditions.

[0098] 1 1 1 85％ 2 0.9 1 86％ 3 0.8 1.02 87％ 4 0.75 1.03 90％ 5 0.7 1.04 91％ 6 0.6 1.05 92％ 7 0.5 1.07 98％ 8 0.375 1.09 98％ 9 0.25 1.136 98％ 10 0.125 1.187 98％

[0099] Table 1

[0100] Furthermore, this embodiment also provides a test method for elution conditions. Multiple reaction vessels injected with 60 μL of lysis reagent were eluted according to the conditions in Table 2 below, and the magnetic bead retrieval rate was calculated after elution. The sample types and volumes used were the same. Results showed that, under the conventional conditions of v3 / v2 = 1 and a1 / a = 1, the magnetic bead retrieval rate after elution was 86%. To achieve a retrieval rate of 90%, v3 / v2 = 0.75 and a3 / a2 = 1.02 were used. Based on v3 / v2 = 0.125-0.5 and a3 / a2 = 1.07-1.187, the magnetic bead retrieval rate after elution reached 98%, achieving an unexpected technical effect. Observations during the experiment indicated that during the lysis process, the repeated injection and aspiration of liquid from the reaction vessel by the pipetting module carried away some magnetic beads, resulting in a 2% magnetic bead loss. Considering the time required, the slower the second pipetting speed and the larger the volume of liquid pipetted, the longer the time required. Therefore, v3 / v2 = 0.5 and a3 / a2 = 1.07 are the optimal conditions.

[0101]

[0102]

[0103] Table 1

[0104] During the elution condition test, it was found that when liquid was continuously injected and aspirated from the reaction vessel for 1 minute, when v3 reached 380.95 μL / s, the liquid in the reaction vessel splashed, and the magnetic beads adhered to the inner wall of the reaction vessel, which was not conducive to the collection of the target analyte. When v3 was 38.10 μL / s, the mixing effect of the liquid in the reaction vessel was poor, and the target analyte was difficult to detach from the magnetic beads, which was also not conducive to the collection of the target analyte. When v3 was between 142.86 μL / s and 238.10 μL / s, the mixing effect of the liquid in the reaction vessel was good, which was conducive to the collection of the target analyte.

[0105] Example 3:

[0106] This embodiment provides an analytical apparatus for performing the above-described liquid processing method.

[0107] Those skilled in the art will clearly understand that, for the sake of convenience and brevity, the specific working process of the analytical device described above can be referred to the corresponding process in the foregoing embodiments, and will not be repeated here.

[0108] Example 4:

[0109] This invention also provides an electronic device for operating the above-described liquid processing method; see [link to previous document]. Figure 16The diagram shows the structure of an electronic device, which includes a memory 100 and a processor 101. The memory 100 is used to store one or more computer instructions, which are executed by the processor 101 to implement the liquid processing method described above.

[0110] Furthermore, Figure 16 The electronic device shown also includes a bus 102 and a communication interface 103, with the processor 101, the communication interface 103 and the memory 100 connected via the bus 102.

[0111] The memory 100 may include high-speed random access memory (RAM) or non-volatile memory, such as at least one disk storage device. Communication between this system network element and at least one other network element is achieved through at least one communication interface 103 (which can be wired or wireless), such as the Internet, wide area network, local area network, or metropolitan area network. The bus 102 may be an ISA bus, PCI bus, or EISA bus, etc. The bus can be divided into address bus, data bus, control bus, etc. For ease of representation, Figure 16 The symbol is represented by a single double-headed arrow, but this does not mean that there is only one bus or one type of bus.

[0112] Processor 101 may be an integrated circuit chip with signal processing capabilities. In implementation, each step of the above method can be completed by the integrated logic circuitry in the hardware of processor 101 or by instructions in software form. Processor 101 can be a general-purpose processor, including a Central Processing Unit (CPU), a Network Processor (NP), etc.; it can also be a Digital Signal Processor (DSP), an Application Specific Integrated Circuit (ASIC), a Field-Programmable Gate Array (FPGA), or other programmable logic devices, discrete gate or transistor logic devices, or discrete hardware components. It can implement or execute the methods, steps, and logic block diagrams disclosed in the embodiments of this invention. The general-purpose processor can be a microprocessor or any conventional processor. The steps of the methods disclosed in the embodiments of this invention can be directly manifested as execution by a hardware decoding processor, or execution by a combination of hardware and software modules in the decoding processor. The software module can reside in a readily available storage medium in the art, such as random access memory, flash memory, read-only memory, programmable read-only memory, electrically erasable programmable memory, or registers. This storage medium is located in memory 100, and processor 101 reads information from memory 100 and, in conjunction with its hardware, completes the steps of the method described in the foregoing embodiments.

[0113] This invention also provides a computer-readable storage medium storing computer-executable instructions. When these computer-executable instructions are invoked and executed by a processor, they cause the processor to implement the aforementioned liquid processing method. For specific implementation details, please refer to the method embodiments, which will not be repeated here.

[0114] The computer program product of the liquid processing method and analysis device provided in the embodiments of the present invention includes a computer-readable storage medium storing program code. The instructions included in the program code can be used to execute the methods in the preceding method embodiments. For specific implementation, please refer to the method embodiments, which will not be repeated here.

[0115] Those skilled in the art will clearly understand that, for the sake of convenience and brevity, the specific working process of the system and / or device described above can be referred to the corresponding process in the foregoing method embodiments, and will not be repeated here.

[0116] Furthermore, in the description of the embodiments of the present invention, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in the present invention based on the specific circumstances.

[0117] If the aforementioned functions are implemented as software functional units and sold or used as independent products, they can be stored in a computer-readable storage medium. Based on this understanding, the technical solution of this invention, essentially, or the part that contributes to the prior art, or a portion of the technical solution, can be embodied in the form of a software product. This computer software product is stored in a storage medium and includes several instructions to cause a computer device (which may be a personal computer, server, or network device, etc.) to execute all or part of the steps of the methods described in the various embodiments of this invention. The aforementioned storage medium includes various media capable of storing program code, such as USB flash drives, portable hard drives, read-only memory (ROM), random access memory (RAM), magnetic disks, or optical disks.

[0118] In the description of this invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0119] Finally, it should be noted that the above-described embodiments are merely specific implementations of the present invention, used to illustrate the technical solutions of the present invention, and not to limit it. The scope of protection of the present invention is not limited thereto. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any person skilled in the art can still modify or easily conceive of changes to the technical solutions described in the foregoing embodiments within the technical scope disclosed in the present invention, or make equivalent substitutions for some of the technical features; and these modifications, changes, or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A liquid treatment method for treating liquid within a reaction vessel, characterized in that, The method includes: Perform multiple first pipetting operations on the liquid in the reaction vessel; wherein the first pipetting operation includes liquid injection and liquid aspiration. A second pipetting operation is performed on the liquid in the reaction vessel; wherein the second pipetting operation includes a liquid injection operation and a liquid aspiration operation, the injection rate of the second pipetting operation is less than the injection rate of the first pipetting operation, and the volume of liquid injected in the second pipetting operation is greater than the volume of liquid injected in the first pipetting operation. The liquid processing method is applied to a nucleic acid extraction process using magnetic beads. The nucleic acid extraction process includes a lysis process, a washing process, and an elution process. During the lysis process and the elution process, a first pipetting operation and a second pipetting operation are performed by a pipetting module. The pipetting module includes a pipetting track fixed on a frame and a pipetting mechanism that moves along the pipetting track. The pipetting mechanism installs or removes a pipetting element, which is a tip. Before performing multiple first pipetting operations on the liquid in the reaction vessel, the method further includes: raising the temperature of the reaction vessel region from a first temperature to a second temperature; wherein the second temperature is greater than the first temperature, the first temperature is not less than 25°C, the first temperature is not greater than 37°C, the second temperature is not less than 25°C, and the second temperature is not greater than 60°C; before performing a second pipetting operation on the liquid in the reaction vessel, the method further includes: lowering the temperature of the reaction vessel region from the second temperature to the first temperature. The ratio of the injection rate of the second pipetting operation to the injection rate of the first pipetting operation is not greater than 0.5, and the ratio of the injection volume of the second pipetting operation to the injection volume of the first pipetting operation is not less than 1.07; the ratio of the injection volume of the second pipetting operation to the injection volume of the first pipetting operation is related to the ratio of the second temperature to the first temperature. After the step of reducing the temperature of the reaction vessel region from the second temperature to the first temperature, the method further includes: raising a magnet disposed on the side of the reaction vessel from an initial position to a first position; before the step of performing a second liquid transfer operation on the liquid in the reaction vessel, the method further includes: raising the magnet from the first position to a second position; after the step of performing a second liquid transfer operation on the liquid in the reaction vessel, the method further includes: lowering the magnet from the second position to the first position.

2. The method according to claim 1, characterized in that, The steps of performing multiple first pipetting operations on the liquid in the reaction vessel include: Determine whether the liquid volume in the reaction vessel is greater than a preset threshold; if yes, control the sampling mechanism to move continuously in the vertical direction; if no, control the sampling mechanism to remain stationary in the vertical direction.

3. The method according to claim 1, characterized in that, The first temperature is 37°C, and the second temperature is 60°C.

4. The method according to claim 1, characterized in that, The step of raising the temperature of the reaction vessel region from a first temperature to a second temperature includes: During the pyrolysis process, the temperature of the reaction vessel region is maintained at a first temperature; the pyrolysis agent is mixed with the sample inside the reaction vessel; and the temperature of the reaction vessel region is raised from the first temperature to a second temperature.

5. The method according to claim 4, characterized in that, The step of raising the temperature of the reaction vessel region from a first temperature to a second temperature includes: During the elution process, the temperature of the reaction vessel region is maintained at a first temperature; the eluent is mixed with the magnetic beads inside the reaction vessel; and the temperature of the reaction vessel region is raised from the first temperature to a second temperature.

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