Trace meter and dynamic ultra-micro pipetting device calibration method

Abstract

A trace meter and a dynamic ultra-micro pipetting device calibration method. The trace meter comprises a measurement chamber (1), a temperature sensor (2), a pressure sensor (3) and a controller (4); the top of the measurement chamber (1) is open; the temperature sensor (2) is arranged at the bottom of the measurement chamber (1); the pressure sensor (3) is arranged below the measurement chamber (1); the temperature sensor (2) and the pressure sensor (3) are both electrically connected to the controller (4); the temperature sensor (2) is used for measuring the temperature of a liquid in the measurement chamber (1); the pressure sensor (3) is used for measuring the weight of the liquid in the measurement chamber (1). The accuracy of ultra-micro dynamic pipetting of a high-throughput nucleic acid extraction device can be conveniently measured and calibrated, so as to improve the metering accuracy of ultra-micro pipetting of the high-throughput nucleic acid extraction device.

Description

A trace meter and a dynamic ultra-micro pipetting device calibration method Technical Field

[0001] The present invention relates to the technical field of dynamic pipetting precision calibration, and in particular to a trace meter and a dynamic ultra-micro pipetting equipment calibration method. Background Art

[0002] A dynamic pipetting device, or automated liquid handling system with high-throughput dynamic pipetting capabilities, is an automated device based on the principles of liquid displacement or gas displacement pipetting, performing liquid handling tasks such as pipetting through robotic operation. Automated pipetting systems are primarily used in biology and clinical medicine, often in processes such as RNA purification, microRNA purification, viral nucleic acid purification, DNA / RNA recovery, and protein purification, primarily responsible for the aliquoting and transfer of samples or reagents. High-throughput dynamic pipetting devices are often used in pre-processing for high-throughput sequencing. High-throughput sequencing technology, also known as "next-generation sequencing" or "massively parallel sequencing," is a technique that, compared to conventional dideoxy sequencing, can perform parallel sequencing on a large number of nucleic acid molecules at once, typically generating at least 100Mb of sequencing data per reaction. A trace amount refers to an extremely small amount, so small that it is barely noticeable. In applied science, a substance present at a concentration below one part per million is considered a trace amount. Trace amounts are also used in chemistry, materials science, and biomedicine.

[0003] A dynamic pipetting device is a device that extracts and purifies components such as nucleic acids by transferring reaction solutions. It usually includes multiple pipetting heads. A single pipetting head is used to quantitatively transfer liquid, while multiple pipetting heads are used to achieve high-throughput extraction of liquid.

[0004] Regarding the above-mentioned related technologies, the applicant believes that in the related technologies, the pipetting accuracy of the pipetting head is affected due to human operation and environmental factors during long-term use, especially the inability to accurately measure ultra-micro dynamic pipetting. The conventional measurement and calibration equipment is not accurate enough in measuring and calibrating the accuracy of ultra-micro dynamic pipetting, resulting in excessive differences between the channels of the pipetting equipment, which in turn affects the measurement results of multiple samples and is not convenient for large-scale application of small samples by the pipetting equipment.

[0005] Summary of the Invention

[0006] The present invention aims to provide a trace meter and a dynamic ultra-micro pipetting device calibration method, which facilitates the measurement and calibration of the accuracy of ultra-micro dynamic pipetting of high-throughput nucleic acid extraction equipment, so as to improve the measurement accuracy of ultra-micro pipetting of high-throughput nucleic acid extraction equipment.

[0007] The present invention is achieved through the following technical solutions:

[0008] The present invention provides a trace meter, comprising a measuring chamber, a temperature sensor, a pressure sensor and a controller. The measuring chamber is provided with an opening at the top, the temperature sensor is provided at the bottom of the measuring chamber, and the pressure sensor is provided below the measuring chamber. The temperature sensor and the pressure sensor are both electrically connected to the controller. The temperature sensor is used to measure the temperature of the liquid inside the measuring chamber, and the pressure sensor is used to weigh the liquid inside the measuring chamber.

[0009] Furthermore, the inner bottom wall of the measuring chamber is provided with a lowest inclination point, and the lowest inclination point is located directly below the center of gravity of the measuring chamber. A measuring hole is opened at the lowest inclination point of the bottom wall of the measuring chamber, and the side of the measuring hole close to the interior of the measuring chamber is covered with a flexible thermally conductive film. The sensing end of the temperature sensor is placed in the measuring hole and abuts against the lower surface of the flexible thermally conductive film.

[0010] Furthermore, an anti-steam screen is provided on the upper cover of the measuring chamber, and a through hole for the pipetting head to pass through is provided on the anti-steam screen.

[0011] Furthermore, it also includes a bottom shell arranged below the measuring chamber, the pressure sensor is arranged in the bottom shell, a wind shield is arranged on the bottom shell, the wind shield is used to cover the measuring chamber, a drip hole for the pipette head to pass through is provided on the top wall of the wind shield, a protective plate is provided above the wind shield, a passage hole is provided on the protective plate, and the apertures of the through hole and the drip hole are larger than the aperture of the passage hole, the center lines of the drip hole, the through hole and the measuring hole coincide with each other, and gaps are left between the measuring chamber and the anti-steam screen and the wind shield respectively.

[0012] Furthermore, a weighing plate is provided below the measuring bin, the pressure sensor is located below the weighing plate, a positioning groove is provided on the weighing plate, the positioning groove includes an inwardly concave conical surface and a horizontal inner bottom wall, and a positioning portion is provided on the outer bottom wall of the measuring bin, the shape of the positioning portion matches the positioning groove, the positioning portion is located in the positioning groove, and the measuring hole of the measuring bin is located directly above the inner bottom wall of the positioning groove.

[0013] The present invention also provides a method for calibrating a dynamic ultra-micro pipetting device, which uses the trace meter described above to calibrate the dynamic ultra-micro pipetting device, comprising the following steps:

[0014] S1: Move the pipetting head to the target position;

[0015] S2: Move the tracer so that the dripping hole is directly below the pipetting head;

[0016] S3: Place the standard liquid at the position to be taken;

[0017] S4: Clear the trace instrument measurement data;

[0018] S5: Operate the same pipetting head to dynamically pipette liquid into the measuring chamber n times, and read the temperature data and weighing data of the liquid after each pipetting, where n is the total number of pipetting times in a single channel;

[0019] S6: The controller obtains n sets of measurement data of the dynamic pipetting process of a single channel, and performs summing, correction and single-channel pipetting consistency calculation on the measurement data;

[0020] S7: remove the standard liquid in the measuring chamber;

[0021] S8: Repeat steps S1 to S7 to measure the next channel;

[0022] S9: After measuring all channels, calculate the total standard deviation of dynamic high-throughput pipetting.

[0023] Furthermore, in step S6, the controller calculates the volume of the liquid in the measuring chamber, and the liquid volume V x is the volume of liquid after each pipetting of a single channel, m x is the mass of the measured liquid, and t is the temperature of the measured liquid.

[0024] Furthermore, in step S6, the controller controls the liquid volume V x Make correction, volume correction value V c =a+b×V x , the corrected volume measurement result V=V c +V x , V is the final measurement result of the liquid volume after the current pipetting, a and b are the correction coefficients for periodic calibration management, and are preset in the controller after measurement and calibration by the upper-level calibration unit.

[0025] Furthermore, in step S6, the single channel pipetting consistency V i is the volume of the i-th pipetting in the current channel, i=1, 2, 3,...n.

[0026] Furthermore, in step S9, the total standard deviation of high-throughput pipetting V ji is the volume of the i-th pipetting of the j-th channel, i = 1, 2, 3, ... n, j = 1, 2, 3, ... m, m is the number of channels being measured, is the mean volume of each pipetting,

[0027] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0028] 1. Multiple pipettings are performed into the measuring chamber through a single channel, and the temperature, mass, and volume of each pipetting are measured and calculated simultaneously, thereby calculating the single-channel pipetting consistency and judging the single-channel metering accuracy. The smaller the single-channel pipetting consistency value, the higher the single-channel pipetting metering accuracy; conversely, the lower the single-channel pipetting metering accuracy. The trace meter structure and measurement method of the present invention can reduce the evaporation and loss of liquid during the pipetting measurement process, which is beneficial to improving the measurement accuracy of ultra-micro pipetting of the pipetting device, thereby facilitating the calibration of the dynamic ultra-micro pipetting accuracy of the pipetting device. The trace meter of the present invention can directly output the volume value of high-precision pipetting, which is convenient for use.

[0029] 2. After performing multiple pipetting measurements on all channels, calculate the total standard deviation of the high-throughput pipetting to facilitate the judgment of the overall pipetting accuracy of the high-throughput pipetting device. The smaller the total standard deviation, the higher the overall pipetting accuracy of the high-throughput pipetting device. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, constitute a part of this application, and do not constitute a limitation of the embodiments of the present invention.

[0031] In the attached figure:

[0032] FIG1 is a schematic structural diagram of embodiment 1 of the present invention;

[0033] FIG2 is a cross-sectional view of a local structure of Example 1 of the present invention;

[0034] FIG3 is a temperature acquisition circuit diagram of Example 1 of the present invention;

[0035] FIG4 is a weighing circuit diagram of Example 1 of the present invention.

[0036] The marks and corresponding component names in the accompanying drawings are: 1. Measuring chamber; 2. Temperature sensor; 3. Pressure sensor; 4. Controller; 5. Measuring hole; 6. Flexible thermal conductive film; 7. Anti-steam screen; 8. Through hole; 9. Bottom shell; 10. Wind shield; 11. Drip hole; 12. Protection plate; 13. Passage hole; 14. Weighing plate; 15. Positioning slot; 16. Pipette head; 17. Host computer; 18. Analog-to-digital converter; 19. Microcontroller unit; 20. External interface; 21. Power module. DETAILED DESCRIPTION

[0037] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the examples and accompanying drawings. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and are not intended to limit the present invention. It should be noted that the present invention is already in the actual development and use stage.

[0038] Example 1

[0039] In this embodiment, a trace meter is provided, referring to Figures 1 and 2, comprising a measuring chamber 1, a temperature sensor 2, a pressure sensor 3 and a controller 4. The measuring chamber 1 is provided with an opening at the top, the temperature sensor 2 is provided at the bottom of the measuring chamber 1, and the pressure sensor 3 is provided below the measuring chamber 1. The temperature sensor 2 and the pressure sensor 3 are both electrically connected to the controller 4. The temperature sensor 2 is used to measure the temperature of the liquid inside the measuring chamber 1, and the pressure sensor 3 is used to measure the weight of the liquid inside the measuring chamber 1. Multiple pipettings are performed in the measuring chamber through a single channel, and the temperature, mass and volume of each pipetting are measured and calculated at the same time. Then, the single-channel pipetting consistency is calculated by the controller 4 to judge the metering accuracy of the single channel. The smaller the single-channel pipetting consistency value, the higher the pipetting metering accuracy of the single channel. Conversely, the lower the pipetting metering accuracy of the single channel, the total standard deviation of the high-throughput pipetting can also be calculated to facilitate judgment and calibration of the overall pipetting accuracy of the high-throughput pipetting device. The smaller the total standard deviation, the higher the overall pipetting accuracy of the high-throughput pipetting device.

[0040] As a preferred embodiment, referring to Figures 1 and 2, the inner bottom wall of the measuring chamber 1 is provided with an inclined lowest point, the inner bottom wall of the measuring chamber 1 is a concave cone, and the inclined lowest point is located directly below the center of gravity of the measuring chamber 1, and a through measuring hole 5 is opened at the inclined lowest point of the bottom wall of the measuring chamber 1. The side of the measuring hole 5 close to the interior of the measuring chamber 1 is covered with a flexible thermally conductive film 6, the sensing end of the temperature sensor 2 is placed in the measuring hole 5 and is tightly pressed against the lower surface of the flexible thermally conductive film 6 (the side of the flexible thermally conductive film 6 facing away from the interior of the measuring chamber 1). After adding liquid to the measuring chamber 1, the liquid gathers at the lowest point inside the measuring chamber 1, which is conducive to maintaining the balance and stability of the measuring chamber 1; the temperature sensor 2 adopts a thermistor temperature sensor, and the sensing part of the temperature sensor 2 measures the liquid in the measuring chamber 1 through the flexible thermally conductive film 6, and has sensitive response and good stability.

[0041] As a preferred embodiment, referring to Figures 1 and 2, a removable cover above the measuring chamber 1 is provided with an anti-steam screen 7. The anti-steam screen 7 and the measuring chamber 1 are both made of ultra-light and low thermal conductivity materials. The ultra-light and low thermal conductivity materials can be aerogel materials, vacuum insulation materials, graphite polystyrene materials and PFT insulation materials, etc. A through hole 8 for the pipetting head 16 to pass through is provided on the anti-steam screen 7. The operating pipetting head 16 extends through the through hole 8 of the anti-steam screen 7 into the measuring chamber 1 to add liquid. The anti-steam screen 7 is beneficial to reducing the evaporation and loss of liquid in the measuring chamber 1, which is of greater significance for the precision measurement of ultra-micro pipetting.

[0042] As a preferred embodiment, referring to Figures 1 and 2, the tracer also includes a bottom shell 9 arranged below the measuring chamber 1, the pressure sensor 3 is arranged in the bottom shell 9, and a windshield 10 is provided on the bottom shell 9 through a snap fastener, which is convenient for disassembling the windshield 10. The windshield 10 is used to cover the measuring chamber 1, and a drip hole 11 for the pipette head 16 to pass through is provided on the top wall of the windshield 10. There is a gap between the measuring chamber 1 and the anti-steam screen 7 and the windshield 10 respectively. The windshield 10 is conducive to reducing the shaking effect of the flow of external air on the measuring chamber 1, which is conducive to ensuring the accuracy of the liquid weight measurement of the measuring chamber 1. The outer top wall of the windshield 10 is also provided with a protective plate 12 through a snap fastener, and the protective plate 12 is used in conjunction with the anti-steam screen 7. A through hole 13 is provided on the protective plate 12 for the pipetting head 16 to pass through. The center lines of the through hole 13, the dripping hole 11, the through hole 8 and the measuring hole 5 are all vertically arranged and coincide with each other. The aperture of the through hole 13 is smaller than the aperture of the through hole 8, and the aperture of the through hole 13 is smaller than the aperture of the dripping hole 11, so as to prevent the pipetting head 16 from touching the anti-steam screen 7 and the windshield 10 after extending into the measuring chamber 1, and facilitate the pipetting head 16 to pass through the through hole 13, the dripping hole 11 and the through hole 8 in turn and directly drop the liquid on the measuring hole 5 of the measuring chamber 1, which is beneficial to maintaining the balance and stability of the measuring chamber 1 and to ensuring the accuracy of the liquid weight data measured by the pressure sensor 3.

[0043] As a preferred embodiment, referring to Figures 1 and 2, a weighing pan 14 is provided below the measuring chamber 1, and the pressure sensor 3 is provided in the middle position just below the weighing pan 14. A positioning groove 15 is provided on the upper surface of the weighing pan 14. The positioning groove 15 is concave inwardly conical. The positioning groove 15 includes a horizontally arranged inner bottom wall and a concave conical surface. The outer bottom wall of the measuring chamber 1 is provided with a positioning portion. The positioning portion is an outwardly convex conical structure, and the positioning portion matches the shape of the positioning groove 15 (that is, the positioning portion includes a convex conical surface and a horizontal bottom surface located below the conical surface). The positioning portion is located in the positioning groove 15. Inside, the positioning part is used to position the measuring chamber 1 on the weighing pan 14, so as to facilitate the rapid and accurate placement of the measuring chamber 1 at the designated position on the weighing pan 14 and ensure the balance of the measuring chamber 1; the measuring hole 5 is located at the horizontal bottom surface of the positioning part, that is, the measuring hole 5 is located directly above the horizontal inner bottom wall of the positioning groove 15. At this time, the sensing end of the temperature sensor 2 is located at the inner bottom wall of the positioning groove 15, so that the center of gravity of the measuring chamber 1 is located directly above the inner bottom wall of the positioning groove 15, which is beneficial to keep the balance and stability of the measuring chamber 1 at all times while adding liquid into the measuring chamber 1.

[0044] 1 and 2 , the housing includes two analog-to-digital converters 18 (ADCs), a microcontroller unit 19 (MCU), and a power supply module 21. One ADC 18 is electrically connected to the temperature sensor 2 via a temperature acquisition circuit, and the other is electrically connected to the pressure sensor 3 via a weighing circuit. Both ADCs 18 are electrically connected to the MCU 19, which reads the temperature data measured by the temperature sensor 2 and the liquid weight data measured by the pressure sensor 3 via the corresponding ADC 18. The housing also includes an external interface 20. The controller 4 transmits power to the external interface 20 via a cable. The power supply module 21 receives input power from the external interface 20 and modulates the power into a reference power supply AVDD, a highly stable differential power supply VCC+, and VCC-. After the system is started, the controller 4 transmits a 5V voltage to the external interface 20 via a connecting cable. The power supply module 21 adjusts and stabilizes the voltage and then supplies power to other modules. After the MCU 19 is powered on, it initializes the peripherals and performs a system self-test. After the self-test is completed, the system enters the startup state.

[0045] 2 and 3 , the temperature acquisition circuit includes a thermistor NTC, a first resistor R1, a second resistor R2, and a capacitor C4. The second resistor R2, the first resistor R1, and the capacitor C4 are connected in series in sequence. The end of the second resistor R2 away from the first resistor R1 is connected to the reference power supply AVDD, and the end of the capacitor C4 away from the first resistor R1 is grounded. The thermistor NTC is connected to the analog-to-digital converter 18 and then connected in parallel with the first resistor R1. The reference power supply AVDD is divided by the second resistor R2 and acts on the thermistor NTC. The high-voltage side of the thermistor NTC is connected to the analog-to-digital converter 18 through the first resistor R1 to realize signal acquisition and AD conversion. The analog-to-digital converter 18 then transmits the temperature data to the microcontroller 19.

[0046] 1, 2 and 4, the weighing circuit includes a third resistor R3, a fourth resistor R4, an operational amplifier U2, a fifth resistor R5, a sixth resistor R6, a seventh resistor R7 and an eighth resistor R8. The third resistor R3 and the eighth resistor R8 are variable resistors, U2 is an operational amplifier, referred to as an operational amplifier, and the operational amplifier U2, the fifth resistor R5, the sixth resistor R6 and the seventh resistor R7 together constitute a current-voltage conversion amplifier circuit. The fifth resistor R5 is connected in parallel between the in-phase input terminal and the output terminal of the operational amplifier U2, and the negative input terminal of the operational amplifier U2 is grounded. The current balancer U1 of the pressure sensor 3 is connected to the weighing circuit, and one end of the current balancer U1 is sequentially connected in series with the fourth resistor R4 and the third resistor R3 (sliding resistor). The high-stable differential power supply VCC+ and VCC- provide a group of high-stable differential power supplies of equal size and opposite phase to the weighing circuit. The end of the third resistor R3 away from the fourth resistor R4 is connected to V CC+ is connected, and the end of the current balancer U1 away from the fourth resistor R4 is electrically connected to the non-inverting input terminal of the operational amplifier U2. The inverting input terminal of the operational amplifier U2 is connected in series with the sixth resistor R6, the seventh resistor R7 and the eighth resistor R8 (sliding resistor). The end of the eighth resistor R8 away from the seventh resistor R7 is connected to VCC-. One end of the analog-to-digital converter 18 is electrically connected to the output terminal of the operational amplifier U2, and the other end is connected to the circuit between the sixth resistor R6 and the seventh resistor R7. By adjusting the third resistor R3 and the eighth resistor R8, the current balancer can reach an initial balance. When the measured liquid is added to the measuring chamber 1, the pressure sensor 3 is subjected to force, and the current balancer U1 outputs a microcurrent. The current-to-voltage conversion amplifier circuit converts the microcurrent output by the current balancer U1 into a voltage signal and amplifies it. The analog-to-digital converter 18 performs analog-to-digital conversion on the converted and amplified voltage signal and outputs the weight data of the liquid to the microcontroller unit 19. After the pipetting head 16 adds the liquid to the measuring chamber 1, the microcontroller unit 19 transmits the measurement data to the controller 4 via the data bus. The controller 4 calculates and corrects the data and then uploads all the data to the host computer 17 for recording and storage.

[0047] This embodiment also provides a method for calibrating a dynamic ultra-micro pipetting device, using the above-mentioned tracer, and referring to Figures 1 to 4, including the following steps:

[0048] S1: Move the pipetting head 16 to the target position;

[0049] S2: Move the tracer so that the dripping hole 11 is located directly below the pipetting head 16;

[0050] S3: Place the standard liquid at the position to be taken, and the standard liquid uses pure water as the medium;

[0051] S4: Clear the temperature and weighing data previously measured by the trace instrument;

[0052] S5: The pipetting head 16 is operated to descend and extend into the measuring chamber, and the same pipetting head 16 is used to dynamically pipette liquid into the measuring chamber 1 10 times, while the microcontroller unit 19 reads the temperature data and weighing data of the liquid after each pipetting;

[0053] S6: The controller 4 obtains the 10 sets of measurement data of the single channel transmitted by the microcontroller unit 19, and performs summation and correction on the 10 sets of measurement data of the single channel, and then calculates the single channel pipetting consistency;

[0054] S7: Remove the standard liquid in the measuring chamber 1;

[0055] S8: Repeat steps S1 to S7 to measure the next channel;

[0056] S9: After measuring all channels, the controller 4 calculates the total standard deviation of the dynamic high-throughput pipetting.

[0057] In steps S6 and S9 , the controller 4 aggregates, corrects and calculates the data and then uploads all the data to the host computer for recording and storage.

[0058] Furthermore, in step S6, the controller 4 calculates the temperature data, and the temperature measurement value is calculated by the formula Calculated, where t is the temperature measurement value, t0 is the temperature measurement lower limit value of temperature sensor 2, in °C; x t is the output value of the analog-to-digital converter (ADC) 18 at the current temperature, is the output value of the corresponding analog-to-digital converter when the temperature measurement lower limit is reached, R t is the resolution of the temperature measurement circuit composed of the temperature sensor 2 and the analog-to-digital converter, that is, the corresponding change in the output data of the analog-to-digital converter when a change of 1°C occurs.

[0059] Furthermore, in step S6, the controller 4 calculates the weighing data, and the weighing value is calculated by the formula Calculated, where m x is the mass measurement value, m0 is the weight measurement lower limit of pressure sensor 3, in μg; x m is the output value of the analog-to-digital converter (ADC) 18 at the current temperature, is the output value of the analog-to-digital converter corresponding to the lower limit of temperature measurement, R m It is the resolution of the weighing circuit composed of the pressure sensor 3, the corresponding analog-to-digital converter and the weighing circuit, that is, the corresponding change in the output data of the analog-to-digital converter when a change of 1 μg occurs.

[0060] Furthermore, in step S6, the controller 4 measures the volume V of the liquid in the chamber 1. x Calculate the liquid volume V x is the volume of liquid after each pipetting of a single channel, m x is the mass of the measured liquid, and t is the temperature of the measured liquid, which can be the temperature value input by the operator or the temperature value obtained in step S5.

[0061] Furthermore, in step S6, the controller controls the liquid volume V x Make correction, volume correction value V c =a+b×V x , the corrected volume measurement result V=V c +V x , V is the final measurement result of the liquid volume after the current pipetting, a and b are correction coefficients for periodic calibration management, and are preset in the controller 4 after the trace instrument is measured and calibrated by the upper-level calibration unit; the correction volume of this embodiment adopts the linear fitting method in order to reduce the comprehensive error of the trace instrument's measurement result of the liquid volume. Conventional correction methods mostly adopt the broken line fitting method. This method requires the standard values ​​and measurement values ​​of all calibration points to be input one by one, which is inconvenient to operate. The linear fitting method only requires the input of two constants for correction. The operation is simple and the correction effect is stable, which is convenient for improving the accuracy of the trace instrument's measurement data.

[0062] Furthermore, in step S6, the controller 4 calculates the single-channel pipetting consistency. V i is the volume of the i-th pipetting in the current channel, i=1, 2, 3, ... n, where n is the total number of pipetting times of the current channel. When 10 pipetting times are performed on a single channel, n=10.

[0063] Furthermore, in step S9, the controller 4 calculates the total standard deviation of the dynamic high-throughput pipetting. V ji is the volume of the i-th pipetting of the j-th channel, i = 1, 2, 3, ... n, n is the total number of pipetting times of a single channel, when a single channel performs 10 pipetting times, n = 10, j = 1, 2, 3, ... m, m is the number of channels being measured, is the mean volume of each pipetting,

[0064] During each pipetting measurement, the system automatically takes ten consecutive readings and calculates the average value. For example, when pipetting the first channel, the average value of 10 consecutive readings is 9.9454μL≈9.945μL. The volume reading data for each pipetting of the first channel is shown in the following table:

[0065] According to the formula Substitute the average volume of each pipetting into the above formula Substitute the volume data of each pipetting into V in the above formula i The Type A uncertainty of the measurement result is calculated as U A =0.08nL, rounded to U A =0.10nL.

[0066] According to the method of this embodiment, a four-channel dynamic pipetting device is calibrated. According to steps S1 to S8, the raw data obtained are as follows: i is the volume of each pipetting of the current channel, i=1, 2, 3, ... n; V is the final measurement result of the total volume of the liquid after the current pipetting.

[0067] When calibrating the four channels, the volume of each pipetting is shown in the following table:

[0068] According to the formula The single-channel pipetting consistency is calculated and shown in the following table:

[0069] According to the formula The absolute total standard deviation of high-throughput pipetting was calculated to be 0.005 μL, and the relative total standard deviation relative to the nominal value of the pipetted volume was 0.5%.

[0070] According to the formula Calculate the mean volume measurement for high-throughput pipetting as

[0071] According to the indication error = measured indication - reference value, the indication error E of the high-throughput pipetting device for this measurement is calculated. a =0.058μL, then at the working point of 10μL, its correction value is C=-0.058μL.

[0072] According to the indicated value range = maximum positive deviation + maximum negative deviation, the corrected range of this measurement is calculated to be R = 0.051 μL.

[0073] It can be seen that the present invention realizes the measurement of single-channel pipetting consistency γ, total standard deviation σ, indication error Ea, range R and other precision-related indicators of trace-level ultra-high-precision high-throughput pipetting equipment, and can provide a correction value C to calibrate the dynamic pipetting equipment. The Class A uncertainty of the measurement result can reach 1nL, which is more than an order of magnitude higher than that of conventional measurement methods.

[0074] The present invention provides a method for calibrating a dynamic ultra-micro pipetting device using a trace meter, which enables single-channel pipetting consistency measurement and total standard deviation measurement of high-throughput pipetting devices. This method provides a high-precision measurement value assurance technology for high-throughput pipetting devices and pipetting operations, and is conducive to the promotion of high-throughput technology in high-precision application fields. The method achieves trace-level ultra-high-precision high-throughput pipetting accuracy measurement, with a volume resolution of 0.1 nL and a measurement uncertainty better than 1 nL, which is more than an order of magnitude higher than conventional measurement methods. Uncertainty refers to the degree of uncertainty about the measured value due to measurement error, and conversely, it also indicates the reliability of the result. It is an indicator of the quality of the measurement result. The smaller the uncertainty, the higher the quality, the higher the level, and the higher its use value. The larger the uncertainty, the lower the quality, the lower the level, and the lower its use value. When reporting the results of physical quantity measurements, the corresponding uncertainty must be given. This not only facilitates the user to assess its reliability, but also enhances the comparability between measurement results. The method also achieves fully automatic calibration of the pipetting accuracy of the pipetting device, greatly improving the calibration efficiency of the pipetting device.

[0075] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A trace meter, characterized in that: The invention comprises a measuring chamber (1), a temperature sensor (2), a pressure sensor (3) and a controller (4); the measuring chamber (1) is provided with an opening at the top, the temperature sensor (2) is provided at the bottom of the measuring chamber (1), the pressure sensor (3) is provided below the measuring chamber (1), the temperature sensor (2) and the pressure sensor (3) are both electrically connected to the controller (4); the temperature sensor (2) is used to measure the temperature of liquid inside the measuring chamber (1), and the pressure sensor (3) is used to weigh the liquid inside the measuring chamber (1).

2. The tracer according to claim 1, characterized in that: The inner bottom wall of the measuring chamber (1) is provided with a lowest inclination point, and the lowest inclination point is located directly below the center of gravity of the measuring chamber (1); a measuring hole (5) is provided at the lowest inclination point of the bottom wall of the measuring chamber (1); a side of the measuring hole (5) close to the inside of the measuring chamber (1) is covered with a flexible heat-conductive film (6); and the sensing end of the temperature sensor (2) is placed in the measuring hole (5) and abuts against the lower surface of the flexible heat-conductive film (6).

3. The tracer according to claim 1, characterized in that: The measuring chamber (1) is covered with an anti-steam screen (7), and the anti-steam screen (7) is provided with a through hole (8) for the pipetting head (16) to pass through.

4. The tracer according to claim 3, characterized in that: The invention also comprises a bottom shell (9) arranged below the measuring chamber (1), wherein the pressure sensor (3) is arranged inside the bottom shell (9); a wind shield (10) is arranged on the bottom shell (9), wherein the wind shield (10) is used to cover the measuring chamber (1); a dripping hole (11) for a pipette head (16) to pass through is provided on the top wall of the wind shield (10); a protective plate (12) is arranged above the wind shield (10), wherein a passage hole (13) is provided on the protective plate (12); the apertures of the through hole (8) and the dripping hole (11) are both larger than the aperture of the passage hole (13); the center lines of the dripping hole (11), the through hole (8) and the measuring hole (5) coincide with each other; and gaps are left between the measuring chamber (1) and the anti-steam screen (7) and the wind shield (10), respectively.

5. The tracer according to claim 1, characterized in that: A weighing pan (14) is arranged below the measuring chamber (1), the pressure sensor (3) is located below the weighing pan (14), a positioning groove (15) is provided on the weighing pan (14), the positioning groove (15) comprises an inwardly concave cone surface and a horizontal inner bottom wall, a positioning portion is arranged on the outer bottom wall of the measuring chamber (1), the shape of the positioning portion matches the positioning groove (15), the positioning portion is located in the positioning groove (15), and the measuring hole (5) of the measuring chamber (1) is located directly above the inner bottom wall of the positioning groove (15).

6. A method for calibrating a dynamic ultra-micropipette device, characterized in that: A method for calibrating a dynamic ultra-micro pipetting device using a trace meter as described in any one of claims 1 to 5 comprises the following steps: S1: Move the pipetting head to the target position; S2: Move the tracer so that the drip hole is directly below the pipetting head; S3: placing the standard liquid at the position to be taken; S4: Clear the trace instrument measurement data; S5: Operate the same pipetting head to dynamically pipette liquid into the measuring chamber n times, and read the temperature data and weighing data of the liquid after each pipetting, where n is the total number of pipetting times for a single channel; S6: The controller obtains the measurement data of n groups of dynamic pipetting processes of a single channel, and performs calculation, correction and single-channel pipetting consistency calculation on the measurement data; S7: remove the standard liquid in the measuring chamber; S8: Repeat steps S1 to S7 to measure the next channel; S9: After measuring all channels, calculate the overall standard deviation of dynamic high-throughput pipetting.

7. The method for calibrating a dynamic ultra-micropipette device according to claim 6, characterized in that: In step S6, the controller calculates the volume of the liquid in the measuring chamber. V x is the liquid volume after each pipetting of a single channel, m x is the mass of the liquid being measured, and t is the temperature of the liquid being measured.

8. The method for calibrating a dynamic ultra-micropipette device according to claim 7, characterized in that: In step S6, the controller controls the liquid volume V x Make correction, volume correction value V c =a+b×V x , the corrected volume measurement result V = V c +V x , V is the final measurement result of the liquid volume after the current pipetting, a and b are the correction coefficients for periodic calibration management, and are preset in the controller after measurement and calibration by the upper-level calibration unit.

9. The method for calibrating a dynamic ultra-micropipette device according to claim 8, characterized in that: In step S6, single channel pipetting consistency V i is the volume of the i-th pipetting in the current channel, 10. The method for calibrating a dynamic ultra-micropipette device according to claim 9, characterized in that: In step S9, the total standard deviation of high-throughput pipetting V ji is the volume of the i-th pipetting of the j-th channel, i = 1, 2, 3, ... n, j = 1, 2, 3, ... m, m is the number of channels measured, is the mean volume of each pipetting,

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