Calibration method of automatic liquid treatment system

Abstract

The invention provides a calibration method of an automatic liquid treatment system, and relates to the field of biochemical analysis instruments, and the calibration method comprises the following steps: S1, preparation before calibration: putting standard water, reagents, instruments and equipment required by calibration and ALHS into a laboratory for full constant temperature; s2, preparing a standard solution: preparing a lemon yellow standard solution according to an ALHS range; s3, checking the airtightness of the ALHS: checking the airtightness of the ALHS through repeated pipetting operation; s4, formal pipetting operation: in the pipetting operation process, obtaining original basic data; s5, data processing is carried out, wherein ALHS pipette volume indication errors, repeatability and channel differences are calculated; s6, recording a calibration result; and S7, making a calibration certificate. The method can solve the problem that there is no unified and normative ALHS calibration method, standard and metrological characteristics in the market; the problems that the current ALHS can not be calibrated, can not be calibrated completely and can not be calibrated accurately are solved; the problem that calibration is difficult in the small-capacity measurement research direction is solved, and the construction cost of calibration items is reduced.

Description

Technical Field

[0001] The invention relates to the field of biochemical analysis instruments, and particularly discloses a calibration method for an automatic liquid processing system. Background Art

[0002] Automatic Liquid Handling Systems (ALHS) are primarily used in the pharmaceutical, food, and disease control sectors, and are used for sampling and liquid addition in biochemical analyses. They typically have 96 or more channels and can quickly and consistently perform liquid sampling, dilution, and other processes. Compared to traditional multichannel pipettes, ALHSs have less variability between channels and require far fewer pipetting cycles, thereby reducing sample variability introduced by pipetting operations. While the primary parameters of ALHSs are similar to those of pipettes, the liquid sampling volume significantly influences test results. Therefore, to ensure accurate test results, it is necessary to calibrate the performance of ALHSs for quality control purposes. However, a search has revealed no publicly available verification procedures or calibration methods for ALHSs.

[0003] As ALHS becomes more popular in the industry, companies in various fields have put forward consistent demands and developed standardized calibration methods to trace ALHS standard data.

[0004] Based on the above analysis, this application proposes a calibration method for an automatic liquid handling system. Summary of the Invention

[0005] In order to solve the problem that the application volume of ALHS market is gradually increasing, but there is no unified and effective calibration method at present, the present invention provides a calibration method for an automatic liquid handling system with the following technical solutions: The following steps are involved: S1. Preparation before calibration: Place the standard water, reagents, instruments and equipment required for calibration in the calibration laboratory together with the ALHS and keep them at a constant temperature. S2. Prepare standard solution: Prepare a certain concentration of tartrazine standard solution according to the ALHS range; S3. Check the airtightness of the ALHS: Check the airtightness of the ALHS by repeated pipetting operations; S4. Formal pipetting operation: During the pipetting operation, obtain the original basic data; S5. Data processing: Calculate the ALHS pipetting volume indication error, repeatability, and channel differences; S6. Record calibration results: record the result data and basic data of ALHS pipetting volume calibration calculation; S7. Prepare calibration certificate: Prepare calibration certificate and fill in information for ALHS holder.

[0006] Furthermore, the instruments and equipment in step S1 are specifically a standard microplate, a microplate reader, an electronic balance, a multi-channel pipette, and a thermometer; the reagent is a tartrazine standard solution; and the constant temperature time is at least 4 hours.

[0007] Furthermore, in step S2, 20%, 50%, and 80% of the range of ALHS are selected as three calibration points, and the absorbance of the tartrazine standard solution after pipetting satisfies one of the following two conditions: a) Calibration point V < 40 μL: Use a multichannel pipette to transfer 100 μL of calibration water to each channel of the standard microplate. Then add the required calibration volume of the tartrazine standard solution so that the absorbance of the diluted tartrazine standard solution is between 0.8 and 1.2. b) Calibration point V ≥ 40 μL: Use a multichannel pipette to directly pipette the volume of tartrazine standard solution to be calibrated so that the absorbance of the tartrazine standard solution is between 0.8 and 1.2.

[0008] Furthermore, the absorbance of the lemon yellow standard solution is calculated according to the following formula:

[0009] In the above formula, A—absorbance of lemon yellow standard solution; A 450 —Absorbance of lemon yellow standard solution under 450nm light source; A 620 —Absorbance of lemon yellow standard solution under 620nm light source; —Absorbance of a standard microplate at 450 nm when blank or pre-filled with 100 μL of calibration water; —Absorbance of a standard microplate under a 620 nm light source when blank or pre-filled with 100 μL of calibration water.

[0010] Furthermore, the ALHS pipetting volume indication error, repeatability and channel difference were calibrated by weighing with an electronic balance and measuring absorbance with a microplate reader. Specifically, the following requirements were followed: Calibration point V < 40 μL: Use a multichannel pipette to transfer 100 μL of calibration water to a standard microplate, then add the required calibration volume of tartrazine standard solution so that the absorbance of the diluted tartrazine standard solution is between 0.8 and 1.2; Calibration point V ≥ 40 μL: Use a multichannel pipette to directly pipette the volume of lemon yellow standard solution to be calibrated so that the absorbance of the lemon yellow standard solution is between 0.8 and 1.2; Weigh the mass of the blank standard microplate, then transfer 20%, 50%, and 80% of the tartrazine standard solution to the standard microplate according to the ALHS range, weigh the mass of the standard microplate after transfer, and measure the absorbance of each channel in the standard microplate. Repeat the measurement for each calibration point 6 times.

[0011] Furthermore, the calculation steps for ALHS pipetting volume indication error, repeatability and channel difference are as follows: (1) Calculate the average actual pipetting volume of ALHS in a standard solution of lemon yellow at 20°C according to the following formula:

[0012] In the above formula, V 20 —The average actual volume of liquid transferred at 20°C, in μL; △m—mass difference of the standard microplate before and after pipetting, in mg; N—the number of channels of ALHS; —weight density, take 8.00g / cm³; —The air density in the laboratory during calibration is 0.0012g / cm³; —density of tartrazine standard solution or diluted tartrazine standard solution at t°C, in g / cm³; β—the volume expansion coefficient of ALHS is 4.5×10 -4 / ℃; t—temperature of the tartrazine standard solution during calibration, in °C; (2) The formula for calculating the single pipetting volume of each channel of ALHS is as follows

[0013] In the above formula, V ij —A single pipetting volume of a certain channel of ALHS, in μL; A ij —Absorbance measurement value of a certain channel of ALHS; Ā—average absorbance of all channels of ALHS; (3) Take the average of the 6 measurement results of each channel of ALHS and calculate the pipetting volume indication error according to the following formula:

[0014] In the above formula, E is the error of the pipetting volume indication,%; —The average value of 6 measurements, in μL; V s—Standard value of pipetting volume, in μL; (4) Calculate the pipetting volume repeatability according to the following formula:

[0015] In the above formula, s r —Pipette volume repeatability, %; V ijk —The kth measurement result of a certain channel of ALHS, in μL; (5) The formula for calculating the average volume of pipetting of all channels is as follows:

[0016] (6) Calculate the pipetting volume difference of each channel of ALHS according to the following formula:

[0017] In the above formula, Z ij —Difference in pipetting volume of a certain channel of ALHS, %; Z ij —The average volume of pipetted across all channels of the ALHS, in μL.

[0018] Furthermore, to simplify the calculation process, the actual average volume of ALHS pipetted at a temperature of 20°C for the lemon yellow standard solution can be written as follows:

[0019] Among them, the K(t) value can be obtained by looking up the table.

[0020] The main difficulty of this method is that the amount of liquid taken in ALHS is very small, and the evaporation of water during the calibration process has a significant impact on the calibration results. This has a greater impact on the calibration results of the sampling volume accuracy and a smaller impact on the channel differences. When studying the calibration method, the R&D team focused on reducing the impact of water evaporation on the calibration results.

[0021] The beneficial effects of the present invention are: 1. The present invention provides a calibration method for an automated liquid handling system. As the market usage of ALHS gradually increases, this method can solve the problem of the lack of unified and standardized ALHS calibration methods, calibration standards, and measurement characteristics in the market.

[0022] 2. The present invention provides a calibration method for an automatic liquid handling system, which solves the current problem of inability to calibrate, incomplete calibration, and inaccurate calibration of ALHS.

[0023] 3. The present invention provides a calibration method for an automatic liquid handling system, which essentially performs capacity calibration through chemical methods, solving the calibration difficulties in small-capacity measurement research. The chemical method also greatly reduces the cost of constructing the calibration project, and enables on-site calibration to a certain extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 1 is a flow chart of the steps of the calibration method of the present invention. DETAILED DESCRIPTION

[0025] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0026] Automatic liquid handling system: a professional name in the industry, the full English name is "Automated Liquid Handling System", and the English abbreviation is "ALHS".

[0027] In one embodiment, a calibration method for an automated liquid handling system is provided, the calibration method comprising the following steps: S1. Preparation before calibration: Place the reagents, instruments and equipment required for calibration in the calibration laboratory together with the ALHS and keep them at a constant temperature.

[0028] S2. Prepare standard solution: Prepare a certain concentration of tartrazine standard solution according to the ALHS range.

[0029] S3. Check the airtightness of the ALHS: Check the airtightness of the ALHS by repeated pipetting operations. Repeat the pipetting operation at least 10 times and there should be no dripping, leakage, or bubbles.

[0030] S4. Formal pipetting operation: During the pipetting operation, the original basic data is obtained.

[0031] S5. Perform data processing: Calculate the ALHS pipetting volume indication error, repeatability, and channel differences.

[0032] S6. Record Calibration Results: Record the results and basic data of the ALHS pipetting volume calibration calculation. This includes the number of channels, calibration point volume range, standard microplate absorbance measurement results, total mass, average absorbance, actual standard microplate volume, room temperature, K(t) value, pipetting volume indication error, pipetting volume repeatability, pipetting volume channel differences, and measurement uncertainty.

[0033] Prepare calibration certificate: Prepare calibration certificate and fill in information for ALHS holder.

[0034] The calibration certificate consists of a cover, inner pages, and calibration data, where the calibration data includes the pipetting volume indication error, pipetting volume repeatability, pipetting volume channel difference, and measurement uncertainty of each calibration point.

[0035] The information on the calibration certificate shall include at least the following: a) Title, such as "Calibration Certificate"; b) Laboratory name and address; c) Unique identification of the certificate or report (such as certificate number), identification of each page and the total number of pages; d) The name and address of the sending institution; e) Description and clear identification of the ALHS (such as model, product number, etc.); f) calibration date; g) Identification of the technical specification on which the calibration is based, including name and code; h) a statement of the traceability and validity of the measurement standards used for calibration; i) Description of the calibration environment; j) calibration results and their measurement uncertainties; k) Signatures of the calibrator and verifier; l) Signature of the person approving the calibration certificate.

[0036] The laboratory environment requirements before calibration are as follows: a) The indoor ambient temperature range is 15°C to 25°C, and the temperature change during calibration is less than 1°C / h; b) The relative humidity range of the indoor environment is 40%~80%; c) During calibration, there must be no direct sunlight, strong electromagnetic interference, vibration, strong airflow, or corrosive gas.

[0037] A better technical solution is that the instruments and equipment in the above step S1 are specifically a standard microplate, a microplate reader, an electronic balance, a multi-channel pipette, and a thermometer, the reagent is a tartrazine standard solution, and the constant temperature time is at least 4 hours.

[0038] Tartrazine: analytical grade; Electronic balance: The graduation value is 0.01 mg and must comply with Level ① in JJG1036-2022 "Electronic Balance Verification Regulations"; ELISA analyzer: The indication stability range is [-0.003, 0.003], the absorbance indication error range is [-0.015, 0.015], and the channel difference is less than 0.006. If the ELISA analyzer is not wavelength-adjustable, filters with wavelengths of 450nm and 620nm should be configured; Multichannel pipette: with 8 or 12 channels, nominal capacity 100μL, the maximum allowable error range of each channel at 100μL is -1μL to 1μL; Calibration water: distilled water or deionized water that meets the requirements of GB / T6682-2008 "Specifications and Test Methods for Water Used in Analytical Laboratories" and should be placed in the laboratory 24 hours in advance so that the temperature difference between it and the room temperature is no more than 2°C. Standard microplate: should be free of obvious scratches or defects that affect use; Thermometer: The measuring range is 0℃~30℃, the graduation value is 0.1℃, and the maximum allowable error range is -0.2℃~0.2℃.

[0039] A better technical solution is to select 20%, 50%, and 80% of the ALHS range as three calibration points in the above step S2, and the absorbance of the lemon yellow standard solution after pipetting meets one of the following two conditions: a) Calibration point V < 40 μL: Use a multichannel pipette to transfer 100 μL of calibration water to each channel of the standard microplate. Then add the required calibration volume of the tartrazine standard solution so that the absorbance of the diluted tartrazine standard solution is between 0.8 and 1.2. b) Calibration point V ≥ 40 μL: Use a multichannel pipette to directly pipette the volume of tartrazine standard solution to be calibrated so that the absorbance of the tartrazine standard solution is between 0.8 and 1.2.

[0040] For a better technical solution, the absorbance of the lemon yellow standard solution is calculated according to the following formula

[0041] In the above formula, A—absorbance of lemon yellow standard solution; A 450 —Absorbance of lemon yellow standard solution under 450nm light source; A 620 —Absorbance of lemon yellow standard solution under 620nm light source; —Absorbance of the standard microplate under a 450nm light source when blank (V>40μL) or pre-filled with 100μL calibration water (V≤40μL); —Absorbance of a standard microplate at 620 nm when blank (V > 40 μL) or pre-filled with 100 μL of calibration water (V ≤ 40 μL).

[0042] A better technical solution is to use an electronic balance and a microplate reader to measure absorbance to calibrate the ALHS pipetting volume indication error, repeatability, and channel difference, specifically: according to the following requirements Calibration point V < 40 μL: Use a multichannel pipette to transfer 100 μL of calibration water to a standard microplate, then add the required calibration volume of tartrazine standard solution so that the absorbance of the diluted tartrazine standard solution is between 0.8 and 1.2; Calibration point V ≥ 40 μL: Use a multichannel pipette to directly pipette the volume of lemon yellow standard solution to be calibrated so that the absorbance of the lemon yellow standard solution is between 0.8 and 1.2; Weigh the mass of the blank standard microplate, then transfer 20%, 50%, and 80% of the tartrazine standard solution to the standard microplate according to the ALHS range, weigh the mass of the standard microplate after transfer, and measure the absorbance of each channel in the standard microplate. Repeat the measurement for each calibration point 6 times.

[0043] For a better technical solution, the calculation steps for ALHS pipetting volume indication error, repeatability, and channel difference are as follows: (1) Calculate the average actual pipetting volume of ALHS in a standard solution of lemon yellow at 20°C according to the following formula:

[0044] In the above formula, V 20 —The average actual volume of liquid transferred at 20°C, in μL; △m—mass difference of the standard microplate before and after pipetting, in mg; N—the number of channels of ALHS; —weight density, take 8.00g / cm³; —The air density in the laboratory during calibration is 0.0012g / cm³; —density of tartrazine standard solution or diluted tartrazine standard solution at t°C, in g / cm³; β—the volume expansion coefficient of ALHS is 4.5×10 -4 / ℃; t—temperature of the tartrazine standard solution during calibration, in °C; (2) The formula for calculating the single pipetting volume of each channel of ALHS is as follows

[0045] In the above formula, V ij —A single pipetting volume of a certain channel of ALHS, in μL; A ij —Absorbance measurement value of a certain channel of ALHS; Ā—average absorbance of all channels of ALHS; (3) Take the average of the 6 measurement results of each channel of ALHS and calculate the pipetting volume indication error according to the following formula:

[0046] In the above formula, E is the error of the pipetting volume indication,%; —The average value of 6 measurements, in μL; V s —Standard value of pipetting volume, in μL; (4) Calculate the pipetting volume repeatability according to the following formula:

[0047] In the above formula, s r —Pipette volume repeatability, %; V ijk —The kth measurement result of a certain channel of ALHS, in μL; (5) The formula for calculating the average volume of pipetting of all channels is as follows:

[0048] (6) Calculate the pipetting volume difference of each channel of ALHS according to the following formula:

[0049] In the above formula, Z ij —Difference in pipetting volume of a certain channel of ALHS, %; —The average volume of pipetted across all channels of the ALHS, in μL.

[0050] A better technical solution is to simplify the calculation process, the actual average volume of ALHS pipetted at the standard solution temperature of 20°C can be written as the following formula:

[0051] Among them, the K(t) value can be obtained by looking up the table.

[0052] According to industry professionals, the use of this method to calibrate ALHS can improve the accuracy of ALHS detection, provide a basis for traceability, and provide technical support for ALHS quality control in various industries.

[0053] Other beneficial effects of the method of the present invention are: 1. As the market usage of ALHS gradually increases, this method can solve the problem of no unified and standardized ALHS calibration method, calibration standard and metrological characteristics in the market.

[0054] 2. The present invention solves the problem that the current ALHS cannot be calibrated, is not fully calibrated, and is not accurate.

[0055] 3. This invention essentially uses chemical methods to perform capacity calibration, solving the calibration difficulties in small-capacity measurement research. Furthermore, the chemical method greatly reduces the cost of the calibration project and enables on-site calibration to a certain extent.

[0056] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims, not the foregoing description, and all variations within the meaning and range of equivalents of the claims are intended to be included therein. Any reference sign in a claim should not be construed as limiting the claim to which it relates.

Claims

1. A calibration method for an automatic liquid handling system, characterized in that: The following steps are involved: S1. Preparation before calibration: Place the standard water, reagents, instruments and equipment required for calibration in the calibration laboratory together with the ALHS and keep them at a constant temperature. S2. Prepare standard solution: Prepare a certain concentration of tartrazine standard solution according to the ALHS range; S3. Check the airtightness of the ALHS: Check the airtightness of the ALHS by repeated pipetting operations; S4. Formal pipetting operation: During the pipetting operation, obtain the original basic data; S5. Data processing: Calculate the ALHS pipetting volume indication error, repeatability, and channel differences; S6. Record calibration results: Record the results and basic data of the ALHS pipetting volume calibration calculation; S7. Prepare calibration certificate: Prepare calibration certificate and fill in information for ALHS holder.

2. The calibration method of the automatic liquid handling system according to claim 1, characterized in that: The instruments and equipment in step S1 are specifically a standard microplate, a microplate reader, an electronic balance, a multi-channel pipette, and a thermometer. The reagent is a tartrazine standard solution, and the constant temperature time is at least 4 hours.

3. The calibration method of the automatic liquid handling system according to claim 2, characterized in that: In step S2, 20%, 50%, and 80% of the ALHS range are selected as three calibration points, and the absorbance of the tartrazine standard solution after pipetting satisfies one of the following two conditions: a) Calibration point V < 40 μL: Use a multichannel pipette to transfer 100 μL of calibration water to each channel of the standard microplate. Then add the required calibration volume of the tartrazine standard solution so that the absorbance of the diluted tartrazine standard solution is between 0.8 and 1.

2. b) Calibration point V ≥ 40 μL: Use a multichannel pipette to directly pipette the volume of tartrazine standard solution to be calibrated, so that the absorbance of the tartrazine standard solution is between 0.8 and 1.

2.

4. The calibration method of the automatic liquid handling system according to claim 3, characterized in that: The absorbance of lemon yellow standard solution is calculated according to the following formula , in the above formula, A—absorbance of lemon yellow standard solution; A 450 —Absorbance of lemon yellow standard solution under 450nm light source; A 620 —Absorbance of lemon yellow standard solution under 620nm light source; —Absorbance of a standard microplate at 450 nm when blank or pre-filled with 100 μL of calibration water; —Absorbance of a standard microplate under a 620 nm light source when blank or pre-filled with 100 μL of calibration water.

5. The calibration method of the automatic liquid handling system according to claim 3, characterized in that: The ALHS pipetting volume indication error, repeatability, and channel difference were calibrated using an electronic balance and a microplate reader to measure absorbance. The specific requirements are as follows: Calibration point V < 40 μL: Use a multichannel pipette to transfer 100 μL of calibration water to a standard microplate, then add the required calibration volume of tartrazine standard solution so that the absorbance of the diluted tartrazine standard solution is between 0.8 and 1.2; Calibration point V ≥ 40 μL: Use a multichannel pipette to directly pipette the volume of lemon yellow standard solution to be calibrated so that the absorbance of the lemon yellow standard solution is between 0.8 and 1.2; Weigh the mass of the blank standard microplate, then transfer 20%, 50%, and 80% of the tartrazine standard solution to the standard microplate according to the ALHS range, weigh the mass of the standard microplate after transfer, and measure the absorbance of each channel in the standard microplate. Repeat the measurement for each calibration point 6 times.

6. The calibration method of any automatic liquid handling system according to claim 2 or 5, characterized in that: The calculation steps for ALHS pipetting volume indication error, repeatability and channel difference are as follows: (1) Calculate the average actual pipetting volume of ALHS in a standard solution of lemon yellow at 20°C according to the following formula: , in the above formula, V 20 —The average actual volume of liquid transferred at 20°C, in μL; △m—mass difference of the standard microplate before and after pipetting, in mg; N—the number of channels of ALHS; —weight density, take 8.00g / cm³; —The air density in the laboratory during calibration is 0.0012g / cm³; —density of tartrazine standard solution or diluted tartrazine standard solution at t°C, in g / cm³; β—the volume expansion coefficient of ALHS is 4.5×10 -4 / ℃; t—temperature of the tartrazine standard solution during calibration, in °C; (2) The formula for calculating the single pipetting volume of each channel of ALHS is as follows , in the above formula, V ij —A single pipetting volume of a certain channel of ALHS, in μL; A ij —Absorbance measurement value of a certain channel of ALHS; Ā—average absorbance of all channels of ALHS; (3) Take the average of the 6 measurement results of each channel of ALHS and calculate the pipetting volume indication error according to the following formula: In the above formula, E is the error of the pipetting volume indication,%; —The average value of 6 measurements, in μL; V s —Standard value of pipetting volume, in μL; (4) Calculate the pipetting volume repeatability according to the following formula: , in the above formula, s r —Pipette volume repeatability, %; V ijk —The kth measurement result of a certain channel of ALHS, in μL; (5) The formula for calculating the average volume of pipetting of all channels is as follows: ； (6) Calculate the pipetting volume difference of each channel of ALHS according to the following formula: , in the above formula, Z ij —Difference in pipetting volume of a certain channel of ALHS, %; —The average volume of pipetted across all channels of the ALHS, in μL.

7. The calibration method of the automatic liquid handling system according to claim 6, characterized in that: To simplify the calculation process, the actual average volume of ALHS pipetted at 20°C of lemon yellow standard solution can be written as follows: , where the K(t) value can be obtained by looking up the table.

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