Sample for verifying fluorescence detection function of multifunctional microplate reader and verification method thereof

Abstract

The invention discloses a sample for verifying the fluorescence detection function of a multifunctional microplate reader, the sample is formed by mixing neutral gum and an environment-friendly transparent agent, and the neutral gum has the performance of spontaneous green fluorescence. Neutral gum is added into an environment-friendly transparent agent to be diluted, the diluted neutral gum is added into a microwell plate according to a set volume gradient, the neutral gum can be regarded as a standard sample to be put on a machine to detect the green fluorescence intensity within at least four months later, the detected fluorescence value and the volume of the diluted neutral gum added into a hole are subjected to correlation analysis, and if the correlation coefficient is larger than 0.9, the neutral gum can be determined. Therefore, the fluorescence detection function of the multifunctional microplate reader is relatively good. The microplate added with the sample provided by the invention has the advantages of low cost, simple configuration, stable performance, convenience in use, repeated use within at least four months and the like, and can be widely applied to accuracy verification of fluorescence (green fluorescence) detection functions of a multifunctional microplate reader.

Description

Technical Field

[0001] The present invention belongs to the field of detection technology of experimental instruments, and in particular relates to a technology for verifying the fluorescence detection function of a multifunctional microplate reader, specifically a sample for verifying the fluorescence detection function of a multifunctional microplate reader and a verification method thereof. Background Art

[0002] The multifunctional microplate reader is the most commonly used instrument in scientific research. It generally uses microplates (96-well plates account for more than 90%) to detect light absorption, fluorescence and other functions. Since the instrument generates test data, there are certain requirements for its detection accuracy. The manufacturer has a test plate that can be used to verify the accuracy of the instrument's detection. Since the test plate is expensive and the whole process of using the test plate to verify the instrument function is relatively complicated, generally colleges and universities or scientific research institutions will not purchase test plates, but regularly (usually once every 1-2 years) ask the manufacturer's engineers to bring test plates to the door for testing. Therefore, in daily work, for the person in charge of the instrument, to determine whether the instrument is accurate or not, other methods need to be used for verification.

[0003] Although light absorption and fluorescence detection are two functions of the multifunctional microplate reader, they are actually two completely independent functions. In terms of medical research, light absorption experiments are far more than fluorescence detection experiments, at least 10-20 times. When a student questions the accuracy of the light absorption function of the instrument, the person in charge of the instrument only needs to ask other students who have used it recently, especially for experiments with standard curves. If other students report that the instrument function is fine and the correlation coefficient of the standard curve is close to 1, it can be determined that the poor data is a problem with the student's sample, not the instrument. However, there are very few experiments with fluorescence detection. When students obtain poor data from fluorescence detection and question the fluorescence detection function of the microplate reader, it is difficult for the person in charge of the instrument to rule out problems with the instrument by asking for data from other recent users.

[0004] Although the manual of the instrument (such as the Tecan Infinite M200 PRO multifunctional microplate reader used in the verification example) also provides a method of "preparing a standard solution and adding the standard solution to a 96-well plate to verify the accuracy and stability of the instrument's fluorescence detection function", the liquid in this method needs to be prepared on the spot, and each time it needs to be weighed and then prepared, which is cumbersome. In addition, the standard solution will slowly evaporate after being added to the 96-well plate, resulting in a different concentration than when it was just prepared. It will completely evaporate in a few days, and it needs to be re-prepared and wells added. Therefore, the prepared standard solution cannot be stored for a long time after being added to the 96-well plate.

[0005] Therefore, if a sample that can be preserved for a long time can be invented, the sample can be tested and verified at any time when students question the fluorescence detection function of the instrument, which can greatly reduce the workload of the instrument manager. FITC and GFP are the most commonly used fluorescence in scientific research (at least accounting for more than 80% of all fluorescence detection experiments). Although the two are not the same fluorescence, the parameters set when the instrument detects the two are the same, so for the instrument, they can be considered the same fluorescence. Given that green fluorescence FITC and GFP detection are the most commonly used fluorescence when using an enzyme-labeled instrument to detect fluorescence, it is only necessary to invent a sample that can verify the green fluorescence detection function of a multifunctional enzyme-labeled instrument. Summary of the invention

[0006] The purpose of the present invention is to address the problems existing in the prior art and to provide a sample for verifying the fluorescence detection function of a multifunctional microplate reader and a verification method thereof. The sample is cheap, easy to prepare, and easy to preserve. It can be reused multiple times within a period of time (at least 4 months) and can be tested on the machine at any time.

[0007] The purpose of the present invention is to be solved by the following technical solutions:

[0008] A sample for verifying the fluorescence detection function of a multifunctional microplate reader, characterized in that the sample is composed of a mixture of neutral gum and an environmentally friendly transparent agent, wherein the neutral gum has the property of spontaneous green fluorescence.

[0009] When the sample is mixed, the volume ratio of the neutral gum to the sample is greater than the volume ratio of the environmentally friendly transparent agent to the sample.

[0010] The sample comprises the following components by volume: 55% to 90% of neutral gum and 15% to 45% of environmentally friendly transparent agent. The sum of the percentages of the above components by volume is 100%.

[0011] The neutral gum is from Shanghai Aladdin Biochemical Technology Co., Ltd., or from Sinopharm Chemical Reagent Co., Ltd., or from Shanghai Huashen Rehabilitation Equipment Co., Ltd.

[0012] The environmentally friendly transparent agent can be any liquid environmentally friendly transparent agent sold on the market. The environmentally friendly transparent agent used in this application is preferably from Wuhan Hongzi Biotechnology Co., Ltd. or from Hangzhou Mount Sinai Biotechnology Co., Ltd.

[0013] The sample can be reused for no less than 4 months.

[0014] When the sample is used for verification, the detection value of the fluorescence signal increases with the increase of the amount of sample added, and the correlation coefficient of the linear relationship presented is greater than 0.95.

[0015] A method for verifying a sample of a multifunctional microplate reader for fluorescence detection, characterized in that the steps of the verification method are as follows:

[0016] S1. Add environmentally friendly transparent agent into neutral gum according to the set ratio, and evenly dilute the neutral gum to obtain a sample;

[0017] S2, respectively aspirate 0 μL, 30 μL, 60 μL, 90 μL, 120 μL, 150 μL, 180 μL, 210 μL, 240 μL, and 270 μL of the sample and add them to the microplate to prepare a microplate with samples added;

[0018] S3. Use the multifunctional microplate reader to be verified to detect the FITC fluorescence signal of the microplate with the sample added, and make a standard curve with the added sample volume as the horizontal axis and the detection value of the FITC fluorescence signal as the vertical axis. When the correlation coefficient of the standard curve is greater than 0.9, it proves that the fluorescence detection function of the multifunctional microplate reader meets the experimental requirements;

[0019] S4. After placing the microplate with the sample in step S2 for a period of time, repeat step S3 to verify whether the fluorescence detection function of the multifunctional microplate reader meets the experimental requirements and at the same time verify the effectiveness of the sample in the microplate after being placed for a period of time.

[0020] The microplate in step S2 is selected from a microplate capable of detecting the fluorescence signal at the top of the sample in the well and a microplate capable of detecting the fluorescence signal at the bottom of the sample in the well.

[0021] The sample in step S2 is added into the microplate according to the requirement of setting at least two parallel wells for each volume.

[0022] The period of time in step S4 is any day from the day when the sample is added to the microplate to four months, or any day exceeding four months.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] The sample raw materials provided by the present invention are simple, and only neutral gum and environmentally friendly transparent agent are used. The environmentally friendly transparent agent is used to reduce the viscosity of the neutral gum and ensure the accurate absorption volume. It has the advantages of low cost, simple configuration, stable performance, and easy use. It can be widely used in the accuracy verification of the fluorescence (green fluorescence) detection function of the multifunctional enzyme labeler.

[0025] After the sample provided by the present invention is added to the microplate, it only needs to be covered with a plate cover (the cover only serves to prevent dust) without having to be specially sealed for storage. The microplate with the sample can be repeatedly used to verify the fluorescence detection function of the multifunctional microplate reader within at least four months, and has the characteristics of long service life and easy use. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Attached Figure 1 A standard curve diagram of the detection data of the FITC fluorescence signal in the top reading mode on the day when the sample is added to the all-black 96-well plate in the verification example provided by the present invention and the added sample volume;

[0027] Attached Figure 2 A standard curve diagram of the detection data of the FITC fluorescence signal in the top reading mode and the added sample volume after the all-black 96-well plate in the verification example provided by the present invention has been added with the sample for one month;

[0028] Attached Figure 3 A standard curve diagram of the detection data of the FITC fluorescence signal in the top reading mode and the added sample volume in the all-black 96-well plate in the verification example provided by the present invention after adding samples for two months;

[0029] Attached Figure 4 A standard curve diagram of the detection data of the FITC fluorescence signal in the top reading mode and the added sample volume in the all-black 96-well plate in the verification example provided by the present invention after adding the sample for three months;

[0030] Attached Figure 5 A standard curve diagram of the detection data of the FITC fluorescence signal in the top reading mode and the added sample volume in the all-black 96-well plate in the verification example provided by the present invention after adding samples for four months;

[0031] Attached Figure 6 A standard curve diagram of the detection data of the FITC fluorescence signal in the bottom reading mode of the black-walled transparent-bottom 96-well plate in the verification example provided by the present invention and the added sample volume on the day when the sample is added;

[0032] Attached Figure 7 A standard curve diagram of the detection data of the FITC fluorescence signal in the bottom reading mode and the added sample volume in the black-walled transparent-bottom 96-well plate in the verification example provided by the present invention after adding the sample for one month;

[0033] Attached Figure 8 A standard curve diagram of the detection data of the FITC fluorescence signal in the bottom reading mode and the added sample volume in the black-walled transparent-bottom 96-well plate in the verification example provided by the present invention after adding samples for two months;

[0034] Attached Fig. 9 A standard curve diagram of the detection data of the FITC fluorescence signal in the bottom reading mode and the added sample volume in the black-walled transparent-bottom 96-well plate in the verification example provided by the present invention after adding the sample for three months;

[0035] Attached Fig.10 This is a standard curve diagram of the detection data of the FITC fluorescence signal in the bottom reading mode and the added sample volume in the black-walled transparent bottom 96-well plate in the verification example provided by the present invention after adding samples for four months. DETAILED DESCRIPTION

[0036] Example embodiments will now be described more fully with reference to the accompanying drawings. However, example embodiments can be implemented in many forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that the present invention will be comprehensive and complete and fully convey the concept of the example embodiments to those skilled in the art.

[0037] The invention provides a sample for verifying the fluorescence detection function of a multifunctional microplate reader. The sample is composed of a mixture of a neutral gum and an environmentally friendly transparent agent, wherein the neutral gum has the performance of spontaneous green fluorescence; when the sample is mixed, the volume ratio of the neutral gum to the sample is greater than the volume ratio of the environmentally friendly transparent agent to the sample; when the sample is used for verification, the detection value of the fluorescence signal increases with the increase of the amount of sample added, and the correlation coefficient of the linear relationship presented is greater than 0.95.

[0038] Adding an environmentally friendly transparent agent to neutral gum can reduce the viscosity of the neutral gum and ensure the accurate absorption volume after dilution.

[0039] Furthermore, the volume content of the components of the sample is: 55% to 90% neutral gum, 15% to 45% environmentally friendly transparent agent, and the sum of the volume content percentages of the components of the above sample is 100%; a preferred solution is: the volume ratio of the neutral gum to the sample is 65% to 80%, and the volume ratio of the environmentally friendly transparent agent to the sample is 20% to 35%.

[0040] Furthermore, the neutral gum comes from Shanghai Aladdin Biochemical Technology Co., Ltd., or from Sinopharm Chemical Reagent Co., Ltd., or from Shanghai Huashen Rehabilitation Equipment Co., Ltd.; the environmentally friendly transparent agent can be any liquid environmentally friendly transparent agent sold on the market, preferably an environmentally friendly transparent agent from Wuhan Hongzi Biotechnology Co., Ltd. or Hangzhou Mount Sinai Biotechnology Co., Ltd.

[0041] Based on the above samples for verifying the fluorescence detection function of the multifunctional microplate reader, the present invention also provides a verification method for whether the above samples can be used to verify the fluorescence detection function of the multifunctional microplate reader and whether they are still effective after long-term storage.

[0042] The steps of a sample verification method for verifying the fluorescence detection function of a multifunctional microplate reader provided by the present invention are as follows:

[0043] S1. Add environmentally friendly transparent agent into neutral gum according to the set ratio, and evenly dilute the neutral gum to obtain a sample;

[0044] S2, respectively aspirate 0 μL, 30 μL, 60 μL, 90 μL, 120 μL, 150 μL, 180 μL, 210 μL, 240 μL, and 270 μL of the sample and add them to the microplate to prepare a microplate with samples added;

[0045] S3. Use the multifunctional microplate reader to be verified to detect the FITC fluorescence signal of the microplate with the sample added, and make a standard curve with the added sample volume as the horizontal axis and the detection value of the FITC fluorescence signal as the vertical axis. When the correlation coefficient of the standard curve is greater than 0.9, it proves that the fluorescence detection function of the multifunctional microplate reader meets the experimental requirements;

[0046] S4. After placing the microplate with the sample in step S2 for a period of time, repeat step S3 to verify whether the fluorescence detection function of the multifunctional microplate reader meets the experimental requirements and at the same time verify the effectiveness of the sample in the microplate after being placed for a period of time.

[0047] In the above verification method, the sample in step S2 is added to the microplate according to the requirement of setting at least two parallel wells for each volume, and the detection value of the FITC fluorescence signal in step S3 is averaged.

[0048] In addition, since the fluorescence signal can be detected by a multifunctional microplate reader using a completely black microplate and a microplate with black walls and a transparent bottom, according to the instrument manual, the completely black microplate is generally used to detect the fluorescence signal at the top of the sample in the well (top reading mode), and the microplate with black walls and a transparent bottom is generally used to detect the fluorescence signal at the bottom of the sample in the well (bottom reading mode). Therefore, the microplate in step S2 is selected from a microplate that can detect the fluorescence signal of the top of the sample in the well and a microplate that can detect the fluorescence signal of the bottom of the sample in the well.

[0049] Verification Example

[0050] To ensure the validity of the experimental results, the Tecan InfiniteM200 PRO multifunctional microplate reader with fluorescence detection performance verified by the test plate was used as the multifunctional microplate reader to be verified, and the microplate was selected as a 96-well plate. Equal amounts of neutral gum from Shanghai Aladdin Biochemical Technology Co., Ltd. (referred to as Brand A), from Sinopharm Chemical Reagent Co., Ltd. (referred to as Brand B), and from Shanghai Huashen Rehabilitation Equipment Co., Ltd. (referred to as Brand C) were selected and diluted with the environmentally friendly transparent agent of Wuhan Hongzi Biotechnology Co., Ltd., and three samples were prepared by diluting according to the volume ratio of neutral gum: environmentally friendly transparent agent 5:2, namely, samples containing Brand A neutral gum (referred to as Sample A), samples containing Brand B neutral gum (referred to as Sample B), and samples containing Brand C neutral gum (referred to as Sample C). Take a completely black 96-well plate (use the top reading mode for detection on the machine) and a black-walled transparent-bottom 96-well plate (use the bottom reading mode for detection on the machine); 0 μL, 30 μL, 60 μL, 90 μL, 120 μL, 150 μL, 180 μL, 210 μL, 240 μL, and 270 μL of sample A, sample B, and sample C are respectively added to the completely black 96-well plate and the black-walled transparent-bottom 96-well plate. Two parallel wells are set for each volume on each microplate, and the layout of the two 96-well plates is the same, as shown in Table 1.

[0051]

[0052] Table 1 Layout of 96-well plates

[0053] 1. Top reading mode detection

[0054] After sample preparation, add the all-black 96-well plate. On the same day, use Tecan Infinite M200 PRO multifunctional microplate reader to detect FITC fluorescence. The software settings are: excitation light 485nm, emission light 535nm, top reading, Z-position item selected calculated from E10 (i.e. the 10th well in the 5th row, generally the well with the strongest fluorescence is selected, and the well with the strongest fluorescence), Gain value selected "optimal" to automatically obtain the best value by the software, the Gain value automatically used by the software is 118, and the rest of the software settings are in accordance with standard operations. After the test, a standard curve is made with the added sample volume as the horizontal axis and the mean of the detected values ​​of the FITC fluorescence signal as the vertical axis. For each type of neutral gum, as the added sample volume increases, the FITC fluorescence signal gradually increases and shows a certain linear relationship (such as Figure 1 As shown), the correlation coefficient R 2 Greater than 0.97.

[0055] The black 96-well plates with samples were placed for 1 month, 2 months, 3 months, and 4 months (respectively Figure 2 ,3 , 4, and 5), and then use the same instrument to detect FITC fluorescence. The software settings are: excitation light 485nm, emission light 535nm, top reading, Z-position selected as calculated from E10, and Gain value selected as "optimal" to automatically obtain the best value by the software. The Gain values ​​automatically used by the software are: 116, 118, 120, and 121. The rest of the software settings are based on the sample configuration and the settings on the day of adding the 96-well plate. After the test, a standard curve was made with the added sample volume as the horizontal axis and the mean of the detected values ​​of the FITC fluorescence signal as the vertical axis. For each type of neutral gum, as the added sample volume increased, the fluorescence signal gradually increased and showed a certain linear relationship. The correlation coefficient R 2 Both are greater than 0.98.

[0056] 2. Bottom Reading Mode Detection

[0057] After the sample was prepared, it was added to a black-walled, transparent-bottomed 96-well plate. On the same day, the FITC fluorescence was detected using a Tecan infinite M200pro multifunctional microplate reader. The software settings were: excitation light 485nm, emission light 535nm, bottom reading, Gain value "optimal" was selected to obtain the automatic optimal value from the software. The Gain value automatically used by the software was 130, and the rest of the software settings were in accordance with standard operations. After the test, a standard curve was drawn with the added sample volume as the horizontal axis and the mean of the FITC fluorescence signal detection value as the vertical axis. For each type of neutral gum, as the added sample volume increased, the fluorescence signal gradually increased and showed a certain linear relationship (such as Figure 6 As shown), the correlation coefficient R 2 Greater than 0.98.

[0058] The black-walled, transparent-bottomed 96-well plates with samples were placed for 1 month, 2 months, 3 months, and 4 months (respectively Figure 7 , 8 , 9, and 10) and then use the same instrument to detect FITC fluorescence. The software settings are: excitation light 485nm, emission light 535nm, bottom reading, Gain value "optimal" is selected to obtain the automatic optimal value by the software. The Gain values ​​automatically used by the software are: 131, 132, 132, 133. The rest of the software settings are based on the sample configuration and the settings on the day of adding the 96-well plate. After the test, a standard curve is made with the added sample volume as the horizontal axis and the mean of the detection value of the FITC fluorescence signal as the vertical axis. For each type of neutral gum, as the added sample volume increases, the fluorescence signal gradually increases. Although the correlation coefficient decreases, it still shows a trend of increasing the detection value with increasing volume. The correlation coefficient R 2 Both are greater than 0.95.

[0059] Correlation coefficient R of bottom reading mode data 2 Correlation coefficient R for data without top reading mode 2 The reason may be: the top reading mode is direct reading, while the bottom reading mode needs to penetrate the bottom of the microplate for reading, so the top reading mode of most microplate readers is better than the bottom reading mode.

[0060] Depend on Figure 1-Figure 10 The data shown show that, whether it is the top-reading test of a completely black 96-well plate or the bottom-reading test of a black-walled transparent-bottom 96-well plate, the FITC fluorescence detection values ​​of the three neutral gums diluted with environmentally friendly transparent agents increase with the increase of the added sample volume over a period of at least four months, and show a good linear relationship, with a correlation coefficient R 2 Both are greater than 0.95.

[0061] Therefore, the instrument manager can use the sample provided by the present invention to verify the fluorescence detection function of the multifunctional microplate reader. The specific operation is as follows: dilute the neutral gum with an environmentally friendly transparent agent, add it to the 96-well plate according to the set volume gradient, and for at least 4 months thereafter, the 96-well plate with the sample can be regarded as a standard, and its green fluorescence intensity can be detected on the machine. It is only necessary to perform a correlation analysis between the detected fluorescence value and the volume of the diluted neutral gum added. If the correlation coefficient is greater than 0.9, it can be considered that the fluorescence detection function of the multifunctional microplate reader is good. This is a simple and convenient method for verifying the fluorescence detection function of the microplate reader.

[0062] Compared with plate reading verification using a test plate or configuring standard reagents to verify the fluorescence detection function of an instrument, after the sample provided by the present invention is added to the 96-well plate, the 96-well plate with the sample has the advantages of low cost, simple configuration, stable performance, easy use, and can be repeatedly used for at least four months. It can be widely used in the accuracy verification of the fluorescence (green fluorescence) detection function of a multifunctional microplate reader.

[0063] In the description of this specification, the description of the terms "one embodiment", "a preferred embodiment", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiment of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0064] The above embodiments are only for illustrating the technical idea of ​​the present invention, and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the present invention; any technology not involved in the present invention can be realized by existing technologies.

Claims

1. A sample for verifying the fluorescence detection function of a multifunctional microplate reader, characterized in that: The sample is composed of a mixture of neutral gum and an environmentally friendly transparent agent, wherein the neutral gum has the property of spontaneous green fluorescence.

2. The sample for verifying the fluorescence detection function of a multifunctional microplate reader according to claim 1, characterized in that: When the sample is mixed, the volume ratio of the neutral gum to the sample is greater than the volume ratio of the environmentally friendly transparent agent to the sample.

3. The sample for verifying the fluorescence detection function of the multifunctional microplate reader according to claim 2, characterized in that: The volume content of the components of the sample is: 55% to 90% of neutral gum, 15% to 45% of environmentally friendly transparent agent, and the sum of the volume content percentages of the components of the above samples is 100%.

4. The sample for verifying the fluorescence detection function of a multifunctional microplate reader according to claim 3, characterized in that: The volume ratio of the neutral gum to the sample is 65% to 80%, and the volume ratio of the environmentally friendly transparent agent to the sample is 20% to 35%.

5. The sample for verifying the fluorescence detection function of a multifunctional microplate reader according to claim 1, characterized in that: The neutral gum is from Shanghai Aladdin Biochemical Technology Co., Ltd., or from Sinopharm Chemical Reagent Co., Ltd., or from Shanghai Huashen Rehabilitation Equipment Co., Ltd.

6. The sample for verifying the fluorescence detection function of a multifunctional microplate reader according to claim 1, characterized in that: The environmentally friendly transparent agent comes from Wuhan Hongzi Biotechnology Co., Ltd. or from Hangzhou Mount Sinai Biotechnology Co., Ltd.

7. The sample for verifying the fluorescence detection function of a multifunctional microplate reader according to claim 1, characterized in that: The sample can be reused for no less than 4 months.

8. The sample for verifying the fluorescence detection function of a multifunctional microplate reader according to any one of claims 1 to 7, characterized in that: When the sample is used for verification, the detection value of the fluorescence signal increases with the increase of the amount of sample added, and the correlation coefficient of the linear relationship presented is greater than 0.

95.

9. A method for verifying a sample using the method of verifying the fluorescence detection function of a multifunctional microplate reader according to any one of claims 1 to 7, characterized in that: The steps of this verification method are as follows: S1. Add an environmentally friendly transparent agent into the neutral gum according to a set ratio, and evenly dilute the neutral gum to obtain a sample; S2, respectively aspirate 0 μL, 30 μL, 60 μL, 90 μL, 120 μL, 150 μL, 180 μL, 210 μL, 240 μL, and 270 μL of the sample and add them to the microplate to prepare a microplate with samples added; S3. Use the multifunctional microplate reader to be verified to detect the FITC fluorescence signal of the microplate with the sample added, and make a standard curve with the added sample volume as the horizontal axis and the detection value of the FITC fluorescence signal as the vertical axis. When the correlation coefficient of the standard curve is greater than 0.9, it proves that the fluorescence detection function of the multifunctional microplate reader meets the experimental requirements; S4. After placing the microplate with the sample in step S2 for a period of time, repeat step S3 to verify whether the fluorescence detection function of the multifunctional microplate reader meets the experimental requirements and at the same time verify the effectiveness of the sample in the microplate after being placed for a period of time.

10. The sample for verifying the fluorescence detection function of a multifunctional microplate reader according to claim 9, characterized in that: The microplate in step S2 is selected from a microplate capable of detecting the fluorescence signal of the top of the sample in the well and a microplate capable of detecting the fluorescence signal of the bottom of the sample in the well; the sample in step S2 is added to the microplate according to the requirement of setting at least two parallel wells for each volume.

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