Concentration detection method for reducing detection limit of perforated plate fluorometer and improving detection repeatability

Abstract

The invention discloses a concentration detection method for reducing the detection limit of a perforated plate fluorometer and improving the detection repeatability. The concentration detection method comprises the following steps: acquiring a background correction coefficient A and a response coefficient K of a perforated plate by using a perforated plate fluorescence detector; and measuring the signal value Bnm of each hole site of the perforated plate without containing the solution and the signal value Snm of each hole site after the to-be-measured solution is added, and calculating the actual concentration of the sample solution in each hole. According to the method, a perforated plate background correction coefficient A is obtained in advance by using a perforated plate fluorescence detector, the contribution of a solvent in holes to a fluorescence background value is quantified, and then in the actual measurement process, empty hole signal values of all hole sites in the perforated plate can be obtained before a sample is filled, and then a zero point value is calculated in a back mode. According to the method, the zero point parameter of each hole in the perforated plate, which is neglected and omitted in a traditional method, is obtained, the analysis signal-to-noise ratio is increased, the detection limit of analysis is reduced, and the measurement repeatability and the measurement precision are improved.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, and in particular to a method for reducing the detection limit of a multi-well plate fluorometer and improving detection repeatability, which is mainly used in analysis fields such as food safety, environmental pollution, and biomolecule detection. Background Art

[0002] A multi-well plate fluorometer is a quantitative analysis instrument used for high-throughput fluorescence analysis. It is widely used in food safety, environmental testing, biomedicine and other fields. For the fluorescence quantitative analysis system, an accurate and stable background value is the basis for ensuring measurement accuracy and high sensitivity. However, in the field of high-throughput fluorescence quantitative analysis of multi-well plates, especially under ultraviolet / deep ultraviolet excitation conditions, the material uniformity, shape, and cleanliness between wells and between plates in the multi-well plate will cause deviations in the intensity of its Raman / Rayleigh scattered light and background fluorescence.

[0003] The multi-well plate is a disposable detection device. If the method of adding solvent to the wells to measure the zero point is adopted, then the solvent is emptied and blown dry, and then the sample is added to measure the zero point, not only the process is cumbersome, but also it is easy to introduce new pollution, such as dust particles, which affects the quantitative accuracy. The traditional zero point selection method generally selects any few wells in the 96 wells to add solvent, and takes the average value as the typical value of the zero point. However, this method ignores the existence of background differences between wells and plates, making the measurement repeatability (RSD) often not less than 5%. In addition, this difference will become the system noise of the analysis process in high-throughput analysis, superimposed on the electrical noise of the instrument, so that the detection limit of the instrument cannot be further improved. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a concentration detection method for reducing the detection limit of a multi-well plate fluorometer and improving the detection repeatability. By obtaining in advance the correlation between the zero-point value of an empty multi-well plate and that after adding a solvent, the correction coefficient of the well plate is obtained and written into the instrument. When the same multi-well plate is subsequently used for quantitative analysis, the zero-point value after adding the solvent can be back-calculated by measuring the empty plate signal only once, that is, only two scans are required during the high-throughput analysis process, and there is no need to perform additional zero-point parameter measurements on the multi-well plate after adding the solvent. This method provides an accurate zero point for high-throughput fluorescence analysis, improves measurement accuracy and repeatability while simplifying the operating process, thereby reducing the detection limit.

[0005] The technical means adopted by the present invention are as follows:

[0006] A concentration detection method for reducing the detection limit of a multi-well plate fluorometer and improving detection repeatability, comprising:

[0007] Using a multi-well plate fluorescence detector to obtain a multi-well plate background correction coefficient A and a response coefficient K, wherein the background correction coefficient A is the difference between the fluorescence detection signal of the multi-well plate after adding a solvent and the fluorescence detection signal of an empty plate, and the response coefficient K is the signal response generated by the multi-well plate fluorescence meter to a unit concentration of a substance to be tested;

[0008] Measure the signal value B of each well of the multi-well plate without solution nm , where n represents the column number and m represents the row number. The signal value S of each well after adding the test solution is measured. nm , calculate the actual concentration of the sample solution in each well according to the following formula:

[0009] C nm =[S nm -(B nm +A)] / K

[0010] Among them, C nm Indicates the actual concentration of the sample solution in each well.

[0011] Furthermore, the values ​​of the background correction coefficient A and the response coefficient K are related to the number of fluorescence detection light paths;

[0012] The number of detection light paths used when calculating the concentration of the solution at the well position to be measured is consistent with the fluorescence detection light path corresponding to the well position.

[0013] Furthermore, the background correction coefficient A is obtained by the following method:

[0014] Obtain the signal values ​​before and after adding solvent in more than 50 groups of different wells;

[0015] Perform a linear fit on the two sets of data, and the intercept of the fitting equation is the background correction coefficient A.

[0016] Furthermore, the response coefficient K is obtained in the following manner:

[0017] The concentration gradient solutions containing the analyte are detected in sequence, and a standard curve is made by comparing the fluorescence detection signal with the solution concentration. The slope of the standard curve is the K value.

[0018] Furthermore, the multi-well plate is a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, a 96-well plate or a 384-well plate.

[0019] Furthermore, the method adopts an ultraviolet fluorescence analysis system with an excitation light wavelength less than 400 nm.

[0020] The main difference between the present invention and the prior art is that the traditional method often chooses to add solvent to one or several wells in the multi-well plate, and uses this value as the measurement zero point of all wells in subsequent measurements, ignoring the existence of differences between wells and plates. However, the present method uses the method of inferring the solvent signal based on the empty well signal to obtain the zero point value after adding solvent to all wells on the multi-well plate, and corrects and offsets the differences between wells and plates ignored by the traditional method. This method not only improves the accuracy of fluorescence analysis, omits the experimental steps of measuring the zero point parameters after adding solvent, but also improves the detection signal-to-noise ratio, reduces the detection limit of analysis, and improves measurement repeatability and measurement accuracy.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] By back-calculating the influence of various scattering and fluorescence effects of the solvent and pool wall in the well on the signal value, the zero point value of each well position on the multi-well plate is obtained, thereby improving the accuracy, repeatability and sensitivity of high-throughput fluorescence quantitative analysis.

[0023] By obtaining in advance the correlation between the zero-point value of the empty porous plate and that after adding the solvent, the experimental step of measuring the zero-point parameter after adding the solvent is eliminated, thereby improving the detection speed.

[0024] The method has good robustness. When using multi-well plates of different specifications / apertures, sample volumes and solvents, the effect of the inner wall surface on the Rayleigh and Raman scattered light and fluorescence of the solvent varies. It is only necessary to re-calibrate the experiment for the 96-well plate of the specification and the fixed experimental parameters to obtain the background correction coefficient A under the 96-well plate of the specification and experimental conditions, and write it into the instrument. The algorithm can be applied to the subsequent analysis under the 96-well plate of the specification and experimental parameters. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0026] Figure 1 The present invention is a flow chart of a concentration detection method for reducing the detection limit of a multi-well plate fluorometer and improving detection repeatability.

[0027] Figure 2 is the correlation between the zero point value of the empty 96-well plate and the zero point value after adding the solvent in each channel of Example 1, and ah corresponds to 8 different background correction coefficient fitting lines.

[0028] Figure 3The results of random sample concentration calculation for 0.6 μg / L coumarin in Example 1, where a is the result of the fixed zero point method and b is the result of the method of this patent. DETAILED DESCRIPTION

[0029] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. 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 creative work should fall within the scope of protection of the present invention.

[0030] like Figure 1 As shown, the present invention provides a method for reducing the detection limit of a multi-well plate fluorometer and improving detection repeatability, characterized in that it comprises the following steps:

[0031] S1. Obtaining the correction coefficient: Use a multi-well plate fluorescence detector to obtain the multi-well plate background correction coefficient A and response coefficient K, wherein the background correction coefficient A is the difference between the fluorescence detection signal of the multi-well plate after adding the solvent and the fluorescence detection signal of the empty plate; the response coefficient K is the signal response generated by the multi-well plate fluorometer to the unit concentration of the substance to be tested.

[0032] Specifically, the number of background correction coefficient A and response coefficient K is related to the number of fluorescence detection light paths, and the parameters used to calculate the concentration of the solution at the well position to be measured should be consistent with the fluorescence detection light path corresponding to the well position.

[0033] Preferably, the background correction coefficient A in the present invention is the contribution of the scattering effect and background fluorescence effect caused by the solvent and the well wall in the detection well to the signal value, and the coefficient is affected by the type of solvent, the well volume of the 96-well plate, the well shape, and the sample volume. The intercept of the fitting equation obtained by measuring the signal values ​​before and after adding the solvent in more than 50 groups of different wells is linearly fitted for the two groups of data, which is the background correction coefficient A.

[0034] Further preferably, the response coefficient K in the present invention is obtained by sequentially detecting concentration gradient solutions containing the analyte, and making a standard curve of the fluorescence detection signal versus the solution concentration, and the slope of the standard curve is the K value.

[0035] S2. Sample analysis test: Measure the signal value B of each well of the multi-well plate without solution nm , where n represents the column number and m represents the row number; measure the signal value S of each well after adding the test solution nm ; The measured signal B nm , S nmThe background correction coefficient A and the response coefficient K are substituted into the formula to calculate the actual concentration C of the sample solution in each well. nm , the formula is C nm =[S nm -(B nm +A)] / K.

[0036] As a preferred embodiment of the present invention, the multi-well plate used in the scheme of the present invention is a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, a 96-well plate or a 384-well plate. The method of the present invention is used in a multi-well plate fluorometer ultraviolet fluorescence analysis system with an excitation light wavelength set to less than 400 nm.

[0037] The scheme and effect of the present invention are further described below through specific application examples.

[0038] Example 1

[0039] This embodiment discloses a concentration detection method for reducing the detection limit of a multi-well plate fluorometer and improving the detection repeatability. A homemade 96-well plate fluorometer is used, and its optical path part includes 8 fluorescence detection modules (excitation wavelength 365nm, detection of fluorescence at 450nm). The 8 detection modules form a detection array. During the measurement process, the fluorescence signal values ​​of 8 wells in each row of the 96-well plate are obtained simultaneously, and the signal values ​​of all 12 rows and 96 wells can be measured along the column direction. The 96-well plate used in the experiment is Corning's 3915 standard 96-well plate (well volume 360μL, sample volume 250μL). Coumarin 460 was quantitatively analyzed using a 30% ethanol-water solution as the blank solvent. The specific steps are as follows:

[0040] 1) Obtaining the correction coefficient:

[0041] Step 1: Establish the correlation between the zero-point value of the 96-well plate and the value after adding solvent. The background correction coefficient A is obtained by the intercept of the correlation curve between the signal values ​​before and after adding solvent in a total of 576 groups of wells in the previous experiment. n (n=1-8, corresponding to fluorescence detection modules 1-8 respectively), such as Figure 2 As shown in ah.

[0042] Step 2: Establish the working curve of 8 fluorescence detection modules for the analyte (coumarin 460), detect the concentration gradient solutions containing the analyte in turn, and make a standard curve of the fluorescence detection signal versus the solution concentration. The slope of the standard curve is the response coefficient K n (n=1-8, corresponding to fluorescence detection modules No. 1-8 respectively).

[0043] 2) Sample analysis and testing:

[0044] Step 1: Scan the well plate before adding samples to obtain the empty plate signal value B of all wells. nm (n=1~8, m=1~12).

[0045] Step 2: Add the sample to be tested to each well and scan it. Add 0.6 μg / L coumarin 460 solution to 8 random wells and add solvent to all other wells to obtain the measured value S of each well. nm (n=1~8,m=1~12), substitute into formula C nm =[S nm -(B nm + A)] / K and then the concentration value was obtained.

[0046] Figure 3 It is the measured concentration value obtained by adding 0.6μg / L coumarin 460 to 8 random wells in a 96-well plate. Table 1 lists the sensitivity, repeatability (RSD) and measurement accuracy calculated by the traditional fixed zero method and the patented method. Compared with the method using a fixed zero point, this method increases the sensitivity, repeatability (RSD) and measurement accuracy by 3.3, 3.9 and 4.5 times respectively.

[0047] Table 1 Sensitivity, repeatability (RSD) and measurement accuracy calculated using two methods

[0048]

[0049] Example 2

[0050] This embodiment discloses a concentration detection method for reducing the detection limit of a multi-well plate fluorometer and improving the detection repeatability. The commercial multi-well plate fluorometer Cary Eclipse is used, and its optical path part includes a fluorescence detection optical path. The instrument excitation wavelength is set to 340nm, and the fluorescence at 450nm is detected. The multi-well plate used in the experiment is Corning's 4517 standard 96-well plate (well volume 205μL sample volume 120μL). Ochratoxin A is quantitatively analyzed with a water blank solvent. The specific steps are as follows:

[0051] 1) Obtaining the correction coefficient:

[0052] Step 1: Establish the correlation between the zero-point value of the 96-well plate and the value after adding solvent. The background correction coefficient A is obtained by the intercept of the correlation curve between the signal values ​​before and after adding solvent in the 96 groups of wells in the previous experiment.

[0053] Step 2: Establish a working curve of the multi-well plate fluorometer for the analyte (ochratoxin A), detect the concentration gradient solutions containing the analyte in sequence, and make a standard curve of the fluorescence detection signal versus the solution concentration. The slope of the standard curve is the response coefficient K.

[0054] 2) Sample analysis and testing:

[0055] Step 1: Scan the well plate before adding samples to obtain the empty plate signal value B of all wells. nm (n=1-8, m=1-12)

[0056] Step 2: Add the sample to be tested to each well and scan it. Add 20 μg / L ochratoxin A to 8 random wells and add solvent to all the other wells to obtain the measured value S of each well. nm (n=1-8, m=1-12), and the concentration value is obtained by substituting into formula (1).

[0057] Compared with the method using a fixed zero point, this method improves the sensitivity, repeatability (RSD), and measurement accuracy by 2.1, 2, and 3 times, respectively.

[0058] Example 3

[0059] This example provides a concentration detection method for reducing the detection limit of a multi-well plate fluorometer and improving detection repeatability. A commercial multi-well plate fluorometer Cary Eclipse is used, and its optical path includes a fluorescence detection optical path. The instrument excitation wavelength is set to 280nm, and the fluorescence at 450nm is detected. The multi-well plate used in the experiment is ThermoScientific TM Armadillo AB2384B standard 384-well plate (well volume 40 μL, sample volume 25 μL). Methanol was used as the blank solvent for quantitative analysis of zearalenone. The specific steps are as follows:

[0060] 1) Obtaining the correction coefficient:

[0061] Step 1: Establish the correlation between the zero-point value of the empty multi-well plate and the value after adding solvent. The background correction coefficient A is obtained by the intercept of the correlation curve between the signal values ​​before and after adding solvent in a total of 384 groups of wells in the previous experiment.

[0062] Step 2: Establish a working curve of the multi-well plate fluorometer for the analyte (zearalenone), detect the concentration gradient solutions containing the analyte in sequence, and make a standard curve of the fluorescence detection signal versus the solution concentration. The slope of the standard curve is the response coefficient K.

[0063] 2) Sample analysis and testing:

[0064] Step 1: Scan the well plate before adding samples to obtain the empty plate signal value B of all wells. nm (n=1-16, m=1-24)

[0065] Step 2: Add the sample to be tested to each well and scan it. Add 500 μg / L zearalenone to 16 random wells and add solvent to all the remaining wells to obtain the measured value S of each well. nm (n=1-16, m=1-24), and the concentration value is obtained by substituting into formula (1).

[0066] Compared with the method using a fixed zero point, this method improves the sensitivity, repeatability (RSD), and measurement accuracy by 3.1, 3.5, and 4 times, respectively.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A concentration detection method for reducing the detection limit of a multi-well plate fluorometer and improving detection repeatability, characterized in that: include: Using a multi-well plate fluorescence detector to obtain a multi-well plate background correction coefficient A and a response coefficient K, wherein the background correction coefficient A is the difference between the fluorescence detection signal of the multi-well plate after adding a solvent and the fluorescence detection signal of an empty plate, and the response coefficient K is the signal response generated by the multi-well plate fluorescence meter to a unit concentration of a substance to be tested; Measure the signal value B of each well of the multi-well plate without solution nm , where n represents the column number and m represents the row number. The signal value S of each well after adding the test solution is measured. nm , calculate the actual concentration of the sample solution in each well according to the following formula: C nm =[S nm -(B nm +A)] / K Among them, C nm Indicates the actual concentration of the sample solution in each well.

2. A concentration detection method for reducing the detection limit of a multi-well plate fluorometer and improving detection repeatability according to claim 1, characterized in that: The values ​​of the background correction coefficient A and the response coefficient K are related to the number of fluorescence detection light paths; The number of detection light paths used when calculating the concentration of the solution at the well position to be measured is consistent with the fluorescence detection light path corresponding to the well position.

3. A concentration detection method for reducing the detection limit of a multi-well plate fluorometer and improving detection repeatability according to claim 1, characterized in that: The background correction coefficient A is obtained by the following method: Obtain the signal values ​​before and after adding solvent in more than 50 groups of different wells; Perform a linear fit on the two sets of data, and the intercept of the fitting equation is the background correction coefficient A.

4. A concentration detection method for reducing the detection limit of a multi-well plate fluorometer and improving detection repeatability according to claim 1, characterized in that: The response coefficient K is obtained in the following way: The concentration gradient solutions containing the analyte are detected in sequence, and a standard curve is made by comparing the fluorescence detection signal with the solution concentration. The slope of the standard curve is the K value.

5. A concentration detection method for reducing the detection limit of a multi-well plate fluorometer and improving detection repeatability according to claim 1, characterized in that: The multi-well plate is a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, a 96-well plate or a 384-well plate.

6. A concentration detection method for reducing the detection limit of a multi-well plate fluorometer and improving detection repeatability according to claim 3, characterized in that: The method adopts an ultraviolet fluorescence analysis system with an excitation light wavelength less than 400nm.

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