A fluorescence quantitative analysis method for accurately correcting the fluorescence secondary inner filter effect
By correcting the fluorescence attenuation absorption index nopt, the fluorescence spectral correction and quantitative analysis problems caused by the fluorescence secondary internal filtration effect are solved, and higher measurement accuracy and accuracy are achieved.
Patent Information
- Application Number
- CN202211465532.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-11-22
- Publication Date
- 2025-06-24
- Estimated Expiration
- 2042-11-22
AI Technical Summary
The prior art is difficult to accurately correct the fluorescence secondary filtration effect, resulting in difficulties in fluorescence spectroscopy correction and quantitative analysis.
By obtaining the fluorescence spectrum and absorbance spectrum of a series of concentrated fluorescent substance solutions, setting the investigation range for correcting the absorption degree coefficient n of the fluorescence attenuation n after pretreatment, using the absorbance spectrum to correct the fluorescence spectrum at different values, calculating the distortion of the fluorescence spectrum of each concentration, determining the secondary internal filtration effect fluorescence attenuation absorption index nopt, and correcting the fluorescence spectrum based on nopt.
Accurate correction of the fluorescence secondary internal filtration effect is achieved, the linear range of fluorescence intensity prediction concentration is expanded, the error of the prediction model is reduced, and the measurement accuracy and accuracy are improved.
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Figure CN115718087B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of quantitative analysis, and in particular, to a fluorescence quantitative analysis method for accurately correcting the fluorescence secondary inner filter effect. Background Art
[0002] Fluorescence spectrometry has the advantages of high sensitivity, high accuracy, low detection limit, good selectivity, non-destructiveness, simplicity, etc., and is one of the most commonly used and reliable analysis methods. It provides rich information about molecular interactions, energy level transitions, and concentration changes of substances, and is widely used in fields such as biochemical analysis, material applications, clinical medical detection, environmental monitoring, food safety, and public security intelligence.
[0003] Fluorescence spectrometry is based on the relationship between fluorescence intensity and substance concentration. When measuring a low-concentration fluorescence solution, the detected fluorescence intensity F of the solution obs is proportional to the concentration c of the fluorescent substance solution. As the solution concentration further increases, there will be a phenomenon that the fluorescence intensity does not increase linearly with the solution concentration, and even decreases with the increase of the concentration, resulting in a deviation from the linear relationship between the fluorescence intensity and the concentration, and even causing distortion of the spectral shape. If the concentration is inversely deduced based on the fluorescence intensity and the linear relationship coefficient at this time, the predicted concentration value will cause a large deviation. This phenomenon can be explained by the inner filter effect of fluorescence. One is the primary inner filter effect (pIFE). When the solution concentration is too high, the incident light is strongly absorbed by the fluorescent substance in the front part of the sample cell, resulting in a decrease in the actual light intensity used for excitation, and thus a decrease in the fluorescence intensity. The other is the secondary inner filter effect (sIFE), which usually appears in a fluorescence solution where there is a large overlap between the absorption spectrum and the fluorescence emission spectrum. The overlap between the absorption spectrum and the fluorescence emission spectrum causes the emitted fluorescence to be reabsorbed by the sample.
[0004] Due to the existence of the fluorescence inner filter effect (IFE), the conventional fluorescence spectrometry cannot accurately quantify the substance concentration. Existing research regards the fluorescence inner filter effect as an error source in fluorescence analysis, resulting in fluorescence spectrum distortion, spectral shape distortion, and non-linearity between the fluorescence signal intensity and the fluorophore concentration. Many researchers have tried to restore the linear relationship between the fluorescence intensity and its concentration by deriving the IFE correction formula. Some researchers have modified the formula for the fluorescence intensity and the concentration of the fluorescent substance solution using absorbance. The modified formula is:
[0005]
[0006] where, F obs is the measured maximum fluorescence intensity, and F corr is the maximum fluorescence intensity after correcting the inner filter effect from F obs ; A ex and A emDenote the absorbance at the excitation wavelength and the emission wavelength respectively; s is the thickness of the excitation beam, g is the distance between the edge of the excitation beam and the edge of the cuvette (in the direction of the receiving end), and d is the width of the cuvette.
[0007] In 1994, Albinsson et al. gave the corrected fluorescence intensity of approximately
[0008]
[0009] Nettles et al. measured the Raman signal of water in a series of reference samples, obtained the change in Raman intensity caused by the inner filter effect in the sample, and made a simple linear fit through the relationship between the Raman intensity of water and absorbance and other quantities to determine the effective excitation path length d of the fluorometer ex and the emission path length d em , and then used formula (3) to more reliably correct the fluorescence and Raman inner filter effects, where L is the width of the cuvette used to measure the absorption spectrum of the sample.
[0010]
[0011] Ma et al. studied the sIFE of the fluorescence excitation spectrum of rhodamine 6G aqueous solution with changing concentration and proposed a mathematical model to solve the inflection point of the fluorescence excitation spectrum caused by sIFE.
[0012] Most of the correction formulas in the above-mentioned studies correct the inner filter effect based on the geometric structure of the cuvette and the geometric parameters of the beam, without considering the influence of the solute-solvent system of the fluorescent substance itself on the inner filter effect, nor considering that for the fluorescent substance with sIFE, as the solution concentration increases, dimers or multimers will be formed. Dimers or multimers have different contributions to the fluorescence spectrum and absorbance spectrum, and also have different influences on the absorption process of the emitted fluorescence. The traditional method of correcting the absorption process cannot meet the correction requirements, and the correction of problems such as the attenuation of the fluorescence spectrum intensity and the red shift of the fluorescence peak remains to be improved. Therefore, the fluorescence intensity formula corrected by absorbance for fluorescence intensity still cannot be used for the measurement and quantitative analysis of fluorescent solutions with sIFE. Summary of the Invention
[0013] Aiming at the defects in the prior art, the purpose of the present invention is to provide a fluorescence quantitative analysis method for accurately correcting the fluorescence secondary inner filter effect to solve the problems of spectral correction and quantification of fluorescent substances with secondary inner filter effect.
[0014] According to one aspect of the present invention, there is provided a fluorescence quantitative analysis method for accurately correcting the fluorescence secondary inner filter effect, the method comprising:
[0015] Obtain the fluorescence spectra and absorbance spectra of a series of solutions of fluorescent substances with different concentrations;
[0016] Preprocess the fluorescence spectrum and the absorbance spectrum, and set the investigation range of the correction fluorescence attenuation absorption degree coefficient n and the investigation segment of the spectral distortion degree;
[0017] According to the secondary inner filter effect fluorescence attenuation absorption index analysis model, use the absorbance spectrum to correct the fluorescence spectrum at all concentrations within the investigation range of n, and obtain the fluorescence spectra of the fluorescence solutions at all concentrations under different n values after correction;
[0018] Calculate the distortion degree of the fluorescence spectra at each concentration within the investigation range of n;
[0019] According to the minimum distortion degree within the full concentration range, determine the correction fluorescence absorption degree coefficient n at this time, which is the secondary inner filter effect fluorescence attenuation absorption index n of the fluorescent substance; opt ;
[0020] Use the absorbance spectrum to calculate the fluorescence spectra at all concentrations when n = n; opt to obtain the fluorescence spectra corrected based on n; opt The fluorescence spectrum after correction.
[0021] According to the fluorescence spectrum corrected based on n; opt to obtain the concentration of the solution to be measured; specifically, during the measurement process, the fluorescence spectrum after correction when n = n; opt is the spectrum that needs to be processed and obtained in both the modeling and prediction stages. In the modeling stage, the solutions at each concentration are processed through the above steps to obtain the fluorescence spectra corrected based on n; opt for modeling, and establish a "fluorescence intensity - concentration" linear relationship with the concentration values of the fluorescence solutions; in the prediction stage, the solutions with unknown concentrations are processed through the above steps to obtain the fluorescence spectra corrected based on n; opt for the solution, and according to the above "fluorescence intensity - concentration" linear relationship, obtain the concentration of the solution to be measured, realizing a more accurate measurement of the concentration of the fluorescence solution.
[0022] Furthermore, when setting the investigation range of the correction fluorescence attenuation absorption degree coefficient n and the investigation segment of the spectral distortion degree, where: the investigation segment of the spectral distortion degree is the wavelength segment where the absorbance spectrum and the fluorescence spectrum overlap.
[0023] Furthermore, when setting the investigation range of the correction fluorescence attenuation absorption degree coefficient n and the investigation segment of the spectral distortion degree, where: the investigation range is set according to the lowest point of the distortion degree, and the investigation range covers the lowest point of the distortion degree.
[0024] Furthermore, according to the secondary inner filter effect fluorescence attenuation absorption index analysis model, where the secondary inner filter effect fluorescence attenuation absorption index analysis model is:
[0025]
[0026] n opt represents the fluorescence attenuation absorption index of the secondary inner filter effect, A em represents the absorbance at the emission wavelength, d em represents the emission path length, L represents the sum of the excitation path length and the emission path length, F obs represents the measured fluorescence intensity, F oorr represents from F obs the maximum fluorescence intensity after correcting the inner filter effect.
[0027] Furthermore, in the fluorescence attenuation absorption index analysis model based on the secondary inner filter effect, where: the determination process of the fluorescence attenuation absorption index analysis model of the secondary inner filter effect includes:
[0028] Based on the fluorescence intensity formula after correcting the fluorescence intensity with absorbance, a coefficient for correcting absorption is added before the absorbance of the emitted fluorescence to characterize the degree of absorption to be corrected. The corresponding formula I is:
[0029]
[0030] where, F obs represents the measured fluorescence intensity, F corr represents from F obs the maximum fluorescence intensity after correcting the inner filter effect; A ex and A em respectively represent the absorbances at the excitation wavelength and the emission wavelength; d ex represents the excitation path length, d em represents the emission path length, L represents the sum of the excitation path length and the emission path length;
[0031] Regarding the influence of the primary inner filter effect and the influence of the secondary inner filter effect as two parts, then formula I is written as:
[0032]
[0033] Determine the optimal value of the fluorescence attenuation absorption degree coefficient n according to the similarity of the fluorescence spectrum shape and whether the peak is corrected at the same wavelength, that is, n opt , then:
[0034]
[0035] Furthermore, calculate the distortion degree of the fluorescence spectra at each concentration within the investigation range of n, where: the distortion degree is represented by any one of the Euclidean distance, the correlation coefficient, and the spectral angle.
[0036] Further, preprocess the fluorescence spectrum and the absorbance spectrum, including: removing the baseline generated by the electrical signal drift and the dark current in the spectrometer, and removing the spectra with gross errors according to the Chauvenet's criterion.
[0037] Further, obtain the fluorescence spectra and absorbance spectra of a series of solutions of fluorescent substances, where: the device for measuring the fluorescence spectrum includes a light source, a collimation system, a cuvette, a converging system, and a detector, and the converging system is disposed in the direction perpendicular to the excitation light in the detection plane and on the center line of the exit surface of the cuvette; the excitation light emitted by the light source is collimated by the collimation system and then enters from the center of the entrance surface of the cuvette to excite the fluorescent solution, and the fluorescence converged and emitted by the converging system is received by the detector.
[0038] Further, obtain the fluorescence spectra and absorbance spectra of a series of solutions of fluorescent substances, where: the device for measuring the absorbance spectrum is a spectrophotometer.
[0039] Compared with the prior art, the present invention has at least one of the following beneficial effects:
[0040] The fluorescence decay absorption index n of the optimized parameter - secondary inner filter effect (sIFE) reflecting the self-absorption effect proposed by the present invention opt , when receiving fluorescence in the direction perpendicular to the incident light, is only related to the solute-solvent system of the fluorescent substance itself, and has nothing to do with the geometric parameters of the cuvette and the light beam and the concentration of the fluorescent substance. n opt Can accurately reflect the fluorescence decay degree caused by the secondary inner filter effect of different fluorescent substances and can correct for any non-ideal excitation or emission beam shape. Based on n opt The fluorescence spectrum correction method can determine n opt , correct the intensity decay and peak red shift of the fluorescence spectrum caused by sIFE, expand the linear range of the fluorescence intensity to predict the concentration, reduce the error of the prediction model, and improve the measurement accuracy and precision. n opt Greatly corrects the fluorescence experiment under the interference of sIFE, solves the problems of spectral correction and quantification of fluorescent substances with secondary inner filter effect, and provides a new means for correcting the inner filter effect in the field of fluorescence quantitative analysis. BRIEF DESCRIPTION OF THE DRAWINGS
[0041] By reading the following detailed description of the non-limiting embodiments with reference to the accompanying drawings, other features, objects, and advantages of the present invention will become more apparent:
[0042] Figure 1 It is a schematic flowchart of a fluorescence quantitative analysis method for accurately correcting the fluorescence secondary inner filter effect according to an embodiment of the present invention;
[0043] Figure 2Schematic diagram of the fluorescence measurement principle according to an embodiment of the present invention;
[0044] Figure 3 Among them, (a) is the uncorrected absorbance spectrum of the sodium fluorescein aqueous solution according to an embodiment of the present invention, and (b) is the uncorrected fluorescence spectrum of the sodium fluorescein aqueous solution according to an embodiment of the present invention;
[0045] Figure 4 Optimization results of n in the sodium fluorescein aqueous solution according to an embodiment of the present invention; wherein, (a) is a three-dimensional color map, and (b) is a two-dimensional curve graph;
[0046] Figure 5 Corrected fluorescence spectrum of the sodium fluorescein aqueous solution according to an embodiment of the present invention;
[0047] Figure 6 Analysis of the correction effect of the sodium fluorescein aqueous solution according to an embodiment of the present invention; wherein, (a) is a graph showing the relationship between the fluorescence intensity before and after correction and the concentration at 505 nm, (b) is a graph showing the relationship between the fluorescence intensity before and after correction and the concentration at 515 nm, (c) is a graph showing the relationship between the integrated fluorescence intensity before and after correction and the concentration from 513 nm to 518 nm, and (d) is a graph showing the relationship between the fluorescence intensity before and after correction and the concentration at 550 nm. Detailed implementation manners
[0048] The present invention will be described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that those of ordinary skill in the art can make several deformations and improvements without departing from the concept of the present invention. These all belong to the protection scope of the present invention.
[0049] An embodiment of the present invention provides a fluorescence quantitative analysis method for accurately correcting the fluorescence secondary inner filter effect. Referring to Figure 1 , this method first determines the optimization parameter of the fluorescence self-absorption effect - the fluorescence attenuation absorption index nopt of the secondary inner filter effect, and corrects the fluorescence spectrum based on this index, including:
[0050] S1, obtaining the fluorescence spectra and absorbance spectra of a series of concentration fluorescence substance solutions;
[0051] S2, preprocessing all the obtained fluorescence spectra and absorbance spectra, and setting the investigation range of the correction fluorescence attenuation absorption degree coefficient n and the investigation section of the spectral distortion degree;
[0052] S3, according to the fluorescence attenuation absorption index analysis model of the secondary inner filter effect, using the absorbance spectrum to correct the fluorescence spectra at all concentrations within the investigation range of n, and obtaining the fluorescence spectra of the fluorescence solutions at all concentrations under different n values after correction;
[0053] S4, calculate the distortion degree of the fluorescence spectrum of each concentration within the investigation range of n;
[0054] S5, according to the minimum distortion within the full concentration range, determine the corrected fluorescence absorption coefficient n at this time, which is the secondary inner filter effect fluorescence attenuation absorption index n of the fluorescent substance. opt ;
[0055] S6, using the absorbance spectrum to calculate all concentrations when n = n opt The fluorescence spectrum based on n opt Corrected fluorescence spectra.
[0056] S7, according to step S6 based on n opt The corrected fluorescence spectrum is used to obtain the concentration of the solution to be tested; specifically, during the measurement process, n = n opt The corrected fluorescence spectrum at the time of n is the spectrum that needs to be processed in both the modeling and prediction stages. In the modeling stage, each concentration solution is processed by the above steps S1-S6 to obtain the modeled n-based opt The corrected fluorescence spectrum is used to establish a linear relationship between fluorescence intensity and concentration with the concentration value of the fluorescent solution. In the prediction stage, the solution of unknown concentration is processed through the above steps S1-S6 to obtain the solution based on n opt The corrected fluorescence spectrum is used to obtain the concentration of the solution to be tested based on the above-mentioned "fluorescence intensity-concentration" linear relationship, thereby achieving more accurate fluorescence solution concentration measurement.
[0057] In some embodiments, in step S1, the device for measuring the fluorescence spectrum includes a light source, a collimation system, a cuvette, a converging system and a detector; wherein the light source may be a light source such as LED, LD, laser, etc., and the wavelength is selected according to the excitation wavelength of the fluorescent substance; the collimation system may be a collimation structure such as a convex lens, a collimation lens barrel, an optical fiber-optic fiber collimator, etc.; the converging system is arranged in the detection plane perpendicular to the direction of the excitation light and located on the center line of the exit surface of the cuvette, and the converging system may be a converging structure such as a convex lens, an optical fiber-optic fiber collimator, etc.; the detector may be a detection structure such as a spectrometer that can obtain full spectrum data. After the excitation light emitted by the light source is collimated by the collimation system, it is incident from the center of the incident surface of the cuvette to excite the fluorescent solution, and the fluorescence emitted by the converging system is transmitted to the detector to be received. It should be noted that in the specific implementation, the specific devices and structures in the above-mentioned light source, collimation system, converging system, and detector are not limited to the above-mentioned ones, and can be determined according to application requirements.
[0058] The fluorescence spectra and absorbance spectra of a series of fluorescent substance solutions with different concentrations are obtained, wherein the device for measuring the absorbance spectrum is a spectrophotometer.
[0059] In some embodiments, in step S2, all the acquired fluorescence spectra and absorbance spectra are preprocessed, including: removing the baseline generated by the electrical signal drift and dark current in the spectrometer, and eliminating the spectra with gross errors according to the Chauvenet's criterion.
[0060] In some embodiments, in step S2, the investigation range of the correction coefficient n for the fluorescence attenuation absorption degree and the investigation segment of the spectral distortion degree are set, where: the investigation segment of the spectral distortion degree is the wavelength segment where the absorbance spectrum and the fluorescence spectrum overlap. The investigation range is set according to the lowest point of the distortion degree. For example, in Figure 4 b, the lowest point is approximately around n = 2.0. The overall distortion degree is a process of first decreasing and then increasing. Therefore, the investigation range needs to cover the range around n = 2.0. The investigation range is 0 - 3.2, and the investigation step size is 0.1, so as to find the n value corresponding to the lowest point of the distortion degree and make n opt as accurate as possible. Those skilled in the art can understand that according to Figure 4 the lowest point of the distortion degree in b, the investigation range can also be 1.5 - 3, or 1.8 - 2.2; in order to make n opt more accurate, the investigation step size can be 0.01 or even 0.001.
[0061] In some embodiments, in step S3, according to the secondary inner filter effect fluorescence attenuation absorption index analysis model, where the secondary inner filter effect fluorescence attenuation absorption index analysis model is:
[0062]
[0063] n opt represents the secondary inner filter effect fluorescence attenuation absorption index, A em represents the absorbance at the emission wavelength, d em represents the emission path length, L represents the sum of the excitation path length and the emission path length, F obs represents the measured fluorescence intensity, F oorr represents the maximum fluorescence intensity after correcting the inner filter effect from F obs .
[0064] Furthermore, according to the secondary inner filter effect fluorescence attenuation absorption index analysis model, where: the determination process of the secondary inner filter effect fluorescence attenuation absorption index analysis model includes:
[0065] S31, for a certain determined fluorescent substance, there is an optimal n, that is, n opt . When receiving fluorescence in the direction perpendicular to the incident light, n opt is only related to the solute-solvent system of the fluorescent substance itself, and has nothing to do with the geometric parameters of the cuvette and the light beam and the concentration of the fluorescent substance. For the convenience of subsequent description, nopt Named "secondary inner filter effect fluorescence attenuation absorption index". n opt It can accurately reflect the fluorescence attenuation degree of different fluorescent substances caused by sIFE and can correct for any non-ideal excitation or emission beam shape.
[0066] Based on the fluorescence intensity formula after correcting the fluorescence intensity using absorbance, a coefficient n for correcting absorption is added before the absorbance of the emitted fluorescence opt to characterize the degree of absorption to be corrected, and the corresponding formula I is:
[0067]
[0068] where, F obs represents the measured fluorescence intensity, F corr represents the maximum fluorescence intensity after correcting the inner filter effect from F obs ; A ex and A em represent the absorbances at the excitation wavelength and the emission wavelength respectively; d ex represents the excitation path length, d em represents the emission path length, and L represents the sum of the excitation path length and the emission path length; the corresponding measurement schematic diagram is as Figure 2 shown.
[0069] S32, considering the influence of the primary inner filter effect and the influence of the secondary inner filter effect as two parts, then formula I is written as:
[0070]
[0071] S33, since pIFE is the main influence on the spectral intensity due to the absorption of the excitation light, the fluorescence spectrum does not change in spectral shape, while sIFE is due to the absorption of the emitted fluorescence, and the superposition of the absorption spectrum and the emission spectrum will cause a change in the spectral shape of the fluorescence spectrum. Intuitively, it is that the wavelength position of the peak moves with the concentration, and the fluorescence intensity also decreases. The optimal value of n can be determined according to the similarity of the fluorescence spectrum shape and whether the peak is corrected at the same wavelength, and is not affected by the previous item. Therefore, when solving for n opt , the latter item can be temporarily focused on. Y Therefore, the optimal value of the fluorescence attenuation absorption degree coefficient n is determined according to the similarity of the fluorescence spectrum shape and whether the peak is corrected at the same wavelength, that is, n opt , then:
[0072]
[0073] In some embodiments, in step S4, the distortion degree of the fluorescence spectra at various concentrations within the investigation range of n is calculated, where: the distortion degree D is a parameter characterizing the spectral similarity, and the distortion degree can be represented by any one of the Euclidean distance, the correlation coefficient, and the spectral angle. In some other embodiments, the distortion degree can also be represented by other parameters, as long as the same functions can be achieved.
[0074] In the above embodiments of the present invention, an optimized parameter reflecting the self-absorption effect of the fluorescent substance - the fluorescence attenuation absorption index n of the secondary inner filter effect (sIFE) is proposed. opt . When receiving fluorescence in the direction perpendicular to the incident light, n opt is only related to the solute-solvent system of the fluorescent substance itself, and is independent of the geometric parameters of the cuvette and the light beam, as well as the concentration of the fluorescent substance. n opt can accurately reflect the degree to which the fluorescence is affected by sIFE and can correct for any non-ideal excitation or emission beam shapes. Therefore, after determining n opt and applying n opt to correct the fluorescence spectrum, the attenuation of the fluorescence spectrum intensity and the peak red shift caused by the secondary inner filter effect can be corrected, the linear range of the concentration predicted according to the fluorescence intensity can be expanded, the relative error of the prediction model can be reduced, and the measurement accuracy and precision can be improved.
[0075] In a specific embodiment, the sample to be measured is an aqueous solution of sodium fluorescein with 10 concentration gradients in the concentration range of 2.5 mg / L - 25 mg / L. In some other embodiments, other fluorescent solutions with sIFE can also be selected, including but not limited to aqueous solutions of rhodamine B, ethanol solutions of rhodamine B, glycerol solutions of rhodamine B, aqueous solutions of rhodamine 6G, and ethanol solutions of chlorophyll, etc.
[0076] The device for measuring the fluorescence spectrum of the fluorescent solution includes a light source, a collimation system, a cuvette, a converging system, and a detector. Specifically, the light source selects an LED with a central wavelength of 460 nm, the collimation system and the converging system both select concave lenses, and the detector selects a spectrometer with the model QE65000 from Ocean Optics. The excitation light emitted by the LED is collimated by a convex lens, enters from the center of the incident surface of a four-way cuvette with an optical path of 10 mm, excites the fluorescent solution, and a convex lens is set on the center line of the exit surface of the cuvette in the direction perpendicular to the excitation light in the detection plane to converge the emitted fluorescence, which is then transmitted through an optical fiber into the spectrometer for reception.
[0077] The device used to measure the absorbance spectrum of the fluorescent solution is a spectrophotometer, specifically, a double-beam UV-visible spectrophotometer of model TU-1901. In some other embodiments, other types of spectrophotometers can also be used, not limited to the UV-visible band. The optical path of the cuvette for absorbance measurement is determined according to the actual fluorescence measurement d em Specifically, when the detected fluorescence is emitted at the center of the fluorescence cuvette, d ex =d em =5mm. Therefore, a cuvette with an optical path of 5mm is selected as the cuvette for absorbance measurement.
[0078] A fluorescence quantitative analysis method for accurately correcting the fluorescence secondary inner filter effect comprises the following steps:
[0079] Step 1: Weigh 12.5 mg of sodium fluorescein (AR, Shanghai MacLean Biochemical Co., Ltd.) powder using a high-precision electronic balance and dissolve it in a 250 mL volumetric flask to prepare a 50 mg / L sodium fluorescein aqueous solution. Dilute the solution with deionized water to form a series of sodium fluorescein aqueous solutions with a concentration gradient of 2.5 mg / L-25 mg / L, with concentrations of 2.5 mg / L, 5 mg / L, 7.5 mg / L, 10 mg / L, 12.5 mg / L, 15 mg / L, 17.5 mg / L, 20 mg / L, 22.5 mg / L, and 25 mg / L, respectively.
[0080] Step 2: Place the 10 prepared sodium fluorescein aqueous solutions of different concentrations into a UV spectrophotometer and measure their absorbance spectra. Figure 3 (a) is shown. Then, the sodium fluorescein aqueous solution is placed in the fluorescence device to measure its fluorescence spectrum. The integration time is set to 100ms. The fluorescence spectrum of each sample concentration is measured 50 times. The spectrum with excessive fluctuation is eliminated, and the remaining spectra are averaged. The measured fluorescence spectrum is shown in Figure 3 (b) as shown.
[0081] Step 3: Preprocess all spectra, remove the baseline generated by the electrical signal drift and dark current in the spectrometer, and eliminate spectra with gross errors according to the Laida criterion; set the investigation range of the correction fluorescence attenuation absorption coefficient n to 0-3.2, and the investigation step to 0.1; set the investigation section of the spectral distortion D to 490nm-506nm;
[0082] Step 4: According to the formula The fluorescence spectra at all concentrations of n within the range of 0-3.2 are corrected using the absorbance spectrum to obtain the corrected fluorescence spectrum;
[0083] Step 5: Calculate the distortion degree D of the fluorescence spectra at each concentration when n is within 0 - 3.2. In this embodiment, the distortion degree D is the Euclidean distance between the normalized fluorescence spectrum corrected for each concentration solution and the standardized fluorescence spectrum corrected for the lowest concentration solution; the results are as Figure 4 (a) and Figure 4 (b) shown;
[0084] Step 6: According to the minimum distortion degree D within the full concentration range min , determine that the corrected fluorescence absorption degree coefficient n at this time is 2.0, that is, the secondary inner filter effect fluorescence attenuation absorption index n opt = 2.0.
[0085] Step 7: According to the formula Use the absorbance spectrum to calculate the fluorescence spectra at all concentrations when n = n opt = 2.0, and obtain the corrected fluorescence spectra, as Figure 5 shown.
[0086] Result analysis:
[0087] From Figure 3 (a) and Figure 3 (b) comparison, it can be seen that the absorption spectrum of sodium fluorescein overlaps with the fluorescence spectrum in the wavelength range of 470 nm - 530 nm. Part of the fluorescence will be absorbed by itself, which means that sIFE occurs. sIFE will not only reduce the peak intensity, but also cause an obvious red shift of the peak wavelength with the increase of concentration. In Figure 3 (b), the 515 nm standard line is marked with a solid line, and the wavelengths of the corrected fluorescence spectrum peaks of different concentration solutions are marked with dotted lines. The red shift of the wavelength can be observed by comparing the dotted line and the solid line.
[0088] In Figure 4 (a), the abscissa represents the concentration of the sodium fluorescein aqueous solution, and the ordinate represents n. The intersection of the concentration and n is the distortion degree D of the fluorescence spectrum. The smaller the distortion degree D, the better, indicating a high spectral similarity and a good correction effect. That is, among all concentrations, the n at the point where D is the smallest is the n opt of the fluorescence substance solution. It can be seen from the color bar on the right that the closer the distortion degree D is to black, the better the result. When the value of n is distributed around 2.0, the distortion degree D within the entire concentration range is the smallest. Observing this result from the two-dimensional curve graph, as Figure 4 (b) shown. The change of the distortion degree D of sodium fluorescein at different concentrations with n is represented by curves of different grayscales. When the value of D is distributed around 2.0, the distortion degree D within the entire concentration range is the smallest.
[0089] Therefore, n optIt can be selected as 2.0. Calibrated fluorescence spectra of aqueous solutions of sodium fluorescein at a series of concentrations are plotted as shown in Figure 5 . The 515 nm standard line is marked with a solid line, and the wavelengths at which the peaks of the calibrated fluorescence spectra of solutions with different concentrations are located are marked with dashed lines. In Figure 5 , the solid line coincides with the dashed line. The peak wavelength for each concentration is 515 nm, and as the concentration increases, the red shift of the peak wavelength is corrected, indicating that the fluorescence peak shift and spectral shape change caused by sIFE are successfully corrected.
[0090] As shown in Figure 6 , it is necessary to show the relationship between the calibrated fluorescence intensity and the concentration to more clearly evaluate the calibration effect. In Figure 6 (b), the linear range of the concentration predicted by the calibrated fluorescence spectrum is extended from 12.5 mg / L to 15 mg / L, an increase of 20%. The linear correlation coefficient R of the fitting curves for the first six groups of concentrations 2 increases from 0.9873 to 0.9920. The linear range of the concentration predicted by the calibrated fluorescence spectrum is extended from 12.5 mg / L to 15 mg / L, an increase of 20%. The linear correlation coefficient R of the fitting curves for the first six groups of concentrations 2 increases from 0.9876 to 0.9921. By comparing Figure 6 (b) and Figure 6 (c), it can be seen that modeling by the fluorescence peak intensity or by integrating the fluorescence intensity within a wavelength range around the fluorescence peak wavelength has little effect on the linear correlation coefficient R 2 . Therefore, to facilitate modeling, the fluorescence peak intensity is selected to characterize the fluorescence intensity of the aqueous sodium fluorescein solution at this concentration.
[0091] However, in Figure 6 (b) and Figure 6 (c), there is still non-linearity at high concentrations. During the experiment, spectral measurements are often inaccurate near the detection upper limit of the spectrometer, resulting in non-linearity. The fluorescence intensity detection upper limit of the QE65000 spectrometer is 65535. In the experiment, all fluorescence data within about 2 / 3 of the detection upper limit are regarded as valid measurement data, which means that the highest peak intensity is about 43690. Fluorescence spectral data at a wavelength of 505 nm are selected to establish the relationship with the concentration to explore this effect. In Figure 6 (a), the linear range of the concentration predicted by the calibrated fluorescence spectrum is extended, and the linear correlation coefficient R of the fitting curves for the first five groups of concentrations 2 increases from 0.9737 to 0.9954. The results show that the non-linearity is not caused by the inaccuracy of spectral measurements near the fluorescence intensity detection upper limit of the spectrometer. If this possibility is excluded, it may be caused by the spatial attenuation of the excitation light in pIFE.
[0092] In the experiment, even if data has been obtained in the absorbance range of 0.2 - 0.8 and the experimental setups for fluorescence and absorbance are separated from each other, pIFE inevitably occurs, resulting in non-linearity at high concentrations. Since the aqueous solution of sodium fluorescein does not absorb light with a wavelength of 550 nm, therefore Figure 6 the fluorescence in (d) is almost the same before and after correction. The linear correlation coefficient R of the concentration fitting curves for the first six groups 2 is 0.989. When the fluorescence intensity in the figure does not exceed 2 / 3 of the detection upper limit of the spectrometer, and after excluding absorbance interference and being corrected by the algorithm proposed in this paper, non-linearity still appears at high concentrations, which indicates that the non-linearity is indeed caused by pIFE and spatial attenuation of the excitation light occurs.
[0093] The above fluorescence quantitative analysis method for correcting the fluorescence secondary inner filter effect determines the n of the aqueous solution of sodium fluorescein in this embodiment opt , and corrects its fluorescence spectrum affected by the secondary inner filter effect, expands the linear range of the predicted concentration of the fluorescence intensity, reduces the error of the prediction model, and improves the measurement accuracy and precision.
[0094] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various deformations or modifications within the scope of the claims, which does not affect the essence of the present invention. The above preferred features can be used in any combination without conflict.
Claims
1. A fluorescence quantitative analysis method for accurately correcting fluorescence secondary inner filter effect, characterized in that, include: Obtain fluorescence spectra and absorbance spectra of fluorescent substance solutions with a series of concentrations; Preprocessing the fluorescence spectrum and the absorbance spectrum, and setting an investigation range for correcting the fluorescence attenuation absorption coefficient n and an investigation section for the spectrum distortion; According to the secondary inner filter effect fluorescence attenuation absorption index analysis model, the fluorescence spectra at all concentrations within the investigated range of n are corrected using the absorbance spectrum to obtain the corrected fluorescence spectra of all concentrations of fluorescent solutions at different n values; Calculate the degree of distortion of the fluorescence spectrum of each concentration within the investigated range of n; According to the minimum distortion degree within the full concentration range, determine the corrected fluorescence absorption degree coefficient n at this time, which is the secondary inner filter effect fluorescence attenuation absorption index n of this fluorescent substance opt ; Calculate the fluorescence spectra at all concentrations using the absorbance spectra when n = n opt to obtain the fluorescence spectra corrected based on n opt ; According to the fluorescence spectrum based on n opt After calibration, the concentration of the solution to be measured can be obtained; Among them: the inspection section of the spectral distortion is the wavelength section where the absorbance spectrum and the fluorescence spectrum overlap; the inspection range is set according to the lowest point of the distortion, and the inspection range covers the lowest point of the distortion; The secondary inner filter effect fluorescence attenuation absorption index analysis model is: n opt represents the fluorescence attenuation absorption index of the secondary inner filter effect, A em represents the absorbance at the emission wavelength, d em represents the emission path length, L represents the sum of the excitation path length and the emission path length, F obs represents the measured fluorescence intensity, F corr represents from F obs the maximum fluorescence intensity after correcting the inner filter effect in 2. The fluorescence quantitative analysis method for accurately correcting the fluorescence secondary inner filter effect according to claim 1, characterized in that, The secondary inner filter effect fluorescence attenuation absorption index analysis model, wherein: the determination process of the secondary inner filter effect fluorescence attenuation absorption index analysis model includes: Based on the fluorescence intensity formula after using absorbance to correct the fluorescence intensity, a correction absorption coefficient is added before the emission fluorescence absorbance to characterize the degree of absorption that needs to be corrected. The corresponding formula I is: Among them, F obs represents the measured fluorescence intensity, and F corr represents the maximum fluorescence intensity after correcting the inner filter effect from F obs ; A ex and A em represent the absorbances at the excitation wavelength and the emission wavelength, respectively; d ex represents the excitation path length, d em represents the emission path length, and L represents the sum of the excitation path length and the emission path length; Considering the influence of the primary inner filter effect and the influence of the secondary inner filter effect as two parts, Formula I can be written as: Determine the optimal value of the fluorescence attenuation absorption degree coefficient n according to the similarity of the fluorescence spectrum shape and whether the peak values are corrected to the same wavelength, that is, n opt , then:
3. The fluorescence quantitative analysis method for accurately correcting the fluorescence secondary inner filter effect according to claim 1, characterized in that, The calculation is performed for the distortion degree of the fluorescence spectrum of each concentration within the investigation range of n, wherein the distortion degree is represented by any one of the Euclidean distance, the correlation coefficient and the spectral angle.
4. The fluorescence quantitative analysis method for accurately correcting the fluorescence secondary inner filter effect according to claim 1, wherein The fluorescence spectrum and the absorbance spectrum are preprocessed, including: removing the baseline generated by the electric signal drift and dark current in the spectrometer, and eliminating the spectrum containing gross errors according to the Laida criterion.
5. The fluorescence quantitative analysis method for accurately correcting the fluorescence secondary inner filter effect according to claim 1, wherein The method obtains fluorescence spectra and absorbance spectra of a series of fluorescent substance solutions with different concentrations, wherein: a device for measuring the fluorescence spectrum includes a light source, a collimation system, a cuvette, a converging system and a detector; the converging system is arranged in a detection plane in a direction perpendicular to the excitation light and located on the center line of the exit surface of the cuvette; the excitation light emitted by the light source is collimated by the collimation system and then incident from the center of the incident surface of the cuvette to excite the fluorescent solution; the fluorescence emitted by the converging system is received by the detector.
6. The fluorescence quantitative analysis method for accurately correcting the fluorescence secondary inner filter effect according to claim 1, wherein The fluorescence spectra and absorbance spectra of a series of fluorescent substance solutions are obtained, wherein the device for measuring the absorbance spectrum is a spectrophotometer.