CN112903644B - Ultra-wide range fluorescence quantitative analysis method and fluorescence measurement system - Google Patents

Abstract

The invention provides a fluorescent quantitative analysis method and a fluorescent measurement system with an ultra-wide range, which comprise the following steps: establishing an analysis model and carrying out fluorescence quantitative analysis in specific measurement by using the analysis model; wherein, the establishing of the analysis model comprises: s1, establishing the relation between the received fluorescence intensity and the concentration of the fluorescent substance: s2, an analytical model is established for the specific fluorescent substance to be measured based on the above-mentioned relationship between the intensity of received fluorescence and the concentration of the fluorescent substance, thereby determining constants in the expression. The invention establishes a fluorescence emission model of a fluorescent substance solution with a certain volume based on the interaction and propagation rules between exciting light and fluorescent substances, deduces and obtains the accurate relation between the fluorescence intensity received in a large range from low concentration to high concentration and the concentration of the fluorescent substances, can solve the difficult problems of high-concentration fluorescent substance quantification and wide-range fluorescent substance quantification, and is not only suitable for low-concentration fluorescent substance quantification but also suitable for high-concentration fluorescent substance quantification.

Description

An ultra-wide range fluorescence quantitative analysis method and fluorescence measurement system

technical field

The invention relates to the technical field of quantitative analysis, in particular, to an ultra-wide-range fluorescence quantitative analysis method and a fluorescence measurement system.

Background technique

Fluorescence quantitative analysis method has the advantages of high sensitivity, good selectivity and simple operation, and is widely used in biochemical analysis, medical detection, environmental monitoring, food safety and other fields. In the conventional fluorescence quantitative analysis method, the solution of the fluorescent substance to be tested is usually placed in a cuvette, the excitation light of a certain wavelength is incident on the cuvette, and the emitted light is collected in a certain angular direction (such as the direction perpendicular to the incident light). Fluorescence signal, when the concentration of the fluorescent substance solution is not too large, the linear relationship between the fluorescence intensity and the concentration of the fluorescent substance solution:

入射光强度I₀。Among them, ε is the molar extinction coefficient, c is the concentration of the fluorescent substance solution, x is the optical path of the excitation light in the solution, and the fluorescence quantum yield of the fluorescent substance

Incident light intensity I ₀ .

Since the linear relationship of Lambert Beer's law is established only when the concentration of the solution is in a small range, and the excitation light is absorbed by the solution during the propagation in the solution and gradually weakened, that is, the excitation light has a spatial attenuation effect. When the concentration of the fluorescent substance is high, the conventional fluorescence quantitative analysis will cause a large error, so it is no longer applicable.

In order to expand the concentration application range of the fluorescence quantitative analysis method, some researchers modified the formula of the fluorescence intensity and the concentration of the fluorescent substance solution by using the absorbance. The revised fluorescence intensity formula is:

Among them, F _obs represents the fluorescence intensity received by the detector; F _ideal represents the fluorescence intensity corrected by the correction formula; A _Ex represents the absorbance value after the excitation light passes through the sample cell; A _Em represents the emission light after passing through the sample cell Absorbance value, s is the diameter when the cross section of the excitation beam is regarded as a uniform cylinder; g is the distance from the edge of the excitation beam to the edge of the sample cell, and d is the width of the sample cell.

In 1994, Albinsson et al. proposed a modified formula that was widely used later. The modified fluorescence intensity formula is:

After correction, the measurement range of fluorescence quantitative analysis has been expanded to a certain extent.

Limited by the linear range of Lambert Beer's law, when the concentration of the fluorescent substance is high, the relationship between the absorbance and the concentration deviates from the linear law. Moreover, due to the influence of the stray light of the instrument, the absorbance of the solution is difficult to be accurately measured at high concentrations. Therefore, the fluorescence intensity formula obtained by using absorbance to correct the fluorescence intensity at high concentration still cannot be used for the measurement and quantitative analysis of high-concentration fluorescent substances.

At present, for some applications where the concentration of fluorescent substances is high or the concentration range is wide, the quantitative monitoring and analysis of the fluorescent substances is still a difficult problem. For example: in the textile printing and dyeing industry, online measurement of a wide range of high-concentration fluorescent dye substances is required. Another example: the total concentration of free amino acids in normal adult plasma is 350-650 mg·L ^-1 , and the concentration varies greatly during the day and night. When using traditional fluorescence quantitative analysis to detect the concentration of free amino acids, it is necessary to dilute the plasma to a low concentration, and the dilution process will change the plasma. the state of certain substances.

SUMMARY OF THE INVENTION

Aiming at the defects in the prior art, the purpose of the present invention is to provide an ultra-wide-range fluorescence quantitative analysis method and a fluorescence measurement system to solve the difficult problems of high-concentration fluorescent substance quantification and wide-range fluorescent substance quantification.

The first aspect of the present invention provides an ultra-wide-range fluorescence quantitative analysis method, including: establishing an analysis model and using the analysis model to perform fluorescence quantitative analysis in specific measurements; wherein,

The establishment of an analysis model includes:

S1, establish the relationship between the received fluorescence intensity and the concentration of the fluorescent substance:

Under the condition of single-wavelength excitation light, the expression of the relationship between the received fluorescence intensity _FR and the concentration of the fluorescent substance is:

为荧光量子产率，ε为摩尔消光系数，c为荧光物质的浓度，ζ＝-2.303ε，k为一个表征荧光物质吸收光能力的常数，N是激发光功率常数，Φ为一个表征荧光物质荧光转化效率的常数，荧光接收区域为x＝a到x＝b的范围，d是微分符号；where λ _Em is the fluorescence emission wavelength,

is the fluorescence quantum yield, ε is the molar extinction coefficient, c is the concentration of the fluorescent substance, ζ=-2.303ε, k is a constant characterizing the ability of the fluorescent substance to absorb light, N is the excitation light power constant, Φ is a characterizing the fluorescent substance The constant of fluorescence conversion efficiency, the fluorescence receiving area is the range from x=a to x=b, and d is the differential symbol;

S2, based on the above-mentioned expression (4), an analytical model is established for the specific fluorescent substance to be measured, so as to determine the constant in the expression.

Preferably, the above S2 includes:

S21, establishing a series of gradient concentration solution samples of the tested fluorescent substance in a wide concentration range, determining the excitation light intensity and the fluorescence receiving range, obtaining the received fluorescence intensity corresponding to different fluorescent substance concentrations through experiments, and using these data as a training set;

S22, using the least squares method and the Levenberg-Marquardt algorithm to perform optimal fitting, thereby determining the parameters Φ·N, k, ζ in the expression of S1;

The Levenberg-Marquardt algorithm combines the steepest descent method and the Taylor series linearization method, and obtains the search direction by solving the optimization model. The optimization model is as follows:

为n维实数域，J_k为雅克比矩阵，r_k为信赖域半径，μ_k为阻尼参数，h为步长；Among them, d _k is the search direction,

is the n-dimensional real number field, J _k is the Jacobian matrix, r _k is the trust region radius, μ _k is the damping parameter, and h is the step size;

Thereby, a relationship model of received fluorescence intensity-fluorescent substance concentration is established.

Preferably, the above-mentioned quantitative fluorescence analysis in the specific measurement includes: measuring the absorbance while measuring the fluorescence, taking the concentration corresponding to the maximum received fluorescence intensity as the concentration threshold, and the corresponding absorbance at this time as the absorbance threshold. Specifically, if the measured absorbance of the solution is less than the absorbance threshold, the fluorescence intensity rule less than the concentration threshold is used to predict the concentration of the fluorescent substance; if the measured absorbance is greater than the absorbance threshold, the fluorescence intensity rule greater than the concentration threshold is used to predict the concentration of the fluorescent substance .

In a second aspect of the present invention, a fluorescence measurement system is provided, which is used in the above-mentioned ultra-wide-range fluorescence quantitative analysis method. Specifically, the fluorescence measurement system includes: an excitation light source, a diaphragm, a first collimating lens, a sample cell, a first concave mirror, a fiber probe, a second concave mirror, a second collimating lens, and a narrow-band filter , Condensing lens, and spectrometer, wherein: the light emitted by the excitation light source is converted into a parallel beam by the first collimating lens after passing through the diaphragm, and the parallel beam irradiates the liquid sample in the sample cell vertically, part of the light is absorbed by the sample, part of the light is transmitted, transmitted The light is reflected by the first concave mirror to reach the fiber probe; after the sample is excited by the excitation light, it emits fluorescence around it, and the fluorescence is collected in the direction perpendicular to the excitation light. The fluorescence emitted to the left is reflected by the second concave mirror and emitted to the right. The fluorescent light is collimated by the second collimating lens, filtered by a narrow-band filter, and finally converged by the converging lens on the fiber probe, and the fluorescence and transmitted light are transmitted to the spectrometer through the fiber.

Preferably, a second concave mirror is placed on the left side of the sample cell to improve the receiving efficiency of fluorescence; a first concave mirror is placed on the rear side of the sample cell, the first concave mirror reflects and converges and transmits through the sample The excitation light of the cell is sent to the fiber optic probe, and the absorbance of the sample is calculated using this part of the light.

Preferably, the second collimating lens, the band-pass filter and the condensing lens are arranged in sequence on the fluorescence receiving light path, and are used for collimation, filtering and condensing.

Compared with the prior art, the present invention has the following beneficial effects:

The above-mentioned ultra-large-scale fluorescence quantitative analysis method of the present invention, based on the interaction and propagation law between the excitation light and the fluorescent substance, establishes a fluorescence emission model of a certain volume of fluorescent substance solution, and deduces the reception in a wide range from low concentration to high concentration. The accurate relationship between the fluorescence intensity and the concentration of fluorescent substances can solve the problem of quantifying high-concentration fluorescent substances and wide-range fluorescent substances, and is not only suitable for the quantification of low-concentration fluorescent substances, but also for high-concentration fluorescent substances.

The above-mentioned fluorescence measurement system for the ultra-large-scale fluorescence quantitative analysis method of the present invention can realize simultaneous measurement of fluorescence intensity and absorbance, and provide relevant measurement data for the above-mentioned ultra-large-scale fluorescence quantitative analysis method.

Description of drawings

Other features, objects and advantages of the present invention will become more apparent by reading the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 is a graph of the relationship between the received fluorescence intensity and the concentration of fluorescent substances according to an embodiment of the present invention;

2 is an ultra-wide-range fluorescence quantitative analysis diagram of an embodiment of the present invention;

3 is a schematic structural diagram of a fluorescence measurement system according to an embodiment of the present invention;

FIG. 4 is a flowchart of data processing for the original spectrum according to an embodiment of the present invention;

Fig. 5 is a graph of the fitting situation of the received fluorescence intensity-tryptophan concentration relationship of the expression of an embodiment of the present invention to 43 groups of tryptophan standard solutions of different concentrations;

Fig. 6 is a quantitative analysis situation diagram of the received fluorescence intensity-tryptophan concentration data of 11 concentration gradients in the test set according to an embodiment of the present invention;

7 is a graph of simulation and experimental results of the fluorescence distribution of tryptophan concentration gradient according to an embodiment of the present invention.

Detailed ways

The present invention will be described in detail below with reference to specific embodiments. The following examples 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, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

In the embodiment of the present invention, in order to solve the problem of high-concentration fluorescent substance quantification and wide-range fluorescent substance quantification, an ultra-large-range fluorescent quantitative analysis method is proposed, which is not only suitable for low-concentration fluorescent substance quantification, but also high-concentration fluorescent substance quantification. Specifically, the ultra-large-scale fluorescence quantitative analysis method in this embodiment is divided into the following three steps:

Step 1: Establish the relationship between the received fluorescence intensity and the concentration of the fluorescent substance

In this step, taking a certain volume of solution (such as the solution in the cuvette) as the research object, the micro-element analysis method is used to divide the solution into many micro-elements, and the micro-elements are selected as the object, as shown in Figure 1. It is shown that considering the interaction and propagation law between the excitation light and the micro-element fluorescent substance, the accurate spatial intensity distribution of fluorescence can be obtained, so as to accurately describe the fluorescence emission intensity and its spatial distribution law.

Assuming that the sample cell contains a certain concentration of fluorescent substance solution, a parallel Gaussian distribution excitation beam passes through the sample cell along the x direction, and a fluorescence emission plane (x, y) is intercepted for analysis and calculation.

After deduction and calculation, under the condition of single-wavelength excitation light, the expression of the relationship between the received fluorescence intensity _FR and the concentration of the fluorescent substance is:

ε是摩尔消光系数，c为荧光物质的浓度，ζ＝-2.303ε，光强的单位为任意单位，样品池长度单位采用归一化单位，k是一个表征荧光物质吸收光能力的常数，N是激发光功率常数，Φ是一个表征荧光物质荧光转化效率的常数，荧光接收区域为x＝a到x＝b的范围，d是微分符号；Among them, the fluorescence emission wavelength λ _Em , and the fluorescence quantum yield is

ε is the molar extinction coefficient, c is the concentration of the fluorescent substance, ζ=-2.303ε, the unit of light intensity is an arbitrary unit, the unit of the sample cell length is a normalized unit, k is a constant characterizing the ability of the fluorescent substance to absorb light, N is the excitation light power constant, Φ is a constant characterizing the fluorescence conversion efficiency of the fluorescent substance, the fluorescence receiving area is in the range from x=a to x=b, and d is the differential symbol;

The above formula (4) accurately reflects the relationship between the received fluorescence intensity and the concentration of the fluorescent substance.

Step 2: Build an analytical data model and determine constants

Based on formula (4), an analysis model is established for the specific fluorescent substance to be measured, and the method is as follows:

First, establish a series of gradient concentration solution samples of the tested fluorescent substance in a wide concentration range, determine the excitation light intensity and the fluorescence receiving range, obtain the received fluorescence intensity corresponding to different fluorescent substance concentrations through experiments, and use this data as a training set.

Then, the least squares method and the Levenberg-Marquardt algorithm are used to perform optimal fitting, thereby determining the parameters Φ·N, k, and ζ in formula (4). The Levenberg-Marquardt algorithm combines the steepest descent method and the Taylor series linearization method to optimize the model by solving:

Thereby obtaining the search direction.

Thereby, the relationship model of the received fluorescence intensity-fluorescent substance concentration can be established.

In this step, any fluorescence quantitative analysis can use this analytical expression and determine the parameters Φ·N, k, ζ in the analytical expression through the Levenberg-Marquardt optimization fitting algorithm, and establish the received fluorescence intensity-fluorescent substance concentration curve.

This model is valid in a wide concentration range, and the constants in it are consistent in the experiment and can be determined, so it is suitable not only for the quantification of low-concentration fluorescent substances, but also for the quantification of high-concentration fluorescent substances.

Step 3: Use a specific test device to perform quantitative fluorescence analysis in specific measurement, apply the above analysis model to the quantification of wide-range fluorescent substances, and analyze the concentration of fluorescent substances based on the above received fluorescence intensity-fluorescent substance concentration relationship.

As shown in FIG. 2 , the absorbance is measured while the fluorescence is measured, and the concentration corresponding to the maximum received fluorescence intensity is taken as the concentration threshold, and the corresponding absorbance at this time is the absorbance threshold. If the measured absorbance of the solution is less than the absorbance threshold, the fluorescence intensity law less than the concentration threshold is used to predict the concentration of the fluorescent substance; if the measured absorbance is greater than the absorbance threshold, the fluorescent intensity law greater than the concentration threshold is used to predict the concentration of the fluorescent substance.

In another embodiment, in order to better realize the simultaneous measurement of fluorescence intensity and absorbance, the following fluorescence measurement system is also designed in the embodiment of the present invention, the structure of which is shown in FIG. 3 . Specifically, in this fluorescence test system: the light emitted by the excitation light source 1 is converted into a parallel beam by the first collimating lens 3 after passing through the diaphragm 2, and the parallel beam irradiates the liquid sample 5 in the sample cell 4 vertically, and a part of the light is absorbed by the sample , part of the light is transmitted, and the transmitted light is reflected by the first concave mirror 6 to reach the fiber probe 7; after the sample is excited by the excitation light, it emits fluorescence around it, and the fluorescence is collected in the direction perpendicular to the excitation light, and the fluorescence emitted to the left passes through the second concave surface. After being reflected by the mirror 8, it is collimated by the second collimating lens 9 together with the fluorescent light emitted to the right, and then filtered by the narrow-band filter 10, and finally converged on the fiber probe 7 by the converging lens 11, and the fluorescent light and the transmitted light pass through the fiber together. Conducted to the spectrometer 12 . These measured parameters will be used in an ultra-wide range of fluorescence quantitative analysis methods.

In order to better illustrate the above-mentioned ultra-wide-range fluorescence quantitative analysis method of the present invention, the following description is given in conjunction with specific application examples to understand the implementation details, but it should be understood that the following examples are not intended to limit the present invention. Specifically, tryptophan is used as a fluorescent substance, and its concentration is measured.

In this application example, the ultra-wide range fluorescence quantitative analysis method is performed according to the following steps:

The first step is to build a fluorescence measurement system.

The structure diagram of the fluorescence measurement system used in this step is shown in FIG. 3 . In order to measure the concentration of tryptophan, the specific structural parameter selection and measurement process of the fluorescence measurement system are as follows:

1) The excitation light source 1 uses a 280nm ultraviolet LED, and the 280nm excitation light excites tryptophan, which can generate 340±40nm fluorescence.

2) The ultraviolet light emitted by the excitation light source 1 is collimated by a plano-convex lens 3 through a circular diaphragm 2 with an aperture of φ=5mm, and becomes a parallel Gaussian beam as the excitation beam.

3) The effective optical power of the LED light source is 2mW.

4) Select a four-sided clear quartz cuvette with an optical path length of L=10mm as the sample cell for containing the tryptophan solution.

5) A second concave mirror 8 is placed on the left side of the quartz cuvette to improve the fluorescence receiving efficiency.

6) A first concave mirror 6 is placed on the rear side of the quartz cuvette. The concave mirror reflects and focuses the excitation light of 280 nm transmitted through the sample cell, and the absorbance of the tryptophan solution can be calculated by using this part of the light.

7) A collimating lens 9 (plano-convex lens), a bandpass filter 10 and a condensing lens 11 (plano-convex lens) are placed on the fluorescence receiving optical path for collimation, filtering and convergence to obtain 340±40nm tryptophan fluorescence.

8) The fluorescence signal and excitation light signal received by the optical fiber probe 7 are transmitted into the spectrometer 12 through the optical fiber.

9) The spectrometer 12 adopts an ultraviolet-visible spectrometer, and the measurement spectral range is 200-900 nm.

In the second step, in order to determine the parameters in the model of the received fluorescence intensity of the tryptophan solution - the concentration of the fluorescent substance, the following operations are performed:

1) Configure 43 tryptophan solutions with different concentration gradients for modeling, and configure 10 samples for each concentration, which is used as the standard solution, and the measured data is used as the training set.

2) Configure 11 concentration gradients of pure tryptophan solution for testing, and configure 10 samples for each concentration, which are used to test whether the model with the determined parameters has the ability to generalize and the instrument's sensitivity to the tryptophan fluorescent chromophore. Responsiveness, the measured data is used as the test set.

3) The tryptophan concentration in the solution measured in each group is shown in Table 1.

Table 1

4) All sample solutions were packed in 15mL reagent tubes and stored in the dark at 4°C for 48 hours before analysis.

5) After 48 hours, the solution to be tested was allowed to reach 25°C in a dark constant temperature chamber, and then transferred to a quartz cuvette for analytical testing.

6) After the test of each sample, wash the quartz cuvette with deionized water five times, and add the next set of samples for testing after the water evaporates.

7) The test time of each sample is guaranteed to be within 1 min to reduce the influence of fluorescence bleaching on the measurement.

In the third step, the original spectrum obtained from the test is preprocessed through the flow chart shown in Figure 4.

1) Remove the baseline generated by the electrical signal drift and dark current of the spectrometer during measurement, and ensure that each spectrum has the same base.

式中，x_b∈{x_I，i∈1,2,…,n}表示每一浓度中的一个光谱样本；

表示样本的算术平均值；|v_b|表示剩余误差的绝对值；σ是由贝塞尔公式算出的标准偏差。如果样本满足上式则认为x_b是含有粗大误差值的数据点，应予以剔除。2) According to the Laida criterion, the spectrum with gross errors is eliminated, and the Laida criterion is expressed as:

where x _b ∈ {x _I , i ∈ 1,2,…,n} represents a spectral sample in each concentration;

represents the arithmetic mean of the sample; |v _b | represents the absolute value of the residual error; σ is the standard deviation calculated by the Bessel formula. If the sample satisfies the above formula, it is considered that x _b is a data point with a gross error value and should be eliminated.

3) The spectral data is filtered by Savitzky-Golay to reduce noise.

Example result analysis:

43 groups of tryptophan standard solutions with different concentrations were measured, and each group contained 10 samples.

The variation of the received fluorescence intensity measured by these concentration gradient solutions to the tryptophan concentration can be fitted by the relationship between the received fluorescence intensity and the concentration of the fluorescent substance.

For the same excitation light source, the same fluorescence receiving range, and the same CCD integration time, the expression (4) given by the relationship between the received fluorescence intensity and the concentration of the fluorescent substance fits the experimental data very well.

In the concentration range of 0.02-250 mg/L, the maximum number of iterations was 1000 for optimal fitting with the Levenberg-Marquardt algorithm at 95% confidence limits.

As shown in (a) of FIG. 6 , the analytical expression (4) fits the received fluorescence intensity-tryptophan concentration relationship to a goodness of fit of R ² =0.9939.

As shown in Fig. 6(b), for the low concentration segment, the linear regression goodness of fit R ² >0.99 ranged from 0.02 to 17.5 mg/L. The measurement sensitivity (slope of linear regression) in the linear range was 454 A.U./(mg·L ⁻¹ ). The blank value of the sensor (the vertical intercept of the linear regression) was 199 A.U. The lowest detection limit of the sensor obtained from the triple variance of the lowest concentration is 20.73 μg/L, and the relative error is within 1%, which meets the requirements of high-precision detection.

The high concentration section also has higher measurement sensitivity. Although the slope in this section is not as good as the linear section, because the concentration in this section is relatively high, the relative error of using this section for concentration prediction is very small.

The model trained from the standard solution training set, combined with the tryptophan concentration prediction method, can predict the tryptophan concentration in a wide concentration range with high precision, and can be extended to a general fluorescent quantitative analysis method.

Figure 6 (a) The received fluorescence intensity-tryptophan concentration data points of the 11 concentration gradients in the test set relative to the received fluorescence intensity-tryptophan concentration model of the determined parameters, they are all closely distributed in the quantitative analysis curve. Around, it can be seen that this curve can well describe the quantitative relationship between the fluorescence intensity and concentration of tryptophan solution.

In combination with the above-described method for predicting the tryptophan concentration, the tryptophan concentration prediction result shown in (b) of FIG. 6 can be obtained. Among them, the black line represents the prediction result obtained by using the quantitative analysis method based on the analysis of the fluorescence intensity distribution law proposed in the embodiment of the present invention, and the red line represents the prediction within the linear range of Lambert Beer's law using the ultraviolet spectrophotometry As a result, the blue line shows the predicted results obtained by conventional fluorescence quantitative analysis that satisfy the linearity of fluorescence-concentration.

Figure 6 (c) shows the relative error of the prediction results obtained by using the quantitative analysis method, ultraviolet spectrophotometry, and conventional fluorescence quantitative analysis based on the analysis of the fluorescence intensity distribution law of the present invention under each tryptophan concentration. It can be seen that the fluorescence quantitative analysis method proposed in the present invention not only has the same prediction accuracy as the UV spectrophotometry and the conventional fluorescence quantitative analysis method, but also has a wider analysis range than the UV spectrophotometry and the conventional fluorescence quantitative analysis method. When the concentration of the target substance is too high or too low, UV spectrophotometry will produce a large analysis error, and the analysis results are easily affected by the interfering substance when there are interfering substances. The emission fluorescence wavelength of the is specific and can avoid interference by interfering substances, but its analyzable linear range is very narrow.

The fluorescence quantitative analysis method proposed by the invention comprehensively considers the omission condition of the high-order term of c in the Taylor expansion of e ^-2.303εcx and the influence of the spatial attenuation effect of the excitation light, and obtains a high-precision ultra-wide concentration range. method for predicting fluorescent substances.

In addition, the fluorescence distribution is summarized by combining the received fluorescence intensity-fluorescent substance concentration distribution law, as shown in Fig. 7(a):

The six pictures are the tryptophan concentrations under the same excitation light intensity: c=0.1mg/L, c=3mg/L, c=10mg/L, c=30mg/L, c=60mg/L, c=120mg Simulation results of the fluorescence intensity distribution of /L.

When the tryptophan concentration is c=0.1mg/L, the fluorescence excited by the solution is relatively weak;

When the concentration of tryptophan is c=3mg/L and c=10mg/L, the concentration is still within the linear range of absorbance-concentration described by Lambert Beer's theorem, the spatial attenuation effect of excitation light is not significant, and the distribution of fluorescence intensity The whole is a bright passband, and the higher the tryptophan concentration, the wider and brighter the bright passband;

When the concentration of tryptophan is c=30mg/L, the absorbance-concentration relationship gradually begins to exceed the linear range described by Lambert Beer's theorem, and the spatial attenuation effect of the excitation light begins to gradually manifest. It can be observed that the fluorescence intensity at the front of the sample cell is obvious. It should be stronger than the fluorescence intensity at the back end of the sample cell;

When the tryptophan concentration is c=60mg/L, with the increase of tryptophan concentration, the increase of the absorbance of the sample is no longer obvious. Due to the existence of the spatial attenuation effect of the excitation light, the fluorescence intensity distribution spreads with the excitation light. The direction is obviously layered, and due to the Gaussian nature of the excitation light intensity distribution, the fluorescence distribution of the same intensity presents a triangular shape;

When the tryptophan concentration is c=120mg/L, with the further increase of the tryptophan concentration, the absorbance of the sample almost no longer changes, the spatial attenuation effect of the excitation light is very significant, and the fluorescence intensity distribution follows the propagation direction of the excitation light. The delamination that occurred became more obvious, the interval between layers became shorter, and the fluorescence was mainly concentrated at the front of the sample cell.

The six graphs in (b) of Fig. 7 correspond to the experimental phenomena of the six concentrations of tryptophan solution calculated by simulation respectively. The photos of each sample cell are the original photos of the camera without any image processing, and the fluorescence distribution of each concentration is consistent with the fluorescence intensity distribution of the calculated analysis.

Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above-mentioned specific embodiments, and those skilled in the art can make various variations or modifications within the scope of the claims, which do not affect the essential content of the present invention. The above-mentioned preferred features can be used in any combination as long as they do not conflict with each other.

Claims (9)

1. an ultra-wide range of fluorescence quantitative analysis method, is characterized in that, comprises: establishes analysis model and utilizes described analysis model to carry out the fluorescence quantitative analysis in concrete measurement; Wherein, The establishment of the analysis model includes: S1, establish the relationship between the received fluorescence intensity and the concentration of the fluorescent substance: Under the condition of single-wavelength excitation light, the expression of the relationship between the received fluorescence intensity _FR and the concentration of the fluorescent substance is:

where λ _Em is the fluorescence emission wavelength,

is the fluorescence quantum yield, ε is the molar extinction coefficient, c is the concentration of the fluorescent substance, ζ=-2.303ε, k is a constant characterizing the ability of the fluorescent substance to absorb light, N is the excitation light power constant, Φ is a characterizing the fluorescent substance The constant of fluorescence conversion efficiency, the fluorescence receiving area is the range from x=a to x=b, and d is the differential symbol; S2, based on the above-mentioned expression (4), establish an analytical model for the specific fluorescent substance to be measured, so as to determine the constant in the expression; The above S2, including: S21, establishing a series of gradient concentration solution samples of the tested fluorescent substance in a wide concentration range, determining the excitation light intensity and the fluorescence receiving range, obtaining the received fluorescence intensity corresponding to different fluorescent substance concentrations through experiments, and using these data as a training set; S22, using the least squares method and the Levenberg-Marquardt algorithm to perform optimal fitting, thereby determining the parameters Φ·N, k, ζ in the expression of S1; The Levenberg-Marquardt algorithm combines the steepest descent method and the Taylor series linearization method, and obtains the search direction by solving the optimization model. The optimization model is as follows:

Among them, d _k is the search direction,

is the n-dimensional real number field, J _k is the Jacobian matrix, r _k is the trust region radius, μ _k is the damping parameter, and h is the step size; Thereby, a relationship model of received fluorescence intensity-fluorescent substance concentration is established. 2. the ultra-wide range fluorescence quantitative analysis method according to claim 1, is characterized in that, the fluorescence quantitative analysis in the described carrying out specific measurement, wherein: measure the absorbance while measuring fluorescence, receive the concentration corresponding to the maximum fluorescence intensity is the concentration threshold, and the corresponding absorbance at this time is the absorbance threshold. 3. the fluorescence quantitative analysis method of ultra-wide range according to claim 2, is characterized in that, if the absorbance of the solution of measurement is less than the absorbance threshold value, then utilizes the fluorescence intensity rule less than the concentration threshold value to predict the fluorescent substance concentration; If the absorbance is greater than the absorbance threshold, the fluorescence intensity law greater than the concentration threshold is used to predict the concentration of the fluorescent substance. 4. The ultra-wide-range fluorescence quantitative analysis method according to claim 1, wherein the performing the fluorescence quantitative analysis in the specific measurement further comprises: preprocessing the original spectrum, and the preprocessing includes removing the Spectral and/or noise reduction for gross errors. 5. The ultra-wide-range fluorescence quantitative analysis method according to claim 4, wherein the preprocessing further comprises: removing the baseline generated by the electrical signal drift and dark current of the spectrometer in the measurement, ensuring that each Spectra have the same base. 6 . The ultra-wide-range fluorescence quantitative analysis method according to claim 4 , wherein the removing the spectrum with gross error comprises: according to the Laida criterion, removing the spectrum containing gross error, wherein the Layi Reach criterion expression:

where x _b ∈{x _I ,i∈1,2,…,n} represents a spectral sample in each concentration;

represents the arithmetic mean of the sample; |v _b | represents the absolute value of the residual error; σ is the standard deviation calculated by the Bessel formula; If the sample satisfies the above formula, it is considered that x _b is a data point with a gross error value, and it is eliminated. 7. A fluorescence measurement system for the method according to claim 1, characterized in that it comprises: an excitation light source, a diaphragm, a first collimating lens, a sample cell, a first concave mirror, a fiber probe, a second Concave mirror, second collimating lens, narrow-band filter, converging lens, spectrometer, wherein: The light emitted by the excitation light source is converted into a parallel beam by the first collimating lens after passing through the diaphragm. The parallel beam irradiates the liquid sample in the sample cell vertically, part of the light is absorbed by the sample, part of the light is transmitted, and the transmitted light is reflected by the first concave mirror Reach the fiber probe; after the sample is excited by the excitation light, it emits fluorescence around it, and the fluorescence is collected in the direction perpendicular to the excitation light. The fluorescence emitted to the left is reflected by the second concave mirror and then goes through the second collimation together with the fluorescence emitted to the right The lens is collimated, filtered by a narrow-band filter, and finally converged by a converging lens on the fiber probe, and the fluorescence and transmitted light are transmitted to the spectrometer through the fiber together. 8 . The fluorescence measurement system according to claim 7 , wherein a second concave reflector is placed on the left side of the sample cell to improve the fluorescence receiving efficiency; the first concave reflector is placed on the back side of the sample cell. 9 . The first concave mirror reflects and condenses the excitation light transmitted through the sample cell to the fiber probe, and uses this part of the light to calculate the absorbance of the sample. 9 . The fluorescence measurement system according to claim 7 , wherein the second collimating lens, the narrow-band filter and the condensing lens are arranged one after the other on the fluorescence receiving light path, and are respectively used for collimating the fluorescence. 10 . Straight, filtered and converged.

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