CN112903644A - Ultra-wide range fluorescence quantitative analysis method and fluorescence measurement system - Google Patents
Ultra-wide range fluorescence quantitative analysis method and fluorescence measurement system Download PDFInfo
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- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 33
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 33
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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
Technical Field
The invention relates to the technical field of quantitative analysis, in particular to a super-wide-range fluorescent quantitative analysis method and a fluorescent measurement system.
Background
The fluorescent quantitative analysis method has the advantages of high sensitivity, good selectivity, simple operation and the like, and is widely applied to the fields of biochemical analysis, medical detection, environmental monitoring, food safety and the like. In a conventional quantitative fluorescence analysis method, a fluorescent substance solution to be measured is usually contained in a cuvette, excitation light having a certain wavelength is incident on the cuvette, and an emitted fluorescence signal is collected in a certain angular direction (e.g., a direction perpendicular to the incident light), and 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 is:
where ε is the molar extinction coefficient, c is the concentration of the solution of the fluorescent substance, x is the optical path length of the excitation light passing through the solution, the fluorescence quantum yield of the fluorescent substance
Intensity of incident light I0。
Because the linear relation of the lambert beer law is established only when the concentration of the solution is in a small range, and the excitation light is gradually weakened by the solution in the process of propagating in the solution, namely the excitation light has a space attenuation effect, when the concentration of the detected fluorescent substance is high, a large error is generated by adopting a conventional fluorescence quantitative method for analysis, and the method is not applicable any more.
In order to expand the applicable range of the concentration of the fluorescence quantitative analysis method, some researchers correct the formula of the fluorescence intensity and the concentration of the fluorescent substance solution by using the absorbance, and the corrected formula of the fluorescence intensity is as follows:
wherein, FobsIndicating the intensity of the fluorescence received by the detector; fidealThen the fluorescence intensity after correction by the correction formula is represented; a. theExRepresenting the absorbance value of the exciting light after passing through the sample cell; a. theEmThen represents the absorbance value of the emitted light after passing through the sample cell, s is the excitation light beamThe diameter in the case of a uniform cylindrical section; g is the distance from the edge of the excitation beam to the edge of the cuvette and d is the width of the cuvette.
Albinsson et al, 1994, proposed a correction formula that was later widely used, the corrected fluorescence intensity formula being:
after the correction, the measurement range of the fluorescence quantitative analysis is expanded to a certain extent.
Limited by the linear range of the lambert beer law, when the concentration of the fluorescent substance is high, the relation between absorbance and concentration deviates from the linear law, and the absorbance of the solution at high concentration is difficult to be accurately measured due to the influence of stray light of the instrument. Therefore, the fluorescence intensity formula after correcting the fluorescence intensity at a high concentration by absorbance cannot be used for measurement and quantitative analysis of a high-concentration fluorescent substance.
At present, for some application occasions with high concentration or wide concentration variation range of fluorescent substances, the quantitative monitoring and analysis of the fluorescent substances are still difficult. For example: in the textile printing and dyeing industry, there is a need for on-line measurement of fluorescent dye materials in a wide range and at high concentrations. For another example: the total concentration of free amino acid in the plasma of normal adult is 350-650 mg.L-1The concentration change is large day and night, when the concentration of free amino acid is detected by adopting the traditional fluorescence quantitative analysis, the plasma needs to be diluted to low concentration, and the state of certain substances in the plasma is changed in the dilution process.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a super-wide-range fluorescence quantitative analysis method and a fluorescence measurement system, which solve the difficult problems of high-concentration fluorescent substance quantification and wide-range fluorescent substance quantification.
In a first aspect of the present invention, there is provided a method for quantitative analysis of fluorescence over a broad range, comprising: 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 the following steps:
s1, establishing the relation between the received fluorescence intensity and the concentration of the fluorescent substance:
receiving fluorescence intensity F under the condition of single-wavelength excitation lightRThe expression for the relationship with the concentration of the fluorescent substance is:
wherein λ isEmIs the wavelength of the fluorescence emission,
in terms of fluorescence quantum yield, epsilon is a molar extinction coefficient, c is the concentration of the fluorescent substance, zeta is-2.303 epsilon, k is a constant for representing the light absorption capability of the fluorescent substance, N is an excitation light power constant, phi is a constant for representing the fluorescence conversion efficiency of the fluorescent substance, a fluorescence receiving area ranges from x to x and b, and d is a differential sign;
s2, an analytical model is established for the specific fluorescent substance to be measured based on the above expression (4), thereby determining constants in the expression.
Preferably, the S2 includes:
s21, establishing a series of gradient concentration solution samples of the detected 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 taking the data as a training set;
s22, performing optimization fitting by using a least square method and a Levenberg-Marquardt algorithm, thereby determining parameters phi · N, k and zeta in the expression of S1;
the Levenberg-Marquardt algorithm integrates a steepest descent method and a Taylor series linearization method, and obtains a search direction by solving an optimization model which is as follows:
wherein d iskIn order to search for the direction(s),
is n-dimensional real number field, JkIs a Jacobian matrix, rkTo trust the radius of the field, mukIs a damping parameter, and h is a step length;
thereby establishing a model of the received fluorescence intensity versus the concentration of the fluorescent substance.
Preferably, the performing of the quantitative analysis of fluorescence in the specific measurement comprises: and measuring the absorbance while measuring the fluorescence, wherein the concentration corresponding to the maximum received fluorescence intensity is taken as a concentration threshold, and the corresponding absorbance at the moment is taken as an absorbance threshold. Specifically, if the measured absorbance of the solution is less than the absorbance threshold, the fluorescent substance concentration is predicted by using the fluorescence intensity rule less than the concentration threshold; and if the measured absorbance is greater than the absorbance threshold, predicting the concentration of the fluorescent substance by using the fluorescence intensity rule greater than the concentration threshold.
In a second aspect of the invention, a fluorescence measurement system is provided for the ultra-wide range fluorescence quantitative analysis method. Specifically, the fluorescence measurement system includes: excitation light source, diaphragm, first collimating lens, sample cell, first concave mirror, fiber probe, second concave mirror, second collimating lens, narrowband optical filter, convergent lens, spectrum appearance, wherein: light emitted by an excitation light source passes through a diaphragm and then is converted into parallel light beams by a first collimating lens, the parallel light beams vertically irradiate a liquid sample in a sample cell, a part of light is absorbed by the sample, a part of light is transmitted, and the transmitted light is reflected by a first concave reflector to reach an optical fiber probe; the sample is excited by exciting light and then emits fluorescence to the periphery, the direction vertical to the exciting light is selected to collect the fluorescence, the fluorescence emitted to the left is reflected by a second concave reflector and then is collimated by a second collimating lens together with the fluorescence emitted to the right, then the fluorescence is filtered by a narrow-band filter, and finally the fluorescence and the transmitted light are converged on an optical fiber probe by a converging lens, and the fluorescence and the transmitted light are conducted to a spectrometer together through an optical fiber.
Preferably, a second concave mirror is arranged at the left side of the sample pool to improve the receiving efficiency of fluorescence; and a first concave reflector is arranged at the rear side of the sample cell, reflects and converges the exciting light transmitted through the sample cell to the optical fiber probe, and the absorbance of the sample is calculated by using the part of light.
Preferably, the second collimating lens, the band-pass filter and the converging lens are sequentially arranged in a front-to-back manner on the fluorescence receiving optical path and are used for collimating, filtering and converging.
Compared with the prior art, the invention has the following beneficial effects:
the fluorescent quantitative analysis method with the ultra-large range establishes a fluorescent emission model of a fluorescent substance solution with a certain volume based on the interaction and propagation rule between exciting light and a fluorescent substance, deduces and obtains the accurate relation between the received fluorescent intensity and the fluorescent substance concentration in a large range from low concentration to high concentration, 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.
The fluorescence measurement system for the ultra-large range fluorescence quantitative analysis method can realize the simultaneous measurement of fluorescence intensity and absorbance, and provides related measurement data for the ultra-large range fluorescence quantitative analysis method.
Drawings
Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:
FIG. 1 is a graph of received fluorescence intensity versus concentration of a fluorescent substance in accordance with an embodiment of the present invention;
FIG. 2 is a diagram of an ultra-wide range fluorescence quantitative analysis according to an embodiment of the present invention;
FIG. 3 is a schematic diagram of a fluorescence measurement system according to an embodiment of the present invention;
FIG. 4 is a flow chart of raw spectra for data processing according to an embodiment of the present invention;
FIG. 5 is a graph of a relationship between received fluorescence intensity and tryptophan concentration for 43 groups of tryptophan standard solutions with different concentrations according to an embodiment of the present invention;
FIG. 6 is a graph of a quantitative analysis of received fluorescence intensity versus tryptophan concentration data for 11 concentration gradients in a test set according to an embodiment of the invention;
FIG. 7 is a graph showing the results of simulation and experiment of the fluorescence distribution of the tryptophan concentration gradient according to one embodiment of the present invention.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.
Aiming at the difficult problems of high-concentration fluorescent substance quantification and wide-range fluorescent substance quantification, the embodiment of the invention provides an ultra-large-range fluorescent quantitative analysis method, which is not only suitable for low-concentration fluorescent substance quantification, but also suitable for high-concentration fluorescent substance quantification. Specifically, the method for quantitative analysis of fluorescence in ultra-large range in this embodiment includes the following three steps:
the method comprises the following steps: establishing a relation between the intensity of received fluorescence and the concentration of the fluorescent substance
In this step, a solution (e.g., a solution in a cuvette) of a certain volume is used as a research object, a infinitesimal analysis method is adopted to divide the solution into a plurality of infinitesimals, the infinitesimals are selected as the object, and as shown in fig. 1, the interaction and propagation rules between the excitation light and the infinitesimal fluorescent substance are considered to obtain accurate spatial intensity distribution of the fluorescence, so that the fluorescence emission intensity and the spatial distribution rules thereof are accurately described.
Assuming that the sample cell is filled with a certain concentration of the 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.
Through derivation calculation, under the condition of single-wavelength exciting light, the intensity of the received fluorescence isDegree FRThe expression for the relationship with the concentration of the fluorescent substance is:
wherein the fluorescence emission wavelength lambdaEmFluorescence quantum yield of
Epsilon is a molar extinction coefficient, c is the concentration of the fluorescent substance, zeta is-2.303 epsilon, the unit of light intensity is an arbitrary unit, the length unit of the sample cell adopts a normalized unit, k is a constant for representing the light absorption capacity of the fluorescent substance, N is an excitation light power constant, phi is a constant for representing the fluorescence conversion efficiency of the fluorescent substance, the fluorescence receiving area is a range from x to b, and d is a differential sign;
the above equation (4) accurately reflects the received fluorescence intensity-fluorescent substance concentration relationship.
Step two: establishing analysis data model and determining constants
Based on formula (4), an analytical model is established for the specific fluorescent substance to be tested, the method is as follows:
firstly, establishing a series of gradient concentration solution samples of the fluorescent substance to be detected in a wide concentration range, determining the excitation light intensity and the fluorescence receiving range, obtaining the receiving fluorescence intensity corresponding to different fluorescent substance concentrations through experiments, and taking the data as a training set.
Then, the parameters Φ · N, k, ζ in equation (4) are determined by performing optimization fitting using the least squares method and Levenberg-Marquardt algorithm. The Levenberg-Marquardt algorithm integrates a steepest descent method and a Taylor series linearization method, and an optimization model is solved:
thereby obtaining a search direction.
Thereby, a model of the relation between the intensity of received fluorescence and the concentration of the fluorescent substance can be established.
In this step, any quantitative fluorescence analysis can use the analytical expression and determine parameters Φ · N, k, ζ in the analytical expression by a Levenberg-Marquardt optimization fitting algorithm to establish a received fluorescence intensity-fluorescent substance concentration curve.
The model is effective in a wide concentration range, and constants in the model are consistent in experiments, so that the model can be determined, and is suitable for quantification of not only low-concentration fluorescent substances but also high-concentration fluorescent substances.
Step three: the fluorescence quantification analysis in the specific measurement was performed using a specific test device, the above analysis model was applied to the wide-range fluorescent substance quantification, and the concentration of the fluorescent substance was analyzed based on the above received fluorescence intensity-fluorescent substance concentration relationship.
As shown in fig. 2, the absorbance is measured simultaneously with the fluorescence, and the concentration corresponding to the maximum received fluorescence intensity is taken as the concentration threshold, and the absorbance corresponding to this time is taken as the absorbance threshold. If the measured absorbance of the solution is smaller than the absorbance threshold, predicting the concentration of the fluorescent substance by using a fluorescence intensity rule smaller than the concentration threshold; and if the measured absorbance is greater than the absorbance threshold, predicting the concentration of the fluorescent substance by using the fluorescence intensity rule greater than the concentration threshold.
In another embodiment, in order to better realize the simultaneous measurement of fluorescence intensity and absorbance, the following fluorescence measurement system is further designed in the embodiment of the present invention, and the structural diagram thereof is shown in fig. 3. Specifically, in the present fluorescence test system: light emitted by an excitation light source 1 passes through a diaphragm 2 and then is converted into parallel light beams by a first collimating lens 3, the parallel light beams vertically irradiate a liquid sample 5 in a sample cell 4, a part of light is absorbed by the sample, a part of light is transmitted, and the transmitted light is reflected by a first concave reflector 6 to reach an optical fiber probe 7; the sample is excited by exciting light and then emits fluorescence to the periphery, the fluorescence is collected in the direction vertical to the exciting light, the fluorescence emitted to the left is reflected by a second concave reflecting mirror 8 and then collimated by a second collimating lens 9 together with the fluorescence emitted to the right, the fluorescence is filtered by a narrow-band filter 10 and finally converged on an optical fiber probe 7 by a converging lens 11, and the fluorescence and transmitted light are transmitted to a spectrometer 12 together through an optical fiber. These measured parameters will be used in a very broad range of fluorescent quantitative analysis methods.
To better illustrate the above-described ultra-broad range fluorescence quantitative analysis method of the present invention, the following description is given in conjunction with specific application examples to understand the details of the implementation, but it should be understood that the following examples are not intended to limit the present invention. Specifically, tryptophan was used as a fluorescent substance, and the concentration thereof was measured.
In the embodiment of the application, the ultra-wide range fluorescence quantitative analysis method is carried out according to the following steps:
firstly, a fluorescence measurement system is set up.
The configuration of the fluorescence measuring system used in this step is shown in FIG. 3. For measuring the concentration of tryptophan, the specific structural parameters of the fluorescence measurement system are selected and measured as follows:
1) an excitation light source 1 adopts a 280nm ultraviolet LED, and the 280nm excitation light excites tryptophan to generate 340 +/-40 nm fluorescence.
2) Ultraviolet light emitted by an excitation light source 1 passes through a circular diaphragm 2 with the aperture phi of 5mm and is collimated by a plano-convex lens 3, and the ultraviolet light becomes a parallel Gaussian beam serving as an excitation beam.
3) The effective luminous power of the LED light source is 2 mW.
4) A four-side light-transmitting quartz cuvette with an optical path L of 10mm is selected as a sample cell for containing tryptophan solution.
5) A second concave reflector 8 is placed on the left side of the quartz cuvette to improve the receiving efficiency of the fluorescence.
6) A first concave mirror 6 is placed on the rear side of the quartz cuvette, and reflects and focuses the excitation light of 280nm transmitted through the sample cell, and the absorbance of the tryptophan solution can be calculated by using the reflected light.
7) And a collimating lens 9 (plano-convex lens), a band-pass filter 10 and a converging lens 11 (plano-convex lens) are arranged on the fluorescence receiving path and are used for collimating, filtering and converging to obtain 340 +/-40 nm tryptophan fluorescence.
8) The fluorescence signal and the excitation light signal received by the fiber optic probe 7 are transmitted into the spectrometer 12 via the optical fiber.
9) The spectrometer 12 adopts an ultraviolet-visible spectrometer, and the measurement spectrum range is 200 and 900 nm.
In a second step, in order to determine the parameters in the model of the received fluorescence intensity-fluorescent substance concentration of the tryptophan solution, the following operations are performed:
1) the tryptophan solutions with 43 different concentration gradients for modeling were prepared, 10 samples were prepared for each concentration, and the measured data were used as a training set.
2) The pure tryptophan solution with 11 concentration gradients, 10 samples for each concentration, was prepared for testing whether the determined parametric model had generalization ability and instrument response ability to the tryptophan fluorescent chromophore, and the measured data were used as a test set.
3) The tryptophan concentrations in the solutions measured for each group are shown in table 1.
TABLE 1
4) All sample solutions were contained in 15mL reagent tubes and analyzed after 48 hours of storage at 4 ℃ in the dark.
5) After 48 hours, the test solution was allowed to be brought to 25 ℃ in a dark thermostated chamber and subsequently transferred to a quartz cuvette for analytical testing.
6) And (4) washing the quartz cuvette five times by using deionized water after the test of each sample is finished, and adding the next group of samples for testing after the water in the quartz cuvette is volatilized.
7) The test time for each sample was guaranteed to be within 1min to reduce the effect of fluorescence bleaching on the measurements.
Third, the original spectrum obtained by the test is preprocessed by the flow chart shown in fig. 4.
1) The baseline of the spectrometer produced by the drift of the electrical signal and the dark current during the measurement is removed, ensuring that the spectra at each time have the same base.
2) Spectra containing gross errors are rejected according to the Laplace criterion, which is expressed as:
in the formula, xb∈{xII ∈ 1,2, …, n } represents one spectral sample in each concentration;
represents the arithmetic mean of the samples; | vb| represents the absolute value of the residual error; σ is the standard deviation calculated by Bessel's equation. Consider x if the sample satisfies the above equationbData points with large error values are to be eliminated.
3) The spectral data is subjected to Savitzky-Golay filtering to reduce noise.
Example analysis of results:
43 sets of tryptophan standard solutions of different concentrations were measured, each set containing 10 samples.
The variation of the received fluorescence intensity versus the tryptophan concentration measured from these concentration gradient solutions can be fitted with a received fluorescence intensity-fluorescent substance concentration relation.
For the same excitation light source, the same fluorescence receiving range and the same CCD integration time, the expression (4) given by the relation of the received fluorescence intensity and the fluorescent substance concentration fits well with experimental data.
The maximum number of iterations for an optimized fit using the Levenberg-Marquardt algorithm at a 95% confidence limit is 1000 over the concentration range of 0.02-250 mg/L.
As shown in (a) of FIG. 6, analytical expression (4) was fitted to the relationship of the received fluorescence intensity-tryptophan concentration to reach R20.9939.
As shown in FIG. 6 (b), for the low concentration section, the linear regression goodness of fit R2Linear range > 0.99 of0.02-17.5 mg/L. The measurement sensitivity (slope of linear regression) of the linear range was 454a.u./(mg · L)-1). The blank value (vertical axis intercept of linear regression) of the sensor was 199a.u. The lowest detection limit of the sensor obtained by triple variance of the lowest concentration is 20.73 mu g/L, the relative error is within 1 percent, and the high-precision detection requirement is met.
The high concentration section also has higher measurement sensitivity, and although the slope in this section is not as good as that in the linear section, because the concentration in this section is higher, the relative error in making a concentration prediction using this section is small.
The model obtained by training the standard solution training set can be used for predicting the tryptophan concentration in a wide concentration range with high precision by combining the tryptophan concentration prediction method, and can be popularized to a general fluorescence quantitative analysis method.
In FIG. 6 (a), the 11 concentration gradients of the received fluorescence intensity-tryptophan concentration data points in the test set are closely distributed around the quantitative analysis curve relative to the parameter-determined received fluorescence intensity-tryptophan concentration model, and it can be seen that the curve can well describe the quantitative relationship between the fluorescence intensity and the concentration of the tryptophan solution.
The prediction result of tryptophan concentration shown in fig. 6 (b) can be obtained by combining the above-described method for predicting tryptophan concentration. The black line represents the prediction result obtained by the quantitative analysis method based on the analysis of the fluorescent intensity distribution rule provided by the embodiment of the invention, the red line represents the prediction result in the lambert beer's law linear range by using the ultraviolet spectrophotometry, and the blue line represents the prediction result meeting the fluorescent-concentration linear rule obtained by using the conventional fluorescent quantitative analysis.
Fig. 6 (c) shows relative errors of the prediction results obtained by the quantitative analysis method based on the analysis of the fluorescent intensity distribution rule of the present invention, the ultraviolet spectrophotometry, and the conventional quantitative fluorescence analysis, respectively, at each tryptophan concentration. It can be seen that the fluorescence quantitative analysis method provided by the invention not only has the equivalent prediction precision of the ultraviolet spectrophotometry and the conventional fluorescence quantitative analysis method, but also has a wider analysis range than the ultraviolet spectrophotometry and the conventional fluorescence quantitative analysis method. The ultraviolet spectrophotometry method can generate larger analysis errors when the concentration of a target substance is too high or too low, and the analysis result is easily influenced by an interference substance when the interference substance exists; although the conventional fluorescence quantitative analysis method has specificity to the emission fluorescence wavelength of a target substance and can avoid interference by an interfering substance, the linear range in which the analysis can be performed is very narrow.
The fluorescence quantitative analysis method provided by the invention comprehensively considers e in principle-2.303εcxThe omission condition of the high-order term of c in the Taylor expansion and the influence of the space attenuation effect of the exciting light are obtained, and the high-precision prediction method of the fluorescent substance with the ultra-wide concentration range is obtained.
Further, the fluorescence distribution was summarized in combination with the law of the received fluorescence intensity-fluorescent substance concentration distribution as shown in (a) of fig. 7:
the results of the simulated calculation of the fluorescence intensity distribution of the six graphs are respectively 0.1mg/L, 3mg/L, 10mg/L, 30mg/L, 60mg/L and 120mg/L of tryptophan concentration under the same excitation light intensity.
When the concentration of tryptophan is 0.1mg/L, the fluorescence excited by the solution is relatively very weak;
when the tryptophan concentration is c-3 mg/L and c-10 mg/L, the concentration is still in the linear range of absorbance-concentration described by Lambert beer's theorem, the excitation light spatial attenuation effect is not significant, the fluorescence intensity distribution integrally presents a bright passband, and the higher the tryptophan concentration is, the wider and brighter the bright passband is;
when the concentration of tryptophan is c-30 mg/L, the absorbance-concentration relationship gradually begins to exceed the linear range described by the Lambert beer theorem, the excitation light spatial attenuation effect gradually shows up, and the fluorescence intensity at the front end of the sample cell is obviously stronger than the fluorescence intensity at the rear end of the sample cell;
when the tryptophan concentration is 60mg/L, the increase of the absorbance of the sample is not obvious any more along with the increase of the tryptophan concentration, the fluorescence intensity distribution is obviously layered along with the propagation direction of the exciting light due to the existence of the space attenuation effect of the exciting light, and the fluorescence distribution with the same intensity presents a triangular shape due to the Gaussian property of the excitation intensity distribution;
when the concentration of tryptophan is 120mg/L, the absorbance of the sample is almost not changed along with the further increase of the concentration of tryptophan, the space attenuation effect of the exciting light is very obvious, the layering of the fluorescence intensity distribution along with the propagation direction of the exciting light is more obvious, the interlayer interval is shortened, and the fluorescence is mainly gathered at the front end of the sample cell.
The six graphs in FIG. 7 (b) correspond to the experimental phenomenon of the tryptophan solutions at six concentrations calculated by simulation, respectively. The picture of each sample cell is the original picture of the camera without any image processing, and the fluorescence distribution of each concentration is consistent with the fluorescence intensity distribution calculated and analyzed.
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention. The above-described preferred features may be used in any combination without conflict with each other.
Claims (10)
1. A method for ultra-wide range quantitative fluorescence analysis, comprising: 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 the following steps:
s1, establishing the relation between the received fluorescence intensity and the concentration of the fluorescent substance:
receiving fluorescence intensity F under the condition of single-wavelength excitation lightRThe expression for the relationship with the concentration of the fluorescent substance is:
wherein λ isEmIs the wavelength of the fluorescence emission,
in terms of fluorescence quantum yield, epsilon is a molar extinction coefficient, c is the concentration of the fluorescent substance, zeta is-2.303 epsilon, k is a constant for representing the light absorption capability of the fluorescent substance, N is an excitation light power constant, phi is a constant for representing the fluorescence conversion efficiency of the fluorescent substance, a fluorescence receiving area ranges from x to x and b, and d is a differential sign;
s2, an analytical model is established for the specific fluorescent substance to be measured based on the above expression (4), thereby determining constants in the expression.
2. The ultra-wide range quantitative fluorescence analysis method of claim 1, wherein said S2 comprises:
s21, establishing a series of gradient concentration solution samples of the detected 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 taking the data as a training set;
s22, performing optimization fitting by using a least square method and a Levenberg-Marquardt algorithm, thereby determining parameters phi · N, k and zeta in the expression of S1;
the Levenberg-Marquardt algorithm integrates a steepest descent method and a Taylor series linearization method, and obtains a search direction by solving an optimization model which is as follows:
wherein d iskIn order to search for the direction(s),
is n-dimensional real number field, JkIs a Jacobian matrix, rkTo trust the radius of the field, mukIs a damping parameter, and h is a step length;
thereby establishing a model of the received fluorescence intensity versus the concentration of the fluorescent substance.
3. The ultrawide range quantitative fluorescence analysis method of claim 1, wherein the performing quantitative fluorescence analysis in a specific measurement, wherein: and measuring the absorbance while measuring the fluorescence, wherein the concentration corresponding to the maximum received fluorescence intensity is taken as a concentration threshold, and the corresponding absorbance at the moment is taken as an absorbance threshold.
4. The ultra-wide range fluorescent quantitative analysis method of claim 3, wherein if the absorbance of the measured solution is less than the absorbance threshold, the fluorescent substance concentration is predicted using the fluorescent intensity law that is less than the concentration threshold; and if the measured absorbance is greater than the absorbance threshold, predicting the concentration of the fluorescent substance by using the fluorescence intensity rule greater than the concentration threshold.
5. The ultrawide range quantitative fluorescence analysis method of claim 1, wherein said performing quantitative fluorescence analysis in a particular measurement further comprises: the raw spectra are pre-processed, which includes rejecting spectra containing gross errors and/or noise reduction.
6. The ultra-wide range fluorescent quantitative analysis method of claim 5, wherein said preprocessing further comprises: the baseline of the spectrometer produced by the drift of the electrical signal and the dark current during the measurement is removed, ensuring that the spectra at each time have the same base.
7. The ultra-wide range quantitative fluorescence analysis method of claim 5, wherein said rejecting spectra containing gross errors comprises: rejecting spectra containing gross errors according to Laplace's criterion, wherein Laplace's criterion is expressed as:
in the formula, xb∈{xII ∈ 1,2, …, n } represents one spectral sample in each concentration;
represents the arithmetic mean of the samples; | vb| represents the absolute value of the residual error; σ is the standard deviation calculated by Bessel's equation;
consider x if the sample satisfies the above equationbThe data points containing the gross error values are eliminated.
8. A fluorescence measurement system for use in the method of claim 1, wherein: the method comprises the following steps: excitation light source, diaphragm, first collimating lens, sample cell, first concave mirror, fiber probe, second concave mirror, second collimating lens, narrowband optical filter, convergent lens, spectrum appearance, wherein:
light emitted by an excitation light source passes through a diaphragm and then is converted into parallel light beams by a first collimating lens, the parallel light beams vertically irradiate a liquid sample in a sample cell, a part of light is absorbed by the sample, a part of light is transmitted, and the transmitted light is reflected by a first concave reflector to reach an optical fiber probe; the sample is excited by exciting light and then emits fluorescence to the periphery, the direction vertical to the exciting light is selected to collect the fluorescence, the fluorescence emitted to the left is reflected by a second concave reflector and then is collimated by a second collimating lens together with the fluorescence emitted to the right, then the fluorescence is filtered by a narrow-band filter, and finally the fluorescence and the transmitted light are converged on an optical fiber probe by a converging lens, and the fluorescence and the transmitted light are conducted to a spectrometer together through an optical fiber.
9. The fluorescence measurement system of claim 8, wherein: a second concave reflecting mirror is arranged on the left side of the sample cell to improve the receiving efficiency of fluorescence; and a first concave reflector is arranged at the rear side of the sample cell, reflects and converges the exciting light transmitted through the sample cell to the optical fiber probe, and the absorbance of the sample is calculated by using the part of light.
10. The fluorescence measurement system of claim 8, wherein: the second collimating lens, the band-pass filter and the converging lens are sequentially arranged in a front-back manner on the fluorescence receiving optical path and are respectively used for collimation, filtering and convergence.
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