CN105300945A - Fluorescence quenching method for quantitative analysis of chitosan - Google Patents
Fluorescence quenching method for quantitative analysis of chitosan Download PDFInfo
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Abstract
The invention belongs to the technical field of detection and discloses a fluorescence quenching method for quantitative analysis of chitosan. In the invention, spectral signature of reactive red 4 associated with chitosan in a weak acid environment and its use in chitosan quantitative analysis are researched. The research result shows that in a weak acid B-R buffer system, chitosan has fluorescence quenching effects on reactive red 4, and at wavelength of lambda ex/lambda em=285nm/341nm, in a concentration scope of 0.050-2.00 gammas per milliliter, the fluorescence quenching degree and the chitosan concentration form a good linear relationship. The linear equation is delta F=68.78c+2.648c(c, microgram/milliliter), R<2>=0.9992 and a detection limit is 0.039 micrograms/milliliter. The fluorescence quenching method utilizing a reactive red 4 as a probe for quantitative analysis of chitosan is built. The method has a yield of 96.27-105.3%.
Description
Technical field
The present invention relates to detection technique field, more specifically, relate to a kind of fluorescence quenching method of quantitative test shitosan.
Background technology
Shitosan (CTS) is chitin deacetylase based products, and its chemical name is Chitosan (Isosorbide-5-Nitrae)-2-amino-2-deoxidation-D-glucosan, is that nature content is only second to cellulosic second largest natural organic high-molecular compound.It is the ectoskeletal principal ingredient of shell-fish (shrimp, crab) animal, insect.Chitosan-phospholipid complex is important bioactivator, have antitumor, anticoagulation, the function such as Weight-reducing and lipid-lowering and develop immunitypty, can be used as the adjuvant of Medicines and Health Product, food, cosmetics etc. and prepares the important source material of artificial skin and operation suture thread etc.Therefore, shitosan is in biomedicine, and the field such as environmental health and food all has broad application prospects.
Along with shitosan widespread use, the accurate quantitative analysis of shitosan seems very important.At present, spectrophotometric method and fluorescence method is mainly concentrated on for the method for shitosan quantitative test both at home and abroad.At present, spectrographic determination shitosan generally have ignored shitosan as macromolecule natural products, and its molecular weight from several thousand to 1,000,000 not etc., when chitosan molecule amount and standard items differ greatly in sample, may not produce certain systematic error, affect Accurate Determining.
Summary of the invention
The present invention, in order to overcome the above-mentioned deficiency of prior art, provides the application of Reactive Red 4 in the probe as quantitative test shitosan.
It take Reactive Red 4 as the method for the quantitative test shitosan of probe that another object of the present invention is to provide a kind of.
Another object of the present invention is to provide a kind of fluorescence quenching method of quantitative test shitosan.
To achieve these goals, the present invention is achieved by following scheme:
The present invention first have studied Reactive Red 4 and shitosan under weakly acidic condition associate after spectral signature, and the application in shitosan quantitative test.Result shows in faintly acid B-R buffer system, fluorescence quenching is there is in shitosan to Reactive Red 4, at λ ex/ λ em=285nm/341nm wavelength place, in the concentration range of 0.050 ~ 2.00 μ g/mL, its fluorescent quenching degree and chitosan concentration are good linear relationship.Linear equation is:
Δ F=68.78
c+ 2.648(
c, μ g/mL),
r 2=0.9992, detect and be limited to 0.039 μ g/mL.
Accordingly, the application of application claims prolection red 4 in the probe as quantitative test shitosan.
In addition, the present invention also protects a kind of take Reactive Red 4 as the method for the quantitative test shitosan of probe.
A fluorescence quenching method for quantitative test shitosan, comprises the following steps:
The drafting of typical curve: configure chitosan solution and 1 blank reagent that n becomes gradient concentration, the B-R buffer solution adding 2 times of volumes in the chitosan solution of gradient concentration and 1 blank reagent is respectively become to n, and then add isopyknic Reactive Red 4 solution respectively, the shitosan solution to be measured of the one-tenth concentration gradient after diluting is obtained after mixing, the fluorescence intensity of shitosan solution to be measured is detected respectively after leaving standstill, drawing standard curve, further linear equation:
Δ F=68.78
c+ 2.648(
c, μ g/mL)
In formula
Δ F=
f 0-
f,
f 0for the fluorescence intensity of blank solution,
ffor the fluorescence intensity of the chitosan solution of variable concentrations; Sample detection: to in the supernatant after acetic acid process testing sample, add the B-R buffer solution of 2 times of volumes, and then add isopyknic Reactive Red 4 solution, mix rear leaving standstill, fluorescence intensity, is calculated the concentration of shitosan in testing sample by linear equation.
Preferably, the described standing time is within 120 minutes.
Preferably, the concentration of described Reactive Red 4 solution is 1.00 × 10
-4the pH of mol/L, B-R buffer solution is 4.5.
Preferably, as follows by the step of acetic acid process testing sample: the acetum of testing sample 0.5mol/L is dissolved, centrifugal until completely dissolved, obtain supernatant, detect for fluorescent quenching after supernatant dilution.
A fluorescence quenching method for quantitative test shitosan, comprises the following steps:
The drafting of typical curve: configuration 0.5,1,2,3, the chitosan solution of 5,10,20 μ g/mL and 1 blank reagent, get chitosan solution and the blank reagent of the above-mentioned variable concentrations of 1mL respectively, the pH adding 2mL is respectively the B-R buffer solution of 4.5, then adds 1.00 × 10 of 1mL respectively
-4mol/L Reactive Red 4 solution, 10mL is settled to respectively with water, the shitosan solution to be measured that concentration is respectively 0.05,0.1,0.2,0.3,0.5,1.0,2.0 μ g/mL is obtained after mixing, leave standstill 15 ~ 120 minutes, detect the fluorescence intensity of shitosan solution to be measured, drawing standard curve, further linear equation:
Δ F=68.78
c+ 2.648(
c, μ g/mL)
In formula
Δ F=
f 0-
f,
f 0for the fluorescence intensity of blank solution,
ffor the fluorescence intensity of the chitosan solution of variable concentrations; Sample detection: the acetum of 0.4g testing sample 0.5mol/L is dissolved, be settled to 100mL, centrifugal until completely dissolved, get 2.5mL supernatant, and be settled to 100mL, as sample operation liquid, get 1mL sample operation liquid, the pH adding 2mL is the B-R buffer solution of 4.5, then adds 1.00 × 10 of 1mL
-4mol/L Reactive Red 4 solution, is settled to 10mL with water, and mix latter standing 15 ~ 120 minutes, fluorescence intensity, is calculated the concentration of shitosan in testing sample by linear equation.Compared with prior art, the present invention has following beneficial effect:
The present invention have studied first Reactive Red 4 and shitosan under weakly acidic condition associate after spectral signature, and the application in shitosan quantitative test.Result shows in faintly acid B-R buffer system, fluorescence quenching is there is in shitosan to Reactive Red 4, at λ ex/ λ em=285nm/341nm wavelength place, in the concentration range of 0.050 ~ 2.00 μ g/mL, its fluorescent quenching degree and chitosan concentration are good linear relationship.Linear equation is:
Δ F=68.78
c+ 2.648(
c, μ g/mL),
r 2=0.9992, detect and be limited to 0.039 μ g/mL.The present invention utilizes the combination of shitosan and Reactive Red 4 accordingly, and establish the fluorescence quenching method analyzing shitosan, the method has simple to operate, fast, accurately, and sensitivity high.
Accompanying drawing explanation
Fig. 1 is ultra-violet absorption spectrum, fluorescence spectrum and Resonance Rayleigh Scattering Spectra.
Fig. 2 is the fluorescence pattern of different chitosan concentration, 1: Reactive Red 4 (1.0 × 10
-4mol/L); 2 ~ 6: Reactive Red 4 (1.0 × 10
-4mol/L)-shitosan (0.1,0.5,1.0,1.5,2.0 μ g/mL) ionic associate.
Fig. 3 is the Stern-Volmer figure under different temperatures.
Fig. 4 is the impact of reactive red solution concentration.
Fig. 5 is the impact of buffer solution consumption.
Embodiment
To make the present invention below in conjunction with Figure of description and specific embodiment and elaborating further, described embodiment, only for explaining the present invention, is not intended to limit scope of the present invention.The test method used in following embodiment if no special instructions, is conventional method; The material used, reagent etc. if no special instructions, are the reagent that can obtain from commercial channels and material.
Shitosan (CTS) standard (when 20 DEG C, 1% is dissolved in 1% acetum): low-molecular-weight is≤200mPa.S; Middle-molecular-weihydroxyethyl is 200 ~ 400mPa.S; High molecular is 400 ~ 1000mPa.S.
Shitosan (CTS) standard solution collocation method: accurately taking 0.0400g shitosan, to be dissolved in certain volume concentration be in 0.5mol/L acetum, is then settled to 100mL in volumetric flask, obtains the Standard Reserving Solution that concentration is 400.0 μ g/mL; Draw storing solution 2.50mL in the volumetric flask of 100mL, with water constant volume to scale, obtain the shitosan standard solution of 10.00 μ g/mL, now with the current.
Reactive Red 4 operation liquid (1.00 × 10
-4mol/L): precision takes 0.0250g Reactive Red 4 (Sigma company) dyestuff, and use water-soluble solution, constant volume in 250mL volumetric flask, shakes up, for subsequent use.
Britton-Robinson (B-R) buffer solution: press different proportion by 0.04mol/L acid mixture [(2.71mL orthophosphoric acid+2.36mL glacial acetic acid+2.47g boric acid)/L] and 0.2mol/LNaOH solution formulated.
embodiment 1
One, spectral analysis: in the color comparison tube of 10mL, add the shitosan standard solution (10.00 μ g/mL) of 1mL, pH be 4.50 B-R buffer solution 2.00mL, concentration be 1.00 × 10
-4mol/L Reactive Red 4 operation liquid 1.00mL, pure water is settled to scale, shakes up.Scan ultravioletvisible absorption light light collection of illustrative plates, excitation spectrum, fluorescence spectrum and Resonance Rayleigh Scattering Spectra respectively, the results are shown in Figure 1.As shown in Figure 1, CTS-Reactive Red 4 is at excitation wavelength 285nm, there is maximum fluorescence value at wavelength of fluorescence 341nm place, excitation spectrum becomes specular relation with fluorescence spectrum, judge the peak that each vibrational energy level that the lowest vibration energy level that fluorescence pattern should be the first excited electronic state gets back to ground state is formed, disturb less, peak value is high.Fluorescence excitation spectrum has good overlapping with ultra-violet absorption spectrum peak shape position herein, illustrates to there occurs energy trasfer between shitosan and Reactive Red 4.
Two, fluorescence spectrum: in the color comparison tube of 6 10mL, adds 1mL concentration is respectively the chitosan solution of 0,1,5,10,15,20 μ g/mL, pH is 4.50 B-R buffer solution 2.00mL successively, concentration is 1.00 × 10
-4the Reactive Red 4 operation liquid 1.00mL of mol/L, is settled to scale mark with water, shakes up and obtain the shitosan solution to be measured that concentration is respectively 0,0.1,0.5,1.0,1.5,2.0 μ g/mL.Use 1cm quartz colorimetric utensil, at λ ex/ λ em=285/341nm place, measure the fluorescence intensity of solution to be measured
f, the results are shown in Figure 2, as seen from Figure 2 in the B-R buffer solution of pH4.50, the fluorescence intensity of shitosan to Reactive Red 4 has obvious quenching effect, and within the scope of finite concentration, its quencher degree is linear with the chitosan concentration added.
Three, fluorescent quenching mode: fluorescent quenching process is divided into static quenching and dynamic quenching, static quenching is because quencher and fluorophor molecule form non-luminous complex or molecular complex in ground state, thus causes the process that light-emitting phosphor intensity reduces; Dynamic quenching is the interaction process between quencher and phosphor excitation state molecule, and this process defers to Stern-Volmer equation:
f 0/
f=1+
k sv[Q].
In formula,
f 0for not adding the fluorescence intensity of quencher,
ffor adding the fluorescence intensity of quencher,
k svfor quenching constant, [Q] is the concentration of quencher shitosan.
With
f 0/
fto [Q] mapping, see Fig. 3, obtain the quenching constant of three temperature (303,313,323) in table 1 by quencher curve, visible, quenching constant raises along with the rising of temperature, Δ
fraise along with the rising of temperature, can judge that the fluorescent quenching of shitosan to Reactive Red 4 belongs to Dynamic Fluorescence quencher.Dynamic quenching is due to relevant with diffusion, and when temperature raises, the viscosity of solution declines, and the motion of molecule is simultaneously accelerated, and molecular diffusivity increases, thus increases quenching constant.
Table 1Stern-Volmer equation constant
| Temperature (T/K) | Stern-Volmer equation ([Q], μ g/mL) | Related coefficient ( R 2） | K SV (mL/μg) |
| 303 | F 0/ F=0.9425+0.2624[Q] | 0.9914 | 0.2624 |
| 313 | F 0/ F =0.9702+0.3024 [Q] | 0.9926 | 0.3024 |
| 323 | F0/F=0.9847+0.3259 [Q] | 0.9918 | 0.3259 |
Four, fluorescence quenching method experimental condition optimization
1, the selection of buffer solution and pH: shitosan is soluble cation fiber in an acidic solution, but acidity also will cause too by force decomposing, therefore research is selected to carry out in weakly acidic solution.This research have selected the B-R buffer solution of buffering range at weakly acidic Acetic acid-sodium acetate, hexamethylenetetramine-hydrochloric acid buffer solution and buffering wider range, investigates the impact of different buffer system on Reactive Red 4 fluorescent quenching intensity.
Get two groups of 10mL color comparison tubes, one group adds 1.00mL shitosan standard solution, and another group is reagent blank.Add each buffer solution 2.00mL of different pH value respectively, 1.00 × 10
-4the Reactive Red 4 solution 1.00mL of mol/L, changes the pH value of B-R, HAc-NaAc and hexamethylenetetramine-hydrochloric acid buffer solution, measures the fluorescence intensity of test fluid during different pH value, and deduction corresponding reagent is blank, investigates the maximum Δ of each buffer solution
ftime pH value, three kinds of buffer solution Optimal pH are respectively 4.50,5.50,5.50.Meanwhile, under the optimal pH condition of above-mentioned three kinds of buffer solution, prepare shitosan typical curve respectively, the related coefficient of combined standard curve, find optimized buffer solution.
Result shows, in B-R buffer solution, shitosan is to the fluorescent quenching degree of Reactive Red 4 is comparatively strong and system sensitivity is the highest, and the typical curve linear relationship of preparation is best.Therefore, the B-R buffer solution system of pH4.50 is selected in follow-up test.
2, the selection of Reactive Red 4 solution usage: get two groups of 10mL color comparison tubes, one group adds 1.00mL shitosan standard solution, and another group is reagent blank, fixing Reactive Red 4 concentration (1.00 × 10
-4mol/L) and B-R buffer solution addition (pH4.50,2.00mL), Reactive Red 4 operation liquid consumption is changed.Measure the fluorescence intensity of corresponding Reactive Red 4 addition
f, reagent blank
f 0, draw
Δ F-
vcurve, measurement result is shown in Fig. 4.Experimental result shows, when Reactive Red 4 solution usage is 1.00mL, and the fluorescence intensity difference DELTA of system
fmaximum.
3, the selection of buffer solution consumption: get two groups of 10mL color comparison tubes, one group adds 1.00mL shitosan standard solution, and another group is reagent blank, fixing Reactive Red 4 (1.00 × 10
-4mol/L) addition is 1.00mL, changes the addition of B-R damping fluid (pH4.50), measures the fluorescence intensity of each pipe
f, reagent blank
f 0, draw
Δ F-
vcurve, measurement result is shown in Fig. 5.As seen from Figure 5, when the addition of buffer solution is 2.00mL, system presents large fluorescence difference
Δ F.Therefore the addition of this experimental selection buffer solution is 2.00mL.
4, reaction time and stabilization time: get two groups of 10mL color comparison tubes (standard pipe and reagent blank pipe), standard pipe adds CTS standard solution 1.00mL, the B-R buffer solution 2.00mL of pH4.50,1.00 × 10
-4mol/L Reactive Red 4 solution 1.00mL, measure placement 5,10,15,20,25,30,40,50,60,90,120,150, system after 180min
Δ F.Experiment shows, fluorescent quenching value substantially constant in 5 ~ 60min, afterwards, along with the reaction time extends,
Δ Fslightly rise.
5, reagent adds the impact of order: in one group of 10mL color comparison tube, set casing glycan standard solution addition (1.00mL), Reactive Red 4 concentration (1.00 × 10
-4mol/L, 1.00mL) and B-R buffer solution addition (pH4.50,2.00mL), what change reagent adds order, measures fluorescent quenching degree Δ
f.Experimental result shows, is mixed by shitosan in the ban with B-R buffer solution, then adds people's Reactive Red 4, its fluorescent quenching degree (Δ
f=74.12) maximum, and relatively stable.So experiment all selects shitosan or shitosan sample test liquid first to mix with buffer solution, then adds Reactive Red 4, afterwards with water constant volume, shake up.
Five, linear relationship and detection limit: in a series of 10mL color comparison tube, adds 1mL concentration sequentially respectively and is respectively (chitosan solution of 0.5,1,2,3,5,10,15,20 μ gmL, the B-R damping fluid 2.00mL of pH4.50,1.00 × 10
-4mol/L Reactive Red 4 operation liquid 1.00mL, with distilled water diluting to scale mark, shakes up, and obtains concentration and is respectively that (the shitosan solution to be measured of 0.05,0.10,0.20,0.30,0.50,1.00,1.50,2.00 μ g/mL, prepares reagent blank simultaneously.Result shows, in 0.050 ~ 2.00 μ g/mL concentration range, and fluorescent quenching intensity Δ
fwith chitosan concentration
cin good linear relation, its linear equation is:
Δ F=68.78
c+ 2.648,
r 2=0.9992.Meanwhile, the least concentration value 0.05 μ g/mL in selection standard curve, prepares 13 parts of parallel standards test fluid, prepares corresponding reagent blank simultaneously, measures each test solution fluorescence intensity, and deduction is blank calculates Δ
fvalue
.according to standard deviation and slope of standard curve, calculate this method detects and is limited to 0.039 μ g/mL.
Six, coexisting substances interference: at present, the common health products containing CTS on market are as chitin capsule, and its major auxiliary burden composition has gelatin, starch, dextrin etc.Be in the solution of 1.00 μ g/mL respectively at chitosan mass concentration, add coexisting substances possible in chitin capsule, carry out interference test mensuration, contrast with the standard solution measurement result not adding interfering material, calculate the concentration adding each coexisting substances when producing about ± 5% relative error, the results are shown in Table 2.
Table 2. coexisting substances interference test result
| Coexisting substances | Coexisting substances concentration (μ g/mL) | Relative error (%) |
| Glucose sugar | 1.80×10 4 | -2.99 |
| Ascorbic acid | 5.00 | -3.42 |
| Citric acid | 0.15×10 3 | 3.72 |
| Β-cyclodextrin | 6.0 | 2.88 |
| Soluble starch | 0.30×10 3 | 5.16 |
| Bovine serum albumin(BSA) | 0.80 | 4.07 |
| Gelatin | 3.00 | 3.24 |
| Glycocoll | 0.40×10 3 | 3.56 |
| L-Leu | 2.00 | 2.19 |
| L-Aspartic acid | 0.40×10 3 | -5.43 |
| 1B | 8.00 | 4.94 |
| K + | 4.00×10 3 | 4.81 |
| Na + | 2.00×10 3 | 4.52 |
| Mg 2+ | 0.01 | 2.94 |
| Ca 2+ | 5.00 | -2.85 |
| Zn 2+ | 3.00 | 4.07 |
| Cu 2+ | 1.50 | -3.16 |
| Fe 3+ | 0.16 | -4.50 |
| NH 4 + | 0.20×10 3 | 5.16 |
Result shows, coexisting substances possible in chitin capsule is as less to measurement result interference in soluble starch, citric acid, glucose, glycocoll and the trace element such as potassium, sodium, corresponding also less on the accuracy impact of CTS assay in sample.Part metals ion is as Ca
2+, Mg
2+, Cu
2+and Fe
3+deng comparatively large to the determination influences of shitosan, the interference of metallic ion can be eliminated by adding appropriate EDTA screening agent in sample test liquid.
Seven, the impact of chitosan molecule amount: get three groups of 10mL color comparison tubes, first group adds low-molecular weight chitoglycan standard, and second group adds middle-molecular-weihydroxyethyl shitosan standard, and the 3rd group adds high molecular weight chitosan standard.The concentration of the shitosan solution to be measured of three groups of different molecular weights is 0.05,0.10,0.20,0.30,0.50,1.00,2.00 μ g/mL, each parallel 3 parts, prepares reagent blank simultaneously.Measure the fluorescence intensity of above each solution, draw the typical curve of different molecular weight shitosan.SPSS20.0 is used to carry out slope significance test to three typical curves, obtain P>0.05, illustrate that the slope of three typical curves does not have difference, and then tentatively can infer that this kind of method measures shitosan content substantially not by the impact of molecular size range.
application examples 1
The fluorescence quenching method that the present invention sets up is detecting the application on market in conventional shitosan medicine, the conventional medication that should use in use-case with Austria's sharp peacekeeping Ai get Lan two brands for representative.Ao Liwei is purchased from Weihai Nan Bowan Bioisystech Co., Ltd, and Ai get Lan is purchased from Shanghai Tongji Biological Product Co., Ltd..The detecting step of two kinds of medicines is as follows:
1, sample pretreatment: accurately take 0.0400g chitosan capsules powder (sharp peacekeeping Ai get Lan difficult to understand two brands) respectively, dissolve with the acetum that certain volume concentration is 0.5mol/L, then 100mL is settled to, until completely dissolved in the centrifugal 15min of 6000r/min, get 2.5mL supernatant, add the EDTA solution 1mL of 0.01mol/L, after be settled to 100mL with pure water, as sample operation liquid.
2, sample size measures: in the color comparison tube of 2 10mL, adds the sample operation liquid of 1mL sharp peacekeeping Ai get Lan difficult to understand two kinds of products respectively, each Guan Zhongzai add pH be 4.50 B-R buffer solution 2.00mL, concentration be 1.00 × 10
-4the Reactive Red 4 operation liquid 1.00mL of mol/L, is settled to scale mark with water, shakes up.Use 1cm quartz colorimetric utensil, at λ ex/ λ em=285/341nm place, measure the fluorescence intensity of each solution
f, and the reagent blank of correspondence
f 0, calculate
Δf=
f 0 -F.Because fluorescence quenching method measures shitosan not by the impact of reference material molecular weight, therefore, when detecting sample mesochite glycan content, selection low-molecular weight chitoglycan is standard.
3, Precision Experiment: according to the method for step 2, adds the sample operation liquid of certain volume in 10mL color comparison tube, parallel 3 parts, prepares reagent blank and low-molecular weight chitoglycan typical curve simultaneously.Calculation sample operation liquid chitosan concentration, the results are shown in Table 3.
Table 3 sample determination result
| Sample | Ao Liwei (mg/g) | Ai get Lan (mg/g) |
| 1 | 872.5 | 997.5 |
| 2 | 890.0 | 977.5 |
| 3 | 922.5 | 960.0 |
| Mean value (mg/g) | 895.0 | 978.3 |
| RSD(%) | 2.83 | 1.92 |
The fluorescence quenching method recovery is tested: in two groups of 10mL color comparison tubes, add Ao Liwei shitosan sample operation liquid and Ai get Lan shitosan sample operation liquid 0.50mL respectively, then add in each pipe the CTS titer 0.30 of 10.00 μ g/mL, 0.50,1.00mL, prepare blank, each parallel 5 parts simultaneously.Measure each pipe fluorescence intensity, calculate recovery of standard addition.The results are shown in Table 4.
Table 4 recovery of standard addition result
Claims (7)
1. the application of Reactive Red 4 in the probe as quantitative test shitosan.
2. be the method for the quantitative test shitosan of probe with Reactive Red 4.
3. a fluorescence quenching method for quantitative test shitosan, is characterized in that, comprises the following steps:
The drafting of typical curve: configure chitosan solution and 1 blank reagent that n becomes gradient concentration, the B-R buffer solution adding 2 times of volumes in the chitosan solution of gradient concentration and 1 blank reagent is respectively become to n, and then add isopyknic Reactive Red 4 solution respectively, the shitosan solution to be measured of the one-tenth concentration gradient after diluting is obtained after mixing, the fluorescence intensity of shitosan solution to be measured is detected respectively after leaving standstill, drawing standard curve, further linear equation:
Δ F=68.78
c+ 2.648(
c, μ g/mL)
In formula
Δ F=
f 0-
f,
f 0for the fluorescence intensity of blank solution,
ffor the fluorescence intensity of the chitosan solution of variable concentrations; Sample detection: to in the supernatant after acetic acid process testing sample, add the B-R buffer solution of 2 times of volumes, and then add isopyknic Reactive Red 4 solution, mix rear leaving standstill, fluorescence intensity, is calculated the concentration of shitosan in testing sample by linear equation.
4. fluorescence quenching method according to claim 3, is characterized in that, the described standing time is within 120 minutes.
5. fluorescence quenching method according to claim 3, is characterized in that, the concentration of described Reactive Red 4 solution is 1.00 × 10
-4the pH of mol/L, B-R buffer solution is 4.5.
6. fluorescence quenching method according to claim 3, it is characterized in that, as follows by the step of acetic acid process testing sample: the acetum of testing sample 0.5mol/L is dissolved, centrifugal until completely dissolved, obtain supernatant, detect for fluorescent quenching after supernatant dilution.
7. a fluorescence quenching method for quantitative test shitosan, is characterized in that, comprises the following steps:
The drafting of typical curve: configuration 0.5,1,2,3, the chitosan solution of 5,10,20 μ g/mL and 1 blank reagent, get chitosan solution and the blank reagent of the above-mentioned variable concentrations of 1mL respectively, the pH adding 2mL is respectively the B-R buffer solution of 4.5, then adds 1.00 × 10 of 1mL respectively
-4mol/L Reactive Red 4 solution, 10mL is settled to respectively with water, the shitosan solution to be measured that concentration is respectively 0.05,0.1,0.2,0.3,0.5,1.0,2.0 μ g/mL is obtained after mixing, leave standstill 15 ~ 120 minutes, detect the fluorescence intensity of shitosan solution to be measured, drawing standard curve, further linear equation:
Δ F=68.78
c+ 2.648(
c, μ g/mL)
In formula
Δ F=
f 0-
f,
f 0for the fluorescence intensity of blank solution,
ffor the fluorescence intensity of the chitosan solution of variable concentrations;
Sample detection: the acetum of 0.4g testing sample 0.5mol/L is dissolved, be settled to 100mL, centrifugal until completely dissolved, get 2.5mL supernatant, and be settled to 100mL, as sample operation liquid, get 1mL sample operation liquid, the pH adding 2mL is the B-R buffer solution of 4.5, then adds 1.00 × 10 of 1mL
-4mol/L Reactive Red 4 solution, is settled to 10mL with water, and mix latter standing 15 ~ 120 minutes, fluorescence intensity, is calculated the concentration of shitosan in testing sample by linear equation.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105486648A (en) * | 2015-11-13 | 2016-04-13 | 广东药学院 | Resonance Rayleigh scattering method for quantitatively analyzing chitosan |
| CN106053399A (en) * | 2016-05-27 | 2016-10-26 | 广东药科大学 | Method for measuring chitosan content by using water-soluble aniline blue as probe |
| CN108225879A (en) * | 2017-12-30 | 2018-06-29 | 广东药科大学 | A kind of method for measuring chitosan content by the use of the carmine resonance rayleigh light scattering method as probe |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2012103014A (en) * | 2010-11-05 | 2012-05-31 | Tokyo Metropolitan Institute Of Medical Science | Fluorescence enhancing agent and fluorescence enhancing method and use thereof |
| CN102527347A (en) * | 2012-01-19 | 2012-07-04 | 上海海洋大学 | Magnetic chitosan/cationic surface active agent modified zeolite adsorbent and preparation method and application thereof |
| CN105486648A (en) * | 2015-11-13 | 2016-04-13 | 广东药学院 | Resonance Rayleigh scattering method for quantitatively analyzing chitosan |
-
2015
- 2015-11-13 CN CN201510774801.8A patent/CN105300945B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2012103014A (en) * | 2010-11-05 | 2012-05-31 | Tokyo Metropolitan Institute Of Medical Science | Fluorescence enhancing agent and fluorescence enhancing method and use thereof |
| CN102527347A (en) * | 2012-01-19 | 2012-07-04 | 上海海洋大学 | Magnetic chitosan/cationic surface active agent modified zeolite adsorbent and preparation method and application thereof |
| CN105486648A (en) * | 2015-11-13 | 2016-04-13 | 广东药学院 | Resonance Rayleigh scattering method for quantitatively analyzing chitosan |
Non-Patent Citations (4)
| Title |
|---|
| 白研等: "《活性红4离子缔合分光光度法测定壳聚糖》", 《食品科学》 * |
| 白研等: "《荧光猝灭法测定壳聚糖含量》", 《分析试验室》 * |
| 穆志英等: "《核固红荧光猝灭法测定壳聚糖含量》", 《分析测试学报》 * |
| 高贵珍等: "《茜素红S荧光猝灭法测定壳聚糖含量》", 《光谱学与光谱分析》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105486648A (en) * | 2015-11-13 | 2016-04-13 | 广东药学院 | Resonance Rayleigh scattering method for quantitatively analyzing chitosan |
| CN105486648B (en) * | 2015-11-13 | 2018-08-24 | 广东药科大学 | A kind of resonance rayleigh light scattering method of quantitative analysis chitosan |
| CN106053399A (en) * | 2016-05-27 | 2016-10-26 | 广东药科大学 | Method for measuring chitosan content by using water-soluble aniline blue as probe |
| CN108225879A (en) * | 2017-12-30 | 2018-06-29 | 广东药科大学 | A kind of method for measuring chitosan content by the use of the carmine resonance rayleigh light scattering method as probe |
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