CN105300945B - A kind of fluorescence quenching method of quantitative analysis chitosan - Google Patents
A kind of fluorescence quenching method of quantitative analysis chitosan Download PDFInfo
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Abstract
The invention belongs to detection technique fields, specifically disclose a kind of fluorescence quenching method of quantitative analysis chitosan.The present invention has studied Reactive Red 4 and the spectral signature after chitosan association, and its application in chitosan quantitative analysis under weakly acidic condition first.The result shows that in faintly acid B R buffer systems, there are fluorescence quenchings to Reactive Red 4 for chitosan, at λ ex/ λ em=285nm/341nm wavelength, in the concentration range of 0.050~2.00 μ g/mL, fluorescent quenching degree is in good linear relationship with chitosan concentration.Linear equation is：ΔF=68.78c+2.648（c, μ g/mL）,R 2=0.9992, detection is limited to 0.039 μ g/mL.Accordingly, the fluorescence quenching method that chitosan is analyzed using Reactive Red 4 as probe quantitative is established, the rate of recovery of this method is 96.27~105.3%.
Description
Technical field
The present invention relates to detection technique fields, more particularly, to a kind of fluorescence quenching method of quantitative analysis chitosan.
Background technology
Chitosan（CTS）It is chitin deacetylation product, its chemical name is Chitosan (Isosorbide-5-Nitrae) -2- amino -2-
Deoxidation-D- glucans are the second largest natural organic polymer compounds that nature content is only second to cellulose.It is shell-fish
The main component of the ectoskeleton of (shrimp, crab) animal, insect.Chitosan and its derivative is important bioactive substance, has
Antitumor, anticoagulation, the functions such as Weight-reducing and lipid-lowering and strengthen immunity can be used as the addition of Medicines and Health Product, food, cosmetics etc.
Agent and the important source material for preparing artificial skin and operation suture thread etc..Therefore, chitosan is in biomedicine, environmental sanitation and food
Equal fields suffer from wide application prospect.
With chitosan extensive use, the accurate quantitative analysis of chitosan seems particularly significant.Currently, being used for chitosan both at home and abroad
The method of quantitative analysis is concentrated mainly on spectrophotometry and fluorescence method.Currently, spectrographic determination chitosan generally has ignored shell
Glycan is differed from thousands of to million as macromolecule natural products, molecular weight, when chitosan molecule amount and standard items in sample
When differing greatly, certain systematic error may be will produce, Accurate Determining is influenced.
Invention content
The present invention provides Reactive Red 4 in the spy as quantitative analysis chitosan to overcome the above-mentioned deficiency of the prior art
Application in needle.
It is a further object to provide a kind of using Reactive Red 4 as the method for the quantitative analysis chitosan of probe.
It is also another object of the present invention to provide a kind of fluorescence quenching methods of quantitative analysis chitosan.
To achieve the goals above, the present invention is achieved by following scheme：
The present invention has studied Reactive Red 4 and the spectral signature after chitosan association under weakly acidic condition first, and its poly- in shell
Application in sugared quantitative analysis.The result shows that in faintly acid B-R buffer systems, there are fluorescent quenchings to Reactive Red 4 for chitosan
Effect, at λ ex/ λ em=285nm/341nm wavelength, in the concentration range of 0.050 ~ 2.00 μ g/mL, fluorescent quenching degree with
Chitosan concentration is in good linear relationship.Linear equation is：ΔF =68.78c+2.648（c, μ g/mL）,R 2=0.9992, inspection
Rising limit is 0.039 μ g/mL.
Accordingly, application of the claimed Reactive Red 4 in the probe as quantitative analysis chitosan.
In addition, the present invention also protect it is a kind of using Reactive Red 4 as the method for the quantitative analysis chitosan of probe.
A kind of fluorescence quenching method of quantitative analysis chitosan, includes the following steps：
The drafting of standard curve：N are configured at the chitosan solution of gradient concentration and 1 blank reagent, to n at gradient
It is separately added into the B-R buffer solutions of 2 times of volumes in the chitosan solution of concentration and 1 blank reagent, is then separately added into again
The Reactive Red 4 solution of volume, the solution to be measured of the chitosan at concentration gradient after being diluted after mixing, after standing respectively
The fluorescence intensity of chitosan solution to be measured is detected, standard curve is drawn, further obtains linear equation：ΔF =68.78c+2.648
（c, μ g/mL）
In formulaΔF = F 0-F,F 0For the fluorescence intensity of blank solution,FFluorescence for the chitosan solution of various concentration is strong
Degree；Sample detection：To in the supernatant after acetic acid processing sample to be tested, the B-R buffer solutions of 2 times of volumes are added, then again
Isometric Reactive Red 4 solution is added, stands after mixing, fluorescence intensity, sample to be tested is calculated by linear equation
The concentration of middle chitosan.
Preferably, the time of the standing is within 120 minutes.
Preferably, a concentration of the 1.00 × 10 of the Reactive Red 4 solution-4The pH of mol/L, B-R buffer solution is 4.5.
Preferably, the step of handling sample to be tested with acetic acid is as follows：Sample to be tested is molten with the acetum of 0.5mol/L
Solution, centrifuges until completely dissolved, obtains supernatant, is detected for fluorescent quenching after supernatant dilution.
A kind of fluorescence quenching method of quantitative analysis chitosan, includes the following steps：
The drafting of standard curve：The chitosan solution and 1 blank reagent of 0.5,1,2,3,5,10,20 μ g/mL are configured, point
The chitosan solution and blank reagent for not taking the above-mentioned various concentrations of 1mL are separately added into the B-R buffer solutions that the pH of 2mL is 4.5,
It is separately added into the 1.00 × 10 of 1mL again-4Mol/L Reactive Red 4 solution is settled to 10mL with water, obtains after mixing dense respectively
Degree is respectively the chitosan solution to be measured of 0.05,0.1,0.2,0.3,0.5,1.0,2.0 μ g/mL, stands 15~120 minutes, inspection
The fluorescence intensity of chitosan solution to be measured is surveyed, standard curve is drawn, further obtains linear equation：ΔF =68.78c+2.648（c,
μg/mL）
In formulaΔF = F 0-F,F 0For the fluorescence intensity of blank solution,FFluorescence for the chitosan solution of various concentration is strong
Degree；Sample detection：The 0.4g samples to be tested acetum of 0.5mol/L is dissolved, 100mL is settled to, until completely dissolved from
The heart takes 2.5mL supernatants, and is settled to 100mL, as sample operation liquid, takes the 1mL sample operation liquid, the pH that 2mL is added to be
4.5 B-R buffer solutions, add the 1.00 × 10 of 1mL-4Mol/L Reactive Red 4 solution, 10mL is settled to water, is uniformly mixed
15~120 minutes are stood afterwards, fluorescence intensity is calculated the concentration of chitosan in sample to be tested by linear equation.With it is existing
Technology is compared, and the present invention has the advantages that：
The present invention has studied Reactive Red 4 and the spectral signature after chitosan association under weakly acidic condition for the first time, and its poly- in shell
Application in sugared quantitative analysis.The result shows that in faintly acid B-R buffer systems, there are fluorescent quenchings to Reactive Red 4 for chitosan
Effect, at λ ex/ λ em=285nm/341nm wavelength, in the concentration range of 0.050~2.00 μ g/mL, fluorescent quenching degree
It is in good linear relationship with chitosan concentration.Linear equation is：ΔF =68.78c+2.648（c, μ g/mL）,R 2=0.9992,
Detection is limited to 0.039 μ g/mL.The present invention utilizes the combination of chitosan and Reactive Red 4 accordingly, establishes the fluorescence of analysis chitosan
Quenching method, this method have it is easy to operate, quickly, accurately, the features such as high sensitivity.
Description of the drawings
Fig. 1 is ultra-violet absorption spectrum, fluorescence spectrum and Resonance Rayleigh Scattering Spectra.
Fig. 2 be different chitosan concentrations fluorescence pattern, 1：Reactive Red 4（1.0×10-4mol/L）；2~6：Reactive Red 4
(1.0×10-4Mol/L)-chitosan (0.1,0.5,1.0,1.5,2.0 μ g/mL) ionic associate.
Fig. 3 is the Stern-Volmer figures under different temperatures.
Fig. 4 is the influence of active red solution concentration.
Fig. 5 is the influence of buffer solution dosage.
Specific implementation mode
The present invention is made with specific embodiment with reference to the accompanying drawings of the specification and further being elaborated, the embodiment
It is served only for explaining the present invention, be not intended to limit the scope of the present invention.Test method used in following embodiments is such as without spy
Different explanation, is conventional method；Used material, reagent etc., unless otherwise specified, for the reagent commercially obtained
And material.
Chitosan (CTS) standard（At 20 DEG C, 1% is dissolved in 1% acetum）：Low molecular weight is≤200
mPa.S；Middle-molecular-weihydroxyethyl is 200 ~ 400mPa.S；High molecular weight is 400 ~ 1000mPa.S.
Chitosan (CTS) standard solution configuration method：It is accurate to weigh 0.0400g chitosans to be dissolved in certain volume a concentration of
In 0.5mol/L acetums, then in volumetric flask constant volume to 100mL, the Standard Reserving Solution of a concentration of 400.0 μ g/mL is obtained；
Storing solution 2.50mL is drawn in the volumetric flask of 100 mL, with water constant volume to scale, obtains the chitosan standard of 10.00 μ g/mL
Solution, it is now with the current.
Reactive Red 4 operates liquid（1.00×10-4mol/L）：Precision weighs 0.0250g Reactive Red 4s（Sigma companies）Dyestuff,
With water dissolution, the constant volume in 250mL volumetric flasks shakes up, spare.
Britton-Robinson (B-R) buffer solution：By 0.04mol/L mixed acid [(2.71mL orthophosphoric acid+
2.36mL glacial acetic acid+2.47g boric acid)/L] it is formulated by different proportion with 0.2mol/L NaOH solution.
Embodiment 1
One, spectrum analysis：In the colorimetric cylinder of 10mL, the chitosan standard solution of 1mL is added（10.00μg/mL）, pH be
4.50 B-R buffer solutions 2.00mL, a concentration of 1.00 × 10-4Mol/L Reactive Red 4s operate liquid 1.00mL, and pure water is settled to quarter
Degree, shakes up.Scanning ultravioletvisible absorption light light collection of illustrative plates, excitation spectrum, fluorescence spectrum and Resonance Rayleigh Scattering Spectra respectively, knot
Fruit sees Fig. 1.As shown in Figure 1, CTS- Reactive Red 4s have maximum fluorescence value at excitation wavelength 285nm, wavelength of fluorescence 341nm, excitation
Spectrum and fluorescence spectrum are mirrored into symmetric relation, judge that fluorescence pattern should be the lowest vibration energy level of the first excited electronic state and return to
Each vibration level of ground state is formed by peak, and interference is less, and peak value is high.Fluorescence excitation spectrum and ultra-violet absorption spectrum peak herein
Morpheme is equipped with preferable overlapping, illustrates that energy transfer has occurred between chitosan and Reactive Red 4.
Two, fluorescence spectrum：In the colorimetric cylinder of 6 10mL, it is respectively 0,1,5,10,15,20 μ to sequentially add 1mL concentration
The chitosan solution of g/mL, the B-R buffer solutions 2.00mL that pH is 4.50, a concentration of 1.00 × 10-4The Reactive Red 4 of mol/L is grasped
Make liquid 1.00mL, graduation mark is settled to water, shakes up to obtain the shell that concentration is respectively 0,0.1,0.5,1.0,1.5,2.0 μ g/mL
Glycan solution to be measured.With 1cm quartz colorimetric utensils the fluorescence intensity of solution to be measured is measured at λ ex/ λ em=285/341nmF,
As a result see Fig. 2, as seen from Figure 2 in the B-R buffer solutions of pH 4.50, chitosan has the fluorescence intensity of Reactive Red 4 apparent
Quenching effect, and within the scope of a certain concentration, the chitosan concentration for quenching degree and addition is in a linear relationship.
Three, fluorescent quenching mode：Fluorescent quenching process is divided into static quenching and dynamic quenching, and static quenching is due to quenching
Agent forms non-luminous complex or molecular complex with fluorophor molecule in ground state, so as to cause light-emitting phosphor strength reduction
Process；Dynamic quenching is the interaction process between quencher and phosphor excitation state molecule, which defers to Stern-
Volmer equations：F 0/F=1+K sv[Q]。
In formula,F 0Not add the fluorescence intensity of quencher,FFor the fluorescence intensity of quencher is added,K svFor quenching constant,
[Q] is the concentration of quencher chitosan.
WithF 0/FIt maps to [Q], sees Fig. 3, three temperature are obtained by quenching curve（303、313、323）Quenching constant see
Table 1, it is seen then that quenching constant is increased with the raising of temperature, ΔFIt is increased with the raising of temperature, it can be determined that chitosan
Dynamic Fluorescence quenching is belonged to the fluorescent quenching of Reactive Red 4.Dynamic quenching is due to related with diffusion, and solution when temperature raising
Viscosity declines, while the movement of molecule accelerates, and coefficient of molecular diffusion increases, to increase quenching constant.
1 Stern-Volmer equation constants of table
| Temperature（T/K） | Stern-Volmer equations ([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：Chitosan is soluble cation fiber in an acidic solution, but acid too strong
It also leads to decompose, therefore studies selection and carried out in weakly acidic solution.This research has selected buffering range in weakly acidic acetic acid-
Sodium acetate, hexamethylenetetramine-hydrochloric acid buffer solution and the B-R buffer solutions for buffering wider range, investigate different buffer systems
Influence to Reactive Red 4 fluorescent quenching intensity.
Two groups of 10mL colorimetric cylinders are taken, one group of addition 1.00mL chitosan standard solution, another group is reagent blank.Respectively plus
Enter each buffer solution 2.00mL of different pH value, 1.00 × 10-4The Reactive Red 4 solution 1.00mL of mol/L changes B-R, HAc-
The pH value of NaAc and hexamethylenetetramine-hydrochloric acid buffer solution, the fluorescence intensity of test fluid when measuring different pH value deduct corresponding
Reagent blank investigates each buffer solution maximum ΔFWhen pH value, three kinds of buffer solution Optimal pHs are respectively 4.50,5.50,
5.50.Meanwhile under the conditions of the optimal pH of above-mentioned three kinds of buffer solutions, chitosan standard curve, combined standard are prepared respectively
The related coefficient of curve finds optimized buffer solution.
The result shows that in B-R buffer solutions, chitosan is sensitive compared with strong and system to the fluorescent quenching degree of Reactive Red 4
Highest is spent, the standard curve linear relationship of preparation is best.Therefore, the B-R buffer solution systems of pH 4.50 are selected in follow-up test.
2, the selection of Reactive Red 4 solution usage：Two groups of 10mL colorimetric cylinders are taken, one group adds 1.00mL chitosan standard solution,
Another group is reagent blank, fixed Reactive Red 4 concentration（1.00×10-4mol/L）With B-R buffer solution additions（PH4.50,
2.00mL）, change Reactive Red 4 and operate liquid dosage.Measure the fluorescence intensity of corresponding Reactive Red 4 additionF, reagent blankF 0,
It drawsΔF-VCurve, measurement result are shown in Fig. 4.The experimental results showed that when Reactive Red 4 solution usage be 1.00mL when, system it is glimmering
Luminous intensity difference DELTAFIt is maximum.
3, the selection of buffer solution dosage：Two groups of 10mL colorimetric cylinders are taken, one group adds 1.00mL chitosan standard solution, another
Group is reagent blank, fixed Reactive Red 4（1.00×10-4mol/L）Addition be 1.00 mL, change B-R buffer solutions
（pH4.50）Addition, measure the fluorescence intensity of each pipeF, reagent blankF 0, drawΔF-VCurve, measurement result are shown in Fig. 5.
As seen from Figure 5, when the addition of buffer solution is 2.00mL, big fluorescence difference is presented in systemΔF.Therefore this experimental selection buffers
The addition of solution is 2.00mL.
4, reaction time and stabilization time：Take two groups of 10mL colorimetric cylinders（Standard pipe and reagent blank pipe）, standard pipe addition
The B-R buffer solutions 2.00mL of CTS standard solution 1.00mL, pH4.50,1.00 × 10-4Mol/L Reactive Red 4 solution
1.00mL measures system after placing 5,10,15,20,25,30,40,50,60,90,120,150,180 minΔF.Experiment
Show the fluorescence quenching value substantially constant in 5~60min, later, as the reaction time extends,ΔFSlightly rise.
5, the influence of order is added in reagent：In one group of 10 mL colorimetric cylinder, fixed chitosan standard solution addition
（1.00mL）, Reactive Red 4 concentration（1.00×10-4Mol/L, 1.00mL）With B-R buffer solution additions（PH4.50,
2.00mL）, change the addition order of reagent, measure fluorescent quenching degree ΔF.The experimental results showed that in the ban by chitosan and B-R
Buffer solution mixes, then adds people's Reactive Red 4, fluorescent quenching degree (ΔF=74.12) maximum, and stablize relatively.So real
It tests and chitosan or chitosan sample test liquid is selected first to be mixed with buffer solution, add Reactive Red 4, it is fixed with water later
Hold, shake up.
Five, linear relationship and detection limit：In a series of 10mL colorimetric cylinders, it is sequentially separately added into 1mL concentration point
Not Wei (chitosan solution of 0.5,1,2,3,5,10,15,20 μ gmL, the B-R buffer solutions 2.00mL of pH4.50,1.00 × 10- 4Mol/L Reactive Red 4s operate liquid 1.00mL, graduation mark is diluted to distilled water, is shaken up, obtain concentration be respectively (0.05,0.10,
0.20, the chitosan solution to be measured of 0.30,0.50,1.00,1.50,2.00 μ g/mL, while reagent preparation blank.The result shows that
In 0.050~2.00 μ g/mL concentration ranges, fluorescent quenching intensity ΔFWith chitosan concentrationcIn good linear relationship, line
Property equation is：ΔF =68.78c+ 2.648,R 2=0.9992.Meanwhile the 0.05 μ g/ of minimum concentration value in selection standard curve
ML prepares 13 parts of parallel standards test fluids, while preparing corresponding reagent blank, measures each test solution fluorescence intensity, deducts blank meter
Calculate ΔFValue。According to standard deviation and slope of standard curve, calculate this method detection be limited to 0.039 μ g/mL.
Six, coexisting substances interfere：Currently, such as crust cellulose capsule of the common health products containing CTS in the market, major auxiliary burden
Ingredient has gelatin, starch, dextrin etc..Respectively in the solution of a concentration of 1.00 μ g/mL of chitosan mass, crust cellulose capsule is added
In possible coexisting substances, carry out interference test measurement, with not plus the standard solution measurement result of interfering substance compare, calculate and produce
The concentration of each coexisting substances is added when raw about ± 5% relative error, the results are shown in Table 2.
2. coexisting substances interference test result of table
| Coexisting substances | Coexisting substances concentration（µg/mL） | Relative error (%) |
| Glucose sugar | 1.80×104 | -2.99 |
| Ascorbic acid | 5.00 | -3.42 |
| Citric acid | 0.15×103 | 3.72 |
| Β-cyclodextrin | 6.0 | 2.88 |
| Soluble starch | 0.30×103 | 5.16 |
| Bovine serum albumin(BSA) | 0.80 | 4.07 |
| Gelatin | 3.00 | 3.24 |
| Glycine | 0.40×103 | 3.56 |
| L-Leu | 2.00 | 2.19 |
| L-Aspartic acid | 0.40×103 | -5.43 |
| L-lysine | 8.00 | 4.94 |
| K+ | 4.00×103 | 4.81 |
| Na+ | 2.00×103 | 4.52 |
| Mg2+ | 0.01 | 2.94 |
| Ca2+ | 5.00 | -2.85 |
| Zn2+ | 3.00 | 4.07 |
| Cu2+ | 1.50 | -3.16 |
| Fe3+ | 0.16 | -4.50 |
| NH4 + | 0.20×103 | 5.16 |
The result shows that possible coexisting substances such as soluble starch, citric acid, glucose, glycine in crust cellulose capsule
And the trace element such as potassium, sodium measurement result is interfered it is smaller, on the accuracy of CTS assays in sample influence it is corresponding also compared with
It is small.Part metals ion such as Ca2+、Mg2+、Cu2+And Fe3+It is larger Deng the determination influences to chitosan, it can be by sample test
Suitable EDTA screening agents are added in liquid to eliminate the interference of metal ion.
Seven, the influence of chitosan molecule amount：Take three groups of 10mL colorimetric cylinders, first group of addition low-molecular weight chitoglycan standard,
High molecular weight chitosan standard is added in second group of addition middle-molecular-weihydroxyethyl chitosan standard, third group.The shell of three groups of different molecular weights
The concentration of glycan solution to be measured is 0.05,0.10,0.20,0.30,0.50,1.00,2.00 μ g/mL, each parallel 3 parts, simultaneously
Reagent preparation blank.The fluorescence intensity for measuring above each solution, draws the standard curve of different molecular weight chitosan.It uses
SPSS20.0 pairs of three standard curves obtain P into line slope significance test>0.05, illustrate that the slope of three standard curves does not have
Difference, and then can tentatively infer that such method measures chitosan content and do not influenced substantially by molecular size range.
Application examples 1
Application of the fluorescence quenching method that the present invention establishes in detection in the market conventional chitosan drug makes in the application example
Conventional medication is using sharp peacekeeping two brands of Ai get Lan difficult to understand as representative.Ao Liwei is purchased from the limited public affairs of Weihai Nan Bowan biotechnologys
Department, Ai get Lan are purchased from Shanghai Tongji Biological Product Co., Ltd..The detecting step of two kinds of drugs is as follows：
1, sample pretreatment：0. 0400g chitosan capsules powder is accurately weighed respectively（Profit peacekeeping two product of Ai get Lan difficult to understand
Board）, dissolved with the acetum of a concentration of 0.5mol/L of certain volume, be then settled to 100mL, until completely dissolved in
6000r/min centrifuges 15min, takes 2.5mL supernatants, the EDTA solution 1mL of 0.01mol/L are added, after with pure water be settled to 100
ML, as sample operation liquid.
2, sample size measures：In the colorimetric cylinder of 2 10mL, it is separately added into 1mL sharp peacekeeping two kinds of products of Ai get Lan difficult to understand
Sample operation liquid, the B-R buffer solutions 2.00mL, a concentration of 1.00 × 10 that pH is 4.50 are added in each pipe-4Mol/L's
Reactive Red 4 operates liquid 1.00mL, is settled to graduation mark with water, shakes up.With 1cm quartz colorimetric utensils, in em=285/ λ ex/ λ
At 341nm, the fluorescence intensity of each solution is measuredFAnd corresponding reagent blankF 0, calculateΔF = F 0 -F.Due to fluorescent quenching
Method measures chitosan not to be influenced by reference substance molecular weight, therefore, when detecting chitosan content in sample, selects low molecular weight shell
Glycan is standard.
3, Precision Experiment：According to the method for step 2, the sample operation liquid of certain volume is added in 10 mL colorimetric cylinders,
Parallel 3 parts, while reagent preparation blank and low-molecular weight chitoglycan standard curve.Calculate sample operation liquid chitosan concentration, knot
Fruit is shown in Table 3.
3 sample measurement result of table
| 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 |
| Average value（mg/g） | 895.0 | 978.3 |
| RSD(%) | 2.83 | 1.92 |
The fluorescence quenching method rate of recovery is tested：In two groups of 10mL colorimetric cylinders, it is separately added into Ao Liwei chitosan sample operations
0.50 mL of liquid and Ai get Lan chitosan sample operations liquid, be then added into each pipe 10.00 μ g/mL CTS titers 0.30,
0.50,1.00mL, while blank is prepared, each parallel 5 parts.Each pipe fluorescence intensity is measured, recovery of standard addition is calculated.It the results are shown in Table 4.
4 recovery of standard addition result of table
Claims (5)
1. a kind of fluorescence quenching method of quantitative analysis chitosan, which is characterized in that include the following steps：
The drafting of standard curve：N are configured at the chitosan solution of gradient concentration and 1 blank reagent, to n at gradient concentration
Chitosan solution and 1 blank reagent in be separately added into the B-R buffer solutions of 2 times of volumes, it is then molten in chitosan respectively again
The Reactive Red 4 solution of same volume, the shell at concentration gradient after being diluted after mixing are added in liquid and blank solution
Glycan solution to be measured detects the fluorescence intensity of chitosan solution to be measured after standing respectively, draws standard curve, further linearly
Equation：ΔF =68.78c+2.648（c, μ g/mL）
In formulaΔF = F 0-F,F 0For the fluorescence intensity of blank solution,FFor the fluorescence intensity of the chitosan solution of various concentration；Sample
Product examine is surveyed：To in the supernatant after acetic acid processing sample to be tested, the B-R buffer solutions of 2 times of volumes are added, then add
The Reactive Red 4 solution of volume, stands, fluorescence intensity after mixing, and calculating shell in sample to be tested by linear equation gathers
The concentration of sugar.
2. fluorescence quenching method according to claim 1, which is characterized in that the time of the standing is within 120 minutes.
3. fluorescence quenching method according to claim 1, which is characterized in that a concentration of the 1.00 of the Reactive Red 4 solution ×
10-4The pH of mol/L, B-R buffer solution is 4.5.
4. fluorescence quenching method according to claim 1, which is characterized in that the step of handling sample to be tested with acetic acid is as follows：
The sample to be tested acetum of 0.5mol/L is dissolved, is centrifuged until completely dissolved, supernatant is obtained, is used for after supernatant dilution
Fluorescent quenching detects.
5. a kind of fluorescence quenching method of quantitative analysis chitosan, which is characterized in that include the following steps：
The drafting of standard curve：The chitosan solution and 1 blank reagent for configuring 0.5,1,2,3,5,10,20 μ g/mL, take respectively
The chitosan solution and blank reagent of the above-mentioned various concentrations of 1mL, the pH for being separately added into 2mL is 4.5 B-R buffer solutions, then divides
Not Jia Ru 1mL 1.00 × 10-4Mol/L Reactive Red 4 solution is settled to 10mL respectively with water, obtains concentration point after mixing
Not Wei 0.05,0.1,0.2,0.3,0.5,1.0,2.0 μ g/mL chitosan solution to be measured, stand 15~120 minutes, detect shell
The fluorescence intensity of glycan solution to be measured draws standard curve, further obtains linear equation：ΔF =68.78c+2.648（c, μ g/
mL）
In formulaΔF = F 0-F,F 0For the fluorescence intensity of blank solution,FFor the fluorescence intensity of the chitosan solution of various concentration；
Sample detection：The 0.4g samples to be tested acetum of 0.5mol/L is dissolved, is settled to 100mL, until completely dissolved
Centrifugation, takes 2.5mL supernatants, and be settled to 100mL, as sample operation liquid, takes the 1mL sample operation liquid, the pH that 2mL is added to be
4.5 B-R buffer solutions, add the 1.00 × 10 of 1mL-4Mol/L Reactive Red 4 solution, 10mL is settled to water, is uniformly mixed
15~120 minutes are stood afterwards, fluorescence intensity is calculated the concentration of chitosan in sample to be tested by linear equation.
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