CN101329279B - Method for rapidly testing cysteine in water solution - Google Patents
Method for rapidly testing cysteine in water solution Download PDFInfo
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- CN101329279B CN101329279B CN2008100554539A CN200810055453A CN101329279B CN 101329279 B CN101329279 B CN 101329279B CN 2008100554539 A CN2008100554539 A CN 2008100554539A CN 200810055453 A CN200810055453 A CN 200810055453A CN 101329279 B CN101329279 B CN 101329279B
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Abstract
The invention provides a method for quickly detecting cysteine in a water solution. The method directly detects the content of the cysteine in a buffer solution (the pH value is 6.0) qualitatively and quantitatively by using xylenol orange as a developer on the basis of cupric ion Cu<2+>; the detection process is simple, the judging is objective and convenient and the method of the invention is easy to be generalized and applied. The method of the invention has exact measurement result and high sensitiveness, is not interfered by other aminophenol and negative ions, and can be generalized andapplied to the determination of cysteine in clinical blood samples, urine samples and food.
Description
Technical field:
The present invention relates to the halfcystine detection technique, specifically belong to the method for halfcystine in a kind of fast detecting aqueous solution, comprise detection kit and detect test paper.
Background technology:
Halfcystine (Cysteine, be called for short Cys) is an important thio-compounds amino acid, has participated in various important cell functions, comprises synthetic, the detoxifcation and the metabolism of protein; In the metabolism of biology, halfcystine participates in the reduction process of cell, has metabolism and the protection liver cell of regulating phosphatide in the liver and avoids functions such as poisonous substance infringement.The metabolism meeting of disorderly halfcystine causes cystinosis---the defective disease that a kind of chromosome recessive inheritance disease is produced.Increasing with hyperhomocysteinemiainjury of cysteine content involves mutually in the body, can cause some life-threatening physiological maladies, and (senile dementia: Lee's Ying is beautiful, Yan Fuling to comprise Alzheimer's disease, modern medicine, 2005,33,195), Parkinson's disease (Wang Wenxia, Zang Weizhou, Feng Yan, Xu Jun, medicine forum magazine, 2005,9,20) and cardiovascular and cerebrovascular disease (favour is eliminated too and is waited for Zhou Xianliang, Hu Aihua. Chinese cardiovascular disease magazine, 1999,27,121).Low-level halfcystine will cause growing slowly, the hair depigmentation, the muscle oedema, lassitude is drowsiness, symptom such as hepatic injury, therefore the detection method of setting up quick, accurate, sensitive halfcystine has great importance, and also is to prevent and monitor one of important measures of the disease that is caused by them.
At present, about measuring the halfcystine in the solution, can reduce following several to the method that detects the halfcystine in the solution both at home and abroad:
1, high performance liquid chromatography ((G.Chwatko, E.Bald, Talanta, 2000,52,509);
2, electrochemical determination (Du Baozhong, Yao Binghua, Xue Li etc. Journal of Analytical Science, 2004,20,622);
3, chemoluminescence method (Zhang Shaohong, Shi Baian, Xi Juan, assay laboratory, 2007,26,5);
4, enzyme process (Han Qinghong, Xu Mingxu, Tan Yuying, Tang Li, Chinese invention patent, 02823051.5.);
5, fluorescence spectrometry method (Qiu Haiou, seat Yongqing, Ceng Fanrong, Yang Ming, Tang Zhiyong, assay laboratory, 2006,25,34)
6, colorimetric detection and analysis method (Guo Yong, Shao Shijun, Jiang Shengxiang, Shi Yanping, Xu Jian, Chinese invention patent, 200410092788.X)
But all there are some unfavorable factors respectively in these above methods, for example:
1, high performance liquid chromatography, electrochemical determination need be by the instruments of complexity, and need pre-service sample and can not be full-automatic, and complicated operation is wasted time and energy, and has limited its application clinically;
Though 2, enzyme is exempted from method sensitivity and specificity are preferably arranged,, cost an arm and a leg, also limited it and applied because be biopreparate;
3, other analytic approach because will organic or semi-aqueous in measure, cause secondary pollution easily, therefore also have certain limitation.
So, develop the method that detects halfcystine in a kind of aqueous solution of simple and sensitive and be very important.
Summary of the invention:
The objective of the invention is to break through the method for traditional in the past detection halfcystine, provide a kind of simple, sensitive, detect the method for halfcystine in the aqueous solution fast.
The method of halfcystine is based on cupric Cu in the fast detecting aqueous solution provided by the invention
2+Ion, be developer with the xylenol orange at pH be direct qualitative, quantitative ground detection halfcystine in 6.0 the HEPES buffer solution, its concrete detection method is as follows:
The method of halfcystine (spectroscopic methodology detection) comprises the steps: in a kind of fast detecting aqueous solution provided by the invention
(1), the HEPES buffer solution of preparation pH5.5-6.5 concentration 1-100mM, and prepare 2 * 10
-3The Cu of M
2+Solution, 2 * 10
-3The xylenol orange solution of M;
(2), the HEPES buffer solution of 2ml is added in the clean ultraviolet cuvette, as blank, with microsyringe absorption 2 * 10
-3The xylenol orange solution 25 μ l of M are added in the above-mentioned cuvette, and solution is detected on the UV, visible light spectrophotometer by colourless flavescence at this moment, and 441nm has absorption maximum;
(3), in the cuvette of step (2), add Cu with xylenol orange equivalent
2+Solution, this moment, solution was purplish red by xanthochromia, detected on the UV, visible light spectrophotometer, found that maximum absorption band becomes 572nm by above-mentioned 441nm;
(4), get the halfcystine aqueous solution, be added in step (3) cuvette with microsyringe gradually, application of sample limit, limit is detected on the UV, visible light spectrophotometer, adding along with the halfcystine aqueous solution, maximum absorption band is become again to 441nm gradually by 572nm again, and the color of solution is also gradually by the purplish red yellow that becomes again, and this moment, the 441nm absorption peak reached maximal value, stop to add the halfcystine aqueous solution, add halfcystine aqueous solution volume and count V this moment
Halfcystine
(5), by [Cu
2+] * 4 * 25 μ l/V
Halfcystineμ l calculates the content of halfcystine.
The method of halfcystine (solution colourimetry-kit method) comprises the steps: in the another kind of fast detecting aqueous solution provided by the invention
(1), the HEPES buffer solution of preparation pH5.5-6.5 concentration 1-100mM, and prepare 2 * 10
-3The Cu of M
2+Solution, 2 * 10
-3The xylenol orange solution of M;
(2), get two clean color comparison tubes, respectively add the HEPES buffer solution of 2ml, respectively add 2 * 10 with microsyringe again
-3The xylenol orange solution 25 μ l of M, solution is designated as R by colourless flavescence at this moment
1, R
2
(3), at R
2Middle adding 2 * 10
-3The Cu of M
2+Solution 25 μ l, this moment, solution was purplish red by xanthochromia;
(4), get the halfcystine aqueous solution, be added to R with microsyringe gradually
2In, add to color and R
1Identical, institute's halfcystine aqueous solution volume is counted V
Halfcystine
(5), by [Cu
2+] * 4 * 25 μ l/V
Halfcystineμ l calculates the content of halfcystine.
The method (test paper colourimetry) of halfcystine comprises the steps: in the another kind of fast detecting aqueous solution provided by the invention
(1), the HEPES buffer solution of preparation pH5.5-6.5 concentration 1-100mM, and prepare 2 * 10
-3The Cu of M
2+Solution, 2 * 10
-3The xylenol orange solution of M;
(2) with the Cu of 25 μ l
2+The xylenol orange solution of solution and 25 μ l is added in the HEPES buffer solution of 2ml, and after stirring, this moment, solution was aubergine;
(3) bulk filter paper is immersed 5-10 second in the purplish red solution of step (2), takes out and in air, dry, soak into 1-3 time repeatedly, dry afterwards halfcystine test paper (color is purple), it is standby that this test paper is cut into strip;
(4) get 5 of step (3) stick form test papers, be infiltrated on concentration respectively and be 0,0.01,0.04,0.08,2-5 second in the halfcystine aqueous solution of 0.1mM, take out, promptly be the halfcystine test paper concentration gradient color range figure of purple (blank), aubergine (0.01mM), purple yellow (0.04mM), khaki (0.08mM) and golden yellow (0.1mM) successively;
(5) get the halfcystine test paper and be infiltrated on and treat that 2-5 takes out second in the test sample,, judge the content for the treatment of the test sample halfcystine according to test paper change color and semicystinol concentration gradient color range figure contrast.
Described Cu
2+Solution is formulated with solubility cupric salt such as cupric chloride, Schweinfurt green, copper sulphate, copper nitrate, cupric perchlorate, copper gluconate etc.
Described HEPES buffer solution can be used acetic acid-sodium acetate buffer solution, buffer solution of sodium phosphate or buffer solution of potassium phosphate.
The method of halfcystine also can be used for the homocysteine fast detecting in the fast detecting aqueous solution of the present invention.
Compared with prior art, the present invention has the following advantages and effect: 1, the present invention detects halfcystine in pure water solution, has avoided the secondary pollution of using organic or semi-aqueous detection to cause, is of value to environmental protection and health; 2, detection architecture cost of manufacture of the present invention is lower, utilizes the uv-spectrophotometric instrument, even can directly detect by visual colorimetric determination by any instrument, and testing process is simple, sensitive, quick; 3, detection method of the present invention has shown high sensitivity and selectivity to halfcystine, and other occurrence of amino acid is interference detection results not; 4, the various negative ion that extensively exist in body fluid also do not disturb our system, have got rid of their influences to the halfcystine qualitative and quantitative detection; 5, the detection of halfcystine is quantitative, the very big range of linearity is arranged, and error is very little; 6, halfcystine test paper provided by the present invention is easy to use, differentiates fast succinctly, and is cheap, is easy to popularize, and is fit to large, medium and small type hospital and the daily detection in clinic, and the very wide scope of application is arranged.
Description of drawings:
Fig. 1 is containing 25 μ M xylenol orange, and pH6.0HEPES (10mM) adds Cu in the buffer solution gradually
2+UV, visible light absorb figure.
Fig. 2 is containing 25 μ M xylenol orange and 25 μ M Cu (NO
3)
23H
2O, pH6.0 HEPES (10mM) add the UV, visible light absorption figure of halfcystine gradually in the buffer solution.
Fig. 3 is containing 25 μ M xylenol orange and 25 μ M Cu (NO
3)
23H
2O, pH6.0 HEPES (10mM) but add the colorimetric view of halfcystine in the buffer solution gradually.
Fig. 4 is containing 25 μ M xylenol orange and 25 μ M Cu (NO
3)
23H
2O, pH6.0 HEPES (10mM) but add the colorimetric view of testing sample in the buffer solution.
Fig. 5 is containing 25 μ M xylenol orange and 25 μ M Cu (NO
3)
23H
2O adds various amino acid whose UV, visible light and absorbs figure in pH6.0 HEPES (10mM) buffer solution.
Fig. 6 is containing 25 μ M xylenol orange and 25 μ M Cu (NO
3)
23H
2O adds various anionic UV, visible light and absorbs figure in pH6.0 HEPES (10mM) buffer solution.
Fig. 7 is containing 25 μ M xylenol orange and 25 μ M Cu (NO
3)
23H
2O, pH6.0 HEPES (10mM) but add various amino acid whose colorimetric views in the buffer solution.
Fig. 8 is containing 25 μ M xylenol orange and 25 μ M Cu (NO
3)
23H
2O, pH6.0 HEPES (10mM) but add various anionic colorimetric views in the buffer solution.
Fig. 9 is containing 25 μ M xylenol orange and 25 μ M Cu (NO
3)
23H
2O, pH6.0 HEPES (10mM) add the UV, visible light absorption figure of homocysteine gradually in the buffer solution.
Figure 10 is containing 25 μ M xylenol orange and 25 μ M Cu (NO
3)
23H
2O, pH6.0 HEPES (10mM) but add the colorimetric view of a large amount of homocysteines in the buffer solution.
Figure 11 is halfcystine test paper concentration gradient color range figure.
Embodiment:
Embodiment 1: the method for halfcystine in the spectroscopic methodology fast detecting aqueous solution
HEPES (10mM) aqueous buffer solution of preparation pH6.0, and prepare 2 * 10
-3The copper nitrate aqueous solution of M, 2 * 10
-3The xylenol orange aqueous solution and 2 * 10 of M
-3The halfcystine aqueous solution of M; The HEPES buffer solution of 2mL is added in the clean ultraviolet cuvette,, draws 2 * 10 with microsyringe as blank
-3The xylenol orange solution 25 μ l of M are added in this cuvette, and solution is detected on the UV, visible light spectrophotometer by colourless flavescence at this moment, and 441nm has absorption maximum; In above-mentioned cuvette, add 2 * 10
-3The copper nitrate solution 25 μ l of M, solution is detected on the UV, visible light spectrophotometer by yellow purpling redness at this moment, finds that maximum absorption band becomes 572nm by above-mentioned 441nm, and ultraviolet-visible absorption spectroscopy is seen Fig. 1; Get the halfcystine aqueous solution, be added in this cuvette with microsyringe gradually, application of sample limit, limit is detected on HP8453 UV, visible light spectrophotometer, adding along with halfcystine, maximum absorption band is become again to 441nm gradually by 572nm again, the color of solution also becomes yellow again by aubergine gradually, when absorption peak when 441nm reaches maximum, this moment, the halfcystine addition was 100 μ l; Calculate: 2 * 10
-3* 100 μ l/2 * 10
-3* 25 μ l=4 calculate and determine that halfcystine is 4 to 1 with the copper ion coordination ratio; UV, visible light absorption figure sees Fig. 2.The content of halfcystine is by [Cu
2+] * 4 * 25 μ l/V
Halfcystineμ l calculates.
Embodiment 2: the method for halfcystine in solution colourimetry (kit method) the fast detecting aqueous solution
The kit preparation:
HEPES (10mM) aqueous buffer solution of preparation pH6.0, and prepare 2 * 10
-3The copper nitrate aqueous solution of M, 2 * 10
-3The xylenol orange aqueous solution and 2 * 10 of M
-3The halfcystine aqueous solution of M; Get five color comparison tubes, add HEPES buffer solution, the 25 μ l 2 * 10 of 2ml respectively
-3The xylenol orange solution of M, solution is designated as R by colourless flavescence at this moment
1, R
2, R
3, R
4, R
5At R
2, R
3, R
4, R
5Middle adding 2 * 10
-3The copper nitrate solution 25 μ l of M, this moment solution by yellow purpling redness, respectively with microsyringe draw 35 μ l, 65 μ l, 100 μ l halfcystines are added to R
3, R
4, R
5In, but the colorimetric view is seen Fig. 3, R
3, R
4, R
5The semicystinol concentration of representative is respectively 35 μ M, 65 μ M, 100 μ M.
The unknown concentration halfcystine is measured:
HEPES (10mM) buffer solution of preparation pH6.0, and prepare 2 * 10
-3The Cu of M
2+Solution, 2 * 10
-3The xylenol orange solution of M; Get color comparison tube, add HEPES buffer solution, the 25 μ l of 2ml, 2 * 10
-3The xylenol orange solution of M, solution adds 2 * 10 by colourless flavescence at this moment
-3The Cu of M
2+Solution 25 μ l, this moment, solution was by yellow purpling redness; Get and treat test sample, add this color comparison tube, add to color and R with microsyringe
1Identically see Fig. 4, used volume is V
Halfcystine(μ l); By [Cu
2+] * 4 * 25 μ l/V
Halfcystineμ l calculates the content of halfcystine.
Embodiment 3: the method for halfcystine in the test paper colourimetry fast detecting aqueous solution
HEPES (10mM) aqueous buffer solution of preparation pH6.0, and prepare 2 * 10
-3The copper nitrate aqueous solution of M, 2 * 10
-3The xylenol orange aqueous solution of M and concentration are 0.01,0.04,0.08, the halfcystine aqueous solution of 0.1mM; Cu with 25 μ l
2+The xylenol orange solution of solution and 25 μ l is added in HEPES (10mM) buffer solution of 2ml, the back solution that stirs is aubergine, bulk filter paper was immersed in this purplish red solution 10 seconds, taking-up is dried in air, soak into repeatedly again dry 3 times available halfcystine test paper, it is stand-by that this test paper is cut into strip;
Get 5 of step stick form test papers, be infiltrated on concentration respectively and be 0,0.01,0.04,0.08, in the halfcystine aqueous solution of 0.1mM 3 seconds, take out, the halfcystine test paper concentration gradient color range figure that promptly obtains purple (blank), aubergine (0.01mM), purple yellow (0.04mM), khaki (0.08mM) and golden yellow (0.1mM) sees Figure 11;
Get the halfcystine test paper and be infiltrated on and treat in the test sample to take out in 3 seconds, contrast with semicystinol concentration gradient color range figure according to the test paper change color, i.e. the content of decidable sample halfcystine to be measured.
Embodiment 4: other amino acid not stray light spectrometry is measured halfcystine
HEPES (10mM) buffer solution of preparation pH6.0, and prepare 2 * 10
-3The Cu of M
2+Solution, 2 * 10
-3The glycocoll (Gly) of the xylenol orange solution of M and 0.1M, serine (Ser), threonine (Thr), methionine (Met), aspartic acid (Asp), arginine (Arg), histidine (His), tyrosine (Tyr), lysine (Lys), tryptophane (Try), phenylalanine (Phe), glutamine (Glu), leucine (Leu), valine (Val), proline (Pro), cystine (Cystine); The HEPES buffer solution of 2ml is added in the clean ultraviolet cuvette,, draws 2 * 10 with microsyringe as blank
-3The xylenol orange solution 25 μ l of M are added in this cuvette, and solution is detected on the UV, visible light spectrophotometer by colourless flavescence at this moment, and 441nm has absorption maximum; In above-mentioned cuvette, add 2 * 10
-3The Cu of M
2+Solution 25 μ l, this moment, solution was red by yellow purpling, detected on the UV, visible light spectrophotometer, found that maximum absorption band 441nm becomes 572nm; Get above-mentioned various Freamine 25 μ l respectively, join one by one in the cuvette, application of sample limit, limit is detected on the UV, visible light spectrophotometer, and along with various amino acid whose addings, maximum absorption band is 572nm, does not change; In above-mentioned solution, add 2 * 10
-3The halfcystine solution 100 μ l of M, solution colour becomes yellow by aubergine, and maximum absorption band is blue shifted to 441nm by 572nm; UV, visible light absorption figure sees Fig. 5.
Embodiment 5: other amino acid does not disturb the colorimetric estimation halfcystine
HEPES (10mM) buffer solution of preparation pH6.0, and prepare 2 * 10
-3The Cu of M
2+Solution, 2 * 10
-3The glycocoll (Gly) of the xylenol orange solution of M and 0.1M, serine (Ser), threonine (Thr), methionine (Met), aspartic acid (Asp), arginine (Arg), histidine (His), tyrosine (Tyr), lysine (Lys), tryptophane (Trp), phenylalanine (Phe), glutamine (Glu), leucine (Leu), valine (Val), proline (Pro), cystine (Cystine), halfcystine (Cys); Get 18 color comparison tubes, add HEPES buffer solution, the 25 μ l 2 * 10 of 2ml respectively
-3The xylenol orange solution, 2 * 10 of M
-3The Cu of M
2+Solution 25 μ l, this moment, solution was purplish red by xanthochromia, got each seed amino acid 25 μ l, be added in the color comparison tube with microsyringe, but the colorimetric view was seen Fig. 7.(other amino acid of 50 times of amounts do not disturb the detection of halfcystine)
Embodiment 6: negative ion does not disturb uv-vis spectra to measure halfcystine
HEPES (10mM) buffer solution of preparation pH6.0, and prepare 2 * 10
-3The Cu of M
2+Solution, 2 * 10
-3The xylenol orange solution of M and oxalate, acetate, nitrate radical, carbonate, hydrogen phosphate, fluorine ion, the chlorion of 0.1M; The HEPES buffer solution of 2ml is added in the clean ultraviolet cuvette,, draws 2 * 10 with microsyringe as blank
-3The xylenol orange solution 25 μ l of M are added in this cuvette, and solution is detected on the UV, visible light spectrophotometer by colourless flavescence at this moment, and 441nm has absorption maximum; In above-mentioned cuvette, add 2 * 10
-3The Cu of M
2+Solution 25 μ l, this moment, solution was purplish red by xanthochromia, detected on the UV, visible light spectrophotometer, found that maximum absorption band 441nm becomes 572nm; Get above-mentioned various anion solutions 25 μ l respectively, join one by one in the cuvette, application of sample limit, limit is detected on the UV, visible light spectrophotometer, and along with various anionic addings, maximum absorption band is 572nm, does not change, and UV, visible light absorption figure sees Fig. 6.(oxalate of 50 times of amounts, acetate, nitrate radical, carbonate, hydrogen phosphate, fluorine ion, chlorion do not disturb the detection of halfcystine)
Embodiment 7: negative ion does not disturb the colorimetric estimation halfcystine
HEPES (10mM) buffer solution of preparation pH6.0, and prepare 2 * 10
-3The Cu of M
2+Solution, 2 * 10
-3The xylenol orange solution of M and oxalate, acetate, nitrate radical, carbonate, hydrogen phosphate, fluorine ion, the chlorion of 0.1M; Get 11 each color comparison tube, add HEPES buffer solution, the 25 μ l 2 * 10 of 2ml respectively
-3The xylenol orange solution, 2 * 10 of M
-3The Cu of M
2+Solution 25 μ l, this moment, solution was purplish red by xanthochromia, got various negative ion 25 μ l, be added in the color comparison tube with microsyringe, but the colorimetric view was seen Fig. 8.
Embodiment 8: the detection of the halfcystine of acetic acid-sodium acetate buffer solution
Acetic acid-sodium-acetate buffer of preparation pH6.0, with the HEPES buffer solution in its alternate embodiment 1, other solution preparations and detection step are identical with embodiment 1, and it is identical with Fig. 1, Fig. 2 that spectroscopic methodology detects ultraviolet-visible absorption spectroscopy; The colourimetry testing process is with embodiment 2, and is more identical with Fig. 3 than chromatic graph;
Embodiment 9: phosphoric acid is received the detection of halfcystine of buffer solution
The 0.1M sodium phosphate buffer liquor of preparation pH6.0, with the HEPES buffer solution in its alternate embodiment 1, other solution preparations and detection step are identical with embodiment 1, and it is identical with Fig. 1, Fig. 2 that spectroscopic methodology detects ultraviolet-visible absorption spectroscopy; The colourimetry testing process is with embodiment 2, and is more identical with Fig. 3 than chromatic graph.
Embodiment 10: homocysteine detects
HEPES (10mM) buffer solution of preparation pH6.0, and prepare 2 * 10
-3The Cu of M
2+Solution, 2 * 10
-3The xylenol orange solution and 2 * 10 of M
-3The homocysteine aqueous solution of M; The HEPES buffer solution of 2mL is added in the clean ultraviolet cuvette,, draws 2 * 10 with microsyringe as blank
-3The xylenol orange solution 25 μ l of M are added in this cuvette, and solution is detected on the UV, visible light spectrophotometer by colourless flavescence at this moment, and 441nm has absorption maximum; In above-mentioned cuvette, add 2 * 10
-3The Cu of M
2+Solution 25 μ l, this moment, solution was purplish red by xanthochromia, detected on the UV, visible light spectrophotometer, found that maximum absorption band becomes 572nm by above-mentioned 441nm, and ultraviolet-visible absorption spectroscopy is seen Fig. 1; Get homocysteine, be added in this cuvette with microsyringe gradually, application of sample limit, limit is detected on HP8453 UV, visible light spectrophotometer, adding along with homocysteine, maximum absorption band is become again to 441nm gradually by 572nm again, the color of solution is also gradually by the purplish red yellow that becomes again, when absorption peak when 441nm reaches maximum, this moment, the homocysteine addition was 320 μ l; Calculate: 2 * 10
-3* 320 μ l/2 * 10
-3* 25 μ l=12.8 illustrate that the coordination relation of homocysteine and copper ion can not science be determined.Though homocysteine also can cause detection architecture ultra-violet absorption spectrum and change in color.But in view of the content of halfcystine in the body fluid be homocysteine content 20-30 doubly, its existence does not influence the detection of this patent system to halfcystine.UV, visible light absorption figure sees Fig. 9.It sees Figure 10 than chromatic graph.
Claims (9)
1. the method for halfcystine in the fast detecting aqueous solution is characterized in that comprising the steps:
(1), the HEPES buffer solution of preparation pH5.5-6.5 concentration 1-100mM, and prepare 2 * 10
-3The Cu of M
2+Solution, 2 * 10
-3The xylenol orange solution of M;
(2), the HEPES buffer solution of 2ml is added in the clean ultraviolet cuvette, as blank, with microsyringe absorption 2 * 10
-3The xylenol orange solution 25 μ l of M are added in the above-mentioned cuvette, and solution is detected on the UV, visible light spectrophotometer by colourless flavescence at this moment, and 441nm has absorption maximum;
(3), in the cuvette of step (2), add Cu with xylenol orange equivalent
2+Solution, this moment, solution was purplish red by xanthochromia, detected on the UV, visible light spectrophotometer, found that maximum absorption band becomes 572nm by above-mentioned 441nm;
(4), get the halfcystine aqueous solution, be added in step (3) cuvette with microsyringe gradually, application of sample limit, limit is detected on the UV, visible light spectrophotometer, adding along with the halfcystine aqueous solution, maximum absorption band is become again to 441nm gradually by 572nm again, and the color of solution is also gradually by the purplish red yellow that becomes again, and this moment, the 441nm absorption peak reached maximal value, stop to add the halfcystine aqueous solution, add halfcystine aqueous solution volume and count V this moment
Halfcystineμ l;
(5), by [Cu
2+] * 4 * 25 μ l/V
Halfcystineμ l calculates the content of halfcystine.
2. the method for halfcystine in the fast detecting aqueous solution is characterized in that comprising the steps:
(1), the HEPES buffer solution of preparation pH5.5-6.5 concentration 1-100mM, and prepare 2 * 10
-3The Cu of M
2+Solution, 2 * 10
-3The xylenol orange solution of M;
(2), get two clean color comparison tubes, respectively add the HEPES buffer solution of 2ml, respectively add 2 * 10 with microsyringe again
-3The xylenol orange solution 25 μ l of M, solution is designated as R by colourless flavescence at this moment
1, R
2
(3), at R
2Middle adding 2 * 10
-3The Cu of M
2+Solution 25 μ l, this moment, solution was purplish red by xanthochromia;
(4), get the halfcystine aqueous solution, be added to R with microsyringe gradually
2In, add to color and R
1Identical, institute adds halfcystine aqueous solution volume and counts V
Halfcystineμ l;
(5), by [Cu
2+] * 4 * 25 μ l/V
Halfcystineμ l calculates the content of halfcystine.
3. the method for halfcystine in the fast detecting aqueous solution is characterized in that comprising the steps:
(1), the HEPES buffer solution of preparation pH5.5-6.5 concentration 1-100mM, and prepare 2 * 10
-3The Cu of M
2+Solution, 2 * 10
-3The xylenol orange solution of M;
(2) with the Cu of 25 μ l
2+The xylenol orange solution of solution and 25 μ l is added in the HEPES buffer solution of 2ml, and after stirring, this moment, solution was aubergine;
(3) bulk filter paper is immersed 5-10 second in the purplish red solution of step (2), takes out and in air, dry, soak into 1-3 time repeatedly, dry afterwards the halfcystine test paper, it is standby that this test paper is cut into strip;
(4) get 5 of step (3) stick form test papers, be infiltrated on concentration respectively and be 0,0.01,0.04,0.08,2-5 second in the halfcystine aqueous solution of 0.1mM, take out, promptly be purple, aubergine, purple yellow, khaki and flavous halfcystine test paper concentration gradient color range figure successively;
(5) get the halfcystine test paper and be infiltrated on and treat that 2-5 takes out second in the test sample,, judge the content for the treatment of the test sample halfcystine according to test paper change color and semicystinol concentration gradient color range figure contrast.
4. the halfcystine test paper that makes in the method for halfcystine in the fast detecting aqueous solution as claimed in claim 3.
5. the halfcystine test paper concentration gradient color range figure that makes in the method for halfcystine in the fast detecting aqueous solution as claimed in claim 3.
6. as the method for halfcystine in claim 1, the 2 or 3 described fast detecting aqueous solution, it is characterized in that described Cu
2+Solution is formulated with the solubility cupric salt.
7. the method for halfcystine in the fast detecting aqueous solution as claimed in claim 6 is characterized in that described solubility cupric salt is selected from cupric chloride, Schweinfurt green, copper sulphate, copper nitrate, cupric perchlorate, copper gluconate.
8. as the method for halfcystine in claim 1, the 2 or 3 described fast detecting aqueous solution, it is characterized in that described HEPES buffer solution replaces with acetic acid-sodium acetate buffer solution, buffer solution of sodium phosphate or buffer solution of potassium phosphate.
9. the method as halfcystine in claim 1, the 2 or 3 described fast detecting aqueous solution is used for the homocysteine fast detecting.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008100554539A CN101329279B (en) | 2008-07-18 | 2008-07-18 | Method for rapidly testing cysteine in water solution |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008100554539A CN101329279B (en) | 2008-07-18 | 2008-07-18 | Method for rapidly testing cysteine in water solution |
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| CN102200514A (en) * | 2010-03-23 | 2011-09-28 | 中国科学院兰州化学物理研究所 | Method for analyzing sulphydryl amino acid by colorimetric detection |
| CN101915760B (en) * | 2010-07-07 | 2013-10-09 | 东华大学 | Method for detecting cysteine content in real time by colorimetric method |
| CN102087219A (en) * | 2010-12-22 | 2011-06-08 | 湖南大学 | Method for detecting specific sulfhydryl-containing amino acid |
| CN102977113A (en) * | 2012-12-11 | 2013-03-20 | 山西大学 | Method for detecting mercaptan |
| CN103217406B (en) * | 2013-03-21 | 2015-09-09 | 上海交通大学 | Based on halfcystine and the Cu of Au/Ag core/shell quantum dot 2+the method for making of fluorescence probe |
| CN105699462B (en) * | 2014-11-28 | 2018-06-08 | 中国科学院青岛生物能源与过程研究所 | A kind of method for quantitatively detecting L-cysteine |
| CN104849271B (en) * | 2015-05-26 | 2017-06-30 | 中国科学院烟台海岸带研究所 | A kind of probe based on flower cyanines is used for the method for detecting trace bivalent cupric ion |
| CN105181666B (en) * | 2015-09-23 | 2018-02-06 | 山西大学 | A kind of reagent and method of fluoroscopic examination cysteine |
| CN110161018B (en) * | 2019-06-25 | 2021-06-25 | 山东大学 | Method for detecting and identifying cysteine and homocysteine based on liquid crystal sensing platform |
| CN110715914B (en) * | 2019-11-05 | 2022-07-12 | 鲁东大学 | Multi-mode detection method of L-cysteine |
| CN111303072A (en) * | 2020-02-27 | 2020-06-19 | 山西大学 | Reagent for distinguishing and detecting cysteine and synthetic method and application thereof |
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