CN100445731C - Method for colorimetric detecting and analysing cysteine - Google Patents
Method for colorimetric detecting and analysing cysteine Download PDFInfo
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- CN100445731C CN100445731C CNB200410092788XA CN200410092788A CN100445731C CN 100445731 C CN100445731 C CN 100445731C CN B200410092788X A CNB200410092788X A CN B200410092788XA CN 200410092788 A CN200410092788 A CN 200410092788A CN 100445731 C CN100445731 C CN 100445731C
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Abstract
The present invention discloses a colorimetric detection and analysis method for cysteine. After the solution of a product to be measured is added into the solution of a colored charge transfer cooperation product, the product to be measured is detected by the color change of the solution. The method uses dipyrrole methane as an electron donor and uses quinone compound as an electron acceptor to generate electron transfer in polar solvent so as to form the detection liquid of the colored charge transfer cooperation product. The product to be measured, which contains sulfhydryl groups, is prepared into the water solution with certain concentration. The water solution of certain volume is mixed with the detection liquid with the same volume, the color of the solution generates obvious changes, and the other amino acid can not influence the detection of the cysteine. The present invention has the unique colorimetric recognizing ability for amino acid molecules containing sulfhydryl functional groups. The present invention has convenient solution preparation, convenient colorimetric detection operation and high color changing speed of colorimetric reaction. The colorimetric detection of the product to be measured can be completed in a short time.
Description
Technical field
The present invention relates to a kind of colorimetric detection and analysis method of halfcystine.
Technical background
Constitute the most basic material of human body, protein, lipid, carbohydrate, inorganic salts, vitamin, water etc. are arranged.As the amino acid that constitutes the protein molecule base unit, constitute one of base substance of each organ in the human body beyond doubt.We can say that amino acid molecular is the fundamental element that constitutes the life mansion, has inseparable relation with the biosome vital movement.It has special physiological function in body, be one of indispensable nutritional labeling in the biosome.
The primary amino acid that constitutes life entity has kind more than 20, if human body lacks any amino acid, just can cause physiological function unusual, influences normally carrying out of organism metabolism, finally causes the generation of disease.Very important as arginine and citrulline to forming urea; The cystine insufficiency of intake will cause that insulin reduces blood sugar increasing etc.Modern medicine study shows, the ANOMALOUS VARIATIONS of certain amino acid concentration also is the root that causes numerous diseases to produce in the body fluid, as homocysteine (Hcy in the blood, Duo a methine than halfcystine in the molecule) raising to promote atherosclerotic, and then it is closely related with the ischemic heart, cranial vascular disease, the detecting homocysteine is an independent reference factor of this type of disease danger of diagnosis in the variation of blood middle concentration at present, and the content that therefore detects homocysteine in the body fluid has exactly become one of key factor of control disease development.At present the detection method of using is loaded down with trivial details and detection speed is slower, develops a kind of convenience, sensitivity, detection means is very important fast.
Contain mercaptoamino acid and biological micromolecule and in the metabolic activity of biological life, playing the part of very important role, the conformation of protein is changed by the redox reaction between micromolecule.
At present, the assay determination of halfcystine generally is after utilizing its reductibility and reacting with some organic reagent, carry out assay determination with galvanochemistry, spectrophotometric method, chemoluminescence method, catalytic kinetics method and fluorescent method, these method complicated operations, sensitivity is not high and selectivity is relatively poor.
Design a kind of system or synthetic compound as acceptor, utilize it to discern certain important biological micromolecule or functional group with special construction.Method the most attractive makes up and uses colorimetric sensor exactly in this technical field.In our known colorimetric sensor, host molecule as sensor is connected with a receptor site and a hyperchromic group by covalent bond, when receptor site and guest molecule are had an effect, hyperchromic group on the host molecule will produce change color, but this detection substantially all is to occur in the non-polar solvent.We find that the charge-transfer complex that quinone and two pyrroles's methane constitute also can be used in colorimetric detection, it is that this provides advantageous environment for detecting water-soluble substances in 1: 1 the water/ethanol, water/acetonitrile mixed solution that this electric charge transfering system can stably be present in volume ratio.Use the detector probe of charge-transfer complex as high selectivity, the change color visual by naked eyes optionally detects halfcystine.Utilize charge-transfer complex colorimetric detection halfcystine not have the pertinent literature report at present.
Summary of the invention
Purpose of the present invention is exactly the weak point that exists in the amino acid detection in order to solve, and a kind of high selectivity of halfcystine detection, convenient and simple analytical approach are provided.
Thinking of the present invention is that the requirement analytical approach is easy and simple to handle, and the selectivity height does not need sample is carried out special processing, does not finish the qualitative analysis of sample at short notice by macroscopic change color by instrument.
The present invention realizes by following measure:
The halfcystine that we use two pyrroles's methane and quinone to form in ethanol or acetonitrile detects liquid, detects the amino acid molecular that contains sulfydryl by macroscopic change color.The blueness that the aqueous solution that adds halfcystine in the ethanol of certain density two pyrroles's methane-quinones or acetonitrile solution, halfcystine detect liquid decorporate become colourless.
A kind of colorimetric detection and analysis method of halfcystine is characterized in that this method may further comprise the steps:
A uses two pyrroles's methane and quinoness, as solvent, makes blue coloured charge-transfer complex as the halfcystine detectable with absolute ethyl alcohol or acetonitrile; Wherein two pyrroles's methane are selected from a kind of in dihydroxy two pyrroles's methane, the dimethyl two pyrroles's methane, and quinones is selected from 2,3,5,6-tetrachloroquinone (TCBQ), 7,7,8, a kind of in the 8-four cyano quinone bismethane (TCNQ);
B is the aqueous buffer solution of 0.005-0.015M with 2-hydroxyethyl-1-piperazinyl ethyl sulfonic acid (HEPES) compound concentration, adds halfcystine and make detection liquid in buffer solution, and the concentration of halfcystine is 1.0 * 10
-3-1.0 * 10
-4M;
The detection liquid that C is identical with volume with the halfcystine detectable mixes, and mixed liquor becomes colorless.
In the halfcystine detectable, the concentration of two pyrroles's methane is 0.001-0.0025M, and the concentration of quinones is 2.5 * 10
-4M-1.0 * 10
-4M.
When containing other Freamine in detecting liquid, change color does not take place.
We have made contrast test with analytical instrument:
Under same condition, make reference with the Freamine of respective concentration, with the absorbance variation at ultraviolet-visible spectrophotometric determination 628nm wavelength place under buffer system.Add the halfcystine aqueous buffer solution in detecting liquid, complex disappears at the absorption peak at 628nm place.
The present invention compares with existing square technology, has following substantive distinguishing features:
1. the present invention need not amino acid is carried out derivatization treatment, and amino acid whose aqueous solution just can satisfy the requirement that detects.
2. detection liquid used in the present invention can mix with arbitrary proportion mutually with water, and solution mixes the back does not have wild effect generations such as precipitation.
3. amino acid detectable used in the present invention, its process for preparation is simple, can be positioned in the refrigerator standby after preparation finishes.
4. the present invention is characterized in that this detection method not by any instrument, detects measured matter by macroscopic change color.
5. the present invention is easy fast, just can detect measured object by change color in the several seconds.
Description of drawings
Fig. 1 is for detecting the ultraviolet spectrogram of halfcystine with the halfcystine detectable.
But Fig. 2 is for detecting the colorimetric view of halfcystine with the halfcystine detectable.
The abbreviation of figure Chinese and English is respectively: glycocoll Gly; Alanine Ala; Valine Val; Leucine Leu; Isoleucine Ile; Aspartic acid Asp; Glutamic acid Glu; Arginine Arg; Lysine Lys; Histidine His; Serine Ser; Threonine Thr; Halfcystine Cys; Methionine Met; Phenylalanine Phe; Tryptophane Try; Proline Pro; Asparagine Asn; Glutamine Gln.
Embodiment
Embodiment 1
Carry out the preparation of detectable: in dissolve measuring bottle, accurately take by weighing a certain amount of 2,3,5,6-tetrachloroquinone (concentration 2 * 10
-4M) and a certain amount of dimethyl two pyrroles's methane (concentration 2.0 * 10
-3M), it is fixed molten to add anhydrous alcohol solution, and solution becomes after the mazarine stand-by.This dark blue solution is for detecting the detectable of halfcystine.
Carry out the preparation that amino acid detects liquid: take by weighing following 20 seed amino acids (glycocoll Gly respectively; Alanine Ala; Valine Val; Leucine Leu; Isoleucine Ile; Aspartic acid Asp; Glutamic acid Glu; Arginine Arg; Lysine Lys; Histidine His; Serine Ser; Threonine Thr; Halfcystine Cys; Methionine Met; Phenylalanine Phe; Tryptophane Try; Proline Pro; Asparagine Asn; Glutamine Gln) in different test tubes, the concentration that pipettes 10ml with transfer pipet is 1.0 * 10
-2The HEPES aqueous buffer solution of M is 2.0 * 10 after the dissolving in amino acid whose test tube is housed respectively
-3The solution of M.
Carry out the detection of halfcystine: with the buffered with amino acid aqueous solution of 2ml respectively with TCBQ-dimethyl two pyrroles's methane (TCBQ=2.0 * 10 of 2ml
-4M, dimethyl two pyrroles's methane=2.0 * 10
-3M) detection agent mixes, and observes the change color of different aminoacids solution, and the gained result as shown in Figure 2.
Embodiment 2
Carry out the preparation of detectable: in dissolve measuring bottle, accurately take by weighing a certain amount of 2,3,5,6-tetrachloroquinone (concentration 2 * 10
-4M) and a certain amount of dihydroxymethyl two pyrroles's methane (concentration 2.0 * 10
-3M), it is fixed molten to add anhydrous alcohol solution, and solution becomes after the mazarine stand-by.This dark blue solution is for detecting the detectable of halfcystine.
Carry out the preparation that amino acid detects liquid: take by weighing following 20 seed amino acids (glycocoll Gly respectively; Alanine Ala; Valine Val; Leucine Leu; Isoleucine Ile; Aspartic acid Asp; Glutamic acid Glu; Arginine Arg; Lysine Lys; Histidine His; Serine Ser; Threonine Thr; Halfcystine Cys; Methionine Met; Phenylalanine Phe; Tryptophane Try; Proline Pro; Asparagine Asn; Glutamine Gln) in different test tubes, the concentration that pipettes 10ml with transfer pipet is 1.0 * 10
-2The HEPES aqueous buffer solution of M is 2.0 * 10 after the dissolving in amino acid whose test tube is housed respectively
-3The solution of M.
Carry out the detection of halfcystine: with the buffered with amino acid aqueous solution of 2ml respectively with TCBQ-dihydroxymethyl two pyrroles's methane (TCBQ=2.0 * 10 of 2ml
-4M, dihydroxymethyl two pyrroles's methane=2.0 * 10
-3M) detection agent mixes, and observes the change color of different aminoacids solution, and the gained result as shown in Figure 2.
Embodiment 3
Carry out the preparation of detectable: in dissolve measuring bottle, accurately take by weighing a certain amount of 7,7,8,8-four cyano quinone bismethane (concentration 1.0 * 10
-4M) and a certain amount of dihydroxymethyl two pyrroles's methane (concentration 1.0 * 10
-3M), add acetonitrile dissolving constant volume, solution becomes after the mazarine stand-by.This dark blue solution is for detecting the detection solution of halfcystine.
Carry out the preparation that amino acid detects liquid: take by weighing following 20 seed amino acids (glycocoll Gly respectively; Alanine Ala; Valine Val; Leucine Leu; Isoleucine Ile; Aspartic acid Asp; Glutamic acid Glu; Arginine Arg; Lysine Lys; Histidine His; Serine Ser; Threonine Thr; Halfcystine Cys; Methionine Met; Phenylalanine Phe; Tryptophane Try; Proline Pro; Asparagine Asn; Glutamine Gln) in different test tubes, the concentration that pipettes 10ml with transfer pipet is 1.0 * 10
-2The HEPES aqueous buffer solution of M is 2.0 * 10 after the dissolving in test tube
-3The solution of M.
Carry out the detection of halfcystine: with the buffered with amino acid aqueous solution of 2ml respectively with TCNQ-dihydroxymethyl two pyrroles's methane (TCNQ=1.0 * 10 of 2ml
-4M, dihydroxymethyl two pyrroles's methane=1.0 * 10
-3M) detect liquid and mix, observe the change color of different aminoacids solution, the gained result as shown in Figure 2.Under same condition, be reference with the amino acid acetonitrile-aqueous solution of respective concentration, change with the absorbance at ultraviolet-visible spectrophotometric determination 628nm wavelength place under buffer system.Measured result adds the halfcystine aqueous solution as shown in Figure 1 in detecting solution, detect liquid and disappear at the absorption peak of 628nm.
Embodiment 4
Carry out the preparation of detectable: in dissolve measuring bottle, accurately take by weighing a certain amount of 7,7,8,8-four cyano quinone bismethane (concentration 1.0 * 10
-4M) and a certain amount of dimethyl two pyrroles's methane (concentration 1.0 * 10
-3M), add acetonitrile dissolving constant volume, solution becomes after the mazarine stand-by.This dark blue solution is for detecting the detection solution of halfcystine.
Carry out the preparation that amino acid detects liquid: take by weighing following 20 seed amino acids (glycocoll Gly respectively; Alanine Ala; Valine Val; Leucine Leu; Isoleucine Ile; Aspartic acid Asp; Glutamic acid Glu; Arginine Arg; Lysine Lys; Histidine His; Serine Ser; Threonine Thr; Halfcystine Cys; Methionine Met; Phenylalanine Phe; Tryptophane Try; Proline Pro; Asparagine Asn; Glutamine Gln) in different test tubes, the concentration that pipettes 10ml with transfer pipet is 1.0 * 10
-2The HEPES aqueous buffer solution of M is 2.0 * 10 after the dissolving in test tube
-3The solution of M.
Carry out the detection of halfcystine: with the buffered with amino acid aqueous solution of 2ml respectively with TCNQ-dimethyl two pyrroles's methane (TCNQ=1.0 * 10 of 2ml
-4M, dimethyl two pyrroles's methane=1.0 * 10
-3M) detect liquid and mix, observe the change color of different aminoacids solution, the gained result as shown in Figure 2.Under same condition, be reference with the amino acid acetonitrile-aqueous solution of respective concentration, change with the absorbance at ultraviolet-visible spectrophotometric determination 628nm wavelength place under buffer system.Measured result adds the halfcystine aqueous solution as shown in Figure 1 in detecting solution, detect liquid and disappear at the absorption peak of 628nm.
Claims (1)
1, a kind of colorimetric detection and analysis method of halfcystine is characterized in that this method may further comprise the steps:
A uses two pyrroles's methane and quinoness, as solvent, makes blue coloured charge-transfer complex as the halfcystine detectable with absolute ethyl alcohol or acetonitrile; Wherein two pyrroles's methane are selected from a kind of in dihydroxy two pyrroles's methane, the dimethyl two pyrroles's methane, and quinones is selected from 2,3,5,6-tetrachloroquinone, 7,7,8, a kind of in the 8-four cyano quinone bismethane; The concentration of two pyrroles's methane is 0.001-0.0025M, and the concentration of quinones is 2.5 * 10
-4M-1.0 * 10
-4M;
B is the aqueous buffer solution of 0.005-0.015M with 2-hydroxyethyl-1-piperazinyl ethyl sulfonic acid compound concentration, adds halfcystine and make detection liquid in buffer solution, and the concentration of halfcystine is 1.0 * 10
-3-1.0 * 10
-4M;
The detection liquid that C is identical with volume with the halfcystine detectable mixes, and mixed liquor becomes colorless.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB200410092788XA CN100445731C (en) | 2004-11-12 | 2004-11-12 | Method for colorimetric detecting and analysing cysteine |
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|---|---|---|---|
| CNB200410092788XA CN100445731C (en) | 2004-11-12 | 2004-11-12 | Method for colorimetric detecting and analysing cysteine |
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|---|---|
| CN1773264A CN1773264A (en) | 2006-05-17 |
| CN100445731C true CN100445731C (en) | 2008-12-24 |
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Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101329279B (en) * | 2008-07-18 | 2010-06-23 | 山西大学 | Method for rapidly testing cysteine in water solution |
| CN101738392B (en) * | 2009-11-12 | 2011-10-05 | 宁夏伊品生物科技股份有限公司 | Method for fast measuring threonine content |
| 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 |
| CN101806724B (en) * | 2010-04-16 | 2011-10-05 | 宁夏伊品生物科技股份有限公司 | Simple and fast method for detecting L-lysine content in 65% L-lysine sulphate finished product |
| CN102087219A (en) * | 2010-12-22 | 2011-06-08 | 湖南大学 | Method for detecting specific sulfhydryl-containing amino acid |
| CN103163121A (en) * | 2013-04-02 | 2013-06-19 | 厦门大学 | Detection method of L-cysteine |
| CN108195803B (en) * | 2017-12-11 | 2020-04-17 | 东莞理工学院 | Method for detecting water body disinfection byproducts |
| CN113075147B (en) * | 2021-02-26 | 2022-09-27 | 南开大学滨海学院 | Sasa class magnesium metal organic complex material, preparation method thereof and application of material in detecting sulfur-containing malodorous substances |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1247449A (en) * | 1968-09-20 | 1971-09-22 | Merck Patent Gmbh | Agent for the determination of lipase |
| SU1191791A1 (en) * | 1984-09-28 | 1985-11-15 | 1-Й Московский Ордена Ленина И Ордена Трудового Красного Знамени Медицинский Институт Им.И.М.Сеченова | Method of qualitative determination of glutamic acid or cysteine |
| CN1435692A (en) * | 2002-01-31 | 2003-08-13 | 李勤 | Fluorescent analysis reagent for testing blood homo cysteine, method for preparing same and use thereof |
| JP2004113138A (en) * | 2002-09-26 | 2004-04-15 | Sangaku Renkei Kiko Kyushu:Kk | Method for measuring homocysteine and reagent for measurement |
-
2004
- 2004-11-12 CN CNB200410092788XA patent/CN100445731C/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1247449A (en) * | 1968-09-20 | 1971-09-22 | Merck Patent Gmbh | Agent for the determination of lipase |
| SU1191791A1 (en) * | 1984-09-28 | 1985-11-15 | 1-Й Московский Ордена Ленина И Ордена Трудового Красного Знамени Медицинский Институт Им.И.М.Сеченова | Method of qualitative determination of glutamic acid or cysteine |
| CN1435692A (en) * | 2002-01-31 | 2003-08-13 | 李勤 | Fluorescent analysis reagent for testing blood homo cysteine, method for preparing same and use thereof |
| JP2004113138A (en) * | 2002-09-26 | 2004-04-15 | Sangaku Renkei Kiko Kyushu:Kk | Method for measuring homocysteine and reagent for measurement |
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|---|---|
| CN1773264A (en) | 2006-05-17 |
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