CN102323233B - Reagent and method for detecting homocysteine (HCY) - Google Patents
Reagent and method for detecting homocysteine (HCY) Download PDFInfo
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- CN102323233B CN102323233B CN 201110191582 CN201110191582A CN102323233B CN 102323233 B CN102323233 B CN 102323233B CN 201110191582 CN201110191582 CN 201110191582 CN 201110191582 A CN201110191582 A CN 201110191582A CN 102323233 B CN102323233 B CN 102323233B
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Abstract
The invention provides a reagent for detecting homocysteine (HCY), which is a chromene derivate: 5-(hydroxylmethyl)-7-methyl-3, 3a-dihydrocyclopenta[b]chromen-1(2H)-one (HMMPC). The HMMPC reagent is prepared from 3-hydroxymethyl-5-methyl-salicylic aldehyde and 2-cyclopentenone in the next step at the room temperature; raw materials are cheap, the reaction condition is simple, and the scale production is easy. The invention provides a quantitative detection method for the homocysteine, which is used for quantitatively detecting the content of the homocysteine in a buffer solution of which the pH is 7.0 on account of HMMPC. In the detection method, high sensitivity and selectivity are shown on the HCY; the detection process is simple and convenient, sensitive and rapid; and the detection result is accurate.
Description
Technical field
The present invention relates to homocysteine and detect analytical technology, specifically belong to a kind of reagent and method that detects fast, quantitatively homocysteine.
Background technology
Cardiovascular and cerebrovascular diseases all is a threat to global people's health.Annual global approximately 1,500 ten thousand people die from cardiovascular and cerebrovascular diseases according to estimates.And homocysteine (Hcy) mass formed by blood stasis is an independent risk factor that causes cardiovascular and cerebrovascular diseases, has at present one of clinical indices that Hcy must have been detected as the diagnosis cardiovascular and cerebrovascular diseases in many laboratories.
The detection method for detection of homocysteine of having reported at present has: high performance liquid chromatography (HPLC) (Kusmierek, K.; Glowacki, R.; Bald, E. Anal.Bioanal. Chem., 2006,385,855.), gas chromatography-mass spectrometry chromatogram (MacCoss, M.J.; Fukagawa, N.K.; Matthews, D.E. Anal.Chem., 1999,71,4527.), enzyme process (Frantzen, F.; Faaren, A.L.; Alfheim, I.; Nordhei, A.K.Clin.Chem., 1998,44,311.), immunoassay (Shiu, H.Y.; Chong, H.C.; Leung, Y.C.; Wong, M.K.; Che, C.M.Chem.Eur.J., 2010,16,3308-3313.), capillary electrophoresis (Causse, E.; Tierrier, R.; Champagne, S.; Nertz, M.; Valdiguie, P.; Salvayre, R.J. Chromatogr.A, 1998,817,181.) and electrochemical process (Melnyk, S.; Pogribna, M.; Pogribny, I.; Hine, R.J.; James, S.J.J.Nutr.Biochem., 1999,10,490.).Yet these detection methods need routine analyzer consuming time and expensive reagent and instrument.So, develop that a kind of testing process is simple, sensitive, cost is low, it is clinical to be fit to, scientific research is used reagent and the method for detection homocysteine very necessary.
Summary of the invention:
The present invention is the problem that exists in the detection of prior art homocysteine in order to overcome, and reagent and the method for the homocysteine detection by quantitative that a kind of system is simple, easy to operate, selectivity is high, highly sensitive is provided.
The reagent of detection homocysteine provided by the invention and method, be based on a kind of chromene derivative 5-(hydroxylmethyl)-7-methyl-3,3a-dihydrocyclopenta[b] chromen-1 (2H)-one (HMMPC), detect homocysteine quantitatively in pH is 7.0 HEPES buffered soln.
A kind of reagent that detects homocysteine provided by the invention: it is 5-(hydroxylmethyl)-7-methyl-3,3a-dihydrocyclopenta[b] chromen-1 (2H)-one (HMMPC).
The synthetic route of HMMPC:
The synthetic method of HMMPC, step was: with 3-methylol-5-cresotinic acid aldehyde, cyclopentenone, imidazoles, tetrahydrofuran (THF) and deionized water 1: 1.5: 1.5 in molar ratio: mix at 85: 85, at room temperature stirred 48 hours, with the 1MHCl dilution that is equivalent to 3-methylol-10 times of molar weights of 5-cresotinic acid aldehyde, and with the ethyl acetate extraction that is equivalent to 3-methylol-155 times of molar weights of 5-cresotinic acid aldehyde three times, with the organic phase evaporated under reduced pressure, then use 1: 4 ethyl acetate of volume ratio and sherwood oil in silica gel chromatographic column drip washing, obtain HMMPC.
A kind of method that detects homocysteine provided by the invention comprises the steps:
(1), preparation pH=6.0-9.0, concentration are the HEPES buffered soln of 1-100mM, and prepare the HMMPC ethanolic soln of 2mM with ethanol;
(2), the HMMPC solution of the HEPES buffered soln of 2ml and 10 μ l is added in the clean ultraviolet cuvette, detect at the UV, visible light spectrophotometer, along with the adding for the treatment of test sample, absorption peak 315nm and 389nm all reduce gradually, when absorption peak no longer reduces, stop to add and treat test sample, add this moment and treat that the volume of test sample counts V
Treat test sample
(3), by 10 * 7 * 2 * 10
-3/ V
Treat test sampleCalculate the concentration (mol/L) for the treatment of Hcy in the test sample.
Described HEPES buffered soln can be used the replacements such as NaAc_HAc buffer solution, Tris-HCl, citric acid, phosphate buffer solution.
The experiment proved that, other amino acid not interference system to the mensuration of homocysteine.
Compared with prior art, the present invention has following advantage and effect: 1, detection system is with low cost, and reagent is by 3-methylol-5-cresotinic acid aldehyde and 2-cyclopentenone, and at room temperature a step makes, and raw material is cheap, and reaction conditions is simple, is easy to scale production; 2, detection method of the present invention has shown high susceptibility and selectivity to Hcy, Hcy and other two kinds of mercaptan small molecules Cys and GSH can be distinguished, and having overcome for a long time can't be with the puzzlement of Hcy and Cys, the two differentiation of GSH; 3, testing process is easy, sensitive, quick, and detected result is accurate; 4, detection means is simple, only need to be by ultraviolet spectrophotometer.
Description of drawings:
The single crystal diffraction structure iron of Fig. 1 HMMPC.
Fig. 2 adds Hcy, the Cys of same amount, the UV, visible light absorption figure of GSH in containing 10 μ l HMMPC and pH7.0HEPES (10mM) buffered soln.
In Fig. 3 HMMPC and pH7.0HEPES (10mM) buffered soln, add respectively various amino acid whose histograms.
Fig. 4 embodiment 4 detects the Hcy UV, visible light and absorbs figure.
Fig. 5 embodiment 5 detects the Hcy UV, visible light and absorbs figure.
Fig. 6 embodiment 6 detects the Hcy UV, visible light and absorbs figure.
Embodiment:
Embodiment 1
HMMPC's is synthetic: 3-methylol-5-cresotinic acid aldehyde 2mmoL, cyclopentenone 3mmoL, imidazoles 3mmoL, tetrahydrofuran (THF) 3mL and deionized water 3mL are mixed, at room temperature stirred 48 hours, dilute with 20mL 1M HCl, and with 30mL ethyl acetate extraction three times, with the organic phase evaporated under reduced pressure, then use 1: 4 ethyl acetate of volume ratio and sherwood oil in silica gel chromatographic column drip washing, obtain HMMPC, productive rate 56%.Growing the grain in ethyl acetate and sherwood oil obtains lurid monocrystalline.
The sign of HMMPC:
1H NMR (600MHz, 25 ℃, DMSO-
D6): δ 7.18 (s, 1H), 7.14 (d, 1H), 7.01 (s, 1H), 5.30 (t, 1H), 2.58-2.63 (m, 1H), 2.39-2.74 (m, 2H), 2.11-2.38 (m, 3H), 2.35 (s, 3H);
13C NMR (150MHz, CDCl
3): δ 21.23,28.92, and 37.88,61.94,76.82,122.48,128.61,129.21,130.90,132.36,133.34,151.60,202.06; Ultimate analysis (calcd.%) C
14H
14O
3: C, 73.03; H, 6.13. records: C, 73.13; H, 6.08. crystal data: C
12H
9ClO
2, FW=230.25, crystal size:0.3 * 0.2 * 0.2mm, Triclinic, space group P-1 (No.2),
α=109.469 (2) °, β=95.240 (2) °, γ=95.950 (2) °,
Z=4, T=296K, θ
Max=25.05 °, R
Int=0.0327, R
1=0.0620, wR
2=0.2108, GOF=0.92. single crystal diffraction structure iron is seen Fig. 1.
Embodiment 2
Be the buffered soln of 2ml pH7.0 HEPES (10mM) and concentration that the HMMPC solution of 2mM, 10 μ l is added to respectively in three different ultraviolet cuvettes, Hcy, the Cys, the GSH that add respectively again 70 μ l, detect at ultraviolet-visual spectrometer, UV, visible light absorption figure sees Fig. 2.
Embodiment 3
Be the buffered soln of 2ml pH7.0HEPES (10mM) and concentration that the HMMPC solution of 2mM, 10 μ l is added to respectively in the different ultraviolet cuvettes, each seed amino acid that adds respectively isodose, measure the absorption value of 389nm at ultraviolet-visual spectrometer, draw the histogram of absorption value corresponding to different aminoacids, see Fig. 3.
Embodiment 4 detects Hcy
Preparation pH=6.0 HEPES (10mM) buffered soln, the Hcy solution of preparation 2mM, and with the HMMPC solution of ethanol preparation 2mM; The HMMPC solution of the HEPES buffered soln of 2ml and 10 μ l is added in the clean ultraviolet cuvette, detects at the UV, visible light spectrophotometer, 315nm 389nm has absorption; Get the solution of Hcy, be added in this cuvette with microsyringe gradually, application of sample limit, limit is detected at the UV, visible light spectrophotometer, adding along with Hcy, absorption peak 315nm, 389nm all become gradually and reduce, when absorption peak no longer reduces, stop to add and treat test sample, add this moment and treat that the volume of test sample counts 70 μ l; By 10 * 7 * 2 * 10
-3/ 70 content that calculate Hcy are 2 * 10
-3(mol/L).UV, visible light absorption figure sees Fig. 4.
Embodiment 5 detects Hcy
Preparation pH=7.0 Tris-HCl (10mM) buffered soln, the Hcy solution of preparation 2mM, and with the HMMPC solution of ethanol preparation 2mM; The HMMPC solution of the Tris-HCl buffered soln of 2ml and 10 μ l is added in the clean ultraviolet cuvette, detects at the UV, visible light spectrophotometer, 315nm 389nm has absorption; Get the solution of Hcy, be added in this cuvette with microsyringe gradually, application of sample limit, limit is detected at the UV, visible light spectrophotometer, adding along with Hcy, absorption peak 315nm, 389nm all become gradually and reduce, when absorption peak no longer reduces, stop to add and treat that test sample, this moment add the volume of Hcy and count 70 μ l; By 10 * 7 * 2 * 10
-3/ 70 content that calculate Hcy are 2 * 10
-3(mol/L).UV, visible light absorption figure sees Fig. 5.
Embodiment 6 detects Hcy
Preparation pH=7.0 acetic acid-sodium-acetate (10mM) buffered soln, the Hcy solution of preparation 2mM, and with the HMMPC solution of ethanol preparation 2mM; The HMMPC solution of the NaAc_HAc buffer solution of 2ml and 10 μ l is added in the clean ultraviolet cuvette, detects at the UV, visible light spectrophotometer, 315nm 389nm has absorption; Get the solution of Hcy, be added in this cuvette with microsyringe gradually, application of sample limit, limit is detected at the UV, visible light spectrophotometer, adding along with Hcy, absorption peak 315nm, 389nm all become gradually and reduce, when absorption peak no longer reduces, stop to add and treat that test sample, this moment add the volume of Hcy and count 70 μ l; By 10 * 7 * 2 * 10
-3/ 70 content that calculate Hcy are 2 * 10
-3(mol/L).UV, visible light absorption figure sees Fig. 6.
Claims (4)
1. reagent that detects homocysteine: be characterised in that it is the 5-(methylol)-7-methyl-3,3a-dihydro cyclopenta [b] chromene-1(2H)-ketone (5-(hydroxyl methyl)-7-methyl-3,3a-dihydrocyclopenta[b] chromen-1 (2H)-one, be abbreviated as HMMPC).
2. a kind of synthetic method that detects the reagent of homocysteine as claimed in claim 1, it is characterized in that step is: with 3-methylol-5-cresotinic acid aldehyde, cyclopentenone, imidazoles, tetrahydrofuran (THF) and deionized water 1: 1.5: 1.5 in molar ratio: mix at 85: 85, at room temperature stirred 48 hours, with the 1M HCl dilution that is equivalent to 3-methylol-10 times of molar weights of 5-cresotinic acid aldehyde, and with the ethyl acetate extraction that is equivalent to 3-methylol-155 times of molar weights of 5-cresotinic acid aldehyde three times, with the organic phase evaporated under reduced pressure, then use 1: 4 ethyl acetate of volume ratio and sherwood oil in silica gel chromatographic column drip washing, obtain HMMPC.
3. a method that detects homocysteine is characterized in that comprising the steps:
(1), preparation pH=6.0-9.0, concentration are 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) buffered soln of 1-100mM, and prepare the HMMPC ethanolic soln of 2mM with ethanol;
(2), the HMMPC solution of the HEPES buffered soln of 2ml and 10 μ l is added in the clean ultraviolet cuvette, detect at the UV, visible light spectrophotometer, along with the adding for the treatment of test sample, absorption peak 315nm and 389nm all reduce gradually, when absorption peak no longer reduces, stop to add and treat test sample, add this moment and treat that the volume of test sample counts V
Treat test sample
(3), by 10 * 7 * 2 * 10
-3/ V
Treat test sampleCalculate the concentration for the treatment of Hcy in the test sample.
4. a kind of method that detects homocysteine as claimed in claim 3 is characterized in that described HEPES buffered soln replaces with NaAc_HAc buffer solution, Tris-HCl, citric acid or phosphate buffer solution.
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| CN105181666B (en) * | 2015-09-23 | 2018-02-06 | 山西大学 | A kind of reagent and method of fluoroscopic examination cysteine |
| CN106950210B (en) * | 2017-03-27 | 2019-09-24 | 山西大学 | A kind of reagent detecting glutathione and its synthetic method and application |
| CN109358017B (en) * | 2018-10-26 | 2021-08-10 | 武汉百德瑞康生物技术有限公司 | Homocysteine determination kit, preparation method and detection method thereof |
| CN111285836A (en) * | 2018-12-06 | 2020-06-16 | 广东轻工职业技术学院 | Preparation and application of near-infrared fluorescent probe |
| CN111303072A (en) * | 2020-02-27 | 2020-06-19 | 山西大学 | Reagent for distinguishing and detecting cysteine and synthetic method and application thereof |
| CN111393430B (en) * | 2020-03-17 | 2021-05-14 | 山西大学 | Stemona alkaloid analog near-infrared fluorescent probe and preparation method thereof |
| CN113929652A (en) * | 2020-07-14 | 2022-01-14 | 兰州大学 | Sulfide-responsive self-releasing linker molecule |
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| CN101430280A (en) * | 2008-12-11 | 2009-05-13 | 复旦大学 | Phosphorescence chemical sensor for qualitative homocysteine detection and use thereof |
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| CN101430280A (en) * | 2008-12-11 | 2009-05-13 | 复旦大学 | Phosphorescence chemical sensor for qualitative homocysteine detection and use thereof |
Non-Patent Citations (6)
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| Colorimetric Detection of Thiols Using a Chromene Molecule;Fang-Jun Huo et al.;《Organic Letters》;20090929;第11卷(第21期);第4918-4921页 * |
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