CN101408509B - Aminothiopropionic acid fast detecting method based on gold nano particle colloidal sols absorption spectrum - Google Patents
Aminothiopropionic acid fast detecting method based on gold nano particle colloidal sols absorption spectrum Download PDFInfo
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Abstract
The invention discloses a cysteine quick detection method based on an absorption spectrum of Au nanoparticle collosol. The invention is characterized in that deionized water, buffer solution with 3.8 to 5.7 pH value and Au nanoparticle collosol stock solution are mixed to be prepared into detection solution, and the concentration of the buffer substance and the element Au in the detection solution is respectively 10 plus or minus 1mm and 0.9 plus or minus 0.1mm; the cysteine-containing liquid to be detected is added to the detection solution, the concentration of the cysteine in the formed detection system is 0.5 to 10mum, and the absorption spectrum is measured within ten minutes after even mixing; and according to the difference of the specific value eta of the absorbance of 700 nanometers and 525 nanometers of the wavelength in the absorption spectrum of the detection system, the concentration of the cysteine can be detected quantitatively. The method greatly reduces the time required for detecting the cysteine, and meanwhile, has the advantages of simple equipment, simple operation and mass detection.
Description
Technical field
The invention belongs to the cysteine detecting method technical field, particularly based on the halfcystine method for quick of gold nano particle colloidal sols absorption spectrum.
Background technology
According to " amino acid " (Wu Xianrong writes, and publishing house of Beijing Agricultural University published the 3rd page in 1988) record, halfcystine is a kind of sulfur-containing amino acid, plays an important role in the oxidation-reduction process of organism metabolism.(Zou Zhongmei, explain long-range chief editor according to " source of human-body biological---amino acid ", Chinese Medicine science and technology publishing house, published in 2000, the 63-67 page or leaf) record, halfcystine and derivant thereof also are the potential nerve toxin and the biomarker of multiple physiological environment, and its content direct relation in biosome cardiovascular function.According to U.S.'s " New England Journal of Medicine " (The NewEngland Journal of Medicine 338:1042-1050,1998) report, halfcystine and homolog thereof---homocysteine can be used as the dangerous index of independence of cardiovascular and cerebrovascular disease such as atherosclerotic in clinical practice, its normal concentration in human body is 5-15 μ M under the situation on an empty stomach.The main method that detects halfcystine and derivant thereof at present has fluorescence polarization immunodetection, high performance liquid chromatography, galvanochemistry voltammetry etc., but these detection methods all depend on the instrument and equipment of complex and expensive, and complex operation step, length consuming time can not realize big flow automated analysis again clinically.Recently, U.S.'s " nanometer wall bulletin " (Nano Letters, 8 (2): 529-533.2008.) reported with finishing have the gold nano particle colloidal sols of dna molecular to detect the method for halfcystine, this method is utilized halfcystine and mercury ion (Hg
2+) characteristics that binding ability is strong, realize the purpose of halfcystine detection by quantitative by the double chain DNA molecule uncoiling variation of temperature that detects the coupling gold nano particle colloidal sols.Yet said method need use the instrument of accurate temperature controlling, operates still more loaded down with trivial detailsly, and consuming time more than 2 hours, still has bigger restriction for clinical practice.Up to the present, do not see that as yet bibliographical information can be ten minutes methods with interior detection halfcystine.
Summary of the invention
The objective of the invention is to propose a kind of method for quick of the halfcystine based on gold nano particle colloidal sols absorption spectrum, instrument and equipment complex and expensive in the existing detection technique, operating process are loaded down with trivial details to overcome, the shortcoming of length consuming time.
The present invention is based on the aminothiopropionic acid fast detecting method of gold nano particle colloidal sols absorption spectrum, it is characterized in that: deionized water and pH value are hybridly prepared into detection liquid in 3.8 to 5.7 buffer solution and gold nano particle colloidal sols stoste, make the buffer substance in this detection liquid and the concentration of gold element be respectively 10 ± 1mM and 0.9 ± 0.1mM; The liquid to be detected that will contain halfcystine joins in the above-mentioned detection liquid, and the concentration of halfcystine is 0.5～10 μ M in the feasible detection architecture that forms, and it is mixed the back measured its absorption spectrum in ten minutes; Be ratio η different of 700 nanometers and the absorbance of 525 nanometers according to the absorption spectrum medium wavelength of detection architecture, promptly detection by quantitative goes out the concentration of halfcystine.
The detection liquid that uses in the inventive method is the water-soluble gold nano particle colloidal sols, and when the pH of colloidal sol value was in 3.8～5.7 scopes, it was red detecting liquid; Contain the liquid to be detected of halfcystine in adding after, (SH) can promptly be connected the gold nano grain surface, the carboxyl of the halfcystine other end (COOH) and amino (NH for the sulfydryl in the halfcystine molecule
2) then outwards stretch, and the carboxyl between the different gold nano grain is (COOH) and amino (NH
2) interact byer force, the gold nano grain that causes the surface to be connected with halfcystine is assembled; Because gold nano grain surface plasma resonance absorption characteristic is subjected to the influence of aggregation extent, the color of whole system changes thereupon and reflect quantitative relationship in absorption spectrum, utilizing the absorption spectrum medium wavelength is ratio η different of 700 nanometers and the absorbance of 525 nanometers, can detection by quantitative goes out the concentration of halfcystine; Because (therefore SH) group all can't directly be connected the gold nano particle colloidal sols surface, also can't come its concentration of detection by quantitative by above-mentioned absorption spectrum detection method not have sulfydryl in other the 19 seed amino acid molecules except that halfcystine.This absorption spectrum according to gold nano particle colloidal sols is to the difference response of 20 kinds of natural amino acids, distinguish halfcystine and other amino acid, and, do not see bibliographical information as yet according to the method that the variation of gold nano particle colloidal sols absorption spectrum comes the detection by quantitative semicystinol concentration.Be different from U.S.'s " nanometer wall bulletin " (Nano Letters, 8 (2): the double chain DNA molecule uncoiling variation of temperature of passing through measurement coupling gold nano particle colloidal sols of report realizes the quantitative detecting method of halfcystine 529-533.2008.), and the inventive method is to realize the detection by quantitative purpose of halfcystine by the variation of measuring gold nano particle colloidal sols absorption spectrum.Compared with prior art, adopt the inventive method can within ten minutes, detect the concentration of halfcystine in the system, reduced greatly and detected the required time of halfcystine, and have that equipment is simple, operation is simple and easy and advantage that can batch detection.
Description of drawings
Fig. 1 is that the pH value is 3.8 the abosrption spectrogram of gold nano particle colloidal sols after adding the variable concentrations halfcystine;
Fig. 2 is to be that the wavelength that 3.8 absorption spectrum obtains is the ratio η of 700 nanometers and 525 nanometer absorbances and the graph of relation between the semicystinol concentration by pH value among Fig. 1.
Fig. 3 is that the pH value is 5.0 the abosrption spectrogram of gold nano particle colloidal sols after adding the variable concentrations halfcystine;
Fig. 4 is to be that the wavelength that 5.0 absorption spectrum obtains is the ratio η of 700 nanometers and 525 nanometer absorbances and the graph of relation between the semicystinol concentration by pH value among Fig. 3.
Fig. 5 is that the pH value is 5.7 the abosrption spectrogram of gold nano particle colloidal sols after adding the variable concentrations halfcystine;
Fig. 6 is to be that the wavelength that 5.7 absorption spectrum obtains is the ratio η of 700 nanometers and 525 nanometer absorbances and the graph of relation between the semicystinol concentration by pH value among Fig. 5.
Fig. 7 is that the pH value is 8.0 the abosrption spectrogram of gold nano particle colloidal sols after adding the variable concentrations halfcystine;
Fig. 8 is to be that the wavelength that 8.0 absorption spectrum obtains is the ratio η of 700 nanometers and 525 nanometer absorbances and the graph of relation between the semicystinol concentration by pH value among Fig. 7.
Fig. 9 is that the pH value is to add the absorbance ratio that concentration is behind 20 kinds of different natural amino acids of 3 μ M in 5.0 the gold nano particle colloidal sols to change promptly | η-η
0| column diagram.
Embodiment
Embodiment 1: with the pH value is that 3.8 gold nano particle colloidal sols detects semicystinol concentration
The preparation of step 1, gold nano particle colloidal sols:
Gold nano particle colloidal sols can directly adopt commercial product, also can make by oneself and gets.
The concrete steps of self-control gold nano particle colloidal sols are: adding 48mL deionized water and 2mL concentration are the chlorauric acid solution of 25mM in the 100mL round-bottomed flask, place 90-95 ℃ of water-bath to reflux 5 minutes, add the citric acid three sodium solution that 5mL concentration is 38.8mM then rapidly, and under vigorous stirring, continue in above-mentioned water-bath, to reflux 20 minutes, obtain the peony sol liquid.Remove water-bath and continue and stir, cool to room temperature promptly obtains gold nano particle colloidal sols.
The pH value is that 3.8 buffer solution can directly adopt commercial product, also can make by oneself and gets.
Self-control pH value is that the concrete steps of 3.8 buffer solution are: take by weighing 0.4102 and restrain the sodium acetate crystal, be dissolved in the 20mL deionized water, and in this dense sodium acetate solution, accurately add 2576 μ L glacial acetic acid, obtain mother liquor, this mother liquor transferred in the 100mL volumetric flask and with deionized water be diluted to scale, promptly obtain 100mL500mM buffer solution A.
Step 3, halfcystine detect the preparation of liquid:
Sequencing according to deionized water, buffer solution A, gold nano particle colloidal sols stoste, and press deionized water: buffer solution A: the volume ratio of gold nano particle colloidal sols stoste=39:1:10, is 3.8 with above-mentioned three kinds of liquid mixing with the pH value that keeps system, this moment, the total concentration of buffer substance acetic acid/sodium acetate was 10mM, and the concentration of gold element is 0.91mM, and this solution is designated as B.It should be noted that, when the buffer substance in detecting liquid and the concentration of gold element are respectively 10 ± 1mM and 0.9 ± 0.1mM, it is to the detection best results of halfcystine, sensitivity is the highest, after exceeding this scope, be subjected to the influence of ion equivalent and gold nano grain interphase interaction, this detects the detection sensitivity obviously reduction of liquid to halfcystine.
The detection of step 5, semicystinol concentration:
Difference is 0 μ L, 1 μ L, 2 μ L, 3 μ L, 3.5 μ L, 4 μ L, 5 μ L and 6 μ L by volume, the concentration of getting above-mentioned preparation is eight parts of the halfcystine solution C of 1mM, be dissolved in respectively in the solution B of preparation in the 2mL above-mentioned steps three, and rapidly it is mixed, the concentration of halfcystine is respectively 0 μ M, 0.5 μ M, 1 μ M, 1.5 μ M, 1.75 μ M, 2 μ M, 2.5 μ M and 3 μ M in the detection architecture at this moment, measure its absorption spectrum in mixing respectively within back ten minutes, gained spectral line a1～a8 is plotted among Fig. 1 successively.It should be noted that: detecting the mixing of liquid and halfcystine solution after ten minutes herein, owing to be subjected to the influence of active function groups in the halfcystine, surface adsorption the gold nano particle colloidal sols of halfcystine can partly be adsorbed onto on glass or the silica ware inwall, spectral measurement is produced interference, the inaccurate of measurement result can be caused, therefore its absorption spectrum need be within ten minutes, measured.Along with the increase of semicystinol concentration, marked change takes place from a1 to a8 the absorption spectrum curve of detection architecture: wavelength is that the absorption peak strength of 525 nanometers descends, and the part absorbance of wavelength more than 650 nanometers rises.
With the absorption spectrum medium wavelength is that the ratio of the absorbance of 700 nanometers and 525 nanometers is designated as η, press of the variation mapping of η value with semicystinol concentration, obtain the ratio η of absorbance and the relation curve between the semicystinol concentration as shown in Figure 2, curve b among the figure shows that the η value and the semicystinol concentration of detection architecture are monotonic relationshi, and this curve b promptly can be used as the typical curve that detects semicystinol concentration by the gold nano particle colloidal sols of pH value under 3.8; As seen be that the concentration range that 3.8 o'clock detection architecture can effectively detect halfcystine is 0.5～3 μ M in the pH value.
Embodiment 2: with the pH value is that 5.0 gold nano particle colloidal sols detects semicystinol concentration
Step 1, with the step 1 among the embodiment 1.
The pH value is that 5.0 buffer solution can directly adopt commercial product, also can make by oneself and gets.Self-control pH value is that the concrete steps of 5.0 buffer solution are: take by weighing 2.7343 gram sodium acetate dissolution of crystals in the 20mL deionized water, and adding 954 μ L glacial acetic acid obtain mother liquor in this dense sodium acetate solution.Mother liquor transferred in the volumetric flask and with deionized water be diluted to 100mL, obtain 500mM buffer solution D.
Step 3, halfcystine detect the preparation of liquid:
The concrete operations step is with the step 3 among the embodiment 1, only need be with the buffer solution A in the buffer solution D alternate embodiment 1, make that the pH value of gold nano particle colloidal sols is 5.0 after three kinds of liquid mixing.This solution is designated as E.
The detection of step 5, semicystinol concentration:
By volume 0 μ L, 2 μ L, 4 μ L, 4.5 μ L, 5 μ L, 5.5 μ L, 6 μ L, 7 μ L, 8 μ L, 10 μ L, 14 μ L and the 20 μ L concentration of getting above-mentioned preparation is 12 parts of the halfcystine solution C of 1mM respectively, be dissolved in respectively in the solution E of 2mL, and rapidly it is mixed, make that the concentration of halfcystine is respectively 0 μ M, 1 μ M, 2 μ M, 2.25 μ M, 2.5 μ M in the detection architecture, 2.75 μ M, 3 μ M, 3.5 μ M, 4 μ M, 5 μ M, 7 μ M and 10 μ M, difference absorbance spectrum in ten minutes, gained spectral line c1～c12 is plotted among Fig. 3 successively.Along with the increase of semicystinol concentration, marked change takes place from c1 to c12 the absorption spectrum curve of detection architecture: wavelength is that the absorption peak strength of 525 nanometers descends, and the part absorbance of wavelength more than 650 nanometers rises.
With the absorption spectrum medium wavelength is that the ratio of the absorbance of 700 nanometers and 525 nanometers is designated as η, press of the variation mapping of η value with semicystinol concentration, obtain the ratio η of absorbance and the relation curve between the semicystinol concentration as shown in Figure 4, curve d among the figure shows that the η value and the semicystinol concentration of detection architecture are monotonic relationshi, and this curve d promptly can be used as the typical curve that detects semicystinol concentration by the gold nano particle colloidal sols of pH value under 5.0; As seen be that the concentration range that 5.0 o'clock detection architecture can effectively detect halfcystine is 1～10 μ M in the pH value.
Embodiment 3: with the pH value is that 5.7 gold nano particle colloidal sols detects semicystinol concentration
Step 1, with the step 1 among the embodiment 1.
The pH value is that 5.7 buffer solution can directly adopt commercial product, also can make by oneself and gets.Self-control pH value is that the concrete steps of 5.7 buffer solution are: take by weighing 3.6914 gram sodium acetate dissolution of crystals in the 20mL deionized water, and adding 286 μ L glacial acetic acid obtain mother liquor in this dense sodium acetate solution.Mother liquor transferred in the volumetric flask and with deionized water be diluted to 100mL, obtain 500mM buffer solution F.
Step 3, halfcystine detect the preparation of liquid:
The concrete operations step is with the step 3 among the embodiment 1, only need be with the buffer solution A in the buffer solution F alternate embodiment 1, make that the pH value of gold nano particle colloidal sols is 5.7 after three kinds of liquid mixing.This solution is designated as G.
The detection of step 5, semicystinol concentration:
Get halfcystine solution C 0 μ L, 4 μ L, 5 μ L, 6 μ L, 8 μ L, 12 μ L, 16 μ L, 20 μ L, be dissolved in the solution G of 2mL respectively, and rapidly it mixed, make that the concentration of halfcystine is respectively 0 μ M, 2 μ M, 2.5 μ M, 3 μ M, 5 μ M, 6 μ M, 8 μ M and 10 μ M in the detection architecture, surveyed absorption spectrum in ten minutes respectively, gained spectral line e1～e8 is plotted among Fig. 5 successively.Along with the increase of semicystinol concentration, marked change takes place from e1 to e8 the absorption spectrum curve of detection architecture: wavelength is that the absorption peak strength of 525 nanometers descends, and the part absorbance of wavelength more than 650 nanometers rises.
With the absorption spectrum medium wavelength is that the ratio of the absorbance of 700 nanometers and 525 nanometers is designated as η, press of the variation mapping of η value with semicystinol concentration, obtain the ratio η of absorbance and the relation curve between the semicystinol concentration as shown in Figure 6, curve f among the figure shows that the η value and the semicystinol concentration of detection architecture are monotonic relationshi, and this curve f promptly can be used as the typical curve that detects semicystinol concentration by the gold nano particle colloidal sols of pH value under 5.7; As seen be that the concentration range that 5.7 o'clock detection architecture can effectively detect halfcystine is 2～10 μ M in the pH value.
Comprehensive above three embodiment of the present invention can illustrate, the gold nano particle colloidal sols of pH value in 3.8～5.7 scopes can the detection by quantitative halfcystine concentration, and the concentration range that halfcystine can be gone out by detection by quantitative is 0.5～10 μ M, and testing process is consuming time less than ten minutes; The sensitivity that detects descends along with the rising of pH.
Illustrate that below by one group of comparative example when the pH of gold nano particle colloidal sols value exceeds above-mentioned scope and is under the weak basic condition, the detection architecture in the inventive method no longer has the ability that detects semicystinol concentration.
Comparative example 1:pH value is 8.0 the gold nano particle colloidal sols and the effect of halfcystine
Step 1, with the step 1 among the embodiment 1.
Step 3, with step 3 among the embodiment 1, compound concentration is the halfcystine solution C of 1mM.
Step 5, data processing:
With the absorption spectrum medium wavelength is that the ratio of the absorbance of 700 nanometers and 525 nanometers is designated as η, presses the variation mapping of η value with semicystinol concentration, as shown in Figure 8.Curve h among Fig. 8 shows: under the pH=8.0 condition, by measuring the η value of detection architecture, can't accurately obtain the concentration information of halfcystine in the system.
Illustrate below by another group comparative example: the inventive method has high selectivity to the detection of halfcystine, promptly can effectively detect under the condition of semicystinol concentration, therefore gold nano particle colloidal sols is extremely low for the spectral response of other 19 kinds of natural amino acids, can get rid of other amino acid whose interference and active zone is told halfcystine.
Comparative example 2: with the pH value is the comparison that 5.0 gold nano particle colloidal sols detects 20 kinds of natural amino acids
Step 1, with the step 1 among the embodiment 1.
Step 3, with the step 3 among the embodiment 2.
Step 5, take by weighing 89 milligrams of alanine (Ala) respectively, 174 milligrams of arginine (Arg), 150 milligrams of asparagines (Asn), 133 milligrams of aspartic acids (Asp), 121 milligrams of halfcystines (Cys), 146 milligrams of glutamine (Gln), 147 milligrams in glutamic acid (Glu), 75 milligrams of glycocoll (Gly), 155 milligrams of histidines (His), 131 milligrams of isoleucines (Ile), 131 milligrams of leucines (Leu), 146 milligrams of lysines (Lys), 149 milligrams of methionine (Met), 165 milligrams of phenylalanines (Phe), 115 milligrams of proline (Pro), 105 milligrams of serines (Ser), 119 milligrams of threonines (Thr), 204 milligrams of tryptophanes (Try), 117 milligrams of 181 milligrams in tyrosine (Tyr) and valines (Val), be dissolved in successively in the deionized water of 100mL, obtain the solution that concentration is 20 seed amino acids of 1mM, be designated as solution L1～L20.
With the absorption spectrum medium wavelength is that the ratio of the absorbance of 700 nanometers and 525 nanometers is designated as η; Add before and after the amino acid, the variation of the absorbance ratio η of detection architecture promptly | η-η
0| corresponding different amino acid is made column diagram, as shown in Figure 9.As seen from Figure 9: be all at amino acid concentration under the situation of 3 μ M, only contain the detection architecture of halfcystine | η-η
0| value takes place obviously to change, and 19 kinds other are amino acid whose | η-η
0| be worth relative halfcystine all less than 2%.The inventive method can be distinguished halfcystine and other 19 seed amino acid effectively in view of the above.
The analysis-by-synthesis explanation:
The inventive method realizes the purpose that cysteine quantitatively detects based on gold nano particle colloidal sols absorption spectrum. According to the difference response of gold nano particle colloidal sols to 20 kinds of natural amino acids, can effectively distinguish cysteine and other amino acid; Under the effect of the cysteine of variable concentrations, its absorption spectrum medium wavelength is ratio η different of 700 nanometers and the absorbance of 525 nanometers according to gold nano particle colloidal sols, can quantitatively detect the concentration of cysteine; Wherein the pH value scope of application of gold nano particle colloidal sols is 3.8～5.7. The method of quantitative detection semicystinol concentration of the present invention has fast, device simple, simple to operate and advantage that can batch detection.
Claims (1)
1. aminothiopropionic acid fast detecting method based on gold nano particle colloidal sols absorption spectrum, it is characterized in that: deionized water and pH value are hybridly prepared into detection liquid in 3.8 to 5.7 hac buffer and gold nano particle colloidal sols stoste, make the buffer substance in this detection liquid and the concentration of gold element be respectively 10 ± 1mM and 0.9 ± 0.1mM; The liquid to be detected that will contain halfcystine joins in the above-mentioned detection liquid, and the concentration of halfcystine is 0.5～10 μ M in the feasible detection architecture that forms, and it is mixed the back measured its absorption spectrum in ten minutes; Be ratio η different of 700 nanometers and the absorbance of 525 nanometers according to the absorption spectrum medium wavelength of detection architecture, promptly detection by quantitative goes out the concentration of halfcystine: the semicystinol concentration scope that the detection architecture during pH=3.8 can effectively detect is 0.5～3 μ M; The semicystinol concentration scope that detection architecture during pH=5.0 can effectively detect is 1～10 μ M; The semicystinol concentration scope that detection architecture during pH=5.7 can effectively detect is 2～10 μ M.
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| CN102087219A (en) * | 2010-12-22 | 2011-06-08 | 湖南大学 | Method for detecting specific sulfhydryl-containing amino acid |
| 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 |
| CN103308512A (en) * | 2013-05-22 | 2013-09-18 | 陕西师范大学 | Application of L-cysteine-enveloped nanogold in chiral recognition of tyrosine |
| CN103512855B (en) * | 2013-09-27 | 2015-12-02 | 湖南大学 | The detection method of reduced glutathione |
| CN105067547B (en) * | 2015-07-22 | 2017-11-10 | 浙江大学 | A kind of amino acid fast optical detection method based on graphene oxide/nanogold |
| CN105403522B (en) * | 2015-10-26 | 2018-11-02 | 南昌大学 | A kind of quickly detection remaining method of Fluorine in Foods quinolones |
| CN105784616B (en) * | 2016-04-08 | 2018-07-17 | 江南大学 | The method for detecting cysteine or acetylcysteine based on bimetal nano cluster |
| CN108776127B (en) * | 2018-08-30 | 2021-02-23 | 河南师范大学 | AuAgNCs @ APAP fluorescent probe, preparation method thereof and application thereof in amino acid determination |
| CN109283146A (en) * | 2018-09-19 | 2019-01-29 | 天津工业大学 | Sensor and detection method for L-cysteine detection |
| CN113324933B (en) * | 2021-06-01 | 2022-07-26 | 浙江大学 | Method for detecting angiotensin converting enzyme and application thereof |
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