CN105784620A - Analysis method for colorimetric detection of artemisinin and derivatives thereof - Google Patents
Analysis method for colorimetric detection of artemisinin and derivatives thereof Download PDFInfo
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- CN105784620A CN105784620A CN201610303933.7A CN201610303933A CN105784620A CN 105784620 A CN105784620 A CN 105784620A CN 201610303933 A CN201610303933 A CN 201610303933A CN 105784620 A CN105784620 A CN 105784620A
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- arteannuin
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- analysis method
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- BLUAFEHZUWYNDE-NNWCWBAJSA-N artemisinin Chemical compound C([C@](OO1)(C)O2)C[C@H]3[C@H](C)CC[C@@H]4[C@@]31[C@@H]2OC(=O)[C@@H]4C BLUAFEHZUWYNDE-NNWCWBAJSA-N 0.000 title claims abstract description 79
- 229960004191 artemisinin Drugs 0.000 title claims abstract description 75
- 238000001514 detection method Methods 0.000 title claims abstract description 57
- 238000004458 analytical method Methods 0.000 title claims abstract description 27
- 229930101531 artemisinin Natural products 0.000 title claims abstract description 8
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 32
- 239000010931 gold Substances 0.000 claims abstract description 30
- 108020004414 DNA Proteins 0.000 claims abstract description 25
- 102000053602 DNA Human genes 0.000 claims abstract description 19
- 238000002835 absorbance Methods 0.000 claims abstract description 14
- 150000003254 radicals Chemical class 0.000 claims abstract description 12
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 claims abstract description 9
- 229910001448 ferrous ion Inorganic materials 0.000 claims abstract description 6
- 239000000084 colloidal system Substances 0.000 claims abstract description 4
- 229930191701 arteannuin Natural products 0.000 claims description 70
- 241001597008 Nomeidae Species 0.000 claims description 44
- 239000000243 solution Substances 0.000 claims description 33
- 108020004682 Single-Stranded DNA Proteins 0.000 claims description 17
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 15
- 239000000203 mixture Substances 0.000 claims description 14
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 239000001509 sodium citrate Substances 0.000 claims description 5
- 239000002245 particle Substances 0.000 claims description 4
- 244000025254 Cannabis sativa Species 0.000 claims description 3
- BLUAFEHZUWYNDE-XRNKLDBLSA-N chembl77 Chemical compound C([C@@](OO1)(C)O2)C[C@H]3[C@H](C)CC[C@@H]4C31[C@@H]2OC(=O)[C@@H]4C BLUAFEHZUWYNDE-XRNKLDBLSA-N 0.000 claims description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 3
- 239000012086 standard solution Substances 0.000 claims description 3
- 238000007689 inspection Methods 0.000 claims description 2
- 239000006193 liquid solution Substances 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 230000009467 reduction Effects 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims 1
- 229910052737 gold Inorganic materials 0.000 abstract description 23
- 238000011161 development Methods 0.000 abstract description 3
- 238000006555 catalytic reaction Methods 0.000 abstract 1
- 230000002596 correlated effect Effects 0.000 abstract 1
- 230000000694 effects Effects 0.000 abstract 1
- 230000035945 sensitivity Effects 0.000 abstract 1
- 230000008859 change Effects 0.000 description 5
- 239000003814 drug Substances 0.000 description 3
- IMBKASBLAKCLEM-UHFFFAOYSA-L ferrous ammonium sulfate (anhydrous) Chemical compound [NH4+].[NH4+].[Fe+2].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O IMBKASBLAKCLEM-UHFFFAOYSA-L 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000004445 quantitative analysis Methods 0.000 description 3
- LOUPRKONTZGTKE-WZBLMQSHSA-N Quinine Chemical compound C([C@H]([C@H](C1)C=C)C2)C[N@@]1[C@@H]2[C@H](O)C1=CC=NC2=CC=C(OC)C=C21 LOUPRKONTZGTKE-WZBLMQSHSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 238000010170 biological method Methods 0.000 description 2
- 238000004737 colorimetric analysis Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 201000004792 malaria Diseases 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 238000004451 qualitative analysis Methods 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 235000003826 Artemisia Nutrition 0.000 description 1
- 235000001405 Artemisia annua Nutrition 0.000 description 1
- 240000000011 Artemisia annua Species 0.000 description 1
- 235000003261 Artemisia vulgaris Nutrition 0.000 description 1
- 235000001258 Cinchona calisaya Nutrition 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 229910004042 HAuCl4 Inorganic materials 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 244000030166 artemisia Species 0.000 description 1
- 235000009052 artemisia Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- LOUPRKONTZGTKE-UHFFFAOYSA-N cinchonine Natural products C1C(C(C2)C=C)CCN2C1C(O)C1=CC=NC2=CC=C(OC)C=C21 LOUPRKONTZGTKE-UHFFFAOYSA-N 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- FDWREHZXQUYJFJ-UHFFFAOYSA-M gold monochloride Chemical compound [Cl-].[Au+] FDWREHZXQUYJFJ-UHFFFAOYSA-M 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229960000948 quinine Drugs 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 238000010183 spectrum analysis Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
Landscapes
- Physics & Mathematics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an analysis method for colorimetric detection of artemisinin and derivatives thereof. The analysis method is characterized in that a peroxide bridge structure contained in the artemisinin and derivatives thereof is utilized to generate a high-activity free radical under the catalysis of ferrous ions, and single-chain DNA (Deoxyribonucleic Acid) for maintaining the stability of nano gold colloids is broken, so that the dispersed nano gold colloids are gathered, and the color is changed from wine red into royal purple. The ratio (A610/A540) of absorbances of a nano gold colloid solution at 610nm and 540nm is linearly correlated to the concentrations of the artemisinin and the derivatives thereof, the detection range is 3 to 40 [mu]M, and the detection limit is 1[mu]M. The colorimetric detection method has the advantages of high color development, easiness in operation, low cost and high sensitivity.
Description
Technical field:
The invention belongs to colorimetric analysis and arteannuin and derivant detection technique field thereof, particularly relate to a kind of use nanometer gold and
Single stranded DNA carrys out qualitative, quantitative colorimetric detection method.
Technical background:
Arteannuin is the effective ingredient of Herba Artemisiae Annuae malaria, is also the initiation material of synthetic artemisinin medicine series, current arteannuin series
Medicine has replaced quinine becomes treatment malaria the most safely and effectively medicine.In recent years owing to arteannuin and derivant thereof are at world wide
Extensive application, promote China's arteannuin and extract the fast development of industry, also driven the development of Herba Artemisiae Annuae plant husbandry simultaneously.Because
The usage amount of Herba Artemisiae Annuae is big, and the market demand is wide, and some illegal retailers also take this opportunity to pretend to be certified products to sell with Herba Artemisiae Annuae inferior or adulterant, give green grass or young crops
Artemisin extracts manufacturer and brings the biggest loss, hence sets up that arteannuin is qualitative and quantitative detecting method is particularly important.When
The detection method of front arteannuin includes biological method, spectra methods, red, orange, green, blue, yellow (ROGBY) etc..Biological method such as enzyme linked immunological
Method (ELISA method), radio immunoassay (RIA method), would ordinarily be encountered cost height, operation again when quantitative analysis arteannuin
The problem such as miscellaneous, does not have universality., there is the shortcoming that operating procedure is loaded down with trivial details, time-consuming in spectra methods such as method of UV spectral analysis.
Red, orange, green, blue, yellow (ROGBY) such as high performance liquid chromatography, though having the advantages such as accuracy is high, specificity is strong, result is accurate, but because instrument is held high
Expensive, cost is the highest and can not popularize and promote.Therefore, a kind of quick, easy, accurate, sensitive arteannuin detection side is set up
Method is imperative.
In recent years, along with the continuous appearance of novel nano-material, portable, real-time, the detection technique of low cost and equipment are sent out rapidly
Exhibition is got up.Wherein, golden nanometer particle receives extensive concern and application because of its special physicochemical properties.Nanometer gold refers to directly
Footpath, at the super micro gold particle of 0.5-250nm, is generally dispersed in water.Nanometer gold colorimetric method is applied to by Mirkin etc. first
Biological active substances is analyzed, and utilizes nano-Au solution assembling and produced color change in disaggregation process, has developed naked eyes
The DNA biosensor of visual analyzing detection oligonucleotide hybridization.This Glasslessization detection side based on nanometer gold
Method, it has the remarkable advantages such as convenient, fast, highly sensitive, low cost, instrument be simple, thus is more suitable for field screening
With modern society's demand to analysis and detection technology.Up to now, nanometer gold is used for the visualization inspection of arteannuin and derivant thereof
Survey research have not been reported.
Arteannuin and derivant thereof contain peroxide bridge group, can generate high mars free radical with ferrous iron after can reacting, and destroy strand
The structure of DNA, and nanometer gold can be played Stabilization by single stranded DNA.The present invention studies based on these, by arteannuin and
Derivant generates free radical with ferrous reaction, destroys single stranded DNA so that it is loses the protective effect to nanometer gold, makes scattered receiving
Rice gold is assembled, and color is become bluish violet from claret, reaches the purpose of open hole detection arteannuin and derivant thereof.Nano-Au solution
The change of color is corresponding to the change of absorbance, and nanometer gold is assembled makes characteristic absorption peak red shift, marks concentration with absorbance ratio
Directrix curve, thus set up a kind of based on nanometer gold colorimetric detection arteannuin and the analysis method of derivant.
Summary of the invention:
It is an object of the invention to overcome instantly that complex operation is time-consuming present in arteannuin and derivant detection process thereof, cost is held high
The problems such as expensive, dependence large-scale instrument and equipment, it is provided that a kind of novel colorimetric detection method based on nanometer gold, make use of nanometer gold
Unique optical characteristics and the special construction of arteannuin, construct a simple and quick sensitive detection system.Nanometer gold colloid divides
Being claret during bulk state, in bluish violet after gathering, single stranded DNA can prevent nanometer gold from assembling in ferrous ions soln, from
And nanometer gold colloid is played a protective role.And arteannuin or derivatives thereof may result from by base under ferrous ion is catalyzed, destroy
Single stranded DNA structure so that it is lose protection to nanometer gold, and then make nanometer gold assemble variable color, it is achieved to arteannuin and spread out
Biological colorimetric detection.Described technical scheme is as follows:
Colorimetric detection arteannuin involved in the present invention and the analysis method of derivant thereof, it is characterised in that comprise the steps:
(1) in A, B centrifuge tube, single stranded DNA solution is added respectively；
(2) the B centrifuge tube solution obtained in step (1) adds the acetone soln containing arteannuin or derivatives thereof, A centrifuge tube
Add equivalent acetone soln；
(3) A, B centrifuge tube solution obtained in step (2) is separately added into ferrous ions soln mix homogeneously, reacts one section
Time, in the presence of arteannuin or derivatives thereof, reaction being produced free radical, thus destroy single stranded DNA, if not existing, not having
Free radical produces, and single stranded DNA structure keeps complete；
(4) A, B centrifuge tube solution obtained in step (3) is separately added into nano Au colloid liquid solution mix homogeneously, it was observed that B
In centrifuge tube, mixture color is become bluish violet from claret, i.e. indicates the existence of arteannuin or derivatives thereof；And A centrifuge tube
There is not arteannuin or derivatives thereof in mixture, do not have free radical to produce, nanometer gold colloid has obtained the strand of structural integrity
The protection of DNA, keeps the claret of dispersity；
(5) by ultraviolet spectrophotometer measure A, B centrifuge tube solution absorbance, when B centrifuge tube solution have arteannuin or
Absworption peak red shift in the presence of its derivant；
(6) standard solution of the arteannuin or derivatives thereof of preparation gradient concentration, sequentially determining absorbance after above-mentioned steps,
With absorbance ratio, concentration is drawn standard curve, utilize this standard curve to realize the content of arteannuin or derivatives thereof in sample
Measure.
Colorimetric detection arteannuin involved in the present invention and the analysis method of derivant thereof, it is characterised in that: described step (4) uses
Nanometer gold colloid be that reduction of sodium citrate method is prepared from, particle diameter 10-20nm, concentration is 35nM, and addition is 360 μ L.
Colorimetric detection arteannuin involved in the present invention and the analysis method of derivant thereof, it is characterised in that the reaction of described step (3)
Time range is 5-10min, preferably 8min.
Colorimetric detection arteannuin involved in the present invention and the analysis method of derivant thereof, it is characterised in that: described step (1) strand
DNA molecular, a length of 7bp, sequence is 5 '～CCAACCA～3 ', and concentration is 20 μMs, and detection usage amount is 5-10 μ L,
Preferably 7 μ L.
Colorimetric detection arteannuin involved in the present invention and the analysis method of derivant thereof, it is characterised in that: described step (2) adds
Acetone volume is 40 μ L.
Colorimetric detection arteannuin involved in the present invention and the analysis method of derivant thereof, it is characterised in that: described step (3) is ferrous
The concentration of ion is 1.0%-1.5%, preferably 1%, and detection usage amount is 16-22 μ L, preferably 18 μ L.
Colorimetric detection arteannuin involved in the present invention and the analysis method of derivant thereof, it is characterised in that: described step (5), (6)
Described wavelength is 610nm and 540nm；With the absorbance ratio (A610/A540) vertical coordinate as standard curve at two wavelength
Do figure.
Colorimetric detection arteannuin involved in the present invention and the analysis method of derivant thereof, it is characterised in that: to arteannuin and spread out
Biological detection is limited to 1 μM.
Colorimetric detection arteannuin involved in the present invention and the analysis method of derivant thereof, it is characterised in that: to detection arteannuin and
The detection range of linearity of its derivant is 3-40 μM.
The invention has the beneficial effects as follows: the colour developing of this colorimetric detection method is fast, easily operation, low cost, highly sensitive, for Huang from now on
The aspects such as the flower determination of Artemisia harvest date, genuine Artemisia annua are identified and screen, the detection of artemisinin derivative are provided convenience,
There are bigger clinical practice potentiality and agricultural production application potentiality.
Accompanying drawing illustrates:
The analysis method schematic diagram of Fig. 1, nanometer gold colorimetric detection arteannuin and derivant thereof；
Fig. 2, nanometer gold colorimetric detection arteannuin and the uv-visible absorption spectra figure of derivant thereof；
Fig. 3, nanometer gold colorimetric detection arteannuin and the canonical plotting of derivant thereof；
The specific detection result figure of Fig. 4, nanometer gold colorimetric detection arteannuin and derivant thereof；
Fig. 5, the chemical constitution of artemisinin derivative I and II.
Detailed description of the invention:
The present invention is explained further below in conjunction with instantiation.Should be understood that these examples are merely to illustrate the present invention rather than limit
The scope of the present invention processed.In addition, it is to be understood that after having read the content that the present invention lectures, those skilled in the art can be to this
Various change or amendment are made in invention, and these equivalent form of values fall within the application appended claims book limited range equally.
A kind of colorimetric detection arteannuin and the analysis method of derivant thereof, concrete steps as shown in Figure 1:
Implement 1: prepared by nanometer gold
By gold chloride (HAuCl4) it is configured to 0.01% aqueous solution, take 100mL and be heated to boiling, under agitation, accurately add 4mL
1% trisodium citrate (Na3C6H5O7·2H2O) solution, continues heated and boiled 10min.Observe flaxen aqueous solution of chloraurate
Quickly gray after sodium citrate adds, continue and change into black, be the most gradually stable into claret, overall process about 2-3min.
Continue stirring 15min after stopping heating, be settled to 100mL with distilled water after being cooled to room temperature, obtain a diameter of 16.7nm,
Concentration is the nano-Au solution of 35nM.
Enforcement 2: arteannuin and the qualitative analysis of derivant detection method thereof
(1) in A, B centrifuge tube, 20 μMs of single stranded DNA solution 7 μ L are added respectively；
(2) it is centrifugal that the B centrifuge tube solution obtained in step (1) adds arteannuin or derivatives thereof acetone soln 40 μ L to be measured, A
Pipe adds same amount of acetone soln；
(3) A, B centrifuge tube solution obtained in step (2) is separately added into 1% l ferrous ammonium sulfate solution 18 μ L, mix homogeneously,
Placing 8min, in the presence of arteannuin or derivatives thereof, reaction produces free radical, thus destroys single stranded DNA, if there is not green grass or young crops
Artemisin does not then have free radical to produce, and single stranded DNA structure keeps complete；
(4) A, B centrifuge tube solution obtained in step (3) is separately added into nanometer gold colloid 360 μ L, and mix homogeneously, observes
In B centrifuge tube, mixture color is become bluish violet from claret, i.e. indicates the existence of arteannuin and derivant thereof.And A from
There is not arteannuin in heart pipe mixture, do not have free radical to produce, nanometer gold colloid has obtained the single stranded DNA of structural integrity
Protection, keeps the claret of dispersity, ultraviolet spectrophotometer measurement result display solution to have obvious absorption peaks (figure at 540nm
2).When B centrifuge tube solution exists arteannuin and derivant thereof, the absorbance at 540nm reduces, and produces strong at 610nm
Absworption peak, be achieved in the qualitative analysis to arteannuin and derivant thereof.
Enforcement 3: arteannuin and the quantitative analysis of derivant detection method thereof
(1) in 8 centrifuge tubes, it is separately added into 20 μMs of single stranded DNA solution 7 μ L；
(2) in step (1) gained centrifuge tube, it is separately added into the arteannuin or derivatives thereof solution 40 μ L of variable concentrations；
(3) in step (2) gained centrifuge tube, it is separately added into 1% l ferrous ammonium sulfate solution 18 μ L, fully mixes, place 8min；
(4) in step (3) gained centrifuge tube, nano-Au solution 360 μ L, mix homogeneously it are separately added into；
(5) difference detecting step (4) gained solution absorbance.With the A610/A540 of standard solution as vertical coordinate, concentration is horizontal seat
Mark, draws standard curve, such as Fig. 3.A610/A540 value according to sample calculates sample concentration on standard curve, it is achieved
Quantitative analysis.
Enforcement 4: arteannuin and the specificity analyses of derivant detection method thereof
(1) in 5 centrifuge tubes, it is separately added into 20 μMs of single stranded DNA solution 7 μ L；
(2) in step (1) gained centrifuge tube, it is separately added into 40 μ L and contains arteannuin, artemisinin derivative (Fig. 5) and chaff interference (Portugal
Grape sugar, fructose) solution；
(3) in step (2) gained centrifuge tube, it is separately added into 1% l ferrous ammonium sulfate solution 18 μ L, fully mixes, place 8min.
(4) in step (3) gained centrifuge tube, nano-Au solution 360 μ L, mix homogeneously it are separately added into；
(5) difference detecting step (4) gained solution absorbance.As shown in Figure 4, this method only has height to arteannuin and derivant thereof
Sensitive response.
Compared with prior art, the invention have the characteristics that: reagent the most easily obtains, develop the color fast, highly sensitive, operation letter
Just, it is independent of large complicated instrument.
Claims (9)
1. a colorimetric detection arteannuin and the analysis method of derivant thereof, it is characterised in that comprise the steps:
(1) in A, B centrifuge tube, single stranded DNA solution is added respectively；
(2) the B centrifuge tube solution obtained in step (1) adds the acetone soln containing arteannuin or derivatives thereof, A centrifuge tube
Add equivalent acetone soln；
(3) A, B centrifuge tube solution obtained in step (2) is separately added into ferrous ions soln mix homogeneously, reacts one section
Time, in the presence of arteannuin or derivatives thereof, reaction being produced free radical, thus destroy single stranded DNA, if not existing, not having
Free radical produces, and single stranded DNA structure keeps complete；
(4) A, B centrifuge tube solution obtained in step (3) is separately added into nano Au colloid liquid solution mix homogeneously, it was observed that B
In centrifuge tube, mixture color is become bluish violet from claret, i.e. indicates the existence of arteannuin or derivatives thereof；And A centrifuge tube
There is not arteannuin or derivatives thereof in mixture, do not have free radical to produce, nanometer gold colloid has obtained the strand of structural integrity
The protection of DNA, keeps the claret of dispersity；
(5) by ultraviolet spectrophotometer measure A, B centrifuge tube solution absorbance, when B centrifuge tube solution have arteannuin or
Absworption peak red shift in the presence of its derivant；
(6) standard solution of the arteannuin or derivatives thereof of preparation gradient concentration, sequentially determining absorbance after above-mentioned steps,
With absorbance ratio, concentration is drawn standard curve, utilize this standard curve to realize the content of arteannuin or derivatives thereof in sample
Measure.
A kind of colorimetric detection arteannuin the most as claimed in claim 1 and the analysis method of derivant thereof, it is characterised in that: described
The nanometer gold colloid that step (4) uses is that reduction of sodium citrate method is prepared from, particle diameter 10-20nm, and concentration is 35nM, Herba Artemisiae Annuae
The detection usage amount of element or derivatives thereof is 360 μ L.
3. a kind of colorimetric detection arteannuin as described in claim 1 and 2 and the analysis method of derivant thereof, it is characterised in that institute
The reaction time range stating step (3) is 5-10min, preferably 8min.
A kind of colorimetric detection arteannuin the most as claimed in claim 1 and the analysis method of derivant thereof, it is characterised in that: described
Step (1) single strand dna, a length of 7bp, sequence is 5 '～CCAACCA～3 ', and concentration is 20 μMs, and detection makes
Consumption is 5-10 μ L, preferably 7 μ L.
A kind of colorimetric detection arteannuin the most as claimed in claim 1 and the analysis method of derivant thereof, it is characterised in that: described
It is 40 μ L that step (2) adds acetone volume.
A kind of colorimetric detection arteannuin the most as claimed in claim 1 and the analysis method of derivant thereof, it is characterised in that: described
The concentration of step (3) ferrous ion is 1.0%-1.5%, preferably 1%, and detection usage amount is 16-22 μ L, preferably 18 μ L.
A kind of colorimetric detection arteannuin the most as claimed in claim 1 and the analysis method of derivant thereof, it is characterised in that: described
Wavelength described in step (5), (6) is 610nm and 540nm；With absorbance ratio (A610/A540) at two wavelength as standard
The vertical coordinate of curve does figure.
A kind of colorimetric detection arteannuin the most as claimed in claim 1 and the analysis method of derivant thereof, it is characterised in that: to green grass or young crops
The detection of artemisin and derivant thereof is limited to 1 μM.
A kind of colorimetric detection arteannuin the most as claimed in claim 1 and the analysis method of derivant thereof, it is characterised in that: to inspection
The detection range of linearity surveying arteannuin and derivant thereof is 3-40 μM.
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Cited By (1)
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| CN112129721A (en) * | 2020-10-23 | 2020-12-25 | 湖南星辰生物科技股份有限公司 | Ultraviolet visible light rapid detection method for artemisinin |
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