CN109187935B - Method for detecting ancient silk fabric based on microwave - Google Patents
Method for detecting ancient silk fabric based on microwave Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 25
- 239000004744 fabric Substances 0.000 title claims abstract description 20
- 108010022355 Fibroins Proteins 0.000 claims abstract description 53
- 235000008708 Morus alba Nutrition 0.000 claims abstract description 31
- 240000000249 Morus alba Species 0.000 claims abstract description 31
- 239000002096 quantum dot Substances 0.000 claims abstract description 23
- 239000011324 bead Substances 0.000 claims abstract description 21
- 238000001514 detection method Methods 0.000 claims abstract description 20
- ZCCIPPOKBCJFDN-UHFFFAOYSA-N calcium nitrate Chemical compound [Ca+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ZCCIPPOKBCJFDN-UHFFFAOYSA-N 0.000 claims abstract description 18
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims abstract description 18
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims abstract description 9
- 235000019253 formic acid Nutrition 0.000 claims abstract description 9
- 239000000243 solution Substances 0.000 claims description 48
- 239000007853 buffer solution Substances 0.000 claims description 44
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 38
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 30
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 30
- 239000008367 deionised water Substances 0.000 claims description 25
- 229910021641 deionized water Inorganic materials 0.000 claims description 25
- 238000005406 washing Methods 0.000 claims description 25
- 102000004190 Enzymes Human genes 0.000 claims description 21
- 108090000790 Enzymes Proteins 0.000 claims description 21
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 20
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 20
- 239000007788 liquid Substances 0.000 claims description 18
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 15
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 15
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 15
- 238000003756 stirring Methods 0.000 claims description 15
- 238000011534 incubation Methods 0.000 claims description 14
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 10
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 10
- 229940098773 bovine serum albumin Drugs 0.000 claims description 10
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 10
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 10
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 10
- 239000013642 negative control Substances 0.000 claims description 10
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 10
- 239000000843 powder Substances 0.000 claims description 10
- 239000011780 sodium chloride Substances 0.000 claims description 10
- 238000005303 weighing Methods 0.000 claims description 10
- UHYPYGJEEGLRJD-UHFFFAOYSA-N cadmium(2+);selenium(2-) Chemical compound [Se-2].[Cd+2] UHYPYGJEEGLRJD-UHFFFAOYSA-N 0.000 claims description 9
- UQMZPFKLYHOJDL-UHFFFAOYSA-N zinc;cadmium(2+);disulfide Chemical class [S-2].[S-2].[Zn+2].[Cd+2] UQMZPFKLYHOJDL-UHFFFAOYSA-N 0.000 claims description 9
- 239000002244 precipitate Substances 0.000 claims description 8
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 5
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 5
- 239000007864 aqueous solution Substances 0.000 claims description 5
- 230000000903 blocking effect Effects 0.000 claims description 5
- 239000001913 cellulose Substances 0.000 claims description 5
- 229920002678 cellulose Polymers 0.000 claims description 5
- 229920001577 copolymer Polymers 0.000 claims description 5
- 238000000502 dialysis Methods 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 5
- 238000001704 evaporation Methods 0.000 claims description 5
- 230000005284 excitation Effects 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 5
- 238000004108 freeze drying Methods 0.000 claims description 5
- 238000000227 grinding Methods 0.000 claims description 5
- 230000007935 neutral effect Effects 0.000 claims description 5
- 229920003229 poly(methyl methacrylate) Polymers 0.000 claims description 5
- 239000004926 polymethyl methacrylate Substances 0.000 claims description 5
- 239000013641 positive control Substances 0.000 claims description 5
- 239000001103 potassium chloride Substances 0.000 claims description 5
- 235000011164 potassium chloride Nutrition 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 4
- 239000011148 porous material Substances 0.000 claims description 4
- 238000007789 sealing Methods 0.000 claims description 3
- 238000000265 homogenisation Methods 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 9
- 230000000694 effects Effects 0.000 abstract description 5
- 239000000872 buffer Substances 0.000 description 6
- 238000002965 ELISA Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000009777 vacuum freeze-drying Methods 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 230000003068 static effect Effects 0.000 description 3
- 239000005083 Zinc sulfide Substances 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000010355 oscillation Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000004753 textile Substances 0.000 description 2
- 229910052984 zinc sulfide Inorganic materials 0.000 description 2
- 108010013296 Sericins Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229920000620 organic polymer Polymers 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/36—Textiles
- G01N33/365—Filiform textiles, e.g. yarns
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
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Abstract
The invention relates to the field of cultural relic detection, and discloses a method for detecting ancient silk fabrics based on microwaves. According to the invention, firstly, a calcium nitrate and formic acid system is adopted to prepare the mulberry silk fibroin, then a quantum dot bead-labeled silk fibroin antibody is prepared, then microwave Elisa detection is carried out on the silk fibroin and silk fabric cultural relic sample, and whether the cultural relic sample contains mulberry silk or not can be judged through a fluorescence intensity value. The method has the characteristics of small sample consumption, rapidness, accuracy and high sensitivity, and has a good detection effect on the decayed silk fabrics.
Description
Technical Field
The invention relates to the field of cultural relic detection, in particular to a method for detecting ancient silk fabrics based on microwaves.
Background
Since ancient China, the fabric is a big textile, and the produced fabric is rich in variety, exquisite in process, comfortable and breathable. Among them, the most popular textile is silk of China, so China is also called "the state of silk". The silk mainly comprises mulberry silk and mainly comprises silk fibroin and sericin, wherein the silk fibroin is the main component of the silk and accounts for about 70% of the total weight. And the mulberry silk as an organic polymer material is easily degraded by the influence of light, heat, acid and alkali, microorganisms and the like, so that the structure and performance of crystallinity, molecular weight and the like are changed, and therefore, the conventional detection method has low sensitivity, is greatly influenced by impurity interference and is not suitable for detecting cultural relics, and therefore, a method for detecting silk fabrics with good sensitivity and strong specificity needs to be developed.
The microwave detection has the characteristics of high sensitivity, convenient operation and the like, and is widely applied to various fields. However, since the silk cultural relics to be detected by the invention are different from the conventional detection samples, the microwave detection method needs to be improved specially for the special object of the silk cultural relics.
Disclosure of Invention
In order to solve the technical problem, the invention provides a method for detecting ancient silk fabrics based on microwaves. The invention provides a plurality of improvements to the technology aiming at a special detection object of silk fabric cultural relics. The method for detecting the ancient silk fabric has the characteristics of intuition, accuracy and high sensitivity.
The specific technical scheme of the invention is as follows: a method for detecting ancient silk fabrics based on microwaves comprises the following steps of:
A) weighing 4-6g of mulberry silk, placing the mulberry silk in 180-220mL of 0.018-0.022M sodium carbonate solution, carrying out water bath for 55-65min at the temperature of 75-85 ℃, taking out, washing with deionized water for more than three times, and drying to obtain the fibroin.
B) Taking 1.8-2.2g of dried silk fibroin and 2.3-2.7g of calcium nitrate, adding 48-52mL of formic acid, stirring for 80-100min, filtering, adding sodium bicarbonate until the solution is neutral, dialyzing, freeze-drying, and grinding the obtained silk fibroin into silk fibroin powder for later use.
The invention uses calcium nitrate and formic acid system to dissolve the fibroin, which can not only increase the solubility of the fibroin, but also reduce the damage to the molecular chain of the fibroin, and the system can complete the dissolution of the fibroin at normal temperature without heating.
C) Weighing 18-22mg of cadmium selenide/zinc sulfide quantum dots, 118-122mg of polymethyl methacrylate and 78-82mg of polymaleic anhydride-octadecene copolymer, adding into 1.8-2.2ml of chloroform, mixing with 4.5-5.5ml of 3mg/ml sodium dodecyl sulfate aqueous solution, carrying out ultrasonic homogenization treatment, and evaporating chloroform; and then centrifugally purifying the obtained water-soluble quantum dot beads, and washing the water-soluble quantum dot beads for 2 to 4 times by using deionized water to obtain the quantum dot beads.
The quantum dot bead prepared by the invention comprises a plurality of cadmium selenide/zinc sulfide quantum dots, the luminous intensity is thousands of times of that of a single cadmium selenide/zinc sulfide quantum dot, the fluorescent signal amplification effect is realized in the detection process, and the detection sensitivity is increased.
D) Adding 0.9-1.1 mu g of 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide and 0.1-0.14mg of the water-soluble quantum dot beads obtained in the step C) into 2.5-3.1ml of PBS 7.4 buffer solution, dropwise adding 90-110 mu l of silk fibroin antibody diluted to 1000 times by 1wt% of bovine serum albumin while slowly stirring, placing for 35-45min at room temperature, centrifuging, taking out precipitate, re-suspending by 600 mu l of PBS 7.4 buffer solution, and then keeping for later use at 1-5 ℃.
E) Dissolving 0.02-0.2g of cultural relic sample in 100ml of CB 9.6 buffer solution, mixing and stirring uniformly, standing, adding 80-120 mu l of supernatant into an enzyme label plate a column, preparing the silk fibroin powder obtained in the step B) into 100 mu g/ml silk fibroin solution by using the CB 9.6 buffer solution, adding 80-120 mu l into enzyme label plates B and c columns, taking the B column as a positive control and the c column as a blank control, adding 80-120 mu l of PBS 7.4 buffer solution into the enzyme label plate d column as a negative control, placing the enzyme label plates in a microwave oven for low-level incubation for 2-4min, sucking out liquid in the pores, and washing by using the PBS 7.4 buffer solution.
F) Adding sealing liquid 180-.
G) Adding 80-120 mul of the solution obtained in the step D) into columns a, b and D of the enzyme label plate, adding 80-120 mul of the blocking solution into the column c, placing the column c in a microwave oven for incubation for 2-4min, sucking out liquid in the holes, and washing the liquid with PBS 7.4 buffer solution.
Compared with the traditional method, the invention uses microwave incubation to promote the antigen-antibody accelerated reaction through high-frequency oscillation, greatly shortens the experimental time and improves the repeatability of the detection result.
H) And (3) measuring the fluorescence value by using a fluorescence microplate reader, taking the OD mean value of the negative control plus 3 SD as cut-off value, and if the fluorescence mean value of the row a is larger than the cut-off value of the row d, judging that the text sample contains mulberry silk.
According to the invention, firstly, a calcium nitrate and formic acid system is adopted to prepare the mulberry silk fibroin, then a quantum dot bead-labeled silk fibroin antibody is prepared, then the silk fibroin and a silk fabric cultural relic sample are subjected to microwave detection, and whether the cultural relic sample contains mulberry silk or not can be judged through a fluorescence intensity value. The method has the characteristics of small sample consumption, rapidness, accuracy and high sensitivity, and has a good detection effect on the decayed silk fabrics.
Preferably, in the step A), the solution is dialyzed in deionized water for 2-3 days by a cellulose dialysis bag with the molecular weight cut-off of 8000-10000, the water is replaced every 5-7h, and the silk fibroin solution is subjected to vacuum freeze drying for 2-3 days.
Preferably, in step C), the centrifugation rate is 8000-12000rpm, and the centrifugation time is 8-12 min.
Preferably, in step E), the CB 9.6 buffer is prepared by: 1.5 g of sodium carbonate and 2.9 g of sodium bicarbonate are weighed and added into 800mL of deionized water to be uniformly stirred until the sodium carbonate and the sodium bicarbonate are completely dissolved, then the volume is adjusted to 1000mL by a volumetric flask, and the pH value of the solution is adjusted to 9.6.
Preferably, in step D) and step E), the PBS 7.4 buffer is prepared by: 0.2g of potassium chloride, 0.27 g of potassium dihydrogen phosphate, 8 g of sodium chloride and 1.42 g of disodium hydrogen phosphate are weighed and added into 800mL of deionized water to be uniformly stirred until the potassium dihydrogen phosphate, the sodium chloride and the disodium hydrogen phosphate are completely dissolved, then the volume is determined to be 1000mL by using a volumetric flask, and the pH value of the solution is adjusted to be 7.4.
Preferably, in step F), the blocking solution is 1wt% bovine serum albumin.
Preferably, in step E), step F), step G), washing is performed 3-5 times for 2-4min each time with PBS 7.4 buffer.
Preferably, in step H), when the fluorescence is detected by a fluorescence microplate reader, the excitation wavelength is 360nm, and the emission wavelength is 500 nm.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention can dissolve the fibroin at normal temperature by using a calcium nitrate and formic acid system, thereby not only increasing the solubility of the fibroin, but also reducing the damage to the molecular chain of the fibroin.
(2) The quantum dot bead prepared by the invention comprises a plurality of cadmium selenide/zinc sulfide quantum dots, the luminous intensity is thousands of times of that of a single cadmium selenide/zinc sulfide quantum dot, the fluorescent signal amplification effect is realized in the detection process, and the detection sensitivity is increased.
(3) The microwave used in the invention promotes the antigen-antibody accelerated reaction through high-frequency oscillation, greatly shortens the experimental time and improves the repeatability of the detection result.
(4) The method has the characteristics of small sample consumption, rapidness, accuracy and high sensitivity, and has a good detection effect on the decayed silk fabrics.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1:
A) weighing 5g of mulberry silk, placing the mulberry silk in 180mL of 0.018M sodium carbonate solution, carrying out water bath for 55min at the water bath temperature of 80 ℃, taking out, washing the mulberry silk with deionized water for more than three times, and placing the mulberry silk in an oven for drying.
B) Taking 2g of dried silk fibroin and 2.3g of calcium nitrate, adding 48mL of formic acid, magnetically stirring for 80min, filtering, adding sodium bicarbonate until the solution is neutral, dialyzing in deionized water for 2 days by using a cellulose dialysis bag with the molecular weight cutoff of 8000, changing water every 5h, freeze-drying the silk fibroin solution in a vacuum freeze-drying machine for 2 days, and grinding the obtained silk fibroin into powder for later use.
C) Weighing 20mg of cadmium selenide/zinc sulfide quantum dots, 118mg of polymethyl methacrylate and 78mg of polymaleic anhydride-octadecene copolymer, adding into 1.8ml of chloroform, mixing with 4.5ml of 3mg/ml sodium dodecyl sulfate aqueous solution, treating by using an ultrasonic homogenizer, and then evaporating the chloroform. And then, carrying out centrifugal purification on the obtained water-soluble quantum dot beads, wherein the centrifugal speed is 8000rpm, the time is 8min, and washing for 2 times by using deionized water to obtain the quantum dot beads.
D) Adding 0.9 mu g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and 0.1mg of the quantum dot beads in the step C) into 2.5 ml of PBS 7.4 buffer solution, then dropwise adding 90 mu l of silk fibroin antibody diluted to 1000 times by 1wt% of bovine serum albumin while slowly stirring, standing at room temperature for 35min, centrifuging, taking out the precipitate, re-suspending the precipitate by 600 mu l of PBS 7.4 buffer solution, and storing in a refrigerator at 4 ℃ for later use.
E) Dissolving 0.02g of cultural relic sample in 100ml of CB 9.6 buffer solution, mixing and stirring uniformly, standing, adding 80 mul of supernatant into an enzyme label plate a column, preparing 100 mul/ml of silk fibroin solution from the mulberry silk fibroin powder in the step B) by using the CB 9.6 buffer solution, adding 80 mul of fibroin solution into enzyme label plates B and c columns, taking the B column as a positive control and the c column as a blank control, adding 80 mul of PBS 7.4 buffer solution into the enzyme label plate d column as a negative control, placing the enzyme label plates in a microwave oven for low-grade incubation for 2min, sucking out liquid in the pores, washing for 3 times by using the PBS 7.4 buffer solution, and carrying out 2min each time.
Preparing a CB 9.6 buffer solution: 1.5 g of sodium carbonate and 2.9 g of sodium bicarbonate are weighed and added into 800mL of deionized water to be uniformly stirred until the sodium carbonate and the sodium bicarbonate are completely dissolved, then the volume is adjusted to 1000mL by a volumetric flask, and the pH value of the solution is adjusted to 9.6.
F) Adding 180 microliters of 1wt% bovine serum albumin into each well, placing the wells in a microwave oven for low-grade incubation for 2min, sucking out liquid in the wells, and washing the wells for 3 times with PBS 7.4 buffer solution for 2min each time.
G) And D), adding 80 mul of the solution obtained in the step D) into columns a, b and D of the enzyme label plates, adding 80 mul of the blocking solution into column c, placing the column c in a microwave oven for low-grade incubation for 2min, sucking out liquid in the holes, and washing for 3 times with PBS 7.4 buffer solution for 2min each time.
Preparation of PBS 7.4 buffer: 0.2g of potassium chloride, 0.27 g of potassium dihydrogen phosphate, 8 g of sodium chloride and 1.42 g of disodium hydrogen phosphate are weighed and added into 800mL of deionized water to be uniformly stirred until the potassium dihydrogen phosphate, the sodium chloride and the disodium hydrogen phosphate are completely dissolved, then the volume is determined to be 1000mL by using a volumetric flask, and the pH value of the solution is adjusted to be 7.4.
H) And (3) measuring the fluorescence value by using a fluorescence microplate reader, wherein the excitation wavelength is 360nm, the emission wavelength is 500nm, the OD mean value of the negative control plus 3 SD (static differential) is taken as the cut-off value, and if the fluorescence mean value of the column a is greater than the cut-off value of the column d, the sample is said to contain the mulberry silk.
Example 2:
A) weighing 5g of mulberry silk, placing the mulberry silk in 200mL of 0.02M sodium carbonate solution, carrying out water bath for 60min at the water bath temperature of 80 ℃, taking out, washing the mulberry silk with deionized water for more than three times, and placing the mulberry silk in an oven for drying.
B) Taking 2g of dried silk fibroin and 2.5g of calcium nitrate, adding 50mL of formic acid, magnetically stirring for 90min, filtering, adding sodium bicarbonate until the solution is neutral, dialyzing in deionized water for 2.5 days by using a cellulose dialysis bag with the molecular weight cutoff of 9000, changing water every 6h, freeze-drying the silk fibroin solution in a vacuum freeze-drying machine for 2.5 days, and grinding the obtained silk fibroin into powder for later use.
C) Weighing 20mg of cadmium selenide/zinc sulfide quantum dots, 120mg of polymethyl methacrylate and 80mg of polymaleic anhydride-octadecene copolymer, adding the weighed materials into 2ml of chloroform, mixing the materials with 5ml of 3mg/ml sodium dodecyl sulfate aqueous solution, treating the mixture by using an ultrasonic homogenizer, and then evaporating the chloroform. And then, the obtained water-soluble quantum dot beads are subjected to centrifugal purification, wherein the centrifugal speed is 10000rpm, and the time is 10 min. And washing with deionized water for 3 times to obtain the quantum dot beads.
D) Adding 1 mu g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and 0.12mg of the quantum dot beads in the step C) into 2..8ml of PBS 7.4 buffer solution, then dropwise adding 100 mu l of silk fibroin antibody diluted to 1000 times by 1wt% of bovine serum albumin while slowly stirring, standing for 40min at room temperature, centrifuging, taking out the precipitate, re-suspending the precipitate by 600 mu l of PBS 7.4 buffer solution, and storing the precipitate in a refrigerator at 4 ℃ for later use.
E) Dissolving 0.11g of cultural relic sample in 100ml of CB 9.6 buffer solution, mixing and stirring uniformly, standing, adding 100 mul of supernatant into an enzyme label plate a column, preparing 100 mul of fibroin solution from the mulberry silk fibroin powder in the step B) by using the CB 9.6 buffer solution, adding 100 mul of fibroin solution into enzyme label plates B and c columns, taking the B column as a positive control and the c column as a blank control, adding 100 mul of PBS 7.4 buffer solution into the enzyme label plate d column as a negative control, placing the enzyme label plates in a microwave oven for low-grade incubation for 3min, sucking out liquid in the pores, washing 4 times by using the PBS 7.4 buffer solution, and 3min each time.
Preparing a CB 9.6 buffer solution: 1.5 g of sodium carbonate and 2.9 g of sodium bicarbonate are weighed and added into 800mL of deionized water to be uniformly stirred until the sodium carbonate and the sodium bicarbonate are completely dissolved, then the volume is adjusted to 1000mL by a volumetric flask, and the pH value of the solution is adjusted to 9.6.
F) Adding 1wt% bovine serum albumin 200 μ l into each hole, placing in a microwave oven for low-grade incubation for 3min, sucking out liquid in the holes, and washing for 4 times with PBS 7.4 buffer solution for 3min each time.
G) And D) adding 100 mul of the solution obtained in the step D) into columns a, b and D of the enzyme label plate, adding 100 mul of the sealing solution into column c, placing the column c in a microwave oven for 3min of low-grade incubation, sucking out liquid in the holes, and washing the holes for 4 times for 3min each time by using PBS 7.4 buffer solution.
Preparation of PBS 7.4 buffer: 0.2g of potassium chloride, 0.27 g of potassium dihydrogen phosphate, 8 g of sodium chloride and 1.42 g of disodium hydrogen phosphate are weighed and added into 800mL of deionized water to be uniformly stirred until the potassium dihydrogen phosphate, the sodium chloride and the disodium hydrogen phosphate are completely dissolved, then the volume is determined to be 1000mL by using a volumetric flask, and the pH value of the solution is adjusted to be 7.4.
H) And (3) measuring the fluorescence value by using a fluorescence microplate reader, wherein the excitation wavelength is 360nm, the emission wavelength is 500nm, the OD mean value of the negative control plus 3 SD (static differential) is taken as the cut-off value, and if the fluorescence mean value of the column a is greater than the cut-off value of the column d, the sample is said to contain the mulberry silk.
Example 3:
A) weighing 5g of mulberry silk, placing the mulberry silk in 220mL of 0.022M sodium carbonate solution, carrying out water bath for 65min at the water bath temperature of 80 ℃, taking out, washing the mulberry silk with deionized water for more than three times, and placing the mulberry silk in an oven for drying;
B) taking 2g of dried silk fibroin and 2.7g of calcium nitrate, adding 52mL of formic acid, magnetically stirring for 100min, filtering, adding sodium bicarbonate until the solution is neutral, dialyzing in deionized water for 3 days by using a cellulose dialysis bag with the molecular weight cutoff of 10000, changing water every 7h, freeze-drying the silk fibroin solution in a vacuum freeze-drying machine for 3 days, and grinding the obtained silk fibroin into powder for later use;
C) weighing 20mg of cadmium selenide/zinc sulfide quantum dots, 122mg of polymethyl methacrylate and 82mg of polymaleic anhydride-octadecene copolymer, adding the weighed materials into 2.2ml of chloroform, mixing the materials with 5.5ml of 3mg/ml sodium dodecyl sulfate aqueous solution, treating the mixture by using an ultrasonic homogenizer, and then evaporating the chloroform. And then, carrying out centrifugal purification on the obtained water-soluble quantum dot beads, wherein the centrifugal speed is 12000rpm, and the centrifugal time is 12 min. Washing with deionized water for 4 times to obtain quantum dot beads;
D) adding 1.1 microgram of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and 0.1-0.14mg of quantum dot beads in the step C) into 3.1ml of PBS 7.4 buffer solution, then dropwise adding 110 microlitres of silk fibroin antibody diluted to 1000 times by 1wt% of bovine serum albumin while slowly stirring, standing for 45min at room temperature, centrifuging, taking out precipitate, re-suspending by 600 microlitres of PBS 7.4 buffer solution, and storing in a refrigerator at 4 ℃ for later use;
E) dissolving 0.2g of cultural relic sample in 100ml of CB 9.6 buffer solution, mixing and stirring uniformly, standing, adding 120 mul of supernatant into an a column of an ELISA plate, preparing 100 mul/ml of silk fibroin solution from the mulberry silk fibroin powder in the step B) by using the CB 9.6 buffer solution, adding 120 mul of fibroin solution into B and c columns of the ELISA plate, taking the B column as a positive control and the c column as a blank control, adding 120 mul of PBS 7.4 buffer solution into d column of the ELISA plate as a negative control, placing the ELISA plate in a microwave oven for low-grade incubation for 4min, sucking out liquid in the hole, washing for 5 times by using the PBS 7.4 buffer solution, and carrying out 4min each time;
preparing a CB 9.6 buffer solution: 1.5 g of sodium carbonate and 2.9 g of sodium bicarbonate are weighed and added into 800mL of deionized water to be uniformly stirred until the sodium carbonate and the sodium bicarbonate are completely dissolved, then the volume is adjusted to 1000mL by a volumetric flask, and the pH value of the solution is adjusted to 9.6.
F) Adding 220 mul of 1wt% bovine serum albumin into each hole, placing the holes in a microwave oven for low-grade incubation for 4min, sucking out liquid in the holes, and washing the holes for 5 times with PBS 7.4 buffer solution for 4min each time;
G) adding 120 mul of the solution obtained in the step D) into columns a, b and D of the enzyme label plate, adding 120 mul of the confining liquid into column c, placing in a microwave oven for low-grade incubation for 4min, sucking out liquid in the holes, and washing for 5 times with PBS 7.4 buffer solution for 4min each time;
preparation of PBS 7.4 buffer: 0.2g of potassium chloride, 0.27 g of potassium dihydrogen phosphate, 8 g of sodium chloride and 1.42 g of disodium hydrogen phosphate are weighed and added into 800mL of deionized water to be uniformly stirred until the potassium dihydrogen phosphate, the sodium chloride and the disodium hydrogen phosphate are completely dissolved, then the volume is determined to be 1000mL by using a volumetric flask, and the pH value of the solution is adjusted to be 7.4.
H) And (3) measuring the fluorescence value by using a fluorescence microplate reader, wherein the excitation wavelength is 360nm, the emission wavelength is 500nm, the OD mean value of the negative control plus 3 SD (static differential) is taken as the cut-off value, and if the fluorescence mean value of the column a is greater than the cut-off value of the column d, the sample is said to contain the mulberry silk.
The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.
Claims (8)
1. A method for detecting ancient silk fabrics based on microwaves is characterized by comprising the following steps:
A) weighing 4-6g of mulberry silk, placing the mulberry silk in 180-220mL of 0.018-0.022M sodium carbonate solution, carrying out water bath for 55-65min at the temperature of 75-85 ℃, taking out, washing the mulberry silk with deionized water for more than three times, and drying to obtain fibroin;
B) taking 1.8-2.2g of dried silk fibroin and 2.3-2.7g of calcium nitrate, adding 48-52mL of formic acid, stirring for 80-100min, filtering, adding sodium bicarbonate until the solution is neutral, dialyzing, freeze-drying, and grinding the obtained silk fibroin into silk fibroin powder for later use;
C) weighing 18-22mg of cadmium selenide/zinc sulfide quantum dots, 118-122mg of polymethyl methacrylate and 78-82mg of polymaleic anhydride-octadecene copolymer, adding into 1.8-2.2ml of chloroform, mixing with 4.5-5.5ml of 3mg/ml sodium dodecyl sulfate aqueous solution, carrying out ultrasonic homogenization treatment, and evaporating chloroform; then centrifugally purifying the obtained water-soluble quantum dot beads, and washing the water-soluble quantum dot beads for 2 to 4 times by using deionized water to obtain the quantum dot beads;
D) adding 0.9-1.1 mu g of 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide and 0.1-0.14mg of the water-soluble quantum dot beads obtained in the step C) into 2.5-3.1ml of PBS 7.4 buffer solution, dropwise adding 90-110 mu l of silk fibroin antibody diluted to 1000 times by 1wt% of bovine serum albumin while slowly stirring, placing for 35-45min at room temperature, centrifuging, taking out precipitate, re-suspending by 600 mu l of PBS 7.4 buffer solution, and then keeping for later use at 1-5 ℃;
E) dissolving 0.02-0.2g of cultural relic sample in 100ml of CB 9.6 buffer solution, mixing and stirring uniformly, standing, adding 80-120 mu l of supernatant into an enzyme label plate a column, preparing the silk fibroin powder obtained in the step B) into 100 mu g/ml silk fibroin solution by using the CB 9.6 buffer solution, adding 80-120 mu l of silk fibroin solution into an enzyme label plate B column and a c column, taking the B column as a positive control and the c column as a blank control, adding 80-120 mu l of PBS 7.4 buffer solution into an enzyme label plate d column as a negative control, placing the enzyme label plate in a microwave oven for low-level incubation for 2-4min, sucking out liquid in the pores, and washing by using the PBS 7.4 buffer solution;
F) adding sealing liquid 180-;
G) adding 80-120 mul of the solution obtained in the step D) into columns a, b and D of the enzyme label plate, adding 80-120 mul of the blocking solution into the column c, placing the column c in a microwave oven for incubation for 2-4min, sucking out liquid in the holes, and washing the liquid with PBS 7.4 buffer solution;
H) and (3) measuring the fluorescence value by using a fluorescence microplate reader, taking the OD mean value of the negative control plus 3 SD as cut-off value, and if the fluorescence mean value of the row a is larger than the cut-off value of the row d, judging that the text sample contains mulberry silk.
2. The method as claimed in claim 1, wherein in step B), the solution is dialyzed in deionized water for 2-3 days by using a cellulose dialysis bag with molecular weight cut-off of 8000-10000, and the water is changed every 5-7h, and the silk fibroin solution is vacuum freeze-dried for 2-3 days.
3. The method as claimed in claim 1, wherein the centrifugation rate is 8000-12000rpm and the centrifugation time is 8-12min in step C).
4. The method for detecting ancient silk fabrics based on microwaves as claimed in claim 1, wherein in the step E), the CB 9.6 buffer solution is prepared by the following steps: 1.5 g of sodium carbonate and 2.9 g of sodium bicarbonate are weighed and added into 800mL of deionized water to be uniformly stirred until the sodium carbonate and the sodium bicarbonate are completely dissolved, then the volume is adjusted to 1000mL by a volumetric flask, and the pH value of the solution is adjusted to 9.6.
5. The method for detecting ancient silk fabrics based on microwaves as claimed in claim 1, wherein in the steps D) and E), the PBS 7.4 buffer solution is prepared by the following method: 0.2g of potassium chloride, 0.27 g of potassium dihydrogen phosphate, 8 g of sodium chloride and 1.42 g of disodium hydrogen phosphate are weighed and added into 800mL of deionized water to be uniformly stirred until the potassium dihydrogen phosphate, the sodium chloride and the disodium hydrogen phosphate are completely dissolved, then the volume is determined to be 1000mL by using a volumetric flask, and the pH value of the solution is adjusted to be 7.4.
6. The method for microwave-based detection of ancient silk according to claim 1, wherein in step F), the blocking solution is 1wt% bovine serum albumin.
7. The method for microwave-based detection of ancient silk according to claim 1, wherein in step E), step F), step G), washing with PBS 7.4 buffer solution is performed 3-5 times for 2-4min each time.
8. The method for detecting the ancient silk fabrics based on the microwaves as claimed in claim 1, wherein in the step H), when the fluorescence is detected by a fluorescence microplate reader, the excitation wavelength is 360nm, and the emission wavelength is 500 nm.
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