Method for detecting ancient silk fabrics based on multi-group modified magnetic beads and fluorescent quantum dots

Abstract

The application relates to the field of archaeology, and discloses a method for detecting ancient silk fabrics based on multi-group modified magnetic beads and fluorescent quantum dots. The carboxyl and amino groups are used to modify the magnetic beads to purify and enrich the silk fibroin in the ancient silk fabrics, the streptavidinized fluorescent quantum dots are combined with the biotinylated silk fibroin antibodies, the carboxyl and amino group modified magnetic bead-silk fibroin complex is added to the fluorescent quantum dot complex reaction combination, the sample to be detected is irradiated with laser, the fluorescent quantum dots marked on the sample emit light, and if no light is emitted, it is indicated that there is no silk fibroin. The multi-group modified magnetic beads are used to improve the extraction amount of the silk fibroin, the silk fibroin has good purification effect, the fluorescent quantum dots are irradiated with laser, the light sensitive element is used to detect the light signal, the detection method is sensitive and specific, and the operation is simple, fast and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of archaeology, in particular to a method for detecting ancient silk fabrics based on multi-group modified magnetic beads and fluorescent quantum dots. BACKGROUND

[0002] Silk is a symbol of ancient Chinese civilization, which not only carries brilliant Chinese culture, but also has a profound impact on the world. It is generally believed that the origin of silk can be traced back to the Yangshao Culture period in the middle reaches of the Yellow River (about 5000 BC-3000 BC), but there is still a lack of reliable evidence.

[0003] Detecting silkworm proteins in ancient silk fabrics can be used to determine the origin of silk. Silkworm silk mainly consists of fibroin and sericin, and during the process of making silk, silk reeling, spinning, weaving or obtaining exquisite and comfortable silk, the sericin is usually removed, leaving only the main component fibroin. Most silk fabrics are buried underground for a long time and are often affected by water, temperature, corrosive substances and microorganisms in the soil, becoming dilapidated and gradually losing their original silk structure, and even some are degraded into small molecular polypeptides remaining in the soil, which are difficult to identify using traditional analysis methods such as infrared spectroscopy, Raman spectroscopy and X-ray. Therefore, finding a method with high specificity, good stability, fast detection speed and high fibroin purification is crucial for studying the origin and degradation mechanism of silk. SUMMARY

[0004] To solve the above technical problems, the present application provides a method for detecting ancient silk fabrics based on multi-group modified magnetic beads and fluorescent quantum dots. The present application uses multi-group modified magnetic beads to improve the extraction amount of fibroin, and the fibroin purification effect is good. The fluorescent quantum dots are irradiated by laser, and the light sensitive element detection light signal detection method is sensitive, specific, simple to operate and fast.

[0005] The specific technical scheme of the present application is as follows: a method for detecting ancient silk fabrics based on multi-group modified magnetic beads and fluorescent quantum dots, comprising the following steps:

[0006] 1) Take 40-50 mg of ancient silk fabrics, crush them, and dissolve and mix them with 5-6 mL of a mixture of calcium chloride, water and ethanol, to obtain a dissolved solution.

[0007] 2) Place the dissolved solution in a 55-70℃ water bath for 0.5-1.5 hours, and centrifuge it.

[0008] 3) Absorb the supernatant into a dialysis membrane with a molecular weight cutoff of 800-1200 Da.

[0009] 4) Take the dialysis solution, centrifuge and freeze dry it at -50℃ to -87℃ and 2-8Pa to obtain silk fibroin.

[0010] 5) Take silk fibroin, add lysis buffer to lyse, mix evenly to obtain lysis buffer, and refrigerate for later use.

[0011] 6) Add the buffer solution containing carboxyl and amino co-modified magnetic beads to the lysis buffer, mix well, and allow to stand for adsorption to obtain a dispersion containing silk fibroin-magnetic bead complex. Store in the refrigerator for later use.

[0012] 7) The precipitate was separated under an external magnetic field, washed with ethanol solution, and the silk fibroin-magnetic bead complex was obtained and stored for later use.

[0013] 8) Add a streptavidin-labeled fluorescent quantum dot solution to a centrifuge tube, then add biotinylated silk fibroin antibody, shake and incubate to obtain a dispersion of fluorescent quantum dot-labeled biotinylated silk fibroin antibody complex.

[0014] 9) The silk fibroin-magnetic bead complex was added to the dispersion of biotinylated silk fibroin antibody complex labeled with fluorescent quantum dots for reaction. After centrifugation and washing, the mixture was dispersed in phosphate buffer solution to obtain the test sample. The test sample was irradiated with a laser, and the presence or absence of fluorescence was used to determine whether the test sample contained silk fibroin.

[0015] The method of this invention utilizes carboxyl and amino co-modified magnetic beads to purify and enrich silk fibroin in ancient silk fabrics. Streptavidin-modified fluorescent quantum dots bind to biotinylated silk fibroin antibodies. The carboxyl and amino co-modified magnetic bead-silk fibroin complex is added to the fluorescent quantum dot complex for reaction and binding. The sample to be tested is irradiated with a laser to make the fluorescent quantum dots labeled on the sample glow. If no glow is emitted, it indicates that there is no silk fibroin.

[0016] Preferably, in step 1), the molar ratio of calcium chloride, water and ethanol is 1:(7-9):(1.5-2.5), the dissolution temperature is 96-99℃, and the dissolution time is 1-2 hours.

[0017] Preferably, in step 2), the centrifugation speed is 10,000-12,000 rpm and the centrifugation time is 5-10 minutes.

[0018] Preferably, in step 3), the water is changed every 4-6 hours during dialysis, and dialysis is performed for 24-48 hours to remove calcium and chloride ions from the solution.

[0019] Preferably, in step 4), the freeze-drying time is 20-40 hours.

[0020] Preferably, in step 5), the lysis buffer is a Tris-HCl solution containing 3-5 wt% SDC and pH=8.0, and the concentration of silk fibroin is 1-1.5 mg / mL.

[0021] Preferably, in step 6), the adsorption time is 2-5 minutes.

[0022] Preferably, in step 7), the concentration of the ethanol solution is 60-80 wt%.

[0023] Preferably, in step 8), the temperature for shaking incubation is 20-50℃ and the time is 10-20 minutes.

[0024] Preferably, in step 9), the reaction time is 10-30 minutes and the pH of the phosphate buffer is 5-8.

[0025] Compared with the prior art, the present invention has the following technical effects:

[0026] (1) In the extraction process, the present invention uses magnetic beads co-modified with carboxyl and amino groups to separate the silk fibroin in ancient silk fabrics from other substances by applying an external magnetic field, thereby increasing the concentration of silk fibroin and without introducing any new impurities. The magnetic beads are easy to clean, and the entire process can be carried out in a single centrifuge tube. The process is simple and does not cause any loss of protein.

[0027] (2) Fluorescent quantum dot technology has the characteristics of wide absorption spectrum, narrow emission spectrum (full width at half maximum is about 25-35nm), good photostability, strong resistance to photobleaching and strong resistance to chemical quenching. The binding of quantum dots with biotinylated silk fibroin antibodies and magnetic beads-silk fibroin reaction has high specificity.

[0028] (3) The present invention uses laser to irradiate the sample to be tested, so that the fluorescent quantum dots bound to the label fluoresce, and then uses a photosensitive element to detect the light signal. It has good stability, simple judgment, and can intuitively observe the experimental results. Moreover, compared with the traditional detection method, the detection time is shorter, which can save a lot of time and manpower. Detailed Implementation

[0029] The present invention will be further described below with reference to embodiments.

[0030] Example 1

[0031] 1) Take 45mg of ancient silk fabric, crush the ancient silk fabric with a mortar and pestle, dissolve it in 5.5mL of calcium chloride, water and ethanol (molar ratio of 1:8:2) at 98℃ for 1.5 hours, and shake the resulting mixture well.

[0032] 2) Place the mixture obtained in 1) in a 65℃ water bath for 1 hour, and centrifuge for 6 minutes at a speed of 11000 rpm;

[0033] 3) Remove the centrifuge tube from the centrifuge and use a pipette to transfer 2 mL of the supernatant into a dialysis membrane with a molecular weight cutoff of 1 kD for dialysis. Change the water every 5 hours during dialysis and dialyze for 36 hours to remove calcium and chloride ions from the solution.

[0034] 4) Take 2 mL of the dialyzed solution and place it in a centrifuge tube. Place the centrifuge tube in a freeze dryer to concentrate the solution. The temperature of the freeze dryer is -65℃, the system vacuum is 5 Pa, and the time is 30 hours to obtain silk fibroin from silk fabrics.

[0035] 5) Take the silk fibroin from step 4) into a centrifuge tube, add 1.5 mL of lysis buffer (containing 4 wt% SDC, pH=8.0 Tris-HCl solution) to make the concentration of silk fibroin 1.25 mg / mL, lyse the silk fibroin, mix well, and store in a refrigerator at 4°C for later use.

[0036] 6) Add the carboxyl and amino modified magnetic bead buffer to the mixture in step 5), mix well, adsorb for 4 minutes, and store in a refrigerator at 4°C for later use.

[0037] 7) Apply an external magnetic field to the centrifuge tube, remove the liquid from the centrifuge tube with a pipette, add 70wt% ethanol solution to the centrifuge tube in step 6), shake gently to clean, repeat 3 times, and obtain the complex of magnetic beads and silk fibroin in ancient silk fabrics, and store in a refrigerator at 4°C for later use.

[0038] 8) Take a centrifuge tube and add a solution of fluorescent quantum dots containing streptavidin, then add 5 μL of biotinylated silk fibroin antibody to the centrifuge tube, shake and incubate at 35°C for 15 minutes to obtain a solution of fluorescent quantum dot-labeled biotinylated silk fibroin antibody complex.

[0039] 9) Add the magnetic bead complex from the centrifuge tube in step 7) to the biotinylated silk fibroin antibody complex solution labeled with fluorescent quantum dots in step 8), and react on a rotary mixer for 20 minutes. After the reaction, centrifuge and wash, repeat the washing 3 times, and then disperse in a phosphate buffer solution with pH=6.5. Then irradiate the sample to be tested with a laser to make the fluorescent quantum dots labeled on the antibody fluoresce. This can be used to determine whether the extracted sample contains silk fibroin.

[0040] Example 2

[0041] 1) Take 40mg of ancient silk fabric, crush the ancient silk fabric with a mortar and pestle, dissolve it in 5mL of calcium chloride, water and ethanol (molar ratio of 1:8:2) at 98℃ for 1 hour, and shake the resulting mixture well.

[0042] 2) Place the mixture obtained in 1) in a 55℃ water bath for 1 hour, and centrifuge for 5 minutes at a speed of 10000 rpm;

[0043] 3) Remove the centrifuge tube from the centrifuge and use a pipette to transfer 2 mL of the supernatant into a dialysis membrane with a molecular weight cutoff of 1 kD for dialysis. Change the water every 4 hours during dialysis and dialysis for 24 hours to remove calcium and chloride ions from the solution.

[0044] 4) Take 2 mL of the dialyzed solution and place it in a centrifuge tube. Place the centrifuge tube in a freeze dryer to concentrate the solution. The temperature of the freeze dryer is -50℃, the system vacuum is 2 Pa, and the time is 40 hours to obtain the silk fibroin in the silk fabric.

[0045] 5) Take the silk fibroin from step 4) into a centrifuge tube, add 1 mL of lysis buffer (containing 3 wt% SDC, pH=8.0 Tris-HCl solution) to make the concentration of silk fibroin 1 mg / mL, lyse the silk fibroin, mix well, and store in a refrigerator at 4℃ for later use.

[0046] 6) Add the carboxyl and amino modified magnetic bead buffer to the mixture in step 5), mix well, adsorb for 2 minutes, and store in a refrigerator at 4°C for later use.

[0047] 7) Apply an external magnetic field to the centrifuge tube, remove the liquid from the centrifuge tube with a pipette, add 60wt% ethanol solution to the centrifuge tube in step 6), shake gently to clean, repeat 3 times, and obtain the complex of magnetic beads and silk fibroin in ancient silk fabrics, and store in a refrigerator at 4°C for later use.

[0048] 8) Take a centrifuge tube and add a solution of fluorescent quantum dots containing streptavidin, then add 5 μL of biotinylated silk fibroin antibody to the centrifuge tube, shake and incubate at 20°C for 20 minutes to obtain a solution of fluorescent quantum dot-labeled biotinylated silk fibroin antibody complex.

[0049] 9) Add the magnetic bead complex from the centrifuge tube in step 7) to the biotinylated silk fibroin antibody complex solution labeled with fluorescent quantum dots in step 8), place it on a rotary mixer and react for 10 minutes. After the reaction, centrifuge and wash, repeat the washing 3 times, and then disperse it in a phosphate buffer solution with pH=7.0. Then irradiate the sample to be tested with a laser to make the fluorescent quantum dots labeled on the antibody fluoresce. This can be used to determine whether the extracted sample contains silk fibroin.

[0050] Example 3

[0051] 1) Take 50mg of ancient silk fabric, crush the ancient silk fabric with a mortar and pestle, dissolve it in 6mL of calcium chloride, water and ethanol (molar ratio of 1:8:2) at 98℃ for 2 hours, and shake the resulting mixture well.

[0052] 2) Place the mixture obtained in 1) in a 70℃ water bath for 1 hour, and centrifuge for 10 minutes at a speed of 12000 rpm;

[0053] 3) Remove the centrifuge tube from the centrifuge and use a pipette to transfer 2 mL of the supernatant into a dialysis membrane with a molecular weight cutoff of 1 kD for dialysis. Change the water every 6 hours during dialysis and dialyze for 48 hours to remove calcium and chloride ions from the solution.

[0054] 4) Take 2 mL of the dialyzed solution and place it in a centrifuge tube. Place the centrifuge tube in a freeze dryer to concentrate the solution. The temperature of the freeze dryer is -87℃, the system vacuum is 8 Pa, and the time is 20 hours to obtain the silk fibroin in the silk fabric.

[0055] 5) Take the silk fibroin from step 4) into a centrifuge tube, add 2 mL of lysis buffer (containing 5 wt% SDC, pH=8.0 Tris-HCl solution) to make the concentration of silk fibroin 1.5 mg / mL, lyse the silk fibroin, mix well, and store in a refrigerator at 4°C for later use.

[0056] 6) Add the carboxyl and amino modified magnetic bead buffer to the mixture in step 5), mix well, adsorb for 5 minutes, and store in a refrigerator at 4°C for later use.

[0057] 7) Apply an external magnetic field to the centrifuge tube, remove the liquid from the centrifuge tube with a pipette, add 80wt% ethanol solution to the centrifuge tube in step 6), shake gently to clean, repeat 3 times, and obtain the complex of magnetic beads and silk fibroin in ancient silk fabrics, and store in a refrigerator at 4°C for later use.

[0058] 8) Take a centrifuge tube and add a solution of fluorescent quantum dots containing streptavidin, then add 5 μL of biotinylated silk fibroin antibody to the centrifuge tube, shake and incubate at 50°C for 10 minutes to obtain a solution of fluorescent quantum dot-labeled biotinylated silk fibroin antibody complex.

[0059] 9) Add the magnetic bead complex from the centrifuge tube in step 7) to the biotinylated silk fibroin antibody complex solution labeled with fluorescent quantum dots in step 8), and react on a rotary mixer for 30 minutes. After the reaction, centrifuge and wash, repeat the washing 3 times, and then disperse in a phosphate buffer solution with pH=6.5. Then irradiate the sample to be tested with a laser to make the fluorescent quantum dots labeled on the antibody fluoresce. This can be used to determine whether the extracted sample contains silk fibroin.

[0060] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0061] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for detecting ancient silk fabrics based on multi-functional modified magnetic beads and fluorescent quantum dots, characterized in that: Includes the following steps: 1) Take 40-50mg of ancient silk fabric, crush it, dissolve it in a mixture of 5-6mL of calcium chloride, water and ethanol, and mix and shake well to obtain a solution; 2) Place the solution in a water bath at 55-70℃ for 0.5-1.5 hours, then centrifuge. 3) Transfer the supernatant to a dialysis membrane with a molecular weight cutoff of 800-1200 Da for dialysis; 4) Take the dialysis solution, centrifuge and freeze-dry it at -50℃ to -87℃ and 2-8Pa to obtain silk fibroin; 5) Take silk fibroin, add lysis buffer to lyse, mix well to obtain lysis buffer, and refrigerate for later use; 6) Add the buffer solution containing carboxyl and amino co-modified magnetic beads to the lysis buffer, mix well, and allow to stand for adsorption to obtain a dispersion containing silk fibroin-magnetic bead complex. Store in the refrigerator for later use. 7) The precipitate was separated under an external magnetic field, washed with ethanol solution, and the silk fibroin-magnetic bead complex was obtained and stored for later use. 8) Add a streptavidin-labeled fluorescent quantum dot solution to a centrifuge tube, then add biotinylated silk fibroin antibody, and incubate with shaking at 20-50℃ for 10-20 minutes to obtain a dispersion of fluorescent quantum dot-labeled biotinylated silk fibroin antibody complex. 9) The silk fibroin-magnetic bead complex was added to the dispersion of biotinylated silk fibroin antibody complex labeled with fluorescent quantum dots for reaction. After centrifugation and washing, the mixture was dispersed in phosphate buffer solution to obtain the test sample. The test sample was irradiated with a laser, and the presence or absence of fluorescence was used to determine whether the test sample contained silk fibroin.

2. The method as described in claim 1, characterized in that: In step 1), the molar ratio of calcium chloride, water and ethanol is 1:(7-9):(1.5-2.5), the dissolution temperature is 96-99℃, and the dissolution time is 1-2 hours.

3. The method as described in claim 1, characterized in that: In step 2), the centrifugation speed is 10,000-12,000 rpm and the centrifugation time is 5-10 minutes.

4. The method as described in claim 1, characterized in that: In step 3), the water is changed every 4-6 hours during dialysis, and dialysis is performed for 24-48 hours to remove calcium and chloride ions from the solution.

5. The method as described in claim 1, characterized in that: In step 4), the freeze-drying time is 20-40 hours.

6. The method as described in claim 1, characterized in that: In step 5), the lysis buffer is a Tris-HCl solution containing 3-5 wt% SDC and pH=8.

0.

7. The method as described in claim 1, characterized in that: In step 6), the adsorption time is 2-5 minutes.

8. The method as described in claim 1, characterized in that: In step 7), the concentration of the ethanol solution is 60-80 wt%.

9. The method as described in claim 1, characterized in that: In step 9), the reaction time is 10-30 minutes, and the pH of the phosphate buffer is 5-8.