CN109540994B - A high-sensitivity detection method for cowhide cultural relics - Google Patents

Abstract

The invention relates to the field of cultural relic detection, and discloses a high-sensitivity detection method for cowhide cultural relics. The method of the invention first prepares pure bovine collagen powder, and simultaneously extracts bovine collagen in cultural relic samples; The layer-by-layer self-assembly method sequentially modifies gold nanoparticles, polyacrylic acid-dopamine complexes with hollow structures, and polymethyl methacrylate-maleic anhydride/1-octadecene alternating copolymers on the surface of cleaned glassy carbon electrodes. Spherical nanobeads, rabbit anti-bovine collagen polyclonal antibody; the modified electrodes are used to detect bovine collagen solution and cultural relic-like solution respectively. The method disclosed by the invention has high sensitivity, low detection limit and good reproducibility, and can carry out ultra-sensitive detection on cowhide cultural relics.

Description

A high-sensitivity detection method for cowhide cultural relics

technical field

The invention relates to the field of cultural relic detection, in particular to a high-sensitivity detection method for leather cultural relics.

Background technique

Humans have a very long history of using leather products. As early as ancient times, our ancestors began to use leather made of various animal skins. In thousands of years of historical precipitation, there are a large number of leather cultural relics left in the world. Leather is a natural polymer fiber, and its main component is collagen protein. After a long-term preservation process, the surface morphology of leather cultural relics is seriously damaged, and it is very difficult to identify its species. Therefore, how to use the advanced means of natural science to establish a technical system for the detection of leather cultural relics and extract information on ancient leather from imprints, residues and soil is crucial to the study of the origin and spread of leather.

The current advanced technology for protein detection is based on antibody-antigen Western Blotting or ELISA, but these methods are still difficult to detect some cultural relics with very low protein content.

SUMMARY OF THE INVENTION

In order to solve the above-mentioned technical problems, the present invention provides a high-sensitivity detection method for cowhide cultural relics. The method of the invention has a low detection limit, high sensitivity and good reproducibility, and can detect extremely low content of bovine collagen in the muddled samples of cultural relics. .

The specific technical scheme of the present invention is: a high-sensitivity detection method for cowhide cultural relics, comprising the following steps:

A) Take part of the cowhide of healthy adult cattle, cut it into small pieces, soak and degrease in an incubator with sodium carbonate aqueous solution; take out the degreased cowhide, degrease it with 14-16 mL mass fraction of 4-6% chlorine per 1g cowhide Mix with sodium chloride aqueous solution, stir slowly at room temperature for 10-15 h to remove salt-soluble non-collagen components, and finally rinse with distilled water and drain.

B) Put the drained cowhide in a container, add pepsin in an amount of 2000-4000 U/g cowhide, and then add 28-32 mL of 0.4-0.6 mol/L acetic acid to 1g cowhide, at 35-39 ℃ The enzymatic hydrolysis reaction was carried out in an incubator for 7-9 h; after the enzymatic hydrolysis was completed, the filtrate was filtered, and the filtrate was collected, salted out by adding sodium chloride, and the sodium chloride concentration was 3-5 mol/L. 0.4-0.6 mol/L of acetic acid was dissolved and put into a dialysis bag with a cut-off of 10,000 for dialysis; finally, the collagen solution in the dialysis bag was taken, freeze-dried, and ground to obtain bovine collagen for later use.

C) Take a glassy carbon electrode, and polish the stained glassy carbon electrode on the chamois with 1.0 μm, 0.3 μm, and 0.05 μm alumina suspension in a figure-8 shape; then in anhydrous ethanol and distilled water in turn Ultrasonic washing.

D) Soak the cleaned glassy carbon electrode in 0.4-0.6M dilute sulfuric acid solution, and activate it by cyclic voltammetry on the electrochemical workstation for 35-45 weeks. The scanning range of cyclic voltammetry is -0.4V- 1.6V, the scan rate is 0.08-0.12V/s.

E) Prepare PBS 7.4 solution, weigh K ₃ [Fe(CN) ₆ ], K ₄ [Fe(CN) ₆ ], KCl, add PBS 7.4 solution to obtain [Fe(CN) ₆ ] ^3-/4- Potassium ferricyanide solution at a concentration of 4.5-5.5 mM.

F) Prepare cetyltrimethylammonium bromide solution, HAuCl ₄ solution and NaBH ₄ solution respectively; add cetyltrimethylammonium bromide solution to the centrifuge tube, and add HAuCl to it under the action of shaking ₄ solution; then under the action of shaking, quickly add NaBH ₄ solution, shake vigorously, and let stand to obtain a gold nanoparticle solution.

The gold nanoparticles prepared by the present invention are used for adsorbing and loading samples. Compared with the traditional method of directly conducting experiments on the electrode surface, the gold nanoparticles have larger specific surface area and better loading performance.

G) Mix 0.15-0.25 mg/mL polyacrylic acid solution, 1.5-2.5 mol/L ammonia water and water in a volume ratio of 50-70:80-100:8-12, ultrasonically shake, then add isopropanol and stir Evenly, store at 1-5°C after standing.

H) Mix the solution obtained in step G), water, and isopropanol in a volume ratio of 5-7:8-12:8-12, then add the dopamine solution, and let it stand to obtain a polyacrylic acid-dopamine complex solution with a hollow structure .

The polyacrylic acid-dopamine complex synthesized by the invention has a hollow structure, which can accommodate more samples compared with a flat structure, and at the same time, a large number of amino groups exist on the surface, which is convenient for coupling with the interface with carboxyl groups by the NHS/EDC method.

1) Weigh polymethyl methacrylate and maleic anhydride/1-octadecene alternating copolymer whose mass ratio is 110-130:70-90, add chloroform and sodium dodecyl sulfonate solution, The obtained mixed solution is ultrasonically oscillated, centrifuged, the supernatant is removed, and the remaining product is washed with water for several times to obtain spherical nano-beads, which are finally added to chloroform to prepare a solution for use.

The spherical nano-beads prepared by the present invention can be modified on the polyacrylic acid-dopamine complex by the NHS/EDC method because the shape and surface contain a large number of carboxyl groups, and the binding rate is high. Antibodies whose end groups are amino groups have higher utilization efficiency of antibodies than the traditional method of direct dropwise addition of antibodies.

J) Take the glassy carbon electrode treated in step D), drop 35-45 μL of the gold nanoparticle solution obtained in step F) on the surface of the glassy carbon electrode, and after drying at room temperature, soak the electrode in 0.2-0.3M mercaptopropionic acid 5-7h, then wash thoroughly, soak the glassy carbon electrode in NHS/EDC solution for 0.5-1.5h; then wash the glassy carbon electrode, drop 25-35 μL of the solution obtained in step H) on its surface, dry at room temperature and wash , soak the glassy carbon electrode in NHS/EDC solution for 20-40min; after thorough washing, continue to drop 25-35μL of the solution obtained in step I) on the surface of the glassy carbon electrode, dry at room temperature, and soak in NHS/EDC solution for 20-40min; Finally, 8-12 μL of rabbit anti-bovine collagen polyclonal antibody was dropped on the surface of the glassy carbon electrode, and incubated at 35-39 °C for 0.5-1.5 h.

K) Prepare solutions of different concentrations with the bovine collagen obtained in step B), add 15-25 μL dropwise to the surface of the glassy carbon electrode treated in step J), incubate at 35-39 °C for 0.5-1.5 h, wash and place in the electrophoresis Differential pulse voltammetry was used to scan the test on the ChemStation, and the scanning range was -0.2V-0.6V.

L) Weigh the cultural relic sample, dissolve it with the acetic acid described in step B), and test it according to the method used in step K).

The invention requires very few cultural relic samples, and can effectively detect the cultural relics even in the case of scarcity, which is of great significance for archaeological identification.

Preferably, in step A), the cowhide is cut into small pieces of (1-3) cm×(1-3) cm, the mass concentration of the sodium carbonate aqueous solution is 5-7%, and the degreasing temperature is 45-55° C. , the degreasing time is 3-5h.

Preferably, in step B), the sodium chloride concentration is 3-5 mol/L, the centrifugal speed is 10000-14000 r/min, the centrifugal time is 8-12 min, and the dialysis is performed for 2-4 days.

Preferably, in step C), the diameter of the glassy carbon electrode is 2-4mm, the grinding time is 8-12min, and the ultrasonic washing time is 8-12min.

Preferably, the specific process of step F) is: take 3.6445 g of cetyltrimethylammonium bromide in a conical flask, add 100 mL of water, stir and dissolve in a water bath at 35-39 °C; take 2.057 mL, 10 g/L HAuCl ₄ in a centrifuge tube, add 2.942 mL of water, shake well, and store in the dark; prepare 0.5-0.7 mL of NaBH ₄ solution according to the ratio of NaBH ₄ to frozen water as 11.4 mg:30 mL; filter to obtain 8-12 mL The cetyltrimethylammonium bromide solution was placed in a centrifuge tube, and 0.2-0.3 mL of HAuCl ₄ solution was added to it under shaking _; Shake for 1-3 min, and stand at 20-30 °C for 1.5-2.5 h to obtain a gold nanoparticle solution.

Preferably, in step G), the ultrasonic vibration time is 0.5-1.5h, and the stirring time is 0.5-1.5h.

Preferably, in step H), the standing temperature is 45-55° C., and the standing time is 2-4 h.

Preferably, in step I), the ultrasonic vibration time is 1-3 min, the centrifugal speed is 8000-12000 rpm, and the centrifugal time is 8-12 min.

Preferably, in step J), the molar ratio of NHS and EDC in the NHS/EDC solution is 0.2-0.4:0.4-0.6.

Preferably, in step L), weigh 0.01-0.03 g of a cultural relic sample and dissolve it with 1-3 mL of the acetic acid described in step B).

Compared with the prior art, the beneficial effects of the present invention are:

1. The gold nanoparticles prepared in the present invention are used for adsorbing and loading samples. Compared with the traditional method of conducting experiments directly on the electrode surface, the gold nanoparticles have a larger specific surface area and better loading performance.

2. The polyacrylic acid-dopamine complex synthesized by the present invention has a hollow structure, which can accommodate more samples than a flat structure, and at the same time, there are a large number of amino groups on the surface, which is convenient for coupling with the interface with carboxyl groups by the NHS/EDC method. .

3. The spherical nanobeads prepared by the present invention, because their shape and surface contain a large number of carboxyl groups, can be modified on the polyacrylic acid-dopamine complex with a hollow structure by the NHS/EDC method, and the binding rate is high. At the same time, due to the existence of surface carboxyl groups, Antibodies with amino groups at the end group can be enriched on the interface, and the utilization efficiency of antibodies is higher than the traditional method of directly adding antibodies.

4. The invention requires very few cultural relic samples, and can effectively detect the cultural relics even in the case of scarcity, which is of great significance for archaeological identification.

Description of drawings

Figure 1 is a graph of the detection results of cultural relic samples;

Figure 2 shows the standard curves obtained from bovine collagen solutions with different concentrations.

Detailed ways

The present invention will be further described below in conjunction with the examples.

Example 1

A) Take 15 g of healthy adult cattle hides, cut them into small pieces of 2 cm × 2 cm, put them in a conical flask, and soak them in a 5% sodium carbonate aqueous solution in a 45°C incubator for 4 h. The degreased cowhide was taken out, and after degreasing, 1 g of cowhide was mixed with 14 mL of a 4% sodium chloride aqueous solution by mass, and slowly stirred at room temperature for 12 h to remove salt-soluble non-collagen components, and then washed with distilled water and drained.

B) Put the drained cowhide in a beaker, add pepsin, the amount of protease is 2000 U/g cowhide, add 28 mL of 0.5 mol/L acetic acid to 1 g cowhide, and carry out enzymatic hydrolysis in a 37 ℃ incubator React for 7 hours. After the enzymatic hydrolysis, filter, collect the filtrate, add sodium chloride for salting out, and the sodium chloride concentration is 3 mol/L. Dialysis bags with a retention volume of 10,000 were dialyzed for 3 days. Finally, the collagen solution in the dialysis bag was poured out, freeze-dried, and ground to obtain bovine collagen for use.

C) Take a glassy carbon electrode with a diameter of 3mm, and use the 1.0, 0.3, and 0.05 μm alumina suspension on the chamois to polish the stained glassy carbon electrode in a figure-eight shape for 10 minutes; then Sequentially ultrasonically washed in absolute ethanol and distilled water for 10 min.

D) Soak the cleaned electrode in 0.5 M dilute sulfuric acid solution, and activate it by cyclic voltammetry on the electrochemical workstation for 40 weeks. The scanning range of cyclic voltammetry is -0.4V-1.6V, and the scanning rate is 0.1V/s.

E) Weigh 0.2 g of KCl, 0.27 g of KH ₂ PO ₄ , 8 g of NaCl and 1.42 g of Na ₂ HPO ₄ into 800 mL of deionized water, and stir until completely dissolved. The pH was adjusted to 7.4 to obtain a PBS7.4 solution; K ₃ [Fe(CN) ₆ ] 1.646 g, K ₄ [Fe(CN) ₆ ] 2.112 g, and KCl 7.45 g were weighed, and 1000 mL of PBS 7.4 solution was added. A solution of [Fe(CN)6] ^3-/4- potassium ferricyanide was obtained at a concentration of 5.0 mM.

F) Take 3.6445 g of cetyl trimethyl ammonium bromide in a 250 mL conical flask, add 100 mL of water, stir and dissolve in a 37 ℃ water bath; take 2.057 mL of HAuCl ₄ (10 g/L) and centrifuge in 50 mL In the tube, add 2.942 mL of water, shake well, and store in the dark; prepare 0.6 mL of NaBH ₄ solution in the ratio of 11.4 mg: 30 mL (NaBH ₄ : frozen water); filter to obtain 10 mL of cetyltrimethylammonium bromide In a 50 mL centrifuge tube, add 0.25 mL of the prepared HAuCl ₄ solution to it under the action of shaking; then under the action of shaking, quickly add 0.6 mL of the prepared NaBH ₄ solution, shake vigorously for 2 min, and let stand at 25 °C After 2 h, gold nanoparticles solution was obtained.

G) Take 60 μL of polyacrylic acid (0.2 mg/mL) solution and 90 μL of ammonia water (2 mol/L), add 10 mL of water, sonicate for one hour, then add 110 mL of isopropanol, stir magnetically for one hour, Store at 4°C after standing.

H) Take 6 mL of the solution obtained in step G), add 10 mL of water, 10 mL of isopropanol, and then add 80 μL of dopamine (0.05 g/mL), and let stand at 50 °C for 3 hours to obtain a hollow-structured polyacrylic acid- Dopamine complex.

1) Weigh 120 mg of polymethyl methacrylate and 80 mg of maleic anhydride/1-octadecene alternating copolymer, add 2 mL of chloroform and 5 mL of sodium dodecyl sulfonate solution (3 mg/ mL). The obtained mixture was ultrasonically shaken for 2 min, then centrifuged at 10,000 rpm for 10 min, the supernatant was removed, and the remaining product was washed with water three times to obtain spherical nanobeads, and finally 1 mL of chloroform was added for use.

J) Take the glassy carbon electrode treated in step D), drop 40 μL of the gold nanoparticle solution obtained in step F) on the surface of the electrode, after drying at room temperature, soak the electrode in 0.25M mercaptopropionic acid for 6 hours, and then thoroughly For washing, the electrode was soaked in NHS/EDC (0.3M/0.5M) solution for 1 hour; 30 μL of the solution obtained in step H) was added dropwise to the surface of the electrode after washing, dried at room temperature and washed, and the electrode was immersed in NHS/EDC (0.3M/0.5M) solution for 0.5 hours; after thorough washing, continue to drop 30 μL of the solution obtained in step I) on the electrode surface, dry at room temperature, and soak in NHS/EDC (0.3M/0.5M) solution for 0.5 hours ; Finally, 10 μL of rabbit anti-bovine collagen polyclonal antibody was dropped on the electrode surface, and incubated at 37°C for 1 hour.

K) Prepare a solution with a concentration of 100 ng/mL of bovine collagen obtained in step B), add 20 μL dropwise to the electrode surface treated in step J), incubate at 37°C for 1 hour, wash it on the electrochemical workstation with differential Pulse voltammetry scanning test, the scanning range is -0.2V-0.6V.

L) Weigh 0.02 g of cultural relic sample, dissolve it with 2 mL of acetic acid in step B), and test it according to the method used in step K).

The method used in the present invention can effectively identify the micro traces of cultural relics in soil samples, as shown in Figure 2 (standard curves obtained from bovine collagen solutions with different concentrations), the linear detection range of the method is 0.1-100 ng/mL , the lowest detection limit was 0.067 ng/mL.

The ancient cultural relic samples were tested according to the method of Example 1, and the test results were shown in Figure 1, and the results showed that the samples contained cowhide components.

Example 2

A) Take 15 g of healthy adult cattle hides, cut them into small pieces of 2 cm × 2 cm, put them in a conical flask, and soak them in a 6% sodium carbonate aqueous solution for 4 h in a 50°C incubator. The degreased cowhide was taken out, mixed with 15 mL of 4-6% sodium chloride aqueous solution per 1 g of cowhide after degreasing, and slowly stirred at room temperature for 12 h to remove salt-soluble non-collagen components, and then washed with distilled water and drained.

B) Put the drained cowhide in a beaker, add pepsin, the amount of protease is 3000 U/g cowhide, add 30 mL of 0.5 mol/L acetic acid to 1 g cowhide, and carry out enzymatic hydrolysis in a 37 ℃ incubator React for 8 hours. After the enzymatic hydrolysis, filter, collect the filtrate, add sodium chloride for salting out, and the sodium chloride concentration is 3-5 mol/L. Dialysis was performed in a dialysis bag with a cut-off volume of 10,000 for 3 days. Finally, the collagen solution in the dialysis bag was poured out, freeze-dried, and ground to obtain bovine collagen for use.

C) Take a glassy carbon electrode with a diameter of 3mm, and use the 1.0, 0.3, and 0.05 μm alumina suspension on the chamois to polish the stained glassy carbon electrode in a figure-eight shape for 10 minutes; then Sequentially ultrasonically washed in absolute ethanol and distilled water for 10 min.

D) Soak the cleaned electrode in 0.5 M dilute sulfuric acid solution, and activate it by cyclic voltammetry on the electrochemical workstation for 40 weeks. The scanning range of cyclic voltammetry is -0.4V-1.6V, and the scanning rate is 0.1V/s.

E) Weigh 0.2 g of KCl, 0.27 g of KH ₂ PO ₄ , 8 g of NaCl and 1.42 g of Na ₂ HPO ₄ into 800 mL of deionized water, and stir until completely dissolved. The pH was adjusted to 7.4 to obtain a PBS7.4 solution; K ₃ [Fe(CN) ₆ ] 1.646 g, K ₄ [Fe(CN) ₆ ] 2.112 g, and KCl 7.45 g were weighed, and 1000 mL of PBS 7.4 solution was added. A solution of [Fe(CN)6] ^3-/4- potassium ferricyanide was obtained at a concentration of 5.0 mM.

F) Take 3.6445 g of cetyl trimethyl ammonium bromide in a 250 mL conical flask, add 100 mL of water, stir and dissolve in a 37 ℃ water bath; take 2.057 mL of HAuCl ₄ (10 g/L) and centrifuge in 50 mL In the tube, add 2.942 mL of water, shake well, and store in the dark; prepare 0.6 mL of NaBH ₄ solution in the ratio of 11.4 mg: 30 mL (NaBH ₄ : frozen water); filter to obtain 10 mL of cetyltrimethylammonium bromide In a 50 mL centrifuge tube, add 0.25 mL of the prepared HAuCl ₄ solution to it under the action of shaking; then under the action of shaking, quickly add 0.6 mL of the prepared NaBH ₄ solution, shake vigorously for 2 min, and let stand at 25 °C After 2 h, gold nanoparticles solution was obtained.

G) Take 60 μL of polyacrylic acid (0.2 mg/mL) solution and 90 μL of ammonia water (2 mol/L), add 10 mL of water, sonicate for one hour, then add 110 mL of isopropanol, stir magnetically for one hour, Store at 4°C after standing.

H) Take 6 mL of the solution obtained in step G), add 10 mL of water, 10 mL of isopropanol, and then add 80 μL of dopamine (0.05 g/mL), and let stand at 50 °C for 3 hours to obtain a hollow-structured polyacrylic acid- Dopamine complex.

1) Weigh 120 mg of polymethyl methacrylate and 80 mg of maleic anhydride/1-octadecene alternating copolymer, add 2 mL of chloroform and 5 mL of sodium dodecyl sulfonate solution (3 mg/ mL). The obtained mixture was ultrasonically shaken for 2 min, then centrifuged at 10,000 rpm for 10 min, the supernatant was removed, and the remaining product was washed with water three times to obtain spherical nanobeads, and finally 1 mL of chloroform was added for use.

J) Take the glassy carbon electrode treated in step D), drop 40 μL of the gold nanoparticle solution obtained in step F) on the surface of the electrode, after drying at room temperature, soak the electrode in 0.25M mercaptopropionic acid for 6 hours, and then thoroughly For washing, the electrode was soaked in NHS/EDC (0.3M/0.5M) solution for 1 hour; 30 μL of the solution obtained in step H) was added dropwise to the surface of the electrode after washing, dried at room temperature and washed, and the electrode was immersed in NHS/EDC (0.3M/0.5M) solution for 0.5 hours; after thorough washing, continue to drop 30 μL of the solution obtained in step I) on the electrode surface, dry at room temperature, and soak in NHS/EDC (0.3M/0.5M) solution for 0.5 hours ; Finally, 10 μL of rabbit anti-bovine collagen polyclonal antibody was dropped on the electrode surface, and incubated at 37°C for 1 hour.

K) Prepare a 10 ng/mL solution with bovine collagen obtained in step B), add 20 μL dropwise to the electrode surface treated in step J), incubate at 37°C for 1 hour, wash and use differential pulses on the electrochemical workstation Voltammetry scan test, the scan range is -0.2V-0.6V.

L) Weigh 0.02 g of cultural relic sample, dissolve it with 2 mL of acetic acid in step B), and test it according to the method used in step K).

Example 3

A) Take 15 g of healthy adult cattle hides, cut them into small pieces of 2 cm × 2 cm, put them in a conical flask, and soak them in a 7% sodium carbonate aqueous solution in a 55 °C incubator for 4 h. The degreased cowhide was taken out, and after degreasing, 1 g of cowhide was mixed with 16 mL of a 4-6% sodium chloride aqueous solution by mass, and slowly stirred at room temperature for 12 h to remove salt-soluble non-collagen components, and then washed with distilled water and drained.

B) Put the drained cowhide in a beaker, add pepsin, the amount of protease is 4000 U/g cowhide, add 32 mL of 0.5 mol/L acetic acid to 1 g cowhide, and carry out enzymatic hydrolysis in a 37 ℃ incubator The reaction was carried out for 9 hours. After the enzymatic hydrolysis, filter, collect the filtrate, add sodium chloride for salting out, and the sodium chloride concentration is 3-5 mol/L. Dialysis was performed in a dialysis bag with a cut-off volume of 10,000 for 3 days. Finally, the collagen solution in the dialysis bag was poured out, freeze-dried, and ground to obtain bovine collagen for use.

C) Take a glassy carbon electrode with a diameter of 3mm, and use the 1.0, 0.3, and 0.05 μm alumina suspension on the chamois to polish the stained glassy carbon electrode in a figure-eight shape for 10 minutes; then Sequentially ultrasonically washed in absolute ethanol and distilled water for 10 min.

D) Soak the cleaned electrode in 0.5 M dilute sulfuric acid solution, and activate it by cyclic voltammetry on the electrochemical workstation for 40 weeks. The scanning range of cyclic voltammetry is -0.4V-1.6V, and the scanning rate is 0.1V/s.

E) Weigh 0.2 g of KCl, 0.27 g of KH ₂ PO ₄ , 8 g of NaCl and 1.42 g of Na ₂ HPO ₄ into 800 mL of deionized water, and stir until completely dissolved. The pH was adjusted to 7.4 to obtain a PBS7.4 solution; K ₃ [Fe(CN) ₆ ] 1.646 g, K ₄ [Fe(CN) ₆ ] 2.112 g, and KCl 7.45 g were weighed, and 1000 mL of PBS 7.4 solution was added. A solution of [Fe(CN)6] ^3-/4- potassium ferricyanide was obtained at a concentration of 5.0 mM.

F) Take 3.6445 g of cetyl trimethyl ammonium bromide in a 250 mL conical flask, add 100 mL of water, stir and dissolve in a 37 ℃ water bath; take 2.057 mL of HAuCl ₄ (10 g/L) and centrifuge in 50 mL In the tube, add 2.942 mL of water, shake well, and store in the dark; prepare 0.6 mL of NaBH ₄ solution in the ratio of 11.4 mg: 30 mL (NaBH ₄ : frozen water); filter to obtain 10 mL of cetyltrimethylammonium bromide In a 50 mL centrifuge tube, add 0.25 mL of the prepared HAuCl ₄ solution to it under the action of shaking; then under the action of shaking, quickly add 0.6 mL of the prepared NaBH ₄ solution, shake vigorously for 2 min, and let stand at 25 °C After 2 h, gold nanoparticles solution was obtained.

G) Take 60 μL of polyacrylic acid (0.2 mg/mL) solution and 90 μL of ammonia water (2 mol/L), add 10 mL of water, sonicate for one hour, then add 110 mL of isopropanol, stir magnetically for one hour, Store at 4°C after standing.

H) Take 6 mL of the solution obtained in step G), add 10 mL of water, 10 mL of isopropanol, and then add 80 μL of dopamine (0.05 g/mL), and let stand at 50 °C for 3 hours to obtain a hollow-structured polyacrylic acid- Dopamine complex.

1) Weigh 120 mg of polymethyl methacrylate and 80 mg of maleic anhydride/1-octadecene alternating copolymer, add 2 mL of chloroform and 5 mL of sodium dodecyl sulfonate solution (3 mg/ mL). The obtained mixture was ultrasonically shaken for 2 min, then centrifuged at 10,000 rpm for 10 min, the supernatant was removed, and the remaining product was washed with water three times to obtain spherical nanobeads, and finally 1 mL of chloroform was added for use.

J) Take the glassy carbon electrode treated in step D), drop 40 μL of the gold nanoparticle solution obtained in step F) on the surface of the electrode, after drying at room temperature, soak the electrode in 0.25M mercaptopropionic acid for 6 hours, and then thoroughly For washing, the electrode was soaked in NHS/EDC (0.3M/0.5M) solution for 1 hour; 30 μL of the solution obtained in step H) was added dropwise to the surface of the electrode after washing, dried at room temperature and washed, and the electrode was immersed in NHS/EDC (0.3M/0.5M) solution for 0.5 hours; after thorough washing, continue to drop 30 μL of the solution obtained in step I) on the electrode surface, dry at room temperature, and soak in NHS/EDC (0.3M/0.5M) solution for 0.5 hours ; Finally, 10 μL of rabbit anti-bovine collagen polyclonal antibody was dropped on the electrode surface, and incubated at 37°C for 1 hour.

K) Prepare a 1 ng/mL solution with the bovine collagen obtained in step B), drop 20 μL on the electrode surface treated in step J), incubate at 37°C for 1 hour, wash and use differential pulses on the electrochemical workstation Voltammetry scan test, the scan range is -0.2V-0.6V.

L) Weigh 0.02 g of cultural relic sample, dissolve it with 2 mL of acetic acid in step B), and test it according to the method used in step K).

The raw materials and equipment used in the present invention, unless otherwise specified, are the common raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are the conventional methods in the art.

The above are only preferred embodiments of the present invention and do not limit the present invention. Any simple modifications, changes and equivalent transformations made to the above embodiments according to the technical essence of the present invention still belong to the technical solutions of the present invention. scope of protection.

Claims (10)

1. a high-sensitivity detection method for cowhide cultural relics, is characterized in that comprising the following steps: A) Cut the cowhide into small pieces, soak and degrease the cowhide in an incubator with a sodium carbonate aqueous solution; take out the degreasing cowhide, and mix it with 14-16 mL mass fraction of 4-6% sodium chloride aqueous solution per 1 g cowhide after degreasing. Stir slowly at room temperature for 10-15 h to remove salt-soluble non-collagen components, and finally rinse with distilled water and drain; B) Put the drained cowhide in a container, add pepsin in an amount of 2000-4000 U/g cowhide, and then add 28-32 mL of 0.4-0.6 mol/L acetic acid to 1g cowhide, at 35-39 ℃ The enzymatic hydrolysis reaction was carried out in an incubator for 7-9 h; after the enzymatic hydrolysis was completed, the filtrate was filtered, and the filtrate was collected, salted out by adding sodium chloride, and the sodium chloride concentration was 3-5 mol/L. 0.4-0.6 mol/L of acetic acid was dissolved and put into a dialysis bag with a cut-off of 10,000 for dialysis; finally, the collagen solution in the dialysis bag was taken, freeze-dried, and ground to obtain bovine collagen, which was used for later use; C) Take a glassy carbon electrode, and polish the stained glassy carbon electrode on the chamois in a figure-8 shape in an alumina suspension of 1.0 μm, 0.3 μm and 0.05 μm in turn; then in anhydrous ethanol and distilled water in turn Medium ultrasonic washing; D) Soak the cleaned glassy carbon electrode in 0.4-0.6M dilute sulfuric acid solution, and activate it by cyclic voltammetry on the electrochemical workstation for 35-45 weeks. The scanning range of cyclic voltammetry is -0.4V- 1.6V, the scan rate is 0.08-0.12V/s; E) Prepare a PBS solution with pH 7.4, weigh K ₃ [Fe(CN) ₆ ], K ₄ [Fe(CN) ₆ ], KCl, and add PBS solution with pH 7.4 to obtain [Fe(CN) ₆ ] ^3-/4- potassium ferricyanide solution at a concentration of 4.5-5.5 mM; F) Prepare cetyltrimethylammonium bromide solution, HAuCl ₄ solution and NaBH ₄ solution respectively; add cetyltrimethylammonium bromide solution to the centrifuge tube, and add HAuCl to it under the action of shaking ₄ solution; then under the action of shaking, quickly add NaBH ₄ solution, shake vigorously, and let stand to obtain a gold nanoparticle solution; G) Mix 0.15-0.25 mg/mL polyacrylic acid solution, 1.5-2.5 mol/L ammonia water and water in a volume ratio of 50-70:80-100:8-12, ultrasonically shake, then add isopropanol and stir Evenly, store at 1-5°C after standing; H) Mix the solution obtained in step G), water and isopropanol in a volume ratio of 5-7:8-12:8-12, then add the dopamine solution and let it stand to obtain a polyacrylic acid-dopamine complex solution with a hollow structure; 1) Weigh polymethyl methacrylate and maleic anhydride/1-octadecene alternating copolymer whose mass ratio is 110-130:70-90, add chloroform and sodium dodecyl sulfonate solution, The obtained mixed solution is ultrasonically vibrated, centrifuged, the supernatant is removed, and the remaining product is washed with water for several times to obtain spherical nano-beads, which are finally added to chloroform to obtain a solution, which is for subsequent use; J) Take the glassy carbon electrode treated in step D), drop 35-45 μL of the gold nanoparticle solution obtained in step F) on the surface of the glassy carbon electrode, and after drying at room temperature, soak the electrode in 0.2-0.3M mercaptopropionic acid After washing the glassy carbon electrode in NHS/EDC solution for 0.5-1.5h, add 25-35 μL of the solution obtained in step H) dropwise to the surface of the glassy carbon electrode, and dry it at room temperature Washing, soak the glassy carbon electrode in NHS/EDC solution for 20-40min; after thorough washing, continue to drop 25-35μL of the solution obtained in step I) on the surface of the glassy carbon electrode, dry at room temperature, and soak in NHS/EDC solution for 20-40min ; Finally, drop 8-12 μL rabbit anti-bovine collagen polyclonal antibody on the surface of the glassy carbon electrode, and incubate at 35-39 °C for 0.5-1.5 h; K) Prepare solutions of different concentrations with the bovine collagen obtained in step B), add 15-25 μL dropwise to the surface of the glassy carbon electrode treated in step J), incubate at 35-39 °C for 0.5-1.5 h, wash and place in the electrophoresis Differential pulse voltammetry scanning test was used on the chemical workstation, and the scanning range was -0.2V-0.6V; L) Weigh the cultural relic sample, dissolve it with the acetic acid described in step B), add 15-25 μL dropwise to the surface of the glassy carbon electrode treated in step J), incubate at 35-39°C for 0.5-1.5h, wash it in the electrochemical The differential pulse voltammetry scan was used on the workstation, and the scan range was -0.2V-0.6V. 2. The high-sensitivity detection method of a cowhide cultural relic as claimed in claim 1, wherein in step A), the cowhide is cut into small pieces of 1-3cm×1-3cm, and the quality of the sodium carbonate aqueous solution is The concentration is 5-7%, the degreasing temperature is 45-55 ℃, and the degreasing time is 3-5h. 3. the high-sensitivity detection method of a kind of cowhide cultural relic as claimed in claim 1, is characterized in that, in step B), described sodium chloride concentration is 3-5 mol/L, and centrifugal speed is 10000-14000 r/ min, centrifugation time 8-12 min, dialysis 2-4 days. 4. the high-sensitivity detection method of a kind of cowhide cultural relic as claimed in claim 1, is characterized in that, in step C), the diameter of glassy carbon electrode is 2-4mm, grinding time is 8-12min; Ultrasonic washing time is 8 -12min. 5. the high-sensitivity detection method of a kind of cowhide cultural relic as claimed in claim 1, is characterized in that, the concrete process of step F) is: get 3.6445 g cetyl trimethyl ammonium bromide in the conical flask, Add 100 mL of water, stir and dissolve in a water bath at 35-39 °C; take 2.057 mL of 10 g/L HAuCl ₄ in a centrifuge tube, add 2.942 mL of water, shake well, and store in the shade; according to the ratio of NaBH ₄ to frozen water Prepare 0.5-0.7 mL of NaBH ₄ solution for 11.4 mg:30 mL; filter to obtain 8-12 mL of cetyltrimethylammonium bromide solution in a centrifuge tube, add 0.2-0.3 mL of HAuCl to it under shaking ₄ solution; then under the action of shaking, quickly add 0.5-0.7 mL of NaBH ₄ solution, shake vigorously for 1-3 min, and stand at 20-30 °C for 1.5-2.5 h to obtain a gold nanoparticle solution. 6 . The high-sensitivity detection method for cowhide cultural relics according to claim 1 , wherein, in step G), the ultrasonic oscillation time is 0.5-1.5h, and the stirring time is 0.5-1.5h. 7 . 7 . The high-sensitivity detection method for cowhide cultural relics according to claim 1 , wherein, in step H), the standing temperature is 45-55° C., and the standing time is 2-4 h. 8 . 8. the high-sensitivity detection method of a kind of cowhide cultural relic as claimed in claim 1, is characterized in that, in step 1), ultrasonic vibration time is 1-3 min, centrifugal speed is 8000-12000 rpm, and centrifugal time is 8-3 min. 12min. 9 . The high-sensitivity detection method for cowhide cultural relics according to claim 1 , wherein in step J), the molar ratio of NHS and EDC in the NHS/EDC solution is 0.2-0.4:0.4-0.6. 10 . 10. The high-sensitivity detection method of a cowhide cultural relic as claimed in claim 1, wherein in step L), 0.01-0.03 g cultural relic sample is weighed and dissolved in 1-3 mL of acetic acid described in step B). .

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