Method for measuring content of polydeoxyribonucleotide in implant containing polydeoxyribonucleotide

Abstract

The invention discloses a method for determining the content of polydeoxyribonucleotide in a polydeoxyribonucleotide-containing implant, and relates to the technical field of nucleotide content determination. The implant contains collagen and polydeoxyribonucleotide, and the determination method comprises the following steps: determining the absorbance of the implant at the ultraviolet light of 260 nm, and recording the absorbance as A260; measuring the absorbance of the implant at the ultraviolet light of 280 nm, and recording the absorbance as A280; and measuring the content of the polydeoxyribonucleotide by calculating the difference value between the A260 of the first preset multiple and the A280 of the second preset multiple. According to the method, the content of the polydeoxyribonucleotide in the implant containing the collagen and the polydeoxyribonucleotide at the same time can be accurately measured.

Description

Technical Field

[0001] The present application relates to the technical field of nucleotide content determination, and in particular, to a method for determining the content of polydeoxyribonucleotides in an implant containing polydeoxyribonucleotides. Background Art

[0002] Polydeoxyribonucleotides belong to the nucleotide class of substances, which have strong absorption of ultraviolet light, especially at 260nm. In the prior art, the ultraviolet absorbance at 260nm is usually used to determine the content of polydeoxyribonucleotides.

[0003] In implants containing both collagen and polydeoxyribonucleotides, not only does polydeoxyribonucleotides have obvious absorption at 260nm, but collagen is a protein and also absorbs at 260nm. This results in the fact that the absorbance of implants containing both collagen and polydeoxyribonucleotides measured at 260nm cannot accurately determine the content of polydeoxyribonucleotides in the implants. Summary of the invention

[0004] The embodiment of the present application provides a method for determining the content of polydeoxyribonucleotides in an implant containing polydeoxyribonucleotides, which can more accurately measure the content of polydeoxyribonucleotides in an implant containing both collagen and polydeoxyribonucleotides.

[0005] The embodiment of the present application is implemented as follows: The present application provides a method for determining the content of polydeoxyribonucleotides in an implant containing polydeoxyribonucleotides, wherein the implant further contains collagen, and the determination method comprises the following steps: Determine the absorbance of the implant at 260 nm of ultraviolet light, recorded as A260; Determine the absorbance of the implant at 280 nm of ultraviolet light, recorded as A280; The content of the polydeoxyribonucleotide is determined by calculating the difference between the A260 of the first preset multiple and the A280 of the second preset multiple.

[0006] In a possible embodiment, the implant is first diluted into a colorless transparent solution, and the absorbance of the colorless transparent solution at ultraviolet light of 260 nm and 280 nm is measured, which are recorded as A1 and A2 respectively, wherein A260=A1*dilution factor, and A280=A2*dilution factor.

[0007] In a possible embodiment, the implant is diluted into a colorless transparent solution with a diluent, and the diluent includes any one of phosphate buffer, hydrochloric acid, sodium hydroxide, Tris-HCl buffer and pure water.

[0008] In one possible embodiment, the pH of the phosphate buffer or the Tris-HCl buffer is 6-8.

[0009] In a possible embodiment, the concentration of the hydrochloric acid or the sodium hydroxide is 0.05-0.5 mol / L.

[0010] In a possible implementation manner, the first preset multiple is 14.5-15.0.

[0011] In a possible implementation manner, the second preset multiple is 18-18.5.

[0012] In a possible implementation manner, the first preset multiple is 14.97, and the second preset multiple is 18.34.

[0013] In a possible implementation manner, the first preset multiple is 14.68, and the second preset multiple is 18.1.

[0014] The embodiments of the present application have at least the following beneficial effects: The method for determining the content of polydeoxyribonucleotides in an implant containing polydeoxyribonucleotides according to an embodiment of the present application determines the content of polydeoxyribonucleotides by determining the absorbance of the implant at 260nm and 280nm of ultraviolet light, and then calculating the difference between A260 of a first preset multiple and A280 of a second preset multiple. After testing, it has been found that the content of polydeoxyribonucleotides in an implant containing both collagen and polydeoxyribonucleotides can be measured more accurately. DETAILED DESCRIPTION

[0015] The embodiments of the present application will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present application and should not be considered as limiting the scope of the present application. In the examples, if specific conditions are not specified, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0016] The following is a specific description of the method for determining the content of polydeoxyribonucleotides in the polydeoxyribonucleotide-containing implant according to the embodiment of the present application: In a first aspect, the present invention provides a method for determining the content of polydeoxyribonucleotides in an implant containing polydeoxyribonucleotides, wherein the implant also contains collagen, and the determination method comprises the following steps: The absorbance of the implant at 260 nm of ultraviolet light was measured and recorded as A260; The absorbance of the implant at 280 nm of ultraviolet light was measured and recorded as A280; The content of polydeoxyribonucleotides is determined by calculating the difference between the first preset multiple of A260 and the second preset multiple of A280. That is, the content of polydeoxyribonucleotides = mA260-nA280.

[0017] Both polydeoxyribonucleotide and collagen have absorption at 260nm and 280nm, among which polydeoxyribonucleotide has more obvious absorption at 260nm and collagen has more obvious absorption at 280nm. The content of polydeoxyribonucleotide is determined by measuring the absorbance of the implant at ultraviolet light at 260nm and 280nm, and then calculating the difference between A260 of the first preset multiple and A280 of the second preset multiple. After testing, it is found that the content of polydeoxyribonucleotide in the implant containing both collagen and polydeoxyribonucleotide can be measured more accurately.

[0018] In some embodiments, the first preset multiple (m) is 14.5-15.0, and the second preset multiple (n) is 18-18.5. Exemplarily, the first preset multiple is any one of 14.5, 14.6, 14.7, 14.8, 14.9, and 15, or a value between any two of them; the second preset multiple is any one of 18, 18.1, 18.2, 18.3, 18.4, and 18.5, or a value between any two of them. According to the inventors of the present application, the first preset multiple is 14.5-15.0, and the second preset multiple is 18-18.5. Combined with the absorbance at 260nm and 280nm, the content of polydeoxyribonucleotides in the implant containing both collagen and polydeoxyribonucleotides can be accurately measured.

[0019] Exemplarily, the first preset multiple is 14.97, and the second preset multiple is 18.34. Exemplarily, the first preset multiple is 14.68, and the second preset multiple is 18.1.

[0020] Furthermore, the implant is first diluted to a colorless transparent solution, and the absorbance of the colorless transparent solution at ultraviolet light of 260 nm and 280 nm is measured, which are recorded as A1 and A2, respectively. A260=A1*dilution factor, and A280=A2*dilution factor.

[0021] When measuring the absorbance of the implant, the transparency of the implant will affect the accurate value of the absorbance measurement of the implant. Therefore, if the implant itself is non-transparent, first dilute the implant into a colorless transparent solution, and then measure the absorbance of the colorless transparent solution at 260nm and 280nm of ultraviolet light, which are recorded as A1 and A2 respectively. A260=A1*dilution factor, A280=A2*dilution factor; if the implant itself is transparent, the absorbance can be measured directly. For example, first dilute the implant 10 times to make it a colorless transparent solution, then A260=A1*10, A280=A2*10.

[0022] In the embodiment of the present application, a diluent is used to dilute the implant into a colorless transparent solution. For example, the diluent includes any one of phosphate buffer, hydrochloric acid, sodium hydroxide, Tris-HCl buffer and pure water. The above diluents can dilute the implant of the present application into a colorless transparent solution.

[0023] Optionally, the pH of the phosphate buffer or Tris-HCl buffer is 6-8, for example, pH 6, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8 or 8.

[0024] Optionally, the concentration of hydrochloric acid or sodium hydroxide is 0.05-0.5 mol / L. For example, the concentration of hydrochloric acid or sodium hydroxide is any one of 0.05 mol / L, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L and 0.5 mol / L, or a value between any two of them.

[0025] The method for determining the content of polydeoxyribonucleotides in the polydeoxyribonucleotide-containing implant of the present application is further described in detail below in conjunction with the examples. Example

[0026] This embodiment provides a method for determining the content of polydeoxyribonucleotides in an implant containing polydeoxyribonucleotides, wherein the implant is compounded with polydeoxyribonucleotides and collagen, the content of polydeoxyribonucleotides in the implant is 10 mg / mL, and the content of collagen is 30 mg / mL.

[0027] The assay method comprises the following steps: The above implant was diluted 100 times with phosphate buffer to obtain a colorless transparent solution, and the absorbance of the diluted colorless transparent solution at ultraviolet light 260nm and 280nm was measured, which were recorded as A1 and A2, respectively, wherein the absorbance of the implant at ultraviolet light 260nm is A260=A1*100, and the absorbance of the implant at ultraviolet light 280nm is A280=A2*100.

[0028] The absorbance A260 of the implant of this embodiment at 260nm ultraviolet light is 1.9591, and the absorbance A280 of the implant at 280nm ultraviolet light is 1.0275. The content of polydeoxyribonucleotides calculated by the formula polydeoxyribonucleotide content = 14.97 A260-18.34A280 is 10.48 mg / mL. The difference with the actual content of polydeoxyribonucleotides in the implant is within 5%.

[0029] The content of polydeoxyribonucleotides was calculated to be 10.16 mg / mL by the formula polydeoxyribonucleotide content = 14.68 A260-18.1 A280, which was within 2% of the actual content of polydeoxyribonucleotides in the implant. Example

[0030] This embodiment provides a method for determining the content of polydeoxyribonucleotides in an implant containing polydeoxyribonucleotides, wherein the implant is compounded with polydeoxyribonucleotides and collagen, the content of polydeoxyribonucleotides in the implant is 10 mg / mL, and the content of collagen is 31 mg / mL.

[0031] The assay method comprises the following steps: The above implant was diluted 100 times with phosphate buffer to obtain a colorless transparent solution, and the absorbance of the diluted colorless transparent solution at ultraviolet light 260nm and 280nm was measured, which were recorded as A1 and A2, respectively, wherein the absorbance of the implant at ultraviolet light 260nm is A260=A1*100, and the absorbance of the implant at ultraviolet light 280nm is A280=A2*100.

[0032] The absorbance A260 of the implant of this embodiment at 260nm ultraviolet light is 1.8087, and the absorbance A280 of the implant at 280nm ultraviolet light is 0.9451. The content of polydeoxyribonucleotides calculated by the formula polydeoxyribonucleotide content = 14.97 A260-18.34A280 is 9.74 mg / mL. The difference with the actual content of polydeoxyribonucleotides in the implant is within 3%.

[0033] The content of polydeoxyribonucleotides was calculated to be 9.45 mg / mL by the formula polydeoxyribonucleotide content = 14.68 A260-18.1 A280, which was within 6% of the actual content of polydeoxyribonucleotides in the implant. Example

[0034] This embodiment provides a method for determining the content of polydeoxyribonucleotides in an implant containing polydeoxyribonucleotides, wherein the implant is compounded with polydeoxyribonucleotides and collagen, the content of polydeoxyribonucleotides in the implant is 10 mg / mL, and the content of collagen is 32 mg / mL.

[0035] The assay method comprises the following steps: The above implant was diluted 100 times with phosphate buffer to obtain a colorless transparent solution, and the absorbance of the diluted colorless transparent solution at ultraviolet light 260nm and 280nm was measured, which were recorded as A1 and A2, respectively, wherein the absorbance of the implant at ultraviolet light 260nm is A260=A1*100, and the absorbance of the implant at ultraviolet light 280nm is A280=A2*100.

[0036] The absorbance A260 of the implant of this embodiment at 260nm ultraviolet light is 2.0136, and the absorbance A280 of the implant at 280nm ultraviolet light is 1.1131. The content of polydeoxyribonucleotides calculated by the formula polydeoxyribonucleotide content = 14.97 A260-18.34A280 is 9.73 mg / mL. The difference with the actual content of polydeoxyribonucleotides in the implant is within 3%.

[0037] The content of polydeoxyribonucleotides was calculated to be 9.41 mg / mL by the formula polydeoxyribonucleotide content = 14.68 A260-18.1 A280, which was within 6% of the actual content of polydeoxyribonucleotides in the implant. Example

[0038] This embodiment provides a method for determining the content of polydeoxyribonucleotides in an implant containing polydeoxyribonucleotides, wherein the implant is compounded with polydeoxyribonucleotides and collagen, the content of polydeoxyribonucleotides in the implant is 10 mg / mL, and the content of collagen is 33 mg / mL.

[0039] The assay method comprises the following steps: The above implant was diluted 100 times with phosphate buffer to obtain a colorless transparent solution, and the absorbance of the diluted colorless transparent solution at ultraviolet light 260nm and 280nm was measured, which were recorded as A1 and A2, respectively, wherein the absorbance of the implant at ultraviolet light 260nm is A260=A1*100, and the absorbance of the implant at ultraviolet light 280nm is A280=A2*100.

[0040] The absorbance A260 of the implant of this embodiment at 260nm ultraviolet light is 2.0592, and the absorbance A280 of the implant at 280nm ultraviolet light is 1.1301. The content of polydeoxyribonucleotides calculated by the formula polydeoxyribonucleotide content = 14.97 A260-18.34A280 is 10.10 mg / mL. The difference with the actual content of polydeoxyribonucleotides in the implant is within 2%.

[0041] The content of polydeoxyribonucleotides was calculated to be 9.77 mg / mL by the formula polydeoxyribonucleotide content = 14.68 A260-18.1 A280, which was within 3% of the actual content of polydeoxyribonucleotides in the implant. Example

[0042] This embodiment provides a method for determining the content of polydeoxyribonucleotides in an implant containing polydeoxyribonucleotides, wherein the implant is compounded with polydeoxyribonucleotides and collagen, the content of polydeoxyribonucleotides in the implant is 10 mg / mL, and the content of collagen is 34 mg / mL.

[0043] The assay method comprises the following steps: The above implant was diluted 100 times with phosphate buffer to obtain a colorless transparent solution, and the absorbance of the diluted colorless transparent solution at ultraviolet light 260nm and 280nm was measured, which were recorded as A1 and A2, respectively, wherein the absorbance of the implant at ultraviolet light 260nm is A260=A1*100, and the absorbance of the implant at ultraviolet light 280nm is A280=A2*100.

[0044] The absorbance A260 of the implant of this embodiment at 260nm ultraviolet light is 1.8319, and the absorbance A280 of the implant at 280nm ultraviolet light is 0.9617. The content of polydeoxyribonucleotides calculated by the formula polydeoxyribonucleotide content = 14.97 A260-18.34A280 is 9.79 mg / mL. The difference with the actual content of polydeoxyribonucleotides in the implant is within 3%.

[0045] The content of polydeoxyribonucleotides was calculated to be 9.49 mg / mL by the formula polydeoxyribonucleotide content = 14.68 A260-18.1 A280, which was within 6% of the actual content of polydeoxyribonucleotides in the implant. Example

[0046] This embodiment provides a method for determining the content of polydeoxyribonucleotides in an implant containing polydeoxyribonucleotides, wherein the implant is compounded with polydeoxyribonucleotides and collagen, the content of polydeoxyribonucleotides in the implant is 10 mg / mL, and the content of collagen is 35 mg / mL.

[0047] The assay method comprises the following steps: The above implant was diluted 100 times with phosphate buffer to obtain a colorless transparent solution, and the absorbance of the diluted colorless transparent solution at ultraviolet light 260nm and 280nm was measured, which were recorded as A1 and A2, respectively, wherein the absorbance of the implant at ultraviolet light 260nm is A260=A1*100, and the absorbance of the implant at ultraviolet light 280nm is A280=A2*100.

[0048] The absorbance A260 of the implant of this embodiment at 260nm ultraviolet light is 1.8852, and the absorbance A280 of the implant at 280nm ultraviolet light is 0.9939. The content of polydeoxyribonucleotides calculated by the formula polydeoxyribonucleotide content = 14.97 A260-18.34A280 is 9.99 mg / mL. The difference with the actual content of polydeoxyribonucleotides in the implant is within 0.1%.

[0049] The content of polydeoxyribonucleotides was calculated to be 9.69 mg / mL by the formula polydeoxyribonucleotide content = 14.68 A260-18.1 A280, which was within 4% of the actual content of polydeoxyribonucleotides in the implant. Example

[0050] This embodiment provides a method for determining the content of polydeoxyribonucleotides in an implant containing polydeoxyribonucleotides, wherein the implant is compounded with polydeoxyribonucleotides and collagen, the content of polydeoxyribonucleotides in the implant is 11 mg / mL, and the content of collagen is 35 mg / mL.

[0051] The assay method comprises the following steps: The above implant was diluted 100 times with phosphate buffer to obtain a colorless transparent solution, and the absorbance of the diluted colorless transparent solution at ultraviolet light 260nm and 280nm was measured, which were recorded as A1 and A2, respectively, wherein the absorbance of the implant at ultraviolet light 260nm is A260=A1*100, and the absorbance of the implant at ultraviolet light 280nm is A280=A2*100.

[0052] The absorbance A260 of the implant of this embodiment at 260nm ultraviolet light is 1.9615, and the absorbance A280 of the implant at 280nm ultraviolet light is 1.0117. The content of polydeoxyribonucleotides calculated by the formula polydeoxyribonucleotide content = 14.97 A260-18.34A280 is 10.81 mg / mL. The difference with the actual content of polydeoxyribonucleotides in the implant is within 2%.

[0053] The polydeoxyribonucleotide content was calculated to be 10.48 mg / mL by the formula polydeoxyribonucleotide content = 14.68 A260-18.1 A280, which was within 6% of the actual polydeoxyribonucleotide content in the implant. Example

[0054] This embodiment provides a method for determining the content of polydeoxyribonucleotides in an implant containing polydeoxyribonucleotides, wherein the implant is compounded with polydeoxyribonucleotides and collagen, the content of polydeoxyribonucleotides in the implant is 12 mg / mL, and the content of collagen is 35 mg / mL.

[0055] The assay method comprises the following steps: The above implant was diluted 100 times with phosphate buffer to obtain a colorless transparent solution, and the absorbance of the diluted colorless transparent solution at ultraviolet light 260nm and 280nm was measured, which were recorded as A1 and A2, respectively, wherein the absorbance of the implant at ultraviolet light 260nm is A260=A1*100, and the absorbance of the implant at ultraviolet light 280nm is A280=A2*100.

[0056] The absorbance A260 of the implant of this embodiment at 260nm ultraviolet light is 2.2485, and the absorbance A280 of the implant at 280nm ultraviolet light is 1.1886. The content of polydeoxyribonucleotides calculated by the formula polydeoxyribonucleotide content = 14.97 A260-18.34A280 is 11.86 mg / mL. The difference with the actual content of polydeoxyribonucleotides in the implant is within 2%.

[0057] The content of polydeoxyribonucleotides was calculated to be 11.50 mg / mL by the formula polydeoxyribonucleotide content = 14.68 A260-18.1 A280, which was within 5% of the actual content of polydeoxyribonucleotides in the implant. Example

[0058] This embodiment provides a method for determining the content of polydeoxyribonucleotides in an implant containing polydeoxyribonucleotides, wherein the implant is compounded with polydeoxyribonucleotides and collagen, the content of polydeoxyribonucleotides in the implant is 13 mg / mL, and the content of collagen is 35 mg / mL.

[0059] The assay method comprises the following steps: The above implant was diluted 100 times with phosphate buffer to obtain a colorless transparent solution, and the absorbance of the diluted colorless transparent solution at ultraviolet light 260nm and 280nm was measured, which were recorded as A1 and A2, respectively, wherein the absorbance of the implant at ultraviolet light 260nm is A260=A1*100, and the absorbance of the implant at ultraviolet light 280nm is A280=A2*100.

[0060] The absorbance A260 of the implant of this embodiment at 260nm ultraviolet light is 2.4465, and the absorbance A280 of the implant at 280nm ultraviolet light is 1.2969. The content of polydeoxyribonucleotides calculated by the formula polydeoxyribonucleotide content = 14.97 A260-18.34A280 is 12.84 mg / mL. The difference with the actual content of polydeoxyribonucleotides in the implant is within 2%.

[0061] The content of polydeoxyribonucleotides was calculated to be 12.44 mg / mL by the formula polydeoxyribonucleotide content = 14.68 A260-18.1 A280, which was within 6% of the actual content of polydeoxyribonucleotides in the implant.

[0062] In summary, the method for determining the content of polydeoxyribonucleotides in an implant containing polydeoxyribonucleotides according to the embodiment of the present application determines the content of polydeoxyribonucleotides by determining the absorbance of the implant at ultraviolet light of 260nm and 280nm, and then determining the content of polydeoxyribonucleotides by calculating the difference between A260 of a first preset multiple and A280 of a second preset multiple. The method can more accurately measure the content of polydeoxyribonucleotides in an implant containing both collagen and polydeoxyribonucleotides, and the determined content of polydeoxyribonucleotides in the implant differs from the actual content of polydeoxyribonucleotides in the implant by 0-6%.

[0063] The above description is only a specific embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for determining the content of polydeoxyribonucleotides in an implant containing polydeoxyribonucleotides, wherein the implant also contains collagen, characterized in that: The determination method comprises the following steps: Determine the absorbance of the implant at 260 nm of ultraviolet light, recorded as A260; Determine the absorbance of the implant at 280 nm of ultraviolet light, recorded as A280; The content of the polydeoxyribonucleotide is determined by calculating the difference between the A260 of the first preset multiple and the A280 of the second preset multiple.

2. The method for determining the content of polydeoxyribonucleotides in the polydeoxyribonucleotide-containing implant according to claim 1, characterized in that: First, the implant is diluted to a colorless transparent solution, and the absorbance of the colorless transparent solution at ultraviolet light of 260 nm and 280 nm is measured, which are recorded as A1 and A2 respectively. The A260 is A1*dilution factor, and the A280 is A2*dilution factor.

3. The method for determining the content of polydeoxyribonucleotides in the polydeoxyribonucleotide-containing implant according to claim 2, characterized in that: The implant is diluted into a colorless transparent solution with a diluent, and the diluent includes any one of phosphate buffer, hydrochloric acid, sodium hydroxide, Tris-HCl buffer and pure water.

4. The method for determining the content of polydeoxyribonucleotide in the polydeoxyribonucleotide-containing implant according to claim 3, characterized in that: The pH of the phosphate buffer or the Tris-HCl buffer is 6-8.

5. The method for determining the content of polydeoxyribonucleotide in the polydeoxyribonucleotide-containing implant according to claim 3, characterized in that: The concentration of the hydrochloric acid or the sodium hydroxide is 0.05-0.5 mol / L.

6. The method for determining the content of polydeoxyribonucleotide in the polydeoxyribonucleotide-containing implant according to any one of claims 1 to 5, characterized in that: The first preset multiple is 14.5~15.

0.

7. The method for determining the content of polydeoxyribonucleotide in the polydeoxyribonucleotide-containing implant according to any one of claims 1 to 5, characterized in that: The second preset multiple is 18~18.

5.

8. The method for determining the content of polydeoxyribonucleotide in the polydeoxyribonucleotide-containing implant according to any one of claims 1 to 5, characterized in that: The first preset multiple is 14.97, and the second preset multiple is 18.

34.

9. The method for determining the content of polydeoxyribonucleotide in the polydeoxyribonucleotide-containing implant according to any one of claims 1 to 5, characterized in that: The first preset multiple is 14.68, and the second preset multiple is 18.1.