CN109468313B - Light-controlled functionalized capillary tube for nucleic acid extraction and preparation method and application thereof - Google Patents
Light-controlled functionalized capillary tube for nucleic acid extraction and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a light-regulated functionalized capillary tube for nucleic acid extraction and a preparation method and application thereof, belonging to the technical field of biological detection. According to the invention, 3-aminopropyltriethoxysilane, 4-bromomethyl-3-nitrobenzoic acid and 4-amino-1-butanol are sequentially modified on the inner surface of the capillary by a chemical modification method, so that the inner surface of the capillary is provided with a large amount of positive charges, and nucleic acid with electronegativity can be adsorbed; and under the irradiation of 302nm ultraviolet light, the chemical bond generated by the reaction of the 4-bromomethyl-3-nitrobenzoic acid and the 4-amino-1-butanol is broken, so that the nucleic acid adsorbed on the inner surface of the capillary is released into the liquid phase again for the next detection. The invention greatly simplifies the operation process of nucleic acid separation and extraction, shortens the time of nucleic acid separation and extraction, and can utilize ultraviolet irradiation to re-release the extracted nucleic acid molecules into the liquid phase for subsequent storage or detection.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a light-controlled functionalized capillary tube for nucleic acid extraction, and a preparation method and application thereof.
Background
The nucleic acid amplification detection has the characteristics of high sensitivity, strong specificity and the like, and is considered as a powerful weapon for early diagnosis of diseases. Nucleic acid extraction is the primary core step in the nucleic acid amplification detection process. The method mainly aims to extract a nucleic acid sample to be detected with high purity from a complex actual sample, eliminate biomacromolecules such as protein, polysaccharide, fat and the like, and avoid the influence of the biomacromolecules on downstream nucleic acid amplification and detection, thereby ensuring the accuracy of nucleic acid analysis to the maximum extent.
The current commercialized nucleic acid extraction methods mainly include a centrifugal column method and a magnetic separation method. The method needs complicated manual operation and professional test equipment, has the defects of long time consumption and the like, and greatly limits the clinical popularization and application of the method. Therefore, there is a need to develop a simple and fast nucleic acid isolation and extraction device.
Disclosure of Invention
In order to overcome the disadvantages and shortcomings of the prior art, the primary object of the present invention is to provide a light-regulated functionalized capillary for nucleic acid extraction. By a chemical modification method, 3-aminopropyltriethoxysilane (3-aminopropy) triethoxysilane, APTES, 4-bromomethyl-3-nitrobenzoic acid (4-bromomethyl-3-nitrobenzoic acid) and 4-Amino-1-butanol (4-Amino-1-butanol) are sequentially modified on the inner surface of a capillary, so that the inner surface of the capillary is provided with a large amount of positive charges, nucleic acid with electronegativity can be adsorbed, and the purposes of simply and quickly separating and extracting the nucleic acid are achieved. And under the irradiation of 302nm ultraviolet light, the chemical bond generated by the reaction of the 4-bromomethyl-3-nitrobenzoic acid and the 4-amino-1-butanol can be broken, so that the DNA adsorbed on the inner surface of the capillary tube is released into the liquid phase again for the next detection.
Another object of the present invention is to provide a method for preparing the above-mentioned light-controlled functionalized capillary for nucleic acid extraction.
Another object of the present invention is to provide the use of the above-mentioned light-regulated functionalized capillary for nucleic acid extraction.
It is still another object of the present invention to provide a kit comprising a light-regulated functionalized capillary for nucleic acid extraction.
The purpose of the invention is realized by the following technical scheme:
a light-controlled functionalized capillary for nucleic acid extraction is characterized in that 3-aminopropyltriethoxysilane (3-aminopropy) triethoxysilane (APTES), 4-bromomethyl-3-nitrobenzoic acid (4-bromomethyl-3-nitrobenzoic acid) and 4-Amino-1-butanol (4-Amino-1-butanol) are sequentially modified on the inner surface of the capillary by a chemical modification method, so that the inner surface of the capillary is provided with a large amount of positive charges, and nucleic acid with electronegativity can be adsorbed; and under the irradiation of 302nm ultraviolet light, the chemical bond generated by the reaction of the 4-bromomethyl-3-nitrobenzoic acid and the 4-amino-1-butanol can be broken, so that the nucleic acid adsorbed on the inner surface of the capillary can be released into the liquid phase again for the next detection. The chemical labeling principle is shown in FIG. 1.
The preparation method of the light-regulated functionalized capillary for nucleic acid extraction comprises the following steps:
(1) cleaning the interior of the capillary tube with hydrochloric acid;
(2) cleaning the interior of the capillary tube with deionized water;
(3) cleaning the interior of the capillary tube with NaOH solution;
(4) cleaning the interior of the capillary tube with deionized water;
(5) placing the cleaned capillary tube in a drying box for drying;
(6) placing the dried capillary tube in an ethanol solution of 3-aminopropyltriethoxysilane, and standing at room temperature in a dark place;
(7) cleaning the interior of the capillary with ethanol;
(8) placing the cleaned capillary tube in a drying box for drying;
(9) continuously introducing a 4-bromomethyl-3-nitrobenzoic acid solution into the dried capillary, and heating the capillary to keep the temperature of the capillary constant;
(10) cleaning the interior of the capillary tube with N, N-dimethylformamide;
(11) continuously introducing a 4-amino-1-butanol solution into the capillary;
(12) cleaning the interior of the capillary tube with N, N-dimethylformamide;
(13) and (3) drying the cleaned capillary in a drying oven to obtain the light-regulated and controlled functionalized capillary for nucleic acid extraction.
The cleaning time from the step (1) to the step (4) is 30min to 90min, preferably 60 min;
the cleaning time in the steps (7), (10) and (12) is 1-20 min, preferably 10 min;
the drying conditions in the steps (5), (8) and (13) are drying at 30-70 ℃ for 10 min-10 h, preferably drying at 50 ℃ for 3 h;
the standing time in the step (6) is 5-24 h, preferably 10 h;
the time for continuously introducing the 4-bromomethyl-3-nitrobenzoic acid solution in the step (9) is 10 to 72 hours, preferably 48 hours;
the time for continuously introducing the 4-amino-1-butanol solution in the step (11) is 10 to 48 hours, preferably 24 hours;
the heating temperature in the step (9) is 40-60 ℃, and the optimal temperature is 50 ℃;
the flow rate of the liquid in the steps (1) to (4) and the steps (7), (10) and (12) is 1 to 100. mu.L/min, preferably 10. mu.L/min;
the flow rate of the liquid in the steps (9) and (11) is 10mL/min to 100mL/min, preferably 50 mL/min;
the concentration of the hydrochloric acid in the step (1) is 0.5M-5M, and preferably 1M;
the concentration of the NaOH solution in the step (3) is 0.5M-5M, and preferably 1M;
the concentration of the ethanol solution of the 3-aminopropyltriethoxysilane in the step (6) is 2-10% (v/v), preferably 5% (v/v);
the 4-bromomethyl-3-nitrobenzoic acid solution in the step (9) adopts N, N-dimethylformamide as a solvent, and the concentration of the N, N-dimethylformamide is 4 mg/mL-100 mg/mL, preferably 40 mg/mL; dicyclohexylcarbodiimide (Dicyclohexylcarbodiimide) is added as a catalyst, and the concentration of the Dicyclohexylcarbodiimide is 3 mg/mL-75 mg/mL, preferably 30 mg/mL;
the 4-amino-1-butanol solution in the step (11) is N, N-dimethylformamide with the concentration of 1.8 mg/mL-45 mg/mL, preferably 18 mg/mL; furthermore, triethylamine is added as a catalyst, and the concentration of the triethylamine is 10-50% (v/v), preferably 25% (v/v).
The light-regulated functionalized capillary for nucleic acid extraction is applied to nucleic acid extraction.
A kit for extracting nucleic acid comprises the light-regulated functionalized capillary for extracting nucleic acid.
The kit further comprises a TE buffer solution.
The kit is used for simple and rapid nucleic acid extraction, and specifically comprises the following steps:
firstly, centrifuging a bacterial culture solution, removing a supernatant, and collecting thalli;
secondly, re-suspending the collected thalli by using a TE buffer solution, and fully and uniformly mixing;
placing the heavy suspension in a water bath, and heating;
fourthly, taking out and standing at room temperature;
carefully sucking supernatant liquid to at least 1/4 position in the tube by using the light-regulated functionalized capillary for nucleic acid extraction, and inclining the capillary left and right to make the solution flow left and right in the capillary; preferably to position 2/3; when the suction range is up to the full suction, the liquid is not allowed to flow after the full suction, and the liquid may be left to stand for 20 seconds or more.
Sixthly, vertically placing the capillary tube, so that most of liquid flows out of the capillary tube under the action of gravity, and forcibly throwing out the residual small amount of liquid;
seventhly, repeating the fifth step and the sixth step once, and separating to obtain nucleic acid molecules with electronegativity under the action of an electrostatic adsorption principle inside the capillary;
absorbing deionized water or TE buffer into the capillary tube by the same method, and irradiating by ultraviolet light of 302nm to release nucleic acid molecules into liquid phase again for subsequent storage or detection.
The heating condition in the step (iii) is heating at 90-100 ℃ for 5-20 min, preferably heating at 95 ℃ for 10 min;
the standing time in the step (iv) is 1min or more, preferably 5 min; the purpose of this step is: in the high concentration bacterial condition, the broken cell wall and other components sufficiently precipitate to improve the purity of the extracted DNA, in the low concentration condition, this step can be omitted.
The time of the left-right flow in the fifth step is 20s or more, preferably 1 min.
The time of the ultraviolet irradiation in the step (viii) is 10min to 1h, preferably 30 min.
Compared with the prior art, the invention has the following advantages and effects:
(1) the invention greatly simplifies the operation process of nucleic acid separation and extraction and shortens the time of nucleic acid separation and extraction.
(2) The device is used for separating and extracting nucleic acid, does not need additional equipment for assistance, and is simple to operate and high in efficiency.
(3) The device is convenient to integrate, and can realize automatic extraction of nucleic acid.
(4) The separated nucleic acid can be directly amplified and detected in situ.
(5) The invention can utilize ultraviolet light irradiation to re-release the extracted nucleic acid molecules into the liquid phase for subsequent storage or detection by a chemical modification method.
Drawings
FIG. 1 is a schematic diagram of the basic principle of the present invention, i.e., a method for marking the inside of a capillary tube.
FIG. 2 shows the results of the verification of the labeling process inside the capillary and the results of the assay which can be used for nucleic acid extraction and release.
FIG. 3 shows the result of nucleic acid extraction using the functionalized capillary, amplification of the extracted nucleic acid molecules, and detection of the amplified nucleic acid molecules.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The reagents used in the examples were purchased from New England Biotechnology, capillary from Western medical university and Vibrio Parahaemolyticus (Vibrio Parahaemolyticus) from institute of microorganisms, Guangdong province.
Example 1
1. Glass capillary internal surface marking
(1) The interior of the capillary was cleaned with 1M hydrochloric acid for 1 hour.
(2) The inside of the capillary was washed with deionized water for 1 hour.
(3) The inside of the capillary was washed with a 1M NaOH solution for 1 hour.
(4) The inside of the capillary was washed with deionized water for 1 hour.
(5) Placing the cleaned capillary tube in a drying oven, and drying at 50 ℃ for 3 h;
(6) placing the dried capillary tube in 5% ethanol solution of 3-aminopropyltriethoxysilane, and standing in dark at room temperature for 10 hr;
(7) cleaning the interior of the capillary tube with ethanol for 10 min;
(8) drying the cleaned capillary tube in a drying oven at 50 ℃ for 3 h;
(9) a solution of 4-bromomethyl-3-nitrobenzoic acid at a concentration of 40mg/mL was continuously introduced into the dried capillary tube in the presence of N, N-dimethylformamide and Dicyclohexylcarbodiimide (Dicyclohexylcarbodiimide) as a catalyst at a concentration of 30 mg/mL. While the capillary was heated to maintain its temperature at 50 ℃. The duration of the process was 48 h.
(10) Cleaning the interior of the capillary tube for 10min by using N, N-dimethylformamide;
(11) 4-amino-1-butanol solution with the concentration of 18mg/mL is continuously introduced into the capillary, the solvent is N, N-dimethylformamide, and triethylamine is required to be added as a catalyst, and the concentration of the triethylamine is 25%. The duration of the process is 24 h;
(12) cleaning the interior of the capillary tube for 10min by using N, N-dimethylformamide;
(13) and (3) drying the cleaned capillary tube in a drying oven at 50 ℃ for 3h to obtain the light-regulated and controlled functionalized capillary tube for nucleic acid extraction.
2. Verification of inner surface modification result of capillary tube
(1) FITC dye with electronegativity is introduced into the labeled capillary, the capillary is tilted left and right, and the FITC solution flows left and right in the capillary for 2 minutes. The interior of the capillary was then rinsed with deionized water.
(2) The capillary was irradiated with 535nm light and the results were observed with a 475nm filter.
The results of the tests are shown in FIG. 2, and the results are now analyzed as follows:
(a) the figure shows the results before (NC) and after (NC) labeling of 3-aminopropyltriethoxysilane. The capillary tube contains a large amount of amino groups inside due to the marking of 3-aminopropyltriethoxysilane. The amino group with positive charge can adsorb FITC dye with negative charge, so that a fluorescence signal is generated inside the capillary.
(b) The figure shows the results after labeling 4-bromomethyl-3-nitrobenzoic acid (NC). After the 4-bromomethyl-3-nitrobenzoic acid is labeled, a large number of amino sites are covered, so that the number of positive charges on the inner surface of the capillary is reduced, and the fluorescence signal intensity is reduced after FITC dye is introduced.
(c) The figure shows the results of labeling 4-amino-1-butanol pre- (NC) and post (+). After the 4-amino-1-butanol is marked, the content of amino groups in the capillary is recovered, so that the fluorescence signal intensity is recovered after FITC is introduced.
(d) The figure shows the results before (NC) and after (+) irradiation with 302nm UV light. After irradiation, the chemical bond linking 4-bromomethyl-3-nitrobenzoic acid to 4-amino-1-butanol is cleaved, allowing the FITC dye to be released back into the liquid phase. At this time, the intensity of the fluorescence signal in the capillary tube is reduced after the capillary tube is cleaned.
The experimental result shows that each chemical modification step can be completed correctly, so that the light-regulated and controlled functionalized capillary tube is obtained and used for separating nucleic acid molecules in sample lysate, and the purified nucleic acid molecules are released into a liquid phase again under the action of ultraviolet light for further storage or detection.
Example 2
1. Vibrio parahaemolyticus DNA extraction
(1) 1mL of overnight-cultured bacterial culture was added to a 1.5mL centrifuge tube, centrifuged at 10000rpm for 2min, and the supernatant was discarded to collect the cells.
(2) The collected cells were resuspended in 400mL of TE buffer and mixed well.
(3) Placing the heavy suspension in a water bath at 95 deg.C, and heating for 10 min.
(4) Taking out the centrifuge tube, and standing for 5min at room temperature.
(5) The supernatant was carefully drawn up by capillary tubing to a position 2/3 in the tube, and the capillary tubing was tilted left and right to allow the solution to flow left and right in the capillary tubing for 1 minute.
(6) The capillary tube is positioned vertically so that most of the liquid flows out of the capillary tube under the influence of gravity. And (4) forcibly throwing the residual small amount of liquid to a waste liquid pool.
(7) Repeating the steps (5) and (6) once, and separating to obtain nucleic acid molecules with electronegativity under the action of electrostatic adsorption principle in the capillary;
(8) the deionized water or TE buffer is absorbed into the capillary by the same method, and the ultraviolet light with the wavelength of 302nm is used for irradiating for 30min, so that the nucleic acid molecules can be released into the liquid phase again and can be used for subsequent storage or detection.
(9) And carrying out PCR amplification on the recovered nucleic acid molecule sample, and then detecting by electrophoresis.
(10) The steps (1) to (9) were repeated using water instead of the bacterial culture as a control experiment.
The results of the detection are shown in FIG. 3. Test results prove that the method can be used for quickly separating and extracting nucleic acid.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A light-modulated functionalized capillary for nucleic acid extraction, characterized in that: sequentially modifying 3-aminopropyltriethoxysilane, 4-bromomethyl-3-nitrobenzoic acid and 4-amino-1-butanol on the inner surface of the capillary by a chemical modification method to ensure that the inner surface of the capillary has a large number of positive charges so as to adsorb nucleic acid with electronegativity; and under the irradiation of 302nm ultraviolet light, the chemical bond generated by the reaction of the 4-bromomethyl-3-nitrobenzoic acid and the 4-amino-1-butanol is broken, so that the nucleic acid adsorbed on the inner surface of the capillary is released into the liquid phase again.
2. The method of preparing a light-regulated functionalized capillary for nucleic acid extraction according to claim 1, wherein the method comprises the steps of: the method comprises the following steps:
(1) cleaning the interior of the capillary tube with hydrochloric acid;
(2) cleaning the interior of the capillary tube with deionized water;
(3) cleaning the interior of the capillary tube with NaOH solution;
(4) cleaning the interior of the capillary tube with deionized water;
(5) drying the cleaned capillary;
(6) placing the dried capillary tube in an ethanol solution of 3-aminopropyltriethoxysilane, and standing for 5-24 h at room temperature in a dark place;
(7) cleaning the interior of the capillary with ethanol;
(8) drying the cleaned capillary;
(9) continuously introducing a 4-bromomethyl-3-nitrobenzoic acid solution into the dried capillary for 10 to 72 hours, and heating the capillary at the same time to keep the temperature of the capillary constant; the heating temperature is 40-60 ℃;
(10) cleaning the interior of the capillary tube with N, N-dimethylformamide;
(11) continuously introducing a 4-amino-1-butanol solution into the capillary for 10 to 48 hours;
(12) cleaning the interior of the capillary tube with N, N-dimethylformamide;
(13) and drying the cleaned capillary to obtain the light-regulated functionalized capillary for nucleic acid extraction.
3. The method of preparing a light-controlled functionalized capillary tube for nucleic acid extraction according to claim 2, wherein:
the cleaning time from the step (1) to the step (4) is 30-90 min;
the cleaning time in the steps (7), (10) and (12) is 1-20 min;
the drying conditions in the steps (5), (8) and (13) are drying for 10 min-10 h at 30-70 ℃.
4. The method of preparing a light-controlled functionalized capillary tube for nucleic acid extraction according to claim 2, wherein:
the time for continuously introducing the 4-bromomethyl-3-nitrobenzoic acid solution in the step (9) is 48 hours;
and (3) continuously introducing the 4-amino-1-butanol solution in the step (11) for 24 hours.
5. The method of preparing a light-controlled functionalized capillary tube for nucleic acid extraction according to claim 2, wherein:
the flow rate of the liquid in the steps (1) to (4) and the steps (7), (10) and (12) is 1-100 mu L/min;
the flow rate of the liquid in the steps (9) and (11) is 10mL/min to 100 mL/min;
the concentration of the ethanol solution of the 3-aminopropyltriethoxysilane in the step (6) is 2-10%.
6. The method of preparing a light-controlled functionalized capillary tube for nucleic acid extraction according to claim 2, wherein:
the 4-bromomethyl-3-nitrobenzoic acid solution in the step (9) adopts N, N-dimethylformamide with the concentration of 4 mg/mL-100 mg/mL; dicyclohexylcarbodiimide was added as a catalyst at a concentration of 3mg/mL to 75 mg/mL.
7. The method of preparing a light-controlled functionalized capillary tube for nucleic acid extraction according to claim 2, wherein:
the 4-amino-1-butanol solution in the step (11) is N, N-dimethylformamide with the concentration of 1.8 mg/mL-45 mg/mL; in addition, triethylamine is required to be added as a catalyst, and the concentration of the triethylamine is 10-50%.
8. Use of the light-regulated functionalized capillary for nucleic acid extraction according to claim 1 in nucleic acid extraction.
9. A kit for extracting nucleic acid, characterized in that: a functionalized capillary for nucleic acid extraction comprising the light modulation of claim 1.
10. A method for extracting nucleic acid using the kit according to claim 9, characterized in that: the method comprises the following steps:
firstly, centrifuging a bacterial culture solution, removing a supernatant, and collecting thalli;
secondly, re-suspending the collected thalli by using a TE buffer solution, and fully and uniformly mixing;
placing the heavy suspension in a water bath, and heating;
fourthly, taking out and standing at room temperature;
fifthly, carefully absorbing the supernatant to at least 1/4 position in the tube by using the light-controlled functionalized capillary for nucleic acid extraction of claim 1, and inclining the capillary left and right to make the solution flow left and right in the capillary; when the suction range is full, the liquid can not flow after full suction, and the liquid is kept still for 20s or more;
sixthly, vertically placing the capillary tube, so that most of liquid flows out of the capillary tube under the action of gravity, and forcibly throwing out the residual small amount of liquid;
seventhly, repeating the fifth step and the sixth step once, and separating to obtain nucleic acid molecules with electronegativity under the action of an electrostatic adsorption principle inside the capillary;
sucking deionized water or TE buffer into the capillary tube by the same method, and irradiating by ultraviolet light of 302nm to release nucleic acid molecules into the liquid phase again.
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| CN101018870A (en) * | 2004-07-15 | 2007-08-15 | 鲁米根公司 | Methods of using cleavable solid phases for isolating nucleic acids |
| CN108456673A (en) * | 2017-02-20 | 2018-08-28 | 华南师范大学 | A kind of functionalization capillary and the preparation method and application thereof for nucleic acid extraction |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101018870A (en) * | 2004-07-15 | 2007-08-15 | 鲁米根公司 | Methods of using cleavable solid phases for isolating nucleic acids |
| CN108456673A (en) * | 2017-02-20 | 2018-08-28 | 华南师范大学 | A kind of functionalization capillary and the preparation method and application thereof for nucleic acid extraction |
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| UV- and visible-excited fluorescence of nucleic acids separated by capillary electrophoresis;Zabzdyr JL 等;《J Chromatogr A》;20011231;第911卷(第2期);第269-276页 * |
| 核酸提取方法的研究进展;张丽 等;《中国动物检疫》;20111231;第28卷(第12期);第75-78页 * |
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