CN114890477B - Organic solvent response type magnetic bead, preparation method and application thereof - Google Patents
Organic solvent response type magnetic bead, preparation method and application thereof Download PDFInfo
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- 239000011737 fluorine Substances 0.000 claims abstract description 8
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- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 3
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- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01G—COMPOUNDS CONTAINING METALS NOT COVERED BY SUBCLASSES C01D OR C01F
- C01G49/00—Compounds of iron
- C01G49/02—Oxides; Hydroxides
- C01G49/08—Ferroso-ferric oxide (Fe3O4)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2002/00—Crystal-structural characteristics
- C01P2002/80—Crystal-structural characteristics defined by measured data other than those specified in group C01P2002/70
- C01P2002/82—Crystal-structural characteristics defined by measured data other than those specified in group C01P2002/70 by IR- or Raman-data
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2004/00—Particle morphology
- C01P2004/01—Particle morphology depicted by an image
- C01P2004/03—Particle morphology depicted by an image obtained by SEM
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2004/00—Particle morphology
- C01P2004/01—Particle morphology depicted by an image
- C01P2004/04—Particle morphology depicted by an image obtained by TEM, STEM, STM or AFM
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2006/00—Physical properties of inorganic compounds
- C01P2006/42—Magnetic properties
Abstract
The invention discloses an organic solvent response type magnetic bead, which comprises ferroferric oxide particles and surfactant molecules wrapped outside the ferroferric oxide particles, wherein the surfactant molecules are chain molecules, one end of each surfactant molecule is carboxyl, the other end of each surfactant molecule is fluoroalkyl, and the carboxyl end of each surfactant molecule is at one side far away from the ferroferric oxide particles. The invention also discloses a preparation method of the organic solvent response type magnetic beads, which comprises the following steps: placing 10-150 parts by mass of fluorine-containing surfactant, 10-120 parts by mass of ferroferric oxide particles, 10-150 parts by mass of film-forming resin and 2-3 parts by mass of dispersing agent into 300 parts by mass of water, heating, stirring at high speed by a magnetic stirrer while heating, and dispersing by ultrasonic after stirring; and magnetically separating the obtained solution to obtain solid precipitate, and drying to obtain the organic solvent response type magnetic beads.
Description
Technical Field
The invention relates to an organic solvent response type magnetic bead, and also relates to a preparation method of the organic solvent response type magnetic bead and application of the organic solvent response type magnetic bead in nucleic acid extraction.
Background
Magnetic nanoparticles have been widely used in biomedical fields in recent years as an important component in materials. This is because it has similar particle size and structure with most biological macromolecules such as cells, viruses, proteins, nucleic acids, etc. that constitute living bodies, and has the advantages of large coupling capacity, large specific surface area, fast diffusion speed, easy modification, high suspension stability, and good biocompatibility. Because the nano magnetic particles have high saturation magnetization when being influenced by a magnetic field and can realize intellectualization and automation, the nano magnetic particles are widely applied to separation and extraction, loading, enrichment, targeted manipulation and the like of nucleic acid, protein, pathogenic bacteria, cells and other substances.
However, for most magnetic beads for nucleic acid extraction, the extraction rate is 70-80%, and about 3 times of elution process are required, which is required for common nucleic acid detection, and the number of copies of nucleic acid in a sample is hundreds of copies to be detected by fluorescent quantitative PCR or digital PCR, so that effective detection cannot be performed on low-abundance samples. In addition, the method has the advantages of complex operation steps and long time consumption due to the large number of times of elution, and is easy to cause loss of nucleic acid, so that the method can not meet the requirement of rapid detection of low-abundance samples.
Disclosure of Invention
The invention aims to: the invention aims to provide an organic solvent response type magnetic bead capable of rapidly detecting the concentration of a low-abundance nucleic acid sample; the invention also aims to provide a preparation method of the organic solvent response type magnetic beads and application of the organic solvent response type magnetic beads in nucleic acid extraction.
The technical scheme is as follows: the organic solvent response type magnetic bead comprises ferroferric oxide particles and surfactant molecules wrapped outside the ferroferric oxide particles, wherein the surfactant molecules are chain molecules, one end of each surfactant molecule is carboxyl, the other end of each surfactant molecule is fluoroalkyl, and the carboxyl end of each surfactant molecule is at one side far away from the ferroferric oxide particles. The loading of the surfactant was 50% of the mass of the magnetic beads.
The preparation method of the organic solvent response type magnetic beads specifically comprises the following steps: placing 10-150 parts by mass of fluorine-containing surfactant, 10-120 parts by mass of ferroferric oxide particles, 10-150 parts by mass of film-forming resin and 2-3 parts by mass of dispersing agent in 300 parts by mass of water, heating, stirring at a high speed by a magnetic stirrer, and performing ultrasonic dispersion after stirring (the effect of ultrasonic dispersion after stirring is that of sterilizing, removing pollution impurities and removing superfluous surfactant at the same time); and magnetically separating the obtained solution to obtain solid precipitate, and drying to obtain the organic solvent response type magnetic beads.
Wherein the stirring time is 40-60 min, the stirring speed is 3000rpm, the original ferroferric oxide particles are easily damaged due to the too high stirring speed, and the stirring speed is too slow to achieve the effect of uniform dispersion.
Wherein the heating temperature is 80-90 ℃, and the heating can lead the surfactant to be dispersed more uniformly in the system.
Wherein the drying temperature is 60 ℃.
Wherein the organic solvent response type magnetic beads are spherical particles with the particle size of 400-600 nm.
Wherein the fluorine-containing surfactant is at least one of cationic surfactant, anionic surfactant, nonionic surfactant and amphoteric surfactant, and the end group of the fluorine-containing surfactant is fluoroalkyl.
The ferroferric oxide particles are prepared by the following method: adding 10 parts by mass of organic ferric salt into 400 parts by mass of dihydric alcohol, and uniformly stirring to obtain a transparent solution; adding 60 parts by mass of unsaturated fatty acid into the transparent solution, and uniformly stirring to obtain a mixed solution; and (3) placing the mixed solution into a reaction kettle to react for 10-12 hours at 160-190 ℃, magnetically separating the solution after the reaction to obtain solid precipitate, and washing and drying the solid precipitate to obtain the ferroferric oxide particles.
The magnetic beads can be used for nucleic acid extraction, the surfactant coated outside the ferroferric oxide spherical particles is rich in carboxyl, the nucleic acid can be extracted in an acid solution based on a positive and negative electric combination mechanism, in a nucleic acid desorption link, ethanol and/or isopropanol is used for treating the magnetic beads, an induction effect (traction effect) can be generated on fluoroalkyl in surfactant molecules by using ethanol and/or isopropanol as an organic solvent, so that the end groups of the surfactant molecules on the outer surfaces of the ferroferric oxide particles are reversed, and the carboxyl which is originally far from the magnetic bead ends and used for preventing the nucleic acid from being desorbed is reversed, namely the carboxyl end is reversed from the side far from the ferroferric oxide particles to the side close to the ferroferric oxide particles, and at the moment, the fluoroalkyl of the surfactant molecules is outwards oriented, so that the magnetic beads are fully desorbed in high efficiency during the nucleic acid desorption under the double effects of positive and negative electric effects and the carboxyl inversion.
The beneficial effects are that: (1) The magnetic beads solve the problems of incomplete desorption and lower desorption efficiency caused by the obstruction of an ion bridge formed by surface carboxyl groups and nucleic acid of the existing nucleic acid extraction magnetic beads, improve the extraction rate of the existing nucleic acid extraction magnetic beads by 12.5%, and improve the detection limit of a nucleic acid digital LAMP detection method based on the magnetic beads by 20 times; (2) The carboxyl turnover is reversible, so that the magnetic beads can be repeatedly used for 20-30 times without affecting the extraction effect, and have good adaptability to complex environments (such as samples of blood, saliva and the like); (3) The magnetic beads can be well combined with fluorescent quantitative PCR and digital PCR technologies, and when the magnetic beads are combined with the digital PCR technologies, the detection sensitivity of the nucleic acid sample with low abundance is improved by more than 10 times, so that the rapid detection of the concentration of the nucleic acid sample with low abundance (less than 100 copies) is realized; (4) The magnetic beads have good superparamagnetism and have good application prospect in vivo radiography and targeted drug delivery.
Drawings
FIG. 1 is an SEM image of the organic solvent-responsive magnetic beads prepared in example 1;
FIG. 2 is a schematic illustration of rapid aggregation of organic solvent responsive magnetic beads in the vicinity of a magnet;
FIG. 3 is a graph of the results of magnetic property testing of organic solvent responsive magnetic beads and original ferroferric oxide particles;
fig. 4a is a static contact angle of a water drop in an initial state of an organic solvent-responsive magnetic bead, and a static contact angle of a water drop after treatment with ethanol and/or isopropanol; FIG. 4b is a static contact angle of a water drop in the initial state of a conventional commercial magnetic bead, after treatment with ethanol and/or isopropanol; FIG. 4c shows the state of the organic solvent-responsive magnetic beads during the nucleic acid desorption process and the state of the conventional commercial magnetic beads;
FIG. 5 is a schematic diagram showing the nucleic acid concentration change of a sample after adsorption and desorption of a sample by using an ultra-micro nucleic acid detector to measure the concentration of nucleic acid in a 100 mu L sample obtained by establishing a standard curve based on the absorbance corresponding to a standard solution and obtaining the standard curve;
FIG. 6 is a diagram showing the nucleic acid concentration change of a sample after adsorption and desorption of a sample by using an ultra-micro nucleic acid detector to determine the concentration of nucleic acid in a 100 mu L sample obtained by establishing a standard curve based on the absorbance corresponding to a standard solution and obtaining the standard curve;
FIG. 7 shows the nucleic acid extraction yield of organic solvent-responsive magnetic beads under different adsorption and desorption conditions;
FIGS. 8a-c are graphs of detection results obtained by diluting low abundance RNA nucleic acid templates into sample templates (5 Copies,10Copies,25Copies,35Copies,45Copies,55Copies,100Copies,200Copies,300Copies,400 Copies) of different concentrations at room temperature, extracting with organic solvent-responsive magnetic beads under optimal extraction conditions, and performing digital LAMP detection; FIG. 8d is a digital LAMP detection contrast plot of organic solvent-responsive magnetic beads and conventional commercial magnetic beads.
FIG. 9 is a schematic diagram showing the process of adsorbing and desorbing nucleic acids by using the organic solvent-responsive magnetic beads prepared in example 1;
FIG. 10 is an infrared spectrum of the organic solvent-responsive magnetic beads and conventional commercial magnetic beads prepared in example 1;
FIG. 11a is a TEM image and element distribution diagram of a conventional commercial magnetic bead; FIG. 11b is a TEM image and an element distribution diagram of the organic solvent-responsive magnetic beads prepared in example 1.
The technical scheme of the invention is further described below with reference to the accompanying drawings.
Example 1
The invention relates to a preparation method of organic solvent response type magnetic beads, which specifically comprises the following steps:
(1) At room temperature, firstly preparing ferroferric oxide spherical particles: adding 10 parts by mass of organic ferric salt into 400 parts by mass of dihydric alcohol, and uniformly stirring to obtain a transparent solution; adding 60 parts by mass of unsaturated fatty acid into the transparent solution, and uniformly stirring to obtain a mixed solution; transferring the obtained mixed solution into a hydrothermal reaction kettle, heating the reaction kettle to 160 ℃ and keeping the temperature for 10 hours to obtain a black mixed solution; performing magnetic separation on the obtained black solution to obtain black solid precipitate, washing the black solid with ethanol for 3 times, and drying in a 60 ℃ oven to obtain ferroferric oxide magnetic spherical particles with the particle size of 300-400 nm;
(2) 10 parts by mass of a fluorine-containing surfactant, 10 parts by mass of ferroferric oxide spherical particles, 10 parts by mass of a film-forming resin and 2 parts by mass of a dispersing agent are placed in 300 parts by mass of water and heated, a magnetic stirrer is used for stirring at a high speed of 3000rpm for 40min while heating, after stirring, ultrasonic dispersion is carried out for 20min, a black solution is obtained, the obtained black solution is magnetically separated, a black solid precipitate is obtained, and the black solid is dried in a 60 ℃ oven, so that the organic solvent response type magnetic beads are obtained. As can be seen from FIG. 1, the size of the obtained magnetic beads is substantially uniform, and the particle size of the magnetic beads is 500-600 nm. The shape of the magnetic beads is not obviously changed from the prior magnetic beads on an SEM image.
The application of the magnetic beads in the aspect of nucleic acid extraction comprises the following specific application processes:
step 1, sample pretreatment and cracking: grinding fresh or ultralow-temperature stored sample (less than or equal to 100 mg) with liquid nitrogen into powder, transferring the powder to an EP tube as completely as possible, adding 1mL of lysate into the EP tube, carrying out vortex oscillation until no obvious particulate matters exist, and standing for 5min at room temperature;
step 2, nucleic acid binding: 400. Mu.L of a nucleic acid binding solution (mainly containing sodium chloride and dithiothreitol) was added to the EP tube, followed by adding 30mg of the organic solvent-responsive magnetic beads prepared in example 1 (as shown in FIG. 9 a) and adjusting the pH to 5, shaking the magnetic bead suspension uniformly, and further subjecting it to high-speed vortex shaking at 3000rpm for 11 minutes to bind the nucleic acid cleaved from the sample to the magnetic beads (as shown in FIG. 9 b);
step 3, magnetic separation: placing the EP tube on a magnetic frame for standing for 20s until the magnetic beads are completely adsorbed, if the magnetic bead residual liquid exists in the inner cover of the EP tube, keeping the EP tube on the magnetic frame, and integrally reversing the EP tube for 2-3 times to ensure that the magnetic beads are completely adsorbed, and sucking the supernatant to avoid contacting the magnetic beads;
step 4, cleaning: adding 600 mu L of cleaning liquid I (the main component of the cleaning liquid I is isopropanol) into the EP pipe, blowing off magnetic beads by using a pipetting gun, carrying out vortex oscillation for 2min, and carrying out magnetic separation again according to the step 3; adding 600 mu L of cleaning liquid II (the main component of the cleaning liquid II is ethanol) into the EP pipe after magnetic separation, blowing off magnetic beads by using a liquid-transferring gun, carrying out vortex oscillation for 2min, carrying out magnetic separation again according to the step 3, adding 600 mu L of cleaning liquid II into the EP pipe after magnetic separation, blowing off magnetic beads by using the liquid-transferring gun, carrying out vortex oscillation for 2min, and carrying out magnetic separation again according to the step 3;
step 5, removing alcohol: the EP tube after the supernatant is discarded is kept on a magnetic rack, the EP tube cover is opened, the EP tube is dried for about 8 minutes at room temperature until no obvious ethanol smell exists, in the step, carboxyl groups on the surface of the magnetic beads are overturned (as shown in fig. 9 c), and the nucleic acid is separated from the magnetic beads;
step 6, elution: adding 30-100 mu L of eluent (mainly deionized water with pH of about 8) into an EP pipe, and carrying out vortex oscillation until magnetic beads are completely dispersed in a liquid phase, carrying out warm bath for 5min at 58 ℃ and carrying out vortex oscillation for 10s every 2min (as shown in figure 9 d);
step 7, nucleic acid transfer: after the elution is finished, the EP tube is placed on a magnetic rack for standing for 20 seconds until the magnetic beads are completely adsorbed, the eluent is transferred into another clean EP tube by a liquid-transferring gun, and the magnetic beads can be discarded after the extraction process is finished.
After the organic solvent-responsive magnetic beads prepared in example 1 were dispersed with deionized water, neodymium-iron-boron magnets were placed outside the glass bottle of well-dispersed magnetic bead aqueous solution, the organic solvent-responsive magnetic beads had superparamagnetism and rapidly accumulated in the vicinity of the magnets, as shown in fig. 2, and after the magnets were removed, the organic solvent-responsive magnetic beads were re-dispersed in deionized water. The magnetic properties of the samples were tested using a vibrating magnetometer function, the test temperature of the system was 50K-100K and the maximum magnetic field was 3T. 5.0mg of organic solvent-responsive magnetic beads and the magnetic beads were weighed separately (prepared in step (1) of example 1), fixed on a sample holder, and subjected to a magnetic property test, and the saturated magnetization of the magnetic beads was 63.4emug, respectively -1 And 39.5emug -1 As shown in fig. 3. The results show that the organic solvent response type magnetic beads have good magnetic response characteristics, and meanwhile, compared with the ferroferric oxide magnetic spherical particles, the reduction of the saturation magnetization value of the organic solvent response type magnetic beads also proves that the surfactant has the effect of resisting Fe 3 O 4 Encapsulation of the particles.
At room temperature, adding 10g of the organic solvent response type magnetic beads prepared in the example 1 into 15g of the aqueous acrylic acid film forming agent, adding the lg dispersing agent polysorbate 80 and 1.5g of the thickening agent polyvinylpyrrolidone, and performing ultrasonic dispersion for 10min to obtain a mixture; spraying the mixture on the surface of a glass substrate, and naturally drying in air for 2 hours to obtain an initial surface (coating); the initial surface is super-hydrophilic super-oleophobic, the static contact angle of water drop is 18.02 degrees (most of carboxyl is outward and CF group is inward at the moment), as shown in the left graph of fig. 4a, and after the glass substrate containing the coating is soaked in ethanol and/or isopropanol organic solvent for nearly 10min at room temperature, the fluoroalkyl group is oriented outwards at the moment, and the static contact angle of water drop is 153.43 degrees at the moment, as shown in the right graph of fig. 4 a. The organic solvent-responsive magnetic beads now exhibit a superhydrophobic state, as shown to the left in fig. 4 b. For traditional commercial magnetic beads, the mixture is prepared by adopting the method, the initial state is that carboxyl faces outwards, the static contact angle of water drops is 19.06 degrees, and after the mixture is soaked in an ethanol and/or isopropanol organic solvent for about 10 minutes, the carboxyl is still outwards, and the static contact angle of water drops is 18.87 degrees. The conventional commercial magnetic beads now exhibit a superhydrophobic state, as shown on the right side of fig. 4 b.
At room temperature, firstly diluting high abundance nucleic acid templates into sample templates with different concentrations according to a ratio of 10 times, measuring different absorbance by using a nucleic acid detector to establish a standard, wherein the background fluorescence intensity is 0.2 ng/. Mu.L (figure 5: left), then measuring the nucleic acid concentration of 4.1 ng/. Mu.L (figure 5: right) in the original sample 100. Mu.L by using the nucleic acid detector based on a standard curve, the nucleic acid content of 400-420 ng in the original sample, then placing 3-5 mg of organic solvent response type magnetic beads into the sample for nucleic acid extraction, after the reaction time is 11min, magnetically separating the magnetic beads, then again placing the sample into the nucleic acid detector for detection, wherein the sample concentration is 0.2 ng/. Mu.L (figure 5: right), the nucleic acid content of 20ng in the sample is obtained, the nucleic acid absorption rate (410-20)/410=95.2% of the organic solvent response type magnetic beads to nucleic acid is measured by using the nucleic acid detector, the eluted nucleic acid concentration of 2 times is measured by using the eluting solvent response type magnetic beads, the nucleic acid concentration of 3-370 = 3.370 ng/3.90% of the nucleic acid is measured by using the eluting solvent response type magnetic beads, the nucleic acid detector after the reaction time is 11min, the sample concentration of the nucleic acid concentration of 3-370 = 3.370 ng/3.370%.
At room temperature, firstly diluting high abundance nucleic acid templates into sample templates with different concentrations according to a ratio of 10 times, using a nucleic acid detector to measure different absorbance of the sample templates to establish a standard curve, wherein the background fluorescence intensity is 0.2 ng/mu L (figure 6: left), then using the nucleic acid detector to measure the nucleic acid concentration in 100 mu L of an original sample based on the standard curve to be 4.1 ng/mu L (figure 6: right), the nucleic acid content in the original sample is 400-420 ng, then placing 3-5 mg of traditional commercial magnetic beads into the sample for nucleic acid extraction, after the reaction time is 11min, magnetically separating the magnetic beads, then again placing the sample into the nucleic acid detector to detect, the sample concentration is 0.23 ng (0.43-0.2) ng/mu L (figure 6: right), the nucleic acid content in the sample is 23ng, the adsorption rate (410-23)/410=94.4% of the nucleic acid on the magnetic beads is obtained, the eluted nucleic acid is subjected to constant volume 100 mu L by using an eluent, the eluted nucleic acid concentration is 3-0.320 ng/320 ng, and the desorption rate is 3.2.0.320 ng/320.
Under the condition of room temperature, referring to the standard step and method for extracting magnetic bead nucleic acid, in the steps of combining and desorbing the nucleic acid and the magnetic bead, the solution formula for strengthening the combination of the nucleic acid is optimized, the influence factors such as the concentration of polyethylene glycol and sodium chloride, the addition content of the magnetic bead, the pH value and the like are adjusted, the single condition is adjusted, the condition of other variables is ensured to be unchanged, the extraction rate of the organic solvent response type magnetic bead on the nucleic acid under different conditions is respectively calculated, the optimal condition for extracting the organic solvent response type magnetic bead nucleic acid is finally determined, wherein the concentration of the organic solvent response type magnetic bead added sample is 30 mg/ml (figure 7 a), the concentration of sodium chloride is 50 mg/ml (figure 7 b), the concentration of dithiothreitol is 20 mg/ml (figure 7 d), the pH value of the combined solution is 5.5 (figure 7 c), the combined time is not less than 11 minutes (figure 7 f), the pH value of the desorption solution is 8.5 (for the desorption of DNA), and the pH value of the desorption solution is 8 (for the desorption of RNA) (figure 7 e).
At room temperature, dilute the RNA nucleic acid template with low abundance into sample templates (5 Copies,10Copies,25Copies,35Copies,45Copies,55Copies,100Copies,200Copies,300Copies,400 Copies) with different concentrations, extract nucleic acid by using organic solvent response type magnetic beads, and under the optimal extraction condition, digital PCR detection is performed on the sample after the extraction of the organic solvent response type magnetic beads, the nucleic acid concentration of the sample which can be detected by the lowest detection limit of the organic solvent response type magnetic beads is found to be 5Copies/mL, 3 fluorescent droplets are provided, 20 corresponding negative positive droplet statistical graphs (FIG. 8 a), the accuracy is low, only 60% when the nucleic acid concentration is 25Copies/mL, 33 corresponding negative positive droplet statistical graphs (FIG. 8 b), 94% when the nucleic acid concentration is 35Copies/mL, and the accuracy is found to be about 95% when the nucleic acid concentration is about 35 Copies/mL. For the traditional commercial magnetic beads, the concentration of the sample nucleic acid is lower than 55Copies/mL, no result can be detected, the result can be obtained when the concentration of the sample nucleic acid reaches 55Copies/mL, the accuracy is lower, and the ideal accuracy can be reached only when the concentration of the sample nucleic acid reaches 500Copies/mL, which is about 80% (as shown in FIG. 8 d).
Claims (9)
1. The application of the organic solvent response type magnetic beads in the aspect of nucleic acid extraction is characterized in that the specific application process is as follows:
step 1, sample pretreatment and cracking: grinding fresh or ultralow-temperature stored sample liquid nitrogen into powder, transferring the powder into an EP pipe, adding 1mL of pyrolysis liquid into the EP pipe, carrying out vortex oscillation until no obvious particulate matters exist, and standing for 5min at room temperature; the sample is less than or equal to 100mg;
step 2, nucleic acid binding: adding 400 mu L of nucleic acid binding solution into an EP tube, then adding 30mg of organic solvent responsive magnetic beads, regulating the pH to 5, shaking the magnetic bead suspension uniformly, and then placing the mixture into a high-speed vortex at 3000rpm for 11 minutes to bind the nucleic acid cleaved from the sample with the magnetic beads; the nucleic acid binding solution mainly contains sodium chloride and dithiothreitol;
step 3, magnetic separation: placing the EP tube on a magnetic frame for standing for 20s until the magnetic beads are completely adsorbed, if the magnetic bead residual liquid exists in the inner cover of the EP tube, keeping the EP tube on the magnetic frame, and integrally reversing the EP tube upside down for 2-3 times to ensure that the magnetic beads are completely adsorbed, and sucking and discarding supernatant fluid to avoid contacting the magnetic beads;
step 4, cleaning: adding 600 mu L of cleaning liquid I into the EP pipe, blowing off the magnetic beads by using a liquid-transferring gun, carrying out vortex oscillation for 2min, and carrying out magnetic separation again according to the step 3; adding 600 mu L of cleaning liquid II into the EP pipe after magnetic separation, blowing off magnetic beads by using a liquid-transferring gun, carrying out vortex oscillation for 2min, carrying out magnetic separation again according to the step 3, adding 600 mu L of cleaning liquid II into the EP pipe after magnetic separation, blowing off magnetic beads by using the liquid-transferring gun, carrying out vortex oscillation for 2min, and carrying out magnetic separation again according to the step 3; the main component of the cleaning liquid I is isopropanol, and the main component of the cleaning liquid II is ethanol;
step 5, removing alcohol: the EP tube after the supernatant is discarded is kept on a magnetic frame, an EP tube cover is opened, the EP tube is dried for 8min at room temperature until no obvious ethanol smell exists, in the step, carboxyl groups on the surface of the magnetic beads can be overturned, and nucleic acid is separated from the magnetic beads;
step 6, elution: adding 30-100 mu L of eluent into the EP tube, and carrying out vortex oscillation until magnetic beads are completely dispersed in a liquid phase, carrying out warm bath for 5min at 58 ℃ and carrying out vortex oscillation for 10s every 2 min; the eluent is deionized water with pH value of 8;
step 7, nucleic acid transfer: after the elution is finished, placing the EP tube on a magnetic rack for standing for 20 seconds until the magnetic beads are completely adsorbed, transferring the eluent into another clean EP tube by using a liquid-transferring gun, and discarding the magnetic beads at the moment after the extraction process is finished;
the organic solvent response type magnetic beads comprise ferroferric oxide particles and surfactant molecules wrapped outside the ferroferric oxide particles, wherein the surfactant molecules are chain molecules, carboxyl is arranged at one end, fluoroalkyl is arranged at one end, and the carboxyl end of the surfactant molecules is arranged at one side far away from the ferroferric oxide particles; the particle size of the ferroferric oxide magnetic spherical particles is 300-400 nm.
2. The use of the organic solvent-responsive magnetic beads according to claim 1 for nucleic acid extraction, wherein: the loading of the surfactant was 50% of the mass of the magnetic beads.
3. The application of the organic solvent response type magnetic beads in the aspect of nucleic acid extraction according to claim 1, wherein the organic solvent response type magnetic beads are prepared by the following method, and specifically: placing 10-150 parts by mass of fluorine-containing surfactant, 10-120 parts by mass of ferroferric oxide particles, 10-150 parts by mass of film-forming resin and 2-3 parts by mass of dispersing agent into 300 parts by mass of water, heating, stirring at high speed by a magnetic stirrer while heating, and dispersing by ultrasonic after stirring; and magnetically separating the obtained solution to obtain solid precipitate, and drying to obtain the organic solvent response type magnetic beads.
4. The use of the organic solvent-responsive magnetic beads of claim 3 for nucleic acid extraction, wherein: the stirring time is 40-60 min, and the stirring speed is 3000rpm.
5. The use of the organic solvent-responsive magnetic beads of claim 3 for nucleic acid extraction, wherein: the heating temperature is 80-90 ℃.
6. The use of the organic solvent-responsive magnetic beads of claim 3 for nucleic acid extraction, wherein: the drying temperature was 60 ℃.
7. The use of the organic solvent-responsive magnetic beads of claim 3 for nucleic acid extraction, wherein: the organic solvent response type magnetic beads are spherical particles, and the particle size is 400-600 nm.
8. The use of the organic solvent-responsive magnetic beads of claim 3 for nucleic acid extraction, wherein: the fluorine-containing surfactant is at least one of cationic, anionic, nonionic or amphoteric surfactant, and the end group of the fluorine-containing surfactant is fluoroalkyl.
9. The use of the organic solvent-responsive magnetic beads of claim 3 for nucleic acid extraction, wherein: the ferroferric oxide particles are prepared by the following method: adding 10 parts by mass of organic ferric salt into 400 parts by mass of dihydric alcohol, and uniformly stirring to obtain a transparent solution; adding 60 parts by mass of unsaturated fatty acid into the transparent solution, and uniformly stirring to obtain a mixed solution; and (3) placing the mixed solution into a reaction kettle to react for 10-12 hours at 160-190 ℃, magnetically separating the solution after the reaction to obtain solid precipitate, and washing and drying the solid precipitate to obtain the ferroferric oxide particles.
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