Carboxyl nano magnetic bead and application thereof in nucleic acid capture and nucleic acid release

Abstract

The invention belongs to the technical field of nano magnetic beads, and relates to a carboxyl nano magnetic bead and application thereof in DNA capture and DNA release. The invention discloses a preparation method of carboxyl nano magnetic beads, which comprises the following steps: dissolving iminodiacetic acid in a sodium hydroxide solution, stirring in an ice bath, dropwise adding a silane coupling agent, carrying out a first heating reaction, cooling in the ice bath, dropwise adding the silane coupling agent again, carrying out a second heating reaction, and cooling in the ice bath to obtain a carboxyl silane coupling agent; and dispersing the ferroferric oxide / silicon dioxide hydroxyl magnetic compound and a carboxyl silane coupling agent in an organic solvent, and stirring for reaction to obtain the carboxyl nano magnetic beads. The carboxyl nano magnetic bead is a nano-scale magnetic bead, is large in specific surface area, good in suspension property and not easy to settle, and has multiple sites for DNA binding; the method can be used for effectively extracting DNA ladders and cfDNA in nucleic acid capture and nucleic acid release.

Description

Technical Field

[0001] The invention belongs to the technical field of nano magnetic beads and relates to carboxyl nano magnetic beads and applications thereof in DNA capture and DNA release. Background Art

[0002] Molecular biology techniques for nucleic acids are one of the commonly used research methods for pathogen detection, species identification, species origin, diversity assessment, kinship, and system evolution. Whether high-quality nucleic acids can be extracted is the key to molecular biology experiments, and the sensitivity and specificity of the extraction method will also be directly related to the success or failure of subsequent experiments. Therefore, nucleic acid extraction is one of the most critical methods in molecular biology.

[0003] With the widespread application of molecular biology, new requirements have been put forward for nucleic acid extraction technology. Efficiency, convenience, environmental protection, high throughput and automation have become the mainstream direction of the development of nucleic acid extraction technology. Traditional nucleic acid extraction methods include phenol-chloroform method, Trizol method and adsorption column method. They use toxic phenol and chloroform reagents and adopt precipitation and centrifugation processes. The extraction process requires a large amount of sample, and the sample processing process is cumbersome and complicated. The recovery efficiency of nucleic acid is low, and it is difficult to achieve high throughput and automated operation. At present, the mainstream method for nucleic acid extraction is the magnetic bead method. The principle of magnetic bead method for extracting nucleic acid is: using magnetic beads as carriers, the magnetic beads can adsorb nucleic acids under high salt and low pH, and separate from nucleic acids under low salt and high pH, ​​and then move the magnetic beads or transfer liquids to achieve the entire extraction and purification process of nucleic acids.

[0004] Magnetic beads are a kind of nano- to micron-scale magnetic biomaterials. After chemical or biological modification on the surface, they have a variety of biological properties and can respond quickly under the condition of an external magnetic field. They are the most widely used core raw materials in the field of in vitro diagnostics. At present, the preparation methods of magnetic beads mainly include interface deposition method, suspension polymerization method, dispersion polymerization method, seed polymerization method, etc. The interface deposition method is to adsorb positively charged magnetic nanoparticles on the surface of negatively charged polymer microspheres through electrostatic action, and then coat a layer of polymer material on the surface; this method can achieve functional modification of magnetic microspheres by introducing different substances at the interface to meet different needs; but since each magnetic microsphere surface can only adsorb one layer of magnetic nanoparticles, the magnetic content of the magnetic beads is low, and the stability of this method depends on the stability of the interface. Some interface deposition methods have complex operation procedures and high preparation costs. The suspension polymerization method is to disperse magnetic nanoparticles in polymer monomers or solutions to form an oil-in-water emulsion, and then perform polymerization reaction in a suspended state to form microspheres. Its disadvantage is that the affinity between polymer materials and magnetic nanoparticles is poor, and the encapsulation efficiency is low during monomer polymerization due to the strong hydrophilicity of magnetic nanoparticles. In addition, a large number of magnetic particles are attached to the surface of the microspheres, which are difficult to elute, affecting the biological activity of bioactive substances during fixation or separation. The dispersion polymerization method can achieve precise control of the size and shape distribution of the prepared magnetic microspheres, but the quality of the magnetic microspheres prepared by this method is uneven, and most of them have the disadvantages of uneven particle size distribution, low magnetic content, and few surface functional groups. The seed polymerization method uses polymer microspheres with a porous structure as a template, sulfonates (-SO3) or nitrates (-NO2) the inside and outside of the microsphere pores to make them have a hydrophilic interface, then immerses the microspheres in an aqueous solution of iron salts, generates superparamagnetic Fe3O4 or γ-Fe3O4 in the pores under appropriate reaction conditions, and finally uses monomers containing active functional groups to swell, polymerize, and coat the microspheres to close the microsphere pores and functionalize the surface. The seed polymerization method has the advantages of narrow particle size distribution, uniform magnetic content, and rich surface functional groups, and is the main production method used by manufacturers with a large market share. However, there are still some disadvantages in the application of magnetic beads prepared by seed polymerization method for enriching cfDNA in biological samples: the particle size is at the micron level, generally in the range of 1 to 4 μm, with poor suspension and easy sedimentation, which affects the interaction with cfDNA in the sample; the magnetic beads have a small specific surface area and fewer sites for DNA binding, so the amount of magnetic beads needs to be increased to achieve the purpose of effectively separating DNA in the sample, which increases the cost; the polymer base material easily causes strong nonspecific adsorption, thereby introducing impurities and affecting downstream DNA detection. Summary of the invention

[0005] The purpose of the present invention is to solve the above problems in the prior art and to propose a method for preparing carboxyl nano-magnetic beads. The prepared carboxyl nano-magnetic beads have uniform particle size and large specific surface area, and can effectively capture and release nucleic acids.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] A method for preparing carboxyl nanomagnetic beads, comprising:

[0008] (1) mixing an iron source, sodium acetate, sodium citrate, and ethylene glycol and heating the mixture to obtain ferroferric oxide magnetic nanoparticles;

[0009] (2) dispersing ferroferric oxide magnetic nanoparticles in an ethanol / water mixture, adding ammonia water and an organosilicon compound, stirring to react, to obtain a ferroferric oxide / silicon dioxide magnetic composite, and performing magnetic separation to obtain a ferroferric oxide / silicon dioxide hydroxyl magnetic composite;

[0010] (3) dissolving iminodiacetic acid in a sodium hydroxide solution, stirring in an ice bath, adding a silane coupling agent dropwise, performing a first heating reaction, cooling in an ice bath, adding the silane coupling agent dropwise again, performing a second heating reaction, cooling in an ice bath, and obtaining a carboxyl silane coupling agent; dispersing the ferroferric oxide / silicon dioxide hydroxy magnetic composite and the carboxyl silane coupling agent in an organic solvent, stirring and reacting to obtain carboxyl nano magnetic beads.

[0011] Preferably, the mass ratio of the iron source, sodium acetate, sodium citrate and ethylene glycol in (1) is (1-10):(6-15):1:(100-1000).

[0012] Preferably, the iron source in (1) includes one or more of ferric chloride, ferric nitrate and ferric sulfate.

[0013] Preferably, the heating reaction temperature in (1) is 160-240° C. and the time is 6-24 h.

[0014] Preferably, the volume ratio of ethanol to water in the ethanol / water mixture in (2) is (10-50):(5-25).

[0015] Preferably, the mass volume ratio of the ferroferric oxide magnetic nanoparticles and the ethanol / water mixture in (2) is (1-5) mg:1 ml.

[0016] Preferably, the mass volume ratio of ferroferric oxide magnetic nanoparticles, ammonia water and organosilicon compound in (2) is (40-80) mg: (0.1-5) ml: (0.1-5) ml.

[0017] Preferably, the stirring reaction temperature in (2) is 10 to 35° C. and the time is 1 to 12 h.

[0018] Preferably, the concentration of sodium hydroxide in (3) is 5 to 20 M.

[0019] Preferably, the mass volume ratio of iminodiacetic acid and sodium hydroxide solution in (3) is (1-10) g: (20-80) ml.

[0020] Preferably, the silane coupling agent in (3) includes one or more of (3-glycidylpropoxy)trimethoxysilane, 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, and γ-(2,3-epoxypropoxy)propyltrimethoxysilane.

[0021] Preferably, the mass ratio of iminodiacetic acid to the silane coupling agent in (3) is (1-10):1.

[0022] Preferably, the mass ratio of the ferroferric oxide / silicon dioxide hydroxy magnetic composite to the carboxyl silane coupling agent in (3) is 1:(100-1000).

[0023] More preferably, the mass ratio of the ferroferric oxide / silicon dioxide hydroxy magnetic composite to the carboxyl silane coupling agent in (3) is 1:(500-800).

[0024] Preferably, the organic solvent in (3) comprises one or more of methanol, ethanol and isopropanol.

[0025] Preferably, the average particle size of the ferroferric oxide / silicon dioxide magnetic composite in (3) is 80 to 130 nm.

[0026] Preferably, the average particle size of the carboxyl nanomagnetic beads in (3) is 90 to 200 nm.

[0027] Preferably, in (3), iminodiacetic acid is dissolved in a sodium hydroxide solution, stirred in an ice bath, and then a silane coupling agent is added dropwise, a first heating reaction is carried out at 50 to 80° C. for 1 to 12 hours, and then the reaction is cooled in an ice bath, and then the silane coupling agent is added dropwise again, a second heating reaction is carried out at 50 to 80° C. for 1 to 12 hours, and then the reaction is cooled in an ice bath to obtain a carboxyl silane coupling agent; the ferroferric oxide / silicon dioxide magnetic composite and the carboxyl silane coupling agent are dispersed in an organic solvent, and the reaction is stirred at 20 to 50° C. for 1 to 48 hours to obtain carboxyl nano magnetic beads.

[0028] Application of carboxyl nanomagnetic beads in nucleic acid capture and nucleic acid release,

[0029] The nucleic acid capture comprises: mixing a diluent containing carboxyl nano-magnetic beads with a sample to be processed and an acidic buffer, washing, and air-drying to obtain a magnetic bead-nucleic acid complex;

[0030] The nucleic acid release comprises: dispersing the magnetic bead-nucleic acid complex in an alkaline buffer and shaking to elute the nucleic acid.

[0031] Preferably, the sample to be processed includes one or more of plasma, serum and urine.

[0032] Preferably, the nucleic acid includes but is not limited to DNA ladder, cfDNA in blood / urine.

[0033] Preferably, the concentration of the carboxyl nano-magnetic beads in the diluent containing the carboxyl nano-magnetic beads is 10-60 mg / ml.

[0034] Preferably, the solvent in the diluent containing carboxyl nano-magnetic beads is deionized water.

[0035] Preferably, the acidic buffer comprises: guanidine thiocyanate, Triton X-100, PEG, NaCl, NaAc-HAc buffer, isopropanol and water.

[0036] More preferably, the acidic buffer comprises 5-6 mol / L guanidine thiocyanate, 0.1-1 mmol / L Triton X-100, 40%-60% PEG, 200-300 mmol / L NaCl, 50-100 mmol / L pH=5.0 NaAc-HAc buffer, 40%-60% (v / v) isopropanol and water.

[0037] Preferably, the alkaline buffer comprises 10-80 mM TE buffer, pH=10.0.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] 1. The carboxyl nanomagnetic beads of the present invention are nanoscale magnetic beads with large specific surface area, good suspension, not easy to settle, and many sites for DNA binding;

[0040] 2. The carboxyl nanomagnetic beads of the present invention can effectively extract DNA ladder and cfDNA during nucleic acid capture and nucleic acid release;

[0041] 3. The carboxyl nanomagnetic beads of the present invention can be used in nucleic acid capture and nucleic acid release. In the presence of high concentrations of PEG and NaCl, the hydration layer of the DNA molecule is destroyed, the conformation changes, and the phosphate groups are exposed, which are negatively charged. These negatively charged phosphate groups react with the positively charged NaCl to release the DNA molecule. + The ions form an "ion bridge" and combine with the carboxyl groups on the surface of the carboxyl nanomagnetic beads, so that the DNA is specifically adsorbed to the surface of the magnetic beads; when the DNA needs to be recovered from the magnetic beads, the PEG and salt ions are removed, and the aqueous molecules are added to quickly and fully hydrate the DNA, eliminating the ionic interaction between it and the magnetic beads, so that the DNA adsorbed on the magnetic beads can be extracted. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a TEM image of the ferroferric oxide magnetic nanoparticles in Example 1 of the present invention.

[0043] Figure 2 TEM image and EDS energy spectrum of carboxyl nanomagnetic beads in Example 1 of the present invention.

[0044] Figure 3 This is the DLS particle size distribution diagram of the carboxyl nanomagnetic beads in Example 1 of the present invention.

[0045] Figure 4 This is a gel run of the DNA extracted in Application Example 1 of the present invention.

[0046] Figure 5 This is a gel run of the DNA extracted in Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0047] The following are specific embodiments of the present invention, which further describe the technical solutions of the present invention, but the present invention is not limited to these embodiments.

[0048] Unless otherwise specified, the materials used in the present invention are conventional commercial products, and the methods used are conventional technical means.

[0049] Example 1

[0050] Weigh 0.292 g of ferric chloride, 0.72 g of sodium acetate, and 0.08 g of sodium citrate, add 12 mL of ethylene glycol and mix thoroughly with the above mixture under ultrasonic conditions until the color becomes clear and transparent, then transfer to a 25 mL hydrothermal reactor, put the reactor into an oven, react at 200 ° C for 10 hours, cool naturally to room temperature, collect magnetic particles by magnetic separation, wash the magnetic particles with deionized water and anhydrous ethanol in turn, and then dry under vacuum conditions at 60 ° C to obtain ferrosoferric oxide magnetic nanoparticles.

[0051] 60 mg of ferroferric oxide magnetic nanoparticles were dispersed in 37 mL of ethanol and water mixture (ethanol: water volume ratio is 25:12), ultrasonically mixed, 0.6 mL of ammonia water was added, and ultrasonication was continued for 30 min. 0.4 mL of tetraethyl orthosilicate was added, and mechanical stirring was carried out at room temperature for 6 h. The ferroferric oxide / silicon dioxide magnetic composite was collected by magnetic separation, washed with deionized water and anhydrous ethanol in turn, and then dried under vacuum conditions at 60°C to obtain the ferroferric oxide / silicon dioxide magnetic composite.

[0052] The ferroferric oxide / silicon dioxide magnetic composite is subjected to magnetic separation and washing to obtain the ferroferric oxide / silicon dioxide hydroxy magnetic composite.

[0053] 4.2g of iminodiacetic acid was dissolved in 50mL of sodium hydroxide solution (10M, pH=10), magnetically stirred in an ice bath for 1h, and then 1.5g of (3-glycidyl propoxy) trimethoxysilane was slowly dripped in, reacted at 65℃ for 6h, and cooled in an ice bath; 1.5g of (3-glycidyl propoxy) trimethoxysilane was slowly dripped into the system, and reacted at 65℃ for 6h to obtain a carboxyl silane coupling agent. Subsequently, 10mg of ferroferric oxide / silicon dioxide hydroxy magnetic composite and 5g of carboxyl silane coupling agent were dispersed in 25mL of ethanol, mechanically stirred at 40℃ for 24h, washed with deionized water and anhydrous ethanol in turn, and then dried under vacuum at 60℃ to obtain carboxyl nanomagnetic beads.

[0054] Figure 1 This is a TEM image of the ferroferric oxide magnetic nanoparticles in this example. Figure 2 , 3 TEM image, EDS energy spectrum and DLS particle size distribution diagram of carboxyl nanomagnetic beads in this embodiment.

[0055] Example 2

[0056] Weigh 0.292 g of ferric chloride, 0.72 g of sodium acetate, and 0.08 g of sodium citrate, add 12 mL of ethylene glycol and mix thoroughly with the above mixture under ultrasonic conditions until the color becomes clear and transparent, then transfer to a 25 mL hydrothermal reactor, put the reactor into an oven, react at 200 ° C for 10 hours, cool naturally to room temperature, collect magnetic particles by magnetic separation, wash the magnetic particles with deionized water and anhydrous ethanol in turn, and then dry under vacuum conditions at 60 ° C to obtain ferrosoferric oxide magnetic nanoparticles.

[0057] 60 mg of ferroferric oxide magnetic nanoparticles were dispersed in 37 mL of ethanol and water mixture (ethanol: water volume ratio is 25:12), ultrasonically mixed, 0.6 mL of ammonia water was added, and ultrasonication was continued for 30 min. 0.4 mL of methyl orthosilicate was added, and mechanical stirring was carried out at room temperature for 6 h. The ferroferric oxide / silicon dioxide magnetic composite was collected by magnetic separation, washed with deionized water and anhydrous ethanol in turn, and then dried under vacuum conditions at 60 ° C to obtain the ferroferric oxide / silicon dioxide magnetic composite.

[0058] The ferroferric oxide / silicon dioxide magnetic composite is subjected to magnetic separation and washing to obtain the ferroferric oxide / silicon dioxide hydroxy magnetic composite.

[0059] 4.2g of iminodiacetic acid was dissolved in 50mL of sodium hydroxide solution (10M, pH=10), magnetically stirred in an ice bath for 1h, and then 1.5g of 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane was slowly dripped into the system, reacted at 65℃ for 6h, and cooled in an ice bath; 1.5g of 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane was slowly dripped into the system, and reacted at 65℃ for 6h to obtain a carboxyl silane coupling agent. Subsequently, 10mg of ferroferric oxide / silicon dioxide hydroxy magnetic composite and 8g of carboxyl silane coupling agent were dispersed in 25mL of ethanol, mechanically stirred at 40℃ for 24h, washed with deionized water and anhydrous ethanol in turn, and then dried under vacuum at 60℃ to obtain carboxyl nanomagnetic beads.

[0060] Comparative Example 1

[0061] Compared with Example 1, the difference is that 4.2g of iminodiacetic acid is dissolved in 50mL of sodium hydroxide solution (10M, pH=10), magnetically stirred in an ice bath for 1h, 1.5g of (3-glycidyl propoxy) trimethoxy silane is slowly dripped, reacted at 65°C for 6h, and cooled in an ice bath; 1.5g of (3-glycidyl propoxy) trimethoxy silane is slowly dripped into the system, reacted at 65°C for 6h, and a carboxyl silane coupling agent is obtained. Subsequently, 10mg of the ferroferric oxide / silicon dioxide hydroxy magnetic composite in Example 1 and 1g of the carboxyl silane coupling agent are dispersed in 25mL of ethanol, mechanically stirred at 40°C for 24h, washed with deionized water and anhydrous ethanol in turn, and then dried under vacuum at 60°C to obtain carboxyl nano magnetic beads.

[0062] Comparative Example 2

[0063] Compared with Example 1, the difference is that 4.2g of iminodiacetic acid is dissolved in 50mL of sodium hydroxide solution (10M, pH=10), magnetically stirred in an ice bath for 1h, 1.5g of (3-glycidyl propoxy) trimethoxy silane is slowly dripped, reacted at 65°C for 6h, and cooled in an ice bath; 1.5g of (3-glycidyl propoxy) trimethoxy silane is slowly dripped into the system, reacted at 65°C for 6h, and a carboxyl silane coupling agent is obtained. Subsequently, 10mg of the ferroferric oxide / silicon dioxide hydroxy magnetic composite in Example 1 and 10g of the carboxyl silane coupling agent are dispersed in 25mL of ethanol, mechanically stirred at 40°C for 24h, washed with deionized water and anhydrous ethanol in turn, and then dried under vacuum at 60°C to obtain carboxyl nano magnetic beads.

[0064] Comparative Example 3

[0065] Compared with Example 1, the difference is that 4.2g of iminodiacetic acid is dissolved in 50mL of sodium hydroxide solution (10M, pH=10), magnetically stirred in an ice bath for 1h, 3g of (3-glycidyl propoxy) trimethoxysilane is slowly dripped, reacted at 65°C for 6h, and cooled in an ice bath to obtain a carboxyl silane coupling agent. Subsequently, 10mg of the ferroferric oxide / silicon dioxide hydroxy magnetic composite in Example 1 and 5g of the carboxyl silane coupling agent are dispersed in 25mL of ethanol, mechanically stirred at 40°C for 24h, washed with deionized water and anhydrous ethanol in turn, and then dried under vacuum at 60°C to obtain carboxyl nanomagnetic beads.

[0066] Application Example 1

[0067] The carboxyl nanomagnetic beads in Example 1 are used to extract the DNA ladder, comprising:

[0068] (1) Take 10 μl of DNA ladder into a 1.5 mL centrifuge tube, add 25 μl of acidic buffer (5 mol / L guanidine thiocyanate, 0.5 mmol / L Triton X-100, 40% PEG, 200 mmol / L NaCl, 50 mmol / L pH = 5.0 NaAc-HAc buffer, 40% (v / v) isopropanol, the rest is water) and 2 μl of dilution solution containing carboxyl nanoparticles (40 mg / mL, deionized water) and mix well; place on a magnetic rack until the solution is clear, and then remove the clear solution;

[0069] (2) Add 100 μl of 80% ethanol to a centrifuge tube, shake well, and centrifuge. Place the centrifuge tube on a magnetic rack until the solution is clarified. After removing the clarified solution, air-dry the magnetic beads.

[0070] (3) Remove the centrifuge tube from the magnetic rack, add 15 μl of alkaline buffer (50 mM TE buffer, pH = 10.0), and mix by pipetting back and forth; shake for 5 minutes and centrifuge, then place the centrifuge tube on the magnetic rack until the liquid clarifies, and then use a pipette to pipette the clarified liquid into a new centrifuge tube to obtain DNA.

[0071] Figure 4 This is a gel run of the DNA extracted in this application example. Lanes 1 and 8 are DNA ladders, lanes 3, 5, 11, and 12 are the supernatants after carboxyl nano-magnetic beads adsorb DNA, and lanes 2, 4, 9, and 11 are the eluates after carboxyl nano-magnetic beads adsorb DNA. The experiment was repeated four times. Figure 4 It can be clearly seen that the band corresponding to the supernatant has no brightness, and the brightness of the band corresponding to the eluate is basically consistent with the brightness of the DNA ladder, indicating that the carboxyl nanomagnetic beads have successfully captured the DNA.

[0072] Application Example 2

[0073] The carboxyl nanomagnetic beads in Example 1 are used to extract cfDNA from human plasma, comprising:

[0074] (1) Take 2 mL of plasma (~20 ng cfDNA / 2 mL of plasma) into a 15 mL centrifuge tube, add 2.5 mL of acidic buffer (5 mol / L guanidine thiocyanate, 0.5 mmol / L Triton X-100, 40% PEG, 200 mmol / L NaCl, 50 mmol / L pH = 5.0 NaAc-HAc buffer, 40% (v / v) isopropanol, the rest is water) and 30 μL of diluent containing carboxyl nano-magnetic beads (40 mg / mL, deionized water) and mix well; place on a magnetic rack until the solution is clear, and then remove the clear solution;

[0075] (2) Add 1 ml of 80% ethanol to a centrifuge tube, shake well, and centrifuge. Place the centrifuge tube on a magnetic rack until the solution is clarified. After removing the clarified solution, air-dry the magnetic beads.

[0076] (3) Remove the centrifuge tube from the magnetic rack, add 15 μl of alkaline buffer (50 mM TE buffer, pH = 10.0), and mix by pipetting back and forth; shake for 5 minutes and centrifuge, then place the centrifuge tube on the magnetic rack until the liquid clarifies, and then use a pipette to aspirate the clarified liquid into a new centrifuge tube to obtain cfDNA.

[0077] The above steps were repeated three times, and the masses of the extracted cfDNA were 20.25 ng / 2 ml plasma, 21.90 ng / 2 ml plasma, and 21.15 ng / 2 ml plasma, respectively.

[0078] Application Example 3

[0079] Compared with Application Example 2, the difference is that the concentration of the diluent containing carboxyl nanomagnetic beads is 10 mg / mL.

[0080] The process was repeated three times, and the masses of the extracted cfDNA were 5.61 ng / 2 ml plasma, 5.90 ng / 2 ml plasma, and 6.05 ng / 2 ml plasma, respectively.

[0081] Application Example 4

[0082] Compared with Application Example 2, the difference is that the concentration of the diluent containing carboxyl nanomagnetic beads is 60 mg / mL.

[0083] The process was repeated three times, and the masses of the extracted cfDNA were 20.63 ng / 2 ml plasma, 19.90 ng / 2 ml plasma, and 20.05 ng / 2 ml plasma, respectively.

[0084] Application Example 5

[0085] Compared with Application Example 2, the difference lies in that the carboxyl nanomagnetic beads in Example 2 are used.

[0086] The process was repeated three times, and the masses of the extracted cfDNA were 20.48 ng / 2 ml plasma, 21.27 ng / 2 ml plasma, and 20.68 ng / 2 ml plasma, respectively.

[0087] Application Comparative Example 1

[0088] Compared with Application Example 1, the difference is that the ferroferric oxide / silicon dioxide magnetic composite in Example 1 is used to prepare a diluent of the same concentration for the extraction of DNA ladder.

[0089] Figure 5 This is a gel image of the DNA extracted from this comparative example. Lane 1 is a DNA ladder, lanes 2 and 4 are the supernatant after the ferroferric oxide / silicon dioxide magnetic complex adsorbs DNA, and lanes 3 and 5 are the elutions after the ferroferric oxide / silicon dioxide magnetic complex adsorbs DNA. The experiment was performed in parallel twice. Figure 5 It can be clearly seen that the band corresponding to the elution solution has no brightness, and the brightness of the band corresponding to the supernatant is basically consistent with the brightness of the DNA ladder, indicating that the ferroferric oxide / silicon dioxide magnetic complex cannot capture DNA.

[0090] Application Comparative Example 2

[0091] Compared with Application Example 2, the difference is that the ferroferric oxide / silicon dioxide magnetic composite in Example 1 is used to prepare a diluent of the same concentration for the extraction of cfDNA in human plasma.

[0092] The above steps were repeated three times, and the concentration of cfDNA extracted was detected by Qubit, and the result was no detection.

[0093] Application Comparative Example 3

[0094] Compared with Application Example 2, the carboxyl nanomagnetic beads in Comparative Example 1 were used to prepare a diluent of the same concentration for the extraction of cfDNA from human plasma.

[0095] The process was repeated three times, and the masses of the extracted cfDNA were 6.04 ng / 2 ml plasma, 5.89 ng / 2 ml plasma, and 5.57 ng / 2 ml plasma, respectively.

[0096] Application Comparative Example 4

[0097] Compared with Application Example 2, the carboxyl nanomagnetic beads in Comparative Example 2 were used to prepare a diluent of the same concentration for the extraction of cfDNA from human plasma.

[0098] The process was repeated three times, and the masses of the extracted cfDNA were 19.43 ng / 2 ml plasma, 18.97 ng / 2 ml plasma, and 20.01 ng / 2 ml plasma, respectively.

[0099] Application Comparative Example 5

[0100] Compared with Application Example 2, the carboxyl nanomagnetic beads in Comparative Example 3 were used to prepare a diluent of the same concentration for the extraction of cfDNA from human plasma.

[0101] The process was repeated three times, and the masses of the extracted cfDNA were 12.93 ng / 2 ml plasma, 11.79 ng / 2 ml plasma, and 13.01 ng / 2 ml plasma, respectively.

[0102] In summary, the carboxyl nanomagnetic beads of the present invention are nanoscale magnetic beads with large specific surface area, good suspension, not easy to settle, and many sites for DNA binding; in nucleic acid capture and nucleic acid release, in the presence of high concentrations of PEG and NaCl, the hydration layer of the DNA molecule is destroyed, the conformation changes, the phosphate groups are exposed, and they are negatively charged; these negatively charged phosphate groups are bound to the positively charged Na + The ions form an "ion bridge" and combine with the carboxyl groups on the surface of the carboxyl nanomagnetic beads, so that the DNA is specifically adsorbed to the surface of the magnetic beads; when the DNA needs to be recovered from the magnetic beads, the PEG and salt ions are removed, and the aqueous molecules are added to quickly and fully hydrate the DNA, eliminating the ionic interaction between it and the magnetic beads, so that the DNA adsorbed on the magnetic beads can be extracted.

[0103] The specific embodiments described herein are merely examples of the spirit of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in similar ways, but they will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims.

Claims

1. A method for preparing carboxyl nanomagnetic beads, characterized in that: include: (1) mixing an iron source, sodium acetate, sodium citrate, and ethylene glycol and heating the mixture to obtain ferroferric oxide magnetic nanoparticles; (2) dispersing ferroferric oxide magnetic nanoparticles in an ethanol / water mixture, adding ammonia water and an organosilicon compound, stirring to react, to obtain ferroferric oxide / silicon dioxide magnetic composites, and magnetically separating to obtain ferroferric oxide / silicon dioxide hydroxy magnetic beads; (3) dissolving iminodiacetic acid in a sodium hydroxide solution, stirring in an ice bath, and then dropping a silane coupling agent to carry out a first heating reaction, cooling in an ice bath, and then dropping a silane coupling agent again to carry out a second heating reaction. The method comprises cooling in an ice bath to obtain a carboxyl silane coupling agent; dispersing the ferroferric oxide / silicon dioxide hydroxy magnetic beads and the carboxyl silane coupling agent in an organic solvent, and stirring the mixture to react to obtain carboxyl nano magnetic beads.

2. The method for preparing carboxyl nano magnetic beads according to claim 1, wherein The mass volume ratio of iminodiacetic acid and sodium hydroxide solution in (3) is (1-10) g: (20-80) ml.

3. The method for preparing carboxyl nano magnetic beads according to claim 1, characterized in that, The mass ratio of iminodiacetic acid to the silane coupling agent in (3) is (1-10):

1.

4. The method for preparing carboxyl nano magnetic beads according to claim 1, characterized in that, The mass ratio of ferroferric oxide / silicon dioxide hydroxy magnetic beads to carboxyl silane coupling agent in (3) is 1: (500-800).

5. The method for preparing carboxyl nano magnetic beads according to claim 1, characterized in that, The average particle size of the carboxyl nano-magnetic beads in (3) is 50 to 200 nm.

6. The method for preparing carboxyl nano magnetic beads according to claim 1, characterized in that, In the step (3), iminodiacetic acid is dissolved in a sodium hydroxide solution, stirred in an ice bath, and then a silane coupling agent is added dropwise. A first heating reaction is carried out at 50 to 80° C. for 1 to 12 hours, and then the reaction is cooled in an ice bath. The silane coupling agent is added dropwise again, and a second heating reaction is carried out at 50 to 80° C. for 1 to 12 hours, and then the reaction is cooled in an ice bath to obtain a carboxyl silane coupling agent. The ferroferric oxide / silicon dioxide magnetic composite and the carboxyl silane coupling agent are dispersed in an organic solvent, and the reaction is stirred at 20 to 50° C. for 1 to 48 hours to obtain carboxyl nano magnetic beads.

7. A carboxyl nanomagnetic bead, characterized in that: The carboxyl nano-magnetic beads are prepared by the preparation method of any one of claims 1 to 6.

8. The use of carboxyl nano-magnetic beads in nucleic acid capture and nucleic acid release according to claim 7, characterized in that: The nucleic acid capture comprises: mixing a diluent containing carboxyl nano-magnetic beads with a sample to be processed and an acidic buffer, washing, and air-drying to obtain a magnetic bead-nucleic acid complex; The nucleic acid release comprises: dispersing the magnetic bead-nucleic acid complex in an alkaline buffer and shaking to elute the nucleic acid.

9. The use according to claim 8, characterized in that: The concentration of the carboxyl nano-magnetic beads in the diluent containing the carboxyl nano-magnetic beads is 10-60 mg / ml.

10. The use according to claim 8, characterized in that: The sample to be processed includes one or more of plasma, serum, and urine.

Images