Method for preparing functionalized nano material and efficiently enriching and recovering extracellular DNA (Deoxyribose Nucleic Acid) in environment by using functionalized nano material

Abstract

The invention relates to a method for preparing a functionalized nano material and efficiently enriching and recovering extracellular DNA (Deoxyribose Nucleic Acid) in an environment by using the functionalized nano material, belonging to the technical field of gene detection. The preparation method of the terbium phosphate nano adsorption material comprises the following steps: weighing a terbium nitrate solution, dropwise adding an ammonium dihydrogen phosphate solution, carrying out a water bath reaction while violently stirring, cooling to room temperature after the reaction is completed, centrifuging, collecting the precipitate, repeatedly washing the precipitate with water, centrifuging, collecting the precipitate, and drying to obtain the terbium phosphate nano adsorption material. The invention also provides a method for eluting, adsorbing and collecting extracellular DNA by using the terbium phosphate nano adsorption material, wherein an eluent comprises 50-95% of 0.067 M PBS (Phosphate Buffer Solution), 5-50% of ethanol and 1-5g / L of glycine. The prepared terbium phosphate nano adsorption material has excellent adsorption performance and recyclability, and the method has high recovery rate for different types of DNA (deoxyribonucleic acid), and the recovery rate is kept at 65% or above.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection, and particularly relates to a method for preparing functionalized nanomaterials and efficiently enriching and recovering extracellular DNA in the environment by using the same. Background Art

[0002] Total DNA in the natural environment consists of intracellular DNA (iDNA) and extracellular DNA (eDNA). Compared with iDNA located in living cells, eDNA is outside the cell and is widely distributed in ecological niches such as soil, sediment, feces, and aquatic ecosystems, and is also found in tissue cultures and the blood of humans and some other animals. Extracellular DNA (eDNA) plays an important role in multiple biological processes, such as the formation and maturation of biofilms, horizontal gene transfer (HGT), and multi-element cycling (including carbon, nitrogen, and phosphate). However, the biological roles of eDNA vary among different species and even change with different life stages of the same organism. In addition, as an important carrier of specific microbial activities and genetic information in the environment, the dynamic distribution and migration and transformation processes of eDNA in the environment profoundly affect key fields such as water ecological safety assessment, tracking of the spread of antibiotic resistance genes, and biodiversity assessment. Especially in artificial aquatic systems such as the effluent of sewage treatment plants, eDNA not only carries the functional gene map of the microbial community, but is more likely to serve as a horizontal transfer vector of antibiotic resistance genes (eARGs), spreading antibiotic resistance among different microorganisms through horizontal gene transfer (HGT) and accelerating the ecological spread of drug-resistant strains. However, there is currently no standardized method for enriching and recovering eARGs in water, and eDNA in the water environment generally has the characteristics of low abundance (in the ng / L - μg / L level), severe fragmentation, and coexistence with interfering substances such as humic acid, resulting in technical bottlenecks such as large reagent consumption and large fluctuations in the recovery rate of target DNA in traditional enrichment methods, causing inaccuracies in subsequent results such as horizontal gene transfer (HGT) of antibiotic resistance genes and microbial community diversity based on eDNA analysis.

[0003] The current mainstream eDNA capture technology in the water environment mainly follows the classic paradigm of biomolecular extraction:

[0004] The hexadecyltrimethylammonium bromide that the CTAB method relies on is a cationic detergent that can dissolve cell membranes and form complexes with nucleic acids. This complex is soluble under high salt conditions, but difficult to dissolve under low salt conditions. In the process of extracting eDNA, CTAB is used to depolymerize nucleoproteins and the like under high salt conditions, so that DNA is released and then forms a complex with CTAB, namely the CTAB-DNA complex. Next, an organic solvent (such as chloroform) is added for extraction to remove impurities such as proteins, polysaccharides, and phenols. During this process, the CTAB-DNA complex will remain in the upper aqueous phase, while the impurities will be extracted to the lower layer by the organic solvent. Then, the CTAB-DNA complex is separated from the solution by adding alcohol or isopropanol for precipitation. Finally, DNA can be precipitated from the CTAB complex by reducing the salt concentration and centrifuging. During the centrifugation process, DNA will precipitate to the bottom of the centrifuge tube, while CTAB and other impurities will remain in the supernatant. Through washing and drying steps, eDNA extraction is achieved. However, the CTAB method relies on the different solubility of the complex formed by hexadecyltrimethylammonium bromide and nucleic acids under different ionic strengths to achieve DNA precipitation and separation. Although the cost is low, toxic reagents such as chloroform and phenol are used during the operation, which are highly toxic. The subsequent extraction steps are prone to result in low DNA recovery rates. A large amount of organic solvents must be consumed when processing large volumes of water samples. Therefore, it is more suitable for the recovery of eARGs in solid samples such as sediments, sludge and biofilms.

[0005] The principle of sodium acetate ethanol precipitation is that ethanol can capture water molecules around DNA, causing DNA to lose water and thus facilitate polymerization; at the same time, nucleic acid is a water-soluble compound of polyanions, which can combine with many 1-valent and 2-valent ions to form salts. Ethanol can eliminate the hydration layer of DNA to expose the negatively charged phosphate groups, making it easier to react with the high concentration of sodium ions (Na + ) to reduce the repulsive force of like charges between DNA molecules and make it easier to aggregate, eventually forming a DNA sodium salt precipitate. In a low temperature environment, this precipitation-promoting effect will be enhanced, and this precipitate will neither dissolve nor denature, and a commercial kit can be used for subsequent DNA extraction. However, the sodium acetate ethanol precipitation method is limited by the salinity of the water sample and the concentration of other impurities. When using ethanol to precipitate DNA, a large amount of ethanol is consumed when processing large-volume water samples. At the same time, impurities such as polysaccharides and proteins in the water will seriously affect the DNA recovery rate. Although using isopropanol instead of ethanol for DNA precipitation can reduce some reagent consumption, salts or organic matter (such as NaCl, sucrose) are easily co-precipitated with DNA during the operation, and isopropanol is difficult to evaporate and remove in the DNA precipitation, and the DNA precipitation needs to be further rinsed with 70% ethanol several times.

[0006] The magnetic bead adsorption-desorption method is based on the principle that magnetic beads have a stronger affinity for DNA than proteins and humic acid, and can reversibly adsorb DNA. eDNA in water is activated by the action of certain concentrations of polyethylene glycol (PEG) and salt ions (such as Na + ) under the condition that the negatively charged phosphate group is released by dissociating salt ions (such as Na + ) forms an ion bridge with the carboxyl groups on the surface of the magnetic beads, allowing the DNA to be specifically adsorbed to the surface of the carboxyl magnetic beads. After PEG and salts are removed, the addition of aqueous molecules will quickly and fully hydrate the DNA, eliminating the ionic interactions between the three, so that the DNA adsorbed to the surface of the magnetic beads is purified, thereby achieving the extraction of eDNA. However, the magnetic bead method is only suitable for the extraction and recovery of eDNA from small-volume water samples (1-5mL) or sludge samples. The recovery effect on large-volume or smaller-volume water samples is poor, and there is some interference. The cost of the magnetic beads themselves and the magnetic separation equipment restricts its large-scale application. Therefore, it is urgent to develop an efficient, stable, economical and simple method for the enrichment and recovery of extracellular DNA in actual water bodies.

[0007] In recent years, rare earth-based nanomaterials have shown breakthrough potential in the field of biomolecule capture due to their unique crystal field effects and surface coordination properties. 0-14 5d 0-1 6s 2 ), especially for Tb element (4f 9 6s 2 ) is an unfilled energy level, which is in an unstable state and is easily coordinated with the negatively charged phosphate groups on the DNA molecules. At the same time, the phosphate of the Tb element has a high degree of stability and biocompatibility, and is suitable for selectively and stably enriching extracellular DNA without causing damage to the aquatic ecological environment. Up to now, there is no patent involving the technology of enriching and recovering environmental DNA using terbium phosphate nanomaterials. It is worth noting that the effect of terbium phosphate nanomaterials in DNA recovery is significantly affected by the material preparation and elution methods. Therefore, the present invention improves and provides a stable and efficient terbium phosphate preparation method, and applies it to the enrichment and recovery of extracellular DNA in an aquatic environment. Summary of the invention

[0008] In view of the defects existing in the above-mentioned prior art, in order to solve the problems of poor selectivity for low-abundance eDNA, long operation cycle, high reagent consumption, and inability to be recycled multiple times in traditional enrichment methods, the present invention designs and provides a terbium phosphate nanomaterial with stable performance, excellent renewable properties, and capable of efficiently recovering extracellular DNA, as well as an efficient method for extracting and enriching and recovering extracellular DNA in the water environment. The present invention clarifies the addition sequence and operation method of ammonium dihydrogen phosphate solution and terbium nitrate solution during the material preparation process: that is, adding ammonium dihydrogen phosphate solution dropwise to the terbium nitrate solution. The adsorption performance of the terbium phosphate material prepared by this method is efficient and can be stabilized above 95%. In addition, the present invention optimizes the adsorption-elution test: Utilizing the influence of pH and temperature on the properties of DNA (ζ potential and solubility), by adjusting the adsorption conditions and the eluent ratio, desorption at 65°C significantly improves the elution and recovery efficiency of DNA. On the premise of ensuring the unchanged DNA recovery rate, the elution time is successfully reduced to 40 minutes, and at the same time, the ultrasonic step is omitted, reducing energy consumption while avoiding the breakage of eDNA and ensuring the integrity of DNA.

[0009] Adding ammonium dihydrogen phosphate solution to terbium nitrate in the present invention can inhibit the hydrolysis of Tb 3+ , reduce the generation of impurity Tb(OH)3. Adding dropwise can control the crystal nucleation rate, reduce local supersaturation, and generate smaller and more uniform nanoparticles. The fewer impurities and the smaller the crystals, the larger the specific surface area, and the easier it is to adsorb.

[0010] To achieve the above object, the present invention adopts the following technical solutions:

[0011] On the one hand, the present invention provides a preparation method of a terbium phosphate nano-adsorption material, comprising the following steps:

[0012] Weigh the terbium nitrate solution, add the ammonium dihydrogen phosphate solution dropwise, carry out a water bath reaction while stirring vigorously, cool to room temperature after the reaction is completed, centrifuge, collect the precipitate, repeat multiple times to wash the precipitate with water and then centrifuge, collect the precipitate, and dry it to obtain the terbium phosphate nano-adsorption material.

[0013] In the described preparation method, the molar ratio of the terbium nitrate solution to the ammonium dihydrogen phosphate solution is 1:(1 - 10).

[0014] In the described preparation method, the water bath reaction conditions are: temperature 25 - 90°C, time 1 - 10 h.

[0015] In the described preparation method, the centrifugation conditions are: rotation speed 4000 - 10000 rpm, time 2 - 10 min.

[0016] In a second aspect, the present invention provides a terbium phosphate nano-adsorbent material, which is obtained by the method described in any one of the above.

[0017] In a third aspect, the present invention provides a method for efficiently enriching and recovering low-concentration free extracellular DNA, comprising the following steps:

[0018] Take a solution to be treated containing extracellular DNA, add the terbium phosphate nano-adsorbent material as described in claim 5, stir, centrifuge, collect the precipitate, add an eluent, stir at a high temperature and then centrifuge, recover the eluent containing the extracellular DNA solution, collect the precipitate, dry it, and recover the terbium phosphate nano-adsorbent material;

[0019] The eluent contains 50-95% 0.067M PBS, 5-50% ethanol, and 1-5 g / L glycine.

[0020] In the method described above, the concentration of DNA in the solution to be treated containing extracellular DNA is 0.5-50 ng / μL.

[0021] In the method described above, the mass-volume ratio of the terbium phosphate nano-adsorbent material to the solution to be treated is 0.04-1:1.

[0022] In the method described above, the stirring is carried out by magnetic stirring for 10-120 min;

[0023] The conditions for the high-temperature stirring are: temperature 40-70°C, time 10-120 min.

[0024] In the method described above, the conditions for centrifugation are: rotation speed 4000-15000 rpm, time 2-10 min.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The present invention is applicable to extracting low-concentration free extracellular DNA in various volumes and various water bodies, has strong operability, no toxic reagents are introduced, and the total extraction process only takes 100 minutes, which is convenient and efficient.

[0027] 2. The present invention can efficiently extract free extracellular DNA. Even for water body samples with a concentration lower than 0.05 ng / μL, the present invention can still achieve an optimal recovery rate of up to 61.71%.

[0028] 3. The terbium phosphate nano-adsorbent material prepared by the present invention has a high recovery rate for different types of DNA (long-chain, short-chain, high-concentration, low-concentration), and the recovery rate remains above 65%.

[0029] 4. The terbium phosphate nano-adsorbent material prepared by the present invention not only has excellent adsorption performance but also has good recyclability. This characteristic makes the material have broad application prospects in the fields of environmental protection, resource recycling, and sustainable development. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the preparation process of the terbium phosphate nano-material;

[0031] Figure 2 is the XRD pattern of TbPO4 prepared in Example 1;

[0032] Figure 3 is the N2 adsorption / desorption isotherm and pore size distribution curve of TbPO4 prepared in Example 1;

[0033] Figure 4 is the adsorption-elution test process of DNA;

[0034] Figure 5 is the comparison of the DNA adsorption performance of the terbium phosphate nano-adsorbent material prepared in Example 1 of the present invention, terbium phosphate prepared by the hydrothermal method, and common silicon-based nucleic acid adsorption particles;

[0035] Figure 6 is the test result of the adsorption rate of the terbium phosphate nano-adsorbent material for free DNA under different ionic conditions; among them, a is the adsorption rate of the terbium phosphate nano-adsorbent material for free DNA under sodium ion conditions; b is the adsorption rate of the terbium phosphate nano-adsorbent material for free DNA under magnesium ion conditions; c is the adsorption rate of the terbium phosphate nano-adsorbent material for free DNA under the coexistence of sodium ions and magnesium ions;

[0036] Figure 7 is the test result of the adsorption rate and recovery rate of the terbium phosphate nano-adsorbent material prepared in Example 1 of the present invention for DNA under different conditions;

[0037] Figure 8 is the test result of the adsorption and recovery performance of the terbium phosphate nano-adsorbent material prepared in Example 1 of the present invention for different types and different concentrations of DNA; among them, a is the recovery rate result when the DNA concentration is 0.5 - 50 ng / μL, and b is the recovery rate result for long-chain DNA and short-chain DNA;

[0038] Figure 9 is the test result of the performance of the terbium phosphate nano-adsorbent material prepared in Example 1 of the present invention for multiple cyclic recovery of extracellular DNA;

[0039] Figure 10 is the flow chart of the enrichment and recovery of eDNA in the environment based on the terbium phosphate-functionalized nano-adsorbent material. DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0041] Example 1:

[0042] As Figure 1 shown, the preparation method of terbium phosphate nanomaterials of the present invention includes the following steps:

[0043] a) Prepare 20 mL of 0.05 M ammonium dihydrogen phosphate solution and terbium nitrate solution respectively;

[0044] b) Pour the terbium nitrate solution into a small flask equipped with a rotor;

[0045] c) Dropwise add the ammonium dihydrogen phosphate solution to the terbium nitrate solution at 80 °C until all is added;

[0046] d) Keep stirring vigorously at 80 °C for 4 h and then take out the small flask from the water bath;

[0047] e) Centrifuge at 8000 rpm for 5 min after the small flask cools to room temperature;

[0048] f) Discard the supernatant after centrifugation, wash the solid with ultrapure water, and centrifuge again for 5 min after the solid is completely dispersed in water;

[0049] g) Repeat the above steps twice;

[0050] h) Discard the supernatant after centrifugation, and place the open centrifuge tube in a vacuum oven to dry (60 °C, 24 h) to obtain the terbium phosphate nano-adsorption material.

[0051] Figure 2 XRD pattern of TbPO4 prepared for Example 1. The X-ray diffraction (XRD) pattern shows that the synthesized TbPO4 is a hexagonal structure. In addition, multiple distinct and sharp diffraction peaks are shown in the figure, indicating that the sample has good crystallinity.

[0052] Figure 3 N2 adsorption / desorption isotherm of TbPO4. It can be seen from the figure that TbPO4 conforms to the quasi-type IV isotherm and has an H3-type adsorption hysteresis loop according to the IUPAC classification, indicating that its pore structure may be irregular. The BET equation is used to calculate its specific surface area of 36.3 m 2 / g, which has a relatively high specific surface area and is beneficial to the contact with DNA in the reaction. The pore size distribution curve is analyzed using the Bareett–Joyner–Halenda adsorption isotherm estimation (BJH) ([[]] Figure 3 inset). TbPO4 shows a pore size of 0.14 cm 3The pore volume of / g and the pore size of 35.0 nm enable TbPO4 to better achieve the mass transfer process with free DNA in water bodies.

[0053] Example 2:

[0054] As Figure 4 shown, an adsorption-elution method for efficiently enriching and recovering low-concentration free extracellular DNA using the terbium phosphate nano-adsorbent material prepared in Example 1 above includes the following steps:

[0055] a) Add 0.04 - 1 g / L of the terbium phosphate nano-adsorbent material prepared in Example 1 above to a DNA solution of 0.05 - 50 ng / μL (using calf thymus DNA as a representative extracellular DNA, adding 100 mM NaCl and 10 mM MgCl2) placed with a rotor.

[0056] b) Place the above solution on a magnetic stirrer and stir for 60 min.

[0057] c) After sucking out the rotor with a magnet, centrifuge at 10000 rpm for 5 min.

[0058] d) Discard the supernatant after centrifugation, recover the terbium phosphate, and add 2 mL of the prepared elution solution (85% 0.067 M PBS + 15% ethanol + 4 g / L glycine).

[0059] e) Stir the above solution at 65 °C for 40 min to recover extracellular DNA.

[0060] f) After the stirring ends, centrifuge at 10000 rpm for 5 min and collect the eluate.

[0061] g) Wash the terbium phosphate with ultrapure water and place it in a vacuum oven to dry (60 °C, 8 h) to recover the terbium phosphate for reuse.

[0062] Using the above adsorption-elution method, the terbium phosphate prepared by the present invention was used to recover DNA in actual sewage at different volumes and different DNA concentrations, and the results are shown in Table 1 below.

[0063] Table 1 Test results of the adsorption performance of the terbium phosphate nano-adsorbent material for DNA in sewage

[0064]

[0065] Comparative Example 1:

[0066] The preparation of the terbium phosphate adsorbent material by the hydrothermal method includes the following steps:

[0067] a) Prepare 20 mL of 0.05 M ammonium dihydrogen phosphate solution and terbium nitrate solution.

[0068] b) Add terbium nitrate solution and diammonium hydrogen phosphate solution into the reaction kettle at a volume ratio of 1:1, and control the filling degree of the reaction kettle to be 70 - 80%;

[0069] c) Stir the above mixed solution at room temperature for 30 minutes;

[0070] d) After sealing the reaction kettle, put it into the oven and keep it at 180 °C for 48 hours;

[0071] e) After the reaction kettle cools down to room temperature, centrifuge the product in the kettle at 8000 rpm for 5 min;

[0072] f) Discard the supernatant after centrifugation, wash the solid with ultrapure water, and centrifuge again for 5 min after the solid is completely dispersed in water;

[0073] g) Repeat the above steps twice;

[0074] h) Discard the supernatant after centrifugation, and put the open centrifuge tube into the vacuum oven to dry (60 °C, 24 h) to obtain the terbium phosphate adsorption material prepared by the hydrothermal method.

[0075] Comparative Example 2:

[0076] The preparation of silicon-based nucleic acid adsorption particles includes the following steps:

[0077] a) Prepare 400 mL of 0.05 M aluminum chloride solution;

[0078] b) Add 18 mL of Na2CO3 (2 M) to the above solution, and adjust the pH to 7.2 with 1 M Na2CO3, and stir evenly;

[0079] c) Let the mixed turbid solution stand at room temperature for 6 - 12 h, centrifuge at 1100×g for 15 min, and discard the supernatant;

[0080] d) Resuspend the precipitate with 100 mL of 0.14 M NaCl, centrifuge at 1100×g for 15 min, and discard the supernatant;

[0081] e) Repeat the above steps;

[0082] f) After resuspending the precipitate with 0.14 M NaCl, mix the turbid solution with 5 wt% silica gel of 60 - 100 mesh at a volume ratio of 48:52 and load it into the reaction kettle, and control the filling degree of the reaction kettle to be 70 - 80%;

[0083] g) After sealing the reaction kettle, put it into the oven and keep it at 121 °C for 20 min;

[0084] h) After the reaction kettle cools down to room temperature, place the product in the kettle in a blast drying oven at 60 °C for 36 h.

[0085] The adsorption materials prepared in Example 1 and Comparative Examples 1 and 2 were tested for their adsorption rates:

[0086] 1) Add 1 g / L of the terbium phosphate nano-adsorption material prepared in the above example, the terbium phosphate adsorption material prepared by hydrothermal method, and the silicon-based nucleic acid adsorption particles to a 4 mL DNA solution of 50 ng / μL (using calf thymus DNA as the representative extracellular DNA, adding 100 mM NaCl and 10 mM MgCl2) placed with a rotor.

[0087] 2) Place the above solution on a magnetic stirrer and stir, and measure the DNA concentration in the solution at the 30th and 60th minutes.

[0088] 3) Calculate the adsorption rate from the DNA concentration obtained by the test: Adsorption rate = (1 - (C t / C0)) × 100%, where C t is the DNA concentration in the solution measured at the t-th minute, and C0 is the initial DNA concentration in the solution (here it is 50 ng / μL).

[0089] From Figure 5 the comparison of the DNA adsorption performance of the terbium phosphate nano-adsorption material prepared in Example 1 with the terbium phosphate prepared by hydrothermal method and the common silicon-based nucleic acid adsorption particles, it can be seen that the terbium phosphate nano-adsorption particles prepared in the present invention reach equilibrium in 60 minutes for DNA adsorption, and the maximum adsorption rate reaches 80.59 ± 3.18%, and the maximum adsorption rate is significantly better than that of the terbium phosphate adsorption material prepared by hydrothermal method and the silicon-based nucleic acid adsorption particles.

[0090] Example 3:

[0091] a) Add 1 g / L of the terbium phosphate nano-adsorption material prepared in the above Example 1 to a 4 mL DNA solution of 50 ng / μL (using calf thymus DNA as the representative extracellular DNA) placed with a rotor.

[0092] b) Add NaCl to the above solution to make its concentration reach 10 - 500 mM respectively to test the influence of Na + ions on the adsorption of DNA by terbium phosphate, and add MgCl2 to the above solution to make its concentration reach 1 - 50 mM respectively to test the influence of Mg 2+ ions on the adsorption of DNA by terbium phosphate;

[0093] c) Place the above solution on a magnetic stirrer and stir, and measure the DNA concentration in the solution at the 30th and 60th minutes.

[0094] d) Calculate the adsorption rate from the DNA concentration obtained by the test: Adsorption rate = (1 - (C t / C0)) × 100%, where Ct C is the DNA concentration in the solution measured at the t-th minute, and C0 is the initial DNA concentration in the solution (here it is 50 ng / μL);

[0095] e) After sucking out the rotor with a magnet, centrifuge at 10,000 rpm for 5 min;

[0096] f) Discard the supernatant after centrifugation, recover terbium phosphate, and add 2 mL of the prepared eluent solution (85% 0.067 M PBS + 15% ethanol + 4 g / L glycine);

[0097] g) Stir the above solution at 65 °C for 40 min to recover extracellular DNA;

[0098] h) After the stirring is completed, centrifuge at 10,000 rpm for 5 min and collect the eluent;

[0099] i) Test the DNA concentration in the eluent to calculate the recovery rate: Recovery rate = (1 - C x ×2 / C0×4)×100%, where C x is the DNA concentration in the eluent, and C0 is the initial DNA concentration in the solution (here it is 50 ng / μL).

[0100] From Figure 6 in (a) and (b), it can be seen that the adsorption of DNA by the terbium phosphate nano-adsorption particles prepared in Example 1 of the present invention shows a trend of first increasing and then decreasing in the adsorption rate and recovery rate with the increase of the ion concentration under the influence of Na + ions and Mg 2+ ions alone. Under the action of 250 mM Na + ions alone, the adsorption rate and recovery rate of the terbium phosphate nano-adsorption particles for DNA reach the maximum, and the adsorption rate is 87.96 ± 2.04% at this time; under the action of 10 mM Mg 2+ ions alone, the adsorption rate and recovery rate of the terbium phosphate nano-adsorption particles for DNA reach the maximum, and the adsorption rate is 94.45 ± 1.71% at this time.

[0101] Considering that monovalent and divalent cations will play a bridging role in the adsorption between the adsorption particles and DNA, we further analyzed the introduction of Na + ions and Mg 2+ ions into the adsorption process simultaneously, as Figure 6(c), considering the reduction of the influence of salt ions on subsequent other analysis processes and elution processes, we set the sodium ion concentration to 100 mM and the magnesium ion concentration to 10 mM. Under this concentration condition, the adsorption of terbium phosphate nanoadsorbent particles on DNA is further enhanced. Finally, the adsorption rate of DNA reaches 92.13 ± 3.83%, which is 1.3 times that of the action of sodium ions alone and 1.1 times that of the action of magnesium ions alone.

[0102] Example 4:

[0103] a) Add 1 g / L of the terbium phosphate nanoadsorbent material prepared in Example 1 above to a 4 mL, 50 ng / μL DNA solution (using calf thymus DNA as representative extracellular DNA, adding 100 mM NaCl and 10 mM MgCl2) containing a rotor.

[0104] b) Add sucrose to the above solution to make its concentration reach 5 - 200 mg / L respectively to test the effect of sucrose on the adsorption of DNA by terbium phosphate; add peptone to the above solution to make its concentration reach 5 - 200 mg / L respectively to test the effect of peptone on the adsorption of DNA by terbium phosphate; adjust the initial pH of the above solution to reach 3 - 7 respectively (where the pH of the original solution without adjustment is 6) to test the effect of pH on the adsorption of DNA by terbium phosphate;

[0105] c) Place the above solution on a magnetic stirrer and stir, and measure the DNA concentration in the solution at the 30th and 60th minutes.

[0106] d) Calculate the adsorption rate from the DNA concentration obtained by testing: Adsorption rate = (1 - (C t / C0)) × 100%, where C t is the DNA concentration in the solution measured at the t-th minute, and C0 is the initial DNA concentration in the solution (here it is 50 ng / μL);

[0107] e) After sucking out the rotor with a magnet, centrifuge at 10000 rpm for 5 minutes;

[0108] f) Discard the supernatant after centrifugation, recover the terbium phosphate and add 2 mL of the prepared eluent solution (85% 0.067 M PBS + 15% ethanol + 4 g / L glycine);

[0109] g) Stir the above solution at 65 °C for 40 minutes to recover extracellular DNA;

[0110] h) After the stirring is completed, centrifuge at 10000 rpm for 5 minutes and collect the eluent;

[0111] i) Measure the DNA concentration in the eluent to calculate the recovery rate: Recovery rate = (1 - C x×2 / C0×4)×100%, where C x is the DNA concentration in the eluent, and C0 is the initial DNA concentration in the solution (here it is 50 ng / μL).

[0112] From Figure 7 it can be seen that sucrose has basically no effect on the enrichment and recovery of extracellular DNA by terbium phosphate; high concentrations of peptone will significantly inhibit the adsorption of extracellular DNA by terbium phosphate through competitive effects, but have basically no effect on the enrichment and recovery of extracellular DNA by terbium phosphate at environmental concentrations (5 mg / L); while the solution pH shows an acidic promotion and alkaline inhibition effect on the adsorption of extracellular DNA by terbium phosphate; the above results indicate that the terbium phosphate prepared in the present invention has a certain ability to resist interference from environmental factors for the enrichment and recovery of extracellular DNA in the environment.

[0113] Example 5:

[0114] a) Add 1 g / L of the terbium phosphate nano-adsorbent material prepared in Example 1 above to a 0.5 - 50 ng / μL DNA solution (using calf thymus DNA as a representative extracellular DNA, adding 100 mM NaCl and 10 mM MgCl2) containing a rotor to analyze the enrichment and recovery of different concentrations of DNA by terbium phosphate. Add 1 g / L of the terbium phosphate nano-adsorbent material prepared in the above example to a 50 ng / μL DNA solution (using calf thymus DNA to simulate long-chain extracellular DNA and the active sludge DNA extracted by a commercial kit to simulate short-chain extracellular DNA, adding 100 mM NaCl and 10 mM MgCl2) containing a rotor to analyze the enrichment and recovery of DNA with different fragment lengths by terbium phosphate;

[0115] b) After stirring for 60 minutes, use a magnet to suck out the rotor and then centrifuge the solution at 10,000 rpm for 5 min;

[0116] c) Discard the supernatant after centrifugation, recover the terbium phosphate and add 2 mL of the prepared eluent solution (85% 0.067 M PBS + 15% ethanol + 4 g / L glycine);

[0117] d) Stir the above solution at 65 °C for 40 min to recover extracellular DNA;

[0118] e) After the stirring is completed, centrifuge at 10,000 rpm for 5 min and collect the eluent;

[0119] f) Test the DNA concentration in the eluent to calculate the recovery rate: Recovery rate = (1 - C x ×2 / C0×V)×100%, where C x is the DNA concentration in the eluent, C0 is the initial DNA concentration in the solution, and V is the volume of the initial DNA solution.

[0120] As can be seen from Figure 8 Example 1 of the present invention, terbium phosphate prepared shows high recovery rates for different types and concentrations of DNA, especially better recovery rates for low-concentration DNA.

[0121] Example 6:

[0122] a) Add 1 g / L of the terbium phosphate nano-adsorbent material prepared in Example 1 above to a 4 mL DNA solution (using calf thymus DNA as a representative extracellular DNA, adding 100 mM NaCl and 10 mM MgCl2) with a concentration of 50 ng / μL placed with a rotor;

[0123] After stirring for 60 minutes, measure the DNA concentration in the solution, and calculate the adsorption rate through the measured DNA concentration: Adsorption rate = (1 - (C / C0)) × 100%, where C is the DNA concentration in the solution measured after 60 minutes, and C0 is the initial DNA concentration in the solution (here it is 50 ng / μL);

[0124] b) After sucking out the rotor with a magnet, centrifuge at 10000 rpm for 5 min; after sucking out the rotor with a magnet, centrifuge the solution at 10000 rpm for 5 min;

[0125] c) Discard the supernatant after centrifugation, recover terbium phosphate and add 2 mL of the prepared eluent solution (85% 0.067 M PBS + 15% ethanol + 4 g / L glycine);

[0126] d) Stir the above solution at 65 °C for 40 min to recover extracellular DNA;

[0127] e) After the stirring ends, centrifuge at 10000 rpm for 5 min, collect the eluent, and measure the DNA concentration in the eluent to calculate the recovery rate: Recovery rate = (1 - C x × 2 / C0 × 4) × 100%, where C x is the DNA concentration in the eluent, and C0 is the initial DNA concentration in the solution (here it is 50 ng / μL);

[0128] f) Recover terbium phosphate, wash terbium phosphate with ultrapure water and place it in a vacuum oven for drying (60 °C, 8 h) to recover terbium phosphate;

[0129] g) Repeat the above operations a) to f) multiple times to judge the recyclability of terbium phosphate.

[0130] As can be seen from Figure 9 it can be known that the terbium phosphate prepared by the present invention has a certain recyclability, and the enrichment recovery rate for extracellular DNA can still reach 50.42 ± 1.78% after being used three times.

Claims

1. A preparation method of a terbium phosphate nano-adsorption material, characterized in that, It includes the following steps: Weigh terbium nitrate solution, and gradually add ammonium dihydrogen phosphate solution while stirring vigorously during the water bath reaction. After the reaction is completed, cool it to room temperature, centrifuge, collect the precipitate, repeat the process multiple times by washing the precipitate with water and then centrifuging, collect the precipitate, and dry it to obtain the terbium phosphate nano-adsorbent material.

2. The preparation method according to claim 1, characterized in that, The molar ratio of the terbium nitrate solution to the ammonium dihydrogen phosphate solution is 1:(1 - 10).

3. The preparation method according to claim 1, characterized in that, The conditions for the water bath reaction are: temperature 25 - 90 °C, time 1 - 10 h.

4. The preparation method according to claim 1, characterized in that, The conditions for centrifugation are: rotational speed 4000 - 10000 rpm, time 2 - 10 min.

5. A terbium phosphate nano-adsorption material, characterized in that, It is prepared by the method described in any one of claims 1 - 4.

6. A method for efficiently enriching and recovering low-concentration free extracellular DNA, characterized in that, It includes the following steps: Take the solution to be treated containing extracellular DNA, add the terbium phosphate nano-adsorbent material described in claim 5, stir, centrifuge, collect the precipitate, add the eluent, stir at high temperature and then centrifuge, recover the eluent containing the extracellular DNA solution, collect the precipitate, dry it, and recover the terbium phosphate nano-adsorbent material; The eluent contains 50 - 95% 0.067M PBS, 5 - 50% ethanol, and 1 - 5 g / L glycine.

7. The method according to claim 6, wherein The concentration of DNA in the solution to be treated containing extracellular DNA is 0.5 - 50 ng / μL.

8. The method according to claim 6, wherein The mass - volume ratio of the terbium phosphate nano-adsorbent material to the solution to be treated is (0.04 - 1):1 g / L.

9. The method according to claim 6, wherein The stirring is carried out by magnetic stirring for 10 - 120 min; The conditions for high - temperature stirring are: temperature 40 - 70 °C, time 10 - 120 min.

10. The method according to claim 6, characterized in that, The conditions for centrifugation are rotational speed 4000 - 15000 rpm, time 2 - 10 min.

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