DNA phosphorus tracer wrapped in iron oxide and its preparation method and application

Abstract

The present invention provides a DNA phosphorus tracer coated with iron oxide, and a preparation method and application thereof. The DNA phosphorus tracer coated with iron oxide of the present invention is composed of a SiO2 core, a SiO2 shell coated in the core, and an iron oxide capsid coated in the outermost layer; the core is a SiO2 microsphere of different particle sizes with nucleic acid adsorbed on the surface, a layer of SiO2 shell is coated on the outer side of the core, and a layer of iron oxide capsid is coated on the SiO2 shell. Based on the characteristic that iron oxide has strong adsorption to phosphorus, the present invention synthesizes DNA phosphorus tracers coated with iron oxide of different sizes to mark phosphates entering the soil and drainage ditches at different times and spatial positions.

Description

Technical Field

[0001] The invention belongs to the field of environmental protection, and in particular relates to a DNA phosphorus tracer wrapped in iron oxide, and a preparation method and application thereof. Background Art

[0002] Lead 210 is a geological fallout radionuclide (t 1 / 2 = 22.3 years), is produced in the decay series of uranium 238. Uranium 238 decays into several short-lived nuclides in turn, until radium 226 (t 1 / 2 = 1600 years), then decays into radon 222, a short-lived gaseous nuclide that escapes into the atmosphere (t 1 / 2 = 3.83 days), which decays through a series of short-lived nuclides until Pb-210, which is strongly adsorbed to particulate matter and aerosols (K d =10 3 -10 6 L / kg), and returns to the surface through dry and wet precipitation. The radium 226 that has not left the terrestrial environment exists in the soil and rocks, and a part of it decays in situ into lead 210, which is in equilibrium with the parent material. Lead 210 and radium 226 are measured at the same time, and the amount of lead 210 in equilibrium with radium 226 is subtracted from the total lead 210, which is the lead 210 that has settled through the atmosphere.

[0003] Cesium 137 is an artificial radionuclide (t 1 / 2 = 30 years), the peak production of cesium-137 occurred in 1963, and the contribution to the atmosphere ceased around 1976. During the period of nuclear bomb testing, cesium-137 was distributed in the stratosphere around the world. Since cesium-137 is strongly adsorbed by particulate matter and aerosols (K d About 10 5 L / kg), and dry and wet deposition introduces cesium 137 to the surface. The unique and well-characterized generation, deposition, and soil profile distribution of cesium 137 make it an ideal radionuclide for sediment dating and tracer techniques.

[0004] Beryllium-7 is a relatively short-lived cosmic radioactive isotope (t 1 / 2 = 53.3 days), which is produced by cosmic rays in the spallation of oxygen and nitrogen in the upper atmosphere. Beryllium 7 is strongly adsorbed on particulate matter (K d =104-105 L / kg), and both wet and dry deposition occurs. Because Be-7 has a much shorter half-life than Cs-137 or Pb-210, it can be used to elucidate patterns on monthly or even single runoff event timescales, whereas Cs-137 and Pb-210 tend to provide information on an annual scale.

[0005] In a small, predominantly agricultural watershed in Wisconsin, USA, during a moderate rainfall event in June 2008, sediment was primarily derived from upstream soil or cultivated surface, with a small contribution from upturned streambed mud and almost no contribution from the streambank. During this event, sediment was deposited on the streambed, forming a temporary reservoir that could later be resuspended, with particle-bound phosphorus accounting for 33-46% of the total phosphorus (TP) transported in the stream.

[0006] The existing tracing methods for phosphorus non-point source pollution can only trace the source type, such as soil surface, riverbed mud, riverbank, etc., but cannot trace the specific spatial location of the source. Therefore, it is urgent to develop a new type of phosphorus tracer. Summary of the invention

[0007] The purpose of the present invention is to provide a DNA phosphorus tracer wrapped by iron oxide and a preparation method and application thereof.

[0008] The concept of the present invention is as follows: Studies have found that soil particles of different sizes contribute differently to non-point source pollution of phosphorus, and the smaller the soil particles, the greater their contribution to non-point source pollution of phosphorus. DNA phosphorus tracers wrapped with iron oxides of different particle sizes are placed at different locations in the irrigation area, and the placement and time of the DNA tracers are encoded with DNA sequences. The DNA tracers placed at the same location and time are a mixture of SiO2 (DNA (SiO2)) microsphere particles wrapped with iron oxides of four different particle sizes (60nm, 250nm, 550nm and 1.2μm). The strong adsorption of iron oxides on phosphorus is used to trace the time and spatial location of the source of phosphorus adsorbed on soil particles of different sizes.

[0009] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides an iron oxide-coated DNA phosphorus tracer, wherein the phosphorus tracer is composed of SiO2 (DNA (SiO2)) microsphere particles coated with iron oxide of four different particle sizes;

[0010] The microsphere particles are composed of a core, an outer shell and an iron oxide capsid wrapped in the outer shell;

[0011] The inner core is SiO2 microspheres of four different sizes with nucleic acid adsorbed on the surface, a layer of SiO2 shell is wrapped on the outer side of the inner core, and a layer of iron oxide capsid is wrapped on the surface of the shell;

[0012] The particle sizes of the SiO2 microspheres are 20 nm, 200 nm, 500 nm and 1000 nm, respectively. Correspondingly, the particle sizes of the iron oxide-coated SiO2 (DNA (SiO2)) microsphere particles are 60 nm, 250 nm, 550 nm and 1.2 μm, respectively.

[0013] In the present invention, the particle size of the SiO2 (DNA (SiO2)) microsphere particles with positive surface charge is mainly controlled by the particle size of the SiO2 microsphere (core) and is fine-tuned by the thickness of the SiO2 shell.

[0014] In the present invention, the nucleic acid may be a single-stranded or double-stranded DNA having a length of 60-120 bp and not containing a stem-loop or hairpin structure.

[0015] In a second aspect, the present invention provides a method for preparing an iron oxide-encapsulated DNA phosphorus tracer, comprising the following steps:

[0016] (1) Preparing SiO2 microspheres of different particle sizes;

[0017] (2) The SiO2 microspheres are contacted with the functionalized reagent TMAPS to make the surface of the SiO2 microspheres positively charged, and then the nucleic acids are electrostatically adsorbed to serve as the core;

[0018] (3) contacting the core with TEOS to catalyze the growth of SiO2, forming a layer of SiO2 shell on the outside of the core, and obtaining SiO2 (DNA (SiO2)) microsphere particles of different particle sizes;

[0019] (4) Preparation of hematite nanoparticles: Adding ferric chloride solution to boiling ultrapure water to obtain hematite nanoparticles with a particle size of about 10 nm;

[0020] (5) mixing the SiO2(DNA(SiO2)) microsphere particles obtained in step (3) with the hematite nanoparticles obtained in step (4) so ​​that the outermost layer of the SiO2(DNA(SiO2)) microsphere particles is coated with an iron oxide capsid, thereby obtaining;

[0021] Furthermore, the preparation method comprises the following steps:

[0022] A. Preparation of SiO2 (DNA (SiO2)) microspheres coated with iron oxide with a particle size of 60 nm

[0023] A1. Preparation of SiO2 core solution: SiO2 microspheres with a particle size of 20 nm were suspended in isopropanol to prepare a SiO2 core solution with a concentration of 50 mg / mL;

[0024] A2. Preparation of TMAPS-functionalized SiO2 microspheres with positive surface charge: Mix the prepared SiO2 core solution, take 1 mL and add it to a 2 mL microcentrifuge tube, and ultrasonicate for 10 minutes to obtain a uniform SiO2 microsphere colloidal solution; add 10 μL of TMAPS solution, and then stir at 900 rpm in a constant temperature box at room temperature for 12 hours, and wash with isopropanol to obtain SiO2 microspheres with positive surface charge;

[0025] A3. Preparation of SiO2 (DNA (SiO2)) microsphere solution with a particle size of about 40 nm: first prepare a nucleic acid solution with a concentration of 50 μg / mL with ultrapure water; then add 700 μL ultrapure water, 320 μL nucleic acid solution and 35 μL SiO2 microspheres with positive surface charge into a centrifuge tube, mix and then ultrasonicate to obtain a uniform solution; wash with ultrapure water by centrifugation and then ultrasonicate to obtain a uniform colloidal solution; then, add 0.5 μL TMAPS solution thereto, mix well, then add 0.5 μL TEOS, and stir at room temperature at a speed of 900 rpm for 4 hours; then add 32.4 μL TEOS to the system, and stir at room temperature at a speed of 900 rpm for 4 days; wash with ultrapure water and mix well with ultrasonicate to obtain a colloidal solution of SiO2 (DNA (SiO2)) microspheres with a particle size of about 40 nm;

[0026] A4. Preparation of SiO2(DNA(SiO2)) microspheres with uniform particle size of about 40 nm: The SiO2(DNA(SiO2)) microsphere colloidal solution was freeze-dried in vacuum to obtain SiO2(DNA(SiO2)) microspheres, and 1 mg of SiO2 microspheres was added to 1 g of zirconium beads in a micro-grinder, and the zirconium beads with a diameter of 0.1 mm were ground for 2 to 8 minutes, and then centrifuged and washed 3 times at 21000 g, the supernatant was removed and ultrapure water was added to obtain SiO2(DNA(SiO2)) microspheres with uniform particle size of about 40 nm;

[0027] A5. Preparation of hematite nanoparticles with a particle size of about 10 nm: adding 100 mL of 1 mol / L ferric chloride solution to 900 mL of boiling ultrapure water through a dropper at a rate of two drops per second to obtain a hematite nanoparticle solution; cooling the hematite nanoparticle solution to room temperature to obtain hematite nanoparticles with a particle size of about 10 nm;

[0028] A6. Preparation of SiO2 (DNA (SiO2)) microsphere particles coated with iron oxide with a particle size of about 60 nm: 1.8-18 g of the hematite nanoparticles obtained in step A5 and 50 mg of the SiO2 (DNA (SiO2)) microspheres obtained in step A4 were mixed, and the mixture was stirred at room temperature for 2 hours. The resulting product was washed to remove salt, and centrifuged and dried under nitrogen to obtain SiO2 (DNA (SiO2)) microsphere particles coated with iron oxide with a particle size of about 60 nm;

[0029] B. Preparation of SiO2 (DNA (SiO2)) microspheres coated with iron oxide with a particle size of 250 nm

[0030] B1. Preparation of SiO2 core solution: SiO2 microspheres with a particle size of 200 nm were suspended in isopropanol to prepare a SiO2 core solution with a concentration of 50 mg / mL;

[0031] B2. Preparation of TMAPS-functionalized SiO2 microspheres with positive surface charge: Mix the prepared SiO2 core solution, take 1 mL and add it to a 2 mL microcentrifuge tube, and ultrasonicate for 10 minutes to obtain a uniform SiO2 microsphere colloidal solution; add 10 μL of TMAPS solution, and then stir at 900 rpm in a constant temperature box at room temperature for 12 hours, and wash with isopropanol to obtain SiO2 microspheres with positive surface charge;

[0032] B3. Preparation of SiO2 (DNA (SiO2)) microsphere solution with a particle size of about 230 nm: first prepare a nucleic acid solution with a concentration of 50 μg / mL with ultrapure water; then add 700 μL ultrapure water, 320 μL nucleic acid solution and 35 μL SiO2 microspheres with positive surface charge into a centrifuge tube, mix and then ultrasonicate to obtain a uniform solution; wash with ultrapure water by centrifugation and then ultrasonicate to obtain a uniform colloidal solution; then, add 0.5 μL TMAPS solution thereto, mix well, then add 0.5 μL TEOS, and stir at room temperature at 900 rpm for 4 hours; then add 2.4 μL TEOS to the system, and stir at room temperature at 900 rpm for 4 days; wash with ultrapure water and mix well with ultrasonicate to obtain a colloidal solution of SiO2 (DNA (SiO2)) microspheres with a particle size of about 230 nm;

[0033] B4. Preparation of SiO2(DNA(SiO2)) microspheres with uniform particle size of about 230 nm: The SiO2(DNA(SiO2)) microsphere colloidal solution was freeze-dried in vacuum to obtain SiO2(DNA(SiO2)) microspheres, and 1 g of zirconium beads was added to 1 mg of SiO2 microspheres in a micro-grinder, and the zirconium beads with a diameter of 0.1 mm were ground for 2 to 8 minutes, and then centrifuged and washed 3 times at 21000 g, the supernatant was removed and ultrapure water was added to obtain SiO2(DNA(SiO2)) microspheres with uniform particle size of about 230 nm;

[0034] B5. Preparation of hematite nanoparticles with a particle size of about 10 nm: adding 100 mL of 1 mol / L ferric chloride solution to 900 mL of boiling ultrapure water through a dropper at a rate of two drops per second to obtain a hematite nanoparticle solution; cooling the hematite nanoparticle solution to room temperature to obtain hematite nanoparticles with a particle size of about 10 nm;

[0035] B6. Preparation of SiO2 (DNA (SiO2)) microsphere particles coated with iron oxide with a particle size of about 250 nm: 35-350 mg of the hematite nanoparticles obtained in step B5 and 50 mg of the SiO2 (DNA (SiO2)) microspheres obtained in step B4 were mixed, and the mixture was stirred at room temperature for 2 hours. The resulting product was washed to remove salt, and centrifuged and dried under nitrogen to obtain SiO2 (DNA (SiO2)) microsphere particles coated with iron oxide with a particle size of about 250 nm;

[0036] C. Preparation of SiO2(DNA(SiO2)) microspheres coated with iron oxide with a particle size of 550 nm

[0037] C1. Preparation of SiO2 core solution: SiO2 microspheres with a particle size of 500 nm were suspended in isopropanol to prepare a SiO2 core solution with a concentration of 50 mg / mL;

[0038] C2. Preparation of TMAPS-functionalized SiO2 microspheres with positive surface charge: Mix the prepared SiO2 core solution, take 1 mL and add it to a 2 mL microcentrifuge tube, and ultrasonicate for 10 minutes to obtain a uniform SiO2 microsphere colloidal solution; add 10 μL of TMAPS solution, and then stir at room temperature in a constant temperature box at 900 rpm for 12 hours, and wash with isopropanol to obtain SiO2 microspheres with positive surface charge;

[0039] C3. Preparation of SiO2 (DNA (SiO2)) microsphere solution with a particle size of about 530 nm: first prepare a nucleic acid solution with a concentration of 50 μg / mL with ultrapure water; then add 700 μL ultrapure water, 320 μL nucleic acid solution and 35 μL SiO2 microspheres with positive surface charge into a centrifuge tube, mix and then treat with ultrasound to obtain a uniform solution; wash with ultrapure water by centrifugation and then treat with ultrasound to obtain a uniform colloidal solution; then, add 0.5 μL TMAPS solution thereto, mix well, then add 0.5 μL TEOS, and stir at 900 rpm for 4 hours at room temperature; then add 0.9 μL TEOS to the system, and stir at 900 rpm for 4 days at room temperature; wash with ultrapure water and mix well with ultrasound to obtain a colloidal solution of SiO2 (DNA (SiO2)) microspheres with a particle size of about 530 nm;

[0040] C4. Preparation of SiO2(DNA(SiO2)) microspheres with uniform particle size of about 530 nm: The SiO2(DNA(SiO2)) microsphere colloidal solution was freeze-dried in vacuum to obtain SiO2(DNA(SiO2)) microspheres, and 1 g of zirconium beads was added to 1 mg of SiO2 microspheres in a micro-grinder, and the zirconium beads with a diameter of 0.1 mm were ground for 2 to 8 minutes, and then centrifuged and washed 3 times at 21000 g, the supernatant was removed and ultrapure water was added to obtain SiO2(DNA(SiO2)) microspheres with uniform particle size of about 530 nm;

[0041] C5. Preparation of hematite nanoparticles with a particle size of about 10 nm: adding 100 mL of 1 mol / L ferric chloride solution to 900 mL of boiling ultrapure water through a dropper at a rate of two drops per second to obtain a hematite nanoparticle solution; cooling the hematite nanoparticle solution to room temperature to obtain hematite nanoparticles with a particle size of about 10 nm;

[0042] C6. Preparation of SiO2 (DNA (SiO2)) microsphere particles coated with iron oxide with a particle size of about 550 nm: 11.8-118 mg of the hematite nanoparticles obtained in step C5 and 50 mg of the SiO2 (DNA (SiO2)) microspheres obtained in step C4 were mixed, and stirred at room temperature for 2 hours. The resulting product was washed to remove salt, and centrifuged and dried under nitrogen to obtain SiO2 (DNA (SiO2)) microsphere particles coated with iron oxide with a particle size of about 550 nm;

[0043] D. Preparation of SiO2 (DNA (SiO2)) microspheres coated with iron oxide with a particle size of 1.2 μm

[0044] D1. Preparation of SiO2 core solution: SiO2 microspheres with a particle size of 1000 nm were suspended in isopropanol to prepare a SiO2 core solution with a concentration of 50 mg / mL;

[0045] D2. Preparation of TMAPS-functionalized SiO2 microspheres with positive surface charge: Mix the prepared SiO2 core solution, take 1 mL and add it to a 2 mL microcentrifuge tube, and ultrasonicate for 10 minutes to obtain a uniform SiO2 microsphere colloidal solution; add 10 μL of TMAPS solution, and then stir at 900 rpm in a constant temperature box at room temperature for 12 hours, and wash with isopropanol to obtain SiO2 microspheres with positive surface charge;

[0046] D3. Preparation of SiO2 (DNA (SiO2)) microsphere solution with a particle size of about 1180nm: first prepare a nucleic acid solution with a concentration of 50μg / mL with ultrapure water; then add 700μL ultrapure water, 320μL nucleic acid solution and 35μL SiO2 microspheres with positive surface charge into a centrifuge tube, mix and then treat with ultrasound to obtain a uniform solution; wash with ultrapure water by centrifugation and then treat with ultrasound to obtain a uniform colloidal solution; then, add 0.5μL TMAPS solution thereto, mix well, then add 0.5μL TEOS, and stir at room temperature at 900rpm for 4 hours; then add 3μLTEOS to the system and stir at room temperature at 900rpm for 4 days; wash with ultrapure water and mix well with ultrasound to obtain a colloidal solution of SiO2 (DNA (SiO2)) microspheres with a particle size of about 1180nm;

[0047] D4. Preparation of SiO2 (DNA (SiO2)) microspheres with uniform particle size of about 1180 nm: The SiO2 (DNA (SiO2)) microsphere colloidal solution was freeze-dried in vacuum to obtain SiO2 (DNA (SiO2)) microspheres, and 1 mg of SiO2 microspheres was added to 1 g of zirconium beads in a micro-grinder, and the zirconium beads with a diameter of 0.1 mm were ground for 2 to 8 minutes, and then centrifuged and washed 3 times at 21000 g, the supernatant was removed and ultrapure water was added to obtain SiO2 (DNA (SiO2)) microspheres with uniform particle size of about 1180 nm;

[0048] D5. Preparation of hematite nanoparticles with a particle size of about 10 nm: adding 100 mL of 1 mol / L ferric chloride solution to 900 mL of boiling ultrapure water through a dropper at a rate of two drops per second to obtain a hematite nanoparticle solution; cooling the hematite nanoparticle solution to room temperature to obtain hematite nanoparticles with a particle size of about 10 nm;

[0049] D6. Preparation of SiO2 (DNA (SiO2)) microsphere particles coated with iron oxide with a particle size of about 1.2 μm: 5.6-56 mg of hematite nanoparticles obtained in step D5 and 50 mg of SiO2 (DNA (SiO2)) microspheres obtained in step D4 were mixed and stirred at room temperature for 2 hours. The resulting product was washed to remove salt, and centrifuged and dried under nitrogen to obtain SiO2 (DNA (SiO2)) microsphere particles coated with iron oxide with a particle size of about 1.2 μm.

[0050] The preparation method of the TMAPS solution is as follows: TMAPS is dissolved in methanol to prepare a solution with a concentration of 50% w / w.

[0051] In the aforementioned method, ultrapure water is used to prepare a double-stranded DNA solution in steps A3, B3, C3 and D3, and the double-stranded DNA is obtained by annealing a single-stranded DNA shown in any one of SEQ ID NOs: 1-4 and its reverse complementary strand.

[0052] In a third aspect, the present invention provides an iron oxide-encapsulated DNA phosphorus tracer prepared according to the method.

[0053] In a fourth aspect, the present invention provides the use of the iron oxide-encapsulated DNA phosphorus tracer in the spatiotemporal tracing of non-point source phosphorus pollution.

[0054] The iron oxide-coated DNA phosphorus tracer is placed in the farmland, and the proportion of the four iron oxide-coated SiO2 (DNA) SiO2 microsphere particles with different particle sizes is determined according to the particle size of the soil colloid particles and their proportion.

[0055] Among them, SiO2 (DNA (SiO2)) microsphere particles wrapped with iron oxide with a particle size of 60nm traced soil colloids of 0.025-0.2μm, SiO2 (DNA (SiO2)) microsphere particles wrapped with iron oxide with a particle size of 250nm traced soil colloids of 0.2-0.45μm, SiO2 (DNA (SiO2)) microsphere particles wrapped with iron oxide with a particle size of 550nm traced soil colloids of 0.45-0.8μm, and SiO2 (DNA (SiO2)) microsphere particles wrapped with iron oxide with a particle size of 1.2μm traced soil particles ≥1μm.

[0056] Preferably, the volume portions of SiO2 (DNA (SiO2)) microsphere particles coated with iron oxide with particle sizes of 60 nm, 250 nm, 550 nm and 1.2 μm are respectively 20-30 portions, 10-20 portions, 0.1-5 portions and 40-60 portions.

[0057] The iron oxide-coated DNA phosphorus tracer is placed in the drainage ditch, and the proportion of four iron oxide-coated SiO2 (DNA (SiO2)) microsphere particles with different particle sizes is determined according to the particle size and proportion of suspended particles in the drainage ditch water.

[0058] Among them, SiO2 (DNA (SiO2)) microsphere particles wrapped with iron oxide with a particle size of 60nm traced suspended particles of 0.025-0.2μm, SiO2 (DNA (SiO2)) microsphere particles wrapped with iron oxide with a particle size of 250nm traced suspended particles of 0.2-0.45μm, SiO2 (DNA (SiO2)) microsphere particles wrapped with iron oxide with a particle size of 550nm traced suspended particles of 0.45-0.8μm, and SiO2 (DNA (SiO2)) microsphere particles wrapped with iron oxide with a particle size of 1.2μm traced suspended particles ≥1μm.

[0059] Preferably, the volume proportions of SiO2 (DNA (SiO2)) microsphere particles coated with iron oxide with particle sizes of 60 nm, 250 nm, 550 nm and 1.2 μm are 0.1-5 parts, 0.1-5 parts, 0.1-2 parts and 90-100 parts respectively.

[0060] The optimal application condition of the tracer of the present invention is pH ≥ 5. If the pH is < 5, the iron oxide capsid will be dissolved to a certain extent, affecting the use effect of the tracer.

[0061] The above technical solution has at least the following advantages and beneficial effects:

[0062] The present invention is based on the fact that iron oxide has a strong adsorption effect on phosphorus. By synthesizing DNA phosphorus tracers wrapped in iron oxides of different sizes, the phosphate radicals entering the soil and drainage ditches at different times and spatial positions are marked. That is, DNA phosphorus tracers of different sizes wrapped in iron oxides are used to adsorb phosphorus, and are placed in different positions of the irrigation area. The placement position and time of the DNA tracer are encoded with a DNA sequence, and the spatial position of the source of phosphorus adsorbed on soil particles of different sizes is traced. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 Schematic diagram of the preparation process of the DNA phosphorus tracer coated with iron oxide in a preferred embodiment of the present invention.

[0064] Figure 2 The DNA phosphorus tracers wrapped with iron oxides of different sizes in the preferred embodiment of the present invention trace the phosphates entering the watershed at different times and spatial locations. Different colors in the figure represent tracers encoded by different DNA sequences. DETAILED DESCRIPTION

[0065] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0066] Experimental reagents: isopropanol (purity>99.8%), TMAPS solution (trimethyl[3-(trimethoxysilyl)propyl]nitrogen chloride; 50% (wt / wt) dissolved in methanol), double-stranded DNA annealed with the single strand and its reverse complementary strand in Table 1, ultrapure water, tetraethyl orthosilicate (TEOS, purity≥99.0%). Annealing buffer (10mM Tris, pH7.5-8.0, 50mM NaCl, 1mM EDTA).

[0067] SiO2 microspheres were purchased from nanoComposix (https: / / nanocomposix.com / collections / solid-silica-spheres / products / nanoxact-silica-nanospheres?variant=15906837954649). The SiO2 microsphere models are 20 nm SiO2 microspheres (SIAN20-25M), 200 nm SiO2 microspheres (SIAN200-25M), 500 nm SiO2 microspheres (SIAN500-25M), and 1000 nm SiO2 microspheres (SIAN1000-25M).

[0068] Deoxyribonuclease I (DNase I) was purchased from Thermo Fisher Scientific (https: / / www.thermofisher.cn / order / catalog / product / EN0521).

[0069] Table 1 DNA sequences (SEQ ID NO: 1-4)

[0070]

[0071] Note: The forward primer is shown in bold underline, and the reverse primer is shown in bold italics.

[0072] Example 1 Preparation of DNA phosphorus tracer wrapped with iron oxide

[0073] In this embodiment, double-stranded DNA is used as an example to prepare DNA phosphorus tracers wrapped with iron oxides of different sizes.

[0074] A. Preparation of SiO2 (DNA (SiO2)) microspheres coated with iron oxide with a particle size of 60 nm

[0075] A1. Preparation of SiO2 core solution: SiO2 microspheres with a particle size of 20 nm were suspended in isopropanol to prepare a SiO2 core solution with a concentration of 50 mg / mL.

[0076] A2. Preparation of TMAPS-functionalized SiO2 microspheres with positive surface charge: Mix the prepared SiO2 core solution, take 1 mL and add it into a 2 mL microcentrifuge tube, and ultrasonicate it for 10 minutes to obtain a uniform colloidal solution of SiO2 microspheres; add 10 μL of TMAPS solution thereto, and then stir it at 900 rpm in a constant temperature box at room temperature for 12 hours. After washing with isopropanol, SiO2 microspheres with positive surface charge are obtained.

[0077] A3. Preparation of SiO2 (DNA (SiO2)) microsphere solution with a particle size of about 40 nm: first prepare a nucleic acid solution with a concentration of 50 μg / mL with ultrapure water; then add 700 μL ultrapure water, 320 μL nucleic acid solution and 35 μL SiO2 microspheres with positive surface charge into a centrifuge tube, mix and then treat by ultrasound to obtain a uniform solution; wash by centrifugation with ultrapure water and then treat by ultrasound to obtain a uniform colloidal solution; then, add 0.5 μL TMAPS solution thereto, mix well, then add 0.5 μL TEOS, and stir at room temperature at 900 rpm for 4 hours; then add 32.4 μL TEOS to the system, and stir at room temperature at 900 rpm for 4 days; wash with ultrapure water and mix by ultrasound to obtain a colloidal solution of SiO2 (DNA (SiO2)) microspheres with a particle size of about 40 nm.

[0078] A4. Preparation of SiO2(DNA(SiO2)) microspheres with uniform particle size of about 40 nm: The SiO2(DNA(SiO2)) microsphere colloidal solution was freeze-dried in vacuum to obtain SiO2(DNA(SiO2)) microspheres. The microspheres were ground in a micro-grinder with zirconium beads of 0.1 mm in diameter at a ratio of 1 mg of SiO2 microspheres to 1 g of zirconium beads for 2 to 8 minutes. The mixture was then centrifuged and washed three times at 21,000 g. The supernatant was removed and ultrapure water was added.

[0079] A5. The size and structure of the SiO2(DNA(SiO2)) microspheres after grinding and washing were observed using a transmission electron microscope (TEM), and the Zeta potential (ζ) and hydrodynamic diameter were measured using a Zetasizer Nano ZS. The particle size of the SiO2(DNA(SiO2)) microspheres was 40 nm.

[0080] A6. To ensure that the DNA is completely encapsulated in the SiO2 (DNA (SiO2)) microspheres and effectively protected, the uniformly ground SiO2 (DNA (SiO2)) microspheres are gently mixed in a 0.2U / μL DNase I solution and incubated at 37°C for 1 hour to digest the DNA fragments that are not completely encapsulated on the surface, thereby obtaining SiO2 (DNA (SiO2)) microspheres with completely protected DNA.

[0081] A7. Preparation of hematite nanoparticles with a particle size of about 10 nm: 100 mL of 1 mol / L ferric chloride solution was slowly added to 900 mL of boiling ultrapure water at a rate of two drops per second through a dropper to prepare a hematite nanoparticle solution. The hematite nanoparticle solution was cooled to room temperature to obtain hematite nanoparticles with a particle size of about 10 nm.

[0082] A8. Preparation of Fe2O3(SiO2(DNA(SiO2))) microsphere particles with a particle size of about 60 nm: 1.8-18 g of hematite nanoparticles obtained in step A7 and 50 mg of SiO2(DNA(SiO2)) microspheres obtained in step A4 were mixed and stirred at room temperature for 2 hours. The obtained solid was washed several times to remove all salts, and centrifuged and dried under N2. Fe2O3(SiO2(DNA(SiO2))) microsphere particles with a particle size of about 60 nm were obtained.

[0083] A9. The size and structure of the DNA phosphorus tracer wrapped in iron oxide were observed using a transmission electron microscope (TEM), and its zeta potential (ζ) and hydrodynamic diameter were measured using a Zetasizer Nano ZS. The DNA phosphorus tracer wrapped in iron oxide prepared in this example had a particle size of about 60 nm.

[0084] B. Preparation of SiO2 (DNA (SiO2)) microspheres coated with iron oxide with a particle size of about 250 nm

[0085] B. Preparation of SiO2 (DNA (SiO2)) microspheres coated with iron oxide with a particle size of 250 nm

[0086] B1. Preparation of SiO2 core solution: SiO2 microspheres with a particle size of 200 nm were suspended in isopropanol to prepare a SiO2 core solution with a concentration of 50 mg / mL.

[0087] B2. Preparation of TMAPS-functionalized SiO2 microspheres with positively charged surface: Mix the prepared SiO2 core solution, take 1 mL and add it to a 2 mL microcentrifuge tube, and ultrasonicate for 10 minutes to obtain a uniform SiO2 microsphere colloidal solution; add 10 μL of TMAPS solution thereto, and then stir at 900 rpm in a constant temperature oven at room temperature for 12 hours. After washing with isopropanol, SiO2 microspheres with positively charged surface are obtained.

[0088] B3. Preparation of SiO2 (DNA (SiO2)) microsphere solution with a particle size of about 230 nm: first prepare a nucleic acid solution with a concentration of 50 μg / mL with ultrapure water; then add 700 μL ultrapure water, 320 μL nucleic acid solution and 35 μL SiO2 microspheres with positive surface charge into a centrifuge tube, mix and then treat by ultrasound to obtain a uniform solution; wash by centrifugation with ultrapure water and then treat by ultrasound to obtain a uniform colloidal solution; then, add 0.5 μL TMAPS solution thereto, mix well, then add 0.5 μL TEOS, and stir at room temperature at 900 rpm for 4 hours; then add 2.4 μL TEOS to the system and stir at room temperature at 900 rpm for 4 days; after washing with ultrapure water and mixing by ultrasound, a colloidal solution of SiO2 (DNA (SiO2)) microspheres with a particle size of about 230 nm is obtained.

[0089] B4. Preparation of SiO2(DNA(SiO2)) microspheres with uniform particle size of about 230 nm: The SiO2(DNA(SiO2)) microsphere colloidal solution is vacuum freeze-dried to obtain SiO2(DNA(SiO2)) microspheres. In a micro grinder, 1 mg of SiO2 microspheres is added to 1 g of zirconium beads. The microspheres are ground with 0.1 mm diameter zirconium beads for 2 to 8 minutes, then centrifuged and washed three times at 21000 g. The supernatant is removed and ultrapure water is added.

[0090] B5. The size and structure of the SiO2(DNA(SiO2)) microspheres after grinding and washing were observed using a transmission electron microscope (TEM), and the Zeta potential (ζ) and hydrodynamic diameter were measured using a Zetasizer Nano ZS. The particle size of the SiO2(DNA(SiO2)) microspheres was 230 nm.

[0091] B6. To ensure that the DNA is completely encapsulated in the SiO2 (DNA (SiO2)) microspheres and effectively protected, the uniformly ground SiO2 (DNA (SiO2)) microspheres are gently mixed in a 0.2U / μL DNase I solution and incubated at 37°C for 1 hour to digest the DNA fragments that are not completely encapsulated on the surface, thereby obtaining SiO2 (DNA (SiO2)) microspheres with completely protected DNA.

[0092] B7. Preparation of hematite nanoparticles with a particle size of about 10 nm: 100 mL of 1 mol / L ferric chloride solution was slowly added to 900 mL of boiling ultrapure water at a rate of two drops per second through a dropper to prepare a hematite nanoparticle solution. The hematite nanoparticle solution was cooled to room temperature to obtain hematite nanoparticles with a particle size of about 10 nm.

[0093] B8. Preparation of Fe2O3(SiO2(DNA(SiO2))) microsphere particles with a particle size of about 250 nm: 35-350 mg of hematite nanoparticles obtained in step B7 and 50 mg of SiO2(DNA(SiO2)) microspheres obtained in step B4 were mixed and stirred at room temperature for 2 hours. The obtained solid was washed several times to remove all salts, and centrifuged and dried under N2. Fe2O3(SiO2(DNA(SiO2))) microsphere particles with a particle size of about 250 nm were obtained.

[0094] B9. The size and structure of the DNA phosphorus tracer wrapped in iron oxide were observed using a transmission electron microscope (TEM), and its zeta potential (ζ) and hydrodynamic diameter were measured using a Zetasizer Nano ZS. The DNA phosphorus tracer wrapped in iron oxide prepared in this example had a particle size of about 250 nm.

[0095] C. Preparation of SiO2(DNA(SiO2)) microspheres coated with iron oxide with a particle size of 550 nm

[0096] C1. Preparation of SiO2 core solution: SiO2 microspheres with a particle size of 500 nm were suspended in isopropanol to prepare a SiO2 core solution with a concentration of 50 mg / mL.

[0097] C2. Preparation of TMAPS-functionalized SiO2 microspheres with positively charged surface: Mix the prepared SiO2 core solution, take 1 mL and add it to a 2 mL microcentrifuge tube, and ultrasonicate it for 10 minutes to obtain a uniform SiO2 microsphere colloidal solution; add 10 μL of TMAPS solution thereto, and then stir it at 900 rpm in a constant temperature box at room temperature for 12 hours. After washing with isopropanol, SiO2 microspheres with positively charged surface are obtained.

[0098] C3. Preparation of SiO2 (DNA (SiO2)) microsphere solution with a particle size of about 530 nm: first prepare a nucleic acid solution with a concentration of 50 μg / mL with ultrapure water; then add 700 μL ultrapure water, 320 μL nucleic acid solution and 35 μL SiO2 microspheres with positive surface charge into a centrifuge tube, mix and then treat by ultrasound to obtain a uniform solution; wash by centrifugation with ultrapure water and then treat by ultrasound to obtain a uniform colloidal solution; then, add 0.5 μL TMAPS solution thereto, mix well, then add 0.5 μL TEOS, and stir at room temperature at 900 rpm for 4 hours; then add 0.9 μL TEOS to the system and stir at room temperature at 900 rpm for 4 days; wash with ultrapure water and mix by ultrasound to obtain a colloidal solution of SiO2 (DNA (SiO2)) microspheres with a particle size of about 530 nm.

[0099] C4. Preparation of SiO2(DNA(SiO2)) microspheres with uniform particle size of about 530 nm: The SiO2(DNA(SiO2)) microsphere colloidal solution is vacuum freeze-dried to obtain SiO2(DNA(SiO2)) microspheres. In a micro grinder, 1 mg of SiO2 microspheres is added to 1 g of zirconium beads. The microspheres are ground with zirconium beads of 0.1 mm in diameter for 2 to 8 minutes, and then centrifuged and washed three times at 21000 g. The supernatant is removed and ultrapure water is added.

[0100] C5. The size and structure of the SiO2(DNA(SiO2)) microspheres after grinding and washing were observed using a transmission electron microscope (TEM), and the Zeta potential (ζ) and hydrodynamic diameter were measured using a Zetasizer Nano ZS. The particle size of the SiO2(DNA(SiO2)) microspheres was 530 nm.

[0101] C6. To ensure that the DNA is completely encapsulated in the SiO2 (DNA (SiO2)) microspheres and effectively protected, the uniformly ground SiO2 (DNA (SiO2)) microspheres are gently mixed in a 0.2U / μL DNase I solution and incubated at 37°C for 1 hour to digest the DNA fragments that are not completely encapsulated on the surface, thereby obtaining SiO2 (DNA (SiO2)) microspheres with completely protected DNA.

[0102] C7. Preparation of hematite nanoparticles with a particle size of about 10 nm: 100 mL of 1 mol / L ferric chloride solution was slowly added to 900 mL of boiling ultrapure water at a rate of two drops per second through a dropper to prepare a hematite nanoparticle solution. The hematite nanoparticle solution was cooled to room temperature to obtain hematite nanoparticles with a particle size of about 10 nm.

[0103] C8. Preparation of Fe2O3(SiO2(DNA(SiO2))) microsphere particles with a particle size of about 550 nm: 11.8-118 mg of hematite nanoparticles obtained in step C7 and 50 mg of SiO2(DNA(SiO2)) microspheres obtained in step C4 were mixed and stirred at room temperature for 2 hours. The obtained solid was washed several times to remove all salts, and centrifuged and dried under N2. Fe2O3(SiO2(DNA(SiO2))) microsphere particles with a particle size of about 550 nm were obtained.

[0104] C9. The size and structure of the DNA phosphorus tracer wrapped in iron oxide were observed using a transmission electron microscope (TEM), and its zeta potential (ζ) and hydrodynamic diameter were measured using a Zetasizer Nano ZS. The DNA phosphorus tracer wrapped in iron oxide prepared in this example had a particle size of about 550 nm.

[0105] D. Preparation of SiO2 (DNA (SiO2)) microspheres coated with iron oxide with a particle size of 1.2 μm

[0106] D1. Preparation of SiO2 core solution: SiO2 microspheres with a particle size of 1000 nm were suspended in isopropanol to prepare a SiO2 core solution with a concentration of 50 mg / mL.

[0107] D2. Preparation of TMAPS-functionalized SiO2 microspheres with positively charged surface: Mix the prepared SiO2 core solution, take 1 mL and add it into a 2 mL microcentrifuge tube, and ultrasonicate it for 10 minutes to obtain a uniform SiO2 microsphere colloidal solution; add 10 μL of TMAPS solution thereto, and then stir it at 900 rpm in a constant temperature box at room temperature for 12 hours. After washing with isopropanol, SiO2 microspheres with positively charged surface are obtained.

[0108] D3. Preparation of SiO2 (DNA (SiO2)) microsphere solution with a particle size of about 1180nm: first prepare a nucleic acid solution with a concentration of 50μg / mL with ultrapure water; then add 700μL ultrapure water, 320μL nucleic acid solution and 35μL SiO2 microspheres with positive surface charge into a centrifuge tube, mix and then treat by ultrasound to obtain a uniform solution; wash by centrifugation with ultrapure water and then treat by ultrasound to obtain a uniform colloidal solution; then, add 0.5μL TMAPS solution thereto, mix well, then add 0.5μL TEOS, and stir at room temperature at 900rpm for 4 hours; then add 3μLTEOS to the system and stir at room temperature at 900rpm for 4 days; wash with ultrapure water and mix by ultrasound to obtain a colloidal solution of SiO2 (DNA (SiO2)) microspheres with a particle size of about 1180nm.

[0109] D4. Preparation of SiO2(DNA(SiO2)) microspheres with uniform particle size of about 1180 nm: The SiO2(DNA(SiO2)) microsphere colloidal solution was freeze-dried in vacuum to obtain SiO2(DNA(SiO2)) microspheres. In a micro grinder, 1 mg of SiO2 microspheres was added to 1 g of zirconium beads. The microspheres were ground with zirconium beads of 0.1 mm in diameter for 2 to 8 minutes, and then centrifuged and washed three times at 21000 g. The supernatant was removed and ultrapure water was added.

[0110] D5. The size and structure of the SiO2(DNA(SiO2)) microspheres after grinding and washing were observed using a transmission electron microscope (TEM), and the Zeta potential (ζ) and hydrodynamic diameter were measured using a Zetasizer Nano ZS. The particle size of the SiO2(DNA(SiO2)) microspheres was 1180 nm.

[0111] D6. To ensure that the DNA is completely encapsulated in the SiO2 (DNA (SiO2)) microspheres and effectively protected, the uniformly ground SiO2 (DNA (SiO2)) microspheres are gently mixed in a 0.2U / μL DNase I solution and incubated at 37°C for 1 hour to digest the DNA fragments that are not completely encapsulated on the surface, thereby obtaining SiO2 (DNA (SiO2)) microspheres with completely protected DNA.

[0112] D7. Preparation of hematite nanoparticles with a particle size of about 10 nm: 100 mL of 1 mol / L ferric chloride solution was slowly added to 900 mL of boiling ultrapure water at a rate of two drops per second through a dropper to prepare a hematite nanoparticle solution. The hematite nanoparticle solution was cooled to room temperature to obtain hematite nanoparticles with a particle size of about 10 nm.

[0113] D8. Preparation of Fe2O3(SiO2(DNA(SiO2))) microsphere particles with a particle size of about 1.2 μm: 5.6-56 mg of hematite nanoparticles obtained in step D7 and 50 mg of SiO2(DNA(SiO2)) microspheres obtained in step D4 were mixed and stirred at room temperature for 2 hours. The obtained solid was washed several times to remove all salts, and centrifuged and dried under N2. Fe2O3(SiO2(DNA(SiO2))) microsphere particles with a particle size of about 1.2 μm were obtained.

[0114] D9. The size and structure of the DNA phosphorus tracer wrapped in iron oxide were observed using a transmission electron microscope (TEM), and its zeta potential (ζ) and hydrodynamic diameter were measured using a Zetasizer Nano ZS. The DNA phosphorus tracer wrapped in iron oxide prepared in this example had a particle size of about 1.2 μm.

[0115] The schematic diagram of the preparation process of the above iron oxide-encapsulated DNA phosphorus tracer is shown in Figure 1 .

[0116] Example 2 Application of DNA phosphorus tracers wrapped with iron oxides of different sizes in farmland

[0117] Existing methods for tracing the sources of phosphorus non-point source pollution can only trace the type of source, such as the soil surface, riverbed mud, riverbank, etc., but cannot trace the specific spatial location of the source. This embodiment takes the tracing of phosphorus non-point source pollution in a watershed as an example to provide the use of DNA phosphorus tracers wrapped in iron oxides of different sizes.

[0118] Different crops require different amounts of phosphorus fertilizer and irrigation water, which results in different levels of phosphorus non-point source pollution. Figure 2As shown, the iron oxide-coated DNA phosphorus tracer of the present invention is encoded with three different DNA sequences to mark the farmland and time of placement, DNA5 marks the tracer placed in the pumpkin field, DNA6 marks the tracer placed in the corn field, and DNA4 marks the tracer placed in the wheat field. The iron oxide-coated DNA phosphorus tracers placed in the same location and time are mixed tracers of approximately 60nm, 250nm, 550nm and 1.2μm in size. The percentage of its application is determined by the proportion of soil colloids and the distribution of soil particle size. For example, the volume percentages of iron oxide-coated DNA phosphorus tracers of about 60nm (tracing soil colloids of 0.025-0.2μm), iron oxide-coated DNA phosphorus tracers of about 250nm (tracing soil colloids of 0.2-0.45μm), iron oxide-coated DNA phosphorus tracers of about 550nm (tracing soil colloids of 0.45-0.8μm), and iron oxide-coated DNA phosphorus tracers of about 1.2μm (tracing soil particles ≥1μm) are 35%, 14%, 1%, and 50% respectively. By using the strong adsorption characteristics of iron oxides to phosphorus, the time and spatial location of the source of phosphorus adsorbed on soil colloid particles of different sizes are traced. When phosphorus fertilizer is applied at different times and in different farmlands, samples are collected from nearby groundwater wells and drainage ditches to detect the concentration changes of iron oxide-coated DNA phosphorus tracers and phosphorus in surface water and groundwater.

[0119] Water sample analysis method:

[0120] 1. Total phosphorus (TP) is measured using the agricultural standard NY / T 88-1988.

[0121] 2. Gradual filtration to separate the phosphorus in the solution of ① <0.025μm that is not adsorbed, ② 0.025-0.2μm (which contains 60nm tracer and phosphorus adsorbed on particles of this level), ③ 0.2-0.45μm (which contains 250nm tracer and phosphorus adsorbed on particles of this level), ④ 0.45-0.8μm (which contains 550nm tracer and phosphorus adsorbed on particles of this level), ⑤ ≥1μm (which contains 1.2μm tracer and phosphorus adsorbed on particles of this level).

[0122] 3. Use ammonium molybdate spectrophotometry (GB / T11893-1989) to measure the phosphorus content of each level ①-⑤ respectively.

[0123] 4. Dissolve iron oxides: Use 0.038 M oxalic acid at 100°C and pH 2.5 to dissolve iron oxides.

[0124] 5. Dissolve SiO2, measure the DNA sequence, and examine the sources of particles of this size level and the proportion of each source.

[0125] Centrifuge 100 μL of iron oxide-encapsulated DNA phosphorus tracer at 21000 g for 3 minutes at room temperature and discard the supernatant. Add 300 μL of NH4FHF / NH4F etching solution to completely dissolve the SiO2 shell and SiO2 core encapsulating the DNA tracer, and ensure that the DNA is not hydrolyzed or denatured during the release step. Elute the DNA using 10 mM Tris-HCl (pH 8.5), and then determine the DNA concentration by qPCR using the Taqman probe method.

[0126] Example 3 Application of DNA phosphorus tracers wrapped with iron oxides of different sizes in drainage ditches

[0127] The drainage conditions of different ditch sections at different irrigation periods are different, taking into account the adsorption and decomposition of phosphorus on particles, the sedimentation of suspended particles in the water, and the scouring and siltation of sediment by water. Figure 2 As shown, the DNA phosphorus tracer of the present invention wrapped in iron oxide and labeled with DNA sequence 3 was placed in the drainage ditch at the same position and time. The percentage of the iron oxide-wrapped DNA phosphorus tracer of four particle sizes was determined according to the proportion of suspended particles in the drainage ditch water at the placement point. Figure 2 In the experiment, the four particle size tracers labeled with DNA3 sequence DNA, including DNA phosphorus tracer wrapped in iron oxide of about 60nm (tracing 0.025-0.2μm colloidal particles), DNA phosphorus tracer wrapped in iron oxide of about 250nm (tracing 0.2-0.45μm colloidal particles), DNA phosphorus tracer wrapped in iron oxide of about 550nm (tracing 0.45-0.8μm colloidal particles), and DNA phosphorus tracer wrapped in iron oxide of about 1.2μm (tracing ≥1μm suspended particles), were placed at 4%, 2%, 0.1%, and 93.9% volume percentages. By using the strong adsorption characteristics of iron oxide to phosphorus, the time and spatial location of the source of phosphorus adsorbed on suspended particles of different sizes were traced, and samples were collected at different locations of the drainage ditch to detect the concentration changes of DNA phosphorus tracer wrapped in iron oxide and phosphorus in different ditch sections.

[0128] Water sample analysis method:

[0129] 1. Total phosphorus (TP) is measured using the agricultural standard NY / T 88-1988.

[0130] 2. Gradual filtration to separate the phosphorus in the solution of ① <0.025μm that is not adsorbed, ② 0.025-0.2μm (which contains 60nm tracer and phosphorus adsorbed on particles of this level), ③ 0.2-0.45μm (which contains 250nm tracer and phosphorus adsorbed on particles of this level), ④ 0.45-0.8μm (which contains 550nm tracer and phosphorus adsorbed on particles of this level), ⑤ ≥1μm (which contains 1.2μm tracer and phosphorus adsorbed on particles of this level).

[0131] 3. Use ammonium molybdate spectrophotometry (GB / T11893-1989) to measure the phosphorus content of each level ①-⑤ respectively.

[0132] 4. Dissolve iron oxides: Use 0.038 M oxalic acid at 100°C and pH 2.5 to dissolve iron oxides.

[0133] 5. Dissolve SiO2, measure the DNA sequence, and examine the sources of particles of this size level and the proportion of each source.

[0134] Centrifuge 100 μL of iron oxide-encapsulated DNA phosphorus tracer at 21000 g for 3 minutes at room temperature and discard the supernatant. Add 300 μL of NH4FHF / NH4F etching solution to completely dissolve the SiO2 shell and SiO2 core encapsulating the DNA tracer, and ensure that the DNA is not hydrolyzed or denatured during the release step. Elute the DNA using 10 mM Tris-HCl (pH 8.5), and then determine the DNA concentration by qPCR using the Taqman probe method.

[0135] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. An iron oxide-encapsulated DNA phosphorus tracer, characterized in that: The phosphorus tracer is composed of SiO2 (DNA (SiO2)) microsphere particles wrapped with iron oxide of four different particle sizes; The microsphere particles are composed of a core, an outer shell and an iron oxide capsid wrapped in the outer shell; The inner core is SiO2 microspheres of four different sizes with nucleic acid adsorbed on the surface, a layer of SiO2 shell is wrapped on the outer side of the inner core, and a layer of iron oxide capsid is wrapped on the surface of the shell; The particle sizes of the SiO2 microspheres are 20 nm, 200 nm, 500 nm and 1000 nm, respectively. Correspondingly, the particle sizes of the iron oxide-coated SiO2 (DNA (SiO2)) microsphere particles are 60 nm, 250 nm, 550 nm and 1.2 μm, respectively. The nucleic acid is a single-stranded or double-stranded DNA with a length of 60-120 bp and without a stem-loop or hairpin structure; The preparation method of the iron oxide-wrapped DNA phosphorus tracer comprises the following steps: (1) Prepare SiO2 microspheres of different particle sizes; (2) The SiO2 microspheres are contacted with the functionalized reagent TMAPS to make the surface of the SiO2 microspheres positively charged, and then the nucleic acids are electrostatically adsorbed to serve as the core; (3) contacting the core with TEOS to catalyze the growth of SiO2, forming a layer of SiO2 shell on the outside of the core, and obtaining SiO2 (DNA (SiO2)) microsphere particles of different particle sizes; (4) Preparation of hematite nanoparticles: Add ferric chloride solution to boiling ultrapure water to obtain hematite nanoparticles with a particle size of 10 nm; (5) The SiO2(DNA(SiO2)) microsphere particles obtained in step (3) are mixed with the hematite nanoparticles obtained in step (4) so ​​that the outermost layer of the SiO2(DNA(SiO2)) microsphere particles is coated with an iron oxide shell.

2. The method for preparing the iron oxide-encapsulated DNA phosphorus tracer according to claim 1, characterized in that: The following steps are involved: (1) Prepare SiO2 microspheres of different particle sizes; (2) The SiO2 microspheres are contacted with the functionalized reagent TMAPS to make the surface of the SiO2 microspheres positively charged, and then the nucleic acids are electrostatically adsorbed to serve as the core; (3) contacting the core with TEOS to catalyze the growth of SiO2, forming a layer of SiO2 shell on the outside of the core, and obtaining SiO2 (DNA (SiO2)) microsphere particles of different particle sizes; (4) Preparation of hematite nanoparticles: Add ferric chloride solution to boiling ultrapure water to obtain hematite nanoparticles with a particle size of 10 nm; (5) mixing the SiO2(DNA(SiO2)) microsphere particles obtained in step (3) with the hematite nanoparticles obtained in step (4) so ​​that the outermost layer of the SiO2(DNA(SiO2)) microsphere particles is coated with an iron oxide shell; The nucleic acid is a single-stranded or double-stranded DNA with a length of 60-120 bp and no stem-loop or hairpin structure.

3. The method according to claim 2, characterized in that A. Preparation of SiO2(DNA(SiO2)) microspheres coated with iron oxide with a particle size of 60 nm A1. Preparation of SiO2 core solution: SiO2 microspheres with a particle size of 20 nm were suspended in isopropanol to prepare a SiO2 core solution with a concentration of 50 mg / mL; A2. Preparation of TMAPS-functionalized SiO2 microspheres with positive surface charge: Mix the prepared SiO2 core solution, take 1 mL and add it to a 2 mL microcentrifuge tube, and ultrasonicate for 10 minutes to obtain a uniform SiO2 microsphere colloidal solution; add 10 µL of TMAPS solution, and then stir at room temperature in a constant temperature box at 900 rpm for 12 hours, and wash with isopropanol to obtain SiO2 microspheres with positive surface charge; A3. Preparation of SiO2 (DNA (SiO2)) microsphere solution with a particle size of 40 nm: first prepare a nucleic acid solution with a concentration of 50 µg / mL with ultrapure water; then add 700 µL ultrapure water, 320 µL nucleic acid solution and 35 µL SiO2 microspheres with positive surface charge into a centrifuge tube, mix and then treat with ultrasound to obtain a uniform solution; then wash with ultrapure water by centrifugation and then treat with ultrasound to obtain a uniform colloidal solution; then, add 0.5 µL TMAPS solution thereto, mix well, then add 0.5 µL TEOS, and stir at 900 rpm for 4 hours at room temperature; then add 32.4 µL TEOS to the system, and stir at 900 rpm for 4 days at room temperature; wash with ultrapure water and mix well with ultrasound to obtain a colloidal solution of SiO2 (DNA (SiO2)) microspheres with a particle size of 40 nm; A4. Preparation of SiO2(DNA(SiO2)) microspheres with uniform particle size of 40 nm: The SiO2(DNA(SiO2)) microsphere colloidal solution was freeze-dried in vacuum to obtain SiO2(DNA(SiO2)) microspheres. In a micro-grinder, 1 mg of SiO2 microspheres was added to 1 g of zirconium beads. The particles were ground with zirconium beads with a diameter of 0.1 mm for 2 to 8 minutes. The particles were then centrifuged and washed three times at 21,000 g. The supernatant was removed and ultrapure water was added to obtain SiO2(DNA(SiO2)) microspheres with uniform particle size of 40 nm. A5. Preparation of hematite nanoparticles with a particle size of 10 nm: adding 100 mL of 1 mol / L ferric chloride solution to 900 mL of boiling ultrapure water through a dropper at a rate of two drops per second to obtain a hematite nanoparticle solution; cooling the hematite nanoparticle solution to room temperature to obtain hematite nanoparticles with a particle size of 10 nm; A6. Preparation of SiO2 (DNA (SiO2)) microsphere particles coated with iron oxide with a particle size of 60 nm: 1.8-18 g of the hematite nanoparticles obtained in step A5 and 50 mg of the SiO2 (DNA (SiO2)) microspheres obtained in step A4 were mixed, and the mixture was stirred at room temperature for 2 hours. The resulting product was washed to remove salt, and centrifuged and dried under nitrogen to obtain SiO2 (DNA (SiO2)) microsphere particles coated with iron oxide with a particle size of 60 nm; B. Preparation of SiO2(DNA(SiO2)) microspheres coated with iron oxide with a particle size of 250 nm B1. Preparation of SiO2 core solution: SiO2 microspheres with a particle size of 200 nm were suspended in isopropanol to prepare a SiO2 core solution with a concentration of 50 mg / mL; B2. Preparation of TMAPS-functionalized SiO2 microspheres with positive surface charge: Mix the prepared SiO2 core solution, take 1 mL and add it to a 2 mL microcentrifuge tube, and ultrasonicate for 10 minutes to obtain a uniform SiO2 microsphere colloidal solution; add 10 µL of TMAPS solution, and then stir at room temperature in a constant temperature box at 900 rpm for 12 hours, and wash with isopropanol to obtain SiO2 microspheres with positive surface charge; B3. Preparation of SiO2 (DNA (SiO2)) microsphere solution with a particle size of 230 nm: first prepare a nucleic acid solution with a concentration of 50 µg / mL with ultrapure water; then add 700 µL ultrapure water, 320 µL nucleic acid solution and 35 µL SiO2 microspheres with positive surface charge into a centrifuge tube, mix and then treat with ultrasound to obtain a uniform solution; then wash with ultrapure water by centrifugation and then treat with ultrasound to obtain a uniform colloidal solution; then, add 0.5 µL TMAPS solution, mix well, then add 0.5 µL TEOS, and stir at 900 rpm for 4 hours at room temperature; then add 2.4 µL TEOS to the system, and stir at 900 rpm for 4 days at room temperature; wash with ultrapure water and mix well with ultrasound to obtain a colloidal solution of SiO2 (DNA (SiO2)) microspheres with a particle size of 230 nm; B4. Preparation of SiO2(DNA(SiO2)) microspheres with uniform particle size of 230 nm: The colloidal solution of SiO2(DNA(SiO2)) microspheres was freeze-dried in vacuum to obtain SiO2(DNA(SiO2)) microspheres. In a micro-grinder, 1 mg of SiO2 microspheres was added to 1 g of zirconium beads. The microspheres were ground with zirconium beads with a diameter of 0.1 mm for 2 to 8 minutes. The micro-grinder was then centrifuged and washed three times at 21,000 g. The supernatant was removed and ultrapure water was added to obtain SiO2(DNA(SiO2)) microspheres with uniform particle size of 230 nm. B5. Preparation of hematite nanoparticles with a particle size of 10 nm: adding 100 mL of 1 mol / L ferric chloride solution to 900 mL of boiling ultrapure water through a dropper at a rate of two drops per second to obtain a hematite nanoparticle solution; cooling the hematite nanoparticle solution to room temperature to obtain hematite nanoparticles with a particle size of 10 nm; B6. Preparation of SiO2(DNA(SiO2)) microsphere particles coated with iron oxide with a particle size of 250 nm: 35-350 mg of the hematite nanoparticles obtained in step B5 and 50 mg of the SiO2(DNA(SiO2)) microsphere particles obtained in step B4 were mixed, and the mixture was stirred at room temperature for 2 hours. The resulting product was washed to remove salt, and centrifuged and dried under nitrogen to obtain SiO2(DNA(SiO2)) microsphere particles coated with iron oxide with a particle size of 250 nm; C. Preparation of SiO2(DNA(SiO2)) microspheres coated with iron oxide with a particle size of 550 nm C1. Preparation of SiO2 core solution: SiO2 microspheres with a particle size of 500 nm were suspended in isopropanol to prepare a SiO2 core solution with a concentration of 50 mg / mL; C2. Preparation of TMAPS-functionalized SiO2 microspheres with positive surface charge: Mix the prepared SiO2 core solution, take 1 mL and add it to a 2 mL microcentrifuge tube, and ultrasonicate for 10 minutes to obtain a uniform SiO2 microsphere colloidal solution; add 10 µL of TMAPS solution, and then stir at room temperature in a constant temperature box at 900 rpm for 12 hours, and wash with isopropanol to obtain SiO2 microspheres with positive surface charge; C3. Preparation of SiO2 (DNA (SiO2)) microsphere solution with a particle size of 530 nm: first prepare a nucleic acid solution with a concentration of 50 µg / mL with ultrapure water; then add 700 µL ultrapure water, 320 µL nucleic acid solution and 35 µL SiO2 microspheres with positive surface charge into a centrifuge tube, mix and then treat with ultrasound to obtain a uniform solution; then wash with ultrapure water by centrifugation and then treat with ultrasound to obtain a uniform colloidal solution; then, add 0.5 µL TMAPS solution, mix well, then add 0.5 µL TEOS, and stir at 900 rpm for 4 hours at room temperature; then add 0.9 µL TEOS to the system, and stir at 900 rpm for 4 days at room temperature; wash with ultrapure water and mix well with ultrasound to obtain a colloidal solution of SiO2 (DNA (SiO2)) microspheres with a particle size of 530 nm; C4. Preparation of SiO2(DNA(SiO2)) microspheres with uniform size and a particle size of 530 nm: The SiO2(DNA(SiO2)) microsphere colloidal solution was freeze-dried in vacuum to obtain SiO2(DNA(SiO2)) microspheres. In a micro-grinder, 1 mg of SiO2 microspheres was added to 1 g of zirconium beads. The zirconium beads with a diameter of 0.1 mm were ground for 2 to 8 minutes. The mixture was then centrifuged and washed three times at 21,000 g. The supernatant was removed and ultrapure water was added to obtain SiO2(DNA(SiO2)) microspheres with uniform size and a particle size of 530 nm. C5. Preparation of hematite nanoparticles with a particle size of 10 nm: adding 100 mL of 1 mol / L ferric chloride solution to 900 mL of boiling ultrapure water through a dropper at a rate of two drops per second to obtain a hematite nanoparticle solution; cooling the hematite nanoparticle solution to room temperature to obtain hematite nanoparticles with a particle size of 10 nm; C6. Preparation of SiO2 (DNA (SiO2)) microsphere particles coated with iron oxide with a particle size of 550 nm: 11.8-118 mg of the hematite nanoparticles obtained in step C5 and 50 mg of the SiO2 (DNA (SiO2)) microspheres obtained in step C4 were mixed, and stirred at room temperature for 2 hours. The resulting product was washed to remove salt, and centrifuged and dried under nitrogen to obtain SiO2 (DNA (SiO2)) microsphere particles coated with iron oxide with a particle size of 550 nm; D. Preparation of SiO2(DNA(SiO2)) microspheres coated with iron oxide with a particle size of 1.2 μm D1. Preparation of SiO2 core solution: SiO2 microspheres with a particle size of 1000 nm were suspended in isopropanol to prepare a SiO2 core solution with a concentration of 50 mg / mL; D2. Preparation of TMAPS-functionalized SiO2 microspheres with positive surface charge: Mix the prepared SiO2 core solution, take 1 mL and add it to a 2 mL microcentrifuge tube, and ultrasonicate for 10 minutes to obtain a uniform SiO2 microsphere colloidal solution; add 10 µL of TMAPS solution, and then stir at room temperature in a constant temperature box at 900 rpm for 12 hours, and wash with isopropanol to obtain SiO2 microspheres with positive surface charge; D3, preparation of SiO2 (DNA (SiO2)) microsphere solution with a particle size of 1180 nm: first prepare a nucleic acid solution with a concentration of 50 µg / mL with ultrapure water; then add 700 µL ultrapure water, 320 µL nucleic acid solution and 35 µL SiO2 microspheres with positive surface charge into a centrifuge tube, mix and then treat with ultrasound to obtain a uniform solution; then wash with ultrapure water by centrifugation and then treat with ultrasound to obtain a uniform colloidal solution; then, add 0.5 µL TMAPS solution, mix well, then add 0.5 µL TEOS, and stir at 900 rpm for 4 hours at room temperature; then add 3 µL TEOS to the system, and stir at 900 rpm for 4 days at room temperature; wash with ultrapure water and mix well with ultrasound to obtain a colloidal solution of SiO2 (DNA (SiO2)) microspheres with a particle size of 1180 nm; D4. Preparation of SiO2(DNA(SiO2)) microspheres with uniform particle size of 1180 nm: The colloidal solution of SiO2(DNA(SiO2)) microspheres was freeze-dried in vacuum to obtain SiO2(DNA(SiO2)) microspheres. In a micro-grinder, 1 mg of SiO2 microspheres was added to 1 g of zirconium beads. The zirconium beads with a diameter of 0.1 mm were ground for 2 to 8 minutes. The mixture was then centrifuged and washed three times at 21,000 g. The supernatant was removed and ultrapure water was added to obtain SiO2(DNA(SiO2)) microspheres with uniform particle size of 1180 nm. D5. Preparation of hematite nanoparticles with a particle size of 10 nm: adding 100 mL of 1 mol / L ferric chloride solution to 900 mL of boiling ultrapure water at a rate of two drops per second through a dropper to obtain a hematite nanoparticle solution; cooling the hematite nanoparticle solution to room temperature to obtain hematite nanoparticles with a particle size of 10 nm; D6. Preparation of SiO2 (DNA (SiO2)) microsphere particles coated with iron oxide with a particle size of 1.2 μm: 5.6-56 mg of hematite nanoparticles obtained in step D5 and 50 mg of SiO2 (DNA (SiO2)) microspheres obtained in step D4 were mixed, and the mixture was stirred at room temperature for 2 hours. The resulting product was washed to remove salt, and centrifuged and dried under nitrogen to obtain SiO2 (DNA (SiO2)) microsphere particles coated with iron oxide with a particle size of 1.2 μm; The preparation method of the TMAPS solution is as follows: TMAPS is dissolved in methanol to prepare a solution with a concentration of 50% w / w.

4. The method according to claim 3, characterized in that In steps A3, B3, C3 and D3, ultrapure water is used to prepare a double-stranded DNA solution, wherein the double-stranded DNA is obtained by annealing a single-stranded DNA shown in any one of SEQ ID NOs: 1-4 and its reverse complementary strand.

5. The DNA phosphorus tracer coated with iron oxide prepared by the method according to any one of claims 2 to 4.

6. Use of the iron oxide-coated DNA phosphorus tracer according to claim 1 or 5 in the spatiotemporal tracing of phosphorus non-point source pollution.

7. The use according to claim 6, characterized in that: Placing the iron oxide-coated DNA phosphorus tracer in the farmland, and the proportion of the four iron oxide-coated SiO2 (DNA (SiO2)) microsphere particles with different particle sizes is determined according to the particle size of the soil colloid particles and their proportion; Among them, SiO2(DNA(SiO2)) microspheres wrapped with iron oxide with a particle size of 60 nm traced soil colloids of 0.025-0.2 μm, SiO2(DNA(SiO2)) microspheres wrapped with iron oxide with a particle size of 250 nm traced soil colloids of 0.2-0.45 μm, SiO2(DNA(SiO2)) microspheres wrapped with iron oxide with a particle size of 550 nm traced soil colloids of 0.45-0.8 μm, and SiO2(DNA(SiO2)) microspheres wrapped with iron oxide with a particle size of 1.2 μm traced soil particles ≥1 μm.

8. The use according to claim 7, characterized in that: The dosage volumes of SiO2 (DNA (SiO2)) microsphere particles coated with iron oxide with particle sizes of 60 nm, 250 nm, 550 nm and 1.2 μm are 20-30 parts, 10-20 parts, 0.1-5 parts and 40-60 parts respectively.

9. The use according to claim 6, characterized in that: The iron oxide-coated DNA phosphorus tracer is placed in the drainage ditch, and the proportion of the four iron oxide-coated SiO2 (DNA (SiO2)) microsphere particles with different particle sizes is determined according to the particle size and proportion of suspended particles in the drainage ditch water body; Among them, SiO2(DNA(SiO2)) microspheres wrapped with iron oxide with a particle size of 60 nm traced suspended particles of 0.025-0.2 μm, SiO2(DNA(SiO2)) microspheres wrapped with iron oxide with a particle size of 250 nm traced suspended particles of 0.2-0.45 μm, SiO2(DNA(SiO2)) microspheres wrapped with iron oxide with a particle size of 550 nm traced suspended particles of 0.45-0.8 μm, and SiO2(DNA(SiO2)) microspheres wrapped with iron oxide with a particle size of 1.2 μm traced suspended particles ≥1 μm.

10. The use according to claim 9, characterized in that: The dosage volumes of SiO2 (DNA (SiO2)) microsphere particles coated with iron oxide with particle sizes of 60 nm, 250 nm, 550 nm and 1.2 μm are 0.1-5 parts, 0.1-5 parts, 0.1-2 parts and 90-100 parts respectively.

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