SiO 2 Encapsulated DNA phosphorus tracer and its preparation method and application

Abstract

The present invention provides a DNA phosphorus tracer wrapped in SiO2, and a preparation method and application thereof. The DNA phosphorus tracer wrapped in SiO2 of the present invention is composed of a SiO2 core and a SiO2 shell wrapped in the core; the core is a SiO2 microsphere of different particle sizes with nucleic acid adsorbed on the surface, and a layer of SiO2 shell is wrapped on the outside of the core, which is modified by a functional reagent TMAPS so that the surface of the shell is positively charged. Based on the fact that phosphate is negatively charged, the present invention synthesizes DNA phosphorus tracers wrapped in SiO2 of different sizes with positive charges on the surface to mark phosphates entering the soil and drainage ditches at different times and spatial positions.

Description

Technical Field

[0001] The present invention belongs to the field of environmental protection, and in particular, relates to a SiO 2 Encapsulated DNA phosphorus tracer and its preparation method and application. 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 present invention aims to provide a SiO 2 Encapsulated DNA phosphorus tracer and its preparation method and application.

[0008] The present invention is conceived as follows: Studies have shown that soil particles of different sizes contribute differently to non-point source pollution of phosphorus. The smaller the soil particles, the greater their contribution to non-point source pollution of phosphorus. 2 The DNA phosphorus tracer encapsulated in the water was placed at different locations in the irrigation area. The placement and time of the DNA tracer were encoded by DNA sequences. The DNA tracers placed at the same location and time were SiO2 with positive charges on the surface and four different particle sizes (60nm, 250nm, 550nm and 1.2μm). 2 (DNA(SiO 2 )) A mixture of microsphere particles. Using the negatively charged phosphate group, which will be adsorbed on the positively charged tracer surface, the time and spatial location of the source of phosphorus adsorbed on soil particles of different sizes can be traced.

[0009] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a SiO 2 DNA-encapsulated phosphorus tracers, which consist of four different particle sizes of positively charged SiO 2 (DNA(SiO 2 ))Microsphere particle composition;

[0010] The SiO 2 (DNA)SiO 2 The microsphere particles consist of a core and SiO 2 Shell composition;

[0011] The core is SiO2 with four different particle sizes on the surface of which nucleic acid is adsorbed. 2 Microspheres, with a layer of SiO wrapped around the outside of the core 2The shell is modified by a functionalizing agent TMAPS so that the surface of the shell is positively charged;

[0012] The SiO 2 The particle sizes of the microspheres are 20 nm, 200 nm, 500 nm and 1000 nm respectively. 2 (DNA(SiO 2 ))The particle sizes of the microsphere particles are 60nm, 250nm, 550nm and 1.2μm respectively.

[0013] In the present invention, the surface of the positively charged SiO 2 (DNA(SiO 2 )) The particle size of the microsphere particles is mainly composed of SiO 2 The particle size of the microspheres (core) is controlled by SiO 2 The thickness of the shell is fine-tuned.

[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 SiO 2 The method for preparing the encapsulated DNA phosphorus tracer comprises the following steps:

[0016] (1) Prepare SiO2 with different particle sizes 2 Microspheres;

[0017] (2) SiO 2 The microspheres were contacted with the functionalization reagent TMAPS to make SiO 2 The surface of the microspheres is positively charged, and then electrostatically adsorbs nucleic acids, which serve as the core;

[0018] (3) contacting the core with TEOS to catalyze SiO 2 Growth, forming a layer of SiO 2 Shell, get different particle sizes of SiO 2 (DNA(SiO 2 )) Microsphere particles;

[0019] (4) SiO obtained in step (3) 2 (DNA(SiO 2 )) The microsphere particles are contacted with the functional reagent TMAPS to make SiO 2 (DNA)SiO 2 The surface of the microsphere particles is positively charged.

[0020] Furthermore, the preparation method comprises the following steps:

[0021] A. Preparation of SiO2 with a particle size of about 60 nm and a positively charged surface 2 (DNA(SiO 2 ))Microsphere particles

[0022] A1、SiO 2 Preparation of core solution: SiO 2 The microspheres were suspended in isopropanol to prepare a SiO 2 Kernel solution;

[0023] A2. TMAPS-functionalized SiO with positive surface charge 2 Preparation of microspheres: Prepared SiO 2 The core solution was mixed, 1 mL was added to a 2 mL microcentrifuge tube, and ultrasonic treatment was performed for 10 minutes to obtain a uniform SiO 2 Microsphere colloidal solution; 10 μL TMAPS solution was added thereto, and then stirred at room temperature at 900 rpm in a constant temperature box for 12 hours, and washed with isopropanol to obtain SiO 2 Microspheres;

[0024] A3、SiO 2 (DNA(SiO 2 Preparation of microsphere solution: First, prepare a nucleic acid solution with a concentration of 50 μg / mL with ultrapure water; then add 700 μL of ultrapure water, 320 μL of nucleic acid solution and 35 μL of positively charged SiO 2 The microspheres were mixed and then ultrasonically treated to obtain a uniform solution; the solution was washed by centrifugation with ultrapure water and then ultrasonically treated to obtain a uniform colloidal solution; then, 0.5 μL of TMAPS solution was added thereto and mixed, and then 0.5 μL of TEOS was added and stirred at room temperature at a speed of 900 rpm for 4 hours; then 120.3 μL of TEOS was added to the system and stirred at room temperature at a speed of 900 rpm for 4 days; the solution was washed with ultrapure water and then ultrasonically treated to obtain SiO 2 (DNA(SiO 2 ))Microsphere colloidal solution;

[0025] A4, SiO with uniform particle size of about 60nm 2 (DNA(SiO 2 Preparation of microspheres: SiO 2 (DNA(SiO 2 )) The microsphere colloidal solution was vacuum freeze-dried to obtain SiO 2 (DNA(SiO 2 )) Microspheres were ground in a micromill at 1 mg SiO 2The microspheres were added with 1g zirconium beads, ground with 0.1mm diameter zirconium beads for 2-8 minutes, then centrifuged and washed 3 times at 21000g, the supernatant was removed and ultrapure water was added to obtain SiO2 with uniform particle size of about 60nm. 2 (DNA(SiO 2 ))Microspheres;

[0026] A5, SiO with a positively charged surface and a particle size of about 60 nm 2 (DNA(SiO 2 Preparation of microsphere particles: Take 50 mg of SiO prepared in step A4 2 (DNA(SiO 2 )) microspheres, 10 μL of TMAPS solution was added thereto, and then stirred at 900 rpm in a constant temperature box at room temperature for 12 hours. After washing with isopropanol, SiO2 microspheres with a particle size of about 60 nm and a positively charged surface were obtained. 2 (DNA(SiO 2 )) Microsphere particles;

[0027] B. Preparation of SiO with a particle size of about 250 nm and a positively charged surface 2 (DNA(SiO 2 ))Microsphere particles

[0028] B1、SiO 2 Preparation of core solution: SiO 2 The microspheres were suspended in isopropanol to prepare a SiO 2 Kernel solution;

[0029] B2. TMAPS-functionalized SiO with positive surface charge 2 Preparation of microspheres: Prepared SiO 2 Mix the core solution, take 1 mL and add it to a 2 mL microcentrifuge tube, and ultrasonicate for 10 minutes to obtain a uniform SiO 2 Microsphere colloidal solution; 10 μL TMAPS solution was added thereto, and then stirred at room temperature at 900 rpm in a constant temperature box for 12 hours, and washed with isopropanol to obtain SiO 2 Microspheres;

[0030] B3、SiO 2 (DNA(SiO 2 Preparation of microsphere solution: First, prepare a nucleic acid solution with a concentration of 50 μg / mL with ultrapure water; then add 700 μL of ultrapure water, 320 μL of nucleic acid solution and 35 μL of positively charged SiO 2The microspheres were mixed and then ultrasonically treated to obtain a uniform solution; the solution was washed by centrifugation with ultrapure water and then ultrasonically treated to obtain a uniform colloidal solution; then, 0.5 μL of TMAPS solution was added thereto and mixed, and then 0.5 μL of TEOS was added and stirred at room temperature at a speed of 900 rpm for 4 hours; then 4.4 μL of TEOS was added to the system and stirred at room temperature at a speed of 900 rpm for 4 days; the solution was washed with ultrapure water and then ultrasonically treated to obtain SiO 2 (DNA(SiO 2 ))Microsphere colloidal solution;

[0031] B4, SiO with uniform particle size of about 250nm 2 (DNA(SiO 2 Preparation of microspheres: SiO 2 (DNA(SiO 2 )) The microsphere colloidal solution was vacuum freeze-dried to obtain SiO 2 (DNA(SiO 2 )) Microspheres were ground in a micromill at 1 mg SiO 2 The microspheres were added with 1g zirconium beads, ground with 0.1mm diameter zirconium beads for 2-8 minutes, then centrifuged and washed 3 times at 21000g, the supernatant was removed and ultrapure water was added to obtain SiO2 with uniform particle size of about 250nm. 2 (DNA(SiO 2 ))Microspheres;

[0032] B5, SiO with a positively charged surface and a particle size of about 250 nm 2 (DNA(SiO 2 Preparation of microsphere particles: Take 50 mg of SiO prepared in step B4 2 (DNA(SiO 2 )) microspheres, 10 μL of TMAPS solution was added thereto, and then stirred at 900 rpm in a constant temperature box at room temperature for 12 hours. After washing with isopropanol, SiO2 with a particle size of about 250 nm and a positively charged surface was obtained. 2 (DNA(SiO 2 )) Microsphere particles;

[0033] C. Preparation of SiO with a particle size of about 550 nm and a positively charged surface 2 (DNA(SiO 2 ))Microsphere particles

[0034] C1、SiO 2 Preparation of core solution: SiO 2 The microspheres were suspended in isopropanol to prepare a SiO 2Kernel solution;

[0035] C2, TMAPS functionalized SiO with positive surface charge 2 Preparation of microspheres: Prepared SiO 2 The core solution was mixed, 1 mL was added to a 2 mL microcentrifuge tube, and ultrasonic treatment was performed for 10 minutes to obtain a uniform SiO 2 Microsphere colloidal solution; 10 μL TMAPS solution was added thereto, and then stirred at room temperature at 900 rpm in a constant temperature box for 12 hours, and washed with isopropanol to obtain SiO 2 Microspheres;

[0036] C3、SiO 2 (DNA(SiO 2 Preparation of microsphere solution: First, prepare a nucleic acid solution with a concentration of 50 μg / mL with ultrapure water; then add 700 μL of ultrapure water, 320 μL of nucleic acid solution and 35 μL of positively charged SiO 2 The microspheres were mixed and then ultrasonically treated to obtain a uniform solution; the solution was washed by centrifugation with ultrapure water and then ultrasonically treated to obtain a uniform colloidal solution; then, 0.5 μL of TMAPS solution was added thereto and mixed, and then 0.5 μL of TEOS was added and stirred at room temperature at a speed of 900 rpm for 4 hours; then 1.5 μL of TEOS was added to the system and stirred at room temperature at a speed of 900 rpm for 4 days; the solution was washed with ultrapure water and then ultrasonically treated to obtain SiO 2 (DNA(SiO 2 ))Microsphere colloidal solution;

[0037] C4, SiO with uniform particle size of about 550nm 2 (DNA(SiO 2 Preparation of microspheres: SiO 2 (DNA(SiO 2 )) The microsphere colloidal solution was vacuum freeze-dried to obtain SiO 2 (DNA(SiO 2 )) Microspheres were ground in a micromill at 1 mg SiO 2 The microspheres were added with 1g zirconium beads, ground with 0.1mm diameter zirconium beads for 2-8 minutes, then centrifuged and washed 3 times at 21000g, the supernatant was removed and ultrapure water was added to obtain SiO2 with uniform particle size of about 550nm. 2 (DNA(SiO 2 ))Microspheres;

[0038] C5, SiO with a positively charged surface and a particle size of about 550 nm 2 (DNA(SiO2 Preparation of microsphere particles: Take 50 mg of SiO prepared in step C4 2 (DNA(SiO 2 )) microspheres, 10 μL of TMAPS solution was added thereto, and then stirred at 900 rpm in a constant temperature box at room temperature for 12 hours. After washing with isopropanol, SiO2 with a particle size of about 550 nm and a positively charged surface was obtained. 2 (DNA(SiO 2 )) Microsphere particles;

[0039] D. Prepare SiO with a particle size of about 1.2 μm and a positively charged surface 2 (DNA)SiO 2 Microsphere particles

[0040] D1、SiO 2 Preparation of core solution: SiO 2 The microspheres were suspended in isopropanol to prepare a SiO 2 Kernel solution;

[0041] D2, TMAPS functionalized SiO with positive surface charge 2 Preparation of microspheres: Prepared SiO 2 The core solution was mixed, 1 mL was added to a 2 mL microcentrifuge tube, and ultrasonic treatment was performed for 10 minutes to obtain a uniform SiO 2 Microsphere colloidal solution; 10 μL TMAPS solution was added thereto, and then stirred at room temperature at 900 rpm in a constant temperature box for 12 hours, and washed with isopropanol to obtain SiO 2 Microspheres;

[0042] D3、SiO 2 (DNA(SiO 2 Preparation of microsphere solution: First, prepare a nucleic acid solution with a concentration of 50 μg / mL with ultrapure water; then add 700 μL of ultrapure water, 320 μL of nucleic acid solution and 35 μL of positively charged SiO 2 The microspheres were mixed and then ultrasonically treated to obtain a uniform solution; the solution was washed by centrifugation with ultrapure water and then ultrasonically treated to obtain a uniform colloidal solution; then, 0.5 μL of TMAPS solution was added thereto and mixed, and then 0.5 μL of TEOS was added and stirred at room temperature at a speed of 900 rpm for 4 hours; then 3.4 μL of TEOS was added to the system and stirred at room temperature at a speed of 900 rpm for 4 days; the solution was washed with ultrapure water and then ultrasonically treated to obtain SiO 2 (DNA(SiO 2 ))Microsphere colloidal solution;

[0043] D4, SiO with uniform particle size of about 1.2 μm 2 (DNA(SiO 2 Preparation of microspheres: SiO 2 (DNA(SiO 2 )) The microsphere colloidal solution was vacuum freeze-dried to obtain SiO 2 (DNA(SiO 2 )) Microspheres were ground in a micromill at 1 mg SiO 2 The microspheres were added with 1g zirconium beads, ground with 0.1mm diameter zirconium beads for 2-8 minutes, then centrifuged and washed 3 times at 21000g, the supernatant was removed and ultrapure water was added to obtain SiO2 with uniform particle size of about 1.2μm. 2 (DNA(SiO 2 ))Microspheres;

[0044] D5, SiO with a positively charged surface and a particle size of about 1.2 μm 2 (DNA(SiO 2 Preparation of microsphere particles: Take 50 mg of SiO prepared in step D4 2 (DNA(SiO 2 )) microspheres, 10 μL of TMAPS solution was added thereto, and then stirred at 900 rpm in a constant temperature box at room temperature for 12 hours. After washing with isopropanol, SiO2 microspheres with a particle size of about 1.2 μm and a positively charged surface were obtained. 2 (DNA(SiO 2 ))Microsphere particles.

[0045] 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.

[0046] 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.

[0047] In a third aspect, the present invention provides SiO prepared according to the method 2 Encapsulated DNA phospho-tracer.

[0048] In a fourth aspect, the present invention provides the SiO 2 Application of encapsulated DNA phosphorus tracers in spatiotemporal tracing of non-point source phosphorus pollution.

[0049] Placing the SiO 2 DNA phosphorus tracer encapsulated in four different particle sizes with positively charged SiO 2(DNA)SiO 2 The proportion of microsphere particles added depends on the particle size of soil colloid particles and their proportion.

[0050] Among them, SiO with a particle size of 60nm and a positively charged surface 2 (DNA(SiO 2 )) Microsphere particles trace soil colloids with a size of 0.025-0.2 μm, SiO2 with a particle size of 250 nm and a positively charged surface 2 (DNA(SiO 2 )) Microspheres trace soil colloids with a size of 0.2-0.45 μm and positively charged SiO 2 (DNA(SiO 2 )) Microspheres trace soil colloids with a size of 0.45-0.8 μm and positively charged SiO 2 (DNA(SiO 2 )) Microspheres are used to trace soil particles ≥1μm.

[0051] Preferably, the SiO2 with a particle size of 60 nm, 250 nm, 550 nm and 1.2 μm and a positively charged surface 2 (DNA(SiO 2 )) The volume portions of the microsphere particles are 20-30 portions, 10-20 portions, 0.1-5 portions and 40-60 portions respectively.

[0052] Place the SiO in the drain 2 DNA phosphorus tracer encapsulated in four different particle sizes with positively charged SiO 2 (DNA)SiO 2 The proportion of microsphere particles added is determined according to the particle size and proportion of suspended particles in the drainage ditch water.

[0053] Among them, SiO with a particle size of 60nm and a positively charged surface 2 (DNA(SiO 2 )) Microspheres trace suspended particles of 0.025-0.2μm, SiO2 with a particle size of 250nm and a positively charged surface 2 (DNA(SiO 2 )) Microspheres trace suspended particles of 0.2-0.45 μm, SiO2 with a particle size of 550 nm and a positively charged surface 2 (DNA(SiO 2 )) Microspheres trace suspended particles of 0.45-0.8μm, SiO2 with a particle size of 1.2μm and a positively charged surface 2 (DNA(SiO 2 )) Microsphere particles track suspended particles ≥1μm.

[0054] Preferably, the SiO2 with a particle size of 60 nm, 250 nm, 550 nm and 1.2 μm and a positively charged surface 2 (DNA(SiO 2 )) The volume proportions of the microsphere particles are 0.1-5, 0.1-5, 0.1-2 and 90-100 respectively.

[0055] The above technical solution has at least the following advantages and beneficial effects:

[0056] The present invention is based on the fact that phosphate radical is negatively charged, and by synthesizing SiO2 with different sizes and positively charged surfaces, 2 The SiO2-encapsulated DNA phosphorus tracer was used to label the phosphate that enters the soil and drains at different times and spatial locations. 2 The encapsulated positively charged DNA tracers of different sizes adsorb phosphorus and are placed in different locations in the irrigation area. The location and time of the DNA tracer placement are encoded with DNA sequences to trace the spatial location of the source of phosphorus adsorbed on soil particles of different sizes. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 In the preferred embodiment of the present invention, the surface of SiO 2 Schematic diagram of the preparation process of encapsulated DNA phospho-tracers.

[0058] Figure 2 In the preferred embodiment of the present invention, the surface of SiO 2 The encapsulated DNA phosphorus tracer tracks the phosphate entering the watershed at different times and spatial locations. The different colors in the figure represent the tracers encoded by different DNA sequences. DETAILED DESCRIPTION

[0059] 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.

[0060] 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).

[0061] SiO 2Microspheres were purchased from nanoComposix (https: / / nanocomposix.com / collections / solid-silica-spheres / products / nanoxact-silica-nanospheres?variant=15906837954649). 2 The microsphere models are: 20nm SiO 2 Microspheres (SIAN20-25M), 200nm SiO 2 Microspheres (SIAN200-25M), 500nm SiO 2 Microspheres (SIAN500-25M), 1000nm SiO 2 Microspheres (SIAN1000-25M).

[0062] Deoxyribonuclease I (DNase I) was purchased from Thermo Fisher Scientific (https: / / www.thermofisher.cn / order / catalog / product / EN0521).

[0063] Table 1 DNA sequences (SEQ ID NO: 1-4)

[0064]

[0065] Note: The forward primer is shown in bold underline, and the reverse primer is shown in bold italics.

[0066] Example 1SiO 2 Method for preparing encapsulated DNA phosphorus tracer

[0067] In this example, double-stranded DNA was used as an example to prepare SiO 2 Encapsulated DNA phospho-tracer.

[0068] A. Preparation of SiO2 with a particle size of about 60 nm and a positively charged surface 2 (DNA(SiO 2 ))Microsphere particles

[0069] A1、SiO 2 Preparation of core solution: SiO 2 The microspheres were suspended in isopropanol to prepare a SiO 2 Core solution.

[0070] A2. TMAPS-functionalized SiO with positive surface charge 2Preparation of microspheres: Prepared SiO 2 The core solution was mixed, 1 mL was added to a 2 mL microcentrifuge tube, and ultrasonic treatment was performed for 10 minutes to obtain a uniform SiO 2 Microsphere colloidal solution; 10 μL TMAPS solution was added thereto, and then stirred at room temperature at 900 rpm in a constant temperature box for 12 hours, and washed with isopropanol to obtain SiO 2 Microspheres.

[0071] A3、SiO 2 (DNA(SiO 2 Preparation of microsphere solution: First, prepare a nucleic acid solution with a concentration of 50 μg / mL with ultrapure water; then add 700 μL of ultrapure water, 320 μL of nucleic acid solution and 35 μL of positively charged SiO 2 The microspheres were mixed and then ultrasonically treated to obtain a uniform solution; the solution was washed by centrifugation with ultrapure water and then ultrasonically treated to obtain a uniform colloidal solution; then, 0.5 μL of TMAPS solution was added thereto and mixed, and then 0.5 μL of TEOS was added and stirred at room temperature at a speed of 900 rpm for 4 hours; then 120.3 μL of TEOS was added to the system and stirred at room temperature at a speed of 900 rpm for 4 days; the solution was washed with ultrapure water and then ultrasonically treated to obtain SiO 2 (DNA(SiO 2 ))Microsphere colloidal solution.

[0072] A4, SiO with uniform particle size of about 60nm 2 (DNA(SiO 2 Preparation of microspheres: SiO 2 (DNA(SiO 2 )) The microsphere colloidal solution was vacuum freeze-dried to obtain SiO 2 (DNA(SiO 2 )) Microspheres were ground in a micromill at 1 mg SiO 2 The microspheres were added with a ratio of 1 g zirconium beads, ground with zirconium beads with a diameter of 0.1 mm for 2 to 8 minutes, and then centrifuged and washed three times at 21,000 g, the supernatant was removed and ultrapure water was added.

[0073] A5. Observe the SiO after grinding and washing using a transmission electron microscope (TEM) 2 (DNA(SiO 2 The size and structure of the microspheres were analyzed and their zeta potential (ζ) and hydrodynamic diameter were determined using a Zetasizer Nano ZS. 2 (DNA(SiO 2 ))The particle size of the microspheres is 60nm.

[0074] A6. To ensure that the DNA is completely encapsulated in the SiO 2 (DNA(SiO 2 )) microspheres are effectively protected, and the uniform SiO 2 (DNA(SiO 2 The microspheres were gently mixed in a 0.2 U / μL DNase I solution and incubated at 37°C for 1 hour to digest the DNA fragments that were not completely coated on the surface, obtaining SiO 2 (DNA(SiO 2 ))Microsphere particles.

[0075] A7, SiO with a positively charged surface and a particle size of about 60 nm 2 (DNA(SiO 2 Preparation of microsphere particles: Take 50 mg of SiO prepared in step A4 2 (DNA(SiO 2 )) microspheres, 10 μL of TMAPS solution was added thereto, and then stirred at 900 rpm in a constant temperature box at room temperature for 12 hours. After washing with isopropanol, SiO2 microspheres with a particle size of about 60 nm and a positively charged surface were obtained. 2 (DNA(SiO 2 )) Microsphere particles;

[0076] A8. Observe SiO using a transmission electron microscope (TEM) 2 Encapsulated DNA phosphorus tracer (SiO 2 (DNA(SiO 2 The size and structure of the SiO2 were determined by Zetasizer Nano ZS. 2 Encapsulated DNA phosphorus tracer (SiO 2 (DNA(SiO 2 ))), particle size is about 60nm.

[0077] B. Preparation of SiO with a particle size of about 250 nm and a positively charged surface 2 (DNA(SiO 2 ))Microsphere particles

[0078] B1、SiO 2 Preparation of core solution: SiO 2 The microspheres were suspended in isopropanol to prepare a SiO 2 Core solution.

[0079] B2. TMAPS-functionalized SiO with positive surface charge2 Preparation of microspheres: Prepared SiO 2 The core solution was mixed, 1 mL was added to a 2 mL microcentrifuge tube, and ultrasonic treatment was performed for 10 minutes to obtain a uniform SiO 2 Microsphere colloidal solution; 10 μL TMAPS solution was added thereto, and then stirred at room temperature at 900 rpm in a constant temperature box for 12 hours, and washed with isopropanol to obtain SiO 2 Microspheres.

[0080] B3、SiO 2 (DNA(SiO 2 Preparation of microsphere solution: First, prepare a nucleic acid solution with a concentration of 50 μg / mL with ultrapure water; then add 700 μL of ultrapure water, 320 μL of nucleic acid solution and 35 μL of positively charged SiO 2 The microspheres were mixed and then ultrasonically treated to obtain a uniform solution; the solution was washed by centrifugation with ultrapure water and then ultrasonically treated to obtain a uniform colloidal solution; then, 0.5 μL of TMAPS solution was added thereto and mixed, and then 0.5 μL of TEOS was added and stirred at room temperature at a speed of 900 rpm for 4 hours; then 4.4 μL of TEOS was added to the system and stirred at room temperature at a speed of 900 rpm for 4 days; the solution was washed with ultrapure water and then ultrasonically treated to obtain SiO 2 (DNA(SiO 2 ))Microsphere colloidal solution.

[0081] B4, SiO with uniform particle size of about 250nm 2 (DNA(SiO 2 Preparation of microspheres: SiO 2 (DNA(SiO 2 )) The microsphere colloidal solution was vacuum freeze-dried to obtain SiO 2 (DNA(SiO 2 )) Microspheres were ground in a micromill at 1 mg SiO 2 The microspheres were added with a ratio of 1 g zirconium beads, ground with zirconium beads with a diameter of 0.1 mm for 2 to 8 minutes, and then centrifuged and washed three times at 21,000 g, the supernatant was removed and ultrapure water was added.

[0082] B5. Observe the SiO after grinding and washing using a transmission electron microscope (TEM) 2 (DNA(SiO 2 The size and structure of the microspheres were analyzed and their zeta potential (ζ) and hydrodynamic diameter were determined using a Zetasizer Nano ZS. 2 (DNA(SiO 2 ))The particle size of the microspheres is 250nm.

[0083] B6. To ensure that the DNA is completely encapsulated in the SiO 2 (DNA(SiO 2 )) microspheres are effectively protected, and the uniform SiO 2 (DNA(SiO 2 The microspheres were gently mixed in a 0.2 U / μL DNase I solution and incubated at 37°C for 1 hour to digest the DNA fragments that were not completely coated on the surface, obtaining SiO 2 (DNA(SiO 2 ))Microspheres.

[0084] B7, SiO with a positively charged surface and a particle size of about 250 nm 2 (DNA(SiO 2 Preparation of microsphere particles: Take 50 mg of SiO prepared in step B4 2 (DNA(SiO 2 )) microspheres, 10 μL of TMAPS solution was added thereto, and then stirred at 900 rpm in a constant temperature box at room temperature for 12 hours. After washing with isopropanol, SiO2 with a particle size of about 250 nm and a positively charged surface was obtained. 2 (DNA(SiO 2 ))Microsphere particles.

[0085] B8. Observe SiO using a transmission electron microscope (TEM) 2 Encapsulated DNA phosphorus tracer (SiO 2 (DNA(SiO 2 The size and structure of the SiO2 were determined by Zetasizer Nano ZS. 2 Encapsulated DNA phosphorus tracer (SiO 2 (DNA(SiO 2 ), particle size is about 250nm.

[0086] C. Preparation of SiO with a particle size of about 550 nm and a positively charged surface 2 (DNA(SiO 2 ))Microsphere particles

[0087] C1、SiO 2 Preparation of core solution: SiO 2 The microspheres were suspended in isopropanol to prepare a SiO 2 Core solution.

[0088] C2, TMAPS functionalized SiO with positive surface charge 2 Preparation of microspheres: Prepared SiO 2 The core solution was mixed, 1 mL was added to a 2 mL microcentrifuge tube, and ultrasonic treatment was performed for 10 minutes to obtain a uniform SiO 2 Microsphere colloidal solution; 10 μL TMAPS solution was added thereto, and then stirred at room temperature at 900 rpm in a constant temperature box for 12 hours, and washed with isopropanol to obtain SiO 2 Microspheres.

[0089] C3、SiO 2 (DNA(SiO 2 Preparation of microsphere solution: First, prepare a nucleic acid solution with a concentration of 50 μg / mL with ultrapure water; then add 700 μL of ultrapure water, 320 μL of nucleic acid solution and 35 μL of positively charged SiO 2 The microspheres were mixed and then ultrasonically treated to obtain a uniform solution; the solution was washed by centrifugation with ultrapure water and then ultrasonically treated to obtain a uniform colloidal solution; then, 0.5 μL of TMAPS solution was added thereto and mixed, and then 0.5 μL of TEOS was added and stirred at room temperature at a speed of 900 rpm for 4 hours; then 1.5 μL of TEOS was added to the system and stirred at room temperature at a speed of 900 rpm for 4 days; the solution was washed with ultrapure water and then ultrasonically treated to obtain SiO 2 (DNA(SiO 2 ))Microsphere colloidal solution.

[0090] C4, SiO with uniform particle size of about 550nm 2 (DNA(SiO 2 Preparation of microspheres: SiO 2 (DNA(SiO 2 )) The microsphere colloidal solution was vacuum freeze-dried to obtain SiO 2 (DNA(SiO 2 )) Microspheres were ground in a micromill at 1 mg SiO 2 The microspheres were added with a ratio of 1 g zirconium beads, ground with zirconium beads with a diameter of 0.1 mm for 2 to 8 minutes, and then centrifuged and washed three times at 21,000 g, the supernatant was removed and ultrapure water was added.

[0091] C5. Observe the SiO after grinding and washing using transmission electron microscopy (TEM) 2 (DNA(SiO 2 The size and structure of the microspheres were analyzed and their zeta potential (ζ) and hydrodynamic diameter were determined using a Zetasizer Nano ZS. 2 (DNA(SiO2 ))The particle size of the microspheres is 550nm.

[0092] C6. To ensure that the DNA is completely encapsulated in the SiO 2 (DNA(SiO 2 )) microspheres are effectively protected, and the uniform SiO 2 (DNA(SiO 2 The microspheres were gently mixed in a 0.2 U / μL DNase I solution and incubated at 37°C for 1 hour to digest the DNA fragments that were not completely coated on the surface, obtaining SiO 2 (DNA(SiO 2 ))Microspheres.

[0093] C7, SiO with a positively charged surface and a particle size of about 550 nm 2 (DNA(SiO 2 Preparation of microsphere particles: Take 50 mg of SiO prepared in step C4 2 (DNA(SiO 2 )) microspheres, 10 μL of TMAPS solution was added thereto, and then stirred at 900 rpm in a constant temperature box at room temperature for 12 hours. After washing with isopropanol, SiO2 with a particle size of about 550 nm and a positively charged surface was obtained. 2 (DNA(SiO 2 ))Microsphere particles.

[0094] C8. Observation of SiO using transmission electron microscopy (TEM) 2 Encapsulated DNA phosphorus tracer (SiO 2 (DNA(SiO 2 The size and structure of the SiO2 were determined by Zetasizer Nano ZS. 2 Encapsulated DNA phosphorus tracer (SiO 2 (DNA(SiO 2 ))), particle size is about 550nm.

[0095] D. Prepare SiO with a particle size of about 1.2 μm and a positively charged surface 2 (DNA(SiO 2 ))Microsphere particles

[0096] D1、SiO 2 Preparation of core solution: SiO 2 The microspheres were suspended in isopropanol to prepare a SiO 2 Core solution.

[0097] D2, TMAPS functionalized SiO with positive surface charge 2 Preparation of microspheres: Prepared SiO 2 The core solution was mixed, 1 mL was added to a 2 mL microcentrifuge tube, and ultrasonic treatment was performed for 10 minutes to obtain a uniform SiO 2 Microsphere colloidal solution; 10 μL TMAPS solution was added thereto, and then stirred at room temperature at 900 rpm in a constant temperature box for 12 hours, and washed with isopropanol to obtain SiO 2 Microspheres.

[0098] D3、SiO 2 (DNA(SiO 2 Preparation of microsphere solution: First, prepare a nucleic acid solution with a concentration of 50 μg / mL with ultrapure water; then add 700 μL of ultrapure water, 320 μL of nucleic acid solution and 35 μL of positively charged SiO 2 The microspheres were mixed and then ultrasonically treated to obtain a uniform solution; the solution was washed by centrifugation with ultrapure water and then ultrasonically treated to obtain a uniform colloidal solution; then, 0.5 μL of TMAPS solution was added thereto and mixed, and then 0.5 μL of TEOS was added and stirred at room temperature at a speed of 900 rpm for 4 hours; then 3.4 μL of TEOS was added to the system and stirred at room temperature at a speed of 900 rpm for 4 days; the solution was washed with ultrapure water and then ultrasonically treated to obtain SiO 2 (DNA(SiO 2 ))Microsphere colloidal solution.

[0099] D4, SiO with uniform particle size of about 1.2 μm 2 (DNA(SiO 2 Preparation of microspheres: SiO 2 (DNA(SiO 2 )) The microsphere colloidal solution was vacuum freeze-dried to obtain SiO 2 (DNA(SiO 2 )) Microspheres were ground in a micromill at 1 mg SiO 2 The microspheres were added with a ratio of 1 g zirconium beads, ground with zirconium beads with a diameter of 0.1 mm for 2 to 8 minutes, and then centrifuged and washed three times at 21,000 g, the supernatant was removed and ultrapure water was added.

[0100] D5. Observe the SiO after grinding and washing using a transmission electron microscope (TEM) 2 (DNA(SiO 2 The size and structure of the microspheres were analyzed and their zeta potential (ζ) and hydrodynamic diameter were determined using a Zetasizer Nano ZS. 2(DNA(SiO 2 ))The particle size of the microspheres is 1.2 μm.

[0101] D6. To ensure that the DNA is completely encapsulated in the SiO 2 (DNA(SiO 2 )) microspheres are effectively protected, and the uniform SiO 2 (DNA(SiO 2 The microspheres were gently mixed in a 0.2 U / μL DNase I solution and incubated at 37°C for 1 hour to digest the DNA fragments that were not completely coated on the surface, obtaining SiO 2 (DNA(SiO 2 ))Microspheres.

[0102] D7, SiO with a positively charged surface and a particle size of about 1.2 μm 2 (DNA(SiO 2 Preparation of microsphere particles: Take 50 mg of SiO prepared in step D4 2 (DNA(SiO 2 )) microspheres, 10 μL of TMAPS solution was added thereto, and then stirred at 900 rpm in a constant temperature box at room temperature for 12 hours. After washing with isopropanol, SiO2 microspheres with a particle size of about 1.2 μm and a positively charged surface were obtained. 2 (DNA(SiO 2 ))Microsphere particles.

[0103] D8. Observe SiO using transmission electron microscopy (TEM) 2 Encapsulated DNA tracer (SiO 2 (DNA(SiO 2 The size and structure of the SiO2 were determined by Zetasizer Nano ZS. 2 Encapsulated DNA phosphorus tracer (SiO 2 (DNA(SiO 2 ), particle size is about 1.2μm.

[0104] The positively charged SiO 2 Schematic diagram of the preparation process of the encapsulated DNA phosphor tracer is shown in Figure 1 .

[0105] Example 2 Different sizes of positively charged SiO 2 Application of Encapsulated DNA Phosphorus Tracers in Farmland

[0106] The existing tracing methods for the sources of phosphorus non-point source pollution can only trace the types of sources, such as soil surface, riverbed mud, riverbank, etc., but cannot trace the specific spatial location of the source. This example takes the tracing of phosphorus non-point source pollution in the watershed as an example, and provides SiO 2 Uses of encapsulated DNA phospho-tracers.

[0107] Different crops require different amounts of phosphorus fertilizer and irrigation water, which results in different levels of phosphorus non-point source pollution. Figure 2 As shown, the SiO 2 The encapsulated DNA phosphorus tracers are encoded with three different DNA sequences, marking the fields and time of placement. DNA1 marks the tracer placed in the pumpkin field, DNA2 marks the tracer placed in the corn field, and GM03 marks the tracer placed in the wheat field. 2 The encapsulated DNA phosphorus tracers are mixed tracers of 60nm, 250nm, 550nm and 1.2μm. The percentage of its placement is determined by the proportion of soil colloids and the soil particle size distribution. For example, the 60nm SiO 2 Encapsulated DNA phosphorus tracer (tracing 0.025-0.2 μm soil colloids), 250 nm SiO 2 Encapsulated DNA phosphorus tracer (tracing 0.2-0.45 μm soil colloids), 550 nm SiO 2 Encapsulated DNA phosphorus tracer (tracing 0.45-0.8 μm soil colloids), 1.2 μm SiO 2 The volume percentages of the encapsulated DNA phosphorus tracer (tracing soil particles ≥1μm) were 35%, 14%, 1%, and 50%, respectively. The time and spatial location of the source of phosphorus adsorbed on soil colloid particles of different sizes were traced by taking advantage of the fact that phosphate is negatively charged and will be adsorbed on the surface of the positively charged tracer. The phosphate fertilizer was applied at different times and in different farmlands, and samples were collected from nearby groundwater wells and drainage ditches to detect SiO 2 Encapsulated DNA phosphorus tracers and phosphorus concentration variations in surface and groundwater.

[0108] Water sample analysis method:

[0109] 1. Total phosphorus (TP) is measured using the agricultural standard NY / T 88-1988.

[0110] 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).

[0111] 3. Use ammonium molybdate spectrophotometry (GB / T11893-1989) to measure the phosphorus content of each level ①-⑤ respectively.

[0112] 4. Dissolve SiO 2 , measure the DNA sequence, and examine the sources of particles of this size level and the proportion of each source.

[0113] Centrifuge 100 μL SiO at 21,000 g at room temperature. 2 The DNA phosphor tracer was encapsulated for 3 minutes and the supernatant was discarded. Then 300 μL NH 4 FHF / NH 4 F etching solution, SiO2 encapsulated with DNA tracer 2 Shell and SiO 2 The core was completely dissolved and the DNA was ensured not to be hydrolyzed or denatured during the release step. The DNA was eluted using 10 mM Tris-HCl (pH 8.5) and the DNA concentration was determined by qPCR using the Taqman probe method.

[0114] Example 3 Different sizes of positively charged SiO 2 Application of Encapsulated DNA Phosphorus Tracers in Drainage Ditches

[0115] 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 SiO2 labeled with T2M sequence DNA was placed in the drainage ditch at the same position and time. 2 Encapsulated DNA phosphorus tracer. SiO2 with positively charged surface in four particle sizes 2 The percentage of the encapsulated DNA phosphorus tracer is determined by the proportion of suspended particulate matter in the drainage ditch water at the point of placement. Figure 2 In the experiment, four tracer particle sizes of 60 nm SiO 2 Encapsulated DNA phosphorus tracer (tracing 0.025-0.2 μm colloidal particles), 250 nm SiO 2Encapsulated DNA phosphorus tracer (tracing 0.2-0.45 μm colloidal particles), 550 nm SiO 2 Encapsulated DNA phosphorus tracer (tracing 0.45-0.8 μm colloidal particles), 1.2 μm SiO 2 The volume percentages of the encapsulated DNA phosphorus tracer (tracing suspended particles ≥ 1 μm) were 4%, 2%, 0.1%, and 93.9%. The negatively charged phosphate group was used to adsorb on the positively charged tracer surface. The time and spatial location of the source of phosphorus adsorbed on suspended particles of different sizes were traced. Samples were taken at different locations in the drainage ditch to detect SiO 2 Encapsulated DNA phosphorus tracer and phosphorus concentration variations in different groove segments.

[0116] Water sample analysis method:

[0117] 1. Total phosphorus (TP) is measured using the agricultural standard NY / T 88-1988.

[0118] 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).

[0119] 3. Use ammonium molybdate spectrophotometry (GB / T11893-1989) to measure the phosphorus content of each level ①-⑤ respectively.

[0120] 4. Dissolve SiO 2 , measure the DNA sequence, and examine the sources of particles of this size level and the proportion of each source.

[0121] Centrifuge 100 μL SiO at 21,000 g at room temperature. 2 The DNA phosphor tracer was encapsulated for 3 minutes and the supernatant was discarded. Then 300 μL NH 4 FHF / NH 4 F etching solution, SiO2 encapsulated with DNA tracer 2 Shell and SiO 2 The core was completely dissolved and the DNA was not hydrolyzed or denatured during the release step. The DNA was eluted using 10 mM Tris-HCl (pH 8.5) and the DNA concentration was determined by qPCR using the Taqman probe method.

[0122] 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. SiO 2 Encapsulated DNA phospho-tracer, It is characterized in that The phosphorus tracer consists of four different particle sizes of positively charged SiO 2 (DNA(SiO 2 ))Microsphere particle composition; The SiO 2 (DNA(SiO 2 )) The microsphere particles consist of a core and SiO2 2 Shell composition; The core is SiO2 with four different particle sizes on the surface of which nucleic acid is adsorbed. 2 Microspheres, with a layer of SiO wrapped around the outside of the core 2 The shell is modified by a functionalizing agent TMAPS so that the surface of the shell is positively charged; The SiO 2 The particle sizes of the microspheres are 20 nm, 200 nm, 500 nm and 1000 nm, respectively. 2 (DNA(SiO 2 ))The particle sizes of the microspheres 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 SiO 2 The method for preparing the encapsulated DNA phosphor tracer comprises the following steps: (1) Prepare SiO particles of different sizes 2 Microspheres; (2) SiO 2 The microspheres were contacted with the functionalization reagent TMAPS to make SiO 2 The surface of the microspheres is positively charged, and then electrostatically adsorbs nucleic acids, which serve as the core; (3) contacting the core with TEOS to catalyze the formation of SiO 2 Growth, forming a layer of SiO 2 Shell, get different particle sizes of SiO 2 (DNA(SiO 2 )) Microsphere particles; (4) SiO obtained in step (3) 2 (DNA(SiO 2 )) The microsphere particles are contacted with the functional reagent TMAPS to make SiO 2 (DNA(SiO 2 ))The surface of the microsphere particles is positively charged.

2. SiO according to claim 1 2 Method for preparing encapsulated DNA phosphorus tracers, It is characterized in that The following steps are involved: (1) Prepare SiO particles of different sizes 2 Microspheres; (2) SiO 2 The microspheres were contacted with the functionalization reagent TMAPS to make SiO 2 The surface of the microspheres is positively charged, and then electrostatically adsorbs nucleic acids, which serve as the core; (3) contacting the core with TEOS to catalyze the formation of SiO 2 Growth, forming a layer of SiO 2 Shell, get different particle sizes of SiO 2 (DNA(SiO 2 )) Microsphere particles; (4) SiO obtained in step (3) 2 (DNA(SiO 2 )) The microsphere particles are contacted with the functional reagent TMAPS to make SiO 2 (DNA(SiO 2 )) The surface of the microsphere particles is positively charged, that is; 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, It is characterized in that A. Preparation of SiO2 with a particle size of 60 nm and a positively charged surface 2 (DNA(SiO 2 ))Microsphere particles A1、SiO 2 Preparation of core solution: SiO 2 The microspheres were suspended in isopropanol to prepare a SiO 2 Kernel solution; A2. TMAPS-functionalized SiO with positive surface charge 2 Preparation of microspheres: Prepared SiO 2 The core solution was mixed, 1 mL was added to a 2 mL microcentrifuge tube, and ultrasonicated for 10 minutes to obtain a uniform SiO 2 Microsphere colloidal solution; 10 µL TMAPS solution was added to it, and then stirred at 900 rpm in a constant temperature box at room temperature for 12 hours. After washing with isopropanol, SiO 2 Microspheres; A3、SiO 2 (DNA(SiO 2 Preparation of microsphere solution: First, prepare a 50 µg / mL nucleic acid solution with ultrapure water; then add 700 µL ultrapure water, 320 µL nucleic acid solution and 35 µL positively charged SiO 2 The microspheres were mixed and ultrasonically treated to obtain a uniform solution; the solution was washed by centrifugation with ultrapure water and ultrasonically treated to obtain a uniform colloidal solution; then, 0.5 μL of TMAPS solution was added thereto and mixed, and then 0.5 μL of TEOS was added and stirred at 900 rpm for 4 hours at room temperature; then 120.3 μL of TEOS was added to the system and stirred at 900 rpm for 4 days at room temperature; the SiO2 solution was washed with ultrapure water and ultrasonically treated to obtain a uniform colloidal solution. 2 (DNA(SiO 2 ))Microsphere colloidal solution; A4, SiO with uniform particle size of 60 nm 2 (DNA(SiO 2 Preparation of microspheres: SiO 2 (DNA(SiO 2 )) The microsphere colloidal solution was vacuum freeze-dried to obtain SiO 2 (DNA(SiO 2 )) Microspheres were ground in a micromill at 1 mg SiO 2 The microspheres were added with 1g zirconium beads, ground with 0.1 mm diameter zirconium beads for 2-8 minutes, and then centrifuged and washed three times at 21000 g. The supernatant was removed and ultrapure water was added to obtain SiO2 with uniform particle size of 60 nm. 2 (DNA(SiO 2 ))Microspheres; A5, SiO with a particle size of 60 nm and a positively charged surface 2 (DNA(SiO 2 Preparation of microsphere particles: Take 50 mg of SiO prepared in step A4 2 (DNA(SiO 2 )) microspheres, 10 μL of TMAPS solution was added to them, and then stirred at 900 rpm in a constant temperature box for 12 hours at room temperature. After washing with isopropanol, SiO2 microspheres with a particle size of 60 nm and positive charge on the surface were obtained. 2 (DNA(SiO 2 )) Microsphere particles; B. Preparation of SiO2 with a particle size of 250 nm and a positively charged surface 2 (DNA(SiO 2 ))Microsphere particles B1、SiO 2 Preparation of core solution: SiO 2 The microspheres were suspended in isopropanol to prepare a SiO 2 Kernel solution; B2. TMAPS-functionalized SiO with positive surface charge 2 Preparation of microspheres: Prepared SiO 2 The core solution was mixed, 1 mL was added to a 2 mL microcentrifuge tube, and ultrasonicated for 10 minutes to obtain a uniform SiO 2 Microsphere colloidal solution; 10 µL TMAPS solution was added to it, and then stirred at 900 rpm in a constant temperature box at room temperature for 12 hours. After washing with isopropanol, SiO 2 Microspheres; B3、SiO 2 (DNA(SiO 2 Preparation of microsphere solution: First, prepare a 50 µg / mL nucleic acid solution with ultrapure water; then add 700 µL ultrapure water, 320 µL nucleic acid solution and 35 µL positively charged SiO 2 The microspheres were mixed and ultrasonically treated to obtain a uniform solution; the solution was washed by centrifugation with ultrapure water and ultrasonically treated to obtain a uniform colloidal solution; then, 0.5 µL of TMAPS solution was added thereto and mixed, and then 0.5 µL of TEOS was added and stirred at room temperature at 900 rpm for 4 hours; then 4.4 µL of TEOS was added to the system and stirred at room temperature at 900 rpm for 4 days; the SiO2 solution was washed with ultrapure water and ultrasonically treated to obtain a uniform colloidal solution. 2 (DNA(SiO 2 ))Microsphere colloidal solution; B4, SiO with uniform particle size of 250 nm 2 (DNA(SiO 2 Preparation of microspheres: SiO 2 (DNA(SiO 2 )) The microsphere colloidal solution was vacuum freeze-dried to obtain SiO 2 (DNA(SiO 2 )) Microspheres were ground in a micromill at 1 mg SiO 2 The microspheres were added with 1 g of zirconium beads, ground with 0.1 mm diameter zirconium beads for 2-8 minutes, and then centrifuged and washed three times at 21000 g. The supernatant was removed and ultrapure water was added to obtain SiO2 microspheres with uniform particle size of 250 nm. 2 (DNA(SiO 2 ))Microspheres; B5, SiO with a particle size of 250 nm and a positively charged surface 2 (DNA(SiO 2 Preparation of microsphere particles: Take 50 mg of SiO prepared in step B4 2 (DNA(SiO 2 )) microspheres, 10 μL of TMAPS solution was added to them, and then stirred at 900 rpm in a constant temperature box for 12 hours at room temperature. After washing with isopropanol, SiO2 microspheres with a particle size of 250 nm and positively charged on the surface were obtained. 2 (DNA(SiO 2 )) Microsphere particles; C. Preparation of SiO with a particle size of 550 nm and a positively charged surface 2 (DNA(SiO 2 ))Microsphere particles C1、SiO 2 Preparation of core solution: SiO 2 The microspheres were suspended in isopropanol to prepare a SiO 2 Kernel solution; C2, TMAPS functionalized SiO with positive surface charge 2 Preparation of microspheres: Prepared SiO 2 The core solution was mixed, 1 mL was added to a 2 mL microcentrifuge tube, and ultrasonicated for 10 minutes to obtain a uniform SiO 2 Microsphere colloidal solution; 10 µL TMAPS solution was added to it, and then stirred at 900 rpm in a constant temperature box at room temperature for 12 hours. After washing with isopropanol, SiO 2 Microspheres; C3、SiO 2 (DNA(SiO 2 Preparation of microsphere solution: First, prepare a 50 µg / mL nucleic acid solution with ultrapure water; then add 700 µL ultrapure water, 320 µL nucleic acid solution and 35 µL positively charged SiO 2 The microspheres were mixed and ultrasonically treated to obtain a uniform solution; the solution was washed by centrifugation with ultrapure water and ultrasonically treated to obtain a uniform colloidal solution; then, 0.5 µL of TMAPS solution was added thereto and mixed, and then 0.5 µL of TEOS was added and stirred at 900 rpm for 4 hours at room temperature; then 1.5 µL of TEOS was added to the system and stirred at 900 rpm for 4 days at room temperature; the solution was washed with ultrapure water and ultrasonically treated to obtain SiO 2 (DNA(SiO 2 ))Microsphere colloidal solution; C4, SiO with uniform particle size of 550 nm 2 (DNA(SiO 2 Preparation of microspheres: SiO 2 (DNA(SiO 2 )) The microsphere colloidal solution was vacuum freeze-dried to obtain SiO 2 (DNA(SiO 2 )) Microspheres were ground in a micromill at 1 mg SiO 2 The microspheres were added with 1 g of zirconium beads, ground with 0.1 mm diameter zirconium beads for 2-8 minutes, and then centrifuged and washed three times at 21000 g. The supernatant was removed and ultrapure water was added to obtain SiO2 microspheres with uniform particle size of 550 nm. 2 (DNA(SiO 2 ))Microspheres; C5, SiO with a particle size of 550 nm and a positively charged surface 2 (DNA(SiO 2 Preparation of microsphere particles: Take 50 mg of SiO prepared in step C4 2 (DNA(SiO 2 )) microspheres, 10 μL of TMAPS solution was added to them, and then stirred at 900 rpm in a constant temperature box for 12 hours at room temperature. After washing with isopropanol, SiO2 microspheres with a particle size of 550 nm and positively charged on the surface were obtained. 2 (DNA(SiO 2 )) Microsphere particles; D. Preparation of SiO with a particle size of 1.2 μm and a positively charged surface 2 (DNA(SiO 2 ))Microsphere particles D1、SiO 2 Preparation of core solution: SiO 2 The microspheres were suspended in isopropanol to prepare a SiO 2 Kernel solution; D2, TMAPS functionalized SiO with positive surface charge 2 Preparation of microspheres: Prepared SiO 2 The core solution was mixed, 1 mL was added to a 2 mL microcentrifuge tube, and ultrasonicated for 10 minutes to obtain a uniform SiO 2 Microsphere colloidal solution; 10 µL TMAPS solution was added to it, and then stirred at 900 rpm in a constant temperature box at room temperature for 12 hours. After washing with isopropanol, SiO 2 Microspheres; D3、SiO 2 (DNA(SiO 2 Preparation of microsphere solution: First, prepare a 50 µg / mL nucleic acid solution with ultrapure water; then add 700 µL ultrapure water, 320 µL nucleic acid solution and 35 µL positively charged SiO 2 The microspheres were mixed and then ultrasonically treated to obtain a uniform solution; the solution was washed by centrifugation with ultrapure water and then ultrasonically treated to obtain a uniform colloidal solution; then, 0.5 µL of TMAPS solution was added thereto and mixed, and then 0.5 µL of TEOS was added and stirred at room temperature at 900 rpm for 4 hours; then 3.4 µL of TEOS was added to the system and stirred at room temperature at 900 rpm for 4 days; the solution was washed with ultrapure water and then ultrasonically treated to obtain SiO 2 (DNA(SiO 2 ))Microsphere colloidal solution; D4, SiO with uniform particle size of 1.2 μm 2 (DNA(SiO 2 Preparation of microspheres: SiO 2 (DNA(SiO 2 )) The microsphere colloidal solution was vacuum freeze-dried to obtain SiO 2 (DNA(SiO 2 )) Microspheres were ground in a micromill at 1 mg SiO 2 The microspheres were added with 1 g zirconium beads, ground with 0.1 mm diameter zirconium beads for 2-8 minutes, and then centrifuged and washed three times at 21000 g. The supernatant was removed and ultrapure water was added to obtain SiO microspheres with uniform particle size of 1.2 μm. 2 (DNA(SiO 2 ))Microspheres; D5, SiO with a positive surface charge and a particle size of 1.2 μm 2 (DNA(SiO 2 Preparation of microsphere particles: Take 50 mg of SiO prepared in step D4 2 (DNA(SiO 2 )) microspheres, 10 μL of TMAPS solution was added to them, and then stirred at 900 rpm in a constant temperature oven for 12 hours at room temperature. After washing with isopropanol, SiO2 microspheres with a particle size of 1.2 μm and a positively charged surface were obtained. 2 (DNA(SiO 2 )) Microsphere particles; 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, It is 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. SiO prepared according to the method according to any one of claims 2 to 4 2 Encapsulated DNA phospho-tracer.

6. SiO according to claim 1 or 5 2 Application of encapsulated DNA phosphorus tracers in spatiotemporal tracing of non-point source phosphorus pollution.

7. The use according to claim 6, It is characterized in that Placing the SiO 2 DNA phosphorus tracer encapsulated in four different particle sizes with positively charged SiO 2 (DNA(SiO 2 )) The proportion of microsphere particles to be added depends on the particle size of soil colloid particles and their proportion; Among them, SiO 2 (DNA(SiO 2 )) Microspheres trace soil colloids with a size of 0.025-0.2 μm, SiO2 with a particle size of 250 nm and a positively charged surface 2 (DNA(SiO 2 )) Microspheres trace soil colloids with a size of 0.2-0.45 μm, SiO2 with a particle size of 550 nm and a positively charged surface 2 (DNA(SiO 2 )) Microspheres trace soil colloids with a size of 0.45-0.8 μm, SiO2 with a particle size of 1.2 μm and a positively charged surface 2 (DNA(SiO 2 )) Microspheres are used to trace soil particles ≥1 μm.

8. The use according to claim 7, It is characterized in that SiO with positive surface charge of 60 nm, 250 nm, 550 nm and 1.2 μm 2 (DNA(SiO 2 )) The volume portions of the microsphere particles are 20-30 portions, 10-20 portions, 0.1-5 portions and 40-60 portions respectively.

9. The use according to claim 6, It is characterized in that Place the SiO in the drain 2 DNA phosphorus tracer encapsulated in four different particle sizes with positively charged SiO 2 (DNA(SiO 2 )) The proportion of microsphere particles to be added is determined according to the particle size and proportion of suspended particles in the drainage ditch water; Among them, SiO 2 (DNA(SiO 2 )) Microspheres trace suspended particles of 0.025-0.2 μm, SiO2 with a particle size of 250 nm and a positively charged surface 2 (DNA(SiO 2 )) Microspheres trace suspended particles of 0.2-0.45 μm, SiO2 with a particle size of 550 nm and a positively charged surface 2 (DNA(SiO 2 )) Microspheres trace suspended particles of 0.45-0.8 μm, SiO2 with a particle size of 1.2 μm and a positively charged surface 2 (DNA(SiO 2 )) Microsphere particles track suspended particles ≥1μm.

10. The use according to claim 9, It is characterized in that SiO with positive surface charge of 60 nm, 250 nm, 550 nm and 1.2 μm 2 (DNA(SiO 2 )) The volume proportions of the microsphere particles are 0.1-5, 0.1-5, 0.1-2 and 90-100 respectively.

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