Tracer particles and their application and preparation methods

Abstract

The present invention provides a tracer particle and its application method and preparation method. The tracer particle includes: a core structure; a nucleic acid molecule fixed on the core structure; and a shell layer covering the core structure and the nucleic acid molecule; wherein the core structure has a first porosity, the shell layer has a second porosity, and the first porosity is greater than the second porosity. The tracer particle of the present invention has a core structure with moderate porosity, which increases the fixed amount of nucleic acid molecules and reduces the thermal conductivity of the particles (reducing thermal resistance), thereby improving the temperature tolerance of the tracer particles. In addition, the tracer particles of the present invention also have properties such as acid resistance and alkali resistance, which can further improve the tolerance and recovery rate of the tracer particles in extreme environments.

Description

Technical Field

[0001] The present invention relates to a tracer particle, and in particular to a tracer particle containing a nucleic acid molecule and an application method thereof, as well as a preparation method of the tracer particle. Background Art

[0002] Tracer technology can be used as a tool to monitor fluids, pollution leaks and product tracking. It is currently widely used in geothermal, natural gas and oil exploration. It can improve the extraction efficiency of the above energy sources. It can also be used as a tracer tool for groundwater source tracking and environmental pollution control.

[0003] Commonly used tracers include radioactive tracers, fluorescent tracers, chemical tracers, etc., but their types are limited, the analysis procedures are complicated, and they are toxic and may cause harm to the environment. In view of this, the development of new tracers has received much attention. Biotracers are a new generation of tracers that use biological materials as the main calibration material (labels, fingerprints), are non-toxic, and are less likely to cause environmental pollution. However, the existing biotracers are not ideal for extreme environmental resistance and are difficult to use in extreme environments such as strong acids, strong alkalis, or high temperatures.

[0004] As mentioned above, although the currently existing tracers can generally meet their originally intended uses, they still do not fully meet the needs in all aspects. Summary of the invention

[0005] According to some embodiments of the present invention, a tracer particle is provided, comprising: a core structure; a nucleic acid molecule fixed on the core structure; and a shell layer covering the core structure and the nucleic acid molecule; wherein the core structure has a first porosity, the shell layer has a second porosity, and the first porosity is greater than the second porosity.

[0006] According to some embodiments of the present invention, a method for applying tracer particles is provided, comprising: providing the aforementioned tracer particles; placing the tracer particles in a fluid to be observed; collecting a sample of the fluid, recovering the tracer particles from the sample, and releasing the nucleic acid molecules from the tracer particles; and analyzing the released nucleic acid molecules.

[0007] According to some embodiments of the present invention, a method for preparing a tracer particle is provided, comprising: forming a core structure; fixing a nucleic acid molecule on the core structure; and forming a shell layer on the core structure to cover the core structure and the nucleic acid molecule. In addition, the step of forming the core structure comprises: providing an oil phase solution, which comprises a silicon precursor and an emulsifier; providing an aqueous phase solution, which comprises water and a surfactant; adding the oil phase solution to the aqueous phase solution to form a mixed solution; adding a catalyst to the mixed solution; and heating the mixed solution.

[0008] The tracer particles of the present invention have a core structure with moderate porosity, which increases the amount of nucleic acid molecules that can be fixed, and reduces the thermal conductivity of the particles (reducing thermal resistance), thereby improving the temperature tolerance of the tracer particles. In addition, the tracer particles of the present invention also have acid and alkali resistance properties, which can further improve the tolerance and recovery rate of the tracer particles in extreme environments.

[0009] In order to make the features and advantages of the present invention more obvious and easy to understand, preferred embodiments are given below and described in detail with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Shows a schematic diagram of the structure of tracer particles according to some embodiments of the present invention;

[0011] Figure 2 Shows a schematic diagram of the structure of tracer particles according to some embodiments of the present invention;

[0012] Figure 3 A flowchart showing the steps of a method for preparing tracer particles according to some embodiments of the present invention;

[0013] Figure 4 Showing a gel electrophoresis analysis diagram of a target nucleic acid molecule label according to some embodiments of the present invention;

[0014] Figure 5A Showing a gel electrophoresis analysis diagram of a plasmid product containing a target nucleic acid molecule tag according to some embodiments of the present invention;

[0015] Figure 5B Showing a gel electrophoresis analysis diagram of target nucleic acid molecule labels in plasmid products according to some embodiments of the present invention;

[0016] Figures 6A to 6D Shows the results of toxicity tests on microorganisms of DNA with sequence identification number: 1 according to some embodiments of the present invention;

[0017] Fig. 7A and 7B The scanning electron microscope (SEM) observation images of the core structure before and after the encapsulation process of the shell layer according to some embodiments of the present invention are shown respectively;

[0018] Fig. 8A and 8B The scanning electron microscope (SEM) observation images of the core structure before and after the encapsulation process of the shell layer according to some embodiments of the present invention are shown respectively;

[0019] Fig. 9A and 9BThe temperature resistance test results of the tracer particles according to some embodiments of the present invention are respectively shown;

[0020] Fig.10 Displaying the acid and alkali resistance test results of tracer particles according to some embodiments of the present invention;

[0021] Fig.11 Displaying the acid and alkali resistance test results of tracer particles according to some embodiments of the present invention;

[0022] Fig.12 Shows the results of column tracing tests of tracer particles according to some embodiments of the present invention.

[0023] Wherein, the reference numerals are:

[0024] 10, 20 tracer particles;

[0025] 102 core structure;

[0026] 102p, 106p holes;

[0027] 102s surface;

[0028] 104 Nucleic acid molecules;

[0029] 106 shell;

[0030] 106a outer shell;

[0031] 106b inner shell;

[0032] 10M Preparation method;

[0033] Steps S12, S14, and S16;

[0034] d1, d2 particle size;

[0035] T Thickness. DETAILED DESCRIPTION

[0036] The following is a detailed description of the tracer particles, the application method of the tracer particles, and the preparation method of the tracer particles according to the embodiments of the present invention. It should be understood that the following description provides many different embodiments or examples for implementing different modes of some embodiments of the present invention. The specific components and arrangements described below are only for a simple and clear description of some embodiments of the present invention. Of course, these are only used for examples and are not limitations of the present invention. In addition, similar and / or corresponding numbers may be used in different embodiments to indicate similar and / or corresponding components in order to clearly describe the present invention. However, the use of these similar and / or corresponding numbers is only for a simple and clear description of some embodiments of the present invention, and does not represent any correlation between the different embodiments and / or structures discussed.

[0037] It should be understood that the components or devices of the drawings may exist in various forms known to those with common knowledge in the art. In addition, it should be understood that although the terms "first", "second", "third", etc. may be used herein to describe various components, assemblies, regions, layers or parts, these components, assemblies, regions, layers or parts should not be limited by these terms. These terms are only used to distinguish different components, assemblies, regions, layers or parts. Therefore, a first component, component, region, layer or part discussed below may be referred to as a second component, component, region, layer or part without departing from the teachings of the present invention.

[0038] Here, the terms "about", "approximately", and "substantially" generally mean within 20%, preferably within 10%, more preferably within 5%, or within 3%, or within 2%, or within 1%, or within 0.5% of a given value or range. The quantities given here are approximate quantities, that is, in the absence of specific description of "about", "approximately", and "substantially", the meanings of "about", "approximately", and "substantially" can still be implied.

[0039] The embodiment of the present invention can be used in conjunction with the attached Figure 1 It is also understood that the drawings of the present invention are also considered as part of the disclosure. It should be understood that the drawings of the present invention are not drawn according to scale, and in fact, the size of the components may be arbitrarily enlarged or reduced to clearly show the features of the present invention.

[0040] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meanings as commonly understood by those skilled in the art to which the present invention belongs. It is understood that these terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning consistent with the background or context of the relevant technology and the present invention, and should not be interpreted in an idealized or overly formal manner unless specifically defined in the embodiments of the present invention.

[0041] An embodiment of the present invention provides a tracer particle, which includes a specific nucleic acid molecule as a calibration substance, uses a core structure with a larger porosity to increase the fixation strength of the nucleic acid molecule and enhance the thermal conductivity of the particle, thereby improving the tracer particle's ability to withstand extreme environments.

[0042] Figure 1 A schematic diagram of the structure of the tracer particle 10 according to some embodiments of the present invention is shown. It should be understood that according to some embodiments, additional features may be added to the tracer particle 10. Figure 1, the tracer particle 10 may include a core structure 102, a nucleic acid molecule 104, and a shell 106. The core structure 102 may serve as a carrier of the tracer particle 10, carrying other structures formed subsequently. The nucleic acid molecule 104 is fixed on the core structure 102. The nucleic acid molecule 104 includes a specific nucleic acid sequence and may serve as a marker for the tracer particle 10. Furthermore, the shell 106 covers the core structure 102 and the nucleic acid molecule 104 and may serve as a protection and packaging structure.

[0043] like Figure 1 As shown, in some embodiments, the core structure 102 includes a plurality of holes 102p, and the nucleic acid molecules 104 are fixed in the holes 102p. In detail, in some embodiments, a portion of the nucleic acid molecules 104 can be fixed in the holes 102 of the core structure 102, and a portion of the nucleic acid molecules 104 can be fixed on the surface 102s of the core structure 102.

[0044] In particular, the core structure 102 having the pores 102p can improve the thermal conductivity of the tracer particle 10, making it suitable for high temperature environments, and can also increase the fixation strength of the nucleic acid molecule 104 on the core structure 102. In some embodiments, the core structure 102 has a first porosity, and the first porosity ranges from about 2 nm to about 100 nm or from about 4 nm to about 40 nm. It should be understood that the porosity of the core structure 102 should not be too large, otherwise the effect of protecting the nucleic acid molecule 104 cannot be achieved. On the contrary, the porosity of the core structure 102 should not be too small, otherwise the nucleic acid molecule 104 may not have sufficient locations for attachment, thereby reducing the fixation efficiency of the nucleic acid molecule 104.

[0045] In some embodiments, the particle size d1 of the core structure 102 ranges from about 20 nm to about 9000 nm, from about 20 nm to about 200 nm, from about 30 nm to about 100 nm, or from about 200 nm to about 9000 nm. According to some embodiments, the aforementioned particle size may be a volume average particle size.

[0046] The core structure 102 may be formed of an inorganic material. In some embodiments, the material of the core structure 102 includes at least one of silicon dioxide, silicate, carbonate (such as calcium carbonate), nano-gold, metal oxide, heat-resistant polymer (such as polyethylene glycol or polystyrene) and high molecular polymer (such as polylactic acid).

[0047] In some embodiments, the surface of the core structure 102 may be modified to immobilize the nucleic acid molecule 104 on the core structure 102. Specifically, the surface of the core structure 102 may be positively charged by adding a quaternary ammonium salt containing chloride, thereby connecting with the negatively charged core structure 102. In some embodiments, the quaternary ammonium salt containing chloride may include trimethoxy[3-(methylamino)propyl]silane (TMAPS).

[0048] In addition, the core structure 102 may include at least one of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and peptide nucleic acid (PNA). In some embodiments, the nucleic acid molecule 104 includes double-stranded helical DNA. In some embodiments, the nucleic acid molecule 104 may include a plasmid.

[0049] In some embodiments, the length of the nucleic acid molecule 104 ranges from about 10 base pairs (bp) to about 2000 base pairs, or preferably from about 50 base pairs to about 500 base pairs. In an embodiment where the nucleic acid molecule 104 is a plasmid, the length of the nucleic acid molecule 104 ranges from about 1500 base pairs to about 10000 base pairs, or from about 2000 base pairs to about 4000 base pairs. It should be understood that when the length of the nucleic acid molecule 104 is too long, the shell 106 cannot completely coat the nucleic acid molecule 104, or it takes a long time to complete the coating, increasing the difficulty of the encapsulation process. On the contrary, when the length of the nucleic acid molecule 104 is too short, it may cause the nucleic acid molecule 104 to be easily decomposed, reduce the sequence specificity of the nucleic acid molecule 104, and make the recognition of the marker worse. In addition, according to some embodiments, the nucleic acid molecule 104 in the form of a plasmid can protect the target nucleic acid fragment, which helps to improve the tolerance and recovery rate of the tracer particle 10 in extreme environments, and can simplify the purification steps and has the advantage of easy operation.

[0050] In some embodiments, a nucleic acid molecule 104 having any suitable sequence may be designed as a marker. For example, in some embodiments, in order to improve the temperature tolerance of the nucleic acid molecule 104, the sequence of the designed nucleic acid molecule 104 may include a partial nucleic acid sequence of a thermophilic bacterium (Thermus thermophilus). For example, in some embodiments, the thermophilic bacteria may include Tepidimonas fonticaldi, Tepidimona signava, Tepidimonas aquatica, Bacillus stearothermophilus, Thermoactinomyces vulgaris, Thermus aquaticus, Thermococcus, Thermotoga, Sulfolobus, Thermoproteus, Desulfurolobus, Acidianus, Pyrodictium occultum, Pyrodictium brockii, Methanopyrus, or Pyrobaculum, but are not limited thereto.

[0051] Furthermore, in some embodiments, the sequence of the designed nucleic acid molecule 104 may include a partial nucleic acid sequence of algae. For example, in some embodiments, the algae may include Chlamydomonas reinhardtii, Chlamydomonas moewusii, Chlamydomonas eugametos, Chlamydomonas segnis, etc. belonging to the genus Chlamydomonas; Dunaliella salina, Dunaliella tertiolecta, Dunaliella primolecta, etc. belonging to the genus Dunaliella; Chlorella vulgaris, Chlorella pyrenoidosa, etc. belonging to the genus Chlorella; Haematococcus pluvialis, etc. belonging to the genus Haematococcus; Chlorococcum littorale, etc. belonging to the genus Chlorococcus; Pseudochoricystis, etc. belonging to the genus Pseudochoricystis. ellipsoidea, etc.; Amphora sp., etc. belonging to the genus Amphora; Nitzschia alba, Nitzschia closterium, Nitzschia laevis, etc. belonging to the genus Nitzschia; Crypthecodinium cohnii, etc. belonging to the genus Crypthecodinium; Euglena gracilis, Euglena proxima, etc. belonging to the genus Euglena; Paramecium bursaria, etc. belonging to the genus Paramecium; Synechococcusaquatilis, Synechococcus elongatus, etc. belonging to the genus Synechococcus; Spirulina platensis, Spirulina subsalsa, etc. belonging to the genus Spirulina; Prochlorococcus marine algae, Prochlorococcus marinus, etc.; Oocystis polymorpha belonging to the genus Oocystis, etc., but not limited thereto.

[0052] In some embodiments, the designed nucleic acid molecule 104 may have a hybrid nucleic acid sequence. In some embodiments, the sequence of the designed nucleic acid molecule 104 may include partial nucleic acid sequences from prokaryotes and eukaryotes at the same time, for example, it may include partial nucleic acid sequences from thermophiles and algae at the same time. For example, in some embodiments, the sequence of the nucleic acid molecule 104 includes a partial sequence of 16S rDNA of thermophiles and a partial sequence of 18S rDNA of algae. Since there should be no organisms in the natural environment that have sequence characteristics of both species, the sequence of the nucleic acid molecule 104 that includes both prokaryotes and eukaryotes should be specific, easy to identify tracer particles, and can reduce the possibility of mutual interference with nucleic acid fragments in the environment. Specifically, in some embodiments, the sequence of the designed nucleic acid molecule 104 may include partial nucleic acid sequences of Tepidimonas fonticaldi and Chlamydomonas reinhardtii.

[0053] In addition, in some embodiments, a sequence region having a higher content of cytosine (C) and guanine (G) than adenine (A) and thymine (T) can be selected as the sequence of the nucleic acid molecule 104. Since the interaction between adenine and thymine is stronger than that between cytosine and guanine, when the GC content of the sequence is higher, its melting temperature is higher and the thermal stability is also better. Specifically, in some embodiments, the GC content ratio of the sequence of the designed nucleic acid molecule 104 can be about 55% to about 70%.

[0054] In some embodiments, the nucleic acid molecule 104 has at least 85%, 90%, or 95% sequence similarity to the sequence shown in SEQ ID No. 1. In some embodiments, the nucleic acid molecule 104 may include sequences shown in SEQ ID Nos. 2 and 3. Furthermore, in embodiments where the nucleic acid molecule 104 is a plasmid, the nucleic acid molecule 104 may include a nucleic acid fragment inserted into the plasmid, and the nucleic acid fragment has at least 85%, 90%, or 95% sequence similarity to the sequence shown in SEQ ID No. 1.

[0055] In addition, the nucleic acid sequence can be designed and the nucleic acid molecule 104 can be manufactured by the techniques known in the art of the invention. For example, the designed nucleic acid sequence can be amplified in large quantities by polymerase chain reaction (PCR) using primers complementary to the designed nucleic acid sequence. In some embodiments, the yield of the nucleic acid molecule 104 can be further increased by using biofermentation technology. In detail, a suitable plasmid can be selected, and the designed nucleic acid fragment (e.g., sequence identification number: 1) can be inserted into the plasmid to construct a recombinant plasmid, and then a fermenter can be used to culture a host cell containing the recombinant plasmid in large quantities. In some embodiments, the host cell can include Escherichia coli. In some embodiments, the recombinant plasmid containing the target nucleic acid fragment can be extracted from the host cell using alkaline lysis. For example, according to some embodiments, the nucleic acid molecule 104 can be prepared in a 4-liter fermenter, and the yield of 8.85 mg / day can be achieved, which is much higher than the yield of the nucleic acid molecule 104 prepared by PCR (about 0.4 mg / day).

[0056] As mentioned above, the shell 106 can be used as a packaging material to cover the core structure 102 and the nucleic acid molecule 104. In some embodiments, the shell 106 has a second porosity. In some embodiments, the first porosity of the core structure 102 is greater than the second porosity of the shell 106. In some embodiments, the second porosity of the shell 106 is substantially 0 nm, that is, the shell 106 substantially has no holes and is a solid or dense shell, which can completely seal the nucleic acid molecule 104 to prevent the nucleic acid molecule 104 from being exposed. In some embodiments where the second porosity of the shell 106 is 0, the tracer particle 10 can be applied to the detection of fluids. In other embodiments, the second porosity of the shell 106 is not 0, for example, it is about 0.5 nm to about 10 nm. In some embodiments where the second porosity of the shell 106 is not 0, the tracer particle 10 can be applied to the detection of air.

[0057] In addition, the shell layer 106 may be a single-layer structure or a multi-layer structure. Figure 1 As shown, in some embodiments where the shell layer 106 is a single-layer structure, the shell layer 106 may have substantially no holes and may be substantially encapsulated.

[0058] In some embodiments, the thickness T of the shell layer 106 ranges from about 10 nm to about 5000 nm, or from about 10 nm to about 150 nm, or from about 50 nm to about 120 nm.

[0059] In some embodiments, the shell layer 106 includes at least one of silicon dioxide, silicate, carbonate (such as calcium carbonate), heat-resistant polymer (such as polyethylene glycol or polystyrene) and high molecular polymer (such as polylactic acid). In some embodiments, the core structure 102 and the shell layer 106 can be formed of the same material.

[0060] In some embodiments, the core structure 102 with the fixed nucleic acid molecule 104 can be modified using a chlorine-containing quaternary ammonium salt so that the shell 106 has a positive charge inside to connect with the negatively charged nucleic acid molecule 104 to form a closed shell-core structure. In some embodiments, the chlorine-containing quaternary ammonium salt can include N-methyl-3-aminopropyltrimethoxy (trimethoxy [3- (methylamino) propyl] silane, TMAPS).

[0061] Furthermore, if Figure 1 As shown, in some embodiments, the particle size d2 of the encapsulated tracer particles 10 ranges from about 30 nm to about 10000 nm, or from about 30 nm to about 300 nm, or from about 50 nm to about 150 nm. According to some embodiments, the aforementioned particle size may be a volume average particle size.

[0062] In addition, in some embodiments, the encapsulated tracer particles 10 have good homogeneity, that is, have uniform shape, size or particle size. In some embodiments, the size variation of the tracer particles 10 ranges from about 0% to about 10%.

[0063] Next, please refer to Figure 2 , Figure 2 The schematic diagram of the structure of the tracer particle 20 according to some other embodiments of the present invention is shown. It should be understood that the components or components that are the same or similar to those in the previous text will be represented by the same or similar reference numerals, and their materials, manufacturing methods and functions are the same or similar to those described in the previous text, so this part will not be repeated in the following text. Figure 2 The tracer particles 20 of the embodiment shown are Figure 1 The tracer particle 10 shown is substantially similar, except that the shell 106 of the tracer particle 20 is a multi-layer structure.

[0064] Specifically, in this embodiment, the shell layer 106 includes an outer shell layer 106a and an inner shell layer 106b. Figure 2As shown, in this embodiment, the inner shell layer 106b may include a plurality of holes 106p. The holes 106p may reduce the thermal conductivity of the shell layer 106, thereby improving the temperature tolerance of the tracer particle 20. In some embodiments, the porosity of the inner shell layer 106b ranges from about 4 nm to about 40 nm. In addition, in this embodiment, the outer shell layer 106a substantially has no holes, and can completely seal the nucleic acid molecule 104 to prevent the nucleic acid molecule 104 from being exposed.

[0065] It should be understood that although Figure 2 In the illustrated embodiment, the shell 106 includes two layers, namely, an outer shell 106a and an inner shell 106b, but in other embodiments, the shell 106 may have other suitable numbers of sub-layers. Figure 2 In the illustrated embodiment, the inner shell layer 106b includes holes 106p. In other embodiments, the inner shell layer 106b may be substantially free of holes.

[0066] According to some embodiments, the tracer particles provided by the present invention can be operated at 120°C for at least 5 hours. According to some embodiments, the tracer particles provided by the present invention can be operated at 120°C for more than 24 hours, and can maintain a recovery rate of more than 80%. According to some embodiments, the tracer particles provided by the present invention can be operated at 120°C for more than 720 hours, and can maintain a recovery rate of more than 20%. According to some embodiments, the tracer particles provided by the present invention can be operated at a pH value of 1 to 13 for at least 720 hours. For example, it can be operated at a pH value of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 for at least 720 hours.

[0067] In addition, according to some embodiments, the tracer particles provided by the present invention can be used for fluid tracking and exploration of geothermal or oil wells. Specifically, the tracer particles can track the movement (flow, migration, migration) and recovery of liquids in the fracture area of ​​the formation, thereby analyzing the distribution and status of oil wells or gas wells. According to some embodiments, the tracer particles provided by the present invention can also be used for tracking pollutants. According to some embodiments, the tracer particles provided by the present invention can be used as anti-counterfeiting labels.

[0068] In addition, according to some embodiments, a method for applying tracer particles is provided, comprising the following steps: providing the tracer particles described in the aforementioned embodiments; placing the tracer particles in a fluid to be observed; collecting a sample of the fluid, recovering the tracer particles from the sample, and releasing nucleic acid molecules from the tracer particles; and analyzing the released nucleic acid molecules. In some embodiments, the tracer particles can operate continuously for at least 720 hours in a fluid at 120°C. According to some embodiments, the tracer particles can operate continuously for at least 720 hours in a fluid with a pH value of 1 to 13.

[0069] In some embodiments, hydrofluoric acid can be used to remove the shell of the tracer particles, so that the nucleic acid molecules are released and desorbed from the tracer particles. In some embodiments, the concentration range of the hydrofluoric acid aqueous solution (HF / NH4F) used is about 0.5 (v / v)% to about 3.0 (v / v)%, for example, about 1.5 (v / v)%. In some embodiments, the released nucleic acid molecules can be analyzed by real-time polymerase chain reaction (q-PCR) to confirm the presence of the designed specific nucleic acid molecules and their concentration.

[0070] Next, please refer to Figure 3 , Figure 3 A flowchart of the steps of the preparation method 10M of the tracer particles according to some embodiments of the present invention is shown. It should be understood that in some embodiments, additional operation steps may be provided before, during and / or after the preparation method of the tracer particles. In some embodiments, some of the operations described may be replaced or deleted as needed. In some embodiments, the order of the operations / steps may be interchangeable. In addition, reference may be made to Figure 2 The structure of the tracer particle 20 is shown to understand the following description of the preparation method.

[0071] like Figure 3As shown, in some embodiments, the preparation method 10M of the tracer particle may include forming a core structure 102 (step S12), fixing the nucleic acid molecule 104 on the core structure 102 (step S14), and forming a shell layer 106 on the core structure 102 (step S16) to cover the core structure 102 and the nucleic acid molecule 104. In detail, in some embodiments, the step of forming the core structure 102 may further include providing an oil phase solution, providing an aqueous phase solution, and adding the oil phase solution to the aqueous phase solution to form a mixed solution. The oil phase solution may include a silicon precursor and an emulsifier. In some embodiments, the silicon precursor may include tetraethoxysilane (TEOS). In some embodiments, the emulsifier may include at least one of a C2-C10 short chain alcohol and a non-ionic surfactant. In some embodiments, the C2-C10 short chain alcohol may include isopropanol. In some embodiments, the ratio (volume ratio) of the silicon precursor to the co-emulsifier is between about 5:1 and about 1:10, or between about 1:1 and about 1:10, for example, about 1:1.

[0072] In some embodiments, the oil phase solution may further include a solvent. In some embodiments, the solvent may include at least one of C6-C18 medium-chain alkanes, C6-C18 medium-chain esters, and toluene. In some embodiments, the C6-C18 medium-chain alkanes may include octane. In some embodiments, the ratio (volume ratio) of the silicon precursor and the solvent in the oil phase solution is between about 1:1 and about 1:15, or between about 1:3 and about 1:10, for example, about 1:7.

[0073] In some embodiments, the ratio (volume ratio) of the silicon precursor, the co-emulsifier, and the solvent in the oil phase solution is between about 3:1:1 and about 15:1:1, or between about 5:1:1 and about 10:1:1, for example, about 7:1:1. It should be understood that the ratio of the silicon precursor, the co-emulsifier, and the solvent needs to be controlled within a specific range so that the formed tracer particles have good homogeneity, that is, have a uniform shape, size, or particle diameter.

[0074] Furthermore, the aqueous phase solution may include water and a surfactant. In some embodiments, the surfactant may include at least one of an organic ammonium salt, an alkyl sulfate, and a fatty acid salt. In some embodiments, the organic ammonium salt may include hexadecyl trimethyl ammonium bromide (CTAB). In some embodiments, the ratio of water to the surfactant in the aqueous phase solution is between about 1:1 and about 10:1, or between about 10:1 and about 30:1.

[0075] In some embodiments, the aqueous solution may be heated to a temperature of about 50° C. to about 80° C., or about 55° C. to about 70° C., such as about 60° C., to dissolve the surfactant in water, and then the oil phase solution is added to the aqueous solution.

[0076] In addition, in some embodiments, the step of forming the core structure 102 may further include adding a catalyst to the mixed solution and heating the mixed solution. In some embodiments, the catalyst comprises an alkaline solution. In some embodiments, the pH value of the catalyst ranges from about 8 to about 14. In some embodiments, the catalyst may include at least one of ammonia, sodium hydroxide, calcium hydroxide, potassium hydroxide, and an alkaline liquid.

[0077] In some embodiments, the temperature range of heating the mixed solution may be about 50° C. to about 80° C., or about 55° C. to about 70° C., for example, about 60° C. Furthermore, in some embodiments, the heating time may be about 2 hours to about 4 hours, for example, about 3 hours. In some embodiments, after heating the mixed solution, the mixed solution may be allowed to stand overnight at room temperature, then centrifuged to remove the supernatant, and ultrasonically extracted with ethanol to obtain the core structure 102 (porous carrier of tracer particles).

[0078] In some embodiments, after the step of heating the mixed solution, a step of adding a surface modifier to the mixed solution may be further included to enhance the dispersibility of the porous carrier. In some embodiments, the surface modifier may include a chlorine-containing quaternary ammonium salt, for example, trimethoxy[3-(methylamino)propyl]silane (TMAPS). In detail, in some embodiments, the core structure 102 obtained in the aforementioned step S12 may be dissolved in an emulsifier (e.g., isopropanol), followed by adding a surface modifier and shaking and centrifuging, after which the supernatant is removed and the product is dissolved in water to achieve dispersion.

[0079] Next, in some embodiments, the core structure 102 obtained in the above steps may be mixed with the nucleic acid molecule 104 and shaken and then centrifuged to fix the nucleic acid molecule 104 on the core structure 102 (step S14). As mentioned above, the nucleic acid molecule 104 may include at least one of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and peptide nucleic acid (PNA). In some embodiments, the length of the nucleic acid molecule 104 ranges from about 10 base pairs to about 2000 base pairs. In some embodiments, the nucleic acid molecule 104 may be a plasmid, and its length ranges from about 1500 base pairs to about 10000 base pairs.

[0080] In some embodiments where the length of the nucleic acid molecule 104 ranges from about 10 base pairs to about 2000 base pairs, the ratio (volume ratio) of the core structure 102 to the nucleic acid molecule 104 is between about 1:1 to about 10:1, or between about 2:1 to about 8:1. In some embodiments where the length of the nucleic acid molecule 104 ranges from about 1500 base pairs to about 10000 base pairs, the ratio (volume ratio) of the core structure 102 to the nucleic acid molecule 104 is between about 1:10000 to about 1:1000, or between about 1:100 to about 1:1000.

[0081] In some embodiments, the core structure 102 fixed with the nucleic acid molecule 104 can be added to an alcohol mixed solution to proceed with the subsequent step of forming the shell layer 106. In some embodiments, the alcohol mixed solution can include glycerol, ethanol, and water. In some embodiments, the ratio (volume ratio) of glycerol, ethanol, and water is between about 100:100:1 and about 300:300:1, or between about 100:100:1 and about 200:200:1.

[0082] Next, a shell layer 106 may be formed on the core structure 102 to which the nucleic acid molecule 104 is fixed. In some embodiments, the step of forming the shell layer 106 on the core structure 102 (step S16) may include mixing and shaking the core structure 102 to which the nucleic acid molecule 104 is fixed with a silicon precursor and a surface modifier. In some embodiments, the silicon precursor and the surface modifier may be added twice and shaken twice.

[0083] In some embodiments, the silicon precursor may include tetraethoxysilane (TEOS). In some embodiments, the surface modifier may include a chlorine-containing quaternary ammonium salt, for example, trimethoxy [3-(methylamino)propyl] silane (TMAPS).

[0084] In some embodiments, the ratio (volume ratio) of the core structure 102 immobilized with nucleic acid molecules, the silicon precursor, and the surface modifier is between 1:1:1 and 100:50:1, or between 10:1:1 and 50:5:1.

[0085] In order to make the above and other purposes, features, and advantages of the present invention more obvious and easy to understand, a number of embodiments, comparative embodiments, preparation examples, and test examples are specifically cited below for detailed description as follows, but they are not intended to limit the contents of the present invention.

[0086] Example 1: Design of specific nucleic acid molecules

[0087] Sequence Design of Hybrid DNA

[0088] A 50 bp fragment of 16S rDNA of Tepidimonas fonticaldi (strain AT-A2) and a 50 bp fragment of 18S rDNA of Chlamydomonas reinhardtii (strain CC-621) were selected and concatenated in the form of 25 bp (16S rDNA)-25 bp (18S rDNA)-25 bp (16S rDNA)-25 bp (18S rDNA) to synthesize a 100 bp hybrid DNA sequence (sequence identification number: 1) with higher temperature resistance than general DNA sequences as a specific nucleic acid molecule.

[0089] Confirmation of uniqueness of heterozygous DNA sequences

[0090] The BLAST (Basic Local Alignment Search Tool) system of the National Center for Biotechnology Information (NCBI) was used to compare and confirm the uniqueness of the synthesized hybrid DNA sequence. The comparison result showed zero correlation (no significant similarity), indicating that there was no similar DNA sequence in the database, proving that the designed hybrid DNA sequence was unique.

[0091] Example 2: Preparation of unique nucleic acid molecules

[0092] The gene synthesis company Integrated DNA Technologies was commissioned to synthesize the DNA sequence of sequence identification number: 1 (100 bp). Based on the sequence of sequence identification number: 1, a pair of primers of sequence identification number: 2 and sequence identification number: 3 were designed. The DNA sequence of sequence identification number: 1 was used as a template, and the DNA sequences of sequence identification number: 2 and 3 were used as primers at the 3-end and 5-end (melting temperatures Tm were 59°C and 63°C, respectively). The PCR procedure was performed to amplify the DNA fragment of sequence identification number: 1 to produce enough specific nucleic acid molecules for the subsequent nucleic acid molecule immobilization step.

[0093] The materials used in the PCR program are as follows: 10 ng (1 μl) of template, 2 μl of 3-terminal and 5-terminal primers (10 μM), 25 μl of 2X Taq MasterMix and 20 μl of ddH2O, and the total reaction volume is 50 μl. The temperature conditions of the PCR amplification reaction are set as follows: 95°C for 1 minute → [95°C for 1 minute → 55°C for 30 seconds → 72°C for 9 seconds] for 12 cycles → 72°C for 1 minute → 12°C for a stay.

[0094] The PCR products were analyzed by gel electrophoresis to confirm whether they were the designed specific nucleic acid molecules. The materials used in the gel electrophoresis procedure were as follows: 2.5% agarose, 10X TBE buffer (Tris-borate-EDTA), 1kb DNA ladder (as a marker (M)) (CLUBIO) and 6X Loading dye (CLUBIO), using the DNA electrophoresis system Mupid-2plus (Mupid). The gel electrophoresis results are shown in Figure 2. Figure 4 As shown in the figure, A to C are all PCR products, M is a 1kb DNA ladder marker, Figure 4 It can be seen that the length of the PCR amplified product is 100 bp, which is consistent with the length of the DNA sequence identification number: 1.

[0095] Next, the electrophoresis gel was purified using a Gel / PCR extraction Kit (Biomate) to remove the unacted dNTPs and primers during the PCR process to avoid affecting the subsequent DNA immobilization step. After the purification step, a DNA product with sequence identification number: 1 was obtained.

[0096] Example 3: Preparation of plasmids containing unique nucleic acid molecules

[0097] Use T&A selection vector ( The target specific sequence (sequence identification number: 1) was cloned (cloning), and the recombinant plasmid (length of about 3kb) was introduced into the host Escherichia coli (DH5α). The Escherichia coli was then cultured in a fermentation tank, and the cells were obtained by centrifugation. The plasmid containing the desired target specific sequence was then extracted from the cells by alkaline lysis.

[0098] The plasmid was cut with restriction enzymes EcoRⅠ and HindⅢ, and analyzed by gel electrophoresis to confirm whether the obtained plasmid length was correct (about 3kb). The materials used in the gel electrophoresis procedure were as follows: 1.5% agarose, 0.5XTAE buffer (Tris-Acetate-EDTA), 1kb DNA ladder (as a marker (M)) (CLUBIO) and 6X Loading dye (CLUBIO), using the DNA electrophoresis system Mupid-2plus (Mupid). The results of gel electrophoresis are shown in the figure. Figure 5A As shown in the figure, A is a plasmid without restriction enzyme cutting, B is a plasmid cut by EcoRⅠ, C is a plasmid cut by HindⅢ, M is a marker 1kb DNA ladder, Figure 5A It can be seen that the length of the plasmid obtained by culturing E. coli is about 3 kb, which is consistent with the length of the originally constructed plasmid.

[0099] Next, the obtained plasmid was used as a template, and the DNA sequence with sequence identification number: 2 and 3 was used as the primers at the 3-end and 5-end to perform the PCR procedure. The materials used in the PCR procedure were as follows: 10ng (1μl) of template, 2μl of 3-end and 5-end primers (10μM), 25μl of 2X Taq MasterMix and 20μl of ddH2O, and the total reaction volume was 50μl. The temperature conditions of the PCR amplification reaction were set as follows: 95℃ reaction for 5 minutes → [95℃ reaction for 30 seconds → 60.7℃ reaction for 30 seconds → 72℃ reaction for 10 seconds] for 29 cycles → 72℃ reaction for 5 minutes → 4℃ stay.

[0100] The PCR product was analyzed by gel electrophoresis to confirm whether it was the target specific sequence (sequence identification number: 1). The materials used in the gel electrophoresis procedure were as follows: 2.5% agarose, 10X TBE buffer (Tris-borate-EDTA), 1kb DNA ladder (as a marker (M)) (CLUBIO) and 6X Loading dye (CLUBIO), using the DNA electrophoresis system Mupid-2plus (Mupid). The gel electrophoresis results are shown in Figure 1. Figure 5B As shown in the figure, A to D are all PCR products, M is a marker 1kb DNA ladder, Figure 5B It can be seen that the length of the PCR amplified product is about 100 bp, which is consistent with the DNA length of sequence identification number: 1.

[0101] Test Example 1: Risk Assessment of Synthetic Specific Nucleic Acid Molecules to the Environment and Human Body

[0102] The environmental strain toxicity test was used to test whether the synthesized DNA sequence with sequence identification number: 1 has an inhibitory effect on microorganisms in the environment. Figures 6A to 6D As shown, Fig. 6A , 6B and 6C respectively show the toxicity test results of the synthesized DNA sequence to E.coli, Bacillus cereus and Pseudomonas putida, Fig.6D The above results show that the synthesized DNA sequence with sequence identification number: 1 will not inhibit common microorganisms in the environment.

[0103] Furthermore, the sequence of sequence identification number: 1 is compared with the sequence of human chromosome (Human G+T). The comparison result shows that E-value>>1 (numerical value), and the similarity is zero (if E-value<10 -5 In addition, the sequence of sequence identification number: 1 was further split into four fragments of 25 bp each for comparison. The comparison results showed that E-value>1, the similarity was extremely low, and the risk of replacing human genes was also close to zero.

[0104] Example 4: Preparation of tracer particles A

[0105] Preparation and surface modification of core structures

[0106] Take corn starch and deionized water to prepare a 35% starch suspension, and stir it at 35°C. Use 0.5N NaOH to adjust the pH value of the starch solution to 9.5. Then, take 20g of sodium hypochlorite and slowly add it to the starch solution (addition time is greater than 30 minutes), and use 1N HCl to maintain the pH value of the starch solution at 9.5. After the addition of sodium hypochlorite, continue stirring for 50 minutes, and use 0.5N NaOH to maintain the pH value of the starch solution at 9.5. After the reaction is completed, use 1N HCl to adjust the pH value of the starch solution to 7, wash it with secondary water and alcohol, then filter it with vacuum, and dry it in an oven at 50°C to obtain modified starch.

[0107] Take 22 mL of tetraethoxysilane (TEOS), add it to 36 mL of deionized water and 1 mL of 2% HCl, and stir until the hydrolysis becomes transparent. Then, after adding 3 g of the modified starch mentioned above, slowly drip 20 mL of 5% NH4OH with a separatory funnel and stir for 40 minutes. Filter out the solid and put it in a 50°C oven to dry for 24 hours. After that, calcine the solid at a high temperature of 550°C and keep the temperature for 3 hours. In this way, a porous carrier (core structure) of the tracer particles can be obtained.

[0108] Take 2 g of the aforementioned porous carrier, add it to 20 mL of isopropanol, and evenly disperse it. Then, add 0.889 mL of N-methyl-3-aminopropyl trimethoxy (trimethoxy [3- (methylamino) propyl] silane, TMAPS) and 1 mL of deionized water, and stir at 40 ° C for 2 hours. Then, centrifuge at 15275 RCF (relative centrifugal force) for 10 minutes, remove the supernatant, and disperse the solid in 40 mL of deionized water. In this way, a surface-modified porous carrier can be obtained.

[0109] Immobilization of nucleic acid molecules

[0110] Take 35 μL of the surface-modified porous carrier and add it to 10 μL of the DNA product of the aforementioned sequence identification number: 1 (300 ppm) (or 1300 ppm), shake it with a shaker, and centrifuge it at 18000 RCF for 10 minutes. Then, take out the supernatant and wash it several times with secondary water, and disperse the solid in 500 μL of secondary water.

[0111] Shell Packaging

[0112] Next, 0.6 μL of N-methyl-3-aminopropyl trimethoxy (trimethoxy [3- (methylamino) propyl] silane, TMAPS) and 0.6 μL of TEOS were added, and the mixture was shaken at 900 rpm for 4 hours. After that, 4 μL of TEOS was added, and the mixture was shaken at 900 rpm for 96 hours. Next, the mixture was centrifuged at 19375 RCF for 10 minutes, the supernatant was removed, and the mixture was washed several times with secondary water, so that the solid was dispersed in 45 μL of secondary water. At this point, the tracer particle A was completed.

[0113] Fig. 7A and 7B The scanning electron microscope (SEM) observations of the porous carrier before and after the encapsulation process of the shell layer are shown. From the SEM analysis results, it can be observed that the particle size of the tracer particles before the encapsulation process is about 40nm to 50nm (e.g. Fig. 7A After the packaging process, it increases to about 60nm to 75nm (as shown in Figure 7B shown).

[0114] Example 5: Preparation of tracer particles B

[0115] Preparation and surface modification of core structures

[0116] Take 2g of hexadecyl trimethyl ammonium bromide (CTAB) and 30ml of secondary water in a serum bottle to prepare an aqueous solution, and heat it to 60°C to dissolve it. In addition, take 7.2ml of octane, 1ml of tetraethoxysilane (TEOS) and 1ml of isopropanol to prepare an oil solution, and add the oil solution to the aqueous solution with a dropper. Then add 0.022ml of 25% ammonia water and react at 60°C for 3 hours. After the reaction is completed, let it stand overnight at room temperature. After that, centrifuge the mixed solution after the reaction is completed and remove the supernatant. Use ethanol for ultrasonic vibration extraction to replace the oil solution. Here, a porous carrier (core structure) of the tracer particles can be obtained.

[0117] After centrifugation, the porous carrier was added to 20 mL of isopropanol and evenly dispersed. Then, 0.222 mL of N-methyl-3-aminopropyltrimethoxyethane (TMAPS) was added and shaken for 18 hours using a shaker. The mixture was centrifuged at 15275 RCF (relative centrifugal force) for 10 minutes, the supernatant was removed, and the solid was dispersed in 20 mL of secondary water. Thus, a surface-modified porous carrier was obtained.

[0118] Immobilization of nucleic acid molecules

[0119] Take 350 μL of the surface-modified porous carrier and add it to the plasmid product (100 μL) containing sequence identification number: 1 prepared in the above Example 3. After centrifugation at 15275 RCF for 10 minutes, remove the supernatant and dissolve the solid in 5 ml of alcohol mixture (glycerol: ethanol: water = 150:150:1).

[0120] Shell Packaging

[0121] Next, add 6 μl of TMAPS and 6 μl of TEOS, shake for 4 hours using a shaker, then add 40 μl of TEOS and shake for 4 days using a shaker. Then, add 24 μl of TMAPS and shake for 18 hours using a shaker, and replace the solution with secondary water. Thus, tracer particle B is completed.

[0122] Fig. 8A and 8B The scanning electron microscope (SEM) observations of the porous carrier before and after the encapsulation process of the shell layer are shown respectively. From the SEM analysis results, it can be observed that the particle size of the tracer particles before the encapsulation process is about 30nm to 40nm (e.g. Fig. 8AAfter the packaging process, it increases to about 50nm to 60nm (as shown in Figure 8B shown).

[0123] Comparative Example 1: Preparation of tracer particles C

[0124] The preparation method of tracer particle C is substantially similar to that of tracer particle A in Example 4, but tracer particle C is not encapsulated with a shell layer, that is, the DNA of tracer particle C is naked.

[0125] Comparative Example 2: Preparation of tracer particles D

[0126] The preparation method of tracer particles D is roughly based on Method (1968), and the research results of Kim et al. (TGKim et al., 2017). First, 50 ml of 95% alcohol and 60 ml of secondary aqueous solution were prepared and stirred at a fixed speed of 450 rpm for 15 minutes, and then 20 ml of TEOS was added and mixed for hydrolysis for 30 minutes. Finally, 6 ml of 25% ammonia water was added and stirred for 2 hours to polymerize. After the reaction, the liquid was centrifuged at 15275 RCF for 15 minutes, and the supernatant was removed. The solid was then washed with 95% alcohol for 3 times and placed in a 50°C oven for drying. Compared with the porous carrier of tracer particle A, the carrier of tracer particle D is a dense solid carrier with a porosity close to 0.

[0127] Example 6: Desorption process of nucleic acid molecules

[0128] Hydrofluoric acid (HF) was used to remove the protective shell of DNA and desorb DNA. 10 μL of the encapsulated tracer particles were added to 40 μL of 1.5% HF / NH4F aqueous solution and mixed by shaking for about 5 minutes. Then, the desorbed DNA was recovered using a DNA purification kit (Bioman Scientific).

[0129] Example 7: Analysis of recovered nucleic acid molecules

[0130] Real-time polymerase chain reaction (q-PCR) analysis was used to quantitatively analyze the recovered DNA to confirm whether it contained the designed specific DNA sequence and to measure its concentration (quality) at the same time. The materials used in the q-PCR program were as follows: 1 μl of template (DNA recovered by desorption), 0.75 μl of 3-terminal primer (10 μM) (sequence identification number: 2), 0.75 μl of 5-terminal primer (10 μM) (sequence identification number: 3), 12.5 μl of 2X SYBR Green Master Mitrix (Thermo Fisher Scientific) and 10 μl of ddH2O, with a total reaction volume of 25 μl. The temperature conditions for the q-PCR amplification reaction were set as follows: 95°C for 10 minutes → [95°C for 15 seconds → 60°C for 9 seconds] for 40 cycles → 12°C for retention.

[0131] Test Example 2: Temperature resistance test of tracer particles

[0132] The tracer particles A prepared in Example 4 and the tracer particles C prepared in Comparative Example 1 were placed in an oil bath at 25° C., 100° C., 120° C., 140° C., 160° C., 180° C. and 200° C. for heating for 20 minutes. Then, the tracer particles were taken out, and the DNA remaining on them was recovered and analyzed by colloid electrophoresis.

[0133] The results are as follows Fig. 9A and 9B As shown, Fig. 9A and 9B The temperature resistance test results of the tracer particles prepared in Comparative Example 1 and Example 4 are shown respectively. 25, 100, 120, 140, 160, 180 and 200 in the figure represent the heating temperature, and M is the marker (1 kb DNA ladder). Fig. 9A , Fig. 9B The results show that the high temperature tolerance of the tracer particle C (naked DNA) of Comparative Example 1 can reach about 100°C, while the high temperature tolerance of the tracer particle A (encapsulated DNA) of Example 4 can reach about 200°C.

[0134] In addition, the temperature resistance of the tracer particles A of Example 4 at 120° C. was further tested. Specifically, the recovery rate of DNA was measured at different time points, and the results are shown in Table 1 below.

[0135] Table 1

[0136]

[0137]

[0138] From the results in Table 1, it can be seen that the DNA recovery rate of the encapsulated tracer particles A can still be maintained at 76.5% after being heated for 5 hours.

[0139] In addition, the temperature resistance of the tracer particles B in Example 5 at 120° C. was further tested. The recovery rate of DNA was measured at different time points, and the results are shown in Table 2 below.

[0140] Table 2

[0141] Time (hours) Recovery rate (%) 0.0 100 3 95.2 6.0 88.1 10.0 86.3 24.0 81.5

[0142] From the results in Table 2, it can be seen that the DNA recovery rate of the encapsulated tracer particles B can still be maintained at 81.5% after being heated for 24 hours.

[0143] Test Example 3: Comparison of the temperature resistance of tracer particles

[0144] The tracer particles A prepared in the above-mentioned Example 4 and the tracer particles D prepared in the comparative example 2 were placed in an oil bath at 120° C. for heating. Then, after heating for 1, 2, and 2.5 hours, the tracer particles were taken out and the DNA remaining thereon was recovered. The residual DNA content was measured and the residual rate was calculated. The results are shown in Table 3 below.

[0145] Table 3

[0146]

[0147] From the results in Table 3, it can be seen that after heating at 120°C for 2.5 hours, the DNA residual rate of the tracer particle A prepared in Example 4 is 79.4%, while the DNA residual rate of the tracer particle D prepared in Comparative Example 2 is 52.4%. It can be seen that compared with the tracer particle D with a solid carrier (without pores), the tracer particle A with a porous carrier structure has better high temperature tolerance.

[0148] Test Example 4: Acid and alkali resistance test of tracer particles

[0149] The tracer particles A prepared in the above-mentioned Example 4 were placed in solutions with different pH values ​​to test the tolerance of the structure of the tracer particles A in acidic and alkaline environments. The acidic solution and the alkaline solvent were prepared with sulfuric acid and an acidic solution, respectively. The tracer particles were placed in solutions of pH 1, pH 3, pH 5, pH 7, pH 9 and pH 13 for 60 minutes, and then the tracer particles were taken out, the DNA remaining thereon was recovered, and colloidal electrophoresis analysis was performed.

[0150] The results are as follows Fig.10 As shown in the figure, M is a marker (1kb DNA ladder), Fig.10The results show that the DNA content of tracer particle A is almost not reduced in the range below pH 9, and is not affected by pH changes, and is not significantly reduced in the range above pH 9. The above results show that the tracer particle A of Example 4 has the ability to resist strong acids and strong bases.

[0151] In addition, the tracer particles B of Example 5 were also tested for acid and alkali resistance. The tracer particles B were placed in solutions with different pH values ​​to test the tolerance of the structure of the tracer particles B in acidic and alkaline environments. The acidic solution and alkaline solvent were prepared with sulfuric acid and acidic solution, respectively. The tracer particles were placed in solutions of pH 1, pH 3, pH 5, pH 7, pH 9 and pH 13 for 24 hours. Then, the tracer particles were taken out and the DNA remaining thereon was recovered. The residual DNA content was measured and the residual rate was calculated. The results are shown in FIG11 .

[0152] Depend on Fig.11 The results show that within 1 hour, the DNA amount of tracer particle B in the range below pH 9 hardly decreased (the lines of pH 3, pH 5 and pH 7 overlap in the figure), and was not affected by the change in pH, and did not decrease significantly in the range above pH 9, indicating that tracer particle B has the ability to resist strong acids and strong bases. It is worth noting that after 24 hours of reaction, the DNA amount of tracer particle B in the range below pH 9 did not decrease significantly, indicating that it has the ability to resist strong acids for a long time.

[0153] Test Example 5: Adding tracer particles to the field hot water temperature resistance test

[0154] The plasmid with the target DNA tag constructed was fixed on the nanoporous carrier prepared in the above-mentioned Example 5, and then the tracer particle B was produced after the packaging process. The tracer particle B was placed in a small reaction tank, and the field hot water was added inside. The field hot water was geothermal water from the volcanic geothermal area (China Datun Mountain geothermal water, pH1.5, total dissolved solids in water ~9200ppm) and geothermal water from the metamorphic rock geothermal area (China Renze geothermal water pH8.8, total dissolved solids in water ~4000ppm). Then, each was placed in a 120°C oil bath for heating. Then, after heating for 480 and 720 hours respectively, the tracer particles were taken out and the residual DNA on them was recovered, the residual DNA content was measured, and the residual rate was calculated. The results are shown in Table 4 below.

[0155] Table 4

[0156]

[0157] From the results in Table 4, it can be seen that after heating at 120°C for 720 hours, the DNA residual rate of tracer particles E in the acidic environment is 10.7%, while the DNA residual rate of tracer particles E in the weakly alkaline environment is 7.5%. Therefore, it can be seen that tracer particles E have the feasibility of preliminary application in geothermal field.

[0158] Test Example 6: Column Tracer Test of Tracer Particles

[0159] In order to simulate the application of tracer particles in the actual field (in soil or rock formation), a quartz sand column with a diameter of 0.8 cm and a length of 10.7 cm was prepared by filling quartz sand (0.84 mm) with a No. 20 sieve into a glass column with a diameter of 0.8 cm and a length of 10.7 cm. In general, the hydraulic conductivity of geothermal fluid channels (fractures) is about 10 -7 ~10 -2 m / sec, the water conductivity of the prepared quartz sand column is about 3.4*10 -5 Next, the tracer particles A prepared in the above-mentioned Example 4 were placed in water and passed into the quartz sand column at a flow rate of 0.1 ml / min, and the samples flowing out of the column were collected.

[0160] The purpose of the column tracer test is to investigate the effect of time relative to the degree of diffusion on the recovery of tracer particles. Fig.12 The results show the DNA content of the recovered tracer particles A over time. From the curve change of the DNA content, it can be seen that after the tracer particles A are injected once, a small part of the tracer particles A flows out of the shortest channel through advection, while most of the tracer particles A flow out of the channel by dispersion and diffusion due to the uneven distribution of the flow field (most of them flow out within 140 minutes). The above transmission behavior is consistent with the common tracer test results. In addition, it has been measured that the recovery rate of the tracer particles A can reach 97.5%, indicating that the tracer particles of this embodiment will not be adsorbed by quartz sand and can flow freely in the low-conductivity fluid channel.

[0161] In summary, according to some embodiments of the present invention, the tracer particles include specific nucleic acid molecules as calibration objects (labels, fingerprints), use inorganic materials as substrates and packaging materials, and use a core structure with moderate porosity to increase the amount of nucleic acid molecules fixed, and reduce the thermal conductivity of the particles (reduce thermal resistance), thereby improving the temperature tolerance of the tracer particles. In addition, the tracer particles also have acid and alkali resistance, which can further improve the tolerance and recovery rate of the tracer particles in extreme environments.

[0162] Although the embodiments of the present invention and their advantages have been disclosed as above, it should be understood that any person with common knowledge in the art can make changes, substitutions and modifications without departing from the spirit and scope of the present invention. In addition, the scope of protection of the present invention is not limited to the processes, machines, manufacturing, material compositions, devices, methods and steps in the specific embodiments described in the specification. Any person with common knowledge in the art can understand the current or future developed processes, machines, manufacturing, material compositions, devices, methods and steps from the disclosure of the present invention, as long as they can implement substantially the same functions or obtain substantially the same results in the embodiments described herein, they can all be used according to the present invention. Therefore, the scope of protection of the present invention includes the aforementioned processes, machines, manufacturing, material compositions, devices, methods and steps. In addition, each claim constitutes a separate embodiment, and the scope of protection of the present invention also includes the combination of each claim and embodiment. The scope of protection of the present invention shall be subject to the scope of protection defined by the attached claims. Sequence Listing <110> Industrial Technology Research Institute <120> Tracer particles and their application and preparation methods <160> 3 <210> 1 <211> 100 <212> DNA <213> Artificial sequence <220> <223> Artificially synthesized sequences <400> 1 taacgggtga cggaggatta gggttgctaa tacctggggc tgatgacggc gattccggag 60 agggagtaac ctgagagtac cgtaagaagc accggctaac 100 <210> 2 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Artificially synthesized sequences <400> 2 taacgggtga cggaggatt 19 <210> 3 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Artificially synthesized sequences <400> 3 accgtaagaa gcaccggcta ac 22

Claims

1. A tracer particle, comprising: A core structure; a nucleic acid molecule fixed to the core structure; as well as a shell layer, covering the core structure and the nucleic acid molecule; The core structure has a first porosity, the shell layer has a second porosity, the first porosity is greater than the second porosity, and the second porosity is substantially 0, and the first porosity ranges from 2 nm to 100 nm.

2. The tracer particle according to claim 1, wherein the material of the core structure and the shell layer comprises at least one of silicon dioxide, silicate, carbonate, nano-gold, metal oxide, polyethylene glycol, polystyrene and polylactic acid. The tracer particle according to claim 1 , wherein the shell layer is a single-layer structure or a multi-layer structure. The tracer particle according to claim 1 , wherein the particle size of the tracer particle ranges from 30 nm to 10000 nm. The tracer particle according to claim 1 , wherein the particle size of the core structure ranges from 20 nm to 9000 nm. The tracer particle according to claim 1 , wherein the thickness of the shell layer ranges from 10 nm to 5000 nm. 7 . The tracer particle according to claim 1 , wherein the core structure comprises a plurality of holes, wherein the nucleic acid molecule is fixed in the plurality of holes. The tracer particle according to claim 1 , wherein the length of the nucleic acid molecule ranges from 1500 base pairs to 10000 base pairs.

9. The tracer particle according to claim 1, wherein the nucleic acid molecule has at least 85% sequence similarity to the sequence shown in SEQ ID NO:

1. 10 . The tracer particle according to claim 1 , wherein the nucleic acid molecule comprises the sequences shown in SEQ ID NO: 2 and SEQ ID NO:

3. The tracer particle according to claim 1 , wherein the length of the nucleic acid molecule ranges from 10 base pairs to 2000 base pairs.

12. A method for applying tracer particles, comprising: Providing the tracer particle according to claim 1; Placing the tracer particle in a fluid to be observed; collecting a sample of the fluid, recovering the tracer particle from the sample, and releasing the nucleic acid molecule from the tracer particle; as well as The released nucleic acid molecules are analyzed.

13. The application method of the tracer particles according to claim 12 is used for fluid tracking in geothermal or oil wells.

14. The method for applying tracer particles according to claim 12, wherein the tracer particles can be operated in a fluid at 120°C for at least 5 hours. 15 . The method for applying tracer particles according to claim 12 , wherein the tracer particles can be operated in a fluid with a pH value of 1 to 13 for at least 60 minutes. 16 . The method for applying tracer particles according to claim 12 , wherein the step of releasing the nucleic acid molecule from the tracer particle comprises using a hydrofluoric acid aqueous solution with a concentration ranging from 0.5 (v / v) % to 3.0 (v / v) %. 17 . The method for using tracer particles according to claim 12 , wherein the step of analyzing the released nucleic acid molecules comprises performing a real-time polymerase chain reaction.

18. A method for preparing the tracer particles according to claim 1, comprising: Forming the core structure, including: Providing an oil phase solution, which includes a silicon precursor and an emulsifier; Providing an aqueous solution comprising water and a surfactant; adding the oil phase solution to the water phase solution to form a mixed solution; adding a catalyst to the mixed solution; and heating the mixed solution; immobilizing a nucleic acid molecule on the core structure; and A shell layer is formed on the core structure to cover the core structure and the nucleic acid molecule. 19 . The method for preparing tracer particles according to claim 18 , wherein the ratio of the silicon precursor to the co-emulsifier is between 5:1 and 1:

10. 20 . The method for preparing tracer particles according to claim 18 , wherein the silicon precursor comprises tetraethoxysilane.

21. The method for preparing tracer particles according to claim 18, wherein the co-emulsifier comprises at least one of a C2-C10 short-chain alcohol and a non-ionic surfactant.

22. The method for preparing tracer particles according to claim 18, wherein the oil phase solution further comprises a solvent, which comprises at least one of C6-C18 medium-chain alkanes, C6-C18 medium-chain esters and toluene. 23 . The method for preparing tracer particles according to claim 22 , wherein the ratio of the silicon precursor to the solvent is between 1:1 and 1:

15.

24. The method for preparing tracer particles according to claim 18, wherein the surfactant comprises at least one of organic ammonium salts, alkyl sulfates and fatty acid salts.

25. The method for preparing tracer particles according to claim 18, wherein the catalyst comprises an alkaline solution. 26 . The method for preparing tracer particles according to claim 18 , further comprising adding a surface modifier into the mixed solution after the step of heating the mixed solution.

27. The method for preparing tracer particles according to claim 18, wherein the nucleic acid molecule comprises at least one of deoxyribonucleic acid, ribonucleic acid and peptide nucleic acid.

28. The method for preparing tracer particles according to claim 18, wherein the nucleic acid molecule comprises a plasmid.

29. The method for preparing the tracer particle according to claim 18, wherein the step of forming the shell layer on the core structure comprises: The core structure fixed by nucleic acid molecules is mixed with a silicon precursor and a surface modifier and shaken, wherein the ratio of the core structure fixed by nucleic acid molecules, the silicon precursor and the surface modifier is between 1:1:1 and 100:50:1.

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