阅读说明：本技术 示踪粒子及其应用方法与制备方法 (Tracer particle and application method and preparation method thereof ) 是由 范愷军 韩吟龙 高珮娟 林永洋 郑捷伦 黄坚昌 张永和 林佳龙 吴意珣 陈柏翰 于 2020-06-02 设计创作，主要内容包括：本发明提供一种示踪粒子及其应用方法与制备方法,该示踪粒子包括：一核心结构；一核酸分子,固定于该核心结构上；以及一壳层,包覆该核心结构及该核酸分子；其中该核心结构具有一第一孔隙度,该壳层具有一第二孔隙度,且该第一孔隙度大于该第二孔隙度。本发明的示踪粒子具有适中的孔隙度核心结构,增加了核酸分子的固定量,并降低粒子的热传导性(降低热阻),进而改善示踪粒子的温度耐受性。此外,本发明的示踪粒子亦具有耐酸及耐碱等性能,可进一步提高示踪粒子于极端环境中的耐受性及回收率。(The invention provides a tracer particle and an application method and a preparation method thereof, wherein the tracer particle comprises the following components: a core structure; a nucleic acid molecule immobilized on the core structure; and a shell layer, which coats the core structure and the nucleic acid molecule; 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 particles have a moderate porosity core structure, so that the fixing amount of nucleic acid molecules is increased, the thermal conductivity (thermal resistance) of the particles is reduced, and the temperature tolerance of the tracer particles is improved. In addition, the tracer particles also have the performances of acid resistance, alkali resistance and the like, and can further improve the tolerance and the recovery rate of the tracer particles in extreme environments.)

1. A tracer particle, comprising:

a core structure;

a nucleic acid molecule immobilized on the core structure; and

a shell layer, which coats the core structure and the nucleic acid molecule; the core structure has a first porosity, the shell layer has a second porosity, and the first porosity is greater than the second porosity.

2. The tracer particle of claim 1, wherein the material of the core structure and the shell layer comprises at least one of silica, silicate, carbonate, nanogold, metal oxide, polyethylene glycol, polystyrene, and polylactic acid.

3. The tracer particle of claim 1, wherein the first porosity ranges from 2nm to 100 nm.

4. The tracer particle of claim 1, wherein the second porosity is substantially 0.

5. The tracer particle of claim 1, wherein the second porosity ranges from 0.5nm to 10 nm.

6. The tracer particle of claim 1, wherein the shell layer is a single layer structure or a multi-layer structure.

7. A tracer particle according to claim 6, wherein the multi-layered structure comprises an outer shell layer and an inner shell layer, the inner shell layer comprising a plurality of pores.

8. The tracer particle of claim 1, wherein the tracer particle has a particle size in a range of 30nm to 10000 nm.

9. Tracer particle according to claim 1, wherein the particle size of the core structure ranges from 20nm to 9000 nm.

10. Tracer particle according to claim 1, wherein the thickness of the shell layer ranges from 10nm to 5000 nm.

11. The tracer particle of claim 1, wherein the core structure comprises a plurality of pores, wherein the nucleic acid molecule is immobilized within the plurality of pores.

12. Tracer particle according to claim 1, wherein the nucleic acid molecule has a length in the range of 1500 base pairs to 10000 base pairs.

13. The tracer particle of claim 1, wherein the nucleic acid molecule is substantially identical to the nucleic acid molecule of seq id no: 1 has at least 85% sequence similarity.

14. Tracer particle according to claim 1, wherein the nucleic acid molecule comprises a sequence identifier such as seq id no: 2 and 3.

15. Tracer particle according to claim 1, wherein the nucleic acid molecule has a length in the range of 10 base pairs to 2000 base pairs.

16. A method of using a tracer particle, comprising:

providing a tracer particle according to claim 1;

placing the trace 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 molecule.

17. The method of using tracer particles according to claim 16, which is for fluid tracing in geothermal or oil wells.

18. The method of using tracer particles of claim 16, wherein the tracer particles can be operated in a fluid at 120 ℃ for at least 5 hours.

19. The method of using tracer particles of claim 16, wherein the tracer particles are operable in a fluid having a pH of 1-13 for at least 60 minutes.

20. The method for using a tracer particle of claim 16, wherein the step of releasing the nucleic acid molecule from the tracer particle comprises using an aqueous hydrofluoric acid solution at a concentration ranging from 0.5 (v/v)% to 3.0 (v/v)%.

21. The method of claim 16, wherein the step of analyzing the released nucleic acid molecules comprises performing a real-time polymerase chain reaction.

22. A method of preparing tracer particles, comprising:

forming a core structure comprising:

providing an oil phase solution which comprises a silicon precursor and a co-emulsifier;

providing an aqueous solution comprising water and a surfactant;

adding the oil phase solution into the water phase solution to form a mixed solution;

adding a catalyst into the mixed solution; and

heating the mixed solution; immobilizing a nucleic acid molecule to the core structure; and

forming a shell layer on the core structure to coat the core structure and the nucleic acid molecule.

23. The method of claim 22, wherein the ratio of the silicon precursor to the co-emulsifier is between 5: 1 to 1: 10, respectively.

24. The method of claim 22, wherein the silicon precursor comprises tetraethoxysilane.

25. The method of claim 22, wherein the co-emulsifier comprises at least one of a C2-C10 short-chain alcohol and a nonionic surfactant.

26. The method of claim 22, wherein the oil phase solution further comprises a solvent comprising at least one of C6-C18 medium-long chain alkanes, C6-C18 medium-long chain esters, and toluene.

27. The method of claim 26, wherein the ratio of the silicon precursor to the solvent is between 1: 1 to 1: 15, respectively.

28. The method of claim 22, wherein the surfactant comprises at least one of an organic ammonium salt, an alkyl sulfate, and a fatty acid salt.

29. The method of claim 22, wherein the catalyst comprises an alkaline solution.

30. The method of claim 22, further comprising adding a surface modifier to the mixed solution after the step of heating the mixed solution.

31. The method of claim 22, wherein the nucleic acid molecule comprises at least one of deoxyribonucleic acid (, ribonucleic acid), and peptide nucleic acid.

32. A method of producing tracer particles according to claim 22, wherein the nucleic acid molecules comprise plasmids.

33. The method of claim 22, wherein the step of forming the shell layer on the core structure comprises:

mixing and oscillating the core structure fixed by nucleic acid molecules, a silicon precursor and a surface modifier, wherein the ratio of the core structure fixed by nucleic acid molecules, the silicon precursor and the surface modifier is 1: 1: 1 to 100: 50: 1.

Technical Field

The present invention relates to a tracer particle, and more particularly, to a tracer particle including a nucleic acid molecule, a method for using the same, and a method for preparing the tracer particle.

Background

The tracing technology can be used as a tool for monitoring fluid, pollution leakage, product tracking and the like, is widely applied to exploration of geothermal heat, natural gas and petroleum at present, can improve the extraction efficiency of the energy, and can also be used as a tracing tool for tracing underground water sources and treating environmental pollution.

The types of tracers commonly used at present include radioactive tracers, fluorescent tracers, chemical tracers, etc., but they are limited in types, complicated in analysis procedures and toxic and may cause harm to the environment. In view of this, the development of new tracers is receiving considerable attention. The biological tracer is a new generation of tracer, takes biological materials as main calibration objects (labels and fingerprints), has no toxicity, and is less prone to cause environmental pollution. However, the existing biological tracer has unsatisfactory ability of resisting extreme environments, and is difficult to be applied to extreme environments such as strong acid, strong alkali or high temperature.

In light of the foregoing, while the tracers currently available can generally meet their originally intended use, they have not been entirely satisfactory in every aspect.

Disclosure of Invention

According to some embodiments of the invention, there is provided a tracer particle comprising: a core structure; a nucleic acid molecule immobilized on the core structure; and a shell layer, which coats the core structure and the nucleic acid molecule; the core structure has a first porosity, the shell layer has a second porosity, and the first porosity is greater than the second porosity.

According to some embodiments of the present invention, there is provided a method of applying a trace particle, including: providing the aforementioned tracer particles; placing the trace 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 molecule.

According to some embodiments of the present invention, there is provided a method of preparing a tracer particle, including: forming a core structure; immobilizing a nucleic acid molecule to the core structure; and forming a shell layer on the core structure to coat the core structure and the nucleic acid molecule. Further, the step of forming the core structure includes: providing an oil phase solution which comprises a silicon precursor and a co-emulsifier; providing an aqueous solution comprising water and a surfactant; adding the oil phase solution into the water phase solution to form a mixed solution; adding a catalyst into the mixed solution; and heating the mixed solution.

The tracer particles have a moderate porosity core structure, so that the fixing amount of nucleic acid molecules is increased, the thermal conductivity (thermal resistance) of the particles is reduced, and the temperature tolerance of the tracer particles is improved. In addition, the tracer particles also have the performances of acid resistance, alkali resistance and the like, and can further improve the tolerance and the recovery rate of the tracer particles in extreme environments.

In order to make the features and advantages of the present invention comprehensible, preferred embodiments accompanied with figures are described in detail below.

Drawings

FIG. 1 shows a schematic view of a structure of a tracer particle according to some embodiments of the invention;

FIG. 2 shows a schematic view of a structure of a tracer particle according to some embodiments of the invention;

FIG. 3 shows a flow chart of steps of a method of making tracer particles according to some embodiments of the invention;

FIG. 4 shows a diagram of a colloidal electrophoretic analysis of a tag of a target nucleic acid molecule, according to some embodiments of the invention;

FIG. 5A shows a diagram of a colloidal electrophoretic analysis of a plasmid product containing a tag of a target nucleic acid molecule, according to some embodiments of the invention;

FIG. 5B shows a diagram of a colloidal electrophoretic analysis of a tag of a target nucleic acid molecule in a plasmid product, according to some embodiments of the invention;

FIGS. 6A-6D show sequence identifiers according to some embodiments of the present invention: 1 on the toxicity of the microorganism;

FIGS. 7A and 7B show Scanning Electron Microscope (SEM) views of a core structure before and after an encapsulation process of a shell, respectively, in accordance with some embodiments of the present invention;

FIGS. 8A and 8B are Scanning Electron Microscope (SEM) views of a core structure before and after an encapsulation process of a shell, respectively, in accordance with some embodiments of the present invention;

FIGS. 9A and 9B respectively show temperature resistance test results for trace particles according to some embodiments of the present disclosure;

FIG. 10 shows the results of acid and alkali resistance tests on tracer particles according to some embodiments of the invention;

FIG. 11 shows the results of acid and alkali resistance tests on tracer particles according to some embodiments of the invention;

FIG. 12 shows results of a tubular column trace test of trace particles according to some embodiments of the invention.

Wherein, the reference numbers:

10. 20 trace particles;

102 a core structure;

102p, 106p holes;

102s surface;

104 a nucleic acid molecule;

106 shell layers;

106a an outer shell layer;

106b inner shell layer;

10M preparation method;

s12, S14 and S16;

d1 and d2 particle size;

t thickness.

Detailed Description

The following description will be made in detail with respect to a tracer particle, a method of using the tracer particle, and a method of preparing the tracer particle according to an embodiment of the present invention. It is to be understood that the following description provides many different embodiments, or examples, for implementing different aspects of embodiments of the invention. The specific components and arrangements described below are simply for clarity and to describe some embodiments of the invention. These are, of course, merely examples and are not intended to be limiting. Moreover, similar and/or corresponding reference numerals may be used to identify similar and/or corresponding components in different embodiments to clearly illustrate the invention. However, the use of such like and/or corresponding reference numerals is merely for simplicity and clarity in describing some embodiments of the invention and does not represent any correlation between the various embodiments and/or structures discussed.

It should be understood that the components or devices of the drawings may exist in a variety of forms well known to those skilled in the art. Further, it will be understood that, although the terms first, second, third, etc. may be used herein to describe various elements, components, regions, layers or sections, these elements, components, regions, layers or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer or section from another. Thus, a first element, component, region, layer or section discussed below could be termed a second element, component, region, layer or section without departing from the teachings of the present invention.

As used herein, the terms "about", "approximately", "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 herein are approximate quantities, that is, the meanings of "about", "about" and "substantially" are implied unless otherwise specified.

The embodiments of the present invention can be understood together with the accompanying drawings, which are also to be considered part of the disclosure. It is to be understood that the drawings of the present invention are not to scale and that in fact any enlargement or reduction of the size of the components is possible in order to clearly show the features of the present invention.

Unless defined otherwise, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present invention and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

The embodiment of the invention provides a tracer particle, which comprises a specific nucleic acid molecule as a calibration object, and utilizes a core structure with larger porosity to increase the fixing strength of the nucleic acid molecule, improve the thermal conductivity of the particle and further improve the extreme environment resistance of the tracer particle.

Fig. 1 shows a schematic view of a structure of a tracer particle 10 according to some embodiments of the invention. It is to be understood that additional features may be added to the tracer particle 10 according to some embodiments. Referring to fig. 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 for the tracer particles 10, carrying other structures that are subsequently formed. The nucleic acid molecule 104 is immobilized on the core structure 102. The nucleic acid molecules 104 comprise 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 molecules 104, and serves as a protection and encapsulation structure.

As shown in fig. 1, in some embodiments, the core structure 102 comprises a plurality of pores 102p, and the nucleic acid molecules 104 are immobilized in the pores 102 p. In detail, in some embodiments, a portion of the nucleic acid molecules 104 can be immobilized in the pores 102 of the core structure 102, and a portion of the nucleic acid molecules 104 can be immobilized on the surface 102s of the core structure 102.

In particular, the core structure 102 with the holes 102p can improve the thermal conductivity of the trace particle 10, so that it can be suitable for high temperature environment, and can also increase the fixing strength of the nucleic acid molecules 104 on the core structure 102. In some embodiments, the core structure 102 has a first porosity ranging from about 2nm to about 100nm or from about 4nm to about 40 nm. It is understood that the porosity of the core structure 102 should not be too great to achieve protection of the nucleic acid molecules 104. Conversely, the porosity of the core structure 102 should not be too low, otherwise the nucleic acid molecule 104 may not have sufficient sites to attach, thereby reducing the efficiency of immobilization of the nucleic acid molecule 104.

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

The core structure 102 may be formed of an inorganic material. In some embodiments, the material of the core structure 102 comprises at least one of silica, a silicate, a carbonate (e.g., calcium carbonate), nanogold, a metal oxide, a heat-resistant polymer (e.g., polyethylene glycol or polystyrene), and a high molecular weight polymer (e.g., polylactic acid).

In some embodiments, the surface of the core structure 102 may be modified to immobilize the nucleic acid molecules 104 on the core structure 102. In detail, the surface of the core structure 102 may be positively charged by adding a chlorine-containing quaternary ammonium salt, thereby being linked to the negatively charged core structure 102. In some embodiments, the aforementioned chlorine-containing quaternary ammonium salt may comprise N-methyl-3-aminopropyltrimethoxy alkane (trimethoxy [3- (methyliminoxy) propyl ] silane, TMAPS).

In addition, the core structure 102 may comprise at least one of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and Peptide Nucleic Acid (PNA). In some embodiments, the nucleic acid molecule 104 comprises double-stranded helical DNA. In some embodiments, the nucleic acid molecule 104 may comprise a plasmid (plasmid).

In some embodiments, the nucleic acid molecule 104 ranges in length from about 10 base pairs (bp) to about 2000 base pairs, or preferably from about 50 base pairs to about 500 base pairs. In embodiments where the nucleic acid molecule 104 is a plasmid, the nucleic acid molecule 104 has a length ranging from about 1500 base pairs to about 10000 base pairs, or from about 2000 base pairs to about 4000 base pairs. It is understood that when the length of the nucleic acid molecule 104 is too long, the shell 106 may not completely cover the nucleic acid molecule 104, or it may take a long time to complete the coating, which increases the difficulty of the packaging process. Conversely, if the length of the nucleic acid molecule 104 is too short, the nucleic acid molecule 104 may be easily decomposed, and the sequence specificity of the nucleic acid molecule 104 may be reduced, thereby deteriorating the recognition of the target. Furthermore, 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, with the advantage of easy operation.

In some embodiments, nucleic acid molecules 104 having any suitable sequence can be designed as calibrators. For example, in some embodiments, the designed sequence of the nucleic acid molecule 104 may comprise a partial nucleic acid sequence of a thermophilic species (Thermus thermophilus) in order to increase the temperature tolerance of the nucleic acid molecule 104. For example, in some embodiments, the thermophilic bacteria may comprise proteus thermophilus (Tepidimonas fontici), hyperthermophilus (Tepidimonas ignova), hyperthermophilus aquaticus (Tepidimonas aquaticus), Bacillus stearothermophilus (Bacillus stearothermophilus), actinomyces vulgaris (Thermoactinomyces vulgaris), Thermus aquaticus (Thermus aquaticus), Thermus thermosphaeus (Thermococcus), Thermus habitatus (Thermotoga), Sulfolobus (Sulfolobus), Thermus thermosphaeus (Thermoproteus), Sulfolobus (Sulfolobus), acidomyces (acididus), Thermus occulta (pyrococcus occulta), pyrexium broccoli (pyrum bryosii), methanococcus (pyrobacterium), or pyrum clavatum (pyrum), but is not limited thereto.

Furthermore, in some embodiments, the designed sequence of the nucleic acid molecule 104 can comprise a portion of a nucleic acid sequence of an alga. For example, in some embodiments, the algae may comprise Chlamydomonas reinhardtii (Chlamydomonas reinhardtii), Chlamydomonas mojavensis (Chlamydomonas moewusii), Chlamydomonas ovalis (Chlamydomonas eugamis), Chlamydomonas inertias (Chlamydomonas segnis), and the like belonging to the genus Chlamydomonas; dunaliella salina (Dunaliella salina), Dunaliella salina (Dunaliella tertiolecta), Dunaliella prorectina (Dunaliella primotea), etc., belonging to the genus Dunaliella; chlorella (Chlorella vulgaris), Chlorella pyrenoidosa (Chlorella pyrenoidosa) and the like, which belong to the genus Chlorococcus; haematococcus pluvialis (Haematococcus pluvialis) belonging to the genus Haematococcus, and the like; marine chlorella (chlorecoccus littorale) belonging to the genus chlorella, and the like; chlorophyceae microfine (Pseudomonas aeruginosa) belonging to the genus Chlorophyceae, and the like; eyebrows belonging to the genus eyebrows (Amphora sp.) and the like; marine diatoms (Nitzschia alba), crescentella (Nitzschia closterium), Nitzschia laevis (Nitzschia laevis), etc., belonging to the genus Nitzschia; crypthecodinium cohnii (Crypthecodinium cohnii) belonging to the genus Crypthecodinium, etc.; euglena gracilis (Euglena gracilis), Euglena paraxial (Euglena proxima), etc., belonging to the genus Euglena; paramecium burstaria belonging to the genus Paramecium, etc.; synechococcus aquaticus (Synechococcus aquatilis), Synechococcus elongatus (Synechococcus elongatus), etc., belonging to the genus Synechococcus; spirulina platensis (Spirulina platensis), Spirulina subsalsa (Spirulina subsalsa) belonging to the genus Spirulina, etc.; marine protochlorella (Prochlorococcus marinus) belonging to the genus protochlorella, and the like; oocystis polymorpha (ooctyis polymorpha) belonging to the genus Oocystis, and the like, but are not limited thereto.

In some embodiments, the designed nucleic acid molecule 104 may have a hybrid (hybrid) nucleic acid sequence. In some embodiments, the designed nucleic acid molecule 104 can comprise a portion of a nucleic acid sequence from both prokaryotes and eukaryotes, e.g., from both thermophiles and algae. For example, in some embodiments, the sequence of the nucleic acid molecule 104 includes a partial sequence of the 16S rDNA of a thermophilic bacterium and a partial sequence of the 18S rDNA of an alga. Since there should be no organism in the natural environment that has the sequence characteristics of both species, the sequence of nucleic acid molecules 104 from both prokaryotes and eukaryotes should be specific, easy to identify the tracer particles, and reduce the likelihood of interference with nucleic acid fragments in the environment. Specifically, in some embodiments, the designed nucleic acid molecule 104 sequence can comprise a portion of the nucleic acid sequence of proteus thermophilus b (Tepidimonas fonticaldi) and Chlamydomonas reinhardtii (Chlamydomonas reinhardtii).

In addition, in some embodiments, a sequence region having a higher content of Cytosine (C) and Guanine (G) than Adenine (Adenine, a) and Thymine (Thymine, T) may be selected as the sequence of nucleic acid molecule 104. Since the action force between adenine and thymine is stronger than that between cytosine and guanine, the melting temperature is higher and the thermal stability is better when the GC content of the sequence is higher. Specifically, in some embodiments, the designed sequence of nucleic acid molecules 104 can have a GC content proportion of about 55% to about 70%.

In some embodiments, the nucleic acid molecule 104 is identical to the sequence recognition number: 1 has at least 85%, 90%, or 95% sequence similarity. In some embodiments, the nucleic acid molecule 104 can comprise a sequence identifier such as seq id no: 2 and 3. Furthermore, in the embodiment where the nucleic acid molecule 104 is a plasmid, the nucleic acid molecule 104 may comprise a nucleic acid fragment inserted into the plasmid, and the nucleic acid fragment has the sequence identification number: 1 has at least 85%, 90%, or 95% sequence similarity.

In addition, the nucleic acid molecules 104 can be made by designing nucleic acid sequences and making them by techniques well known in the art to which the invention pertains. 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 a biological fermentation technique. In detail, a suitable plasmid may be selected and a designed nucleic acid fragment (e.g., SEQ ID NO: 1) may be inserted into the plasmid to construct a recombinant plasmid, and then a host cell containing the recombinant plasmid may be mass-cultured using a fermenter. In some embodiments, the host cell can comprise Escherichia coli. In some embodiments, recombinant plasmids comprising the nucleic acid fragments of interest can be extracted from the host cells using alkaline lysis. For example, according to some embodiments, a 4 liter fermentor can be used to produce nucleic acid molecules 104 with a yield of 8.85 mg/day, which is much greater than the yield of nucleic acid molecules 104 produced by PCR (about 0.4 mg/day).

As mentioned above, the shell 106 may serve as an encapsulation material to encapsulate the core structure 102 and the nucleic acid molecules 104. In some embodiments, shell 106 has a second porosity. In some embodiments, the first porosity of core structure 102 is greater than the second porosity of shell layer 106. In some embodiments, the second porosity of the shell 106 is substantially 0nm, i.e., the shell 106 is substantially free of pores and is a solid or dense shell that completely seals the nucleic acid molecule 104 and prevents 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 may be applied for detection of a fluid. In other embodiments, the second porosity of shell 106 is other than 0, for example, from about 0.5nm to about 10 nm. In some embodiments where the second porosity of the shell 106 is other than 0, the tracer particle 10 may be applied for detection in air.

Further, the shell layer 106 may be a single layer structure or a multi-layer structure. In some embodiments, as shown in fig. 1, where the shell 106 is a single layer, the shell 106 may be substantially free of voids and substantially sealed.

In some embodiments, the shell layer 106 has a thickness T in the range of about 10nm to about 5000nm, or about 10nm to about 150nm, or about 50nm to about 120 nm.

In some embodiments, shell layer 106 comprises at least one of silica, a silicate, a carbonate (e.g., calcium carbonate), a heat resistant polymer (e.g., polyethylene glycol or polystyrene), and a high molecular weight polymer (e.g., polylactic acid). In some embodiments, the core structure 102 and the shell layer 106 may be formed of the same material.

In some embodiments, the core structure 102 of the immobilized nucleic acid molecule 104 can be modified with a chlorine-containing quaternary ammonium salt such that the shell 106 is positively charged internally to link with the negatively charged nucleic acid molecule 104 to form a closed shell-core structure. In some embodiments, the aforementioned chlorine-containing quaternary ammonium salt may comprise N-methyl-3-aminopropyltrimethoxy alkane (trimethoxy [3- (methyliminoxy) propyl ] silane, TMAPS).

Furthermore, as shown in fig. 1, in some embodiments, the particle size d2 of the encapsulated tracer particle 10 ranges from about 30nm to about 10000nm, or from about 30nm to about 300nm, or from about 50nm to about 150 nm. According to some embodiments, the aforementioned particle size may be a volume average particle size.

Furthermore, in some embodiments, the encapsulated tracer particles 10 have good homogeneity, i.e., have a uniform shape, size, or particle size. In some embodiments, the variation in the size of the tracer particles 10 ranges from about 0% to about 10%.

Referring to fig. 2, fig. 2 is a schematic diagram illustrating a structure of a trace particle 20 according to another embodiment of the present invention. It should be understood that the same or similar components or elements are denoted by the same or similar reference numerals, and the materials, manufacturing methods and functions thereof are the same or similar to those described above, so that the detailed description thereof will be omitted. The tracer particle 20 of the embodiment shown in fig. 2 is substantially similar to the tracer particle 10 shown in fig. 1, except that the shell 106 of the tracer particle 20 is a multi-layered structure.

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

It should be understood that although in the embodiment shown in FIG. 2, the shell layer 106 includes two layers, an outer shell layer 106a and an inner shell layer 106b, in other embodiments, the shell layer 106 may have other suitable numbers of sub-layers. Furthermore, although the inner shell layer 106b includes holes 106p in the embodiment shown in FIG. 2, in other embodiments, the inner shell layer 106b may be substantially free of holes.

According to some embodiments, the tracer particles provided herein can be operated at 120 ℃ for at least 5 hours or more. According to some embodiments, the tracer particles provided by the present invention can be operated at 120 ℃ 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 ℃ 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 invention can be operated for at least 720 hours under the environment with the pH value of 1-13. For example, the operation may be performed at pH1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 for at least 720 hours or more.

Furthermore, according to some embodiments, the tracer particles provided by the present invention may be used for geothermal or oil well fluid tracking and exploration. In particular, the tracer particles can track the movement (flow, mobilization, migration) and recovery of fluids in fractured regions in the formation, thereby analyzing the distribution and status of the oil or gas well, among other things. According to some embodiments, the tracer particles provided by the present invention may also be used for contaminant tracking. According to some embodiments, the tracer particles provided by the present invention can be used as anti-counterfeit labels.

Further, according to some embodiments, there is provided a method of using a tracer particle, comprising the steps of: providing a tracer particle as described in the previous 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 the nucleic acid molecules from the tracer particles; and analyzing the released nucleic acid molecules. In some embodiments, the tracer particles can be operated in a 120 ℃ fluid for at least 720 hours. According to some embodiments, the tracer particles may be operated in a fluid at a pH of 1-13 for at least 720 hours.

In some embodiments, hydrofluoric acid may be used to remove the shell of the tracer particle, allowing the nucleic acid molecule to be released and desorbed from the tracer particle. In some embodiments, an aqueous hydrofluoric acid solution (HF/NH) is used₄F) Is in the range of about 0.5 (v/v)% to about 3.0 (v/v)%, e.g., about 1.5 (v/v)%. In some embodiments, the released nucleic acid molecule can be analyzed by real-time polymerase chain reaction (q-PCR), confirming the presence of the designed specific nucleic acid molecule, andthe concentration was confirmed.

Referring next to fig. 3, fig. 3 is a flow chart illustrating steps of a method 10M for preparing tracer particles according to some embodiments of the invention. It is to be understood that in some embodiments, additional operational steps may be provided before, during and/or after the preparation method of the tracer particles is performed. In some embodiments, some of the operations described may be replaced or deleted as desired. In some embodiments, the order of operations/steps may be interchangeable. Further, the following description of the preparation method can be understood with reference to the structure of the tracer particle 20 shown in fig. 2.

As shown in fig. 3, in some embodiments, the method 10M for preparing a tracer particle may include forming a core structure 102 (step S12), fixing a nucleic acid molecule 104 to the core structure 102 (step S14), and forming a shell layer 106 on the core structure 102 (step S16) to encapsulate 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 a co-emulsifier. In some embodiments, the precursor of silicon may comprise Tetraethoxysilane (TEOS). In some embodiments, the co-emulsifier may comprise at least one of a C2-C10 short chain alcohol and a nonionic surfactant. In some embodiments, the C2-C10 short chain alcohol may comprise isopropanol. In some embodiments, the ratio (volume ratio) of the precursor of silicon and the co-emulsifier is between about 5: 1 to about 1: 10, or between about 1: 1 to about 1: between 10, for example, about 1: 1.

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

in some embodiments, the ratio (volume ratio) of the silicon precursor, co-emulsifier, and solvent in the oil phase solution is between about 3: 1: 1 to about 15: 1: 1, or between about 5: 1: 1 to about 10: 1: 1, e.g., about 7: 1: 1. it is understood that the ratio of the silicon precursor, co-emulsifier and solvent needs to be controlled in a specific range so that the formed tracer particles have good homogeneity, i.e. have a uniform shape, size or particle size.

Further, the aqueous solution may comprise water and a surfactant. In some embodiments, the surfactant may comprise at least one of an organic ammonium salt, an alkyl sulfate, and a fatty acid salt. In some embodiments, the organic ammonium salts can include cetyltrimethylammonium bromide (CTAB). In some embodiments, the ratio of water to surfactant in the aqueous phase solution is between about 1: 1 to about 10: 1, or between about 10: 1 to about 30: 1.

In some embodiments, the surfactant can be dissolved in water by first heating the aqueous phase solution to a temperature of from about 50 ℃ to about 80 ℃, or from about 55 ℃ to about 70 ℃, for example, about 60 ℃, before adding the oil phase solution to the aqueous phase solution.

Further, in some embodiments, the step of forming the core structure 102 may further comprise adding a catalyst to the mixed solution, and heating the mixed solution. In some embodiments, the catalyst comprises a basic solution. In some embodiments, the catalyst has a pH in the range of about 8 to about 14. In some embodiments, the catalyst may comprise at least one of aqueous ammonia, sodium hydroxide, calcium hydroxide, potassium hydroxide, and alkali based liquids.

In some embodiments, the temperature at which the mixed solution is heated can range from about 50 ℃ to about 80 ℃, or from about 55 ℃ to about 70 ℃, e.g., about 60 ℃. Further, in some embodiments, the heating time may be from 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 left standing overnight at room temperature, and then centrifuged to remove the supernatant, and extracted by ultrasonic vibration using ethanol to obtain the core structure 102 (porous carrier of labeled particles).

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 improve the dispersibility of the porous carrier. In some embodiments, the surface modifier may comprise a chlorine-containing quaternary ammonium salt, for example, may comprise N-methyl-3-aminopropyltrimethoxy propane (TMAPS). In detail, in some embodiments, the core structure 102 obtained in the previous step S12 may be dissolved in a co-emulsifier (e.g., isopropanol), followed by adding a surface modifier and shaking and centrifuging, followed by removing the supernatant and dissolving the product in water to achieve dispersion.

Next, in some embodiments, the core structure 102 obtained in the previous step and the nucleic acid molecule 104 may be mixed and centrifuged after shaking, so as to fix the nucleic acid molecule 104 on the core structure 102 (step S14). As mentioned above, the nucleic acid molecule 104 may comprise at least one of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and Peptide Nucleic Acid (PNA). In some embodiments, the nucleic acid molecule 104 ranges from about 10 base pairs to about 2000 base pairs in length. In some embodiments, the nucleic acid molecule 104 can be a plasmid that ranges in length from about 1500 base pairs to about 10000 base pairs.

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 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: between 1000, or about 1: 100 to about 1: 1000.

In some embodiments, the core structure 102 immobilized by the nucleic acid molecule 104 may be added to the alcohol mixed solution to continue the subsequent step of forming the shell 106. In some embodiments, the alcohol mixed solution may include glycerol, ethanol, and water. In some embodiments, the ratio (volume ratio) of glycerol, ethanol, and water is between about 100: 100: 1 to about 300: 300: 1, or between about 100: 100: 1 to about 200: 200: 1.

Next, a shell 106 may be formed on the core structure 102 immobilized with the nucleic acid molecule 104. In some embodiments, the step of forming the shell 106 on the core structure 102 (step S16) may include mixing and oscillating the core structure 102 immobilized with the nucleic acid molecule 104, a silicon precursor, and a surface modifier. In some embodiments, the silicon precursor and the surface modifier may be added in two separate portions and the two separate portions may be shaken.

In some embodiments, the precursor of silicon may comprise Tetraethoxysilane (TEOS). In some embodiments, the surface modifier may comprise a chlorine-containing quaternary ammonium salt, for example, may comprise N-methyl-3-aminopropyltrimethoxy propane (TMAPS).

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

In order to make the aforementioned and other objects, features and advantages of the present invention comprehensible, several embodiments, comparative embodiments, preparation examples and test examples are described in detail below, but the present invention is not limited thereto.

Example 1: design of specific nucleic acid molecules

Sequence design of hybrid DNA (hybrid DNA)

A50 bp fragment of the 16S rDNA of Thermomyces B (Tepidemonas fonticaldi, strain AT-A2) and a 50bp fragment of the 18S rDNA of Chlamydomonas reinhardtii (strain CC-621) were selected. The DNA fragments are connected in series in a way of 25bp (16S rDNA) -25bp (18S rDNA) -25bp (16S rDNA) -25bp (18S rDNA) to synthesize a 100bp hybrid DNA sequence (sequence identification number: 1) with higher temperature resistance than a general DNA sequence, and the hybrid DNA sequence is used as a specific nucleic acid molecule.

Confirmation of the uniqueness of hybrid DNA sequences

The uniqueness of the hybrid DNA sequence synthesized above was confirmed by Alignment using the BLAST (basic Local Alignment Search tool) system of the National Center for Biotechnology Information (NCBI), and the Alignment result showed zero correlation (no Alignment), which indicates that no similar DNA sequence existed in the database, thus proving that the designed hybrid DNA sequence has uniqueness.

Example 2: preparation of unique nucleic acid molecules

Integrated DNA Technologies synthesis sequence identification by entrusted gene synthesis: 1 (100 bp). According to the sequence identification number: 1, design sequence identification number: 2 and sequence identification number: 3, and a set of primer pairs. Identifying the sequence by a sequence identification number: 1 as a template and the DNA sequences are respectively identified by sequence identification numbers: 2 and 3 as primers at the 3 and 5 ends (melting temperatures Tm of 59 ℃ and 63 ℃ respectively), performing PCR, and identifying the amplified sequences: 1 to produce a nucleic acid molecule of sufficient specificity to carry out a subsequent nucleic acid molecule immobilization step.

The materials used for the PCR procedure were as follows: 10ng (1. mu.l) of template, 2. mu.l of 3-and 5-terminal primers (10. mu.M), 25. mu.l of 2 XTaq MasterMix and 20. mu.l of ddH₂O, total volume of reaction 50. mu.l. The temperature conditions for the PCR amplification reaction were set as: 95 ℃ reaction for 1 min → [95 ℃ reaction for 1 min → 55 ℃ reaction for 30 sec → 72 ℃ reaction for 9 sec]Circulation 12 times → reaction at 72 ℃ for 1 min → residence at 12 ℃.

The PCR product was analyzed by colloidal electrophoresis to determine whether it was a designed specific nucleic acid molecule, and the materials used in the procedure of colloidal electrophoresis were as follows: 2.5% agarose (agarose), 10 XTBE buffer (Tris-rate-EDTA), 1kb DNA ladder (as marker (M)) (CLUBIO) and 6 XTLoaddy (CLUBIO) using the DNA electrophoresis system Mupid-2plus (Mupid). The results of the gel electrophoresis are shown in FIG. 4, in which A-C are PCR products, M is a 1kb DNA ladder as a marker, and it can be seen from FIG. 4 that the length of the PCR amplified product is 100bp, and the sequence identification number: 1 are identical in length.

Then, Gel/PCR extraction Kit (Biomate) was used to purify the electrophoresis Gel, remove the dNTPs and primers that were not used in the PCR process, and avoid affecting the subsequent DNA immobilization step. After the purification step, the sequence identification number: 1.

Example 3: preparation of plasmids comprising an unique nucleic acid molecule

Using T&A cloning vector (cloning vector, yeaster biotech) the target specific sequence (seq id no: 1) cloning (cloning), sending the recombinant plasmid (about 3kb in length) into host Escherichia coli (DH 5 alpha), culturing the Escherichia coli in a fermentation tank in large quantity, obtaining thalli by centrifugation, and extracting the plasmid containing the required target specific sequence from the thalli by an alkaline lysis method.

The plasmid was cleaved with restriction enzymes EcoRI and HindIII and analyzed by colloidal electrophoresis to confirm that the plasmid was correctly sized (about 3kb), using the following materials: 1.5% agarose (agarose), 0.5 XTAE buffer (Tris-Acetate-EDTA), 1kb DNA ladder (as marker (M)) (CLUBIO) and 6 XTLoaddy (CLUBIO) using the DNA electrophoresis system Mupid-2plus (Mupid). The results of the colloidal electrophoresis are shown in FIG. 5A, in which A is a plasmid which has not been cleaved with restriction enzymes, B is a plasmid which has been cleaved with EcoRI, C is a plasmid which has been cleaved with Hind III, and M is a DNA ladder labeled with 1kb, and it can be seen from FIG. 5A that the plasmid obtained by culturing E.coli is about 3kb in length, which is consistent with the length of the originally constructed plasmid.

Then, using the obtained plasmid as a template, and using the sequence identification number: 2 and 3 as primers at the 3 and 5 ends, and performing a PCR program using the following materials: 10ng (1. mu.l) of template, 2. mu.l of 3-and 5-terminal primers (10. mu.M), 25. mu.l of 2 XTaq MasterMix and 20. mu.l of ddH₂O, total volume of reaction 50. mu.l. The temperature conditions for the PCR amplification reaction were set as: reaction at 95 ℃ for 5 minutes → [95 ℃ for 30 seconds → 60.7 ℃ for 30 seconds → 72 ℃ for 10 seconds]Circulation 29 times → reaction at 72 ℃ for 5 minutes → residence at 4 ℃.

The PCR product obtained was analyzed by colloidal electrophoresis to confirm whether it was a target specific sequence (SEQ ID NO: 1), and the materials used in the colloidal electrophoresis procedure were as follows: 2.5% agarose (agarose), 10 XTBE buffer (Tris-rate-EDTA), 1kb DNA ladder (as marker (M)) (CLUBIO) and 6 XTLoaddy (CLUBIO) using the DNA electrophoresis system Mupid-2plus (Mupid). The results of the gel electrophoresis are shown in FIG. 5B, in which A-D are PCR products, M is a 1kb DNA ladder as a marker, and it can be seen from FIG. 5B that the length of the PCR amplified product is about 100bp, and the sequence identification number: 1 are identical in length.

Test example 1: risk assessment of synthetic specific nucleic acid molecules to environment and human body

The synthetic sequence identification number was tested by environmental strain toxicity testing: 1 has an inhibitory effect on the microorganisms in the environment. As shown in fig. 6A to 6D, fig. 6A, 6B and 6C show the results of toxicity experiments of the synthesized DNA sequences against e.coli, Bacillus cereus and Pseudomonas putida, respectively, and fig. 6D is a control group in which e.coli is inhibited. From the above results, the sequence identifiers synthesized were: 1 does not have an inhibitory effect on microorganisms commonly found in the environment.

Further, the sequence identification number: 1 with the sequence of the Human chromosome (Human G + T), and the alignment result shows E-value>>1 (numerical value), similarity is zero (if E-value)<10^-5Representing high homology) and therefore the risk of replacing a human gene is close to zero. In addition, the sequence identification number is further: 1 into four fragments of 25bp respectively, and the comparison result shows that the E-value>1, the similarity is very low, and the risk of replacing human genes is close to zero.

Example 4: preparation of Trace particle A

Preparation of core structure and surface modification

Taking corn starch and deionized water to prepare 35% starch suspension, and stirring at 35 ℃. The pH of the starch solution was adjusted to 9.5 using 0.5N NaOH. Then, 20g of sodium hypochlorite was slowly added to the starch solution (addition time was more than 30 minutes) and the pH of the starch solution was maintained at 9.5 using 1N HCl. After sodium hypochlorite addition, stirring was continued for 50 minutes and the pH of the starch solution was maintained at 9.5 using 0.5N NaOH. After the reaction is finished, adjusting the pH value of the starch solution to 7 by using 1N HCl, cleaning the starch solution by using secondary water and alcohol, then pumping and filtering the starch solution, and drying the starch solution in a 50 ℃ drying oven to obtain the modified starch.

22mL of Tetraethoxysilane (TEOS) is added to 36mL of deionized water and 1mL of 2% HCl, and stirred until the mixture is hydrolyzed to be transparent. Then, after 3g of the modified starch was added, 20mL of 5% NH was slowly dropped into the mixture using a separatory funnel₄OH, stirring for 40 minutes. The solid was filtered off and dried in an oven at 50 ℃ for 24 hours. Thereafter, the solid was calcined at 550 ℃ for 3 hours. In this way, a porous carrier (core structure) of the tracer particles can be obtained.

2g of the porous carrier was added to 20mL of isopropanol and uniformly dispersed, and then 0.889mL of N-methyl-3-aminopropyl trimethoxy alkane (trimethoxy [3- (methylamino) propyl ] silane, TMAPS) and 1mL of deionized water were added thereto and stirred at 40 ℃ for 2 hours. Next, the supernatant was removed by centrifugation at 15275RCF (relative centrifugal force) for 10 minutes, and the solid was dispersed in 40mL of deionized water. In this way, a surface-modified porous support can be obtained.

Immobilization of nucleic acid molecules

Take 35 μ L of surface modified porous support, add to 10 μ L of the aforementioned sequence identifier: 1 (300ppm) (or 1300ppm) was centrifuged at 18000RCF for 10 minutes after shaking with a shaker. Thereafter, the supernatant was taken out and washed several times with secondary water, and the solid was dispersed in 500. mu.L of secondary water.

Encapsulation of shell layers

Next, 0.6. mu.L of N-methyl-3-aminopropyltrimethoxysilane (trimethoxy [3- (methylamino) propyl ] silane, TMAPS) and 0.6. mu.L of TEOS were added and shaken at 900rpm for 4 hours. Thereafter, 4. mu.L of TEOS was added and shaken at 900rpm for 96 hours. Then, centrifugation was carried out at 19375 RCF for 10 minutes, the supernatant was removed, and the solid was dispersed in 45. mu.L of secondary water by washing with secondary water several times. In this way, the trace particle a can be completed.

Fig. 7A and 7B show Scanning Electron Microscope (SEM) views of the porous carrier before and after the encapsulation process of the shell, respectively. From the SEM analysis, it was observed that the particle size of the tracer particles was about 40nm to 50nm before the encapsulation process (as shown in FIG. 7A), and increased to about 60nm to 75nm after the encapsulation process (as shown in FIG. 7B).

Example 5: preparation of Trace particle B

Preparation of core structure and surface modification

An aqueous solution was prepared by dissolving 2g of hexadecyltrimethylamine bromide (CTAB) in 30ml of secondary water in a serum bottle and heating to 60 ℃. Further, an oil phase solution was prepared from 7.2ml of octane, 1ml of Tetraethoxysilane (TEOS), and 1ml of isopropyl alcohol, and the oil phase solution was added dropwise to the aqueous phase solution with a dropper. Then, 0.022ml of 25% aqueous ammonia was added and reacted at 60 ℃ for 3 hours, and after completion of the reaction, it was allowed to stand overnight at normal temperature. And centrifuging the reacted mixed solution, removing the supernatant, performing ultrasonic vibration extraction by using ethanol, and replacing the oil phase solution to obtain the porous carrier (core structure) of the tracer particles.

After the porous carrier was centrifuged, it was added to 20mL of isopropanol and uniformly dispersed, and then 0.222mL of N-methyl-3-aminopropyltrimethoxysilane (TMAPS) was added thereto, and after shaking for 18 hours using a shaker, it was centrifuged at 15275RCF (relative centrifugal force) for 10 minutes to remove the supernatant, and the solid was dispersed in 20mL of secondary water. In this way, a surface-modified porous support can be obtained.

Immobilization of nucleic acid molecules

350 μ L of the surface-modified porous carrier was added to the sample containing the sequence identifier prepared in example 3 above: 1 (100 μ l), after centrifugation for 10 minutes at 15275RCF, the supernatant was removed and the solid was dissolved in 5ml of an alcohol mixture (glycerol: ethanol: water: 150: 1).

Encapsulation of shell layers

Next, 6. mu.l of TMAPS and 6. mu.l of TEOS were added, and the mixture was shaken for 4 hours using a shaker, and then 40. mu.l of TEOS was added and shaken for 4 days using a shaker. Thereafter, 24. mu.l of TMAPS was added and shaken for 18 hours using a shaker, and the solution was exchanged for secondary water. In this way, the trace particle B can be completed.

Fig. 8A and 8B show Scanning Electron Microscope (SEM) views of the porous carrier before and after the encapsulation process of the shell, respectively. From the SEM analysis, it was observed that the particle size of the tracer particles was about 30nm to 40nm before the encapsulation process (as shown in FIG. 8A), and increased to about 50nm to 60nm after the encapsulation process (as shown in FIG. 8B).

Comparative example 1: preparation of Trace particle C

The preparation method of the tracer particle C is substantially similar to that of the tracer particle A in example 4, but the shell of the tracer particle C is not encapsulated, i.e., the DNA of the tracer particle C is naked.

Comparative example 2: preparation of tracer particle D

Method for preparing tracer particles DMethod (1968), and Kim et al (t.g.kim et al, 2017). First, 50ml of 95% alcohol plus 60ml of the secondary aqueous solution were prepared and stirred at a fixed rotation speed of 450rpm for 15 minutes, then 20ml of TEOS was added and mixed and hydrolyzed for 30 minutes, and finally 6ml of 25% ammonia was added and stirred for 2 hours to polymerize. After the reaction is finished, the liquid is centrifuged for 15 minutes at the rotating speed of 15275RCF, the supernatant is removed, the solid is washed for 3 times by 95 percent alcohol, and the mixture is placed in an oven at 50 ℃ for drying. Compared with the porous carrier of the tracer particle A, the carrier of the tracer particle D is a compact solid carrier, and the porosity is close to 0.

Example 6: nucleic acid molecule desorption process

The shell protecting the DNA was removed with hydrofluoric acid (HF), the DNA was desorbed, 10 μ Ι _ of the encapsulated tracer particles were added to 40 μ Ι _ of a 1.5% aqueous HF/NH4F solution, shake-mixed for about 5 minutes using a shaker, and then the desorbed DNA was recovered using a DNA purification kit (Bioman Scientific).

Example 7: analysis of recovered nucleic acid molecules

Quantitative analysis of the recovered DNA was performed using real-time polymerase chain reaction (q-PCR) analysis to confirm whether it contained the designed specific DNA sequence and to measure its concentration (mass) at the same time. The materials used for the q-PCR procedure were as follows: mu.l of template (DNA recovered by desorption), 0.75. mu.l of 3-terminal primer (10. mu.M) (SEQ ID NO: 2), 0.75. mu.l of 5-terminal primer (10. mu.M) (SEQ ID NO: 3), 12.5. mu.l of 2X SYBR Green Master Mitrix (Thermo Fisher Scientific) and 10. mu.l of ddH₂O, total volume of reaction 25. mu.l. The temperature conditions for the q-PCR amplification reaction were set as follows: reaction at 95 ℃ for 10 minutes → [95 ℃ for 15 seconds → 60 ℃ for 9 seconds]Cycle 40 times → 12 ℃ dwell.

Test example 2: temperature resistance test of trace particles

The tracer particle A prepared in example 4 and the tracer particle C prepared in comparative example 1 were heated in an oil bath at 25 ℃, 100 ℃, 120 ℃, 140 ℃, 160 ℃, 180 ℃ and 200 ℃ for 20 minutes. Subsequently, the labeled particles were taken out, and the residual DNA was recovered separately and analyzed by colloidal electrophoresis.

As a result, as shown in FIGS. 9A and 9B, FIGS. 9A and 9B show the results of temperature resistance tests of tracer particles prepared in comparative example 1 and example 4, respectively, in which 25, 100, 120, 140, 160, 180 and 200 represent heating temperatures, and M is a marker (1kb DNA ladder). As can be seen from the results of fig. 9A and 9B, the high temperature resistance of the tracer particle C (naked DNA) of comparative example 1 was about 100 ℃, and the high temperature resistance of the tracer particle a (encapsulated DNA) of example 4 was about 200 ℃.

In addition, the tracer particle a of example 4 was further tested for its temperature resistance at 120 ℃. Specifically, the recovery rate of DNA was measured at different time points, and the results are shown in Table 1 below.

TABLE 1

From the results in table 1, it is clear that the DNA recovery of the encapsulated tracer particle a after 5 hours of heating is maintained at 76.5%.

In addition, the tracer particle B of example 5 was further tested for its temperature resistance at 120 ℃. The recovery of DNA was measured at different time points and the results are shown in Table 2 below.

TABLE 2

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

From the results in table 2, it is clear that the DNA recovery of the encapsulated tracer particle B after heating for 24 hours is maintained at 81.5%.

Test example 3: comparison of temperature resistance of tracer particles

The tracer particle A prepared in example 4 and the tracer particle D prepared in comparative example 2 were heated in an oil bath at 120 ℃. Subsequently, after heating for 1, 2, and 2.5 hours, respectively, the tracer particles were removed and the residual DNA was recovered, and the content of the residual DNA was measured to calculate the residual rate, and the results are shown in table 3 below.

TABLE 3

As is clear from the results in Table 3, the DNA remaining ratio of the tracer particle A prepared in example 4 was 79.4% and the DNA remaining ratio of the tracer particle D prepared in comparative example 2 was 52.4% after heating at 120 ℃ for 2.5 hours. Thus, the tracer particle a having a porous carrier structure has better high temperature resistance compared to the tracer particle D having a solid (non-porous) carrier.

Test example 4: acid and alkali resistance test of tracer particles

The tracer particles a prepared in the previous example 4 were placed in solutions with different pH values to test the resistance of the structure of the tracer particles a in acidic and basic environments. Preparing an acidic solution and an alkaline solvent by using sulfuric acid and an acidic solution respectively. The tracer particles were placed in a solution of pH1, pH 3, pH 5, pH 7, pH 9 and pH 13 for 60 minutes, and then the tracer particles were removed, and the residual DNA was recovered and analyzed by colloidal electrophoresis.

As a result, as shown in FIG. 10, M in the figure is a marker (1kb DNA ladder), and it is understood from the result of FIG. 10 that the amount of DNA of the tracer particle A hardly decreases in the range of pH 9 or less, and is not affected by the change in pH, and does not significantly decrease in the range of pH 9 or more. From the above results, it was found that the tracer particle a of example 4 has strong acid resistance and strong base resistance.

In addition, the acid and alkali resistance of the tracer particle B of example 5 was also tested, and the tracer particle B was placed in solutions of different pH values to test the resistance of the structure of the tracer particle B in acidic and alkaline environments. Preparing an acidic solution and an alkaline solvent by using sulfuric acid and an acidic solution respectively. The tracer particles were placed in a solution at pH1, pH 3, pH 5, pH 7, pH 9 and pH 13 for 24 hours, and then the tracer particles were removed and the DNA remaining thereon was recovered, the content of the remaining DNA was measured, and the remaining percentage was calculated, and the results are shown in fig. 11.

From the results of fig. 11, it is seen that the amount of DNA of the tracer particle B was hardly reduced in the range of pH 9 or less (the lines of pH 3, pH 5, and pH 7 overlap in the figure) within 1 hour, and was not affected by the change in pH, and was not significantly reduced in the range of pH 9 or more, indicating that the tracer particle B has the ability to resist strong acid and strong base. It is noted that the amount of DNA of the tracer particle B did not decrease significantly in the range below pH 9 after 24 hours of reaction, indicating that it has the ability to withstand strong acids for a long period of time.

Test example 5: addition of tracer particles to test temperature resistance of hot water in real field

The plasmid constructed with the target DNA tag is immobilized on the nanoporous carrier prepared in example 5, and the tracer particle B is produced after the encapsulation process. The tracer particles B are placed in a small reaction tank, and real-field geothermal water which is volcano type geothermal water (China Datun mountain geothermal water, pH1.5, total dissolved solids in water-9200 ppm) and metamorphic rock geothermal water (China Renzhou geothermal water, pH8.8, total dissolved solids in water-4000 ppm) are respectively added into the small reaction tank. Then, the resulting mixture was placed in an oil bath at 120 ℃ for heating. Next, after heating for 480 and 720 hours, respectively, the tracer particles were removed and the DNA remaining thereon was recovered, the content of the remaining DNA was measured, and the remaining rate was calculated, and the results are shown in table 4 below.

TABLE 4

From the results in Table 4, it was found that the DNA remaining ratio of the tracer particle E in the acidic environment was 10.7% and the DNA remaining ratio of the tracer particle E in the weakly alkaline environment was 7.5% after heating at 120 ℃ for 720 hours. It can be seen that the tracer particles E have been of primary feasibility for practical geothermal applications.

Test example 6: tubular column tracing test of tracing particles

In order to simulate the case where tracer particles are applied in an actual field (in soil or rock formations), a quartz sand column was prepared by filling quartz sand (0.84mm) of a No. 20 sieve in a glass column having a diameter of 0.8cm and a length of 10.7 cm. Generally, the degree of hydration (conductivity) of geothermal fluid channels (fissures) is about 10^-7～10^-2m/sec, the water conductivity of the prepared quartz sand column is about 3.4 x 10^-5m/sec. Next, the tracer particle A prepared in the foregoing example 4 was placed in water and introduced into a quartz sand column at a flow rate of 0.1ml/min, and a sample flowing out of the column was collected.

The purpose of the column tracer test was to investigate the effect of time on the recovery of tracer particles relative to the extent of diffusion. The results in fig. 12 show the DNA content of the recovered trace particles a over time, and from the curve change of the DNA content, after one injection of the trace particles a, a small part of the trace particles a flow out from the shortest channel by advection, and a large part of the trace particles a flow out from the channel by spreading and diffusion behavior due to the maldistribution of the flow field (most of the trace particles a flow out in 140 minutes). The transmission behavior described above corresponds to common tracer test results. In addition, the recovery rate of the trace particles a was measured to be 97.5%, which indicates that the trace particles of this example are not adsorbed by quartz sand and can freely flow in the low conductivity fluid channel.

In summary, according to some embodiments of the present invention, the tracer particles include specific nucleic acid molecules as a marker (label, fingerprint), and inorganic materials as a substrate and a packaging material, and the core structure with moderate porosity is used to increase the immobilization amount of the nucleic acid molecules and reduce the thermal conductivity (reduce thermal resistance) of the particles, thereby improving the temperature tolerance of the tracer particles. In addition, the tracer particles also have the performances of acid resistance, alkali resistance and the like, and the tolerance and the recovery rate of the tracer particles in an extreme environment can be further improved.

Although embodiments of the present invention and their advantages have been described above, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification, but it is to be understood that any process, machine, manufacture, composition of matter, means, method and steps, presently existing or later to be developed, that will be obvious to one skilled in the art from this disclosure may be utilized according to the present application as many equivalents of the presently available embodiments of the present application and are capable of performing substantially the same function or achieve substantially the same result as the presently available embodiments. Accordingly, the scope of the present application includes the processes, machines, manufacture, compositions of matter, means, methods, and steps described in the specification. In addition, each claim constitutes a separate embodiment, and the scope of protection of the present invention also includes combinations of the respective claims and embodiments. The scope of the present invention is defined by the appended claims.

Sequence listing

<110> institute of Industrial and technology of financial group legal

<120> tracer particle, and application method and preparation method thereof

<160> 3

<210> 1

<211> 100

<212> DNA

<213> Artificial sequence

<220>

<223> artificially synthesized sequence

<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 sequence

<400> 2

taacgggtga cggaggatt 19

<210> 3

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> artificially synthesized sequence

<400> 3

accgtaagaa gcaccggcta ac 22