CN113759076B

CN113759076B - Tracer particles and their application and preparation methods

Info

Publication number: CN113759076B
Application number: CN202010489308.2A
Authority: CN
Inventors: 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
Original assignee: Industrial Technology Research Institute ITRI
Current assignee: Industrial Technology Research Institute ITRI
Priority date: 2020-06-02
Filing date: 2020-06-02
Publication date: 2025-01-24
Anticipated expiration: 2040-06-02
Also published as: CN113759076A

Abstract

The present invention provides a tracer particle and its application method and preparation method, wherein the tracer particle comprises: 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

Trace particle and application method and preparation method thereof

Technical Field

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

Background

The tracing technology can be used as a tool for monitoring fluid, pollution leakage, product tracing 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 sources, and can also be used as a tracing tool for tracing underground water sources and environmental pollution remediation.

The types of tracers commonly used at present include radioactive tracers, fluorescent tracers, chemical tracers and the like, but they are of limited variety, are complex in analysis procedure and are toxic and can be harmful to the environment. In view of this, the development of novel tracers is gaining importance. The biological tracer is a new generation tracer, takes biological materials as main calibration objects (labels and fingerprints), has no toxicity and is less liable to cause environmental pollution. However, existing biological tracers are not very ideal for extreme environment resistance and are difficult to apply to extreme environments such as strong acids, strong bases or high temperatures.

As noted above, while currently available tracers generally meet their intended use, they are not fully satisfactory in all respects.

Disclosure of Invention

According to some embodiments of the present invention, there is provided a trace particle comprising a core structure, a nucleic acid molecule immobilized on the core structure, and a shell layer encapsulating 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.

According to some embodiments of the present invention, there is provided a method of using a tracer particle comprising providing a tracer particle as described above, 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, and analyzing the released nucleic acid molecule.

According to some embodiments of the present invention, a method for preparing a trace particle includes forming a core structure, fixing a nucleic acid molecule on the core structure, and forming a shell layer on the core structure to encapsulate the core structure and the nucleic acid molecule. In addition, the step of forming the core structure comprises providing an oil phase solution comprising a silicon precursor and a co-emulsifier, providing an aqueous phase solution comprising 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.

The tracer particles of the invention have a moderate porosity core structure, increase the fixed amount of 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 trace particles of the invention also have acid resistance, alkali resistance and other properties, and can further improve the tolerance and recovery rate of the trace particles in extreme environments.

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

Drawings

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

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

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

FIG. 4 is a diagram showing a gel electrophoresis analysis of a target nucleic acid molecule tag according to some embodiments of the present invention;

FIG. 5A is a diagram showing a gel electrophoresis analysis of a plasmid product containing a tag of a target nucleic acid molecule according to some embodiments of the present invention;

FIG. 5B is a diagram showing a gel electrophoresis analysis of a tag of a target nucleic acid molecule in a plasmid product according to some embodiments of the present invention;

FIGS. 6A-6D are graphs showing the results of toxicity tests of DNA having sequence ID No. 1 on microorganisms according to some embodiments of the present invention;

FIGS. 7A and 7B are views of a Scanning Electron Microscope (SEM) view of a core structure before and after encapsulation of a shell layer, respectively, according to some embodiments of the present invention;

FIGS. 8A and 8B are views of a Scanning Electron Microscope (SEM) view of a core structure before and after encapsulation of a shell layer, respectively, according to some embodiments of the present invention;

FIGS. 9A and 9B illustrate temperature resistance test results of trace particles, respectively, according to some embodiments of the invention;

FIG. 10 shows acid and base resistance test results of trace particles in accordance with some embodiments of the invention;

FIG. 11 shows acid and base resistance test results of trace particles in accordance with some embodiments of the invention;

Fig. 12 shows a column tracer test result for tracer particles in accordance with some embodiments of the invention.

Wherein, the reference numerals:

10. 20 tracer particles;

102. A core structure;

102p, 106p holes;

102s surface;

104. A nucleic acid molecule;

106. A shell layer;

106a an outer shell layer;

106b an inner shell layer;

a 10M preparation method;

S12, S14 and S16;

d1 and d2 particle diameters;

t thickness.

Detailed Description

The following describes the tracer particles, the application method of the tracer particles, and the preparation method of the tracer particles according to the embodiments of the invention in detail. It is to be understood that the following description provides many different embodiments, or examples, for implementing different aspects of some embodiments of the invention. The specific components and arrangements described below are only for simplicity and clarity in describing some embodiments of the present invention. These are, of course, merely examples and are not intended to be limiting. Moreover, similar and/or corresponding reference numerals may be used in different embodiments to identify similar and/or corresponding components in order to clearly describe the present invention. However, the use of such similar and/or corresponding reference numerals is merely for simplicity and clarity in describing some embodiments of the present invention and is not intended to represent any relevance between the various embodiments and/or structures discussed.

It is to be understood that the components or arrangements of the figures may exist in various forms well known to those skilled in the art. Furthermore, 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 between different components, regions, layers or sections. 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.

The terms "about", "approximately", "substantially" and "substantially" herein 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. Where a given quantity is about, i.e., where "about", and "substantially" are not specifically recited, the meaning of "about", and "substantially" may be implied.

Embodiments of the invention may be understood in conjunction with the accompanying drawings, which are incorporated in and form a part of the disclosure. It should be understood that the drawings of the present invention are not drawn to scale and that virtually any enlargement or reduction of the size of the components is possible in order to clearly demonstrate 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 appreciated 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 trace particle, which comprises specific nucleic acid molecules as a marker, and uses a core structure with larger porosity to increase the fixation strength of the nucleic acid molecules, improve the heat conductivity of the particle and further improve the extreme environment resistance of the trace particle.

Fig. 1 shows a schematic diagram of the structure of a trace particle 10 according to some embodiments of the invention. It should be appreciated that additional features may be added to trace particles 10 according to some embodiments. Referring to FIG. 1, the trace particle 10 may include a core structure 102, a nucleic acid molecule 104, and a shell layer 106. The core structure 102 may serve as a carrier for the trace particles 10, carrying further structures to be formed later. Nucleic acid molecule 104 is immobilized on core structure 102. The nucleic acid molecule 104 comprises a specific nucleic acid sequence that serves as a marker for the trace particle 10. Furthermore, the shell layer 106 encapsulates the core structure 102 and the nucleic acid molecule 104, and may serve as a protective 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 may be immobilized in the pores 102 of the core structure 102, and a portion of the nucleic acid molecules 104 may be immobilized on the surface 102s of the core structure 102.

In particular, the core structure 102 with the holes 102p may improve the thermal conductivity of the tracer particle 10, making it suitable for use in high temperature environments, and may also increase the fixation strength of the nucleic acid molecules 104 to the core structure 102. In some embodiments, the core structure 102 has a first porosity in a range of about 2nm to about 100nm or about 4nm to about 40nm. It should be understood that the porosity of the core structure 102 should not be too great to achieve the effect of protecting the nucleic acid molecule 104. Conversely, the porosity of the core structure 102 should not be too small, 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 9000nm. According to some embodiments, the foregoing 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, silicate, carbonate (e.g., calcium carbonate), nanogold, metal oxide, heat resistant polymer (e.g., polyethylene glycol or polystyrene), and high molecular 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 quaternary ammonium salt containing chlorine, whereby it may be connected with the negatively charged core structure 102. In some embodiments, the aforementioned chlorine-containing quaternary ammonium salt can comprise N-methyl-3-aminopropyl trimethoxyalkane (trimethoxy [3- (methylamino) propyl ] silane, TMAPS).

In addition, the core structure 102 may include at least one of deoxyribonucleic acid (deoxyribonucleic acid, DNA), ribonucleic acid (RNA), and peptide nucleic acid (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 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 embodiments 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 appreciated that when the length of the nucleic acid molecule 104 is too long, the shell 106 may not completely encapsulate the nucleic acid molecule 104 or may take a longer time to complete the encapsulation process, which may increase the difficulty of the encapsulation process. Conversely, when 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, so that the identification degree of the marker may be deteriorated. Furthermore, according to some embodiments, the nucleic acid molecule 104 in the form of a plasmid may protect the target nucleic acid fragment, help to increase the tolerance and recovery of the tracer particle 10 in extreme environments, and may simplify the purification steps, with the advantage of ease of handling.

In some embodiments, nucleic acid molecules 104 having any suitable sequence may be designed as a calibrator. For example, in some embodiments, to increase the temperature tolerance of the nucleic acid molecule 104, the sequence of the designed nucleic acid molecule 104 may comprise a portion of the nucleic acid sequence of a thermophilic bacterial species (Thermus thermophilus). For example, in some embodiments, the thermophilic bacteria may include Proteus thermophilus (Tepidimonas fonticaldi), microthermophilus tarda (Tepidimonas ignava), microthermophilus aquaticus (Tepidimonas aquatica), bacillus stearothermophilus (Bacillus stearothermophilus), thermoactinomyces vulgaris (Thermoactinomyces vulgaris), thermus aquaticus (Thermus aquaticus), thermococcus (Thermococcus), thermotoga (Thermotoga), zosterus sulfureus (Sulfolobus), thermomyces (Thermoproteus), zygophyllum thiofidobacterium (Desulfurolobus), acidomycota (Acidianus), thermomyces crypticum (Pyrodictium occultum), thermomyces brucei (Pyrodictium brockii), methanothermophilus (Methanopyrus), or Thermomyces (Pyrobaculum), but are not limited thereto.

Furthermore, in some embodiments, the sequence of the designed nucleic acid molecule 104 may comprise a partial nucleic acid sequence of algae. For example, in some embodiments, the algae may include Chlamydomonas reinhardtii (Chlamydomonas reinhardtii), chlamydomonas moellendori (Chlamydomonas moewusii), chlamydomonas oomycetes (Chlamydomonas eugametos), chlamydomonas oomycetes (Chlamydomonas segnis), dunaliella (Dunaliella salina), dunaliella (Dunaliella tertiolecta), puff (Dunaliella primolecta), etc., chlorella (Chlorella vulgaris), chlorella pyrenoidosa (Chlorella pyrenoidosa), etc., haematococcus pluvialis (Haematococcus pluvialis) belonging to the genus Haematococcus, chlorella gracilis (Chlorococcum littorale) belonging to the genus Chlorella, etc., chlorella minutissima (Pseudochoricystis ellipsoidea) belonging to the genus Chlorella, etc., chlamydomonas bisporus (Amphora) belonging to the genus Bitsuga, celasta (Nitschezia), celasta (Nitzschia closterium), leucopia left-hand-eye (Dunaliella primolecta), etc., chlorella (Crypthecodinium cohnii) belonging to the genus Chlorella, etc., euglena (35), euglena (43), etc., a (43), euglena (43), etc., belonging to the genus Euglena, euglena (53), etc., but is not limited thereto.

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 comprise a portion of the nucleic acid sequences from both prokaryotes and eukaryotes, e.g., a portion of the nucleic acid sequences from both thermophiles and algae. For example, in some embodiments, the sequence of the nucleic acid molecule 104 comprises a partial sequence of 16S rDNA of thermophilic bacteria and a partial sequence of 18S rDNA of algae. Since organisms having sequence properties of both species should not be present in the natural environment, sequences comprising nucleic acid molecules 104 from both prokaryotes and eukaryotes should be specific, easy to identify trace particles, and less likely to interfere with nucleic acid fragments in the environment. Specifically, in some embodiments, the sequence of the designed nucleic acid molecule 104 may comprise a partial nucleic acid sequence of Proteus thermophilus (Tepidimonas fonticaldi) and Chlamydomonas reinhardtii (Chlamydomonas reinhardtii).

In addition, in some embodiments, regions of the sequences having higher levels of cytosine (Cytosine, C) and guanine (Guanine, G) than levels of adenine (Adenine, A) and thymine (Thymine, T) may be selected as the sequences of the nucleic acid molecule 104. Since the forces between adenine and thymine are stronger than 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 GC content ratio of the sequences of the designed nucleic acid molecules 104 may be about 55% to about 70%.

In some embodiments, nucleic acid molecule 104 has at least 85%, 90%, or 95% sequence similarity to the sequence set forth in sequence ID No. 1. In some embodiments, the nucleic acid molecule 104 may comprise sequences as shown in sequence identification numbers 2 and 3. Furthermore, in embodiments 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 at least 85%, 90% or 95% sequence similarity to the sequence set forth in SEQ ID No. 1.

In addition, nucleic acid sequences can be designed and nucleic acid molecules 104 can be made by techniques known in the art to which the invention pertains. For example, the designed nucleic acid sequence may be amplified in large amounts by a polymerase chain reaction (polymerase chain reaction, PCR) using primers complementary to the designed nucleic acid sequence. In some embodiments, the biological fermentation technique may be further coupled to increase the yield of nucleic acid molecules 104. In detail, a suitable plasmid may be selected and a designed nucleic acid fragment (e.g., sequence identification number: 1) may be inserted into the plasmid to construct a recombinant plasmid (recombinant plasmid), and then a host cell containing the recombinant plasmid may be mass-cultured using a fermentation tank. In some embodiments, the host cell may comprise E.coli (ESCHERICHIA COLI). In some embodiments, the recombinant plasmid comprising the nucleic acid fragment of interest can be extracted from the host cell using an alkaline lysis method. For example, according to some embodiments, a 4 liter fermentor may be used to produce nucleic acid molecule 104 at a yield of 8.85 mg/day, which is far greater than the yield of nucleic acid molecule 104 produced by PCR (about 0.4 mg/day).

As described above, the shell 106 may serve as an encapsulation material that encapsulates the core structure 102 and the nucleic acid molecules 104. In some embodiments, the shell layer 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 layer 106. In some embodiments, the second porosity of the shell layer 106 is substantially 0nm, i.e., the shell layer 106 is substantially void-free and is a solid or dense shell layer that can completely encapsulate the nucleic acid molecule 104 from exposure to the nucleic acid molecule 104. In some embodiments where the second porosity of the shell layer 106 is 0, the trace particles 10 may be used for detection of fluids. In other embodiments, the second porosity of the shell layer 106 is other than 0, for example, from about 0.5nm to about 10nm. In some embodiments where the second porosity of the shell layer 106 is not 0, the trace particle 10 may be used for air detection.

In addition, the shell layer 106 may have a single-layer structure or a multi-layer structure. In some embodiments, in which the shell layer 106 is a single layer structure, as shown in fig. 1, the shell layer 106 may be substantially void-free and substantially sealed (encapsulated).

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

In some embodiments, the shell layer 106 comprises at least one of silica, silicate, carbonate (e.g., calcium carbonate), heat resistant polymer (e.g., polyethylene glycol or polystyrene), and high molecular 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 may be modified with a chlorine-containing quaternary ammonium salt such that the interior of the shell layer 106 is positively charged to connect with the negatively charged nucleic acid molecule 104, forming a closed shell-core structure. In some embodiments, the aforementioned chlorine-containing quaternary ammonium salt can comprise N-methyl-3-aminopropyl trimethoxyalkane (trimethoxy [3- (methylamino) propyl ] silane, TMAPS).

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

Furthermore, in some embodiments, the finished encapsulated trace 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 trace particles 10 ranges from about 0% to about 10%.

Referring next to fig. 2, fig. 2 is a schematic diagram illustrating a trace particle 20 according to another embodiment of the invention. It should be understood that the same or similar components or elements as described above will be denoted by the same or similar reference numerals, and the materials, manufacturing methods and functions thereof are the same as or similar to those described above, so that the description thereof will not be repeated. The embodiment of the trace particle 20 shown in fig. 2 is substantially similar to the trace particle 10 shown in fig. 1, except that the shell layer 106 of the trace particle 20 is a multi-layer structure.

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

It should be appreciated that while in the embodiment shown in FIG. 2, the shell layer 106 comprises 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. Moreover, although in the embodiment shown in FIG. 2, the inner shell layer 106b includes holes 106p, in other embodiments, the inner shell layer 106b may be substantially devoid of holes.

According to some embodiments, the trace particles provided herein may be operated at 120 ℃ for at least 5 hours or more. According to some embodiments, the trace particles provided by the present invention may be operated at 120 ℃ for more than 24 hours, and may maintain a recovery rate of more than 80%. According to some embodiments, the trace particles provided by the present invention may be operated at 120 ℃ for more than 720 hours, and may maintain recovery rates of more than 20%. According to some embodiments, the tracer particles provided by the invention can be operated for at least 720 hours in an environment with a pH value of 1-13. For example, the operation may be performed at a pH of 1,2,3, 4, 5, 6, 7, 8, 9,10, 11, 12 or 13 for at least 720 hours or more.

In addition, according to some embodiments, the trace particles provided by the present invention may be used for fluid tracking and exploration of geothermal or oil wells. Specifically, the trace particles may track the movement (flow, migration, migration) and recovery of fluids in a fracture zone in the formation, thereby analyzing the distribution and status of an oil or gas well, etc. According to some embodiments, the trace particles provided by the present invention may also be used for contaminant tracking. According to some embodiments, the trace particles provided by the invention can be used as anti-counterfeit labels.

Furthermore, according to some embodiments, a method of using a tracer particle is provided comprising the steps of providing a tracer particle as described in the previous embodiments, 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, and analyzing the released nucleic acid molecule. In some embodiments, the tracer particles can be operated in a fluid at 120 ℃ for at least 720 hours. According to some embodiments, the tracer particles can be operated in a fluid having 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, releasing, desorbing the nucleic acid molecules from the tracer particle. In some embodiments, aqueous hydrofluoric acid (HF/NH ₄ F) is used at a concentration in the range of 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 may be analyzed by real-time polymerase chain reaction (real-time polymerase chain reaction, q-PCR), to confirm the presence of the designed specific nucleic acid molecules, and to confirm their concentration.

Next, referring to fig. 3, fig. 3 is a flowchart illustrating steps of a method 10M for preparing trace particles according to some embodiments of the present invention. It will be appreciated that in some embodiments, additional operational steps may be provided before, during and/or after the process for preparing the trace particles. 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. In addition, the following description of the preparation method can be understood with reference to the structure of the trace particle 20 shown in fig. 2.

As shown in fig. 3, in some embodiments, the method 10M for preparing the trace particles may include forming the core structure 102 (step S12), fixing the nucleic acid molecules 104 on the core structure 102 (step S14), and forming the shell layer 106 on the core structure 102 (step S16) to encapsulate the core structure 102 and the nucleic acid molecules 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 contain a precursor of silicon 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 to the co-emulsifier is between about 5:1 to about 1:10, or between about 1:1 to about 1:10, 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 a C6-C18 medium-long chain alkane, a C6-C18 medium-long chain ester, 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 to the solvent in the oil phase solution is between about 1:1 to about 1:15, or between about 1:3 to about 1:10, for example, about 1:7.

In some embodiments, the ratio (volume ratio) of the precursor of silicon, 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 will be appreciated that the proportions of the silicon precursor, co-emulsifier and solvent need to be controlled within specific ranges so that the trace particles formed have good homogeneity, i.e. have a uniform shape, size or particle size.

Furthermore, 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 salt may comprise cetyltrimethylammonium bromide (hexadecyl trimethyl ammonium bromide, CTAB). In some embodiments, the ratio of water to surfactant in the aqueous solution is between about 1:1 to about 10:1, or between about 10:1 to about 30:1.

In some embodiments, the aqueous phase solution may be heated to a temperature of about 50 ℃ to about 80 ℃, or about 55 ℃ to about 70 ℃, such as about 60 ℃, to dissolve the surfactant in the water prior to adding the oil phase solution to the aqueous phase solution.

Furthermore, 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 an alkaline solution. In some embodiments, the pH of the catalyst ranges from about 8 to about 14. In some embodiments, the catalyst may comprise at least one of ammonia, sodium hydroxide, calcium hydroxide, potassium hydroxide, and an alkaline liquid.

In some embodiments, the temperature of the heated mixed solution may range from about 50 ℃ to about 80 ℃, or from about 55 ℃ to about 70 ℃, for example, 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 allowed to stand at room temperature overnight, after which the supernatant is removed by centrifugation, and subjected to ultrasonic shock extraction using ethanol to obtain the core structure 102 (porous carrier of trace 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 enhance the dispersibility of the porous carrier. In some embodiments, the surface modifying agent may comprise a chlorine-containing quaternary ammonium salt, for example, may comprise N-methyl-3-aminopropyl trimethoxyalkane (trimethoxy [3- (methylamino) propyl ] silane, TMAPS). In detail, in some embodiments, the core structure 102 obtained in the step S12 may be dissolved in a co-emulsifier (e.g., isopropanol), followed by adding a surface modifier and performing shaking and centrifugation, and then 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 may be mixed with the nucleic acid molecule 104, and centrifuged after shaking, so as to fix the nucleic acid molecule 104 on the core structure 102 (step S14). With the foregoing in mind, the nucleic acid molecule 104 may comprise at least one of deoxyribonucleic acid (deoxyribonucleic acid, DNA), ribonucleic acid (RNA), and peptide nucleic acid (peptide nucleic acid, PNA). In some embodiments, the nucleic acid molecule 104 ranges in length from about 10 base pairs to about 2000 base pairs. 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 1:1000, or between 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 an alcohol mixture solution, followed by a step of forming the shell layer 106. In some embodiments, the alcohol-mixed solution may comprise glycerin, ethanol, and water. In some embodiments, the ratio (volume ratio) of glycerin, 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.

Then, a shell layer 106 may be formed on the core structure 102 after immobilization of the nucleic acid molecules 104. In some embodiments, the step of forming the shell layer 106 on the core structure 102 (step S16) may comprise mixing and shaking the core structure 102 immobilized with the nucleic acid molecules 104 with a precursor of silicon and a surface modifier. In some embodiments, the silicon precursor and the surface modifier may be added in two portions and subjected to two shaking.

In some embodiments, the precursor of silicon may comprise Tetraethoxysilane (TEOS). In some embodiments, the surface modifying agent may comprise a chlorine-containing quaternary ammonium salt, for example, may comprise N-methyl-3-aminopropyl trimethoxyalkane (trimethoxy [3- (methylamino) propyl ] silane, TMAPS).

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

In order to make the above and other objects, features and advantages of the present invention more comprehensible, several embodiments, comparative examples, preparation examples and test examples are described in detail below, but are not to be construed as limiting the scope of the present invention.

EXAMPLE 1 design of specific nucleic acid molecules

Sequence design of heterozygous DNA (hybrid DNA)

A50 bp fragment of 16S rDNA of Proteus thermophilus (Tepidimonas fonticaldi, strain AT-A2) and a 50bp fragment of 18S rDNA of Chlamydomonas reinhardtii (Chlamydomonas reinhardtii, strain CC-621) were selected. The hybrid DNA sequence (sequence identification number: 1) with the temperature resistance capability higher than that of the common DNA sequence of 100bp is synthesized as a specific nucleic acid molecule by connecting in series in a mode of 25bp (16S rDNA) -25bp (18S rDNA) -25bp (16S rDNA) -25bp (18S rDNA).

Confirmation of heterozygous DNA sequence uniqueness

The uniqueness of the aforementioned synthetic hybrid DNA sequences was confirmed by alignment using the BLAST (Basic Local ALIGNMENT SEARCH Tool) system of the National Center for Biotechnology Information (NCBI), which showed zero correlation (no SIGNIFICANT SIMILARITY), representing the absence of any similar DNA sequences in the database, demonstrating that the designed hybrid DNA sequences were unique.

EXAMPLE 2 preparation of unique nucleic acid molecules

The DNA sequence (100 bp) with sequence identification number 1 was synthesized by the trusted Gene Synthesis company INTEGRATED DNA Technologies. According to the sequence of the sequence identification number 1, a group of primer pairs of the sequence identification number 2 and the sequence identification number 3 are designed. The DNA sequence of SEQ ID No. 1 is used as a template, and the DNA sequences of SEQ ID Nos. 2 and 3 are used as primers at 3 and 5 ends (melting temperature Tm is 59 ℃ and 63 ℃ respectively), and PCR is performed to amplify the DNA fragment of SEQ ID No. 1 to generate enough specific nucleic acid molecules for the subsequent nucleic acid molecule immobilization step.

The PCR procedure was performed using 10ng (1. Mu.l) template, 2. Mu.l 3-and 5-terminal primers (10. Mu.M), 25. Mu.l 2 XTaq Mastermix and 20. Mu.l ddH ₂ O in a total reaction volume of 50. Mu.l. The temperature conditions for the PCR amplification reaction were set such that the reaction was carried out at 95℃for 1 minute to [95℃for 1 minute to 55℃for 30 seconds to 72℃for 9 seconds ] and the reaction was cycled 12 times to 72℃for 1 minute to 12℃for residence.

The PCR products obtained were analyzed by gel electrophoresis using the materials of 2.5% agarose (agarose), 10 XTBE buffer (Tris-borate-EDTA), 1kb DNA LADDER (as a marker (M)) (CLUBIO) and 6X Loading dye (CLUBIO) and using the DNA electrophoresis system Mupid-2plus (Mupid) to confirm whether the PCR products were designed specific nucleic acid molecules. As shown in FIG. 4, the results of the gel electrophoresis are shown in FIG. 4, wherein A-C are all PCR products, M is DNA LADDER kb of the marker, and the length of the PCR amplified product is 100bp, which is consistent with the length of DNA of sequence identification number 1.

Then, gel/PCR extraction Kit (Biomate) is used for purifying the electrophoresis colloid, and dNTPs and primers which are not used in the PCR process are removed, so that the subsequent DNA immobilization step is prevented from being influenced. After the purification step, a DNA product of SEQ ID No. 1 can be obtained.

EXAMPLE 3 preparation of plasmids containing unique nucleic acid molecules

Using T & A cloning vectorCloning vector Yeastern biotech cloning a target specific sequence (SEQ ID NO: 1), introducing the recombinant plasmid (about 3kb in length) into a host E.coli (ESCHERICHIA COLI, DH 5. Alpha.), culturing the E.coli in a fermentation tank in large quantities, obtaining cells by centrifugation, and extracting the plasmid containing the desired target specific sequence from the cells by alkaline lysis.

The plasmid was cut with restriction enzymes EcoRI and HindIII, and the obtained plasmid was analyzed by gel electrophoresis to confirm whether the length of the obtained plasmid was correct (about 3 kb), and the gel electrophoresis procedure was performed using 1.5% agarose (agarose), 0.5 XTAE 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). As a result of the gel electrophoresis, FIG. 5A shows that A is a plasmid which is not cleaved by restriction enzyme, B is a plasmid which is cleaved by EcoRI, C is a plasmid which is cleaved by HindIII, M is DNA LADDER kb which is a marker 1kb, and it is clear from FIG. 5A that the plasmid obtained by culturing Escherichia coli has a length of about 3kb which corresponds to the length of the plasmid originally constructed.

Next, a PCR procedure was performed using the obtained plasmid as a template and DNA sequences of SEQ ID Nos. 2 and 3 as primers of 3 and 5 ends, and the PCR procedure was performed using the following materials: 10ng (1. Mu.l) of the template, 2. Mu.l of the primers of 3 and 5 ends (10. Mu.M), 25. Mu.l of 2 XTaq Mastermix, and 20. Mu.l of ddH ₂ O, with a total reaction volume of 50. Mu.l. The temperature conditions for the PCR amplification reaction were set to be 95℃for 5 minutes to [95℃for 30 seconds to 60.7℃for 30 seconds to 72℃for 10 seconds ] and 29 cycles to 72℃for 5 minutes to 4℃for residence.

The PCR products obtained were analyzed by gel electrophoresis to determine whether they were target specific sequences (SEQ ID NO: 1), and the materials used for the gel electrophoresis procedure were 2.5% agarose (agarose), 10 XTBE buffer (Tris-buffer-EDTA), 1kb DNA LADDER (as a marker (M)) (CLUBIO) and 6X Loading dye (CLUBIO), and the DNA electrophoresis system Mupid-2plus (Mupid) was used. As shown in FIG. 5B, the results of the gel electrophoresis are shown in FIG. 5B, wherein A-D are all PCR products, M is DNA LADDER kb of the marker, and the length of the PCR amplified product is about 100bp, which is consistent with the length of DNA of sequence identification number 1.

Test example 1 evaluation of risk of synthesized specific nucleic acid molecules on environmental and human bodies

The DNA sequence of the synthesized sequence No. 1 was tested for inhibition of microorganisms in the environment by environmental strain toxicity test. The results are shown in FIGS. 6A-6D, in which FIGS. 6A, 6B and 6C show the toxicity test results of the synthesized DNA sequences on E.coli, bacillus cereus and Pseudomonas putida, respectively, and FIG. 6D shows a control group in which E.coli was inhibited. As is clear from the above results, the DNA sequence of SEQ ID NO. 1 thus synthesized does not inhibit microorganisms common in the environment.

Furthermore, the sequence of sequence identification number 1 was aligned with the sequence of Human chromosome (Human G+T), and the alignment showed E-value >1 (numerical value), the similarity was zero (if E-value <10 ^-5 represents high homology), and therefore the risk of substituting the Human gene was close to zero. In addition, the sequence of the sequence identification number 1 is further split into four fragments of 25bp each for comparison, and the comparison result shows that the E-value is more than 1, the similarity is extremely 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

Corn starch and deionized water were taken to prepare a 35% starch suspension and stirred at 35 ℃. The pH of the starch solution was adjusted to 9.5 using 0.5N NaOH. Next, 20g of sodium hypochlorite was slowly added to the starch solution (addition time greater 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 completed, the pH value of the starch solution is adjusted to 7 by using 1N HCl, and the starch solution is cleaned by secondary water and alcohol, pumped and filtered, and dried in a 50 ℃ oven to obtain the modified starch.

22ML of Tetraethoxysilane (TEOS) was added to 36mL of deionized water and 1mL of 2% HCl and stirred until the hydrolysis was clear. Then, after adding 3g of the modified starch, 20mL of 5% NH ₄ OH was slowly dropped into the vessel via a separating funnel, and stirred for 40 minutes. The solid was filtered off and placed in a 50 ℃ oven for drying for 24 hours. Thereafter, the solid was calcined at a high temperature of 550℃for 3 hours. In this case, a porous carrier (core structure) of the trace particles can be obtained.

2G of the porous carrier was added to 20mL of isopropanol, and the mixture was uniformly dispersed, followed by adding 0.889mL of N-methyl-3-aminopropyl trimethoxyalkane (trimethoxy [3- (methylamino) propyl ] silane, TMAPS) and 1mL of deionized water, and stirring the mixture 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 case, a surface-modified porous carrier can be obtained.

Immobilization of nucleic acid molecules

Mu.L of the surface-modified porous carrier was added to 10. Mu.L of the DNA product (300 ppm) (or 1300 ppm) of the aforementioned sequence No. 1, and after shaking using a shaker, the mixture was centrifuged at 18000RCF for 10 minutes. Thereafter, the supernatant was taken out and washed with secondary water several times, and the solid was dispersed in 500. Mu.L of secondary water.

Encapsulation of shell layer

Next, 0.6. Mu.L of N-methyl-3-aminopropyl trimethoxyalkane (trimethoxy [3- (methylamino) propyl ] silane, TMAPS) and 0.6. Mu.L of TEOS were added thereto and the mixture was shaken at 900rpm for 4 hours. After that, 4. Mu.L of TEOS was added thereto and the mixture was shaken at 900rpm for 96 hours. Next, the supernatant was removed by centrifugation at 19375 RCF for 10 minutes and washed several times with secondary water to disperse the solids in 45. Mu.L of secondary water. In this case, trace particle a can be completed.

Fig. 7A and 7B show scanning electron microscope (scanning electron microscope, SEM) observations of the porous support before and after encapsulation of the shell layer, respectively. From the SEM analysis results, it was observed that the particle size of the trace particles was about 40nm to 50nm (as shown in FIG. 7A) before the encapsulation process, and increased to about 60nm to 75nm (as shown in FIG. 7B) after the encapsulation process.

Example 5 preparation of Trace particle B

Preparation of core Structure and surface modification

An aqueous solution was prepared from 2g of cetyltrimethylammonium bromide (hexadecyl trimethyl ammonium bromide, CTAB) and 30ml of secondary water in a serum bottle and heated to 60℃to dissolve. 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 at room temperature overnight. Then, the mixed solution after completion of the reaction was centrifuged to remove the supernatant, and ultrasonic vibration extraction was performed using ethanol to displace the oil phase solution, whereby a porous carrier (core structure) of the trace particles was obtained.

After centrifugation of the porous carrier, the porous carrier was added to 20mL of isopropyl alcohol and uniformly dispersed, then, 0.222mL of N-methyl-3-aminopropyl Trimethoxyalkane (TMAPS) was added, and after shaking for 18 hours using a shaker, the porous carrier was centrifuged at 15275RCF (relative centrifugal force ) for 10 minutes, the supernatant was removed, and the solid was dispersed in 20mL of secondary water. In this case, a surface-modified porous carrier can be obtained.

Immobilization of nucleic acid molecules

350. Mu.L of the surface-modified porous carrier was added to the plasmid product (100. Mu.L) containing sequence No. 1 prepared in the above example 3, and after centrifugation at 15275RCF for 10 minutes, the supernatant was removed and the solid was dissolved in 5ml of an alcohol mixture (glycerol: ethanol: water=150:150:1).

Encapsulation of shell layer

Next, 6. Mu.l TMAPS and 6. Mu.l TEOS were added and the mixture was shaken for 4 hours using a shaker, and then 40. Mu.l TEOS was added and the mixture was shaken for 4 days using a shaker. Then, 24. Mu.l TMAPS was added and the solution was changed to secondary water by shaking with a shaker for 18 hours. In this case, the trace particle B can be completed.

Fig. 8A and 8B show scanning electron microscope (scanning electron microscope, SEM) observations of the porous support before and after encapsulation of the shell layer, respectively. From the SEM analysis results, it was observed that the particle size of the trace particles was about 30nm to 40nm (as shown in FIG. 8A) before the encapsulation process, and increased to about 50nm to 60nm (as shown in FIG. 8B) after the encapsulation process.

Comparative example 1 preparation of tracer particle C

The preparation of trace particle C was substantially similar to trace particle A of example 4, except trace particle C was not encapsulated in a shell, i.e., the DNA of trace particle C was exposed.

Comparative example 2 preparation of tracer particle D

Method for preparing tracer particle D is generally referred toMethod (1968), and Kim et al (T.G.Kim et al, 2017). Firstly, 50ml of 95% alcohol and 60ml of secondary aqueous solution are prepared and stirred for 15 minutes at a fixed rotation speed of 450rpm, then 20ml of TEOS is added for mixed hydrolysis for 30 minutes, and finally 6ml of 25% ammonia water is added and stirred for 2 hours to polymerize. After the reaction, the liquid was centrifuged at 15275 RCF for 15 minutes, the supernatant was removed, and the solid was washed 3 times with 95% alcohol and dried in a50 ℃ oven. Compared with the porous carrier of the trace particle A, the carrier of the trace particle D is a compact solid carrier, and the porosity is close to 0.

Example 6 desorption procedure of nucleic acid molecules

The DNA-protecting shell was removed with hydrofluoric acid (HF), the DNA was desorbed, 10 μl of the encapsulated tracer particles were added to 40 μl of a 1.5% aqueous HF/NH4F solution and mixed with shaking using a shaker for about 5 minutes, 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 a real-time polymerase chain reaction (q-PCR) analysis, confirming whether it contained the designed specific DNA sequence, and simultaneously measuring its concentration (mass). The q-PCR procedure was performed using 1. Mu.l of template (desorbed DNA), 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 ddH ₂ O, and the total volume of reaction was 25. Mu.l. The temperature conditions for the q-PCR amplification reaction were set such that the reaction was carried out at 95℃for 10 minutes to [95℃for 15 seconds to 60℃for 9 seconds ] and the reaction was cycled 40 times to 12 ℃.

Test example 2 temperature resistance test of tracer particles

The tracer particles a prepared in example 4 and the tracer particles C prepared in comparative example 1 were heated in oil baths at temperatures of 25 ℃, 100 ℃, 120 ℃, 140 ℃, 160 ℃, 180 ℃ and 200 ℃ for 20 minutes, respectively. Then, the trace particles were removed, and the DNA remaining thereon was recovered, and subjected to colloidal electrophoresis analysis.

The results are shown in FIGS. 9A and 9B, and FIGS. 9A and 9B show the results of the temperature resistance test of the trace particles prepared in comparative example 1 and example 4, respectively, in which 25, 100, 120, 140, 160, 180 and 200 represent the heating temperature and M represents the marker (DNA LADDER of 1 kb). As can be seen from the results of fig. 9A and 9B, the high temperature resistance of the trace particle C (naked DNA) of comparative example 1 can be up to about 100 ℃, whereas the high temperature resistance of the trace particle a (packaged DNA) of example 4 can be up to about 200 ℃.

Furthermore, the test was further carried out for the temperature resistance of the trace particle a of example 4 at 120 ℃. Specifically, the recovery rate of DNA was measured at various time points, and the results are shown in Table 1 below.

TABLE 1

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

Furthermore, the test was further conducted for the temperature resistance of trace particle B of example 5 at 120 ℃. The recovery of DNA was measured at various 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 can be seen that the DNA recovery of the encapsulated tracer particle B after 24 hours of heating can still be maintained at 81.5%.

Test example 3 temperature resistance test of tracer particles

The tracer particle a prepared in the foregoing example 4 and the tracer particle D prepared in the comparative example 2 were placed in an oil bath at 120 ℃ and heated. Then, after heating for 1,2 and 2.5 hours, the trace particles were taken out 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 3 below.

TABLE 3 Table 3

As is clear from the results in Table 3, the DNA residue ratio of the trace particle A prepared in example 4 was 79.4% and the DNA residue ratio of the trace particle D prepared in comparative example 2 was 52.4% after heating at 120℃for 2.5 hours. Therefore, the trace particle A with the porous carrier structure has better high temperature resistance than the trace particle D with the solid carrier (without pores).

Test example 4 acid and alkali resistance test of tracer particles

The tracer particles a prepared in example 4 above were placed in solutions having different pH values to test the resistance of the structure of the tracer particles a in acidic and alkaline environments. The preparation of the acidic solution and the alkaline solvent is carried out by sulfuric acid and the 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 removed, and the DNA remaining thereon was recovered and analyzed by colloidal electrophoresis.

As shown in FIG. 10, M in the figure is a marker (DNA LADDER of 1 kb), and as is clear from the results in FIG. 10, the amount of DNA of the trace particle A was hardly reduced in the range of pH 9 or less, was not affected by pH change, and was not significantly reduced in the range of pH 9 or more. From the above results, it is clear that the trace particle a of example 4 has strong acid and strong alkali resistance.

In addition, the trace particle B of example 5 was also tested for acid and alkali resistance, and the trace particle B was placed in solutions of different pH values to test the structural resistance of the trace particle B in acidic and alkaline environments. The preparation of the acidic solution and the alkaline solvent is carried out by sulfuric acid and the acidic solution respectively. The trace particles were placed in the solutions of pH 1, pH 3, pH 5, pH 7, pH 9 and pH 13 for 24 hours, then, the trace particles were taken out and the DNA remaining thereon was recovered, the content of the remaining DNA was measured, and the remaining rate was calculated, and the result was shown in FIG. 11.

As is clear from the results of fig. 11, the DNA amount of the trace 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), was not affected by the pH change, and was not significantly reduced in the range of pH 9 or more, indicating that the trace particle B had strong acid and strong alkali resistance. Notably, after 24 hours of reaction, the DNA content of trace particle B was also not significantly reduced in the range below pH 9, indicating its long-term strong acid resistance.

Test example 5 tracer particle addition in field Hot Water temperature resistance test

The nano-porous carrier prepared in the previous example 5 is fixed with the plasmid of the constructed target DNA label, and then the trace particle B is produced after the encapsulation procedure. The trace particles B are placed in a small reaction tank, and solid hot water is added into the small reaction tank, wherein the solid hot water is respectively volcanic type geothermal zone (China Datun mountain geothermal water, pH1.5 and total dissolved solids in water-9200 ppm) geothermal water and metamorphic rock type geothermal zone geothermal water (China Renze geothermal water, pH8.8 and total dissolved solids in water-4000 ppm). And then individually placed in an oil bath at 120 ℃ for heating. Then, after 480 and 720 hours of heating, the trace particles were taken out and the residual DNA was recovered, and the residual DNA content was measured to calculate the residual rate, and the results are shown in Table 4 below.

TABLE 4 Table 4

As is clear from the results in Table 4, the DNA residue ratio of the trace particle E in the acidic environment was 10.7% and the DNA residue ratio of the trace particle E in the weakly alkaline environment was 7.5% after heating at 120℃for 720 hours. It follows that trace particles E have been shown to be viable for applications that are preliminary to geothermal fields.

Test example 6 column Trace test of Trace particles

To simulate the application of the tracer particles in the actual field (in the soil or formation), a silica sand column was prepared by filling a silica sand column of 0.8cm in diameter and 10.7cm in length with silica sand (0.84 mm) of a No. 20 screen. Generally, the geothermal fluid passageways (slots) have a water conductance (hydraulic conductivity) of about 10 ^-7～10^-2 m/sec and the silica sand column is prepared with a water conductance of about 3.4 x 10 ^-5 m/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 aim of the column tracer test was to investigate the effect of time on the recovery of tracer particles with respect to the extent of diffusion. The results of fig. 12 show the DNA content of the recovered trace particle a over time, and from the change in the DNA content curve, it is clear that, after one injection, a small portion of trace particle a flows out of the shortest channel by advection, while a large portion of trace particle a flows out of the channel (mostly flows out at 140 minutes) in a spreading and diffusion behavior due to maldistribution of the flow field. The above-mentioned transmission behavior corresponds to the usual tracer test results. In addition, the recovery rate of the trace particle A can reach 97.5% by measurement, which means that the trace particle of the embodiment cannot be adsorbed by quartz sand and can flow freely in a low-conductivity fluid channel.

In summary, according to some embodiments of the present invention, the trace particles comprise specific nucleic acid molecules as labels, fingerprints, inorganic materials as substrates and packaging materials, and core structures with moderate porosity are used to increase the fixed amount of nucleic acid molecules and reduce the thermal conductivity (reduce thermal resistance) of the particles, thereby improving the temperature tolerance of the trace particles. In addition, the tracer particles also have acid resistance, alkali resistance and other properties, and the tolerance and recovery rate of the tracer particles in extreme environments can be further improved.

Although embodiments and advantages of the present invention have been disclosed, 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. Furthermore, 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, which will be readily apparent to those skilled in the art from the present disclosure, unless otherwise specified in the present disclosure, such that the process, machine, manufacture, composition of matter, means, methods and steps described in the specification are performed by substantially the same function or achieve substantially the same result as the function described in the embodiments. Accordingly, the present invention is intended to cover such departures from the present disclosure as come within known or customary practice in the art to which this invention pertains. In addition, each claim constitutes a separate embodiment, and the scope of the invention also includes combinations of the individual claims and embodiments. The protection scope of the present invention is defined by the appended claims.

Sequence listing

<110> Institute of technology of financial legal industry

<120> Tracer particle and method of use and preparation thereof

<160> 3

<210> 1

<211> 100

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic 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> Synthetic sequences

<400> 2

taacgggtga cggaggatt 19

<210> 3

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic 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; and

A shell layer covers 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, and the second porosity is substantially 0, and the first porosity ranges from 2nm to 100nm.

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; and

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.