DNA-labeled polyacrylamide nanospheres for displacement and tracing and preparation method and application thereof

Abstract

The present invention relates to the technical field of oil production, and provides a DNA-labeled polyacrylamide nanosphere for oil adjustment and tracing, and a preparation method and application thereof. The polyacrylamide nanosphere has both the oil adjustment and tracing effects of polyacrylamide nanospheres, and can also overcome the problem of limited available quantity of fluorescein and high detection limit, thereby achieving the purpose of multi-source tracing of oil adjustment and tracing agents put into different oil wells and multiple tracing of oil adjustment and tracing agents put into the same oil well at different time periods.

Description

Technical Field

[0001] The invention relates to the technical field of oil production, in particular to a DNA-labeled polyacrylamide nanosphere for adjustment, displacement and tracing, and a preparation method and application thereof. Background Art

[0002] The primary recovery of underground crude oil refers to the crude oil being driven from the underground reservoir to the surface, and the secondary recovery is to provide the pressure required for the crude oil to be driven from the underground to the surface by water injection to recover more crude oil. However, the rock formation after the secondary recovery still contains a lot of oil reservoirs. Water well chemical profile control technology can control the depth profile of the reservoir, directly block the high permeability layer, and increase the coverage range of the liquid entering the formation, so as to achieve the purpose of increasing the ultimate recovery rate of crude oil. Polymer microspheres are a commonly used displacement agent, and polymer microspheres are mainly polyacrylamide microspheres.

[0003] In order to realize the tracing of polyacrylamide nanospheres on the basis of the displacement effect of polyacrylamide nanospheres, the existing polyacrylamide nanospheres are fluorescently functionalized and the polyacrylamide nanospheres are traced by fluorescent indicators. However, the fluorescent indicators have the problems of high detection limit, limited available types and inability to trace multiple sources, which cannot achieve the purpose of multi-source tracing of displacement agents put into different injection wells and multiple tracing of displacement agents put into the same injection well at different times. Therefore, it is urgent to develop a new type of polyacrylamide nanospheres. Summary of the invention

[0004] The purpose of the present invention is to provide a DNA-labeled polyacrylamide nanosphere for regulation, displacement and tracing, and a preparation method and application thereof.

[0005] The concept of the present invention is as follows: The present invention provides a DNA profiler tracer wrapped in polyacrylamide (i.e., DNA-labeled polyacrylamide nanospheres for profiler and tracer) and its preparation method and application. The tracer of the present invention is a DNA-labeled polyacrylamide nanosphere. The present invention provides a DNA profiler tracer wrapped in polyacrylamide, which has both the profiler and tracer effects of polyacrylamide nanospheres, and can also overcome the problem of limited available amount of fluorescein and high detection limit, thereby achieving the purpose of multi-source tracing of profilers put into different oil wells and multiple tracing of profilers put into the same oil well at different time periods.

[0006] Specifically, a hybrid DNA molecule formed by combining a DNA marker and a reverse primer pair and a fluorescent indicator derivative containing a carbon-carbon double bond are first prepared, and then introduced into the synthesis process of polyacrylamide nanospheres to prepare a polyacrylamide-encapsulated DNA modulator tracer by reverse microemulsion polymerization.

[0007] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a method for preparing DNA-labeled polyacrylamide nanospheres for modulation, displacement and tracing, comprising the following steps:

[0008] S1. Preparation of hybrid DNA molecules: prepare a single-stranded DNA solution modified with an acrylamide group at the 5′ end and a reverse primer solution modified with an acrylamide group at the 5′ end respectively with ultrapure water, and mix the single-stranded DNA solution and the reverse primer solution in an equimolar ratio to obtain a hybrid DNA molecule solution;

[0009] S2. Preparation of fluorescent indicator derivatives: reacting 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (Solvent Green 7) with an equimolar amount of halogenated olefins under strong alkaline conditions to generate fluorescent agent derivatives containing carbon-carbon double bonds;

[0010] S3, preparation of fluorescent polyacrylamide nanospheres containing DNA markers, comprising the following sub-steps:

[0011] S3a, dissolving 12 g of a mixture of Span-80 and TX-10 in a mass ratio of 3:1 in 100 g of white oil (mineral oil) to obtain an oil phase;

[0012] S3b, dissolving the fluorescent agent derivative containing a carbon-carbon double bond and acrylamide in the hybrid DNA molecule solution to form an aqueous phase, and adjusting the pH of the system to 7 with NaOH; wherein the molar ratio of the fluorescent agent derivative containing a carbon-carbon double bond, acrylamide and the hybrid DNA molecule is (0.0001-0.01):1:(0.0001-0.01);

[0013] S3c. Under stirring conditions, slowly drop the aqueous phase into the oil phase to form an oil-in-water system; the polymerization reaction is initiated by an initiator under nitrogen protection at 30°C and reacted at 55°C for 3 hours; after the reaction, the reaction product is washed with anhydrous ethanol to obtain a fluorescent polyacrylamide nanosphere emulsion containing DNA markers, which is filtered and dried to obtain a solid powder of fluorescent polyacrylamide nanospheres containing DNA markers.

[0014] Furthermore, the strong alkaline condition in step S2 refers to controlling the pH of the reaction system to 8-10 in the presence of NaOH.

[0015] Preferably, the halogenated olefin is bromobutylene.

[0016] Furthermore, the initiator in sub-step S3c is (NH4)2S2O8-NaHSO3; the added initiator is 0.04-0.10% by mass of acrylamide, preferably 0.08%.

[0017] Furthermore, in sub-step S3c, drying is performed at 60° C. for 24 hours.

[0018] Furthermore, the length of the single-stranded DNA in step S1 is 40-200 bp and does not contain a stem-loop or hairpin structure;

[0019] The reverse primer is 10-30 bp in length and does not contain a stem-loop or hairpin structure;

[0020] The reverse primer is complementary to the single-stranded DNA.

[0021] The structure and synthesis route of the fluorescent polyacrylamide nanospheres containing DNA markers prepared by the present invention are shown in Figure 4 .

[0022] In a second aspect, the present invention provides polyacrylamide nanospheres prepared according to the method.

[0023] Furthermore, the particle size of the polyacrylamide nanospheres is x, 50nm≤x≤60nm.

[0024] In a third aspect, the present invention provides a composition consisting of the polyacrylamide nanospheres, and the hybrid DNA molecules used to prepare the polyacrylamide nanospheres are different.

[0025] In a fourth aspect, the present invention provides the use of the polyacrylamide nanospheres or the composition as an oilfield displacement agent.

[0026] In a fifth aspect, the present invention provides the polyacrylamide nanospheres or the composition for use in oilfield adjustment and displacement, which can improve crude oil recovery and realize multi-source tracing of adjustment and displacement agents.

[0027] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0028] The DNA-labeled fluorescent polyacrylamide nanospheres provided by the present invention have both the oil-displacement effect and the tracing effect on the polyacrylamide nanospheres, and can also overcome the problems of limited available quantity of fluorescein and high detection limit, thereby achieving the purpose of multi-source tracing of oil-displacement agents put into different oil wells and multiple tracing of oil-displacement agents put into the same oil well at different time periods. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a schematic diagram of the hybrid DNA molecules in the present invention connecting polymer chains into a network structure.

[0030] Figure 2 Schematic diagram of a U-shaped device for simulating single-well displacement and tracing in a preferred embodiment of the present invention.

[0031] Figure 3This is a conceptual diagram of a preferred embodiment of the present invention in which a DNA profiler tracer wrapped in polyacrylamide is placed in different water injection wells during crude oil production to perform multi-well joint profile and multi-source tracing.

[0032] Figure 4 The structure and synthesis route of the fluorescent polyacrylamide nanosphere containing DNA markers of the present invention are shown in which the acrylamide group used to modify the 5' end of the DNA chain in the hybrid DNA molecule connects the polymer long chain into a network structure, n is an integer between 10000-40000, and m is an integer between 1-400. DETAILED DESCRIPTION

[0033] Aiming at the shortcomings of existing displacement agent tracers: fluorescent indicators have a high detection limit and limited available types, which make it impossible to achieve the purpose of multi-source tracing of displacement agents put into different oil wells and multiple tracing of displacement agents put into the same oil well at different times. The present invention provides a polyacrylamide-wrapped DNA displacement agent tracer (i.e., DNA-labeled polyacrylamide nanospheres for displacement and tracing), which are polyacrylamide nanospheres with fluorescent labels and containing DNA labels and reverse primer pairs, and are prepared by polymerization of acrylamide as the main polymerization monomer.

[0034] The present invention adopts the following technical solution:

[0035] The polyacrylamide nanosphere is a fluorescence-functionalized nanosphere containing a DNA marker, and the particle size of the fluorescence-functionalized polyacrylamide nanosphere containing a DNA marker is x, 50nm≤x≤60nm.

[0036] The fluorescent indicator used in the nanospheres is 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (Solvent Green 7) (purchased from https: / / www.medchemexpress.cn / Pyranine.html). The derivative containing a carbon-carbon double bond generated by the reaction is introduced into the nanospheres during the polymerization of acrylamide monomers to visualize the penetration of polyacrylamide nanospheres and assist in indicating the sampling time and location.

[0037] The DNA marker of the nanosphere is a single-stranded DNA with a length of 40-200 bp modified by acrylamide at the 5' end. The DNA marker and a reverse primer with a length of 10-30 bp modified by acrylamide at the 5' end are combined to generate a hybrid DNA molecule, which participates in the polymerization reaction during the synthesis of the polyacrylamide nanosphere and becomes a part of the polymer. By introducing different DNA markers and reverse primer pairs into the polymerization process of the polyacrylamide nanosphere, the labeling of different batches of polyacrylamide nanospheres is achieved.

[0038] In the present invention, the DNA sequence introduced in the process of synthesizing the polyacrylamide nanospheres is mainly used as a tracer when the polyacrylamide nanospheres are put into use in oil fields.

[0039] In a specific embodiment of the present invention, the prepared polyacrylamide-wrapped DNA displacement agent tracer is put into the oil field. On the basis of achieving deep displacement, the penetration of polyacrylamide nanospheres is visualized by the fluorescent signal of the fluorescent indicator to assist in determining the sampling time and position. The DNA concentration is further detected to obtain the concentration of the nanospheres at different positions, and the spatiotemporal distribution thereof is obtained to achieve tracing of the polyacrylamide nanospheres.

[0040] In specific applications, the tracer of the present invention can achieve the purpose of multi-temporal and spatial tracing of polyacrylamide nanospheres by introducing polyacrylamide nanospheres labeled with different DNA sequences and reverse primer pairs according to the number of target sources and the different nanosphere delivery times.

[0041] The present invention also provides a method for preparing the polyacrylamide-wrapped DNA modulator tracer, comprising the following steps:

[0042] (1) Preparation of DNA markers and reverse primer pairs: Use ultrapure water to prepare a single-stranded DNA solution modified with an acrylamide group at the 5′ end and a reverse primer solution modified with an acrylamide group at the 5′ end. Add the single-stranded DNA solution and the reverse primer solution in an equimolar ratio into the same centrifuge tube and mix them thoroughly to generate hybrid DNA molecules in which the DNA marker and the reverse primer pair are combined in pairs and both have an acrylamide group at the 5′ end, so as to be introduced in the subsequent synthesis process of polyacrylamide nanospheres.

[0043] (2) Preparation of fluorescent indicator derivatives: 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (Solvent Green 7) was reacted with an equimolar amount of halogenated olefins under strong alkaline conditions (pH 8-10) to generate fluorescent agent derivatives containing carbon-carbon double bonds for subsequent introduction into the synthesis of polyacrylamide nanospheres.

[0044] (3) Preparation of fluorescent polyacrylamide nanospheres containing DNA markers:

[0045] a. Add 12 g of a mixture of Span-80 and TX-10 in a mass ratio of 3:1 into 100 g of white oil (mineral oil) to obtain an oil phase solution into a four-necked flask.

[0046] b. Dissolve the fluorescent agent derivative containing a carbon-carbon double bond and AM in ultrapure water containing DNA to form an aqueous phase, and adjust the pH of the system to 7 with NaOH. The molar ratio of the fluorescent agent derivative containing a carbon-carbon double bond, AM and hybrid DNA molecule is (0.0001-0.01):1:(0.0001-0.01).

[0047] c. Slowly drop the water phase into the stirred oil phase to form an oil-in-water system. The polymerization reaction was initiated by the initiator (NH4)2S2O8-NaHSO3 under nitrogen protection at 30°C and continued to react at 55°C for 3 hours. After the reaction, the obtained DNA-labeled fluorescent polyacrylamide nanosphere emulsion was washed with anhydrous ethanol and filtered with qualitative filter paper. Dry at 60°C for 24 hours to obtain a solid powder of DNA-labeled fluorescent polyacrylamide nanospheres.

[0048] The added initiator is 0.04-0.10% by mass of acrylamide, preferably 0.08%.

[0049] d. By introducing hybrid DNA molecules of different sequences into the polymerization process of polyacrylamide nanospheres, labeling of different batches of polyacrylamide nanospheres is achieved.

[0050] The Span-80 (sorbitol fatty acid ester) and TX-10 (alkylphenol polyoxyethylene ether) are used as emulsifiers, AM (acrylamide) is used as the main polymerization monomer, the fluorescent derivative containing carbon-carbon double bonds participates in the polymerization process of the acrylamide monomer and becomes a part of the polyacrylamide polymer chain, and the single-stranded DNA modified by the 5' end and the reverse primer modified by the 5' end are combined to form a hybrid DNA molecule that connects the linear polymer to form a network structure. The DNA marker is introduced into the polyacrylamide nanosphere when the linear polymer is connected into a network, and is used as a tracer ( Figure 1 ).

[0051] Furthermore, the preparation method comprises the following steps:

[0052] 1) Preparation of DNA markers and reverse primer pairs: Use ultrapure water to prepare a single-stranded DNA solution modified with an acrylamide group at the 5′ end and a reverse primer solution modified with an acrylamide group at the 5′ end. Add the single-stranded DNA solution and the reverse primer solution in an equimolar ratio into the same centrifuge tube to allow them to fully combine to generate hybrid DNA molecules in which the DNA marker and the reverse primer pair are combined in pairs and both have acrylamide groups at the 5′ end, so as to be introduced in the subsequent synthesis process of polyacrylamide nanospheres.

[0053] 2) Preparation of fluorescent indicator derivatives: 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (Solvent Green 7) was reacted with an equimolar amount of halogenated olefins under strong alkaline conditions (pH 8-10). The reaction was carried out in a 100mL four-necked flask at 60°C and stirred for 6 hours to fully react to generate fluorescent agent derivatives containing carbon-carbon double bonds. A condensation device was used in the experiment to prevent the volatilization of allyl chloride. After the reaction was completed, it was dried in a vacuum oven for 24 hours to obtain a dry powder of fluorescent indicator derivatives containing carbon-carbon double bonds, which was prepared for subsequent introduction into the synthesis process of polyacrylamide nanospheres.

[0054] 3) Preparation of fluorescent polyacrylamide nanospheres containing DNA markers:

[0055] a. Add 12 g of a mixture of Span-80 and TX-10 in a mass ratio of 3:1 into 100 g of white oil (mineral oil) to obtain an oil phase solution into a four-necked flask.

[0056] b. Dissolve the fluorescent agent derivative containing a carbon-carbon double bond and AM in ultrapure water containing DNA to form an aqueous phase, and adjust the pH of the system to 7 with NaOH. The molar ratio of the fluorescent agent derivative containing a carbon-carbon double bond, AM and hybrid DNA molecule is (0.0001-0.01):1:(0.0001-0.01).

[0057] c. Slowly drop the water phase into the stirred oil phase to form an oil-in-water system. The polymerization reaction was initiated by the initiator (NH4)2S2O8-NaHSO3 under nitrogen protection at 30°C and continued to react at 55°C for 3 hours. After the reaction, the obtained DNA-labeled fluorescent polyacrylamide nanosphere emulsion was washed with anhydrous ethanol and filtered with qualitative filter paper. Dry at 60°C for 24 hours to obtain a solid powder of DNA-labeled fluorescent polyacrylamide nanospheres.

[0058] The added initiator is 0.04-0.10% by mass of acrylamide, preferably 0.08%.

[0059] d. By introducing hybrid DNA molecules of different sequences into the polymerization process of polyacrylamide nanospheres, labeling of different batches of polyacrylamide nanospheres is achieved.

[0060] DNA chain is used as a tracer for tracing polyacrylamide nanospheres. Because of its large number of optional sequences and low detection limit, it can achieve the purpose of multi-source tracing of the displacement agents put into different oil wells and multiple tracing of the displacement agents put into the same oil well at different times.

[0061] Furthermore, in step 1), the hybrid DNA molecule is prepared by combining a single-stranded DNA whose 5′ end is modified with an acrylamide group and a reverse primer whose 5′ end is modified with an acrylamide group.

[0062] The present invention also provides a DNA modulator tracer wrapped in polyacrylamide prepared according to the method, whose particle size x can be in the range of 50-60nm. According to the number of target sources to be traced and the different time periods for releasing the nanospheres, polyacrylamide nanospheres that can achieve multi-temporal and spatial tracing purposes can be prepared by introducing different DNA sequences.

[0063] The present invention also provides that the displacement agent tracer can simultaneously realize displacement and tracer of polyacrylamide nanospheres in the crude oil production process.

[0064] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0065] Experimental reagents: acrylamide (AM), sorbitan fatty acid ester (Span-80) and alkylphenol polyoxyethylene ether (TX-10), sodium hydroxide (NaOH, 99.7%), 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (Solvent Green 7) (pyranine, purchased from https: / / www.medchemexpress.cn / Pyranine.html), anhydrous ethanol, white oil (purchased from Guangzhou Yuting Chemical Technology Co., Ltd., website http: / / www.gzytchem.com / products_detail.asp?id=376), bromobutylene (purchased from Merck, website h ttps: / / www.sigmaaldrich.cn / CN / zh / product / aldrich / 167851? utm_campaign=LR2_774_Chemical%20Synthesis%2010%20-%20China&utm_medium=cpc&utm_source=baidu&u tm_content=aldrich / 167851&utm_term=%E6%BA%B4%E4%B8%81%E7%83%AF&bd_vid=11386344030092193536).

[0066] The single-stranded DNA with different sequences of 5′-end modified by acrylamide and the reverse primer sequence with 5′-end modified by acrylamide are shown in Table 1.

[0067] Table 1 Sequences and probe sequences used to prepare hybrid DNA molecules (SEQ ID NO: 1-24)

[0068]

[0069]

[0070] Note: The bold underline indicates the forward primer, and the italic underline indicates the reverse primer.

[0071] Example 1 Preparation method of polyacrylamide nanospheres containing DNA markers

[0072] This example aims at preparing DNA profiler tracers wrapped in polyacrylamide for deep profile adjustment and tracing in crude oil production. This example takes the hybrid DNA molecules of the five sequences in Table 1 and their reverse primers as examples. In a specific implementation, DNA molecules of different sequences in Table 1 can be selected to prepare different batches of polyacrylamide nanospheres to obtain polyacrylamide nanospheres labeled with different hybrid DNA molecules. I. Preparation method of polyacrylamide nanospheres containing DNA-1 markers

[0073] 1. Preparation of DNA-1 marker and reverse primer pair: Use ultrapure water to prepare single-stranded DNA solution modified with acrylamide at 5′ end and reverse primer solution modified with acrylamide at 5′ end, add single-stranded DNA-1 solution and DNA-1 reverse primer solution in Table 1 into the same centrifuge tube in equimolar ratio to make them fully combine, generate DNA-1 marker and reverse primer pair combined in pairs, and hybrid DNA-1 molecules with acrylamide at 5′ end, so as to be introduced in the subsequent synthesis process of polyacrylamide nanospheres.

[0074] 2. Preparation of fluorescent indicator derivatives: 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (Solvent Green 7) was reacted with equimolar bromobutylene under strong alkaline conditions (pH 9). The reaction was carried out in a 100mL four-necked flask at 60°C and stirred for 6 hours to fully react to generate fluorescent agent derivatives containing carbon-carbon double bonds. A condensation device was used in the experiment to prevent the volatilization of allyl chloride. After the reaction was completed, it was dried in a vacuum oven for 24 hours to obtain a dry powder of fluorescent indicator derivatives containing carbon-carbon double bonds, which was prepared for subsequent introduction into the synthesis process of polyacrylamide nanospheres.

[0075] 3. Preparation of fluorescent polyacrylamide nanospheres labeled with DNA-1:

[0076] (1) Add 12 g of a mixture of Span-80 and TX-10 in a mass ratio of 3:1 into 100 g of white oil and add the oil phase solution into a four-necked flask.

[0077] (2) Dissolving the fluorescent agent derivative containing a carbon-carbon double bond and AM in ultrapure water containing DNA-1 to form an aqueous phase, and adjusting the pH of the system to 7 with NaOH. The molar ratio of the fluorescent agent derivative containing a carbon-carbon double bond, AM and hybrid DNA-1 molecules is 0.001:1:0.001.

[0078] (3) Slowly drop the water phase into the stirred oil phase to form a water-in-oil system. The polymerization reaction was initiated by the initiator (NH4)2S2O8-NaHSO3 at 30°C under nitrogen protection and continued at 55°C for 3 hours.

[0079] The added initiator is 0.08% based on the mass of acrylamide.

[0080] 4. Wash the emulsion with anhydrous ethanol and filter it with qualitative filter paper.

[0081] 5. Take part of the solution as the sample for TEM measurement, observe the size and structure of the nanospheres with a transmission electron microscope (TEM), and measure their Zeta potential (ζ) and hydrodynamic diameter with a Zetasizer Nano ZS. The particle size of the nanospheres is about 55 nm.

[0082] 6. The remaining polyacrylamide nanosphere solution was dried at 60° C. for 24 hours to obtain a solid powder containing fluorescent polyacrylamide nanospheres labeled with DNA-1.

[0083] 7. Quantitative detection of DNA-1 labeled polyacrylamide nanospheres.

[0084] 10 mg of DNA-1 labeled polyacrylamide nanospheres were dissolved in 1 mL of ultrapure water without DNase, and diluted 10 times. 10 μL of probe supermix (SsoAdvanced Universal Probes Supermix) was added to the 96-well plate, and 6 μL of the diluted sample was added to the premixed solution composed of 1 μL ultrapure water, 1 μL DNA-1 probe, 1 μL DNA-1 forward primer, 1 μL DNA-1 reverse primer and probe supermix and mixed thoroughly to form a 20 μL reaction system in each well plate. The 96-well plate was placed in a real-time quantitative PCR instrument with a set program for the experiment, and the DNA-1 copy number concentration of the sample was obtained by comparison with the standard curve. Among them, the purpose of the gradient dilution is to ensure that the DNA concentration dissolved in the polyacrylamide nanospheres can fall within the range of the standard curve of known concentration. The program setting of the real-time quantitative PCR instrument is as follows: keep it at 50°C for 2 minutes and then heat it to 95°C for 10 minutes. Then, the mixture was kept at 95°C for 15 s and then cooled to 56°C for 1 min (40 cycles).

[0085] 8. The relationship between the tracer mass and the number of encapsulated DNA-1 copies can be obtained from the number of DNA-1 copies at the end of step 7 and the mass of the weighed polyacrylamide nanospheres, that is, how many DNA-1 chains are encapsulated per unit mass of polyacrylamide nanospheres.

[0086] II. Preparation method of polyacrylamide nanospheres containing DNA-2 markers

[0087] 1. Preparation of DNA-2 marker and reverse primer pair: Use ultrapure water to prepare single-stranded DNA solution modified with acrylamide at 5′ end and reverse primer solution modified with acrylamide at 5′ end, add single-stranded DNA-2 solution and DNA-2 reverse primer solution in Table 1 into the same centrifuge tube in an equimolar ratio to allow them to fully combine, thereby generating hybrid DNA-2 molecules in which DNA-2 marker and reverse primer pair are combined in pairs and both have acrylamide groups at 5′ end, so as to be introduced in the subsequent synthesis process of polyacrylamide nanospheres.

[0088] 2. Preparation of fluorescent indicator derivatives: 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (Solvent Green 7) was reacted with equimolar bromobutylene under strong alkaline conditions (pH 9). The reaction was carried out in a 100mL four-necked flask at 60°C and stirred for 6 hours to fully react to generate fluorescent agent derivatives containing carbon-carbon double bonds. A condensation device was used in the experiment to prevent the volatilization of allyl chloride. After the reaction was completed, it was dried in a vacuum oven for 24 hours to obtain a dry powder of fluorescent indicator derivatives containing carbon-carbon double bonds, which was prepared for subsequent introduction into the synthesis process of polyacrylamide nanospheres.

[0089] 3. Preparation of fluorescent polyacrylamide nanospheres labeled with DNA-2:

[0090] (1) Add 12 g of a mixture of Span-80 and TX-10 in a mass ratio of 3:1 into 100 g of white oil and add the oil phase solution into a four-necked flask.

[0091] (2) Dissolving the fluorescent agent derivative containing a carbon-carbon double bond and AM in ultrapure water containing DNA-2 to form an aqueous phase, and adjusting the pH of the system to 7 with NaOH. The molar ratio of the fluorescent agent derivative containing a carbon-carbon double bond, AM and hybrid DNA-2 molecules is 0.001:1:0.001.

[0092] (3) Slowly drop the water phase into the stirred oil phase to form a water-in-oil system. The polymerization reaction was initiated by the initiator (NH4)2S2O8-NaHSO3 at 30°C under nitrogen protection and continued at 55°C for 3 hours.

[0093] The added initiator is 0.08% based on the mass of acrylamide.

[0094] 4. Wash the emulsion with anhydrous ethanol and filter it with qualitative filter paper.

[0095] 5. Take part of the solution as the sample for TEM measurement, observe the size and structure of the nanospheres with a transmission electron microscope (TEM), and measure their Zeta potential (ζ) and hydrodynamic diameter with a Zetasizer Nano ZS. The particle size of the nanospheres is about 55 nm.

[0096] 6. The remaining polyacrylamide nanosphere solution was dried at 60° C. for 24 hours to obtain a solid powder containing fluorescent polyacrylamide nanospheres labeled with DNA-2.

[0097] 7. Quantitative detection of DNA-2 labeled polyacrylamide nanospheres.

[0098] 10 mg of DNA-2 labeled polyacrylamide nanospheres were dissolved in 1 mL of ultrapure water without DNase, and diluted 10 times. 10 μL of probe supermix (SsoAdvanced Universal Probes Supermix) was added to the 96-well plate, and 6 μL of the diluted sample was added to the premixed solution composed of 1 μL ultrapure water, 1 μL DNA-2 probe, 1 μL DNA-2 forward primer, 1 μL DNA-2 reverse primer and probe supermix and mixed thoroughly to form a 20 μL reaction system in each well plate. The 96-well plate was placed in a real-time quantitative PCR instrument with a set program for the experiment, and the DNA-2 copy number concentration of the sample was obtained by comparison with the standard curve. Among them, the purpose of the gradient dilution is to ensure that the DNA concentration dissolved in the polyacrylamide nanospheres can fall within the range of the standard curve of known concentration. The program setting of the real-time quantitative PCR instrument is as follows: keep it at 50°C for 2 minutes and then heat it to 95°C for 10 minutes. Then, the mixture was kept at 95°C for 15 s and then cooled to 56°C for 1 min (40 cycles).

[0099] 8. The relationship between the tracer mass and the number of encapsulated DNA-2 copies can be obtained from the number of DNA-2 copies at the end of step 7 and the mass of the weighed polyacrylamide nanospheres, that is, how many DNA-2 chains are encapsulated per unit mass of polyacrylamide nanospheres.

[0100] III. Preparation method of polyacrylamide nanospheres containing DNA-3 markers

[0101] 1. Preparation of DNA-3 marker and reverse primer pair: Use ultrapure water to prepare single-stranded DNA solution modified with acrylamide at 5′ end and reverse primer solution modified with acrylamide at 5′ end, add single-stranded DNA-3 solution and DNA-3 reverse primer solution in Table 1 into the same centrifuge tube in an equimolar ratio to allow them to fully combine, thereby generating hybrid DNA-3 molecules in which DNA-3 marker and reverse primer pair are combined in pairs and both have acrylamide groups at 5′ end, so as to be introduced in the subsequent synthesis process of polyacrylamide nanospheres.

[0102] 2. Preparation of fluorescent indicator derivatives: 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (Solvent Green 7) was reacted with equimolar bromobutylene under strong alkaline conditions (pH 9). The reaction was carried out in a 100mL four-necked flask at 60°C and stirred for 6 hours to fully react to generate fluorescent agent derivatives containing carbon-carbon double bonds. A condensation device was used in the experiment to prevent the volatilization of allyl chloride. After the reaction was completed, it was dried in a vacuum oven for 24 hours to obtain a dry powder of fluorescent indicator derivatives containing carbon-carbon double bonds, which was prepared for subsequent introduction into the synthesis process of polyacrylamide nanospheres.

[0103] 3. Preparation of fluorescent polyacrylamide nanospheres labeled with DNA-3:

[0104] (1) Add 12 g of a mixture of Span-80 and TX-10 in a mass ratio of 3:1 into 100 g of white oil and add the oil phase solution into a four-necked flask.

[0105] (2) Dissolving the fluorescent agent derivative containing a carbon-carbon double bond and AM in ultrapure water containing DNA-3 to form an aqueous phase, and adjusting the pH of the system to 7 with NaOH. The molar ratio of the fluorescent agent derivative containing a carbon-carbon double bond, AM and hybrid DNA-3 molecules is 0.001:1:0.001.

[0106] (3) Slowly drop the water phase into the stirred oil phase to form a water-in-oil system. The polymerization reaction was initiated by the initiator (NH4)2S2O8-NaHSO3 at 30°C under nitrogen protection and continued at 55°C for 3 hours.

[0107] The added initiator is 0.08% based on the mass of acrylamide.

[0108] 4. Wash the emulsion with anhydrous ethanol and filter it with qualitative filter paper.

[0109] 5. Take part of the solution as the sample for TEM measurement, observe the size and structure of the nanospheres with a transmission electron microscope (TEM), and measure their Zeta potential (ζ) and hydrodynamic diameter with a Zetasizer Nano ZS. The particle size of the nanospheres is about 55 nm.

[0110] 6. The remaining polyacrylamide nanosphere solution was dried at 60° C. for 24 hours to obtain a solid powder containing fluorescent polyacrylamide nanospheres labeled with DNA-3.

[0111] 7. Quantitative detection of DNA-3 labeled polyacrylamide nanospheres.

[0112] 10 mg of DNA-3 labeled polyacrylamide nanospheres were dissolved in 1 mL of ultrapure water without DNase, and diluted 10 times. 10 μL of probe supermix (SsoAdvanced Universal Probes Supermix) was added to the 96-well plate, and 6 μL of the diluted sample was added to the premixed solution composed of 1 μL ultrapure water, 1 μL DNA-3 probe, 1 μL DNA-3 forward primer, 1 μL DNA-3 reverse primer and probe supermix and mixed thoroughly to form a 20 μL reaction system in each well plate. The 96-well plate was placed in a real-time quantitative PCR instrument with a set program for the experiment, and the DNA-3 copy number concentration of the sample was obtained by comparison with the standard curve. Among them, the purpose of the gradient dilution is to ensure that the DNA concentration dissolved in the polyacrylamide nanospheres can fall within the range of the standard curve of known concentration. The program setting of the real-time quantitative PCR instrument is as follows: keep it at 50°C for 2 minutes and then heat it to 95°C for 10 minutes. Then, the mixture was kept at 95°C for 15 s and then cooled to 56°C for 1 min (40 cycles).

[0113] 8. The relationship between the tracer mass and the number of encapsulated DNA-3 copies can be obtained from the number of DNA-3 copies at the end of step 7 and the mass of the weighed polyacrylamide nanospheres, that is, how many DNA-3 chains are encapsulated per unit mass of polyacrylamide nanospheres.

[0114] IV. Preparation method of polyacrylamide nanospheres containing DNA-4 markers

[0115] 1. Preparation of DNA-4 marker and reverse primer pair: prepare single-stranded DNA solution modified with acrylamide at 5′ end and reverse primer solution modified with acrylamide at 5′ end with ultrapure water, add single-stranded DNA-4 solution and DNA-4 reverse primer solution in Table 1 into the same centrifuge tube in equimolar ratio to make them fully combine, generate DNA-4 marker and reverse primer pair combined in pairs, and hybrid DNA-4 molecules with acrylamide at 5′ end, so as to be introduced in the subsequent synthesis process of polyacrylamide nanospheres.

[0116] 2. Preparation of fluorescent indicator derivatives: 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (Solvent Green 7) was reacted with equimolar bromobutylene under strong alkaline conditions (pH 9). The reaction was carried out in a 100mL four-necked flask at 60°C and stirred for 6 hours to fully react to generate fluorescent agent derivatives containing carbon-carbon double bonds. A condensation device was used in the experiment to prevent the volatilization of allyl chloride. After the reaction was completed, it was dried in a vacuum oven for 24 hours to obtain a dry powder of fluorescent indicator derivatives containing carbon-carbon double bonds, which was prepared for subsequent introduction into the synthesis process of polyacrylamide nanospheres.

[0117] 3. Preparation of fluorescent polyacrylamide nanospheres labeled with DNA-4:

[0118] (1) Add 12 g of a mixture of Span-80 and TX-10 in a mass ratio of 3:1 into 100 g of white oil and add the oil phase solution into a four-necked flask.

[0119] (2) Dissolving the fluorescent agent derivative containing a carbon-carbon double bond and AM in ultrapure water containing DNA-4 to form an aqueous phase, and adjusting the pH of the system to 7 with NaOH. The molar ratio of the fluorescent agent derivative containing a carbon-carbon double bond, AM and hybrid DNA-4 molecules is 0.001:1:0.001.

[0120] (3) Slowly drop the water phase into the stirred oil phase to form a water-in-oil system. The polymerization reaction was initiated by the initiator (NH4)2S2O8-NaHSO3 at 30°C under nitrogen protection and continued at 55°C for 3 hours.

[0121] The added initiator is 0.08% based on the mass of acrylamide.

[0122] 4. Wash the emulsion with anhydrous ethanol and filter it with qualitative filter paper.

[0123] 5. Take part of the solution as the sample for TEM measurement, observe the size and structure of the nanospheres with a transmission electron microscope (TEM), and measure their Zeta potential (ζ) and hydrodynamic diameter with a Zetasizer Nano ZS. The particle size of the nanospheres is about 55 nm.

[0124] 6. The remaining polyacrylamide nanosphere solution was dried at 60° C. for 24 hours to obtain a solid powder containing fluorescent polyacrylamide nanospheres labeled with DNA-4.

[0125] 7. Quantitative detection of DNA-4 labeled polyacrylamide nanospheres.

[0126] 10 mg of DNA-4 labeled polyacrylamide nanospheres were dissolved in 1 mL of ultrapure water without DNase, and diluted 10 times. 10 μL of probe supermix (SsoAdvanced Universal Probes Supermix) was added to the 96-well plate, and 6 μL of the diluted sample was added to the premixed solution composed of 1 μL ultrapure water, 1 μL DNA-4 probe, 1 μL DNA-4 forward primer, 1 μL DNA-4 reverse primer and probe supermix and mixed thoroughly to form a 20 μL reaction system in each well plate. The 96-well plate was placed in a real-time quantitative PCR instrument with a set program for the experiment, and the DNA-4 copy number concentration of the sample was obtained by comparison with the standard curve. Among them, the purpose of the gradient dilution is to ensure that the DNA concentration dissolved in the polyacrylamide nanospheres can fall within the range of the standard curve of known concentration. The program setting of the real-time quantitative PCR instrument is as follows: keep it at 50°C for 2 minutes and then heat it to 95°C for 10 minutes. Then, the mixture was kept at 95°C for 15 s and then cooled to 56°C for 1 min (40 cycles).

[0127] 8. The relationship between the tracer mass and the number of encapsulated DNA-4 copies can be obtained from the number of DNA-4 copies at the end of step 7 and the mass of the weighed polyacrylamide nanospheres, that is, how many DNA-4 chains are encapsulated per unit mass of polyacrylamide nanospheres.

[0128] V. Preparation method of polyacrylamide nanospheres containing DNA-5 markers

[0129] 1. Preparation of DNA-5 marker and reverse primer pair: Use ultrapure water to prepare single-stranded DNA solution modified with acrylamide at 5′ end and reverse primer solution modified with acrylamide at 5′ end, add single-stranded DNA-5 solution and DNA-5 reverse primer solution in Table 1 into the same centrifuge tube in equimolar ratio to make them fully combine, generate DNA-5 marker and reverse primer pair combined in pairs, and hybrid DNA-5 molecules with acrylamide at 5′ end, so as to be introduced in the subsequent synthesis process of polyacrylamide nanospheres.

[0130] 2. Preparation of fluorescent indicator derivatives: 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (Solvent Green 7) was reacted with equimolar bromobutylene under strong alkaline conditions (pH 9). The reaction was carried out in a 100mL four-necked flask at 60°C and stirred for 6 hours to fully react to generate fluorescent agent derivatives containing carbon-carbon double bonds. A condensation device was used in the experiment to prevent the volatilization of allyl chloride. After the reaction was completed, it was dried in a vacuum oven for 24 hours to obtain a dry powder of fluorescent indicator derivatives containing carbon-carbon double bonds, which was prepared for subsequent introduction into the synthesis process of polyacrylamide nanospheres.

[0131] 3. Preparation of fluorescent polyacrylamide nanospheres labeled with DNA-5:

[0132] (1) Add 12 g of a mixture of Span-80 and TX-10 in a mass ratio of 3:1 into 100 g of white oil and add the oil phase solution into a four-necked flask.

[0133] (2) Dissolving the fluorescent agent derivative containing a carbon-carbon double bond and AM in ultrapure water containing DNA-5 to form an aqueous phase, and adjusting the pH of the system to 7 with NaOH. The molar ratio of the fluorescent agent derivative containing a carbon-carbon double bond, AM and hybrid DNA-5 molecules is 0.001:1:0.001.

[0134] (3) Slowly drop the water phase into the stirred oil phase to form a water-in-oil system. The polymerization reaction was initiated by the initiator (NH4)2S2O8-NaHSO3 at 30°C under nitrogen protection and continued at 55°C for 3 hours.

[0135] The added initiator is 0.08% based on the mass of acrylamide.

[0136] 4. Wash the emulsion with anhydrous ethanol and filter it with qualitative filter paper.

[0137] 5. Take part of the solution as the sample for TEM measurement, observe the size and structure of the nanospheres with a transmission electron microscope (TEM), and measure their Zeta potential (ζ) and hydrodynamic diameter with a Zetasizer Nano ZS. The particle size of the nanospheres is about 55 nm.

[0138] 6. The remaining polyacrylamide nanosphere solution was dried at 60° C. for 24 hours to obtain a solid powder containing fluorescent polyacrylamide nanospheres labeled with DNA-5.

[0139] 7. Quantitative detection of DNA-5 labeled polyacrylamide nanospheres.

[0140] 10 mg of DNA-5 labeled polyacrylamide nanospheres were dissolved in 1 mL of ultrapure water without DNase, and diluted 10 times. 10 μL of probe supermix (SsoAdvanced Universal Probes Supermix) was added to the 96-well plate, and 6 μL of the diluted sample was added to the premixed solution composed of 1 μL ultrapure water, 1 μL DNA-5 probe, 1 μL DNA-5 forward primer, 1 μL DNA-5 reverse primer and probe supermix and mixed thoroughly to form a 20 μL reaction system in each well plate. The 96-well plate was placed in a real-time quantitative PCR instrument with a set program for the experiment, and the DNA-5 copy number concentration of the sample was obtained by comparing with the standard curve. Among them, the purpose of the gradient dilution is to ensure that the DNA concentration dissolved in the polyacrylamide nanospheres can fall within the range of the standard curve of known concentration. The program setting of the real-time quantitative PCR instrument is as follows: keep it at 50°C for 2 minutes and then heat it to 95°C for 10 minutes. Then, the mixture was kept at 95°C for 15 s and then cooled to 56°C for 1 min (40 cycles).

[0141] 8. The relationship between the tracer mass and the number of encapsulated DNA-5 copies can be obtained from the number of DNA-5 copies at the end of step 7 and the mass of the weighed polyacrylamide nanospheres, that is, how many DNA-5 chains are encapsulated per unit mass of polyacrylamide nanospheres.

[0142] Example 2 Single and multiple displacement tracing methods of DNA-labeled polyacrylamide nanospheres

[0143] The DNA-labeled polyacrylamide nanospheres prepared in Example 1 are used to simultaneously realize deep displacement and tracing in the crude oil production process, thereby increasing the oil recovery rate by 8%-16% compared with water flooding, and realizing multiple displacement and tracing.

[0144] The oil-bearing layer core with a length of 30 cm and a diameter of 5 cm is collected from the oil field where the tertiary oil recovery is planned. The horizontal section of the U-shaped device simulates the underground oil-bearing layer. The vertical section with a height of 10 cm and a diameter of 2 cm on the left simulates the water injection well, and the vertical section with a height of 10 cm and a diameter of 2 cm on the right simulates the oil production well ( Figure 2 ).

[0145] Tracing effect: 1 pore volume of 1.5g / L DNA-1 labeled polyacrylamide nanospheres are continuously injected into the U-shaped device from the water injection end, and samples are collected from the oil production end. During the sampling process, a visible light irradiation device is used to observe the position and time of green fluorescence. The concentration of DNA-1 labeled polyacrylamide nanospheres in the sample is measured by the method of steps 7-8 in Example 1, and the penetration curve of polyacrylamide nanospheres in the core is drawn. According to the position and time of fluorescence appearance and the penetration curve of polyacrylamide nanospheres, the relationship between the penetration time of polyacrylamide nanospheres and the green fluorescence appearance time of fluorescein can be established, and the time difference between observing green fluorescence and sampling in the field field experiment can be obtained.

[0146] Multiple tracing effect: First, 1 pore volume of 1.5 g / L DNA-2 labeled polyacrylamide nanospheres were injected into the U-shaped device from the water injection end, and then 1 pore volume of 1.5 g / L DNA-3 labeled polyacrylamide nanospheres were injected into the U-shaped device from the water injection end, and then 1 pore volume of 1.5 g / L DNA-4 labeled polyacrylamide nanospheres were injected into the U-shaped device from the water injection end, and samples were continuously collected from the oil production end during the injection process. The concentrations of DNA-2, DNA-3, DNA-4 and DNA-5 labeled polyacrylamide nanospheres in the sample were measured using the method of steps 7-8 in Example 1, and the penetration curves of multiple displacement and tracing polyacrylamide nanospheres labeled with DNA-2, DNA-3, DNA-4 and DNA-5 in the core were drawn.

[0147] Example 3 Multi-well joint flooding tracing method containing DNA-labeled polyacrylamide nanospheres

[0148] The DNA-labeled polyacrylamide nanospheres prepared in Example 1 were used for multi-well joint flooding and tracing, which could increase the oil recovery rate by 8%-16% compared with water flooding and achieve multi-well joint flooding and multi-source tracing.

[0149] Select the oil field where tertiary oil recovery is planned. Figure 3 The water injection wells No. 1-8 and the oil production wells No. 1-7 shown in the figure were injected with the DNA-labeled polyacrylamide nanospheres in Example 1, and the DNA-labeled polyacrylamide nanospheres in Example 1 were subjected to multi-well joint flooding and tracing for field evaluation ( Figure 3 ).

[0150] DNA-1 to DNA-8 labeled polyacrylamide nanospheres prepared by the method in Example 1 were injected into water injection wells 1-8, and samples were collected from oil production wells 1-7. Visible light was used to irradiate the oil production wells, and the relationship between the penetration time of DNA-labeled polyacrylamide nanospheres and the appearance time of green fluorescence established in Example 2 was used, and the green fluorescence signal was used as an auxiliary to determine the sampling time on site. The concentration of DNA-1 to DNA-8 labeled polyacrylamide nanospheres in the samples collected from oil production wells 1-7 was determined by the method of steps 7-8 in Example 1, and the penetration curves of DNA-1 to DNA-8 labeled polyacrylamide nanospheres injected from water injection wells 1-8 and penetrated from oil production wells 1-7 were drawn, thereby realizing multi-well joint adjustment and tracing between multiple water injection wells and multiple oil production wells.

[0151] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing DNA-labeled polyacrylamide nanospheres for regulation, displacement and tracing, characterized in that: The following steps are involved: S1. Preparation of hybrid DNA molecules: prepare a single-stranded DNA solution modified with an acrylamide group at the 5' end and a reverse primer solution modified with an acrylamide group at the 5' end with ultrapure water, respectively, and mix the single-stranded DNA solution and the reverse primer solution in an equimolar ratio to obtain a hybrid DNA molecule solution; S2. Preparation of fluorescent indicator derivatives: reacting 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt with an equimolar amount of halogenated olefins under strong alkaline conditions to generate fluorescent agent derivatives containing carbon-carbon double bonds; S3, preparation of fluorescent polyacrylamide nanospheres containing DNA markers, comprising the following sub-steps: S3a, dissolving 12 g of a mixture of Span-80 and TX-10 in a mass ratio of 3:1 in 100 g of white oil to obtain an oil phase; S3b, dissolving the fluorescent agent derivative containing a carbon-carbon double bond and acrylamide in the hybrid DNA molecule solution to form an aqueous phase, and adjusting the pH of the system to 7 with NaOH; wherein the molar ratio of the fluorescent agent derivative containing a carbon-carbon double bond, acrylamide and the hybrid DNA molecule is (0.0001-0.01):1:(0.0001-0.01); S3c, under stirring conditions, slowly dripping the water phase into the oil phase to form an oil-in-water system; the polymerization reaction is initiated by an initiator under nitrogen protection at 30°C and reacted at 55°C for 3 hours; after the reaction is completed, the reaction product is washed with anhydrous ethanol to obtain a fluorescent polyacrylamide nanosphere emulsion containing DNA markers, which is filtered and dried to obtain a solid powder of fluorescent polyacrylamide nanospheres containing DNA markers; The length of the single-stranded DNA in step S1 is 40-200 bp and does not contain a stem-loop or hairpin structure; The reverse primer is 10-30 bp in length and does not contain a stem-loop or hairpin structure; The reverse primer is complementary to the single-stranded DNA.

2. The method according to claim 1, characterized in that The strong alkaline condition in step S2 refers to controlling the pH of the reaction system to 8-10 in the presence of NaOH; The halogenated olefin is bromobutylene.

3. The method according to claim 1, characterized in that The initiator in sub-step S3c is (NH4)2S2O8-NaHSO3, and the added initiator is 0.04-0.10% based on the mass of acrylamide.

4. The method according to claim 3, characterized in that: The initiator in sub-step S3c is (NH4)2S2O8-NaHSO3, and the added initiator is 0.08% based on the mass of acrylamide.

5. The method according to claim 1, characterized in that In sub-step S3c, the product is dried at 60°C for 24 hours.

6. Polyacrylamide nanospheres prepared according to the method according to any one of claims 1 to 5.

7. The polyacrylamide nanospheres according to claim 6, characterized in that: Its particle size is x, 50 nm≤x≤60nm.

8. A composition, characterized in that The polyacrylamide nanospheres are composed of the polyacrylamide nanospheres according to claim 6 or 7, and the hybrid DNA molecules used to prepare the polyacrylamide nanospheres are different.

9. Use of the polyacrylamide nanospheres according to claim 6 or 7 or the composition according to claim 8 as an oilfield displacement agent.

10. The polyacrylamide nanospheres according to claim 6 or 7 or the composition according to claim 8 are used for oil field adjustment and displacement, and are used in multi-source tracing of adjustment and displacement agents while improving crude oil recovery.

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