Preparation method and application of auxiliary broken wall nanometer magnetic beads for extracting archaea DNA

Abstract

The application relates to the technical field of archaea DNA extraction, in particular to a preparation method and application of auxiliary wall-breaking nanometer magnetic beads for archaea DNA extraction. The method comprises the following steps: (1) constructing an inverse emulsion droplet microfluidic chip: washing, drying and hydrophobic modification treatment are performed on a PDMS base material to prepare a microfluidic chip; (2) preparing an inverse microemulsion system: the continuous phase and the dispersed phase are respectively pumped into the microfluidic chip at a predetermined flow rate, so that Fe 2+ and Fe 3+ undergo a coprecipitation reaction to generate Fe3O4 nanoparticles; (3) maturation crystallization; (4) demulsification separation and washing; and (5) drying to obtain auxiliary wall-breaking nanometer magnetic beads. The method has the beneficial effect of solving the problems in the prior art that the cell wall is difficult to effectively break, the DNA release rate is low, the extraction purity is poor, the repeatability is insufficient and the standardization and continuous preparation are difficult to realize when DNA extraction is performed on archaea samples in extreme environments.

Description

Technical Field

[0001] This invention relates to the field of archaic DNA extraction technology, and in particular to a method for preparing and applying auxiliary cell-wall-breaking magnetic nanobeads for archaic DNA extraction. Background Technology

[0002] Currently, most existing kits are designed for DNA extraction from common bacteria, fungi, viruses, and mammalian cells. However, there are relatively few kits specifically designed for DNA extraction from archaea in extreme environments (such as extremophiles). The unique characteristics of archaea (such as harsh growth environments and unique cell walls) mean that different processing methods are required during cell disruption and extraction compared to those used for conventional microorganisms.

[0003] DNA extraction is a routine procedure in molecular biology, typically used to extract and purify DNA samples from cells or tissues. Common DNA extraction methods include: The phenol / chloroform method: This is a traditional chemical method often used for large-scale DNA extraction. By using a mixed solvent of phenol and chloroform, DNA can be effectively separated from other cellular components. However, this method is toxic and cumbersome. Commercial kit methods: Currently, there are various commercial DNA extraction kits available on the market, such as those from QIAGEN and Thermo Fisher, suitable for DNA extraction from cells and tissues. These kits typically utilize magnetic bead technology to efficiently extract nucleic acids through magnetic separation. These methods are suitable for DNA extraction from common biological samples such as mammalian cells, bacteria, and fungi, but are often less effective for DNA extraction from archaea in extreme environments.

[0004] Archaea are microorganisms capable of surviving in extreme environments, generally classified into thermophilic archaea, halophilic archaea, and acidophilic archaea. They exhibit extremely high tolerance to environmental conditions such as temperature, salinity, and pH. Because the cell wall and cell membrane structures of these archaea differ from those of conventional bacteria, their DNA extraction process faces the following challenges: **Unique Cell Walls:** Archaea have complex cell wall structures, containing relatively robust layers of glycans or proteins. This makes it difficult for traditional cell wall disruption methods (such as enzymatic hydrolysis or physical disruption) to efficiently break down their cell walls, thus limiting DNA extraction efficiency. **Growth Characteristics in Extreme Environments:** Many extreme-environment archaea grow in environments with temperatures exceeding 80°C, extremely high salinity, or extremely acidic conditions. Their cell walls and membranes are much more robust and tolerant of extreme environments compared to ordinary microorganisms, making their DNA extraction process very difficult.

[0005] While several methods exist for DNA extraction from bacteria, fungi, and mammalian cells, these methods face numerous challenges when extracting DNA from archaea grown in extreme environments. Conventional enzymatic digestion and ultrasonic cell disruption methods are ineffective for archaea, especially those grown in high-temperature or high-salt conditions. Traditional methods have limited effectiveness against the rigid cell walls of archaea, resulting in low extraction efficiency. Furthermore, most commercially available DNA extraction kits are designed for conventional bacteria or mammalian cells; there are no dedicated kits for archaea in extreme environments, particularly thermophilic and halophilic archaea. Researchers must rely on non-specific methods, making the extraction process complex and inefficient. Even when a certain amount of DNA is extracted, incomplete cell disruption leads to low DNA purity, and some DNA fragments may be degraded or lost. Summary of the Invention

[0006] The purpose of this invention is to address the aforementioned deficiencies in the existing technology by providing a method for preparing and applying auxiliary cell wall-breaking magnetic nanobeads for archaeal DNA extraction. This addresses the problems in the existing technology for DNA extraction from archaeal samples in extreme environments, such as difficulty in effectively breaking cell walls, low DNA release rate, poor extraction purity, insufficient reproducibility, and difficulty in achieving standardized and continuous preparation.

[0007] The present invention discloses a method for preparing auxiliary cell-wall-breaking magnetic nanobeads for archaeal DNA extraction, the technical solution of which includes the following steps: (1) Constructing a microfluidic chip for reverse emulsion droplets: The PDMS substrate was cleaned, dried and hydrophobically modified to prepare a microfluidic chip with a continuous phase oil phase channel, a dispersed phase aqueous phase channel, a shrinkage zone channel and an outlet zone channel, so as to achieve stable formation and particle size preparation of reverse emulsion droplets; (2) Preparation of a reverse microemulsion system: FeCl2 and FeCl3 were dissolved in high-purity water with dissolved oxygen removed at a molar ratio of 1:2 to obtain an iron salt dispersed phase; Span80, n-hexanol, and cyclohexane were mixed to obtain a continuous oil phase; an alkaline solution was prepared separately as a precipitant; the continuous phase and dispersed phase were pumped into a microfluidic chip at a predetermined flow rate to form monodisperse droplets under constant temperature and inert gas protection conditions, and the pH value of the reaction system was adjusted to the alkaline range by introducing an alkaline solution, so that Fe 2+ and Fe 3+ A coprecipitation reaction occurs to generate Fe3O4 nanoparticles; (3) Maturation and crystallization: The reverse microemulsion droplets collected at the outlet of the microfluidic chip are further matured at 50°C and maintained for a certain time under stirring and nitrogen protection to promote the full formation of Fe3O4 crystals and improve the crystallinity, dispersibility and magnetic response performance of the particles. (4) Demulsification and washing: Demulsifier was added to the matured reverse microemulsion system to separate the oil and water phases. The lower aqueous phase was taken out for low-temperature high-speed centrifugation to obtain Fe3O4 nanoparticle precipitate. Then, anhydrous ethanol and ultrapure water were used for alternating washing, and ultrasonic dispersion was combined to remove residual surfactants and impurities. (5) Drying to obtain auxiliary cell wall breaking magnetic beads: The washed Fe3O4 nanoparticles were freeze-dried under low temperature vacuum conditions to obtain Fe3O4 auxiliary cell wall breaking magnetic beads with uniform particle size, good dispersibility and strong magnetic response performance.

[0008] Preferably, the fabrication process of the above-mentioned reverse emulsion droplet microfluidic chip is as follows: Step 1: After curing the PDMS film, rinse it sequentially with analytical grade anhydrous ethanol and ultrapure water, and then dry it in a nitrogen atmosphere. Step 2: The above-mentioned PDMS sheets are further subjected to surface hydrophobic treatment by completely immersing the PDMS sheets in trimethylchlorosilane for a period of time; after removing the PDMS sheets, they are dried in an oven at 40-60℃. Step 3: Fix the PDMS sheet on the water platform of the high-resolution camera objective lens, drop a drop of water on the surface of the PDMS sheet using a microsyringe, capture the outline of the droplet with the camera, and measure the tangent of the contact point between the water droplet and the surface of the PDMS sheet to calculate the contact angle of the hydrophobic sheet. Step 4: Design the continuous phase oil phase channel size to be 200×80 μm, the dispersed phase aqueous phase channel size to be 100×80 μm, the contraction zone channel size to be 80×80 μm, the outlet zone channel size to be 100×80 μm, and the collection section channel length to be 15cm. Step 5: Photolithographically etch the microfluidic chip channels from Step 4 onto the hydrophobically treated PDMS wafer, and complete the encapsulation.

[0009] Preferably, the contact angle of the hydrophobic sheet is greater than 120°.

[0010] Preferably, the process for preparing the reverse microemulsion system described above is as follows: Step 1: Connect the cleanroom hose to the high-purity nitrogen cylinder and introduce high-purity water to blow out the dissolved oxygen. Step 2: Weigh 0.00033 mol of spectroscopically pure anhydrous FeCl2 and 0.00066 mol of spectroscopically pure anhydrous FeCl3 using an analytical balance, dissolve them in the above high-purity water, and dilute to 100 ml using a volumetric flask. Seal and set aside for later use. Step 3: Dissolve 10.000g of spectroscopically pure Span 80 and 5.000ml of n-hexanol in 95.240ml of spectroscopically pure cyclohexane; Step 4: Dissolve 11.999g of spectroscopically pure sodium hydroxide in high-purity water, and bring the volume to 100ml using a volumetric flask. Seal and set aside for later use. Step 5: The microfluidic chip is placed in a constant temperature water bath. The emulsified oil solution formed in step 3 is pumped into the continuous phase injection channel of the microfluidic chip as the continuous phase, and the iron salt aqueous solution in step 2 is pumped into the dispersed phase channel of the microfluidic chip as the dispersed phase. The residence time of the fluid in the microfluidic chip exceeds 20 minutes. Step 6: The droplet generation frequency is 120 / min, the droplet size is 20nm, and the monodispersity is greater than 95%; Step 7: Pump the microfluidic chip into the microchannel reactor at a rate of 2 μL / min using a three-channel pump, adjust the pH of the microchannel reactor to 10 ± 0.2, collect the droplets in a brown bottle, maintain a 50°C water bath and stir with a magnetic stirrer at 350 r / min, and continuously bubble with nitrogen for protection. Step 8: Continue crystallization and ripening in a brown bottle for 90 minutes.

[0011] Preferably, the microfluidic chip is placed in a constant temperature water bath at 50±1℃. The emulsified oil solution formed in step three is used as the continuous phase and pumped into the continuous phase injection channel of the microfluidic chip at a rate of 10 μL / min. The iron salt aqueous solution from step two is used as the dispersed phase and pumped into the dispersed phase channel of the microfluidic chip at a rate of 1 μL / min. The pumping pressure is kept below 1.0 bar, and the residence time of the fluid in the microfluidic chip is more than 20 min.

[0012] Preferably, the post-treatment and drying process of the above Fe3O4 nanoparticles is as follows: 1. Add 50% by volume of spectroscopically pure anhydrous ethanol as a demulsifier to the matured reverse microemulsion crystals, stir under magnetic stirring, let stand and separate into layers, and take the lower aqueous phase for later use. 2. Use a pipette to draw up the lower aqueous phase from the above-mentioned layering and inject it into an anti-adsorption PTFE centrifuge tube. Centrifuge with a high-speed refrigerated centrifuge, discard the liquid phase after centrifugation, and keep the solid phase for later use. 3. Wash with anhydrous ethanol and ultrapure water in sequence, alternating multiple times, and disperse with an ultrasonic disperser after each wash. 4. Dry in a vacuum freeze dryer at -55℃ and a vacuum degree <10Pa for 24 hours; 5. Obtain a reddish-brown powder, wherein the iron oxide powder with a particle size of 100nm or larger is black, and the iron oxide powder with a particle size of 25-100nm is dark brown, and store it in a brown bottle in a sealed container. 6. Take a small amount of sample and place it in a PTFE centrifuge tube. Disperse it intermittently with 19 times the mass of anhydrous ethanol by ultrasonication. Then, drop it onto a silicon wafer for spectroscopy and perform SEM and TEM observations. 7. Characterize the reddish-brown powder.

[0013] Preferably, the lower aqueous phase of the above-mentioned layer is aspirated with a pipette and injected into an anti-adsorption PTFE centrifuge tube. The tube is then centrifuged twice at 12000 r / min for 15 min each time at 4°C using a high-speed refrigerated centrifuge. The liquid phase after centrifugation is discarded, and the solid phase is kept for later use.

[0014] Preferably, the extreme environment archaea in step (6) above are thermophilic archaea, halophilic archaea, and / or acidophilic archaea.

[0015] The application of Fe3O4-assisted cell wall disruption nanomagnetic beads for archaeological DNA extraction mentioned in this invention involves the following technical solution: Fe3O4-assisted cell wall disruption nanomagnetic beads are applied to the separation and DNA extraction of archaea in extreme environments within fluid inclusions. Under the action of mechanical disturbance, chemical lysis, or extraction buffer system, the Fe3O4 nanomagnetic beads, through contact and collision with the archaeological cell wall / cell surface and the assisted cell wall disruption effect, improve the cell wall rupture efficiency, promote DNA release, and increase the amount, purity, and stability of extracted DNA.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention utilizes magnetite (Fe3O4) magnetic nanoparticles as the core material, combined with reverse emulsion droplet microfluidic technology, to achieve controllable preparation of magnetic beads. By precisely controlling the microfluidic chip structure, oil-water two-phase system composition, iron salt concentration, flow rate, reaction temperature, pH, and ripening conditions, this invention produces Fe3O4 nanoparticles with uniform particle size, good dispersibility, and excellent magnetic response performance. After demulsification, centrifugation, washing, ultrasonic dispersion, and freeze-drying, auxiliary cell wall disruption magnetic beads are obtained. These magnetic beads can enhance cell wall disruption efficiency, improve DNA release rate, extraction purity, and operational stability during the extraction of archaea DNA in extreme environments, and are particularly suitable for thermophilic, halophilic, and acidophilic archaea samples. Compared with existing technologies, this invention has advantages such as high cell wall disruption efficiency, good extraction effect, strong reproducibility, ease of standardization, and continuous production, and can be widely applied in extreme environment microbiology research, environmental monitoring, metagenomic sequencing, and archaea resource development. Attached Figure Description

[0017] Figure 1 These are morphological characterization images of iron oxide particles at different magnifications and a 500nm scale; Figure 2 These are morphological characterization images of iron oxide particles at different magnifications and a 300nm scale; Figure 3 These are morphological characterization images of iron oxide particles at different magnifications and a 200nm scale; Figure 4 The XRD pattern of Fe3O4-assisted cell wall disruption nanoparticles is shown. Figure 5This is the XPS full spectrum of Fe3O4-assisted cell wall disruption nanoparticles; Figure 6 This is the high-resolution XPS spectrum of Fe 2p in Fe3O4-assisted cell wall breaking nanoparticles; Figure 7 This is the O 1s high-resolution XPS spectrum of Fe3O4-assisted cell wall breaking nanoparticles; Detailed Implementation

[0018] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0019] Example 1: A method for preparing auxiliary cell-wall-breaking magnetic nanobeads for archaeal DNA extraction mentioned in this invention includes the following steps: (1) Constructing a microfluidic chip for reverse emulsion droplets: The PDMS substrate was cleaned, dried and hydrophobically modified to prepare a microfluidic chip with a continuous phase oil phase channel, a dispersed phase aqueous phase channel, a shrinkage zone channel and an outlet zone channel, so as to achieve stable formation and particle size preparation of reverse emulsion droplets; (2) Preparation of a reverse microemulsion system: FeCl2 and FeCl3 were dissolved in high-purity water with dissolved oxygen removed at a molar ratio of 1:2 to obtain an iron salt dispersed phase; Span80, n-hexanol, and cyclohexane were mixed to obtain a continuous oil phase; an alkaline solution was prepared separately as a precipitant; the continuous phase and dispersed phase were pumped into a microfluidic chip at a predetermined flow rate to form monodisperse droplets under constant temperature and inert gas protection conditions, and the pH value of the reaction system was adjusted to the alkaline range by introducing an alkaline solution, so that Fe 2+ and Fe 3+ A coprecipitation reaction occurs to generate Fe3O4 nanoparticles; (3) Maturation and crystallization: The reverse microemulsion droplets collected at the outlet of the microfluidic chip are further matured at 50°C and maintained for a certain time under stirring and nitrogen protection to promote the full formation of Fe3O4 crystals and improve the crystallinity, dispersibility and magnetic response performance of the particles. (4) Demulsification and washing: Demulsifier was added to the matured reverse microemulsion system to separate the oil and water phases. The lower aqueous phase was taken out for low-temperature high-speed centrifugation to obtain Fe3O4 nanoparticle precipitate. Then, anhydrous ethanol and ultrapure water were used for alternating washing, and ultrasonic dispersion was combined to remove residual surfactants and impurities. (5) Drying to obtain auxiliary cell wall breaking magnetic beads: The washed Fe3O4 nanoparticles were freeze-dried under low temperature vacuum conditions to obtain Fe3O4 auxiliary cell wall breaking magnetic beads with uniform particle size, good dispersibility and strong magnetic response performance.

[0020] Specifically, the fabrication process of the reverse emulsion droplet microfluidic chip mentioned in this invention is as follows: Step 1: After curing the PDMS film, rinse it sequentially with analytical grade anhydrous ethanol and ultrapure water, and then dry it in a nitrogen atmosphere. Step 2: The above-mentioned PDMS sheets are further subjected to surface hydrophobic treatment by completely immersing the PDMS sheets in trimethylchlorosilane for 30 minutes; after removing the PDMS sheets, they are dried in an oven at 40-60℃ for 2 hours. Step 3: Fix the PDMS sheet on the water platform of the high-resolution camera objective lens, drop a drop of water on the surface of the PDMS sheet using a microsyringe, capture the outline of the droplet with the camera, and measure the tangent of the contact point between the water droplet and the surface of the PDMS sheet to calculate the contact angle of the hydrophobic sheet. Step 4: Design the continuous phase oil phase channel size to be 200×80 μm, the dispersed phase aqueous phase channel size to be 100×80 μm, the contraction zone channel size to be 80×80 μm, the outlet zone channel size to be 100×80 μm, and the collection section channel length to be 15cm. Step 5: Photolithographically etch the various channels of the microfluidic chip mentioned in Step 4 onto the hydrophobically treated PDMS wafer, and complete the packaging.

[0021] Specifically, the process for preparing the reverse microemulsion system mentioned in this invention is as follows: Step 1: Connect the cleanroom hose to the high-purity nitrogen cylinder and introduce high-purity water to blow out the dissolved oxygen. Step 2: Weigh 0.00033 mol of spectroscopically pure anhydrous FeCl2 and 0.00066 mol of spectroscopically pure anhydrous FeCl3 using an analytical balance, dissolve them in the above high-purity water, and dilute to 100 ml using a volumetric flask. Seal and set aside for later use. Step 3: Dissolve 10.000g of spectroscopically pure Span 80 and 5.000ml of n-hexanol in 95.240ml of spectroscopically pure cyclohexane; Step 4: Dissolve 11.999g of spectroscopically pure sodium hydroxide in high-purity water, and bring the volume to 100ml using a volumetric flask. Seal and set aside for later use. Step 5: The microfluidic chip is placed in a constant temperature water bath. The emulsified oil solution formed in step 3 is pumped into the continuous phase injection channel of the microfluidic chip as the continuous phase, and the iron salt aqueous solution in step 2 is pumped into the dispersed phase channel of the microfluidic chip as the dispersed phase. The residence time of the fluid in the microfluidic chip exceeds 20 minutes. Step 6: The droplet generation frequency is 120 / min, the droplet size is 20nm, and the monodispersity is greater than 95%; Step 7: Pump the microfluidic chip into the microchannel reactor at a rate of 2 μL / min using a three-channel pump, adjust the pH of the microchannel reactor to 10 ± 0.2, collect the droplets in a brown bottle, maintain a 50°C water bath and stir with a magnetic stirrer at 350 r / min, and continuously bubble with nitrogen for protection. Step 8: Continue crystallization and ripening in a brown bottle for 90 minutes.

[0022] Specifically, the post-treatment and drying process of the Fe3O4 nanoparticles mentioned in this invention is as follows: 1. Add 50% by volume of spectroscopically pure anhydrous ethanol as a demulsifier to the matured reverse microemulsion crystals, stir under magnetic stirring, let stand and separate into layers, and take the lower aqueous phase for later use. 2. Use a pipette to draw up the lower aqueous phase from the above-mentioned layering and inject it into an anti-adsorption PTFE centrifuge tube. Centrifuge with a high-speed refrigerated centrifuge, discard the liquid phase after centrifugation, and keep the solid phase for later use. 3. Wash with anhydrous ethanol and ultrapure water in sequence, alternating multiple times, and disperse with an ultrasonic disperser after each wash. 4. Dry in a vacuum freeze dryer at -55℃ and a vacuum degree <10Pa for 24 hours; 5. Obtain a reddish-brown powder, wherein the iron oxide powder with a particle size of 100nm or larger is black, and the iron oxide powder with a particle size of 25-100nm is dark brown, and store it in a brown bottle in a sealed container. 6. Take a small amount of sample and place it in a PTFE centrifuge tube. Disperse it intermittently with 19 times the mass of anhydrous ethanol by ultrasonication. Then, drop it onto a silicon wafer for spectroscopy and perform SEM and TEM observations. 7. Characterize the reddish-brown powder.

[0023] Use a pipette to draw the lower aqueous phase from the above separation and inject it into an anti-adsorption PTFE centrifuge tube. Centrifuge twice at 12000 r / min for 15 min each time at 4°C using a high-speed refrigerated centrifuge. Discard the liquid phase after centrifugation and keep the solid phase for later use.

[0024] The application of Fe3O4-assisted cell wall disruption nanomagnetic beads for archaeal DNA extraction mentioned in this invention involves using Fe3O4-assisted cell wall disruption nanomagnetic beads to separate and extract DNA from archaea in extreme environments within fluid inclusions. Under the action of mechanical disturbance, chemical lysis, or extraction buffer system, the Fe3O4 nanomagnetic beads improve cell wall rupture efficiency and promote DNA release through contact collision and assisted cell wall disruption with the archaeal cell wall / cell surface, thereby increasing the amount, purity, and stability of extracted DNA.

[0025] The aforementioned archaea in extreme environments are thermophilic archaea.

[0026] Example 2, a method for preparing auxiliary cell-wall breaking magnetic nanobeads for archaeal DNA extraction mentioned in this invention, includes the following steps: (1) Constructing a microfluidic chip for reverse emulsion droplets: The PDMS substrate was cleaned, dried and hydrophobically modified to prepare a microfluidic chip with a continuous phase oil phase channel, a dispersed phase aqueous phase channel, a shrinkage zone channel and an outlet zone channel, so as to achieve stable formation and particle size preparation of reverse emulsion droplets; (2) Preparation of a reverse microemulsion system: FeCl2 and FeCl3 were dissolved in high-purity water with dissolved oxygen removed at a molar ratio of 1:2 to obtain an iron salt dispersed phase; Span80, n-hexanol, and cyclohexane were mixed to obtain a continuous oil phase; an alkaline solution was prepared separately as a precipitant; the continuous phase and dispersed phase were pumped into a microfluidic chip at a predetermined flow rate to form monodisperse droplets under constant temperature and inert gas protection conditions, and the pH value of the reaction system was adjusted to the alkaline range by introducing an alkaline solution, so that Fe 2+ and Fe 3+ A coprecipitation reaction occurs to generate Fe3O4 nanoparticles; (3) Maturation and crystallization: The reverse microemulsion droplets collected at the outlet of the microfluidic chip are further matured at 50°C and maintained for a certain time under stirring and nitrogen protection to promote the full formation of Fe3O4 crystals and improve the crystallinity, dispersibility and magnetic response performance of the particles. (4) Demulsification and washing: Demulsifier was added to the matured reverse microemulsion system to separate the oil and water phases. The lower aqueous phase was taken out for low-temperature high-speed centrifugation to obtain Fe3O4 nanoparticle precipitate. Then, anhydrous ethanol and ultrapure water were used for alternating washing, and ultrasonic dispersion was combined to remove residual surfactants and impurities. (5) Drying to obtain auxiliary cell wall breaking magnetic beads: The washed Fe3O4 nanoparticles were freeze-dried under low temperature vacuum conditions to obtain Fe3O4 auxiliary cell wall breaking magnetic beads with uniform particle size, good dispersibility and strong magnetic response performance.

[0027] The difference from Example 1 is: The fabrication process of the reverse emulsion droplet microfluidic chip mentioned in this invention is as follows: Step 1: After curing the PDMS film, rinse it sequentially with analytical grade anhydrous ethanol and ultrapure water, and then dry it in a nitrogen atmosphere. Step 2: The above-mentioned PDMS sheets are further subjected to surface hydrophobic treatment by completely immersing the PDMS sheets in trimethylchlorosilane for 30 minutes; after removing the PDMS sheets, they are dried in an oven at 40-60℃ for 2 hours. Step 3: Fix the PDMS sheet on the water platform of the high-resolution camera objective lens, drop a drop of water on the surface of the PDMS sheet using a microsyringe, capture the outline of the droplet with the camera, and measure the tangent at the contact point between the water droplet and the PDMS sheet surface to calculate the contact angle of the hydrophobic sheet; the contact angle of the hydrophobic sheet must be greater than 120°.

[0028] Step 4: Design the continuous phase oil phase channel size to be 200×80 μm, the dispersed phase aqueous phase channel size to be 100×80 μm, the contraction zone channel size to be 80×80 μm, the outlet zone channel size to be 100×80 μm, and the collection section channel length to be 15cm. Step 5: Photolithographically etch the various channels of the microfluidic chip mentioned in Step 4 onto the hydrophobically treated PDMS wafer, and complete the packaging.

[0029] In addition, the archaea mentioned above in extreme environments are halophilic archaea.

[0030] Example 3, a method for preparing auxiliary cell-wall-breaking magnetic nanobeads for archaeal DNA extraction mentioned in this invention, includes the following steps: (1) Constructing a microfluidic chip for reverse emulsion droplets: The PDMS substrate was cleaned, dried and hydrophobically modified to prepare a microfluidic chip with a continuous phase oil phase channel, a dispersed phase aqueous phase channel, a shrinkage zone channel and an outlet zone channel, so as to achieve stable formation and particle size preparation of reverse emulsion droplets; (2) Preparation of a reverse microemulsion system: FeCl2 and FeCl3 were dissolved in high-purity water with dissolved oxygen removed at a molar ratio of 1:2 to obtain an iron salt dispersed phase; Span80, n-hexanol, and cyclohexane were mixed to obtain a continuous oil phase; an alkaline solution was prepared separately as a precipitant; the continuous phase and dispersed phase were pumped into a microfluidic chip at a predetermined flow rate to form monodisperse droplets under constant temperature and inert gas protection conditions, and the pH value of the reaction system was adjusted to the alkaline range by introducing an alkaline solution, so that Fe 2+ and Fe 3+ A coprecipitation reaction occurs to generate Fe3O4 nanoparticles; (3) Maturation and crystallization: The reverse microemulsion droplets collected at the outlet of the microfluidic chip are further matured at 50°C and maintained for a certain time under stirring and nitrogen protection to promote the full formation of Fe3O4 crystals and improve the crystallinity, dispersibility and magnetic response performance of the particles. (4) Demulsification and washing: Demulsifier was added to the matured reverse microemulsion system to separate the oil and water phases. The lower aqueous phase was taken out for low-temperature high-speed centrifugation to obtain Fe3O4 nanoparticle precipitate. Then, anhydrous ethanol and ultrapure water were used for alternating washing, and ultrasonic dispersion was combined to remove residual surfactants and impurities. (5) Drying to obtain auxiliary cell wall breaking magnetic beads: The washed Fe3O4 nanoparticles were freeze-dried under low temperature vacuum conditions to obtain Fe3O4 auxiliary cell wall breaking magnetic beads with uniform particle size, good dispersibility and strong magnetic response performance.

[0031] The difference from Example 1 is: The process for preparing the reverse microemulsion system mentioned in this invention is as follows: Step 1: Connect the cleanroom hose to the high-purity nitrogen cylinder and introduce high-purity water to blow out the dissolved oxygen. Step 2: Weigh 0.00033 mol of spectroscopically pure anhydrous FeCl2 and 0.00066 mol of spectroscopically pure anhydrous FeCl3 using an analytical balance, dissolve them in the above high-purity water, and dilute to 100 ml using a volumetric flask. Seal and set aside for later use. Step 3: Dissolve 10.000g of spectroscopically pure Span 80 and 5.000ml of n-hexanol in 95.240ml of spectroscopically pure cyclohexane; Step 4: Dissolve 11.999g of spectroscopically pure sodium hydroxide in high-purity water, and bring the volume to 100ml using a volumetric flask. Seal and set aside for later use. Step 5: The microfluidic chip is placed in a constant temperature water bath. The emulsified oil solution formed in step 3 is pumped into the continuous phase injection channel of the microfluidic chip as the continuous phase, and the iron salt aqueous solution in step 2 is pumped into the dispersed phase channel of the microfluidic chip as the dispersed phase. The residence time of the fluid in the microfluidic chip exceeds 20 minutes. Step 6: The droplet generation frequency is 120 / min, the droplet size is 20nm, and the monodispersity is greater than 95%; Step 7: Pump the microfluidic chip into the microchannel reactor at a rate of 2 μL / min using a three-channel pump, adjust the pH of the microchannel reactor to 10 ± 0.2, collect the droplets in a brown bottle, maintain a 50°C water bath and stir with a magnetic stirrer at 350 r / min, and continuously bubble with nitrogen for protection. Step 8: Continue crystallization and ripening in a brown bottle for 90 minutes.

[0032] The microfluidic chip mentioned in this invention is placed in a constant temperature water bath at 50±1℃. The emulsified oil solution formed in step three is used as the continuous phase and pumped into the continuous phase injection channel of the microfluidic chip at a rate of 10 μL / min. The iron salt aqueous solution from step two is used as the dispersed phase and pumped into the dispersed phase channel of the microfluidic chip at a rate of 1 μL / min. The pumping pressure is kept below 1.0 bar, and the residence time of the fluid in the microfluidic chip exceeds 20 min.

[0033] In addition, the archaea mentioned above in extreme environments are acidophilic archaea.

[0034] Additionally, it should be noted that: Figure 1 – Figure 3 Morphology characterization images of Fe3O4-assisted cell wall disruption nanoparticles at different magnifications and scales. Depend on Figure 1 – Figure 3 It can be seen that the obtained Fe3O4 particles are in a nanoscale aggregate state. As the observation scale is gradually reduced from 500 nm to 300 nm and 200 nm, the local morphology of the particles becomes clearer, and it can be observed that the particles are mainly composed of fine near-spherical nanoparticles. Figure 2 and Figure 3 The morphological characteristics displayed were generally consistent, indicating good morphological repeatability of the sample in different observation areas. Overall, the particle size was small and the distribution was relatively uniform. However, due to the magnetic interactions and high surface energy of the Fe3O4 nanoparticles, a certain degree of aggregation occurred in the sample. This type of nanoscale aggregation structure is beneficial for increasing the specific surface area of ​​the material, enabling it to fully contact the archaea cell wall or cell surface during archaea DNA extraction in extreme environments. This enhances the auxiliary cell disruption effect under mechanical disturbance, chemical lysis, or extraction buffer systems.

[0035] Depend on Figure 4 As can be seen, the sample exhibited characteristic diffraction peaks of Fe3O4 at the corresponding diffraction angles, which can mainly be attributed to the (220), (311), (400), (422), (511), and (440) crystal planes of Fe3O4. The positions of each diffraction peak are basically consistent with those of the Fe3O4 standard card, indicating that the main crystalline phase of the prepared sample is iron(III) oxide and that it has a certain degree of crystallinity, proving that the preparation method can successfully obtain Fe3O4 magnetic nanoparticles.

[0036] Figure 5 The results show that Fe and O were the main elemental signals detected on the sample surface, indicating that the material surface has the typical elemental composition characteristics of iron oxides. Meanwhile, trace amounts of C, N, and Cl signals may be present in the spectrum, presumably related to surfactants, solvent residues, adsorbates from the sample preparation process, or surface modification components. These results further confirm that the obtained material is mainly composed of Fe and O, consistent with the surface elemental characteristics of Fe3O4 nanoparticles.

[0037] Depend on Figure 6 It can be seen that characteristic peaks of Fe can be observed in the Fe 2p spectral region, and the peak shape can reflect the characteristics of Fe. 2+ and Fe 3+ The coexistence of two valence states. Fe 2+ / Fe 3+ Coexistence is one of the key characteristics that distinguishes Fe3O4 from iron oxides with a single valence state. Figure 6The formation of the Fe3O4 structure in the sample was further confirmed from the perspective of surface chemical valence state. This result is consistent with the XRD analysis results, indicating that the obtained particles have the crystal structure and surface valence state characteristics of iron(III) oxide.

[0038] Figure 7 The 1s O peak reflects the form of oxygen on the sample surface, typically including Fe–O lattice oxygen and surface hydroxyl groups or adsorbed oxygen. The Fe–O bond corresponds to the ferro-oxygen bond in the Fe3O4 crystal structure and is an important basis for determining the formation of magnetite (Fe3O4). Surface hydroxyl groups or adsorbed oxygen indicate that the particle surface possesses a certain degree of hydrophilicity and interfacial activity. This surface characteristic is beneficial for improving the dispersibility of nanoparticles in aqueous DNA extraction systems and provides a good interfacial basis for their contact with archaea cell walls, participation in assisted cell disruption, and subsequent magnetic separation.

[0039] The above description is merely a partial preferred embodiment of the present invention. Any person skilled in the art can modify the above-described technical solutions or modify them into equivalent technical solutions. Therefore, any simple modifications or equivalent transformations made based on the technical solutions of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing auxiliary cell-wall-breaking magnetic nanobeads for archaeal DNA extraction, characterized in that... Includes the following steps: (1) Constructing a microfluidic chip for reverse emulsion droplets: The PDMS substrate was cleaned, dried and hydrophobically modified to prepare a microfluidic chip with a continuous phase oil phase channel, a dispersed phase aqueous phase channel, a shrinkage zone channel and an outlet zone channel, so as to achieve stable formation and particle size preparation of reverse emulsion droplets; (2) Preparation of a reverse microemulsion system: FeCl2 and FeCl3 were dissolved in high-purity water with dissolved oxygen removed at a molar ratio of 1:2 to obtain an iron salt dispersed phase; Span80, n-hexanol, and cyclohexane were mixed to obtain a continuous oil phase; an alkaline solution was prepared separately as a precipitant; the continuous phase and dispersed phase were pumped into a microfluidic chip at a predetermined flow rate to form monodisperse droplets under constant temperature and inert gas protection conditions, and the pH value of the reaction system was adjusted to the alkaline range by introducing an alkaline solution, so that Fe 2+ and Fe 3+ A coprecipitation reaction occurs to generate Fe3O4 nanoparticles; (3) Maturation and crystallization: The reverse microemulsion droplets collected at the outlet of the microfluidic chip are further matured at 50°C and maintained for a certain time under stirring and nitrogen protection to promote the full formation of Fe3O4 crystals and improve the crystallinity, dispersibility and magnetic response performance of the particles. (4) Demulsification and washing: Add demulsifier to the matured reverse microemulsion system to separate the oil and water phases. Take the lower aqueous phase for low-temperature high-speed centrifugation to obtain Fe3O4 nanoparticle precipitate. Subsequently, the residual surfactant and impurities were removed by alternating washing with anhydrous ethanol and ultrapure water, combined with ultrasonic dispersion. (5) Drying to obtain auxiliary cell wall breaking magnetic beads: The washed Fe3O4 nanoparticles were freeze-dried under low temperature vacuum conditions to obtain Fe3O4 auxiliary cell wall breaking magnetic beads with uniform particle size, good dispersibility and strong magnetic response performance.

2. The method for preparing auxiliary cell-wall-breaking magnetic nanobeads for archaeal DNA extraction according to claim 1, characterized in that: The fabrication process of the reverse emulsion droplet microfluidic chip is as follows: Step 1: After curing the PDMS film, rinse it sequentially with analytical grade anhydrous ethanol and ultrapure water, and then dry it in a nitrogen atmosphere. Step 2: The above-mentioned PDMS sheets are further subjected to surface hydrophobic treatment by completely immersing the PDMS sheets in trimethylchlorosilane for a period of time; after removing the PDMS sheets, they are dried in an oven at 40-60℃. Step 3: Fix the PDMS sheet on the water platform of the high-resolution camera objective lens, drop a drop of water on the surface of the PDMS sheet using a microsyringe, capture the outline of the droplet with the camera, and measure the tangent of the contact point between the water droplet and the surface of the PDMS sheet to calculate the contact angle of the hydrophobic sheet. Step 4: Design the continuous phase oil phase channel size to be 200×80 μm, the dispersed phase aqueous phase channel size to be 100×80 μm, the contraction zone channel size to be 80×80 μm, the outlet zone channel size to be 100×80 μm, and the collection section channel length to be 15 cm. Step 5: Photolithographically etch the microfluidic chip channels from Step 4 onto the hydrophobically treated PDMS wafer, and complete the encapsulation.

3. The method for preparing auxiliary cell-wall-breaking magnetic nanobeads for archaeal DNA extraction according to claim 2, characterized in that: The contact angle of the hydrophobic sheet must be greater than 120°.

4. The method for preparing auxiliary cell-wall-breaking magnetic nanobeads for archaeal DNA extraction according to claim 3, characterized in that: The process for preparing the reverse microemulsion system is as follows: Step 1: Connect the cleanroom hose to the high-purity nitrogen cylinder and introduce high-purity water to blow out the dissolved oxygen. Step 2: Weigh 0.00033 mol of spectroscopically pure anhydrous FeCl2 and 0.00066 mol of spectroscopically pure anhydrous FeCl3 using an analytical balance, dissolve them in the above high-purity water, and dilute to 100 ml using a volumetric flask. Seal and set aside for later use. Step 3: Dissolve 10.000g of spectroscopically pure Span 80 and 5.000ml of n-hexanol in 95.240ml of spectroscopically pure cyclohexane; Step 4: Dissolve 11.999g of spectroscopically pure sodium hydroxide in high-purity water, and bring the volume to 100ml using a volumetric flask. Seal and set aside for later use. Step 5: The microfluidic chip is placed in a constant temperature water bath. The emulsified oil solution formed in step 3 is pumped into the continuous phase injection channel of the microfluidic chip as the continuous phase, and the iron salt aqueous solution in step 2 is pumped into the dispersed phase channel of the microfluidic chip as the dispersed phase. The residence time of the fluid in the microfluidic chip exceeds 20 minutes. Step 6: The droplet generation frequency is 120 / min, the droplet size is 20nm, and the monodispersity is greater than 95%; Step 7: Pump the microfluidic chip into the microchannel reactor at a rate of 2 μL / min using a three-channel pump, adjust the pH of the microchannel reactor to 10 ± 0.2, collect the droplets in a brown bottle, maintain a 50°C water bath and stir with a magnetic stirrer at 350 r / min, and continuously bubble with nitrogen for protection. Step 8: Continue crystallization and ripening in a brown bottle for 90 minutes.

5. The method for preparing auxiliary cell-wall-breaking magnetic nanobeads for archaeal DNA extraction according to claim 4, characterized in that: The microfluidic chip is placed in a constant temperature water bath at 50±1℃. The emulsified oil solution formed in step three is used as the continuous phase and pumped into the continuous phase injection channel of the microfluidic chip at a rate of 10 μL / min. The iron salt aqueous solution from step two is used as the dispersed phase and pumped into the dispersed phase channel of the microfluidic chip at a rate of 1 μL / min. The pumping pressure is kept below 1.0 bar and the residence time of the fluid in the microfluidic chip is more than 20 min.

6. The method for preparing auxiliary cell-wall-breaking magnetic nanobeads for archaeal DNA extraction according to claim 5, characterized in that: The post-treatment and drying process of the Fe3O4 nanoparticles is as follows:

1. Add 50% by volume of spectroscopically pure anhydrous ethanol as a demulsifier to the matured reverse microemulsion crystals, stir under magnetic stirring, let stand and separate into layers, and take the lower aqueous phase for later use.

2. Use a pipette to draw up the lower aqueous phase from the above-mentioned layering and inject it into an anti-adsorption PTFE centrifuge tube. Centrifuge with a high-speed refrigerated centrifuge, discard the liquid phase after centrifugation, and keep the solid phase for later use.

3. Wash with anhydrous ethanol and ultrapure water in sequence, alternating multiple times, and disperse with an ultrasonic disperser after each wash.

4. Dry in a vacuum freeze dryer at -55℃ and a vacuum degree <10Pa for 24 hours; 5. Obtain a reddish-brown powder, wherein, Iron oxide powder with a particle size of 100nm or larger is black, while iron oxide powder with a particle size of 25-100nm is dark brown. It should be stored in a brown bottle and sealed.

6. Take a small amount of sample and place it in a PTFE centrifuge tube. Disperse it intermittently with 19 times the mass of anhydrous ethanol by ultrasonication. Then, drop it onto a silicon wafer for spectroscopy and perform SEM and TEM observations.

7. Characterize the reddish-brown powder.

7. The method for preparing auxiliary cell-wall-breaking magnetic nanobeads for archaeal DNA extraction according to claim 6, characterized in that: Use a pipette to draw the lower aqueous phase from the above separation and inject it into an anti-adsorption PTFE centrifuge tube. Centrifuge twice at 12000 r / min for 15 min each time at 4°C using a high-speed refrigerated centrifuge. Discard the liquid phase after centrifugation and keep the solid phase for later use.

8. The method for preparing auxiliary cell-wall-breaking magnetic nanobeads for archaeal DNA extraction according to claim 1, characterized in that: The extreme environment archaea in step (6) are thermophilic archaea, halophilic archaea, and / or acidophilic archaea.

9. An application of the auxiliary cell-wall-breaking magnetic nanobeads for archaeal DNA extraction as described in claim 1, characterized in that: Fe3O4-assisted cell wall disruption nanobeads were applied to the isolation and DNA extraction of archaea in extreme environments within fluid inclusions. Under the action of mechanical disturbance, chemical lysis, or extraction buffer system, the Fe3O4 nanobeads, through contact and collision with the archaea cell wall / cell surface and the assisted cell wall disruption effect, improved cell wall rupture efficiency, promoted DNA release, and increased DNA extraction yield, purity, and stability.

Images