CN110124543A - A kind of method and apparatus generating body phase nano grade air bubbles - Google Patents

Abstract

The invention relates to a method for generating bulk nano-scale bubbles. A liquid reaction medium is accommodated in a chamber, and a gas with a controllable composition is introduced into the chamber for pressurization, so that the pressure in the chamber is greater than 1ATM and does not exceed 1ATM. If it exceeds 20ATM, keep it for 5-60min, then deflate and depressurize for 5s-30min to normal pressure, the solubility of the gas in the reaction medium decreases and gradually forms gas nuclei, and gradually generates bulk nano-sized bubbles inside the reaction medium with degassing. The present invention also relates to a device for generating bulk nano-scale bubbles. The chamber is connected to different gases through a booster valve and communicated with the outside world through a gas release valve. The pressure in the chamber is controlled through the booster valve and the gas release valve. A liquid reaction medium is accommodated in the chamber and generates bulk nano-bubbles in response to changes in pressure, and the observation device observes the bulk nano-bubbles. According to the method of the invention, bulk nano-bubbles with definite components can be produced, have good reproducibility, and are easy to operate.

Description

A method and device for generating bulk nanoscale bubbles

technical field

The invention relates to nano-bubbles in physical chemistry, more particularly to a method and device for generating bulk nano-bubbles.

Background technique

The earliest reported research on bulk nanobubbles comes from the research on ultrasonic and cavitation effects. Since 2000, there have been a large number of studies on bulk bubbles, which focus on the physical properties of bulk bubbles and their concentrations in solutions. At present, because the existence of bulk nanobubbles may have an impact on many fields, more and more scientific researchers in various fields have begun to have a strong interest in bulk nanobubbles. For example, bulk nanobubbles are used in the field of surface cleaning. By absorbing the pollution aroused by mechanical agitation, they can be prevented from re-precipitating to achieve the cleaning effect. In the field of water treatment, due to the smaller diameter and larger specific surface of nanobubbles, they exist in water for a longer time, which can effectively improve the aeration efficiency, greatly improve the efficiency of sewage treatment and promote ecological restoration. In the field of biomedicine, although the application research of nano-bubbles has just started, it has broad prospects. Micro-nano bubbles have strong echo reflection performance in ultrasonic imaging and ultrasonic contrast agents, which can significantly enhance medical ultrasonic detection signals and make up for traditional ultrasonic imaging. The sensitivity and resolution of diagnostic techniques are not high enough, and microbubbles are limited by their large size, and their application range is limited in their blood vessels. However, nanobubbles can not only pass through the blood vessel wall to reach the interior of the tissue, but also break through Brain-blood barrier can be used as a new type of ultrasound contrast agent for molecular imaging and drug delivery. It also has obvious advantages in the diagnosis and targeted therapy of cancer. Nano-scale bubbles can reach the interior of the tissue and can exist stably for a long time within the temperature range of the human body. Nano-bubbles pass through capillaries that become enlarged due to cancer lesions. Cells gather at this time, and ultrasound imaging can be clearly observed at this time, and then increase the frequency of ultrasound, and the bubbles will burst, so as to achieve the effect of targeted therapy.

However, it is difficult to explain the stable existence of nanobubbles theoretically. According to the interpretation of classical thermodynamics: the smaller the volume of the bubble, the greater the internal pressure, and the high pressure will inevitably cause the bubble to burst. For example, for a bubble with a diameter of 10 nm, its internal pressure calculated according to the Laplace equation is 144 atmospheres (1.44×10 ⁷ Pa) (expressed by physical parameters), such a high internal pressure makes the bubble disappear instantly. In addition, according to molecular dynamics simulation studies, the existence time of nanobubbles in water at room temperature is only several to hundreds of picoseconds, and nanobubbles existing in such a short period of time are often not observed in experiments. But in recent years, several research groups have directly observed nanobubbles on the solid-liquid interface with atomic force microscopy, which provides direct evidence for the existence of nanobubbles. In addition, the existence of nanobubbles was also predicted by cryo-etching method and neutron diffraction method. Therefore, in order to study nanobubbles systematically and deeply, and to resolve the inconsistency between theory and experiment, it is necessary to establish a simple and controllable method to form and deeply study the stability mechanism of nanobubbles. In addition, the source of the gas that forms the bubbles is also difficult to control, resulting in poor repeatability of the experiment. At the same time, nanobubbles have a variety of potential biomedical applications. In order to be applied to biological experiments or clinical medical research, we need to develop methods that can produce nanobubble water with clean gas types and ratios that can be precisely adjusted under laboratory conditions. Most of the existing methods are to generate nanobubbles at the solid-liquid interface.

Contents of the invention

In order to solve the problem of inability to stably generate bulk nanobubbles in the prior art and better promote the important application of nanobubbles in various fields, the present invention aims to provide a method and device for generating bulk nanobubbles.

The method for generating bulk nano-scale bubbles according to the present invention, wherein the liquid reaction medium is accommodated in a chamber, and a gas with a controllable composition is introduced into the chamber for pressurization, so that the pressure in the chamber is greater than 1ATM And do not exceed 20ATM, keep for 5-60min, the solubility of gas in the reaction medium increases, forming a supersaturated state, then degassing and reducing pressure for 5s-30min to normal pressure, the solubility of gas in the reaction medium decreases and gradually forms gas nuclei, with the degassing Bulk nanoscale bubbles are gradually generated inside the reaction medium.

Preferably, deflate and decompress for 15 minutes to normal pressure.

Preferably, the gas is a single gas or a mixture of gases. Preferably, the gas is Kr, H ₂ , N ₂ , He, Ne, Ar, Xe, CH ₄ , O ₂ , CH ₄ , SF ₆ , CF ₄ , C ₅ F ₁₂ , and/or any of the above gases. Proportionally mixed gas mixture.

Preferably, the reaction medium is water, acid solution, alkali solution, salt solution, buffer, and/or protein solution. Preferably, the pH value of the acid solution is hydrochloric acid of 4-7. Preferably, the alkaline solution is an alkaline solution of alkali metal or alkaline earth metal. Preferably, the alkaline solution is sodium hydroxide, potassium hydride, calcium hydroxide, and/or magnesium hydroxide with a pH of 7-10. Preferably, the buffer is a buffer commonly used in biological experiments. Preferably, the buffer is PBS, TE, TAE, TBE, TBS, and/or Tris-Glycine. Preferably, the protein solution is a serum albumin solution. Preferably, the protein solution is bovine serum albumin, and/or pepsin and lysozyme.

Preferably, the reaction medium is pre-degassed to remove dissolved gas therein, so as to obtain a degassed reaction medium. The degassed reaction medium can be used as a reference solution for a control group that is pressurized and then depressurized.

Preferably, the cleaning operation is not only clean and dust-free, but also free from microbial contamination, and nanobubbles with medical grade cleanliness can be obtained. In particular, water vapor is introduced to completely kill the mold spores in the pipeline, remove the most difficult to kill microorganisms, and ensure that other microorganisms are completely eliminated, so as to obtain bulk nanobubbles with medical-grade cleanliness. The generated sparkling water meets the cleanliness specified by the national standard to ensure that the equipment used in production can achieve the required cleanliness (see GB/T6682).

The device for generating bulk nano-bubbles according to the present invention includes a liquid reaction medium, a chamber and an observation device. A valve and an air release valve are used to control the pressure in the chamber. The liquid reaction medium is accommodated in the chamber and generates bulk nano-bubbles in response to changes in pressure. The observation device observes the bulk nano-bubbles.

Preferably, the observation device is a nanoparticle tracking analysis system (Nanoparticle Tracking Analysis, NTA). Specifically, the pressurized and decompressed water is injected into the sample pool of the nano-tracing analysis system, and a microscope is used to observe and a high-resolution camera is used to record the particle trajectory, and the Stokes-Einstein equation can be used to obtain the The particle size of the particle, and the real concentration of the particle can be measured.

Preferably, the observation device adjusts the pressure, holding time and deflation time in the chamber according to the quantity and particle size distribution of bulk nanobubbles, so as to finally control the quantity and size of bulk nanobubbles. For example, the observation device can adjust the pressurization time, and/or deflation time to control the number and size of bulk nanobubbles. Specifically, the gas injected into the control group is a single-component high-purity gas or a gas mixed with a variety of single-component high-purity gases in any proportion. It is compared with the blank group without gas to reflect the generated bulk nanobubbles. quantity and concentration distribution.

According to the method of the invention, single-component bulk nano-bubbles or multi-component mixed gas nano-bubbles can be produced, which has good reproducibility and is easy to operate. In addition, the bulk nano-bubbles produced by the method of the present invention have high cleanliness, can be used in biomedical research, and even reach medical grade cleanliness. Moreover, according to the method of the present invention, the gas type of the nanobubbles, the size and the number of the nanobubbles can be controlled. According to the device of the present invention, it can be further developed into various new equipment such as medical equipment, and has wide application in many fields.

Description of drawings

Fig. 1 is the quantity distribution figure of the bulk phase nanobubble according to embodiment 1 of the present invention;

Fig. 2A is a microscopic result diagram of the buffer solution before pressurization of the ventilator according to Example 2 of the present invention;

Fig. 2B is a microscopic result diagram of the buffer solution pressurized by the ventilator according to Example 2 of the present invention;

Fig. 2C is a microscopic result diagram of a buffer solution degassed after nanobubbles are generated by pressurizing the ventilator according to Example 2 of the present invention;

Fig. 3 shows the particle size distribution and nanobubble concentration of the bulk nanobubbles in the buffer solution degassed after the venting gas is pressurized, pressurized, and pressurized to generate nanobubbles according to Embodiment 2 of the present invention;

Fig. 4 shows the number distribution of the bulk nanobubbles in the buffer solution degassed after the pressurization of the ventilating gas according to Embodiment 2 of the present invention before, after pressurization, and after nanobubbles are generated;

FIG. 5 shows bulk nanobubbles in bovine serum albumin solution according to Example 5 of the present invention.

Detailed ways

Below in conjunction with the drawings, preferred embodiments of the present invention are given and described in detail.

The nanoparticle analyzer used in the embodiment is NanoSight NS300 system (Malvern Instruments, Inc.), equipped with sample cell (blue laser), microscope frame, high-resolution camera sCMOS, nano tracer analysis system; water is Millipore ultrapure Water, the sample cell is washed alternately with water, ethanol, and water before use. Before use, the glass reagent bottle and the glass syringe were washed with deionized water three times, cleaned with ultrasonic waves, and dried in a drying oven.

Make degassed water, use degassed water to configure various liquid reaction media. Steps for making degassed water: Take water, put it in a clean glass reagent bottle with a lid, and freeze it. The purpose of freezing is to remove the gas dissolved in the water as much as possible and carry out deep degassing. Take off the cover, seal it with a parafilm, and punch four small holes on the parafilm, place it in a vacuum drying oven, evacuate it, keep it at 0.1ATM for 10 hours, take it out and repeat the above operation three times, the solution is degassed water , the configuration of other solutions should be non-corrosive. Corrosive solutions will corrode the coating on the stage and make it uneven. The uneven coating will refract the laser and reduce the signal-to-noise ratio.

Embodiment 1 generates krypton nano-bubbles in degassed water

Add krypton gas to the deaerated water to 6 standard atmospheres and keep it for 30 minutes. The distribution of the number of nanobubbles in the bulk phase is shown in Figure 1. The number of bubbles per ml can reach the order of 10 ⁸ .

The specific measurement steps for the number of nanobubbles are as follows:

Open Nanosight and start the temperature control software NTA temperature controller; check whether the connection is intact, and start the main program NTA program. Use a clean glass syringe to take the nano-bubble water prepared by the pressurization and decompression method, inhale about 1ml of the liquid, discharge the air at the top of the syringe, and "slowly" inject it into the sample pool until the liquid flows out from the "outlet end". To prevent the liquid from dripping at the outlet, place a small piece of roll paper. Place the sample cell on the microscope stage, the blue laser light should be in the luminous state (if it is not on, click the camera level in the Capture interface of the NTA software to adjust to a higher level to activate the laser), adjust the position of the sample cell front and back, left and right, up and down The focal length is until the blue laser beam can be observed, and the focal length and position can be finely adjusted by using the observation image of the NTA software. If the field of view is bright, the sample is too concentrated and needs to be re-diluted. The highlight needs to be diluted, set the Camera duration to 60s, and record 5 times to get the average value. As shown in Figure 1, within the time range of 5s<t≦15min, the longer the deflation time, the greater the number of nanobubbles generated. After degassing the nano-bubble water in a vacuum drying oven, it was found that the number of nano-bubbles decreased significantly, so it can be explained that the bubbles generated in the experiment are gas. specific gas components. In addition, the pressurization is realized by increasing the gas content in the cavity with a fixed volume. Before pressurization, the cavity is purged with a reaction gas to blow out the air in the sample cavity. It shows that the generated bubbles are gas nano-bubbles with controllable specific composition, rather than other substances.

The observation process is as above, set the camera level to 9, the screen brightness to 9, and the particle size threshold to 5. Then take the remaining nano-bubble water, seal it with a parafilm, make several small holes, and place it in a vacuum drying oven for degassing for one hour. The observation process is as above, set the camera level to 8, the screen brightness to 10, and the particle size threshold to 5.

Embodiment 2 generates krypton nano bubbles in TBE solution

Prepare the TBE solution with deionized water, use a glass syringe to take the solution in the glass reagent bottle about 2cm away from the gas-liquid interface, and inject it into the sample cell of the nanotracer molecular system. Set the camera level to 8, the screen brightness to 10, and the particle size threshold to 5. Then make nano-bubble water, use a pipette to take 3mL of TBE solution and place it in the sample pool of the three-way valve, introduce krypton gas to make the pressure in the cavity reach 6 standard atmospheres, keep it for 30 minutes, and then the slow degassing process continues 15 minutes.

It was observed that the number of particles in the TBE solution after pressurization and decompression increased significantly to 1.6×10 ⁸ /ml, and the number of particles decreased significantly after degassing to 1.2×10 ⁷ /ml, which was even less than that of the initial untreated solution. The number of particles in any treated TBE solution, ie 1.3 x 10 ⁷ /ml. It shows that the particles increased in the nitrogen nanobubble TBE solution produced by the pressurization and decompression method are nanobubbles, and the number of particles decreases significantly after degassing, which shows that the composition of the nanobubbles increased in the nanobubble TBE solution is indeed gas.

As shown in Figures 2A-2C respectively, the TBE buffer solution before gas pressurization, the TBE buffer solution after gas pressurization, and the TBE buffer solution degassed after gas pressurization to generate nanobubbles. The trajectory of the nanobubbles is recorded by a high-speed camera, and one frame is taken, and the size distribution and concentration of the nanobubbles are obtained by NTA. The trajectory of the refracted spot of the nanobubbles is recorded by a high-speed camera, and the Einstein-Stokes equation is used to solve the problem according to the speed of movement. And got it. It can be seen from the figure that the number of nanobubbles in the TBE buffer solution that has not been subjected to pressure and decompression treatment is small, but the number of nanobubbles in the TBE buffer solution that has been subjected to pressure and decompression treatment increases significantly. Figure 2C is based on Figure 2B After decompression and deep degassing treatment, the number of nano bubbles is significantly reduced. Figure 3 shows the particle size distribution and nanobubble concentration of the bulk nanobubbles in the degassed TBE buffer solution before, after pressurization, after pressurization to generate nanobubbles, as shown in the figure, without The number of particles in the treated water is less than ^{10 5} , while the content of nano-bubbles in krypton nano-bubble water generated by the pressure and decompression method is significantly increased. The number of nanobubbles between 100nm and 200nm is close to 2.0×10 ⁶ /ml, and the number of nanobubbles with an average particle size close to 200nm is far more than 10 ⁶ . Figure 4 shows the distribution of the number of bulk nanobubbles in the degassed TBE buffer solution before, after pressurization, and after pressurization to generate nanobubbles. The nanoparticles that can be detected in the solution before pressurization are very small. However, after the pressure and decompression treatment, the number of nanobubbles increased significantly, and the number exceeded 10 ⁶ , which was an order of magnitude higher than that before untreated. In order to prove that the increased nanoparticles are all nanobubbles, we used the method of decompression Gas, pump out the bubbles in the bubble water, the number of nanoparticles in the solution is significantly reduced, down to an order of magnitude before using the pressure and decompression method, which shows that this method can produce a large number of nanobubbles, and the extra Nanoparticles of an order of magnitude are nanobubbles.

Embodiment 3 generates nitrogen nanobubbles in dilute potassium hydroxide solution

The device and operation steps are as in Example 1, but use 0.001-1.0mol/L potassium hydroxide solution as a solvent, add 6 standard atmospheric pressure, keep for 30 minutes, slow deflation time continues for 15 seconds, and the obtained nanobubble particle size range 50-300nm. The number of bubbles can reach 2.5×10 ⁸ /ml, and the error range is ±5.9×10 ⁷ /ml.

Embodiment 4 generates nitrogen nanobubbles in sodium chloride solution

The device and operation steps are as in Example 1, but using 0.001-0.1 mol/L sodium chloride solution, the size of the generated nanobubbles is similar to that of TBE and potassium hydroxide solution. The number of bubbles can reach 2.13×10 ⁸ /ml, and the error range is ±6.6×10 ⁷ /ml.

Embodiment 5 generates nitrogen nanobubbles in BSA (bovine serum albumin) solution

Pressurize and feed nitrogen into the BSA solution of 0.1mg/mL, so that the internal pressure of the pressurization and decompression device reaches 6ATM, keep it for 30 minutes, and release the gas for 5 minutes. The particle size and number distribution of the nanobubbles produced, as shown in Figure 5 Show, also can produce a large amount of nano-bubbles to bovine serum albumin solution by the method of pressurization and decompression. The number of particles in the bovine serum albumin solution that has not been processed by the pressurization and decompression method is compared as a blank background, showing that a large number of nano bubbles can be produced in the bovine serum albumin solution by pressurization and decompression.

What is described above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Various changes can also be made to the above embodiments of the present invention. That is to say, all simple and equivalent changes and modifications made according to the claims and description of the application for the present invention fall within the protection scope of the claims of the patent of the present invention. What is not described in detail in the present invention is conventional technical contents.

Claims (8)

1. a kind of method for generating body phase nano grade air bubbles, which is characterized in that the reaction medium of liquid is placed in a chamber, to It is passed through the controllable gas of ingredient in chamber to pressurize, so that the indoor pressure of chamber is greater than 1ATM and is no more than 20ATM, keeps 5- 60min, gas solubility increases in reaction medium, forms hypersaturated state, and the decompression 5s-30min that then deflates is to normal pressure, reaction Gas solubility reduction gradually forms gas core in medium, gradually generates body phase nanoscale gas in the inside of reaction medium with deflating Bubble.

2. the method according to claim 1, wherein the gas is pure gas or mixed gas.

3. the method according to claim 1, wherein the reaction medium be water, acid solution, aqueous slkali, salting liquid, Buffer, and/or protein solution.

4. according to the method described in claim 3, it is characterized in that, the acid solution is hydrochloric acid；The aqueous slkali is sodium hydroxide, hydrogen Changeization potassium, calcium hydroxide, and/or magnesium hydroxide；The buffer is PBS, TE, TAE, TBE, TBS, and/or Tris-Glycine； The protein solution is bovine serum albumin(BSA), and/or pepsin and lysozyme.

5. the method according to claim 1, wherein the reaction medium first passes through degassing process in advance to remove wherein The gas of dissolution, to obtain degassing reaction medium.

6. a kind of device for generating body phase nano grade air bubbles, which is characterized in that the device include the reaction medium of liquid, chamber and Observation device, wherein the chamber connects different gas by pressure charging valve and is in communication with the outside by vent valve, passes through the pressurization Valve and vent valve control the pressure in chamber, and it is raw that the reaction medium of liquid is placed in the variation in the chamber and in response to pressure Adult phase nano grade air bubbles, observation device are observed body phase nano bubble.

7. device according to claim 6, which is characterized in that the observation device is nanometer tracer analysis system.

8. device according to claim 6, which is characterized in that observation device is according to the quantity and partial size of body phase nano bubble Distribution is to adjust the pressure in chamber, retention time and deflation time, with the quantity and size of final control volume phase nano bubble.