Preparation Of Bulk Nanobubbles And Investigation Of Their Effect On Protease Activity

Bulk nanobubbles are generally considered to be bubbles of tens to hundreds of nanometers in diameter that occur in liquid solutions or solids.The existence and stability of nanobubbles,especially in the bulk phase,is still a mystery,and there are still controversies on this issue.According to the theory proposed by Epstein-Plesset in1950 to predict the lifetime of individual bubbles.In which we describe lifetime as a function of bubble radius and saturation.For bubbles with a diameter of about 1000 nm,the internal pressure is about 3.9bar at 25 ° C,and this pressure increases as the size of the bubble decreases,making it easy to dissolve quickly.Based on the facts,experimental evidence observed contradicts theoretical predictions describing bubble stability,proving that our assumptions about the continuity of theoretical descriptions should be questioned.Especially for bulk phase nanobubbles,which are soft materials with large two-phase interfaces,their properties cannot be simplified by simple classical theoretical models.Although the theoretical development lags far behind the application,researchers are more and more interested in it.While new production methods are being developed,novel applications are also being explored,including reprogramming genomes using oxygen nanobubbles as drug delivery vehicles;It is used as an ultrasound contrast agent supplemented by high energy focused ultrasound ablation to eliminate cancer cells at a specific point;It is used to improve the microenvironment of the digestive tract and change our microbial genome to assist weight loss and kill helicobacter pylori and other harmful bacteria in the digestive tract;Developing hydrogen nanobubble gasoline mixture as new energy to improve engine performance;It can be used in mineral flotation to improve the enrichment and recovery of fine mineral particles.It can be used in the recovery of micro and nano plastics to improve the concentration of heavy metal biofilm in water.Cultivation of plants and animals for agriculture,fishing,etc.Its application involves all aspects of the wellbeing of mankind for the generations to come,playing a role with huge potential influence.While benefiting from the development of characterization methods,we can roughly measure the particle size distribution,surface potential and other properties of bulk nanobubbles,but its internal chemical information cannot be determined by dynamic light scattering technology,Therefore,in order to distinguish nanobubbles,nanodroplets,and nano solid particles,it is necessary to combine advanced synchrotron radiation technology to confirm.Its stability remains an open question.One of the main reasons for its poor stability is the fusion between bubbles,so how to avoid the fusion is the key to preparing high concentration bulk phase nanobubbles.Bulk nanobubbles with high stability and high concentration have tremendous potential in application in biology.For example,inert gas nanobubbles can be developed for the treatment of decompression sickness,ischemia-reperfusion injury,and developed as anesthetics,etc.Therefore,for its preparation method,it is required that the concentration of the gas source can be controlled,and the cleanliness must meet the medical standards.Therefore,this study started with the development of a bulk nanobubble preparation scheme with controllable gas source type,controllable concentration and high cleanliness standard.Further introduced a protein with better biocompatibility with multiple hydrophobic domains,and changed the solution p H,viscosity and other means to prepare bulk nanobubbles with higher concentration and higher stability.The nanoparticles were characterized by a combination of nanoparticle tracking analysis system,hard X-ray fluorescence imaging,and absorption spectroscopy to determine their concentration,size distribution,and internal chemical information,and through strict degassing control to exclude the pollution of nano-droplets,nano-solid particles and other pollutants.Later,it was applied to regulate the activity of protease,which provided a deeper understanding of the mechanism of inert gas-induced anesthesia.The main progress of this study includes the following parts.Firstly,according to Henry’s law,local gas supersaturation in the liquid phase is caused by pressure and decompression,and bulk nanobubbles with alternative gas sources are generated.By combining the nanoparticle tracking analysis system with hard X-ray fluorescence imaging and absorption spectroscopy to characterize the nanobubbles to determine their concentration,size distribution,and internal chemical information,it is proved that the method can indeed produce bulk nanobubbles,and its concentration depends on the rate of restoring atmospheric pressure.The advantages of this method are that the gas source is controllable,the operation is simple,the concentration can be adjusted by changing the rate of restoring astompheric pressure,and the cleanliness is high.The bulk nanobubbles prepared by this method are suitable for biological research.Secondly,we put forward higher requirements for bulk nanobubbles prepared by pressure and decompression method,hoping for a higher concentration,which is more convenient to observe the effect and conduct quantitative research when applied to biological research.And the disadvantages of this method are that the concentration of the prepared bulk nanobubbles is limited,and the fusion between the bubbles makes its stability not high enough,and it is easy to form large bubbles that float up and disappear.To solve this problem,we introduced proteins with different numbers of hydrophobic domains to attach and modify the bubbles to prevent gas exchange and fusion between bubbles,And the liquid viscosity was changed to reduce the liquid discharge rate between homogeneous bubbles to avoid their fusion.The experimental results showed that compared with the bulk nanobubbles produced in the buffer solution commonly used in biological research,the method can improve the concentration of bulk nanobubbles and prolong their half-life,which is beneficial to the subsequent application.At the same time,we know the physiological inert gas has many important biological effects,but we have not known the working mechanism,because its chemical properties are very stable,not easy to react,and extremely susceptible to slight disturbance hence easy to be detached from the target receptor.This study speculates that the inert gas acts in the form of nanobubbles.Therefore,in this study,the bulk nanobubbles prepared were introduced to interact with protease to study the effect of krypton gas nanobubbles on protease activity.The results showed that nanobubbles could significantly inhibit pepsin activity,and the degree of inhibition depends on the concentration of nanobubbles.The inhibition effect is reversible,and degassing can restore the activity almost to that before inhibition.This study opens a new horizon for our understanding of the mechanism underlying many biological effects of inert gases.It also provides new ideas for potential applications of bulk nanobubbles.