CN101474541A

CN101474541A - Integrated chip and device thereof, and method for preparing micrometre level dispersoid

Info

Publication number: CN101474541A
Application number: CNA2008102412602A
Authority: CN
Inventors: 郑海荣; 姜春香; 靳巧峰; 王战会
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Zhongke Medical Technology Co Ltd
Priority date: 2008-12-16
Filing date: 2008-12-16
Publication date: 2009-07-08
Anticipated expiration: 2028-12-16
Also published as: CN101474541B

Abstract

The invention discloses an integrated chip, a device adopting the integrated chip and a method for preparing micron-sized dispersoid; the substrate of the integrated chip is the substrate with hydrophilic surface; two layers of adjacent structures are a group of structures, and the integrated chip comprises at least one group of structures; in one group of structures, the first structure is provided with M levels of gradients, and the second structure is provided with N levels of gradients; wherein, M and N are natural numbers, and M is less than N; among the gradients, the primary gradient is provided with H1 channel 1, H2 branches are led out from each channel 1 in the secondary gradient, and H1*H2...*HN channel N are formed; one side which is far away from each channel M is provided with a connecting channel respectively; the integrated chip also comprises at least a collecting groove; the integrated chip also comprises at least one collecting groove which is connected with the output end of each collecting channel for collecting and exporting products. The invention has very high productivity and absolute advantage in the aspect of the large scale production of contrast agent.

Description

Integrated chip and device thereof, and method for preparing micron-sized dispersion

[ technical field ] A method for producing a semiconductor device

The invention relates to the contrast imaging technology of contrast agents, in particular to a large-scale integrated chip for manufacturing micron-sized dispersoids by adopting a microfluidic technology; the device adopting the chip can be used for preparing an ultrasonic contrast agent; and a method for preparing a micro-sized dispersion using the apparatus, the micro-sized dispersion being useful as an ultrasound contrast agent.

[ background of the invention ]

In clinical diagnostics, it has become increasingly important to detect structural and functional information about tissues non-invasively, in real time, and dynamically. The ultrasonic diagnosis technology not only has the characteristics, but also has the advantages of safety, wide application range, capability of repeated examination, strong identification capability on soft tissues, strong flexibility, low price and the like compared with the X-ray and nuclear magnetic resonance imaging technologies, and is the most widely used imaging technology in the world.

The use of contrast agents for contrast imaging is central to the development of imaging technology. Compared with various imaging technologies, ultrasound is the imaging technology which has the widest application range but cannot conventionally use contrast agents, and the core of the contrast ultrasound technology is the ultrasound contrast agent.

In 1968, the right heart was visualized by Gramiak et al, who first observed enhanced visualization of the right heart by catheter injection of gas-containing saline, revealing a sequential screen for acoustic visualization of the heart. At this stage, ultrasound contrast agents have been produced mainly by shaking physiological saline or CO2 foaming agent by hand. The microbubbles have large diameters and poor uniformity and are difficult to circulate through the lungs. In addition, because of the absence of the shell, the microbubbles have a short survival time due to the rapid diffusion of the gas in the blood, and the contrast agent in this period is called a 0 th generation ultrasound contrast agent by some researchers.

Feinstein et al really enter the age of left cardiography only by preparing stable microbubbles by a sound vibration method in 1984. Air-encapsulated microbubbles are the first to occur, and the air-encapsulated shell materials are widely available and can be roughly classified into: protein shells, liposome shells, surfactant shells, high polymer shells, and the like, are referred to as first generation ultrasound contrast agents. Due to the high solubility and dispersivity of air in blood, air in the microbubbles is easily dispersed into the blood through the shell, so that the microbubbles of the contrast agent become small or disappear. In addition, since the scattered signal of the microbubble is proportional to the 6 th power of the diameter, the echo signal of the microbubble is attenuated obviously as long as a small amount of air overflows and the diameter of the microbubble becomes slightly smaller. The representative products are albonex in the united states and Levovist of Schering company in germany, the stability and survival time of the first generation of ultrasonic contrast agent are not long enough, which greatly limits the application of the first generation of ultrasonic contrast agent, and the contrast agent market and research field are gradually withdrawn after 2000 years.

Since 1995, the survival time and stability of the ultrasound contrast agent were significantly improved due to the introduction of fluorocarbon-sulfur gas with high molecular weight, low blood solubility and dispersibility, which is called a second generation ultrasound contrast agent. Representative ultrasound contrast agents, Optison, usa,

EchoGen and AI-700, SonoVue (BR1) and BR14 from Bracco, Italy, and the like.

At present, various new-generation products are on the market abroad, while no products are on the market autonomously at home, and the only ultrasonic contrast agent which is approved and commercialized by the Ministry of health of China is produced by Bracco corporation of Italy, so that the price is very high.

In summary, the conventional ultrasound contrast agent is a suspension containing high concentration of microbubbles, which are enveloped by albumin, lipid, surfactant or polymer, have an average diameter of about 2-4 μm and are smaller than red blood cells, and can stably pass through the pulmonary circulation to reach the left heart system and further reach all organs of the whole body after peripheral intravenous injection. The ultrasonic contrast agent microbubbles have great difference in acoustic impedance with blood and human tissues, have very complex action with ultrasonic waves, can change the backscattering coefficient, attenuation coefficient, sound velocity and other acoustic characteristics of the part where the microbubbles are located, and can enhance the ultrasonic echo capability of local tissues after being injected into blood vessels, thereby obviously improving the development effect of ultrasonic images on tissue blood perfusion, obviously improving the sensitivity and specificity of ultrasonic diagnosis, breaking through the bottleneck of conventional ultrasonic development, and further expanding the field of ultrasonic diagnosis.

In addition, the ultrasonic contrast agent can be used for conventional contrast enhancement and also can be used for targeted diagnosis and interventional therapy, and a specific ligand is combined on the surface of a microbubble of the contrast agent so as to reach a tissue or an organ of interest and be selectively combined with a corresponding receptor, so that the aim of specifically enhancing an ultrasonic signal of a target area or locally targeted therapy is fulfilled. The research of interventional therapy continuously expands the application range of the ultrasonic contrast agent and continuously improves the application value.

For example, lowering the ultrasound cavitation threshold with contrast agent microbubbles, thereby promoting sonothrombolysis or enhancing the therapeutic effect of high power focused ultrasound; or the ultrasonic wave is utilized to destroy the microbubbles of the contrast agent carrying the gene or the drug, so that the gene or the drug is released in a targeted way and the penetration of the gene or the drug is promoted, and the gene transfer or the drug targeted therapy is mediated; or utilizing contrast to guide radio frequency or microwave treatment and evaluating treatment effect in real time.

The study of targeted ultrasound contrast agents is becoming a hotspot in the field of ultrasound imaging. The invention of active group selection and modification method, the detection of targeting effect, the research of in vivo drug effect and clinical application all achieve very favorable results. With the rapid development of high molecular materials and the continuous improvement of microbubble preparation processes, microbubble ultrasound contrast agents are bound to develop towards more individuation, and the future microbubble ultrasound contrast agents can be applied to specific ultrasound imaging under different physiological and pathological states, can also be used as carriers carrying drugs or therapeutic genes, and open up a new world in the field of treatment. Therefore, foreign medical institutions predict that the ultrasonic contrast agent will become a common reagent in ultrasonic diagnosis or treatment in the near future, and have great social benefit and economic value.

Currently, the focus of ultrasound field research is focused on two aspects: one is various imaging techniques to enhance ultrasound contrast, such as: triggering imaging technology, second harmonic imaging technology, subharmonic imaging technology, fundamental wave pulse cancellation imaging technology, energy pulse reverse imaging technology, low mechanical index real-time imaging technology and the like; on the other hand, a new more stable and reliable ultrasound contrast agent is researched.

An ideal ultrasound contrast agent should have the following properties: (1) the red blood cell suspension is safe and nontoxic, has low viscosity and no biological activity, has no physiological influence on a circulating system, and does not obviously dilute the concentration of red blood cells; (2) the size of the microbubbles meets the requirement, and the microbubbles have certain bubble pressure, so that the microbubbles can keep enough time and enough strong echo intensity after being perfused; (3) the reflectivity is good, the attenuation artifact is small, the echo signals and Doppler signals are obviously enhanced, and harmonic resonance can be generated to increase the back scattering area; (4) the intravenous injection can be carried out, the in vivo stability is good, the use is convenient, no special requirements are required on the conditions of contrast imaging instruments, and the popularization is convenient; (5) has stable concentration and dosage form, is easy to disinfect, sterilize, store and transport, and can be produced in batch.

The most commonly used methods for preparing ultrasound contrast agent microbubbles at present are vibroacoustic methods and mechanical oscillation methods.

The sono-vibration method is a method of using positive and negative sound pressures generated by high frequency conversion during ultrasonic oscillation, in which the negative sound pressure expands gas existing in a contrast agent preparation liquid to form micro bubbles, and at this time, lipid or albumin, a surfactant, a polymer, etc. in the preparation liquid wrap the micro bubbles on the machine to form stable contrast agent microbubbles. The preparation process of albumin, lipid, polymer, surfactant, etc. in the ultrasonic contrast agent adopts a sound vibration method.

However, the vibroacoustic method has the following disadvantages: (1) the technological parameters of the probe type acoustic vibrometer, including power, position and depth of the probe on the liquid level, are not easy to control, and the process reproducibility is influenced to a certain extent; (2) because the probe is placed in the preparation liquid, aseptic operation is difficult to achieve in the sound vibration process, and the possibility of metal pollution exists, so that certain difficulty is added to the quality control and preparation process of the contrast agent; (3) more heat is generated in the process of the sound vibration, and the activity of the lipid, especially when the contrast agent carrying certain ligands, medicines or genes is prepared, the activity of the simultaneously sound-vibrated ligands, medicines or genes is greatly influenced.

The mechanical oscillation method is characterized in that different positive and negative pressures are generated by different stress time phases of each point in the preparation liquid when high-frequency mechanical oscillation is utilized, wherein the negative pressure can enable gas in the preparation liquid to form micro bubbles. The higher the frequency is, the faster the positive and negative pressure is converted, the shorter the negative pressure time is, the smaller the gas expansion is, and the smaller the formed bubbles are; the smaller the amplitude, the smaller the negative pressure generated, and the smaller the expansion of the gas generated by the negative pressure at the same time, the smaller the bubbles formed, and the higher the frequency and the lower the amplitude are required for the mechanical oscillation device for preparing the ultrasound contrast agent because the smaller microbubbles need to be formed.

However, the mechanical oscillation method has the following disadvantages: (1) the particle size of the microbubbles of the contrast agent cannot be accurately controlled; (2) the formed microbubbles have wide particle size distribution and unstable acoustic characteristics, all the microbubbles contain a certain number of microbubbles larger than 10 microns, and the large microbubbles can cause local vessel blockage or rupture, so that the use of the ultrasonic contrast agent has certain risk; (3) the shell thickness of the formed microbubbles is not uniform; (4) the mechanical oscillation method is used for generating enough force to introduce gas into the liquid solution from the surrounding environment to form bubbles, the oscillation speed largely determines the quantity and the size of formed microbubbles, for example, a contrast agent SonoVue which is clinically approved in China is prepared in a hand-shaking mode, and the repeatability is not good enough.

Accordingly, the prior art is deficient and needs improvement.

[ summary of the invention ]

The technical problem to be solved by the invention is to provide a practical large-scale microfluidic flow focusing device and a preparation method of an ultrasonic contrast agent with uniform, controllable and monodisperse acoustic properties, so as to prepare the ultrasonic contrast agent with controllable particle size and monodispersity. On this basis, an integrated chip, a device using the integrated chip and a method for preparing micron-sized dispersion using the device are provided.

The technical scheme of the invention is as follows:

one technical scheme of the integrated chip is that two layers of adjacent structures are taken as a group of structures, and the integrated chip comprises at least one group of structures; in one group of structures, a first layer structure is provided with M-level gradients, and a second layer structure is provided with N-level gradients; wherein M, N is a natural number, M is less than N; in each stage of gradient, a first stage of gradient is provided with H1 channels 1, a second stage of gradient leads out H2 branches from each channel 1 to form H1 × H2 channels 2, and so on, and an Nth stage of gradient leads out HN branches from each channel (N-1) to form H1 × H2.

In one set of structures, for any specific channel M of the first layer structure, a specific channel N with the number of H (M +1) multiplied by H (M +2) is formed corresponding to the N-level gradient led out from the channel M by the second layer structure;

wherein, the outlets of each specific channel N are equidistantly distributed on the circumference of a circle with a preset radius; for the specific channel M, the output direction is vertical to the circle, the outlet of the specific channel M and the vertical line of the circle pass through the center of the circle, and the distance between the outlet of the specific channel M and the center of the circle is a specific length; the included angle between the output direction of each specific channel N and the vertical line is the same acute angle; along the output direction of the specific channel M, a collecting channel is arranged on one side of the circle away from the specific channel M; the integrated chip also comprises at least one collecting groove connected with the output end of each collecting channel and used for collecting and exporting the products.

Another solution of the integrated chip is that M ═ N-1.

In another embodiment of the integrated chip, the output direction of each specific channel N passes through the center of the circle.

The other technical scheme of the integrated chip is that the cross sections of the channel M and the channel N are rectangular; the rectangle has a height of 20 to 30 μm and a width of 30 to 50 μm, 50 to 100 μm, respectively, or 50 to 100 μm, 30 to 50 μm, respectively.

In another embodiment of the integrated chip, the structure of the collecting channel at least includes one of the following structures: cone, sphere, hemisphere or frustum body with outward amplification, wherein the pipe diameter of the input end is 7-25 μm, and the height is 20-30 μm; or a cylinder or a cuboid, the cross section area of which along the output direction of the corresponding specific channel M is slightly smaller than that of the specific channel M along the direction, and the height is 20-30 μ M.

In yet another embodiment of the integrated chip, the substrate is a surface hydrophilic substrate.

In another embodiment of the integrated chip, the substrate is silicon, glass, polydimethylsiloxane, polymethyl methacrylate, or polycarbonate.

In yet another embodiment of the integrated chip, the combination of the first layer structure and the second layer structure is a combination of a gas channel structure and a liquid channel structure.

One technical scheme of the device is that the device comprises: the device comprises a first substance input unit, a second substance input unit, an integrated chip array formed by at least one integrated chip, and a storage unit; wherein, the integrated chip is the integrated chip corresponding to any of the above-mentioned integrated chip technical schemes;

the collecting tanks of all the integrated chips are respectively connected with the storage units and used for collecting and exporting products; the first substance input unit is respectively connected with each channel 1 of the layer structure of each integrated chip and is used for inputting a first substance; the second substance input units are respectively connected with the channels 1 of the other layer structure of each integrated chip and used for inputting the second substance.

The combination of the first substance input unit and the second substance input unit is the combination of a gas input unit and a liquid input unit; or, the first substance input unit and the second substance input unit are both liquid input units.

The device has the technical scheme that the gas input unit comprises a pressure gas storage tank, a pressure reducing valve, a first transmission pipe, a micro-flowmeter, a pressure regulating valve and a second transmission pipe which are sequentially connected, wherein the second transmission pipe is respectively connected with each channel 1 of one layer of structure of each integrated chip; the liquid input unit comprises a liquid storage device, a third transmission pipe, a digital control type injection pump and a fourth transmission pipe which are sequentially connected, and the fourth transmission pipe is respectively connected with each channel 1 of the other layer structure of each integrated chip.

One technical solution of the method for preparing a micron-sized dispersion is to apply the method to a device comprising an array of integrated chips, wherein the array of integrated chips is composed of integrated chips, and the integrated chips are corresponding to any one of the technical solutions of the integrated chips; the device is any one of the technical schemes related to the device and corresponds to the device;

the method comprises the following steps: a1, inputting a first substance to each channel 1 of a layer structure of each integrated chip according to a first preset condition; inputting a second substance to each channel 1 of the other layer structure of each integrated chip according to a second preset condition; a2, adjusting the input conditions of the first substance and/or the second substance to enable the substance output by each channel M of each first layer structure to be wrapped by the substance of each corresponding specific channel N in a coaxial flow mode to form product streams to be collected respectively, wherein the axial direction of the product streams is coincident with the vertical line; and A3, collecting and leading out products from the output end of each collecting channel.

Another embodiment of the method for preparing the micron-sized dispersion is that the combination of the first substance and the second substance is a combination of gas and liquid; the gas at least comprises one of nitrogen, fluorocarbon gas and fluorine-sulfur gas; the liquid includes at least one of a phospholipid liquid, a surfactant liquid, and a modified liquid of the above liquids.

In another embodiment of the method for preparing the micron-sized dispersion, in step a1, a first liquid is supplied to each channel 1 of the first layer structure of each integrated chip, and a gas or a second liquid is supplied to each channel 1 of the second layer structure of each integrated chip; in step a2, the first liquid output from each channel M of each first layer structure is made to flow coaxially, and the first liquid is wrapped axially by gas or second liquid to form a stream of droplets to be collected, respectively.

In another technical scheme of the method for preparing the micron-sized dispersion, in the step A1, gas is input into each channel 1 of one layer structure of each integrated chip, the pressure is lower than 10psi, and liquid is input into each channel 1 of the other layer structure of each integrated chip, and the flow rate is lower than 3 muL/s; in step a2, the gas and/or liquid input conditions are adjusted.

Another embodiment of the process for preparing the micron-sized dispersion is that in step A1, the pressure of the input gas is less than 5psi and the flow rate of the input liquid is less than 1.5. mu.L/s.

Another technical solution of the method for preparing the micro-sized dispersion is that, in step a1, the liquid is added with specific ligand in advance; alternatively, after step a3, a specific ligand is added to the product.

In another embodiment of the method for preparing the micron-sized dispersion, in step a1, a gas is supplied to each channel 1 of the first layer structure of each integrated chip, and a liquid is supplied to each channel 1 of the second layer structure of each integrated chip; in step a2, the input conditions of gas and/or liquid are adjusted so that the gas output from each channel M of each first layer structure wraps the liquid in a coaxial flow manner in the axial direction to form the microsphere flow to be collected respectively.

In another technical scheme of the method for preparing the micron-sized dispersion, in the step a2, the gas output from each channel M of each first layer structure forms a stable inverted cone with a central axis located at the position of the vertical line under the focusing action of the liquid flowing at high speed at two sides, and the tip of each inverted cone is opposite to the position of each collecting channel.

With the adoption of the scheme, the invention provides an integrated chip, a device adopting the integrated chip and a method for preparing micron-sized dispersoids by adopting the device. On the basis, the invention provides a practical large-scale microfluidic flow focusing device and a preparation method of an ultrasonic contrast agent with uniform, controllable and monodisperse acoustic properties, so as to prepare the ultrasonic contrast agent with controllable particle size and monodispersity; the ultrasonic contrast agent is simple and convenient to prepare, good in effect and safe, and the prepared microbubble shell is uniform in thickness and has high application value.

The most remarkable advantage is that the prepared ultrasound contrast agent microbubble has high monodispersity and controllable particle size, and meets the requirements of the ultrasound contrast imaging technology. The polydispersity index of the microbubble particle size is less than 2%, the particle size is increased along with the increase of gas pressure and is reduced along with the increase of liquid flow rate, and the control is very flexible; meanwhile, the micro-nozzle array formed by a plurality of collecting channels greatly improves the preparation efficiency of the micro-bubbles; the production device has reusability, and reduces production cost.

Also, the apparatus and methods of the present invention can be used to prepare various types of micro-sized dispersions, including ultrasound contrast agent microbubbles, as well as microdroplets or other micro-mixtures. Conventional ultrasonic contrast agents can be prepared directly by the device, such as lipids, albumins, polymers, surfactants and the like; the targeted ultrasound contrast agent can be prepared by adding the specific ligand, and some shell materials such as protein molecules are inactivated under the conditions of high temperature and ultrasound, so the method solves the technical problem which cannot be solved by the traditional sound vibration method. The device of the invention is suitable for both methods, and particularly has advantages when the latter option is adopted, wherein the advantages of the device are that the preparation link is reduced, and the damage of the microbubbles in the preparation link is reduced. Moreover, the device generates less heat in the preparation process of the contrast agent, so the device is particularly suitable for preparing the ultrasonic contrast agent which is also used as a medicine or a gene targeting vector.

[ description of the drawings ]

FIG. 1 is a schematic view of an ultrasound contrast agent preparation apparatus according to an embodiment of the present invention;

FIG. 2 is a schematic diagram of the bottom channel structure of a microfluidic LSI chip according to an embodiment of the present invention;

FIG. 3 is a schematic diagram of a top channel structure of a microfluidic LSI chip according to an embodiment of the present invention;

FIG. 4 is an enlarged schematic view of the nozzle structure shown in phantom in FIG. 2;

FIG. 5 is a schematic illustration of microbubble formation according to an embodiment of the present invention;

FIG. 6 is a schematic diagram of a microfluidic LSI chip according to another embodiment of the present invention;

FIG. 7 is an enlarged view of the structure shown in phantom in FIG. 6;

FIG. 8 is a photograph of microbubbles generated in experiment one of the present invention;

FIG. 9 is a schematic view of an alternative nozzle configuration of an apparatus according to an embodiment of the present invention;

FIG. 10 is a schematic view of the plane of the outlet of each specific channel N in the communicating chamber according to the embodiment of the present invention;

fig. 11 is a schematic view of the longitudinal plane shown in fig. 10.

[ detailed description ] embodiments

The following detailed description is to be read in connection with the drawings and the detailed description.

The particle size and distribution of the ultrasound contrast agent microbubbles are important indexes of the ultrasound contrast agent, however, the microbubble particle size distribution range generated by the current ultrasound contrast agent manufacturing methods such as a sound vibration method and a mechanical oscillation method is wide, the microbubbles are mainly formed by hand shaking or oscillation, and the acoustic property is unstable. Therefore, the preparation method and the device for the large-scale microfluidic ultrasonic contrast agent are provided, a microfluidic large-scale integrated chip is manufactured by combining the mature micro-nano processing technology at present, the microbubbles with controllable particle size and high monodispersity are prepared by utilizing the flow focusing principle, the shell thickness of the microbubbles is uniform, not only can stable acoustic properties be obtained, but also the possibility of fusing different sizes of microbubbles into large microbubbles can be reduced, and the preparation method and the device have higher effectiveness and safety, and better repeatability and controllability in operation.

The present invention therefore proposes an integrated chip using microfluidic technology, which allows the preparation of micron-sized dispersions, including ultrasound contrast agents or other micromixing agents, in a microfluidic manner on a large scale.

The substrate of the integrated chip is a surface hydrophilic substrate, and the surface hydrophilic substrate comprises a hydrophilic substrate and a hydrophobic substrate the surface of which is subjected to hydrophilic treatment; it should be noted that the material of the channel does not necessarily have hydrophilicity, but the effect of the hydrophobic material is not as good as that of the hydrophilic material, and for the hydrophobic material, the surface of the hydrophobic material can be treated to be hydrophilic, and the effect similar to that of the hydrophilic material can also be obtained; the surface is a hydrophilic matrix, and microbubbles can be generated very smoothly.

An example is a substrate of silicon, glass, polydimethylsiloxane, polymethylmethacrylate or polycarbonate. Wherein, the material which can meet the structural requirement in the process and is not easy to react with the gas and the liquid in the channel can be used as the substrate, and the invention does not have any additional limitation; for example, polydimethylsiloxane has good insulating properties and can withstand high voltages; the thermal stability is high, and the method is suitable for processing various reaction chips; the optical detection system has excellent optical characteristics and can be applied to various optical detection systems; in addition, the nano-porous silicon dioxide can form good sealing with a plurality of materials such as silicon, silicon nitride, silicon oxide, glass and the like, and has high hydrophobicity and strong surface adsorption property to biomacromolecules, so that the nano-porous silicon dioxide is widely applied to the research field of microfluidic chips.

Two adjacent structures are taken as a group of structures, and the integrated chip comprises at least one group of structures; i.e. the integrated chip may comprise a two-layer structure or a four-layer structure, a six-layer structure, an eight-layer structure or even more. For example, the integrated chip is provided with only one set of structures. That is, the integrated chip has an even number of layers and is formed by stacking up and down or stacking obliquely, and the specific features of each layer are as follows: the edge layer structure at any end of the integrated chip and the layer structure adjacent to the edge layer structure form a group of structures; the integrated chip can have one group of structures or a plurality of groups of structures. The integrated chips may also be formed in parallel arrangement, i.e. one set of structures or a plurality of sets of stacked multi-layer structures are arranged in parallel.

In one group of structures, two layers of adjacent structures are provided, wherein the first layer of structure is provided with M-level gradient, and the second layer of structure is provided with N-level gradient; wherein M, N is a natural number, M is less than N; in each stage of gradient, the first stage of gradient is provided with H1 channels 1, the second stage of gradient leads out H2 branches from each channel 1 to form H1 × H2 channels 2, and so on, and the Nth stage of gradient leads out HN branches from each channel (N-1) to form H1 × H2. Preferably, each branch is distributed uniformly with respect to the branch of the previous branch, mainly to ensure the same liquid flow rate or the same gas flow rate from the last channels; that is, the Nth order gradient leads HN branches from each channel (N-1), and the HN branches from a certain channel (N-1) are uniformly distributed with respect to the channel.

Wherein each channel 1 of the first layer structure is used for inputting a first substance; each channel 1 of the second layer structure is used for inputting a second substance. The first substance and the second substance may be different gases or liquids, or may be two different liquids, or the first substance may be a gas and the second substance may be a liquid, or the first substance may be a liquid and the second substance may be a gas. For example, the first layer structure acts as a gas channel and the second layer structure acts as a liquid channel; or the first layer structure is used as a liquid channel, and the second layer structure is used as a gas channel; alternatively, the first layer structure acts as a first liquid channel and the second layer structure acts as a second liquid channel.

For example, the first layer structure acts as a gas channel and the second layer structure acts as a liquid channel; at this time, H1 channels 1 of the first layer structure are respectively connected to the gas input ports, and H1 channels 1 of the second layer structure are respectively connected to the liquid input ports; the H1 × H2.. multidot.hm channels M of the first layer structure are respectively connected to the gas outlet, and the H1 × H2.. multidot.hn channels N of the second layer structure are connected to the liquid outlet. Or, the first layer structure serves as a liquid channel, the second layer structure serves as a gas channel, and the rest are similar and are not described in detail.

In a preferred example, M is equal to N-1, i.e., M is 1 less than N, and the gradient number of the first layer structure is one layer less than the gradient number of the second layer structure. For example, N is 8 and M is 7.

Alternatively, M is 2, 3, 4, etc. less than N, e.g., N is 8, M is 4; as another example, N is 6, M is 3; for another example, N is 9 and M is 7; as another example, N is 21 and M is 12. The invention does not make additional limitation, and only needs to realize the difference of the gradient number of the first layer structure and the second layer structure.

Wherein, H1, H2, H3.. For example, H1, H2, H3... HM, H (M +1), H (M + 2.. HN are each 2, and M ═ N-1, such that the number of channels N is 2 times that of channels M; in this case, since H1 is 2, there are two gas inlets and two liquid inlets; alternatively, H1 is 2, and H2 to HN are all 3; alternatively, H1 is 1, H2 is 2, H3 is 3, H4 is 4.. HN is N; alternatively, they are all different, such as H1 being 4, H2 being 1, H3 being 6, H4 being 5. The invention does not make additional limitation to this, and only needs to realize the branch structures of all levels.

For the shape of the channels, the cross-section of each channel is preferably rectangular; wherein, the cross section of the channel M and the cross section of the channel N can be rectangles with the same or different shapes, for example, the height of the rectangle is 20 μ M to 30 μ M, the width of the cross section of the channel M is 30 μ M to 50 μ M, and the width of the cross section of the channel N is 50 μ M to 100 μ M; or the width of the cross section of the channel M is 50-100 μ M, and the width of the cross section of the channel N is 30-50 μ M; alternatively, the width of the channel M cross section and the width of the channel N cross section are both 30 μ M to 50 μ M.

An example is a channel which is a uniform or non-uniform cuboid but which must be rectangular in cross-section, for example a channel which is a regular quadrangular frustum, a quadrangular frustum with a similar rectangular cross-section, or a trapezoidal frustum. In a preferred example, each channel is a rectangular parallelepiped, and the cross section of the channel is a rectangle having a height of 20 μm to 30 μm and a width of 30 μm to 100 μm. For example, the rectangle has a height of 25 μm and a width of 40 μm; as another example, the rectangle has a height of 28 μm and a width of 35 μm; as another example, the rectangle has a height of 22 μm and a width of 45 μm; as another example, the rectangle has a height of 21 μm and a width of 85 μm; as another example, the rectangle has a height of 29 μm and a width of 65 μm; as another example, the rectangle has a height of 29 μm and a width of 75 μm.

In a preferred embodiment, the channel M has a cross-section that is rectangular and the channel N has a cross-section that is the same height.

That is to say, for a certain channel, the cross section perpendicular to the channel direction thereof, that is, the channel cross section of the channel is a square or a rectangle as the cross section, it should be noted that the channel cross section may also be a triangle, a diamond, a pentagon or other shapes, which is not limited in this respect; for each channel, it is only necessary that the area of the channel cross section is at least 600 square microns, for example, 800, 900 or 1000 square microns. For another example, each channel is a cylinder or other body; or, each channel may also be a square body, a circular body or a spiral shape which is bent and changed, for example, in an L shape, a Y shape, etc.; in this case, the output direction of the channel is the direction of the last unbent channel, for example, the output direction of the L-shaped channel is the direction of the channel with the outlet after the direction change.

In one set of structures, for any particular channel M of the first layer structure, it corresponds to a certain branch of the HM branches of the second layer structure from a channel M; the branches form H (M +1) branches at the M +1 level gradient, and specific channels N with the number of H (M +1) multiplied by H (M +2) are formed at the N level gradient; that is, for any channel M of the first layer structure, in the second layer structure, the second layer structure channel M corresponding to the first layer structure channel M will lead out H (M +1) branches at the next-level gradient, and these branches finally form H (M +1) × H (M +2) ·.

The outlets of the specific channels N are equidistantly distributed on the circumference of a circle with a preset radius, namely, the outlets of the channels N are arranged to form a regular polygon and are positioned at each vertex of the regular polygon; for example, the outlets of the channels N form a regular triangle, square, regular pentagon, regular hexagon. In particular, when there are only two channels N, the outlets are located at both ends of a certain diameter of the preset radius circle. The preset radius can be set according to actual conditions, for example, the size of the circular radius is preset according to different reactants and different reaction conditions. The radius of the circle is related to the size of the channel, taking the first layer channel for inputting gas and the second layer channel for inputting liquid as an example, when the gas outlet is vertical to the circle, the liquid outlets are uniformly distributed on the periphery of the gas outlet. For example, the radius of the circle is 150 μm to 1500 μm, and more specifically, the radius of the circle may be 250 μm, 500 μm, 700 μm, 1100 μm, 1200 μm, 1400 μm, or the like. Preferably, the radius of the circle corresponds to the width of the channel, the larger the radius of the circle.

It should be noted that when the dimensions are small, for example, measured in the micron scale, the outlet of the channel, and the input of the collection channel, are often a one-sided concept; in each embodiment of the invention, all the connection lines between the points are referred to as the central point of the outlet surface or the central point of the surface of the input end; for example, the geometric center point of a triangle or rectangle; or approximately the center point of the exit face, or the center point of the face of the input end. In this way, the outlets are arranged to form a regular polygon, which can achieve better accuracy. And the connecting line between the outlets and the input end of the collecting channel can be more accurate, so as to obtain better effect.

One example is shown in fig. 10, there are six specific channels N, outlets of which are equidistantly distributed on the circumference of a circle with a preset radius, the six outlets form a regular hexagon and are located in a transverse plane, the transverse plane is located in a communicating cavity, and liquid flows from the outlets of each specific channel N to the center of the circle with the preset radius, i.e. the distance from the liquid outlet to the center of the circle is the same and the liquid outlets are arranged in a regular hexagon; in the regular hexagon, any two opposite specific channels N are positioned in a longitudinal plane; for example, any two opposing specific channels N, form a symmetrical or substantially symmetrical relationship with the center of the circle of the preset radius.

In the above example, any longitudinal plane is as shown in fig. 11, and two opposite specific channels N form an axisymmetric relationship with respect to the outlet of the specific channel M and the perpendicular line of the circle; after the first layer of gas is output from the outlet, the first layer of gas continues to flow for a distance, i.e. a certain length, which is mainly related to the height or width of the rectangular cross section of the liquid channel of the second layer, for example, the certain length is 25% to 75% of the width or height of the cross section of the liquid channel, such as 30%, 40%, 50%, 60%, 70% of the height of the cross section of the channel, and the preferred example is that when there are only two liquid channels and one gas channel, all the outlets are in a plane, as shown in fig. 4 and 5, the distance from the center of the circle of the gas channel outlet is half of the width of the cross section of the liquid channel; when a plurality of liquid channels are arranged, and the outlet of the gas channel is vertical to the plane of the outlet of the liquid channel, the distance between the outlet of the gas channel and the circle center is half of the height of the cross section of the liquid channel. Then mixing the gas and the liquid, forming a product flow to be collected before entering the input end of the collecting channel, and collecting the product flow through the collecting channel; for example, the product stream to be collected, which forms an inverted cone or other shape, is collected by the collection channel.

In both of the above examples, an even number of specific channels N are described, with either longitudinal plane having a pair of specific channels N forming an axisymmetric relationship or substantially symmetric; it should be noted that, if there are an odd number of specific passages N, although a pair of specific passages N cannot be formed in the longitudinal plane, the effects of the present invention can be achieved as well. For example, the outlets of five specific channels N are equally distributed on the circumference of a circle with a preset radius, the five outlets form a regular pentagon and are located in a transverse plane, the transverse plane is located in a communicating cavity, and the liquid flows from the outlet of each specific channel N to the center of the circle with the preset radius.

The output direction of the specific channel M is perpendicular to the circle, the outlet of the specific channel M passes through the center of the circle with the perpendicular to the circle, that is, the output direction of the specific channel M is perpendicular to the plane of the circle, and the projection of the outlet of the specific channel M on the plane is located at the center of the circle. The output direction of each specific channel N is the same as the included angle of the vertical line, and the included angles are acute angles; for example, the output direction of each specific channel N passes through the center of the circle, each specific channel N is located on each bisector of the circle according to the number of the specific channels N, and if 3 specific channels N are provided, the included angle between two adjacent specific channels N is 120 degrees; or, each specific channel N may form a straight cone, the straight cone uses the output direction of the specific channel M as a rotation axis, the included angles of each specific channel N and the rotation axis are the same, and the included angles are acute angles; for example, the included angles are all 30 degrees, 45 degrees, 60 degrees, 75 degrees, 80 degrees, and so on. For example, since the outlets of the specific channels N are equidistantly distributed on the circumference of a circle with a preset radius, when the number of the specific channels N is even, the outlets of the two specific channels N symmetrical to the rotation axis form a plane including the rotation axis, and the two specific channels N form an isosceles trapezoid in the output direction. For example, each specific channel N forms a symmetrical shape; for example, the hexagonal pyramid is a half-section hexagonal pyramid, that is, a hexagonal frustum, the bottom surface of which is a regular hexagon, and the hexagonal pyramid can form three isosceles trapezoids.

In this way, in a group of structures, by adjusting the input conditions of one layer of structure and the other layer of structure, the substance output by each channel M of each first layer of structure is in a coaxial flow form, the substance output by a certain channel M is wrapped by the substance of each corresponding specific channel N in the axial direction, that is, the substance output by each corresponding specific channel N is output by taking the flow direction of the substance output by a certain channel M as an axis, so as to form a layer of substance output by each corresponding specific channel N; or the substance output by each specific channel N is covered on the substance output by the channel M in a concentric circle mode along the axial direction; respectively forming a product flow to be collected, for example, forming an inverted cone by the product flow to be collected; the axial direction of which coincides with the axis of rotation. That is, the material output from a certain channel M of a certain first layer structure flows along the output direction thereof all the time or flows spirally around the output direction thereof; the output direction is taken as the axial direction, and the material of each specific channel N corresponding to the output direction is wrapped by the material of each specific channel in the axial direction.

The outlet of each specific channel N is communicated with the outlet of the specific channel M; for example, the outlet of each specific channel N is communicated with the outlet of the specific channel M through a communicating cavity; in each embodiment of the invention, the communicating cavity is not limited at all, and only a product flow to be collected is formed and output through the collecting tank, or the communicating cavity is not arranged.

Along the output direction of the specific channel M, a collecting channel is correspondingly arranged on one side of the circle away from the specific channel M; the collecting channel is used for forming a product flow to be collected before the input end of the collecting channel, collecting the product at the input end of the collecting channel, and outputting the product to the collecting tank through the collecting channel; that is, the same number of collecting channels as the number of channels M are provided, and for each specific channel M, a collecting channel is provided along the output direction thereof, and the output directions of the specific channel M and the collecting channel thereof are the same and on the same straight line.

As an example, the structure of the collection channel may comprise one or more of the following structures: cone, sphere, hemisphere or frustum body with outward amplification, wherein the frustum body is a cone with a residual frustum body part with a cut tip, the pipe diameter of the input end of the frustum body is 7-25 μm, and the height of the frustum body is 20-30 μm; or, a cylinder or a cuboid, as shown in fig. 9, the area of the channel cross section along the output direction of the corresponding specific channel M is slightly smaller than the area of the channel cross section of the specific channel M along the output direction, and the height is 20 μ M to 30 μ M, at this time, the length of the cylinder or the cuboid channel needs to be controlled so that the microbubbles are just generated in the collecting tank to have a velocity gradient, thereby generating the shearing force. For example, the structure of the collecting channel is an outwardly enlarged cone, or the structure of the collecting channel is a combination of an outwardly enlarged frustum and a cylinder.

The integrated chip also comprises at least one collecting groove connected with the output end of each collecting channel and used for collecting and exporting the products.

In addition, by using the existing micro-nano processing technology, through the steps of spin coating, exposure, development, casting, peeling, adhesion and the like, a micro-fluidic large-scale integrated chip based on Polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA) or Polycarbonate (PC) can be manufactured.

For example, H1 is 1, and H2 to HN are all 2, as shown in fig. 11, a connection line between an outlet of the specific channel M and an input end of its corresponding collection channel is perpendicular to a connection line between outlets of two specific channels N, that is, each specific channel M corresponds to two specific channels N, respective outlets of the two specific channels N, and a central line connecting the outlets of the specific channels M and the input ends of the corresponding collection channels is a connection line; and, in general, these two specific channels N are arranged symmetrically with respect to the above-mentioned median line; that is, this is a special case, at this time, the two outlets cannot form a certain circle, but are still equidistantly distributed on the circumference of a circle with a preset radius, so that the midperpendicular of the line segment connecting the outlets of the two specific channels N is the connecting line of the outlet of the specific channel M and the input end of the corresponding collecting channel. Since the circle formed by the outlets of the two specific channels N is not unique, the connecting line is also one of the perpendicular lines between the outlet of the specific channel M and the center of the circle.

Thus, when N is 9 and M is 8, the first layer structure finally forms the (M-1) power of 2, namely, the 7 powers of 2, namely, the 128 channel outlets, and the second layer structure finally forms the (N-1) power of 2, namely, the 8 powers of 2, namely, the 256 channel outlets; that is, it has 128 channels M, each channel M corresponding to two channels N, for a total of 128 outputs; for example, the channel M1 corresponds to the channel N11 and the channel N12, and the output directions of the channel N11 and the channel N12 are opposite and are positioned on the same straight line; the distance from the outlet of the channel N11 to the outlet of the channel M1 is the same as the distance from the outlet of the channel N12 to the outlet of the channel M1.

In the above examples, the device is typically characterized by the integration of 128 expanding nozzle chambers, e.g., as shown in fig. 3, a first layer of structure for gas input, as shown in fig. 2, and a second layer of structure for liquid input; the nozzle structure is shown in fig. 4, each gas output channel port corresponds to two opposite liquid output channel ports; the gas has the largest shearing force at the narrowest point of the nozzle, namely the nozzle opening, and the narrowest point of the collecting channel, and then a velocity gradient is generated at the expanded nozzle as shown in fig. 5, so that the focusing and the shedding of the micro-bubbles are facilitated; therefore, the production efficiency of microbubbles can be improved by using the nozzle array.

More specifically, in some examples of the present invention, the flow-focusing principle is used to generate microbubbles, the gas is in the coaxial flow center position under the coating of the liquid on both sides, i.e. the central axis, the gas is focused by the liquid moving at high speed to form a stable cone and is aligned with the nozzle opening position, the nozzle opening is narrowest and has the largest shearing force, as shown in fig. 5, the subsequent expanding nozzle generates a velocity gradient, and the microjet ejected from the cone tip falls off at the nozzle opening to form microbubbles with uniform size.

With the above embodiments, the microfluidic large-scale integrated chip can be used for preparing the ultrasound contrast agent, and the integrated chip described in the above embodiments can also be used for preparing other micro-sized mixtures, such as micro-droplets and the like.

Moreover, the invention also provides a device by adopting the integrated chip of any one of the embodiments. The device comprises a first substance input unit, a second substance input unit, a storage unit and at least one integrated chip as described in any of the above embodiments, which integrated chips form at least one integrated chip array. The collecting tanks of all the integrated chips are respectively connected with the storage units and used for collecting and exporting products; the first substance input unit is respectively connected with each channel 1 of the layer structure of each integrated chip and is used for inputting a first substance; the second substance input units are respectively connected with the channels 1 of the other layer structure of each integrated chip and used for inputting the second substance. The apparatus can be used to prepare micron-sized dispersions, including ultrasound contrast agents or other micro-mixtures.

The combination of the first substance input unit and the second substance input unit is the combination of a gas input unit and a liquid input unit; that is, the first substance input unit is a gas input unit and the second substance input unit is a liquid input unit, or the first substance input unit is a liquid input unit and the second substance input unit is a gas input unit. Or, the first substance input unit and the second substance input unit are both liquid input units.

For example, the device comprises a gas input unit, a liquid input unit, a storage unit and at least one integrated chip as described in any of the above embodiments, which integrated chips form at least one integrated chip array. Alternatively, the apparatus comprises: meters for controlling flow parameters of gases, liquids, and chips for passing gases and liquids through an array of micro-nozzles having substantially uniform diameters.

For example, the whole ultrasonic contrast agent preparation device is shown in fig. 1, a gas source outputs gas, the flow is controlled by a flowmeter or a pressure gauge, and the gas is output to a large-scale microfluidic chip; the liquid source outputs liquid, the flow is controlled by a numerical control injection pump and the liquid is output to the large-scale microfluidic chip; the reaction produces a product, which is collected, for example, by a product collection device. For example, the product is collected and removed by means of collecting tanks of the individual integrated chips, which collecting tanks are connected to the storage units, respectively, and the final product is introduced into the storage units; this allows efficient collection of the final product.

For example, the gas input units are respectively connected with the channels 1 of the layer structure of each integrated chip and used for inputting gas; the liquid input units are respectively connected with the channels 1 of the other layer structure of each integrated chip and are used for inputting liquid.

For example, the gas input units are respectively connected with the channels 1 of the first layer structure of each integrated chip, and the liquid input units are respectively connected with the channels 1 of the second layer structure of each integrated chip; or, the gas input units are respectively connected with the channels 1 of the second layer structure of each integrated chip, and the liquid input units are respectively connected with the channels 1 of the first layer structure of each integrated chip.

As an example, the gas input unit includes a pressure gas tank, a pressure reducing valve, a first transmission pipe, a micro flow meter, a pressure regulating valve, and a second transmission pipe, which are connected in series, and the second transmission pipe is connected to each channel 1 of the one-layer structure of each integrated chip, respectively.

As another example, the liquid input unit includes a liquid storage, a third transfer tube, a digitally controlled syringe pump, and a fourth transfer tube connected in sequence, and the fourth transfer tubes are respectively connected to the channels 1 of the other layer structure of each integrated chip.

In a preferred example, each channel in each integrated chip of the device has a rectangular cross section and a height of 20 μm to 30 μm; the width of the channel M cross section is 30-50 μ M, the width of the channel N cross section is 50-100 μ M, or the width of the channel M cross section is 50-100 μ M, and the width of the channel N cross section is 30-50 μ M; wherein the structure of the collecting channel at least comprises one of the following structures: cone, sphere, hemisphere or frustum body with outward amplification, wherein the pipe diameter of the input end is 7-25 μm, and the height is 20-30 μm; or a cylinder or a cuboid, the cross section area of which along the output direction of the corresponding specific channel M is slightly smaller than that of the specific channel M along the direction, and the height is 20-30 μ M. The substrate of the integrated chip is silicon, glass, polydimethylsiloxane, polymethyl methacrylate or polycarbonate. The combination of the first layer structure and the second layer structure is the combination of a gas channel structure and a liquid channel structure. H1 is 1, and H2 to HN are both 2; n is 8, M is 7, the first layer structure and the second layer structure are both symmetrically provided with two initial channels at two sides of the collecting channel, namely 2 gas inlets and 2 liquid inlets, so that the first layer structure finally forms 2 (M-1) powers of 2, namely 128 channel outlets, and the second layer structure finally forms 2 (N-1) powers of 2, namely 256 channel outlets; in this example, the typical feature of the device is that 128 expanding nozzle cavities are integrated, the gas has the largest shearing force at the narrowest point of the nozzle, namely the narrowest point of the collecting channel, and the subsequent expanding nozzle generates a velocity gradient which is gradually decreased, so that the focusing and shedding of the micro-bubbles are facilitated; the nozzle array can improve the production efficiency of the microbubbles.

More specifically, as shown in fig. 1, gas is delivered from a pressure tank to a gas inlet of a chip through a pressure reducing valve, the flow of the gas is monitored by a micro-flowmeter and a pressure regulating valve, continuous liquid is pumped into the liquid inlet at a constant flow rate by a digital control type injection pump, and microbubbles generated by the micro-fluidic lsi chip are stored in a special storage bottle, which is the prior art.

The microfluidic large-scale integrated chip is manufactured by utilizing a micro-nano processing technology, and has an upper layer structure and a lower layer structure.

As a more specific example, the chip infrastructure is shown in fig. 2. The channel starts to expand step by step from two symmetrically arranged liquid inlets, finally passes through 8-step gradients, and expands in 7 steps to form 256 branches, namely N is 8. Every two branches belonging to one initial inlet serve as liquid inlets of the micro-nozzles, thus forming 128 micro-nozzles. The position a' indicated by the chain line in fig. 2 is a gas inlet of one micro nozzle, 128 gas inlets in total, and the generated micro bubbles are collected and stored by the collecting tank.

The structure of the channel at the top layer of the chip is shown in fig. 3, and similar to the bottom layer structure, the channel is expanded step by step from two symmetrically arranged gas inlets, has 7 gradients, and is expanded by 6 steps, namely M is 7, and 64 branches are finally formed by a single initial gas inlet. Thus, a total of 128 branches are formed by the expansion, and 128 gas inlets corresponding to the bottom layer are communicated, such as a and a'.

Wherein, the bottom layer and the top layer can be interchanged, as shown in fig. 6, which is an alternative form of the microfluidic lsi chip of the present invention, the top layer is a liquid channel structure, the bottom layer is mainly a gas channel, and two initial inlets of the bottom layer are expanded into 128 branches through 6 stages to serve as gas inlets of the micro-nozzles. Fig. 7 is an enlarged view of a micro-nozzle shown in a dashed box in fig. 6, the main difference from fig. 4 being that two liquid inlets communicate with the top layer liquid channel.

Fig. 4 is an enlarged view of the dotted line portion of fig. 2, which is a schematic view of a micro-nozzle in the array of the integrated chip, wherein the micro-nozzle is a basic unit for micro-bubble formation, wherein the arrows represent the flowing direction of gas and liquid, a gas inlet is communicated with the gas channel of the top layer, and the shearing force is greatest at the narrowest part of the collecting channel in the shape of large arrow. The aperture of the collection channel inlet is in the range of 7-25 μm, the channel height is 25 μm, preferably the same as the height of other channels in the same layer structure, the gas inlet width is 30-50 μm, the liquid inlet width is 50-100 μm, the gas forms a stable cone at the central axis position under the liquid focusing action of high-speed flow at both sides, as shown in fig. 5, and is aligned with the nozzle opening position, because the nozzle opening is narrowest and has the largest shearing force, the subsequent expanding nozzle generates a velocity gradient, the micro-jet flow emitted from the cone top end falls off at the nozzle opening to form micro-bubbles with uniform size, and the micro-bubble forming schematic diagram is shown in fig. 5, and the cone and the micro-bubbles formed by flow focusing can be visually seen.

As shown in fig. 9, the shape of the micro-nozzle of the device can be replaced by other geometric shapes forming a velocity gradient, such as a rectangular structure, and only one velocity gradient needs to be generated to make the micro-jet emitted from the cone tip fall off at the nozzle.

It should be noted that the drawings are enlarged in part and are for illustrative purposes and are not drawn to strict scale. These drawings are not intended to be strictly limiting and do not set any additional limitations on the various aspects and embodiments of the present invention.

With the above embodiments, it is possible to integrate the extended micro nozzle array on a large scale, and by inputting gas and liquid at different channel inlets, it is possible to realize and advantageous for the mass production of microbubbles. The device can be used for preparing various types of ultrasonic contrast agent microbubbles by adopting various gases and liquids and inputting the gases from the channels 1 of the first layer structure of each group of structures of the integrated chip.

Exchanging gas and liquid inlets, namely inputting liquid from each channel 1 of the first layer structure of each group of structures of the integrated chip, and generating a large amount of micro-droplets; or two incompatible liquids are respectively introduced, so that micro-droplets of the liquid at the bottom layer wrapping the liquid at the upper layer can be generated, and the method is very suitable for the food industry and the cosmetic industry.

The micro-nozzle array formed by a plurality of collecting channels is adopted, the number of the micro-nozzles can be increased or decreased, for a group of structures, at least one nozzle is arranged, and when N-level gradients are arranged on the first layer of structure, the expansion stage number of the channels is N-1, namely 1 less than the gradient number N. In the case of only one initial inlet, the last branch is 2N-1 and the number of nozzles is typically 2N-2. In the case of two symmetrically arranged initial inlets, the number of nozzles is typically 2N-1. For example, when N is 8, there are 8 levels of gradients, which are expanded by 7 levels, each level is expanded by one time, the first level of gradients is provided with 1 channel 1, the eighth level of gradients is provided with 128 channels 8, and in the case of only one initial inlet, there are 64 nozzles; in the case of two symmetrically arranged initial inlets, there are 128 nozzles. On the premise of easily controlling the flow rate of the fluid and the gas pressure, the larger the number of the nozzles, the better.

Also, a method of preparing micron-sized dispersions that are immiscible; the method is applied to a device comprising an array of integrated chips, wherein the array of integrated chips is composed of at least one integrated chip as described in any one of the above embodiments; the device is the device of any one of the above embodiments. The method comprises the following steps.

A1, inputting a first substance to each channel 1 of a layer structure of each integrated chip according to a first preset condition; and inputting a second substance into each channel 1 of the other layer structure of each integrated chip according to a second preset condition. Wherein, the first substance and the second substance can be different liquids; alternatively, the first substance may be a liquid and the second substance may be a gas; alternatively, the first substance may be a gas and the second substance may be a liquid.

For example, in step a1, gas is fed into each channel 1 of one layer structure of each ic at a pressure lower than 10psi and higher than 0, and liquid is fed into each channel 1 of the other layer structure of each ic at a flow rate lower than 3 μ L/s and higher than 0; for example, the pressure of the input gas is less than 5psi, and the flow rate of the input liquid is less than 1.5 μ L/s; as another example, the pressure of the input gas is less than 8psi, and the flow rate of the input liquid is less than 2.5 μ L/s; as another example, the pressure of the input gas is less than 7.5psi, and the flow rate of the input liquid is less than 2.0 μ L/s; as another example, the pressure of the input gas is less than 4psi and the flow rate of the input liquid is less than 2.8 μ L/s. In this way, uniform microbubbles can be prepared, which can be used in ultrasound contrast agents.

In a specific application, the gas at least comprises one of nitrogen, fluorocarbon gas and fluorine-sulfur gas; the liquid includes at least one of a phospholipid liquid, a surfactant liquid, and a modified liquid of the above liquids.

In order to ensure that the microbubbles have a longer survival time in vivo, the gas materials that can be used in the device are mainly fluorocarbon, fluorine-sulfur type gases with low solubility in plasma, such as C3F8, C4F10, SF6, etc., and also combinations of these gas components or combinations thereof with nitrogen, such as nitrogen and C4F10, nitrogen and C3F 8.

Preparing vacuole, i.e. microvesicle, whose shell material is derived from liquid mainly comprising phospholipids such as DPPC, DPPA, DPPE, DSPC, DSPE, DSPA, etc.; surfactants such as Span20, Span60, Span 80, etc., Tween20, Tween 60, Tween80, etc., and combinations of phospholipids, surfactants, or phospholipids and surfactants; the materials can obviously reduce the surface tension of a gas-liquid interface and have good biocompatibility and blood compatibility.

Or the shell material for preparing the vacuole is a material with special functions obtained by modifying the shell material on the basis of the shell material, and comprises DSPE-PEG2000, DSPE-PEG5000, DSPE-PEG (2000) Biotin, DSPE-PEG (2000) Carboxylic Acid and the like. The molecular structure of the hydrophobic long chain and the hydrophilic polar head of the phospholipid enables the molecular structure of the phospholipid to be self-adjusted in an aqueous environment, the hydrophobic chains are closely arranged together, the hydrophilic head is exposed in an aqueous phase to finally form a vesicular structure, the structure can prevent gas from diffusing out of formed microbubbles, and the hydrophilic shell enables the phospholipid to have excellent biocompatibility. Meanwhile, the phospholipids can have different charge properties due to different polar heads, and the stability of the phospholipid microbubbles can be adjusted by adjusting the components and the proportion of the charged phospholipids. Phospholipids containing long chains of PEG, such as DSPE-PEG2000, have the ability to resist their capture by the reticuloendothelial system during circulation in vivo, prolonging the time of circulation in vivo. Phospholipids containing Biotin ligands, such as DSPE-PEG (2000) Biotin, have the ability to recognize antibody avidin and thus have targeting functions. Thus, by combining different phospholipid components and adjusting the ratio of the components, microbubbles with different functions and effects can be obtained.

A2, adjusting the input conditions of the first substance and/or the second substance, so that the substance output by each channel M of each first layer structure is in a coaxial flow form, and the substance output by each corresponding specific channel N wraps the substance output by the channel M in the axial direction to form product streams to be collected respectively, wherein the axial direction of the product streams to be collected is coincident with the vertical line.

For example, in step a2, the input conditions of different gases or different liquids are adjusted individually, or the input conditions of gases or liquids are adjusted individually, or the input conditions of gases and liquids are adjusted simultaneously, so that the substance output from each channel M of each first layer structure is wrapped by the substance output from each corresponding specific channel N in a coaxial flow manner, and the substance output from each corresponding specific channel M is wrapped in an axial direction, so as to form product streams to be collected, wherein the axial direction of each product stream is coincident with the vertical line; for example, the product streams to be collected each form an inverted cone, the axial direction of which coincides with the vertical.

And A3, collecting and leading out products from the output end of each collecting channel.

For example, a gas is supplied to each channel 1 of the first layer structure of each integrated chip, and a liquid is supplied to each channel 1 of the second layer structure of each integrated chip; thus, a method of forming microbubbles at the nozzle by gas focusing from two side fluid flows can be achieved, forming highly monodisperse microbubbles suitable for ultrasound contrast imaging techniques. Or, liquid is input into each channel 1 of the first layer structure of each integrated chip, and gas is input into each channel 1 of the second layer structure of each integrated chip; in this way, a method of forming micro-droplets at the nozzle by focusing of the liquid from a two-sided gas flow can be obtained.

As mentioned above, a preferred example is the integrated chip, H1 is 1, H2 to HN are 2, N is 9, and M is 8. The device is provided with 128 channels M, each channel M corresponds to two channels N, and 128 output ends are provided; for example, the channel M1 corresponds to the channel N11 and the channel N12, and the output directions of the channel N11 and the channel N12 are opposite and are positioned on the same straight line; the distance from the outlet of the channel N11 to the outlet of the channel M1 is the same as the distance from the outlet of the channel N12 to the outlet of the channel M1.

As another example, in step a1, a gas is supplied to each channel 1 of the first layer structure of each integrated chip, and a liquid is supplied to each channel 1 of the second layer structure of each integrated chip; in step a2, the gas output from each channel M of each first layer structure is made to flow coaxially through each channel N of each second layer structure corresponding to a certain channel M, and the flowing-out liquid axially wraps the gas corresponding to the channel M to form the microspherical flow to be collected. In which the coaxial flow pattern has been described previously.

On the basis of the above example, in step a2, the gas output from each channel M of each first layer structure forms a stable inverted cone with its central axis located at the position of the vertical line under the focusing action of the liquid flowing at high speed on both sides, and the tip of each inverted cone is opposite to each collecting channel.

As another example, in step a1, a first liquid is supplied to each channel 1 of the first layer structure of each integrated chip, and a gas or a second liquid is supplied to each channel 1 of the second layer structure of each integrated chip; in the step a2, the first liquid output from each channel M of each first layer structure is wrapped by gas or second liquid in an axial direction in a coaxial flow manner to form streams of droplets to be collected respectively; for example, a stream of droplets to be collected each forming an inverted cone.

In each of the above examples, in step a1, the liquid may be added with a specific ligand in advance; alternatively, after step a3, a specific ligand is added to the product. Thus, different specific ligands can be added according to requirements, and the targeted ultrasound contrast agent is prepared.

The details of the integrated chip, apparatus and method, and particularly the method of preparing an ultrasound contrast agent, continue to be described below.

In the ultrasound contrast imaging technology, the size of the microbubble contrast agent influences its ability to pass through pulmonary microcirculation and the reflectivity to ultrasound, and the diameter must be less than 7 μm to safely pass through pulmonary arterioles without causing blockage, wherein the scattering intensity and the incident intensity of ultrasound have the following relational formula (1):

wherein, I, I₀Scattering intensity and incident intensity of ultrasound respectively; n is the number of scattering particles; v is the scattering volume; k is the wave number; r is the particle radius, i.e. the microbubble radius; gamma ray_cIs a compression term (gamma)_c＝(k_s-k_m)/k_m，k_s、k_mRespectively, the compressibility of the scattering particles and the compressibility of the medium, known techniques); gamma ray_dIs the density term (gamma)_d＝(3ρ_s-3ρ_m)/(2ρ_s+ρ_m)，ρ_s、ρ_mDensity of scattering particles and medium, respectively, known in the art); θ is the scattering angle; d is the distance to the scattering particle. As can be seen from equation (1), the scattering ratio of the microbubble is a function of the sixth power of the microbubble radius, i.e., the scattering intensity of the ultrasound contrast agent microbubble is proportional to the sixth power of the bubble radius, which means that the smaller the microbubble, the worse the reflectivity, and therefore the optimal diameter size of the microbubble cannot be too small. Therefore, clinical applications require that the optimal diameter size of the microbubbles be between 2 μm and 5 μm.

The diameter of the micro-bubble generated by the micro-fluidic focusing system is mainly related to the flow rate of gas and liquid, and the influence of surface tension is small and can be ignored, as shown in the following formula (2):

d_b/D∝(Q_g/Q_l)^0.4 (2)

wherein Q is_g、Q_lFlow rates of gas, liquid respectively; d is the nozzle opening diameter; q_g/Q_l<1。

Regulating gas pressure by using a pressure regulating valve, setting flow rate parameters of an injection pump to control the flow rate of liquid, wherein the gas pressure P is generally used<5psi (sinks per square inch, 1psi ═ 6.895kPa), liquid flow rate Q<1.5. mu.L/s, the diameter d can be obtained_b<5 μm microbubbles, polydispersity index of the particle size of the microbubbles<2％。

In order to confirm the particle size and production efficiency of microbubbles prepared by the device of the present invention, the following experiment was performed using the above-described integrated chip, in which each layer structure had two symmetrical initial inlets, H1 was 1, H2 to HN were 2, N was 8, M was 7, the first layer structure had 128M channels, and the second layer structure had 256N channels; it integrates 128 expanding nozzle cavities, and the first layer structure is filled with gas and output at the narrowest point of the nozzle, as shown in fig. 4 and 5. The concrete description is as follows.

Experiment one:

the gas is nitrogen, and the liquid is prepared as follows: phosphate Buffered Saline (PBS)8ml, pH 7.4, Tween80 (Tween80)1ml were prepared as a homogeneous mixture, gas pressure was adjusted to 1.8psi, and liquid flow rate was adjusted to 2.0. mu.L^-1The diameter of the microbubbles is about 3.6 μm, and about 7X 109 microbubbles can be generated per minute. As shown in fig. 8, which is a photograph of the generated microbubbles taken under an olympus inverted microscope, it can be seen that the microbubble particle size distribution has a high monodispersity, the particle size distribution is relatively uniform, and the shell thickness is relatively uniform.

Experiment two: on the basis of experiment one, the liquid flow rate was unchanged, the gas pressure was increased to 4.5psi, and the diameter of the microbubbles was about 6 μm, and from the results obtained by microscopy, the particle size distribution was also relatively uniform, and comparing experiment one with experiment two, it was possible to obtain: the size of the particle size of the micro-bubbles can be flexibly controlled by changing the gas pressure; similarly, the liquid flow rate can be varied to achieve the same effect.

Experiment three: on the basis of experiment one, the gas pressure is unchanged, and the liquid flow rate is increased to 2.7 mu Ls^-1The diameter of the obtained microbubbles is about 3 μm, the particle size distribution is also uniform, and the following results can be obtained by comparing the second experiment with the third experiment: the flow rate of the liquid can be changed, and the size of the particle size of the micro-bubbles can be flexibly controlled.

Experiment four: the gas adopts Perfluorocarbon (PFC), and the liquid adopts the following method: lipid dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidic acid polyethylene glycol (DPPA), 5000 dipalmitoyl phosphatidylethanolamine (DPPE-PEG5000) at a molar ratio of 81: 8: 10 were dissolved in chloroform (CHCl3), a homogeneous mixture was formed under vacuum and nitrogen conditions, 1 mol% of fluorescence was addedPolish (DiI-C18) and 4mg/ml NaCl solution into a tube containing the phospholipid mixture, sonicated at room temperature for 20 minutes and mixed with a 10% strength mixture of glycerol and 1, 2 propylene Glycol (GPW), gas pressure 10psi, liquid flow rate 1.0. mu.ls^-1The diameter of the micro-bubble is 5 μm, and about 8X 109 micro-bubbles can be generated per minute.

The invention has the following advantages:

the most remarkable advantage is that the prepared ultrasound contrast agent microbubble has high monodispersity and controllable particle size, and meets the requirements of the ultrasound contrast imaging technology. The polydispersity index of the microbubble particle size is less than 2%, the particle size is increased along with the increase of gas pressure and is reduced along with the increase of liquid flow rate, and the control is very flexible. Moreover, the micro-nozzle array is adopted, so that the preparation efficiency of the micro-bubbles is greatly improved; meanwhile, the device has reusability, and the production cost is reduced.

The device can be used for preparing various types of ultrasonic contrast agent microbubbles, and the prepared microbubbles have uniform shell thickness.

Conventional ultrasonic contrast agents can be prepared directly by the device, such as lipids, albumins, polymers, surfactants and the like; as mentioned above, there are two options for the preparation of targeted ultrasound contrast agents, one of which is currently most commonly used: adding a specific ligand after the preparation of the conventional ultrasonic contrast agent is finished, and preparing the specific ligand additionally; the other method is that the specific ligand is added in advance into the preparation liquid before the contrast agent is prepared, the specific ligand is embedded on the shell of the microbubble when the contrast agent microbubble is formed, and some shell materials such as protein molecules are inactivated under the conditions of high temperature and ultrasound, so that the method cannot be realized by the conventional sound vibration method. The device is suitable for both methods, and particularly has advantages when the latter option is adopted, so that the preparation link is reduced, and the damage of microbubbles in the preparation link is also reduced.

In addition, the device and the method generate less heat in the preparation process of the contrast agent, so the device and the method are particularly suitable for preparing the ultrasonic contrast agent which is also used as a medicine or a gene targeting carrier. The way in which the microbubbles of the contrast agent carry genes or drugs is divided into two categories: adhesion and integration methods, wherein integration methods have significant advantages due to: the adhesion method only adheres the drugs or genes to the surface of the contrast agent microbubble through simple mixing, so that on one hand, the bonding amount is small, on the other hand, the adhered drugs or genes are not firmly bonded with the microbubble, and after intravenous injection, the adhered drugs or genes are easy to fall off under the impact of blood flow, and the targeting property is poor; the integration method can adhere the medicine or gene on the surface of the micro bubble, and integrate the medicine or gene on the micro bubble membrane or wrap the micro bubble, so that the combination amount is increased, and the targeting property is better. Generally, when the sound vibration method is adopted, higher temperature is generated in the preparation process, medicines or genes cannot be added simultaneously during preparation, and the ultrasonic contrast agent can be prepared only by adopting an adhesion method.

The ultrasonic contrast agent is simple and convenient to prepare, good in effect, safe and high in application value. Meanwhile, as described above, the present invention can also be used to prepare micro droplets.

Aiming at the simplest microbubble preparation unit consisting of one gas inlet and two liquid inlets, the production rate of the microbubble preparation unit is about 107/min, the large-scale microfluidic chip can remarkably improve the yield, for example, the yield can be improved to 109/min by integrating 128 microbubble preparation units, in addition, compared with the existing commercial ultrasonic contrast agent, the yield of the ultrasonic contrast agent Difinity approved by the U.S. Food and Drug Administration (FDA) for clinical use is about 109/45 seconds, the yield of the large-scale microfluidic chip can be comparable with the yield, more importantly, the particle size distribution of the Difinity contrast agent has polydispersity, the particle size distribution is wide, the average diameter is about 1.8 μm, the standard deviation of the diameter is 1.5 μm, but the maximum microbubble diameter reaches 20 μm, thereby leading the resonance frequency range of microbubbles to be larger, the bandwidth of the existing ultrasonic imaging system is limited to detect only a narrow resonance frequency range, so that about only 18% of microbubble signals can be detected, in other words, 82% of microbubbles do not work and are wasted, and the sensitivity of the imaging system is reduced.

It will be understood that modifications and variations can be made by persons skilled in the art in light of the above teachings and all such modifications and variations are intended to be included within the scope of the invention as defined in the appended claims.

Claims

1. An integrated chip is characterized in that two adjacent structures are taken as a group of structures, and the integrated chip comprises at least one group of structures;

in one group of structures, a first layer structure is provided with M-level gradients, and a second layer structure is provided with N-level gradients; wherein M, N is a natural number, M is less than N; in each stage of gradient, a first stage of gradient is provided with H1 channels 1, a second stage of gradient leads out H2 branches from each channel 1 to form H1 × H2 channels 2, and so on, and an Nth stage of gradient leads out HN branches from each channel (N-1) to form H1 × H2.

wherein,

the outlets of the specific channels N are equidistantly distributed on the circumference of a circle with a preset radius;

for the specific channel M, the output direction is vertical to the circle, the outlet of the specific channel M and the vertical line of the circle pass through the center of the circle, and the distance between the outlet of the specific channel M and the center of the circle is a specific length;

the included angle between the output direction of each specific channel N and the vertical line is the same acute angle;

along the output direction of the specific channel M, a collecting channel is arranged on one side of the circle away from the specific channel M;

2. The ic of claim 1, wherein M-N-1.

3. The IC of claim 1 wherein the output direction of each particular channel N passes through the center of the circle.

4. The integrated chip of claim 1, wherein the channels M and N have rectangular cross-sections; the rectangle has a height of 20 to 30 μm and a width of 30 to 50 μm, 50 to 100 μm, respectively, or 50 to 100 μm, 30 to 50 μm, respectively.

5. The integrated chip of claim 1, wherein the structure of the collection channel comprises at least one of: cone, sphere, hemisphere or frustum body with outward amplification, wherein the pipe diameter of the input end is 7-25 μm, and the height is 20-30 μm; or a cylinder or a cuboid, the cross section area of which along the output direction of the corresponding specific channel M is slightly smaller than that of the specific channel M along the direction, and the height is 20-30 μ M.

6. The integrated chip of claim 1, wherein the substrate is a surface hydrophilic substrate.

7. The integrated chip of claim 6, wherein the substrate is silicon, glass, polydimethylsiloxane, polymethyl methacrylate, or polycarbonate.

8. The integrated chip of claim 1, wherein the combination of the first layer structure and the second layer structure is a combination of a gas channel structure and a liquid channel structure.

9. An apparatus, comprising: a first substance input unit, a second substance input unit, at least one integrated chip according to any one of claims 1 to 8 forming an integrated chip array, a storage unit;

the collecting tanks of all the integrated chips are respectively connected with the storage units and used for collecting and exporting products;

the first substance input unit is respectively connected with each channel 1 of the layer structure of each integrated chip and is used for inputting a first substance;

the second substance input units are respectively connected with the channels 1 of the other layer structure of each integrated chip and used for inputting the second substance.

10. The apparatus of claim 9, wherein the combination of the first substance input unit and the second substance input unit is a combination of a gas input unit and a liquid input unit; or, the first substance input unit and the second substance input unit are both liquid input units.

11. The apparatus of claim 10, wherein the gas input unit comprises a pressure gas tank, a pressure reducing valve, a first transmission pipe, a micro flow meter, a pressure regulating valve, and a second transmission pipe connected in sequence, the second transmission pipe being connected to each channel 1 of the one-layer structure of each integrated chip, respectively;

the liquid input unit comprises a liquid storage device, a third transmission pipe, a digital control type injection pump and a fourth transmission pipe which are sequentially connected, and the fourth transmission pipe is respectively connected with each channel 1 of the other layer structure of each integrated chip.

12. A method of preparing a micron-sized dispersion for use in an apparatus comprising an array of integrated chips, said array of integrated chips consisting of at least one integrated chip according to any one of claims 1 to 8;

the method comprises the following steps:

a1, inputting a first substance to each channel 1 of a layer structure of each integrated chip according to a first preset condition; inputting a second substance to each channel 1 of the other layer structure of each integrated chip according to a second preset condition;

a2, adjusting the input conditions of the first substance and/or the second substance to enable the substance output by each channel M of each first layer structure to be wrapped by the substance of each corresponding specific channel N in a coaxial flow mode to form product streams to be collected respectively, wherein the axial direction of the product streams is coincident with the vertical line;

13. The method of claim 12, wherein the combination of the first substance and the second substance is a combination of a gas and a liquid; the gas at least comprises one of nitrogen, fluorocarbon gas and fluorine-sulfur gas; the liquid includes at least one of a phospholipid liquid, a surfactant liquid, and a modified liquid of the above liquids.

14. The method according to claim 12, wherein in step a1, a first liquid is supplied to each channel 1 of the first layer structure of each integrated chip, and a gas or a second liquid is supplied to each channel 1 of the second layer structure of each integrated chip;

in step a2, the first liquid output from each channel M of each first layer structure is made to flow coaxially, and the first liquid is wrapped axially by gas or second liquid to form a stream of droplets to be collected, respectively.

15. The method of claim 12, wherein in step a1, gas is fed to each channel 1 of one layer structure of each integrated chip at a pressure of less than 10psi, and liquid is fed to each channel 1 of the other layer structure of each integrated chip at a flow rate of less than 3 μ L/s; in step a2, the gas and/or liquid input conditions are adjusted.

16. The method of claim 15, wherein in step a1, the pressure of the input gas is less than 5psi and the flow rate of the input liquid is less than 1.5 μ L/s.

17. The method according to claim 15, wherein in step a1, the liquid is pre-added with a specific ligand; alternatively, after step a3, a specific ligand is added to the product.

18. The method according to claim 15, wherein in step a1, gas is supplied to each channel 1 of the first layer structure of each integrated chip, and liquid is supplied to each channel 1 of the second layer structure of each integrated chip;

in step a2, the input conditions of gas and/or liquid are adjusted so that the gas output from each channel M of each first layer structure wraps the liquid in a coaxial flow manner in the axial direction to form the microsphere flow to be collected respectively.

19. The method according to claim 18, wherein in step a2, the gas output from each channel M of each first layer structure forms a stable inverted cone with its central axis at the position of the vertical line under the focusing action of the high-speed flowing liquid on both sides, and the tip of each inverted cone is opposite to the position of each collecting channel.