CN114225794A

CN114225794A - T-shaped micro mixer and application thereof

Info

Publication number: CN114225794A
Application number: CN202111444407.XA
Authority: CN
Inventors: 李颖; 李哲宇; 杨运煌; 胡锐; 刘买利
Original assignee: Institute of Precision Measurement Science and Technology Innovation of CAS
Current assignee: Institute of Precision Measurement Science and Technology Innovation of CAS
Priority date: 2021-11-30
Filing date: 2021-11-30
Publication date: 2022-03-25

Abstract

The T-shaped micro mixer provided by the invention is characterized in that a plurality of asymmetrically disturbed microstructures are designed in a single mixing chamber, each microstructure comprises a vertical baffle and two arc baffles to form an inverted gamma shape, so that fluid is divided when contacting the vertical baffle of the microstructure, the arc baffles disturb and reflux the laminar flow divided in front, and the plurality of mixing chambers are arranged so that the fluid is subjected to multiple shunting and merging cycles, so that the laminar flow is better disturbed to form a mixed flow, and sufficient turbulent flow is generated to rapidly realize complete mixing. The T-shaped micro mixer has the characteristics of simple structure, high mixing efficiency, short time, easiness in preparation and the like, can realize the mixing of viscous solution without applying external energy, particularly can realize the uniform mixing of high-viscosity liquid in microsecond time scale, is favorable for maintaining the stability of a sample, and can be widely applied to the biochemical research fields of preparation of residual dipole coupling directional medium samples and the like.

Description

T-shaped micro mixer and application thereof

Technical Field

The invention relates to the technical field of biochemical detection and analysis, in particular to a T-shaped micro mixer and application thereof.

Background

Nuclear magnetic resonance spectroscopy (NMR) is a powerful technique for determining the structure of proteins and organic molecules at the atomic level. NMR relies on various NOE experiments to determine molecular structure, with Residue Dipole Coupling (RDC) being one of the most commonly used anisotropic NMR parameters. The method allows the targeted arrangement of protein molecules through different targeting media and provides information about long range constraints, conformational changes and kinetics (Angew Chem Int Ed Engl, 2020, 59, 17097). The oriented media of RDCs can be broadly classified into the following types: polyacrylamide gel, C_iE_jA/n-hexanol/water system (PEG), a phospholipid bilayer (bicell), a fibrillar viral medium (e.g., filamentous phage pf1/fd), a squalamine system, DNA nanotubes, and the like. To obtain the independent alignment tensor of a particular protein, several different media are required for the RDC measurement. For example, the determination of 5 alignment tensors of ubiquitin protein molecules uses up to 11 different targeting media (j.am. chem. soc., 2003, 125, 10164). At present, among the RDC alignment media, the widely used media are high viscosity PEG system and bicell (viscosity about 25-30 times of pure water).

Currently, PEG media are typically prepared by manual mixing, which leads to two major problems. First, the high viscosity of PEG makes it easy to adhere to the pipette, resulting in inaccuracies in the sample preparation process. Secondly, it is difficult to obtain uniform mixing and form a uniform liquid crystal phase by batch addition, shaking and centrifugation. These operations make the sample preparation unstable, depend on experiential experience, may result in poor sample state stratification, and may also cause abnormality in nuclear magnetic spectrum. Similarly, the preparation of bicell samples also requires complex manipulations involving multiple thermal cycles between 0 ℃ and 40 ℃ to gradually align the lipid molecules into a phospholipid bilayer and form a homogeneous liquid crystal phase. At the same time, too long a bicell-based sample preparation time may lead to hydrolysis of the phospholipid molecules and further affect the authenticity of the following NMR experiment. In short, the high viscosity and special chemistry of PEG and bicell media may result in unstable samples during manual preparation and result in low success and poor controllability of subsequent NMR measurements. Meanwhile, the traditional preparation method is time-consuming and labor-consuming, and the preparation time of two medium samples is about 1h and 2-3h respectively.

Disclosure of Invention

The present invention is directed to a T-type micromixer and its application, which overcome the above-mentioned disadvantages of the prior art.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a T-shaped micro mixer, which comprises an inlet channel, a mixing channel and an observation channel; one end of the mixing channel is connected and communicated with the inlet channel through a first connecting channel to form a T-shaped structure, and the observation channel is connected and communicated with the other end of the mixing channel through a second connecting channel; liquid inlets are formed in two ends of the inlet channel, the two liquid inlets are symmetrically arranged by taking the mixing channel as a central axis, and a liquid outlet is formed in one end of the observation channel;

the mixing channel is formed by connecting a plurality of mixing chambers in series through a third connecting channel, a plurality of microstructures are arranged in each mixing chamber, each microstructure comprises a vertical baffle and two arc baffles, and the two arc baffles are arranged on two sides of the vertical baffle to form an inverted gamma-shaped structure; gaps are reserved between the plurality of microstructures and the side wall of the channel in the mixing chamber where the microstructures are located, and the widths of the first connecting channel, the second connecting channel and the third connecting channel are smaller than the widths of the inlet channel and the observation channel.

Furthermore, the two arc-shaped baffles of the microstructure are respectively a first baffle and a second baffle, and the chord length of the first baffle is larger than that of the second baffle.

Furthermore, four microstructures are arranged in each mixing chamber, and are arranged in a cross shape; the first baffles of the two horizontal micro structures are positioned on the same side; the first baffles of the two vertical microstructures are opposite to the first baffles of the two horizontal microstructures.

Furthermore, four microstructures in two adjacent mixing chambers are arranged in mirror symmetry.

Furthermore, the cross section of the mixing chamber is spindle-shaped, the width of the mixing chamber is 100-300 mu m, and the length of the mixing chamber is 100-300 mu m.

Furthermore, the widths of the first connecting channel, the second connecting channel and the third connecting channel are all 20-45 micrometers.

Furthermore, the width of the inlet channel is the same as that of the observation channel, and the width of the inlet channel is 100-300 mu m.

Further, at least 6 mixing chambers are provided.

The invention also provides application of the T-shaped micro mixer, which is used for quickly preparing a plurality of high-viscosity solutions to be mixed.

Further, the high viscosity solution to be mixed is a PEG medium and a bicell medium.

The technical scheme provided by the invention has the beneficial effects that:

(1) the T-shaped micro mixer provided by the invention designs a plurality of asymmetrically disturbed microstructures in a single mixing chamber, and designs the microstructures into an inverted gamma-shaped microstructure, wherein the microstructures comprise a vertical baffle and two arc baffles, so that fluid which is firstly contacted with a linear structure is divided, the asymmetrically bent shape of the rear arc baffle carries out disturbed reflux on the laminar flow which is divided in front, the laminar flow is effectively disordered, and the diffusion between the laminar flows is greatly enhanced by turbulent flow; the fluid is re-gathered after being divided for multiple times by arranging the multiple mixing chambers, and the laminar flow is better disturbed to form mixed flow after multiple times of flow distribution and combination circulation; by designing a gap between the microstructure baffle and the side wall of the channel in the mixing chamber where the microstructure baffle is located and designing a narrow channel between two adjacent mixing chambers for communication, fluid is extruded to pass through, and thus the diffusion distance is remarkably reduced; the T-shaped micromixer of the invention can realize the circulation of the laminar flow of the segmentation and agglomeration, and generate enough turbulent flow to quickly realize the complete mixing of viscous fluid.

(2) The T-shaped micro mixer has the characteristics of simple structure, high mixing efficiency, short time, easiness in preparation and the like, can mix viscous liquid without applying external energy, and can effectively prevent debris from blocking.

(3) The T-shaped micro mixer provides a new method for preparing RDC (radio radical distribution) oriented media. In the preparation of the sample for RDC analysis, the RDC oriented medium can be mixed in a scale closer to the molecular layer, so that the formation of oriented arrangement of protein molecules in the medium is facilitated, the mixing time is short, the stability of the sample is ensured, and the accuracy of NMR detection is improved.

Drawings

FIG. 1 is a schematic structural diagram of a T-shaped micro mixer according to the present invention;

FIG. 2 is a schematic diagram of a mixing chamber structure of the T-shaped micromixer of the present invention;

FIG. 3a is a photomicrograph of a T-shaped micromixer structure in a microfluidic chip;

FIG. 3b is a diagram of a microfluidic chip prepared by using the T-shaped micro mixer of the present invention;

FIG. 4a is a fluorescence image of a mixing process of fluorescein and sulforhodamine B at a flow rate of 0.003 mL/min;

FIG. 4B is a fluorescence image of a mixing process of fluorescein and sulforhodamine B at a flow rate of 0.03 mL/min;

FIG. 4c is a fluorescence image of a mixing process of fluorescein and sulforhodamine B at a flow rate of 0.10 mL/min;

FIG. 4d is a fluorescence image of the mixing process of fluorescein and sulforhodamine B at a flow rate of 0.23 mL/min;

FIG. 5a is a graph showing the fluorescence distribution in the observation channel (position P7 in FIG. 4 d) at different flow rates;

FIG. 5b is a graph showing fluorescence distribution at P0, P2, P4, P6 and P7 in FIG. 4d at a flow rate of 0.23 mL/min;

FIG. 6a is a graph of the mixing efficiency of pure water, 4% PEG400, 8% PEG400 and 16% PEG400 observed in channels at different flow rates;

FIG. 6b is a phase diagram of mixing efficiency versus flow rate and position (position is indicated by a white dotted line along the outlet, where mixing efficiency C is indicated_mContours of 0.25, 0.4, 0.5, 0.6, 0.75, and 0.9);

fig. 7a is a preparation program diagram for preparing a PEG media RDC sample using the microfluidic chip of the present invention;

FIG. 7b is a diagram of a preparation procedure for preparing a sample of the RDC of the PEG media using a conventional method;

fig. 7c shows deuterium spectra and split widths of PEG media RDC samples prepared by the microfluidic chip of the present invention at different storage time points after preparation: 28.97Hz, 29.03Hz, 29.01Hz, 29.46Hz, 29.52Hz at 20min, 1h, 2h, 12h and 24 h;

fig. 7d is the deuterium spectra of PEG media RDC samples prepared by the conventional method at different storage time points after preparation, split width: 23.85Hz, 23.90Hz, 22.94Hz and 24.24Hz at 20min, 1h, 2h and 12h, and deuterium spectrum split distortion of the sample at 24 h;

FIG. 8a is a preparation process diagram of a bicell medium RDC sample prepared by using the microfluidic chip of the invention;

FIG. 8b is a diagram of a preparation procedure for preparing a bicell media RDC sample using a conventional method;

fig. 8c shows deuterium spectra of RDC samples prepared by the microfluidic chip of the present invention at different storage time points at 0 ℃ after preparation, split widths: 6.79Hz, 6.81Hz and 6.81Hz at 20min, 2h, 12h and 24 h;

FIG. 8d is the deuterium spectra of the bicell medium RDC samples prepared by the traditional method at different storage time points at 0 ℃ after preparation, the split width: at 20min, 2h and 12h, the splitting widths are respectively 4.83Hz, 5.09Hz and 5.04Hz, and the deuterium spectrum of the sample after 24h has split distortion.

1. A liquid inlet; 2. an inlet channel; 3. a mixing channel; 31. a mixing chamber; 32. a microstructure; 321. an arc-shaped baffle plate; 322. a vertical baffle; 4. an observation channel; 5. a liquid outlet; 6. a first connecting channel; 7. a second connecting channel; 8. and a third connecting channel.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention are described in detail below with reference to the accompanying drawings and specific examples.

Example 1:

as shown in fig. 1, a T-type micromixer of the present invention comprises an inlet channel 2, a mixing channel 3 and an observation channel 4; one end of the mixing channel 3 is connected and communicated with the inlet channel 2 through a first connecting channel 6 to form a T-shaped structure, and the observation channel 4 is connected and communicated with the other end of the mixing channel 3 through a second connecting channel 7; liquid inlets 1 are respectively arranged at two ends of the inlet channel 2, the two liquid inlets 1 are symmetrically arranged by taking the mixing channel 3 as a central shaft, and a liquid outlet 5 is arranged at one end of the observation channel 4; the mixing channel 3 is formed by connecting a plurality of mixing chambers 31 in series through a third connecting channel 8, a plurality of microstructures 32 are arranged in each mixing chamber 31, each microstructure 32 comprises a vertical baffle 322 and two arc baffles 321, and the two arc baffles 321 are arranged on two sides of the vertical baffle 322 to form an inverted gamma-shaped structure; a gap is left between the plurality of microstructures 32 and the side wall of the channel in the mixing chamber 31, and the widths of the first connecting channel, the second connecting channel and the third connecting channel are all smaller than the widths of the inlet channel 2 and the observation channel 4. The idea of the invention is that a plurality of asymmetrically disturbed microstructures 32 are designed in a single mixing chamber 31, the microstructures 32 are designed into an inverted gamma shape, the microstructures 32 comprise a vertical baffle 322 and two arc baffles 321, so that a linear structure which contacts fluid firstly divides laminar flow, the asymmetrically bent shape of the rear arc baffle 321 disturbs and reflows the laminar flow divided in front, the laminar flow is effectively disordered, and the turbulent flow greatly enhances the diffusion between the laminar flows; by arranging the mixing chambers 31, the fluid is reunited after being divided for multiple times, and the laminar flow is better disturbed to form mixed flow after being divided and combined for multiple times; by designing a gap between the microstructure 32 and the side wall of the channel in the mixing chamber 31, and designing a third connecting channel 8 between two adjacent mixing chambers 31, the width of the third connecting channel is narrower than that of the mixing channel 3, so that the fluid is extruded to pass through, thereby obviously reducing the diffusion distance; the T-shaped micromixer of the invention can realize the circulation of the laminar flow of the segmentation and agglomeration, and generate enough turbulent flow to quickly realize the complete mixing of viscous fluid.

In order to prepare a microfluidic chip with wider application range, the liquid inlet 1, the inlet channel 2 and the mixing channel 3 can be communicated in a Y-shaped or arrow-shaped manner.

To better realize the asymmetric disrupting structure, the microstructure 32 can include two arc-shaped baffles 321, a first baffle and a second baffle, wherein the chord length of the first baffle is greater than that of the second baffle, and the chord length is the linear distance between two end points of the arc.

In order to ensure good mixing effect of the high-viscosity liquid, four microstructures 32 can be arranged in each mixing chamber 31, and the four microstructures 32 can be arranged in a cross shape; the first baffles of the curved baffles 321 of the two microstructures 32 in the horizontal row are located on the same side; the first stops of two microstructures 32 in a vertical column are positioned opposite the first stops of two microstructures 32 in a row. The four baffles 321 are distributed in a cross shape in each mixing chamber 31 to disturb the fluid laterally and longitudinally, so that the fluid is better turbulent and mixing is accelerated.

In order to improve the mixing efficiency and speed, the four microstructures 32 in two adjacent mixing chambers 31 may be arranged in mirror symmetry. Two adjacent mixing chambers 31 can be classified into a period, when the fluid flows through one mixing chamber 31, the flow direction of the fluid is blocked by the microstructures 32 in the mixing chamber 31 to change, the fluid circulates repeatedly, the laminar flow is effectively disordered along with the multiple flow splitting and merging of the fluid, the formed turbulent flow greatly enhances the diffusion between the laminar flows, the mixing effect is better, and the mixing time is also shortened.

For more beautiful and practical structure, the cross section of the mixing chamber 31 can be spindle-shaped, the width is 100-300 μm, and the length is 100-300 μm; the width of the narrow channel 6 can be 20-45 μm; the inlet channel 2 and the observation channel 4 have the same size and the width of 100-300 μm.

In order to increase the mixing efficiency and reduce the mixing time, at least 6 mixing chambers 31 may be provided in the mixing channel 3.

The T-shaped micromixer of the present invention, which can be used for rapidly mixing high viscosity solutions, will be described in further detail with reference to the following specific examples.

Example 2:

1. the T-shaped micro mixer is used for preparing the micro-fluidic chip

(1) Designing a mask: as shown in fig. 2, the dimensions of the structure design of the T-shaped micro mixer provided in example 1 are: the width of the inlet channel 1 is 200 μm, the width of the observation channel 4 is 200 μm, the mixing chambers 31 are spindle-shaped, the width is 207 μm, the height is 200 μm, each mixing chamber 31 is internally provided with 4 microstructures 32, the microstructures 32 are provided with two arc baffles 321 which are respectively a first baffle and a second baffle, the width of the first baffle and the width of the second baffle are 10 μm, the chord length of the first baffle is larger than that of the second baffle, and the 4 microstructures 32 are arranged in a cross shape; the first baffles of the arcuate baffles 321 of two microstructures 32 in a row are both located on the right side; the first of the arcuate baffles 321 of two of the microstructures 32 in the column are both on the left side. The width of the third connecting channel 8 of two adjacent mixing chambers 31 is 30 μm.

(2) Preparation of PDMS thin layer: firstly, an SU-8 male mold is manufactured by using a soft lithography technology. The SU-8 photoresist (3025) is spun off uniformly (800rpm 40s, 4500rpm 60s) on a cleaned and dried silicon wafer, and the solvent in the photoresist is removed by pre-baking on a hot plate (65 deg.C for 10min, 90 deg.C for 25min) to perform photolithography (8s, 5 mJ/cm)²) And then placing the mold on a hot plate for post-baking (2 min at 65 ℃ and 8min at 90 ℃), developing by PGMEA (polymer pre-preg-membrane electrode) and fixing by isopropanol, and then performing die hardening (120 min at 135 ℃) on the hot plate to obtain the male die with the microstructure. After the male die is manufactured, the structure of the male die is copied to the PMDS sheet by using a rapid forming method. Firstly, mixing PDMS with a curing agent 10:1, removing gas to obtain a precursor, pouring the precursor on a male mold, heating for 2h at 65 ℃ by using an oven, and lifting the cured PDMS to obtain a PDMS sheet with the thickness of about 5 mm.

As shown in fig. 3a, is a micrograph of a T-shaped micromixer structure on a PDMS slab.

(3) The PDMS thin layer is combined with the glass substrate to form a microfluidic chip: a 1mm diameter manual punch was used to punch holes at the sample inlet and outlet. Cleaning the PDMS thin sheet with alcohol and drying with nitrogen to ensure that the cleaning scraps are clean, cleaning the glass slide substrate with alcohol and drying, placing the glass slide substrate and the glass slide substrate into a plasma cleaner for treatment (800V 70s), adhering the surface of the PDMS thin layer with the structure to the glass slide for bonding after aligning, and heating at 65 ℃ for 2h to obtain the PDMS chip.

(4) Assembling a micro mixer: in order to introduce the exogenous sample fluid into the chip for mixing, a conduit needs to be connected thereto. Firstly, two ends of a silica gel hose are respectively connected with a stainless steel tube and a flat-head needle tube, the two ends of the silica gel hose are sequentially washed by alcohol and water and then are blown clean by nitrogen, the other end of the stainless steel tube is inserted into a punched hole in PDMS, a PDMS sheet is stuck to the whole silica gel hose by AB glue and then heated at 65 ℃ for 2h to solidify the AB glue to ensure that no leakage is possible, and finally, the flat-head needle tube is connected with an upper filter head opposite to one end of an injector.

As shown in fig. 3b, is a real image of the prepared microfluidic chip.

2. Investigating the mixing effect of the prepared microfluidic chip

The mixing effect of the microfluidic chip is preliminarily characterized by using sulforhodamine B (red fluorescence) dissolved in PEG400 high-viscosity solutions and fluorescein (green fluorescence) solutions with different volume fractions. The above solutions were mixed in a microfluidic chip at a range of flow rates from 0.003mL/min to 0.23 mL/min.

As a result, as shown in FIGS. 4a, 4b, 4c and 4d, the interface between red and green was clear at 0.003 mL/min. As shown in FIG. 4b, as the flow rate increases to 0.03mL/min, the two colors red and green merge slightly, and the mixing increases a little bit. When the flow rate was set to 0.1mL/min, better mixing was achieved. When the flow rate was further increased to 0.23mL/min, the solution appeared to be uniformly colored when viewed through the channel.

As shown in fig. 5a, the red fluorescence distribution across the viewing channel at each flux is shown;

as shown in FIG. 5b, positioned along the mixing channel at a rate of 0.23mL/min, showing the fluorescence distribution at different locations;

in addition, applicants have also calculated different concentrations of PEG400Efficiency of mixing of the observation channel 4 at different flow rates of the liquid (C)_mThe calculation formula is as follows), as shown in fig. 6a, the micro-fluidic chip of the invention can realize complete mixing at the flow rate of 0.23mL/min, and water (C)_m＝0.924)、4％PEG400(C_m0.915) and 8% PEG400 (C)_m0.918), the applicant found that complete mixing can also be achieved at a flow rate of 0.25mL/min, a 16% PEG solution (C) due to the higher viscosity of the 16% PEG solution_m＝0.912)。

Wherein X_iAnd

respectively are the fluorescence value and the average value of the corresponding position in the special picture, and N is the number of pixels of the corresponding analysis area.

To further investigate the combination of flow rate and position along the mixing channel 2 to achieve thorough mixing, a phase diagram of the mixing efficiency of a 4% PEG400 solution was plotted as shown in fig. 6 b. The applicant has determined, based on the phase diagram shown in fig. 6b, that a flow rate of 0.23mL/min and 6 mixing chambers 31 are selected as mixing structures for preparing the samples.

The applicant then calculated the mixing dead time of the microfluidic chip of the invention according to the experimental conditions. The mixing time is determined as t ═ V_mixV, wherein V_mixIs the total volume (V) of the mixing zone_mix＝0.1466mm²X 20 μm-2.932 nL), ν is the total flow rate of the two injection ports 1 (0.46 mL/min). The T-shaped micro mixer of the microfluidic chip can uniformly mix 16% of PEG400 at the flow rate of 0.23mL/min, and the mixing time is 380 mus.

Therefore, the T-type micromixer of the present invention can be further applied to study some rapid biochemical reactions, such as the folding kinetics and enzyme kinetics of early biomolecules.

3. Preparation of PEG Medium RDC samples Using microfluidic chips

(1) Preparation procedure

Fig. 7a is a schematic diagram of a preparation process for preparing a PEG media RDC sample by using the microfluidic chip of the present invention. And (3) injecting the PEG medium mother liquor and the protein sample from two inlets by using an injector at the flow rate of 0.23mL/min through a mechanical injection pump, and then collecting, namely completing the preparation of the PEG system sample, wherein the required time is 2 min.

As shown in fig. 7b, is a diagram of a preparation procedure for preparing a PEG media RDC sample using a conventional method. By the process of manually preparing PEG media samples, a minimum of 10 times of PEG solution addition to the sample and rotation are required, 1h to complete the preparation.

Obviously, microfluidic chip mixing can produce a more uniform solution than conventional methods, and the T-type micromixer-based method not only simplifies the operating procedure, but also effectively reduces the sample preparation time.

(2) And respectively carrying out NMR detection on the PEG medium RDC samples prepared by the two methods, and acquiring a deuterium spectrum.

The deuterium spectrum of the RDC sample prepared by using the microfluidic chip of the present invention is shown in fig. 7c, and the split is 28.97Hz, 29.03Hz, 29.01Hz, 29.46Hz, and 29.52Hz at 20min, 1h, 2h, 12h, and 24 h.

The deuterium spectra of the RDC samples prepared by the conventional method are shown in fig. 7d, with split at 23.85Hz, 23.90Hz, 22.94Hz, 24.24Hz at 20min, 1h, 2h and 12h, and split distortion at 24 h.

Usually by deuterium spectroscopy (²H NMR) to determine the degree of alignment of the orienting medium and thus the quality of the sample preparation. In the deuterium spectrum²H fragmentation abnormalities, such as no quadrupolar fragmentation or more than two peaks, indicate that the medium fails to form a stable alignment; when there are only two peaks, it is shown that the prepared sample is good, and the closer the heights of the two peaks are, the better the uniformity and alignment of the sample. The results show that RDC samples prepared using the microfluidic chip of the present invention split consistently and the splitting distance remained stable when the storage time was extended to 24h, and furthermore, the two peaks of the deuterium spectra had similar heights (difference) starting from t ═ 20 minutes<10%) and held for 24 hours. Whereas the RDC samples prepared using the conventional method,the splitting distance of the deuterium spectrum of the liquid firstly keeps stable, but the splitting disappears after the liquid is stored for 24 hours, and the liquid is irreversibly split into two layers; at the same time, the heights of the two peaks are 20min (-20%), 1h (-15%) and 2h (>10%) indicating that the samples prepared in this manner did not completely complete alignment within the first few hours, requiring a longer time for self-assembly.

The above data reveal the superiority of using the T-type micromixer of the present invention in the preparation of PEG media RDC samples: 1) the time to complete alignment is shorter (<20 minutes >2 hours compared to traditional methods); 2) the prepared samples were more stable (the samples remained stable within 24 hours), whereas the deuterium spectrum split of the manually prepared samples disappeared after 24 hours. The possible reason for the difference in sample stability may be due to the more tendency of the T-type micromixer to divide the microstructure, preventing the presence of large "nuclei" which would cause the liquid crystal phase to age and lose its alignment. The preparation of stable samples in shorter time is very important in the calculation of NMR protein structure. On one hand, the samples prepared by the T-shaped micromixer of the invention can rapidly realize the effective alignment of proteins in the liquid crystal phase, which enables the RDC experiment to be carried out earlier. Therefore, the nuclear magnetic resonance result is more real, more accurate and more reliable, and the probability of impurity peaks caused by protein degradation can be greatly inhibited. On the other hand, protein samples in nuclear magnetic resonance analysis are always isotopically labelled, expensive and time consuming. A stable RDC sample can complete a long set of NMR data acquisitions, thus greatly reducing cost and time.

4. Preparation of bicell medium RDC sample by using microfluidic chip

(1) Preparation procedure

Fig. 8a is a schematic diagram of a preparation process for preparing a bicell dielectric RDC sample by using the microfluidic chip of the present invention. Since the bicell medium appears as a clear liquid at 0 ℃ and a milky viscous liquid phase at 40 ℃, the applicant believes that 0 ℃ is a temperature at which bicell is more easily mixed. Injecting the bicell mother liquor and the protein sample from two inlets at a flow rate of 0.23mL/min by a mechanical injection pump at 0 ℃, collecting, passing through a water bath at 40 ℃ for 10min, and storing at 0 ℃ to finish the preparation, wherein the required time is 12 min.

FIG. 8b is a diagram of a preparation procedure for preparing a bicell media RDC sample using a conventional method. In the process of manually preparing the bicell medium sample, a bicell solution is added into the sample, and the sample is prepared by carrying out thermal cycle for 4-5 times at 0-40 ℃, wherein the preparation time is up to 2 hours.

(2) And respectively carrying out NMR detection on bicell medium RDC samples prepared by the two methods, and acquiring a deuterium spectrum.

The deuterium spectrum of the bicell medium RDC sample prepared by using the microfluidic chip of the invention is shown in figure 8c, and the splitting distance is as follows: 6.79Hz, 6.81Hz and 6.81Hz at 20min, 2h, 12h and 24 h.

The deuterogram of the bicell medium RDC sample prepared using the traditional method is shown in fig. 8d, the split distance: at 20min, 2h and 12h, the splitting widths are respectively 4.83Hz, 5.09Hz and 5.04Hz, and the deuterium spectrum of the sample after 24h has split distortion.

The result shows that the splitting stability of the spectrum of the sample prepared by the microfluidic chip is greatly improved, and the stable deuterium spectrum is shown within 24 hours, so that the sample prepared by the method has good stability and directional arrangement efficiency; the sample prepared by the traditional method has a relatively large half-peak width of the deuterium spectrum after being stored for 2 hours, which indicates that the uniformity of the solution is poor, the uniformity of the magnetic field is further influenced, and after 24 hours, an abnormal spectrum appears, namely the directional medium fails. The phospholipid molecule, a component in the bicell sample, is easy to hydrolyze, and the hydrolysis of the phospholipid molecule in the bicell medium sample can be accelerated at the temperature of 310K during NMR detection. The method based on the T-shaped micro mixer can complete the whole mixing process in 12 minutes, and greatly reduces the hydrolysis of phospholipid molecules in the bicell sample. For the preparation by the traditional method, phospholipid molecules in a bicell sample are hydrolyzed within 2-3 hours, so that the authenticity of an NMR spectrum is influenced. In addition, the success rate of preparing samples by adopting the traditional method is only about 50 percent, and at least two samples are prepared in the experimental process to prevent the experiment from failing; in contrast, the sample preparation based on the T-shaped micro mixer is simple and stable, and the success rate is high.

In conclusion, the advantage of using the microfluidic chip of the present invention to prepare RDC samples is related to the good control of T-type micro-mixer on microfluid. In the T-type micromixer, the inverted y-type microstructure disturbs laminar flow, creates effective turbulence and greatly enhances mixing. In addition, the fluid is broken down into minute amounts of liquid within the microstructure, giving the protein molecules more opportunity to contact and form an oriented alignment with the liquid crystal phase structure.

The features of the embodiments and embodiments described herein above may be combined with each other without conflict.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A T-shaped micromixer is characterized in that: comprises an inlet channel (2), a mixing channel (3) and an observation channel (4); one end of the mixing channel (3) is connected and communicated with the inlet channel (2) through a first connecting channel (6) to form a T-shaped structure, and one end of the observation channel (4) is connected and communicated with the other end of the mixing channel (3) through a second connecting channel (7); liquid inlets (1) are formed in two ends of the inlet channel (2), the two liquid inlets (1) are symmetrically arranged by taking the mixing channel (3) as a central shaft, and a liquid outlet (5) is formed in the other end of the observation channel (4);

the mixing channel (3) is formed by connecting a plurality of mixing chambers (31) in series through a third connecting channel (8), a plurality of microstructures (32) are arranged in each mixing chamber (31), each microstructure (32) comprises a vertical baffle (322) and two arc-shaped baffles (321), and the two arc-shaped baffles (321) are arranged on two sides of the vertical baffle (322) to form an inverted-Y-shaped structure; gaps are reserved between the plurality of microstructures (32) and the side wall of the channel in the mixing chamber (31) where the microstructures are located, and the widths of the first connecting channel, the second connecting channel and the third connecting channel are smaller than the widths of the inlet channel (2) and the observation channel (4).

2. A T-type micromixer as defined in claim 1, wherein: the two arc-shaped baffles (321) of the microstructure (32) are respectively a first baffle and a second baffle, and the chord length of the first baffle is larger than that of the second baffle.

3. A T-type micromixer as defined in claim 1, wherein: four microstructures (32) are arranged in each mixing chamber (31), and the four microstructures (32) are arranged in a cross shape; the first baffles of two microstructures (32) in the transverse row are positioned on the same side; the first baffles of two microstructures (32) in a vertical column are opposite to the first baffles of two microstructures (32) in a horizontal row.

4. A T-type micromixer as defined in claim 3, wherein: the four microstructures (32) in two adjacent mixing chambers (31) are arranged in mirror symmetry.

5. A T-type micromixer as defined in claim 1, wherein: the cross section of the mixing chamber (31) is spindle-shaped, the width of the mixing chamber is 100-300 mu m, and the length of the mixing chamber is 100-300 mu m.

6. A T-type micromixer as defined in claim 1, wherein: the widths of the first connecting channel (6), the second connecting channel (7) and the third connecting channel (8) are all 20-45 mu m.

7. A T-type micromixer as defined in claim 1, wherein: the width of the inlet channel (2) is the same as that of the observation channel (4), and the width of the inlet channel and the width of the observation channel are both 100-300 mu m.

8. A T-type micromixer as defined in claim 1, wherein: at least 6 mixing chambers (31) are provided.

9. Use of a T-type micromixer according to any of claims 1 to 8, characterized in that: for the rapid preparation of a variety of high viscosity solutions that require mixing.

10. The use of claim 9, wherein: the high-viscosity solution to be mixed is PEG medium and bicell medium.