A microfluidic chip for gradient elution liquid chromatography analysis

Abstract

The application provides a microfluidic chip for gradient elution liquid chromatography analysis, and relates to the field of microfluidic chips, and comprises: upper, middle and lower layer chips which are stacked in sequence; the upper surface of the upper layer chip is sequentially provided with a washing liquid inlet, a microfluidic channel, a micro-mixer, a double-T type sample inlet channel, a sample inlet hole, a sample outlet hole, a multi-well sieve plate, a micro-chromatographic column, a packaged micro-valve, a filling port, a filling channel, a Z type detection cell and a waste liquid outlet; the lower surface of the upper layer chip is processed to be the upper layer of the micro-mixer; the upper surface of the middle layer chip is processed to be the microfluidic channel, the lower layer of the micro-mixer, the double-T type sample inlet channel, the filling channel and the micro-chromatographic column; and the middle layer chip is processed to be the Z type detection cell. The application can realize gradient elution mode liquid chromatography separation, improve chromatography separation efficiency and effectively reduce sample usage.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microfluidic chips, in particular to a microfluidic chip for gradient elution liquid chromatography analysis. BACKGROUND

[0002] A microfluidic chip is a scientific technology that mainly features the manipulation of fluid in a micron-scale space. It is an important development frontier in many fields such as analytical science, micro-electro-mechanical processing, life science, chemical engineering, analytical instruments, and environmental science in the new century. Liquid chromatography analysis technology, as one of the important means of modern analysis, can analyze and purify molecules such as polypeptides, proteins, and other substances, and has important applications in the fields of information science, life science, environmental science, and material science.

[0003] Among them, transferring liquid chromatography analysis technology to a microfluidic chip can realize small sample size and low reagent consumption analysis, greatly improving the analysis efficiency. Integrating multiple functional units of chromatography analysis on a chip using microfluidic technology has the advantages of reducing reagent consumption, shortening analysis time, and improving detection sensitivity, which can reduce costs and mass production.

[0004] When performing liquid chromatography separation, two elution methods are commonly used. One is isocratic elution, and the other is gradient elution. During isocratic elution analysis, the mobile phase remains unchanged throughout the process. When using gradient elution for chromatography separation, the composition of different polarity mobile phases is continuously or intermittently changed, which improves the separation degree between components in the sample, thereby greatly improving the analysis efficiency.

[0005] However, no microfluidic chip capable of gradient elution liquid chromatography analysis has been found on the market. In recent years, the microfluidic chip for liquid chromatography analysis proposed in patents CN115228522A and CN110568087A can only perform isocratic elution. Therefore, there is an urgent need to develop a microfluidic chip that can realize gradient elution liquid chromatography analysis to improve analysis capability. SUMMARY

[0006] The purpose of the present application is to provide a microfluidic chip for gradient elution liquid chromatography analysis, which realizes gradient elution mode liquid chromatography separation, improves chromatography separation efficiency, and effectively reduces sample usage.

[0007] To achieve the above purpose, the present application provides the following solutions:

[0008] A microfluidic chip for gradient elution liquid chromatography analysis, comprising: an upper layer chip, a middle layer chip and a lower layer chip stacked in sequence;

[0009] The upper surface of the upper chip has, in sequence, a washing liquid inlet, a micro-channel, a micro-mixer, a double-T-shaped sample injection channel, a sample injection hole, a sample outlet hole, a multi-well screen plate, a micro-chromatographic column, a packaging micro-valve, a filling hole, a filling channel, a Z-shaped detection cell and a waste liquid outlet; the lower surface of the upper chip is processed to be the upper layer of the micro-mixer; the upper surface of the middle chip is processed to be the micro-channel, the lower layer of the micro-mixer, the double-T-shaped sample injection channel, the filling channel and the micro-chromatographic column; the middle chip is processed to be the Z-shaped detection cell;

[0010] The washing liquid inlet has two, the micro-channel has two sections, one of the washing liquid inlets is connected with one of the micro-channels, and the two sections of the micro-channels are connected with the inlet of the micro-mixer; the two sections of the micro-channels have the same width; the sum of the widths of the two sections of the micro-channels is the same as the width of the inlet of the micro-mixer;

[0011] Two kinds of washing liquids enter from the two washing liquid inlets, flow through the two sections of the micro-channels and converge at the inlet of the micro-mixer, and then flow through the micro-mixer to obtain mixed washing liquids;

[0012] The micro-mixer is connected with the double-T-shaped sample injection channel, and the double-T-shaped sample injection channel is connected with the sample injection hole and the sample outlet hole, respectively; a to-be-tested substance enters from the sample injection hole, passes through the double-T-shaped sample injection channel and flows out from the sample outlet hole, and the double-T-shaped sample injection channel is filled with fixed to-be-tested substances to be detected;

[0013] Two multi-well screen plates are used to package two ends of the micro-chromatographic column; the double-T-shaped sample injection channel is connected with the multi-well screen plate at the front end of the micro-chromatographic column;

[0014] The front end of the micro-chromatographic column is connected with the packaging micro-valve, the filling channel and the filling hole of the micro-chromatographic column, the filling hole is connected with the filling channel, the filling micro-valve is arranged on the filling channel, and the edge of the packaging micro-valve is tangent to the micro-chromatographic column;

[0015] The multi-well screen plate at the rear end of the packaging micro-chromatographic column is connected with the Z-shaped detection cell.

[0016] Optionally, the fixed distance between the double-T-shaped sample injection channels is calculated according to chromatography theory.

[0017] Optionally, the multi-well screen plate is in a cylindrical shape and is embedded in the micro-fluidic chip.

[0018] The aperture of the multi-well screen plate is 2 μm.

[0019] Optionally, the aperture of the Z-shaped detection cell is 500 μm.

[0020] Optionally, the middle layer chip is processed with a detection channel; the detection channel is arranged in a Z-shaped detection pool; and the detection channel is Z-shaped.

[0021] Optionally, the upper layer chip, the middle layer chip and the lower layer chip are processed with positioning holes at the same positions.

[0022] Optionally, the thickness of the upper layer chip is 5 mm; the thickness of the middle layer chip is 3 mm; and the thickness of the lower layer chip is 2 mm.

[0023] Optionally, the microfluidic chip has a length of 70 mm and a width of 30 mm.

[0024] Optionally, the microfluidic chip is made of thermoplastic polymer.

[0025] According to the embodiments of the present application, the following technical effects are achieved: the present application provides a microfluidic chip for gradient elution liquid chromatography analysis, which comprises an upper layer chip, a middle layer chip and a lower layer chip stacked in sequence; the upper surface of the upper layer chip is sequentially provided with a washing liquid inlet, a microfluidic channel, a micro-mixer, a double-T-shaped sample inlet channel, a sample inlet hole, a sample outlet hole, a multi-well sieve plate, a micro-chromatographic column, a packaged micro-valve, a filling port, a filling channel, a Z-shaped detection pool and a waste liquid outlet; the lower surface of the upper layer chip is processed with an upper layer of the micro-mixer; the upper surface of the middle layer chip is processed with the microfluidic channel, a lower layer of the micro-mixer, the double-T-shaped sample inlet channel, the filling channel and the micro-chromatographic column; and the middle layer chip is processed with the Z-shaped detection pool. Two kinds of washing liquids enter the microfluidic chip provided by the present application from two washing liquid inlets, so that gradient elution mode liquid chromatography separation can be achieved, the chromatography separation efficiency is improved, and the use amount of the to-be-tested substances is effectively reduced. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments. Obviously, the drawings described below only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor based on these drawings.

[0027] Figure 1 It is a top view schematic diagram of the microfluidic chip provided by the present application;

[0028] Figure 2 It is an exploded view schematic diagram of the microfluidic chip provided by the present application;

[0029] Figure 3 It is a front view schematic diagram of the microfluidic chip provided by the present application;

[0030] Figure 4A microfluidic chip detection flow diagram provided by the present application is shown. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative efforts fall within the scope of the present application.

[0032] The present application aims to provide a microfluidic chip for gradient elution liquid chromatography analysis, which can realize gradient elution mode liquid chromatography separation, improve chromatography separation efficiency, and effectively reduce sample usage.

[0033] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the present application will be further described in detail below with reference to the drawings and specific embodiments.

[0034] As shown in the drawings, Figure 1 The present application provides a microfluidic chip for gradient elution liquid chromatography analysis, comprising: upper layer chip, middle layer chip and lower layer chip stacked in sequence; wherein the same positions of the upper layer chip, the middle layer chip and the lower layer chip are processed with positioning holes 101, and the three layer chips are fixed through the positioning holes.

[0035] The upper surface of the upper layer chip has, in sequence, eluent inlet 102, microfluidic channel 103, micro-mixer 104, double-T type sample injection channel 106, sample injection hole 105, sample outlet hole 107, multi-well screen plate 108, micro-chromatographic column 109, encapsulated micro-valve 110, filling channel 111, filling port 112, Z-type detection cell 113 and waste liquid outlet; the lower surface of the upper layer chip is processed with the upper layer of the micro-mixer 104; the upper surface of the middle layer chip is processed with the microfluidic channel 103, the lower layer of the micro-mixer 104, the double-T type sample injection channel 106, the filling channel 111 and the micro-chromatographic column 109; the middle layer chip is processed with the Z-type detection cell 113.

[0036] The eluent inlet 102 is connected with the microfluidic channel 103, two sections of the microfluidic channel 103 are connected with the inlet of the micro-mixer 104 after merging, the two sections of the microfluidic channel 103 have the same width, and the sum of the widths is the same as the width of the inlet of the micro-mixer 104; two kinds of eluent enter from two eluent inlets, flow through the microfluidic channel 103, merge at the inlet of the micro-mixer 104, and get mixed eluent after flowing through the micro-mixer 104.

[0037] The end of the micro-mixer 104 is connected with the double-T type sample inlet channel 106, the double-T type sample inlet channel 106 is connected with the sample inlet hole 105 and the sample outlet hole 107 respectively, and the fixed distance between the double-T type sample inlet channels 106 is calculated according to the chromatography theory. The to-be-tested substance enters from the sample inlet hole 105, passes through the double-T type sample inlet channel 106 and flows out from the sample outlet hole 107, and the to-be-tested substance between the double-T type sample inlet channels 106 is filled and will be detected.

[0038] The end of the double-T type sample inlet channel 106 is connected with the porous screen plate 108, two porous screen plates 108 are used for packaging the two ends of the micro-chromatographic column 109, the porous screen plate 108 is embedded in the chip in a cylindrical shape, the porous screen plate 108 can withstand tens of megapascals pressure, the aperture is 2 mu m, and the porous screen plate 108 with a small hole diameter can package the chromatographic filling microspheres with a diameter greater than 2 mu m.

[0039] The front end of the micro-chromatographic column 109 is connected with the packaged micro-valve 110 filled with the chromatographic column, the filling channel 111 and the filling port 112, the filling port 112 is connected with the filling channel 111, the filling micro-valve is arranged on the filling channel 111, and the edge of the packaged micro-valve 110 is tangent to the micro-chromatographic column 109.

[0040] The porous screen plate 108 packaging the micro-chromatographic column 109 is connected with the Z type detection pool 113, the Z type detection pool 113 has a hole diameter of 500 mu m, is similar to the Z type detection pool 113 of a commercial chromatograph, and is processed on the micro-fluidic chip, so that the efficiency of chromatographic analysis is improved.

[0041] The length of the micro-fluidic chip is 70 mm, and the width is 30 mm.

[0042] As shown in the drawings, Figures 2-3 The elution inlet 102, the sample inlet hole 105, the sample outlet hole 107, the filling port 112 and the packaged micro-valve 110 are processed on the upper chip, the micro-mixer upper layer 301 is further processed on the lower surface of the upper chip, the micro-flow channel 103, the micro-mixer lower layer 302, the double-T type sample inlet channel 106, the filling channel 111 and the micro-chromatographic column 109 are processed on the upper surface of the middle layer chip, the Z type detection pool 113 is further processed on the middle layer chip, the detection channel 303 is processed in the middle layer chip, the detection channel 303 is arranged in the Z type detection pool 113, and the detection channel 303 is Z type.

[0043] The thickness of the upper chip is 5 mm, the thickness of the middle layer chip is 3 mm, and the thickness of the lower layer chip is 2 mm.

[0044] The material of the micro-fluidic chip provided by the application is a thermoplastic polymer.

[0045] As a specific embodiment of the application, the thermoplastic polymer material is a cyclic olefin copolymer (COC).

[0046] After the microstructure processing on the three-layer chip, the three-layer chip is bonded by laser fusion bonding process under the irradiation of 9.5KW power laser after the light-absorbing agent is applied on the surface of the chip.

[0047] Figure 4 The microfluidic chip detection process provided by the application is shown in the figure. Figure 4 Two injection pumps 401 pump two kinds of eluent into the eluent inlet 102 of the microfluidic chip 404 provided by the application (different flow rate ratios 1:9, 2:8, 3:7… can be set). The two kinds of eluent are fully mixed after flowing through the micro-mixer 104. The two injection pumps 401 are closed, and the sample is injected from the double-T-shaped sample inlet channel 106 by using the syringe 402. The injection pump 401 is opened, and the fully mixed eluent pushes the fixedly trapped sample into the micro-chromatographic column 109. After separation by the chromatographic column, the detected components flow through the Z-shaped detection cell 113, where the sample components are detected by the absorbance detector, and the results are obtained by computer data analysis. The application also includes a sample waste collection bottle 403, a light source 405, a detector 406 and a data processor 407.

[0048] Using the chip, the proportion of HbA, HbA2 and HbF components of hemoglobin (Hb) can be detected under 415nm light, and the glycosylated hemoglobin HbA1c component can also be accurately detected. The content of bilirubin can be accurately detected under 445nm light using the chip.

[0049] The gradient elution liquid chromatography analysis microfluidic chip provided by the application can realize absorbance detection.

[0050] The detection wavelength range is 320-700nm.

[0051] The sample injection volume is 0.62μL.

[0052] The gradient elution mode liquid chromatography analysis is realized on the microfluidic chip 404, which can effectively improve the efficiency of chromatography and reduce the sample usage.

[0053] Each embodiment in the specification is described in a progressive manner, and each embodiment focuses on the difference from other embodiments. The same or similar parts of each embodiment can be referred to each other.

[0054] The principles and implementation modes of the application are described by using specific examples in this paper. The above embodiment description is only used to help understand the method and core idea of the application; meanwhile, according to the idea of the application, the specific implementation mode and application range can be changed by those skilled in the art. In summary, the content of the specification should not be understood as a limitation of the application.

Claims

1. A microfluidic chip for gradient elution liquid chromatography analysis, characterized in that, The application relates to a microfluidic chip. The upper layer chip, the middle layer chip and the lower layer chip are stacked in sequence. The upper surface of the upper layer chip is provided with, in sequence, a washing liquid inlet, a micro flow channel, a micro mixer, a double-T type sample injection channel, a sample injection hole, a sample outlet hole, a multi-hole screen plate, a micro chromatographic column, a packaging micro valve, a filling channel, a Z type detection pool and a waste liquid outlet; the lower surface of the upper layer chip is provided with an upper layer of the micro mixer; the upper surface of the middle layer chip is provided with the micro flow channel, a lower layer of the micro mixer, the double-T type sample injection channel, the filling channel and the micro chromatographic column; the middle layer chip is provided with the Z type detection pool; the middle layer chip is provided with a detection channel; the detection channel is arranged in the Z type detection pool; and the detection channel is Z type. The washing liquid inlet has two washing liquid inlets, the micro flow channel has two sections, one washing liquid inlet is connected with one section of the micro flow channel, and the two sections of the micro flow channel are connected with the inlet of the micro mixer; the widths of the two sections of the micro flow channel are the same; and the sum of the widths of the two sections of the micro flow channel is the same as the width of the inlet of the micro mixer. Two kinds of washing liquids enter from the two washing liquid inlets, flow through the two sections of the micro flow channel, converge at the inlet of the micro mixer, and then flow through the micro mixer to obtain mixed washing liquids. The micro mixer is connected with the double-T type sample injection channel, and the double-T type sample injection channel is connected with the sample injection hole and the sample outlet hole respectively; a to-be-detected substance enters from the sample injection hole, flows through the double-T type sample injection channel and then flows out from the sample outlet hole, and the double-T type sample injection channel is filled with fixed to-be-detected substances to be detected. Two multi-hole screen plates are used for packaging two ends of the micro chromatographic column; the double-T type sample injection channel is connected with the multi-hole screen plate at the front end of the micro chromatographic column. The front end of the micro chromatographic column is connected with the packaging micro valve, the filling channel and the filling port of the micro chromatographic column, the filling port is connected with the filling channel, a filling micro valve is arranged on the filling channel, and the edge of the packaging micro valve is tangent to the micro chromatographic column. The multi-hole screen plate at the rear end of the packaging micro chromatographic column is connected with the Z type detection pool.

2. The microfluidic chip for gradient elution liquid chromatography according to claim 1, wherein, The fixed distance between the double-T type sample injection channels is calculated according to chromatographic theory.

3. The microfluidic chip for gradient elution liquid chromatography according to claim 1, wherein, The multi-hole screen plate is in a cylindrical shape and is embedded in the microfluidic chip. The aperture of the multi-hole screen plate is 2 mu m.

4. The microfluidic chip for gradient elution liquid chromatography according to claim 1, wherein, The aperture of the Z type detection pool is 500 mu m.

5. The microfluidic chip for gradient elution liquid chromatography according to claim 1, wherein, Positioning holes are arranged at the same positions of the upper layer chip, the middle layer chip and the lower layer chip.

6. The microfluidic chip for gradient elution liquid chromatography according to claim 1, wherein, The thickness of the upper layer chip is 5 mm, the thickness of the middle layer chip is 3 mm, and the thickness of the lower layer chip is 2 mm.

7. The microfluidic chip for gradient elution liquid chromatography according to claim 1, wherein, The length of the microfluidic chip is 70 mm, and the width is 30 mm.

8. The microfluidic chip for gradient elution liquid chromatography according to claim 1, wherein, The material of the microfluidic chip is a thermoplastic polymer.

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