Liquid chromatography array column chip, its preparation method and application

Abstract

The application belongs to the technical field of chemical separation, and particularly relates to a liquid chromatography array column chip, a preparation method and application thereof. The application directly processes a chromatography column on a chip, is small in size, light in weight and low in cost, and a small chromatography column volume makes a required sample and reagent amount less; meanwhile, the chromatography column chip can greatly reduce the existence of dead volume, reduces column-in and column-out effects, and further improves the sensitivity of detection; furthermore, compared with a traditional packed column, the liquid chromatography array column chip provided by the application has lower back pressure, greatly reducing the requirements of the entire liquid chromatography system for driving pressure and pressure resistance. The application chemically grafts sulfonic acid groups on a separation column, has strong cation exchange function, so that the liquid chromatography array column chip provided by the application can perform ion exchange chromatography separation, and can be used for efficiently separating amino acids, polypeptides, proteins and protein derivatives.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of chemical separation, and particularly relates to a liquid chromatography array column chip and a preparation method and application thereof. BACKGROUND

[0002] Liquid chromatography is the most commonly used separation and analysis method in laboratories, which can separate sample components to realize qualitative and quantitative analysis. However, the traditional liquid chromatography system is large in size, heavy in weight, high in cost, and generally needs to send samples to laboratories for detection, which has problems of long analysis cycle, information lag and the like, and cannot meet the needs of on-site real-time detection of samples. In order to further meet this demand, the liquid chromatography system is developing towards miniaturization, and microfluidic chip technology is one of the important ways. The miniaturization of the chromatography column as the core of the chromatography system is the top priority of the research.

[0003] At present, the chromatography column realized on a chip can be divided into open-tube column, packed column, monolithic column and array column according to the processing method. At present, the most common one is the packed column chip. Chinese patent CN1900712A discloses a liquid chromatography chip with a flexible interface, which mainly consists of a chromatography packed column and a through hole for operating fluid; US08323488B2 discloses a chip-based chromatography based on Parylene, which integrates a sample injector, a packed chromatography column and a detection electrode on a 2cm×2cm chip. However, the above types of chromatography packed column chips or chromatography column packing are not dense enough, and the separation performance is low; or the packed chromatography column chip with high density will generate extremely high back pressure, which has extremely high requirements on the structural strength of the chip itself and the driving pressure of the whole system, which is obviously not conducive to the integration of the miniaturized liquid chromatography system.

[0004] “An array of ordered pillars with retentive properties for pressure-driven liquid chromatography fabricated directly from an unmodified cycloolefin polymer[J]”(Illa X,De Malsche W,Bomer J,et al.,Lab on a Chip,2009,9(11):1511-1516.) discloses that an array column is processed by taking cycloolefin polymer (COP) as a base material, and reverse phase chromatography separation is carried out by using the surface hydrophobicity. However, it cannot be applied to the separation of amino acids, polypeptides, proteins and protein derivatives (glycated hemoglobin) and the like, and cannot realize the detection of separated samples. SUMMARY

[0005] The application aims to provide a liquid chromatography array column chip, a preparation method and application thereof.

[0006] To achieve the above-mentioned purpose, the application provides the following technical scheme.

[0007] The application provides a liquid chromatography array column chip, which comprises a separation substrate 200 and a cover substrate 100 fixedly connected with the separation substrate 200.

[0008] The separation substrate 200 is provided with a main channel, a sample inflow channel branch 203 and a sample outflow channel branch 204 in communication with the main channel.

[0009] In the flow direction of the buffer in the main channel, the communication position of the sample outflow channel branch 204 with the main channel is located upstream of the communication position of the sample inflow channel branch 203 with the main channel, the main channel is sequentially divided into a buffer inflow channel 201, a quantitative sample pool 205, a separation channel 206 and a downstream region of the separation channel 206 along the flow direction of the buffer, the downstream region of the separation channel 206 comprises a buffer outflow channel 202; the buffer inflow channel 201 is a main channel region located upstream of the sample outflow channel branch 204, the quantitative sample pool 205 is a main channel region located between the sample outflow channel branch 204 and the sample inflow channel branch 203, the separation channel 206 and the downstream region of the separation channel 206 are main channel regions located downstream of the sample inflow channel branch 203, the inner surface of the separation channel 206 is provided with array-arranged separation columns, the separation columns are perpendicular to the fluid flow direction in the main channel, and the surface of the separation columns is chemically grafted with sulfonic acid groups.

[0010] The cover substrate 100 is provided with a buffer inlet 101, a buffer outlet 102, a sample inlet 103 and a sample outlet 104 corresponding to the buffer inflow channel 201, the buffer outflow channel 202, the sample inflow channel branch 203 and the sample outflow channel branch 204 on the separation substrate 200.

[0011] The buffer inlet 101 is in communication with the buffer inflow channel 201, the buffer outlet 102 is connected with the buffer outflow channel 202, the sample inlet 103 is in communication with the sample inflow channel branch 203, and the sample outlet 104 is in communication with the sample outflow channel branch 204.

[0012] Preferably, the downstream region of the separation channel 206 further comprises an optical detection cell 207; the optical detection cell 207 is located upstream of the buffer outflow channel 202 along the flow direction of the buffer in the main channel; the material of the separation substrate 200 and the cover substrate 100 is a light-transmitting material.

[0013] Preferably, the material of the separation substrate 200 is cyclic olefin copolymer; the material of the cover substrate 100 is polymethyl methacrylate.

[0014] Preferably, the separation columns are arranged in a regular triangle staggered array on the inner surface of the separation channel 206.

[0015] Preferably, the cross-sectional dimension of the main channel is 400 μm x 15 μm.

[0016] The diameter of the separation column is 15 μm.

[0017] Preferably, the cross-sectional dimension of the buffer inlet 101, the buffer outlet 102, the sample inlet 103 and the sample outlet 104 is 800 μm x 800 μm.

[0018] Preferably, the size of the separation substrate 200 and the cover substrate 100 is 26 mm x 15 mm x 2 mm.

[0019] The present application provides a preparation method of the liquid chromatography array column chip as described in the above technical solution, comprising the following steps:

[0020] The buffer inlet 101, the buffer outlet 102, the sample inlet 103 and the sample outlet 104 are prepared on the first substrate by using numerical control micro-milling forming method, to obtain the cover substrate 100.

[0021] The main channel, the sample inflow channel branch 203, the sample outflow channel branch 204 and the separation column arranged on the inner surface of the separation channel 206 are prepared on the second substrate by using hot stamping method; the initial separation substrate.

[0022] The initial separation substrate is soaked in a grafting solution, and a grafting reaction is carried out under ultraviolet light irradiation, to obtain the separation substrate 200; the grafting solution comprises a reaction monomer containing a sulfonic acid group, a photoinitiator and an organic solvent.

[0023] The separation substrate 200 and the cover substrate 100 are heat-press bonded, so that the buffer inlet 101 is communicated with the buffer inflow channel 201, the buffer outlet 102 is communicated with the buffer outflow channel 202, the sample inlet 103 is communicated with the sample inflow channel branch 203, and the sample outlet 104 is communicated with the sample outflow channel branch 204, so as to obtain the liquid chromatography array column chip.

[0024] The application provides application of the liquid chromatography array column chip in separation of amino acids, polypeptides, proteins and protein derivatives.

[0025] The application provides a liquid chromatography detection system, which comprises the liquid chromatography array column chip or the liquid chromatography array column chip prepared by the preparation method and a pump and a detector.

[0026] The application provides a liquid chromatography array column chip, which comprises a separation substrate 200 and a cover substrate 100 fixedly connected with the separation substrate 200; the separation substrate 200 is provided with a main channel, a sample inflow channel branch 203 and a sample outflow channel branch 204 communicated with the main channel; along the flow direction of the buffer in the main channel, the communication position of the sample outflow channel branch 204 with the main channel is located upstream of the communication position of the sample inflow channel branch 203 with the main channel; the main channel is sequentially divided into a buffer inflow channel 201, a quantitative sample cell 205, a separation channel 206 and a downstream region of the separation channel 206 along the flow direction of the buffer, and the downstream region of the separation channel 206 comprises a buffer outflow channel 202; the buffer inflow channel 201 is a main channel region located upstream of the sample outflow channel branch 204, the quantitative sample cell 205 is a main channel region located between the sample outflow channel branch 204 and the sample inflow channel branch 203, and the separation channel 206 and the downstream region of the separation channel 206 are main channel regions located downstream of the sample inflow channel branch 203; the inner surface of the separation channel 206 is provided with array-arranged separation columns, the separation columns are perpendicular to the fluid flow direction in the main channel, and the surface of the separation columns is chemically grafted with sulfonic acid groups; the cover substrate 100 is provided with a buffer inlet 101, a buffer outlet 102, a sample inlet 103 and a sample outlet 104 corresponding to the buffer inflow channel 201, the buffer outflow channel 202, the sample inflow channel branch 203 and the sample outflow channel branch 204 on the separation substrate 200; the buffer inlet 101 is communicated with the buffer inflow channel 201, the buffer outlet 102 is connected with the buffer outflow channel 202, the sample inlet 103 is communicated with the sample inflow channel branch 203, and the sample outlet 104 is communicated with the sample outflow channel branch 204. When the liquid chromatography array column chip works, first, the buffer inlet 101 and the buffer outlet 102 are closed, the sample inlet 103 flows into the chip, flows through the quantitative sample cell 205, and then flows out of the chip through the sample outlet 104, so that the sample injection of the liquid chromatography array column chip is completed; the buffer inlet 101 and the buffer outlet 102 are opened, and the sample inlet 103 and the sample outlet 104 are closed, the buffer flows into the chip through the buffer inlet 101, pushes the sample in the quantitative sample cell 205 to the separation channel 206 for separation, and then the separated components flow out of the separation channel (206) and flow out of the chip through the buffer outlet 102, so that the separation of the sample is completed.Compared with a traditional liquid chromatography column, the liquid chromatography array column chip provided by the application directly processes the chromatography column on a chip, has small volume, light weight and low cost, and the small chromatography column volume makes the required sample and reagent amount less; meanwhile, the chromatography column chip can greatly reduce the existence of dead volume, reduces the column-in effect and column-out effect, and further improves the sensitivity of detection. Moreover, the liquid chromatography array column chip provided by the application uses orderly arranged separation columns to replace the randomly filled filler microspheres as the stationary phase, which can well reduce the vortex diffusion effect caused by the sample flow in the chromatography column, thereby reducing peak expansion; meanwhile, the liquid chromatography array column chip provided by the application has low back pressure, reduces the requirements of the whole chip liquid chromatography system for driving pressure and pressure resistance, and is conducive to the further integration of the system. Finally, the application chemically grafts sulfonic acid groups on the separation column, which has strong cation exchange function, so that the chip provided by the application can perform ion exchange chromatography separation, and can be used for high-efficiency separation of amino acids, polypeptides, proteins and protein derivatives (such as glycosylated hemoglobin).

[0027] In the application, further, the downstream region of the separation channel 206 also comprises an optical detection pool 207; along the flow direction of the buffer in the main channel, the optical detection pool 207 is located upstream of the buffer outflow channel 202; the material of the separation substrate 200 and the cover substrate 100 is light-transmitting material. In the application, after the separation components flow out of the separation channel 206, they are first subjected to optical detection in the optical detection pool 207 at the rear end thereof, and then flow out from the buffer outlet 102, so that the separation and in-situ detection of the sample can be simultaneously completed.

[0028] In the application, further, the material of the separation substrate 200 is cyclic olefin copolymer; the material of the cover substrate 100 is polymethyl methacrylate. The application adopts cyclic olefin copolymer as the separation substrate 200 and polymethyl methacrylate as the cover substrate 100, and the two materials can meet the requirements of chip absorbance detection. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The schematic diagram of the separation substrate 200 and the cover substrate 100 of the liquid chromatography array column chip provided by the embodiment of the application;

[0030] Figure 2 The structural schematic diagram of the cover substrate 100 provided by the embodiment of the application;

[0031] Figure 3 The structural schematic diagram of the separation substrate 200 provided by the embodiment of the application;

[0032] Figure 4 The overall schematic diagram of the liquid chromatography array column chip provided by the embodiment of the application;

[0033] Figure 5 A liquid chromatography system diagram provided for the application example of the present application;

[0034] Figure 6 A detection diagram of glycated hemoglobin separated for the application example of the present application;

[0035] In the figure: 100 is a cover substrate, 200 is a separation substrate, 101 is a buffer inlet, 102 is a buffer outlet, 103 is a sample inlet, 104 is a sample outlet, 203 is a sample inflow channel branch, 204 is a sample outflow channel branch, 201 is a buffer inflow channel, 205 is a quantitative sample cell, 206 is a separation channel, 202 is a buffer outflow channel, 207 is an optical detection cell, a buffer storage container 300, 400 is a buffer delivery pump, 1000 is a buffer inflow micro valve, 500 is a sample storage container, 600 is a sample injection pump, 1100 is a sample inflow micro valve, 1200 is a sample outflow micro valve, 700 is a sample waste cell, 800 is a spectrometer, 900 is a buffer waste cell, and 1300 is a buffer outflow micro valve. DETAILED DESCRIPTION

[0036] The present application provides a liquid chromatography array column chip, comprising a separation substrate 200 and a cover substrate 100 fixedly connected with the separation substrate 200.

[0037] The separation substrate 200 is provided with a main channel and a sample inflow channel branch 203 and a sample outflow channel branch 204 in communication with the main channel;

[0038] In the flow direction of the buffer in the main channel, the communication position of the sample outflow channel branch 204 with the main channel is located upstream of the communication position of the sample inflow channel branch 203 with the main channel, and the main channel is divided into a buffer inflow channel 201, a quantitative sample cell 205, a separation channel 206 and a downstream region of the separation channel 206 in the flow direction of the buffer, the downstream region of the separation channel 206 comprising a buffer outflow channel 202; the buffer inflow channel 201 is a main channel region located upstream of the sample outflow channel branch 204, the quantitative sample cell 205 is a main channel region located between the sample outflow channel branch 204 and the sample inflow channel branch 203, and the separation channel 206 and the downstream region of the separation channel 206 are main channel regions located downstream of the sample inflow channel branch 203, the inner surface of the separation channel 206 is provided with an array of separation columns, the separation columns are perpendicular to the direction of fluid flow in the main channel, and the surface of the separation columns is chemically grafted with sulfonic acid groups;

[0039] The cover substrate 100 is provided with a buffer inlet 101, a buffer outlet 102, a sample inlet 103 and a sample outlet 104 corresponding to the buffer inflow channel 201, the buffer outflow channel 202, the sample inflow channel branch 203 and the sample outflow channel branch 204 on the separation substrate 200.

[0040] The buffer inlet 101 is in communication with the buffer inflow channel 201, the buffer outlet 102 is connected with the buffer outflow channel 202, the sample inlet 103 is in communication with the sample inflow channel branch 203, and the sample outlet 104 is in communication with the sample outflow channel branch 204.

[0041] In the present application, all the preparation raw materials / components are commercially available products well known to those skilled in the art, unless otherwise specified.

[0042] The liquid chromatography array column chip provided by the present application comprises a separation substrate 200.

[0043] In the present application, the separation substrate 200 is used for chromatographic separation and detection of samples.

[0044] As one or more embodiments of the present application, the material of the separation substrate 200 is a light-transmitting material.

[0045] As one or more embodiments of the present application, the material of the separation substrate 200 is cyclic olefin copolymer (COC).

[0046] As one or more embodiments of the present application, the size of the separation substrate 200 is 26mm×15mm×2mm.

[0047] As one or more embodiments of the present application, the cross-sectional size of the main channel is 400μm×15μm.

[0048] As one or more embodiments of the present application, the downstream region of the separation channel 206 further comprises an optical detection cell 207; along the flow direction of the buffer in the main channel, the optical detection cell 207 is located upstream of the buffer outflow channel 202.

[0049] As one or more embodiments of the present application, the separation columns are arranged in a regular triangular staggered array on the inner surface of the separation channel 206.

[0050] As one or more embodiments of the present application, the cross section of the separation column is circular, rhombic or regular hexagonal.

[0051] As one or more embodiments of the present application, the cross section of the separation column is circular, and the cross-sectional diameter of the separation column is 15μm.

[0052] In the present application, the function of the quantitative sample injection pool 205 is to quantify the sample injection amount. For a liquid chromatography column, the sample injection amount should be controlled within a certain range, and too large sample injection amount will cause the column to be overloaded, reducing the performance of the column; too small sample injection amount will easily lead to too low peak shape, which is not conducive to analysis. The maximum sample injection amount of the chromatography array column chip proposed in the present application is only about 15 nL, and the traditional quantitative sample injection technology cannot be realized, so the quantitative sample injection pool is directly processed on the chip.

[0053] The liquid chromatography array column chip provided by the present application comprises a cover substrate 100 fixedly connected with the separation substrate 200.

[0054] As one or more embodiments of the present application, the material of the cover substrate 100 is a light-transmitting material.

[0055] As one or more embodiments of the present application, the material of the cover substrate 100 is polymethyl methacrylate.

[0056] As one or more embodiments of the present application, the size of the cover substrate 100 is 26 mm x 15 mm x 2 mm.

[0057] As one or more embodiments of the present application, the cross-sectional size of the buffer inlet 101, the buffer outlet 102, the sample inlet 103 and the sample outlet 104 is 800 μm x 800 μm.

[0058] The present application provides a preparation method of the liquid chromatography array column chip as described in the above technical solution, comprising the following steps:

[0059] The buffer inlet 101, the buffer outlet 102, the sample inlet 103 and the sample outlet 104 are prepared on the first substrate by using numerical control micro-milling forming method, to obtain the cover substrate 100;

[0060] The main channel, the sample inflow channel branch 203, the sample outflow channel branch 204 and the separation column arranged on the inner surface of the separation channel 206 are prepared on the second substrate by using hot stamping method, to obtain the initial separation substrate;

[0061] The initial separation substrate is soaked in a grafting solution, and a grafting reaction is carried out under ultraviolet light irradiation, to obtain the separation substrate 200; the grafting solution comprises a reaction monomer containing a sulfonic acid group, a photoinitiator and an organic solvent.

[0062] The separation substrate 200 and the cover substrate 100 are heat-pressed and bonded, so that the buffer inlet 101 is communicated with the buffer inflow channel 201, the buffer outlet 102 is connected with the buffer outflow channel 202, the sample inlet 103 is communicated with the sample inflow channel branch 203, and the sample outlet 104 is communicated with the sample outflow channel branch 204, thereby obtaining the liquid chromatography array column chip.

[0063] The cover substrate 100 is prepared by adopting a numerical control micro-milling forming method on a first substrate to form the buffer inlet 101, the buffer outlet 102, the sample inlet 103 and the sample outlet 104.

[0064] In the present application, the material of the first substrate is preferably polymethyl methacrylate (PMMA).

[0065] The present application does not have special requirements for the specific implementation of the numerical control micro-milling forming method.

[0066] As one or more embodiments of the present application, the main working parameters of the numerical control micro-milling forming method are shown in Table 1.

[0067] Table 1 Main working parameters of numerical control micro-milling forming method

[0068]

[0069] The initial separation substrate is prepared by adopting a hot-pressing method on a second substrate to form a main channel, a sample inflow channel branch 203, a sample outflow channel branch 204 and a separation column arranged on the inner surface of the separation channel 206.

[0070] In the present application, the material of the second substrate is preferably COC, and more preferably Topas8007.

[0071] In the present application, the specific implementation method of the hot-pressing method preferably comprises the following steps: the microstructure of the separation substrate 200 is microstructured on a silicon wafer by a photolithography or etching method to obtain a silicon wafer template; the second substrate is placed between the silicon wafer template and a blank silicon wafer, and a hot-pressing bonding machine is used for hot-pressing.

[0072] As one or more embodiments of the present application, the specific operation process of the hot-pressing method is as follows: the temperature is raised to 70℃ (about 10℃ lower than the COC heat deformation temperature), then a pressure of 1.5MPa is applied and maintained; the temperature is raised to 100℃ (about 20℃ higher than the COC heat deformation temperature) again, then the pressure is raised to 2.5MPa, and the temperature and pressure are maintained for 600s; the heating is stopped and the temperature is naturally cooled to 50℃, then the pressure is removed for demolding, and after the excess part of the edge is cut off, the initial separation substrate is obtained.

[0073] After obtaining the initial separation substrate, the initial separation substrate is immersed in a grafting solution to perform a grafting reaction under irradiation of ultraviolet light, so as to obtain the separation substrate 200. The grafting solution comprises a reaction monomer containing a sulfonic acid group, a photoinitiator and an organic solvent.

[0074] In the present application, the COC material has a density of about 1.01 g / cm 3 , which is slightly smaller than the grafting solution. In the present application, only the part of the initial separation substrate having the separation column in the main channel is contacted with the grafting solution, and a photo-induced reaction is performed.

[0075] In the present application, the reaction monomer containing a sulfonic acid group comprises one or more of sodium styrene sulfonate, sodium methacrylate sulfonate, 2-ethyl sulfonate methacrylic acid sodium salt and 2-acrylamido-2-methylpropane sulfonic acid, and is more preferably 2-acrylamido-2-methylpropane sulfonic acid (AMPS).

[0076] In the present application, 2-acrylamido-2-methylpropane sulfonic acid is preferably used as the reaction monomer. The 2-acrylamido-2-methylpropane sulfonic acid has good thermal stability, and can avoid damage to the polymer after grafting in the process of thermal compression bonding. Meanwhile, the sulfonic acid group in the structure of the 2-acrylamido-2-methylpropane sulfonic acid is not on the benzene ring, and has high freedom and strong dissociation ability, and is more easily exchanged with other cations.

[0077] In the present application, the photoinitiator is preferably benzophenone (BP).

[0078] In the specific embodiments of the present application, the photoinitiator (benzophenone) is used as a photo initiator in the photochemical grafting process. Under the catalysis of ultraviolet light irradiation, the benzophenone is converted into an excited state, and a Norrish type II hydrogen abstraction process occurs with the COC, the carbon-carbon double bond in the AMPS is broken, and a chain polymerization reaction occurs at the hydrogen loss site of the COC, so as to graft the AMPS monomer on the surface of the COC.

[0079] In the present application, the organic solvent is preferably ethanol.

[0080] In the present application, in the grafting solution, the mass percentage content of the reaction monomer containing a sulfonic acid group is preferably 25%, the mass percentage content of the photoinitiator is preferably 0.25%, and the mass percentage content of the organic solvent is preferably 74.75%.

[0081] In the present application, the wavelength of the ultraviolet light is 365 nm, and the intensity of the ultraviolet light is preferably 10 mW / cm 2The UV irradiation time of the grafting reaction is preferably 120 seconds. In the present application, the temperature of the grafting reaction is preferably 55°C.

[0082] The obtained separation substrate 200 is preferably washed, which preferably includes washing with ethanol, deionized water and ethanol in turn.

[0083] After obtaining the separation substrate 200 and the cover substrate 100, the separation substrate 200 and the cover substrate 100 are thermally bonded to make the buffer inlet 101 communicate with the buffer inflow channel 201, the buffer outlet 102 communicate with the buffer outflow channel 202, the sample inlet 103 communicate with the sample inflow channel branch 203, and the sample outlet 104 communicate with the sample outflow channel branch 204, thereby obtaining the liquid chromatography array column chip.

[0084] In the present application, the specific implementation of the thermal bonding is preferably as follows: after aligning the microchannel ports of the separation substrate 200 and the cover substrate 100, a pressure of 3.5 MPa is applied at 60°C (about 20°C lower than the thermal deformation temperature of COC), and the pressure is maintained for 600 seconds. Subsequently, it is placed in an incubator and annealed at 85°C (about 5°C higher than the thermal deformation temperature of COC) for 600 seconds, thereby eliminating the residual stress in the bonding process.

[0085] The present application provides the application of the liquid chromatography array column chip in the separation of amino acids, polypeptides, proteins and protein derivatives.

[0086] The present application provides the application of the liquid chromatography array column chip in the separation and detection of amino acids, polypeptides, proteins and protein derivatives.

[0087] In the present application, the protein derivative is specifically preferably glycated hemoglobin.

[0088] The liquid chromatography array column chip provided by this invention, during operation, uses an external microvalve to seal the buffer inlet 101 and buffer outlet 102. The sample flows into the chip through the sample inlet 103 driven by the infusion pump, flows through the quantitative sample cell 205, and then flows out of the chip through the sample outlet 104, thus completing the sample injection of the liquid chromatography array column chip. When the external microvalve at the buffer inlet 101 and buffer outlet 102 is opened, and the sample inlet 103 and sample outlet 104 are sealed by the external microvalve, the buffer flows into the buffer inlet 101 driven by the infusion pump, pushing the sample in the quantitative sample cell 205 to the chromatographic array column 206 for separation. The separated components flow out of the chromatographic array column 206 and are optically detected in the optical detection cell 207 at its rear end, and then flow out from the buffer outlet 102, thus completing the separation and detection of the sample.

[0089] The present invention provides a liquid chromatography detection system, comprising a liquid chromatography array column chip as described in the above technical solution or a liquid chromatography array column chip prepared by the preparation method described in the above technical solution, as well as a pump and a detector.

[0090] In this invention, the pump is a commercial pump.

[0091] In this invention, such as Figure 5 As shown, the liquid chromatography detection system preferably includes: a liquid chromatography array column chip; a buffer delivery pump 400 connected to the buffer inlet 101 of the liquid chromatography array column chip, wherein a buffer inflow microvalve 1000 is provided on the pipeline connecting the buffer delivery pump 400 and the buffer inlet 101; and a buffer storage container 300 connected to the buffer delivery pump 400.

[0092] A buffer waste liquid tank 900 is connected to the buffer outlet 102 of the liquid chromatography array column chip, and a buffer outflow micro valve 1300 is provided on the pipe connecting the buffer waste liquid tank 900 and the buffer outlet 102.

[0093] A sample injection pump 600 is connected to the sample inlet 103 of the liquid chromatography array column chip, and a sample inflow microvalve 1100 is provided on the pipe connecting the sample injection pump 600 and the sample inlet 103; a sample storage container 500 is connected to the sample injection pump 600.

[0094] A sample waste liquid pool 700 is connected to the sample outlet 104 of the liquid chromatography array column chip, and a sample outflow microvalve 1200 is provided on the pipe connecting the sample waste liquid pool 700 and the sample outlet 104.

[0095] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0096] Example 1

[0097] according to Figure 2 The schematic diagram of the cover plate 100 shown is obtained by using a CNC micro-milling method to form polymethyl methacrylate sheet according to the working parameters in Table 1. The cross-sectional dimensions of the buffer inlet 101, buffer outlet 102, sample inlet 103 and sample outlet 104 are all 800μm×800μm, and the cover plate 100 is obtained with a size of 26mm×15mm×2mm.

[0098] according to Figure 3 The schematic diagram of the separated substrate 200 shown is illustrated by photolithography. Figure 3 The aforementioned microstructure is fabricated on a silicon wafer to obtain a silicon wafer template. A COC sheet is placed between the silicon wafer template and a blank silicon wafer, and a hot-press bonding machine is used for hot pressing. The hot pressing procedure is as follows: the temperature is raised to 70°C (approximately 10°C below the COC heat distortion temperature), and then a pressure of 1.5 MPa is applied and maintained; the temperature is raised again to 100°C (approximately 20°C above the COC heat distortion temperature), and then the pressure is raised to 2.5 MPa, and the temperature and pressure are maintained for 600 seconds; heating is stopped and the temperature is allowed to cool naturally to 50°C, and then the pressure is removed for demolding. After removing the excess portion at the edges, the initial separation substrate is obtained. The cross-sectional dimensions of the main channel of the initial separation substrate are 400 μm × 15 μm, and the separation columns are arranged in an equilateral triangular staggered array on the inner surface of the separation channel 206, with a diameter of 15 μm.

[0099] The initial separated substrate was immersed in a grafting solution, wherein the grafting solution consisted of 25% by mass of 2-acrylamido-2-methylpropanesulfonic acid (AMPS), 0.25% by mass of benzophenone (BP), and 74.75% by mass of ethanol; and the grafting was performed at 55°C with a wavelength of 365 nm and an intensity of 10 mW / cm². 2 The separation column was subjected to a chemical grafting reaction by irradiating it with ultraviolet light for 120 seconds. Sulfonic acid groups were grafted onto the surface of the separation column. After the grafting reaction was completed, the column was washed with ethanol, deionized water and ethanol in sequence to obtain a separation substrate 200 with dimensions of 26mm×15mm×2mm.

[0100] After aligning the microchannel ports of the cover substrate 100 and the separation substrate 200, a pressure of 3.5 MPa is applied at 60°C (approximately 20°C below the COC heat distortion temperature) and held at that temperature and pressure for 600 seconds. Then, the substrate is placed in a constant temperature oven and annealed at 85°C (approximately 5°C above the COC heat distortion temperature) for 600 seconds to eliminate residual stress from the bonding process, thus obtaining a liquid chromatography array column chip.

[0101] Application examples

[0102] This application example utilizes the liquid chromatography array column chip prepared in Example 1 in a liquid chromatography system for detecting human blood glucose levels, thereby separating glycated hemoglobin. Normal human hemoglobin is mainly composed of HbA (95-97%), HbA2 (<3%), and HbF (<1%). HbF is primarily found in the fetal stage, while HbA is mainly found in adults. HbA can be further divided into HbA0 (87-89%) and HbA1 (6-8%). HbA0 is the unglycosylated portion, while HbA1 is the glycosylated portion. Based on the different protein chains it contains, HbA1 is further divided into HbA1a (<1%), HbA1b (<1%), and HbA1c (4-6%), with HbA1a and HbA1b present in very low amounts, and HbA1c being the main component. The results of glycated hemoglobin (HbA1c) determination are expressed as a percentage, referring to the concentration of HbA1c relative to the total hemoglobin (Hb) concentration, thus indicating the blood glucose level in the human body. In this application example, a liquid chromatography array column chip is used to separate HbA1(a+b), HbA1c, and HbA0 in a hemoglobin sample.

[0103] like Figure 5 As shown, the constructed liquid chromatography system mainly includes: liquid chromatography array column chips 100 and 200, buffer storage container 300, buffer delivery pump 400 (commercial pump A), sample storage container 500, sample injection pump 600 (commercial pump B), sample waste reservoir 700, spectrometer 800, buffer waste reservoir 900, buffer inflow microvalve 1000 (commercial microvalve A), sample inflow microvalve 1100 (commercial microvalve B), sample outflow microvalve 1200 (commercial microvalve C), and buffer outflow microvalve 1300 (commercial microvalve D). For better illustration of the connection methods, Figure 5 The middle cover substrate 100 and the separation substrate 200 are in a separated state. In actual operation, the liquid chromatography array column chip has completed thermo-press bonding.

[0104] The working steps of a liquid chromatography system are as follows:

[0105] (1) Connect micro valves A and D, disconnect micro valves B and C, turn on commercial pump A, and pump the buffer solution into the liquid chromatography array column chip to rinse for more than 10 minutes until the absorbance baseline of the commercial spectrometer at the back-end optical detection cell is stable.

[0106] (2) Turn off commercial pump A, disconnect micro valves A and D, connect micro valves B and C, turn on commercial pump B, and inject the sample into the quantitative injection cell on the liquid chromatography array column chip.

[0107] (3) Turn off commercial pump B, disconnect micro valves B and C, connect micro valves A and D, turn on commercial pump A, use buffer to carry the sample into the chromatographic array column for separation, and finally use commercial spectrometer to detect absorbance at the optical detection cell to obtain absorbance chromatogram.

[0108] The optimized condition parameters are as follows:

[0109] (1) Column temperature: 25℃;

[0110] (2) Buffer composition: 100mM NaCl phosphate solution, pH=6.0;

[0111] (3) Buffer flow rate: 300 nL / min.

[0112] like Figure 6 As shown, under optimized conditions, the liquid chromatography array column chip proposed in this invention achieves the separation of glycated hemoglobin, and peak 2 is the main component of glycated hemoglobin, HbA1c.

[0113] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A liquid chromatography array column chip, characterized in that, It includes a separation substrate (200) and a cover substrate (100) fixedly connected to the separation substrate (200). The separation substrate (200) is provided with a main channel and a sample inflow channel branch (203) and a sample outflow channel branch (204) connected to the main channel. Along the flow direction of the buffer solution in the main channel, the connection point between the sample outflow channel branch (204) and the main channel is located upstream of the connection point between the sample inflow channel branch (203) and the main channel. The main channel is divided into a buffer inflow channel (201), a quantitative sample cell (205), a separation channel (206), and a downstream region of the separation channel (206) along the flow direction of the buffer solution. The downstream region of the separation channel (206) includes the buffer outflow channel (202). The buffer inflow channel (201) is located upstream of the sample outflow channel (202). The main channel region upstream of the sample outflow channel branch (204) is the main channel region between the sample outflow channel branch (204) and the sample inflow channel branch (203). The separation channel (206) and the downstream region of the separation channel (206) are the main channel regions downstream of the sample inflow channel branch (203). The inner surface of the separation channel (206) is provided with an array of separation columns. The separation columns are perpendicular to the direction of fluid flow in the main channel. The surface of the separation columns is chemically grafted with sulfonic acid groups. The cover substrate (100) is provided with a buffer inlet (101), a buffer outlet (102), a sample inlet (103), and a sample outlet (104) corresponding to the buffer inflow channel (201), the buffer outflow channel (202), the sample inflow channel branch (203), and the sample outflow channel branch (204) on the separation substrate (200). The buffer inlet (101) is connected to the buffer inflow channel (201), the buffer outlet (102) is connected to the buffer outflow channel (202), the sample inlet (103) is connected to the sample inflow channel branch (203), and the sample outlet (104) is connected to the sample outflow channel branch (204).

2. The liquid chromatography array column chip according to claim 1, characterized in that, The downstream region of the separation channel (206) also includes an optical detection cell (207); along the flow direction of the buffer solution in the main channel, the optical detection cell (207) is located upstream of the buffer solution outflow channel (202); the materials of the separation substrate (200) and the cover substrate (100) are light-transmitting materials.

3. The liquid chromatography array column chip according to claim 1 or 2, characterized in that, The material of the separation substrate (200) is a cyclic olefin copolymer; the material of the cover substrate (100) is polymethyl methacrylate.

4. The liquid chromatography array column chip according to claim 1, characterized in that, The separation columns are arranged in an alternating array of equilateral triangles on the inner surface of the separation channel (206).

5. The liquid chromatography array column chip according to claim 1, characterized in that, The cross-sectional dimensions of the main channel are 400μm × 15μm; The separation column has a diameter of 15 μm.

6. The liquid chromatography array column chip according to claim 1, characterized in that, The cross-sectional dimensions of the buffer inlet (101), the buffer outlet (102), the sample inlet (103), and the sample outlet (104) are all 800μm×800μm.

7. The liquid chromatography array column chip according to claim 1, characterized in that, The dimensions of both the separating substrate (200) and the cover substrate (100) are 26mm × 15mm × 2mm.

8. The method for preparing the liquid chromatography array column chip according to any one of claims 1 to 7, characterized in that, Includes the following steps: The buffer inlet (101), the buffer outlet (102), the sample inlet (103), and the sample outlet (104) are prepared on the first substrate using a CNC micro-milling method to obtain the cover substrate (100). Using a hot embossing method, a main channel, a sample inflow channel branch (203), a sample outflow channel branch (204), and a separation column disposed on the inner surface of the separation channel (206) are prepared on a second substrate to obtain an initial separation substrate; The initial separation substrate is immersed in a grafting solution and a grafting reaction is carried out under ultraviolet light irradiation to obtain the separation substrate (200). The grafting solution includes a reactive monomer containing sulfonic acid groups, a photoinitiator and an organic solvent. The separation substrate (200) and the cover substrate (100) are hot-pressed together to connect the buffer inlet (101) with the buffer inflow channel (201), the buffer outlet (102) with the buffer outflow channel (202), the sample inlet (103) with the sample inflow channel branch (203), and the sample outlet (104) with the sample outflow channel branch (204), thereby obtaining the liquid chromatography array column chip.

9. The application of the liquid chromatography array column chip according to any one of claims 1 to 7 or the liquid chromatography array column chip prepared by the preparation method according to claim 8 in the separation of amino acids, peptides, proteins and protein derivatives.

10. A liquid chromatography detection system, characterized in that, Includes the liquid chromatography array column chip according to any one of claims 1 to 7 or the liquid chromatography array column chip prepared by the preparation method according to claim 8, as well as a pump and a detector.

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