An automated on-line extraction-mass spectrometry detection system and method

Abstract

The application discloses an automatic online extraction-mass spectrum detection system and method based on an integrated microfluidic chip, relates to the fields of microfluidic processing and mass spectrum analysis, and automatically integrates a microfluidic chip unit by bonding multiple layers of PDMS and using glass packaging. An online solid-phase extraction desalination unit performs automatic online extraction desalination operation, so that mass spectrum detection of target components in different solutions is realized. The mass spectrum online detection unit is connected with a mass spectrum spray needle through a capillary, so that online real-time mass spectrum detection is realized. The application realizes multi-step automatic operation of extraction only through the chip, and the sample consumption is lower. The sample for mass spectrum detection has high throughput, high selectivity and high sensitivity analysis, and online quantitative analysis of biological samples can be realized. Compared with a traditional detection method, the application automatically performs sample extraction desalination treatment, is efficient and fast, reduces manual operation errors, has high throughput, and has shorter analysis time, so that the demand of instant high-throughput analysis of biological samples is met.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microfluidic processing and mass spectrometry, and more particularly to an automatic online extraction-mass spectrometry detection system and method based on an integrated microfluidic chip. BACKGROUND

[0002] At present, microfluidic technology is a system science and technology focusing on the manipulation of microfluids, and its core implementation platform is a microfluidic chip. The main feature of a microfluidic chip is that the structure for containing fluid reaches the nanoscale in at least one dimension. With the wide application of semiconductor microfabrication technology in the manufacturing of microfluidic chips and the development of new processes such as multi-layer construction of elastic materials, various functional elements and structures have been highly integrated into small chips. Such chips with multiple functions, complex structures and precise and controllable operations are called "integrated microfluidic chips".

[0003] Integrated microfluidic chips, especially those with high density, large scale, high throughput and multi-functionality, have shown important value in the fields of chemistry and biology. Compared with traditional macroscopic experimental devices, microfluidic technology not only significantly reduces sample consumption, but also increases the surface area of the fluid, thereby improving reaction efficiency and reducing environmental pollution caused by waste generation. At the same time, the parallelism advantage of its operation can realize high-throughput and automatic control of experiments. In addition, through the precise control of micro-valves, micro-pumps and other micro-structures, microfluidic chips can flexibly improve the time and spatial resolution of life science research, showing irreplaceable technical advantages.

[0004] Mass spectrometry detection technology plays an important role in the detection of proteins, signaling molecules, drugs and metabolites in cell-related analysis due to its wide detection range, rich molecular information, ability to handle complex samples and provide molecular structure information. With the rapid development of biological and pharmaceutical technology, the demand for new drug development continues to grow, and drug toxicity detection and metabolism analysis have become an indispensable part of the development process. Compared with traditional animal models and two-dimensional cell culture, in vitro models are more suitable for large-scale compound screening due to their advantages of low sample consumption, short experimental period, accurate dose response, high throughput, etc. In the process of mass spectrometry detection, the traditional pretreatment process requires the removal of salts from the sample solution being measured, which is usually done using solid-phase microextraction technology. However, traditional solid-phase extraction microfluidic chips rely on manual operation and cannot achieve complete automation, resulting in large sample consumption, complicated procedures and reliance on injection pumps for sample injection, which is large in volume and low in efficiency.

[0005] Therefore, how to realize automatic online solid-phase extraction desalination, real-time online mass spectrometry detection, and meet the detection of target substances such as drugs, protein factors and nucleic acids in different biological sample solutions is a problem that needs to be solved by those skilled in the art. SUMMARY

[0006] Therefore, the application provides an automatic online extraction-mass spectrum detection system and method based on an integrated microfluidic chip, realizes automatic online solid phase extraction desalination and mass spectrum real-time online detection, meets the detection of target substances such as drugs, protein factors and nucleic acids in different biological sample solutions, and provides convenience for clinical and drug research and development.

[0007] In order to achieve the above object, the application adopts the following technical scheme:

[0008] An automatic online extraction-mass spectrum detection system based on an integrated microfluidic chip comprises an automatic integrated microfluidic chip unit, an online solid phase extraction desalination unit, an automatic control unit, a pressure unit and a mass spectrum online detection unit.

[0009] The automatic integrated microfluidic chip unit has multiple layers of PDMS, each layer has a different function, and specifically comprises a fluid layer, a control layer and a glass packaging layer; the fluid layer is provided with a solid phase extraction column interface.

[0010] The online solid phase extraction desalination unit is provided with a C 18 The gun head of the solid phase extraction column is connected to the automatic control unit through a rubber tube at the other end, and the control layer of the automatic integrated microfluidic chip unit is connected to the automatic control unit.

[0011] The automatic control unit is connected to the pressure unit.

[0012] The mass spectrum online detection unit is connected to the automatic integrated microfluidic chip unit.

[0013] Optionally, the fluid layer has multiple sample injection ports, multiple sample outlets and a solid phase extraction column interface, the control layer is provided with multiple valves at different positions, and the valves are connected to the automatic control unit.

[0014] Optionally, the automatic integrated microfluidic chip unit has a three-layer structure; the upper layer is a fluid layer, the middle layer is a control layer, and the lower layer is a glass packaging layer; the materials of the upper layer and the middle layer are PDMS, and the material of the lower layer is glass; the upper layer and the middle layer are bonded through different proportions of PDMS, the proportion of the upper layer PDMS is 5:1, the proportion of the middle layer PDMS is 20:1, and the PDMS solidification mode is 80 DEG C heating for 20 minutes; the middle layer and the lower layer are plasma bonded, a plasma cleaning machine is used, vacuum is drawn for 150S, air is introduced for 50S, and the surface is hydrophilic treated for 90S, and the glass is cleaned before bonding.

[0015] Optionally, the automatic control unit comprises a computer, a controller, and an electromagnetic valve, which are connected in sequence through a circuit; one end of the electromagnetic valve is connected to the controller, and the other end is connected to the automatic integrated microfluidic chip unit through a plastic hose.

[0016] Optionally, the pressure unit is one of a steel cylinder and a gas pump; the pressure unit provides pressure for the automatic integrated microfluidic chip unit, and the control layer pressure is 20 psi and the fluid layer pressure is 5 psi.

[0017] Optionally, the mass spectrometry online detection unit comprises a capillary tube with an outer diameter of 669 µm, a 360 µm spray needle, a liquid chromatography two-way PEEK variable-diameter 1 / 16'~1 / 32', and a mass spectrometer; the automatic integrated microfluidic chip unit is connected to the capillary tube, and the capillary tube is connected to the mass spectrometry spray needle through the variable-diameter two-way.

[0018] Optionally, the sample collection process is to use gas pressure to elute the sample from the solid-phase extraction column at the end of the gun head into the capillary tube, the sample passes through the capillary tube and forms a spray by electrifying the mass spectrometry spray needle, and finally the mass spectrometry online detection is completed.

[0019] An automatic online extraction-mass spectrometry detection method based on an integrated microfluidic chip, comprising the following steps:

[0020] Sample preparation: first prepare a 1M capecitabine stock solution with DMSO as the solvent, dilute 500µM capecitabine from the stock solution with complete culture medium, and add 0.1% formic acid to make the PH<4;

[0021] Activate the solid-phase extraction column: connect the 100% acetonitrile stock solution pool to the pressure, make it pass through the sample inlet 1, open the valves ①, ②, and ⑦, and close the other valves, so that the channel is filled with acetonitrile, then open ①, ⑦, and ③, and close the other valves, acetonitrile passes through the fluid channel into the gun head loaded with the solid-phase extraction column, the solution reaches 10 µL, and the sample inlet is stopped; open ③ and ⑦, and close the other valves, the pressure pushes the solution in the gun head into the fluid channel and through the waste liquid collection port, and the waste liquid is recovered, the above operation is repeated three times;

[0022] Balance the solid-phase extraction column: give pressure to the 0.1% formic acid stock solution pool, make it pass through the sample inlet 2, open the valves ⑤, ②, and ⑦, and close the other valves, so that the channel is filled with formic acid, then open ⑤, ②, and ③, and close the other valves, formic acid passes through the fluid channel into the gun head loaded with the solid-phase extraction column, the solution reaches 10 µL, and the sample inlet is stopped; open ③ and ⑦, and close the other valves, the pressure pushes the solution in the gun head into the fluid channel and through the waste liquid collection port, and the waste liquid is recovered, the above operation is repeated three times;

[0023] Loading: The sample stock solution is given pressure to pass through the sample inlet 4, the valve 4 is opened, and the other valves are closed, the sample passes through the fluid channel into the gun head loaded with the solid phase extraction column, the solution reaches 10 μL, and the sample loading is stopped; the valves 3 and 6 are opened, and the other valves are closed, the pressure drives the solution in the gun head into the fluid channel and passes through the waste liquid collection port, and the waste liquid is recovered, and the above operation is repeated 7-10 times;

[0024] Washing the solid phase extraction column: The 0.1% formic acid stock solution is given pressure to pass through the sample inlet 2, the valves 5, 2 and 6 are opened, and the other valves are closed, so that the channel is filled with formic acid, then the valves 5, 2 and 3 are opened, and the other valves are closed, the formic acid passes through the fluid channel into the gun head loaded with the solid phase extraction column, the solution reaches 10 μL, and the sample loading is stopped; the valves 3 and 6 are opened, and the other valves are closed, the pressure drives the solution in the gun head into the fluid channel and passes through the waste liquid collection port, and the waste liquid is recovered, and the above operation is repeated three times;

[0025] Sample elution: The 1% formic acid / 50% methanol stock solution is given pressure to pass through the sample inlet 3, the valves 6, 2 and 6 are opened, and the other valves are closed, so that the channel is filled with the eluent, then the valves 6, 2 and 3 are opened, and the other valves are closed, the eluent passes through the fluid channel into the gun head loaded with the solid phase extraction column, the solution reaches 10 μL, and the sample loading is stopped; the valves 3, 6 and 7 are opened, and the other valves are closed, the pressure drives the solution in the gun head into the fluid channel and passes through the mass spectrometry sample inlet, and passes through the two-way into the mass spectrometry spray needle, and the mass spectrometry spray needle is electrified to form an electrospray, so as to directly perform online mass spectrometry detection.

[0026] According to the technical solution, compared with the prior art, the application discloses an automatic online extraction-mass spectrometry detection system and method based on an integrated microfluidic chip, and has the following beneficial effects:

[0027] 1. The multi-layer structure of the integrated microfluidic chip is combined with the electromagnetic valve control, and the multi-step automatic operation of extraction can be realized only through the chip, and the sample consumption is lower.

[0028] 2. The application adopts the integrated microfluidic chip online extraction-mass spectrometry combination, the chip is automatically processed and directly enters the mass spectrometry tip through the instruction, the tip is electrified, the sample forms an electrospray, the mass spectrometry detection sample has high throughput, high selectivity and high sensitivity analysis, and the online quantitative analysis of the biological sample can be realized.

[0029] 3. Compared with the traditional detection method, the sample extraction desalting treatment is automatically performed, which is efficient and fast, gets rid of manual operation, reduces the manual operation error, has high throughput, and has shorter analysis time, so that the demand for instant high-throughput analysis of biological samples is met. DETAILED DESCRIPTION

[0030] 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 or prior art description. Obviously, the drawings described below only constitute a part of the embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of the provided drawings.

[0031] Figure 1 The detection system structure schematic diagram provided by the present application is shown in the figure.

[0032] Figure 2 The automatic integrated microfluidic chip unit structure schematic diagram provided by the present application is shown in the figure.

[0033] Figure 3 The online mass spectrum detection diagram of the drug capecitabine provided by the present application is shown in the figure. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments only constitute a 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 labor fall within the scope of protection of the present application.

[0035] The embodiments of the present application disclose an automatic online extraction-mass spectrum detection system based on integrated microfluidic chip, as shown in the figure, comprising: an automatic integrated microfluidic chip unit, an online solid phase extraction desalination unit, an automatic control unit, a pressure unit, and a mass spectrum online detection unit. Figure 1

[0036] The automatic integrated microfluidic chip unit has multiple layers of PDMS, each layer has different functions, specifically including a fluid layer, a control layer, and a glass packaging layer; the fluid layer is provided with a solid phase extraction column interface;

[0037] The online solid phase extraction desalination unit is inserted with a sharp-end C 18 The gun head of the solid phase extraction column, the other end of the gun head is connected to the automatic control unit through a rubber tube, and the control layer of the automatic integrated microfluidic chip unit is connected to the automatic control unit.

[0038] The automatic control unit is connected to the pressure unit.

[0039] The mass spectrum online detection unit is connected to the automatic integrated microfluidic chip unit.

[0040] In a specific embodiment, as shown in the figure, Figure 2 ​As shown, the automated integrated microfluidic chip unit includes three layers, namely the fluid layer, the control layer, and the glass packaging layer. The fluid layer has four sample inlets, two outlets, and one solid-phase extraction column interface. The sample inlets 1 to 3 are respectively 100% acetonitrile, 0.1% formic acid, and 1% formic acid / 50% methanol. The sample inlet 4 is a biological sample (capecitabine) inlet. The outlets are respectively waste liquid collection ports and mass spectrometry sample inlets. The solid-phase extraction column interface selects a C 18 The gun head of the solid-phase extraction column. The control layer includes eight valves, i.e., also includes eight sample inlets. The control valves ①, ②, ③, ④, ⑤, ⑥, ⑦, and ⑧ are arranged in the fluid channel part, and the purpose is to block the flow of fluid.

[0041] In a specific embodiment, the automated integrated microfluidic chip unit is a three-layer structure; the upper layer is a fluid layer, the middle layer is a control layer, and the lower layer is a glass packaging layer. The materials of the upper layer and the middle layer are PDMS, and the material of the lower layer is glass. The upper layer and the middle layer are bonded by PDMS with different proportions. The PDMS proportion of the upper layer is 5:1, and the PDMS proportion of the middle layer is 20:1. The PDMS solidification method is heating at 80°C for 20 minutes. The middle layer and the lower layer are plasma bonded. A plasma cleaning machine is used to vacuum for 150S, air for 50S, and surface hydrophilic treatment for 90S. The glass is cleaned before bonding.

[0042] In a specific embodiment, the automated control unit includes a computer, a controller, and solenoid valves, which are connected in sequence through a circuit. One end of the solenoid valve is connected to the controller, and the other end is connected to the automated integrated microfluidic chip unit through a plastic hose. The solenoid valve is connected to the computer through a multi-channel controller. The computer issues instructions to the multi-channel controller through a program, and finally the solenoid valve implements the opening and closing of the gas path valve.

[0043] In a specific embodiment, the pressure unit is one of a steel cylinder and a gas pump. It provides pressure for the automated integrated microfluidic chip unit. The pressure of the control layer is 20 psi, and the pressure of the fluid layer is 5 psi. Two flow meters are needed to control the pressure of the control layer and the fluid layer. Importantly, before the experiment, the eight control valves of the control layer need to be filled with solution. When the pressure is provided to the control valve, the solution in the control valve chamber impacts the control layer membrane, which arches upward, thereby compressing the fluid layer channel and blocking the flow of fluid.

[0044] In a specific embodiment, the mass spectrometry online detection unit includes a capillary tube with an outer diameter of 669 µm, a 360 µm spray needle, a liquid chromatography two-way PEEK reducer 1 / 16'~1 / 32', and a mass spectrometer. The automated integrated microfluidic chip unit is connected to the capillary tube, and the capillary tube is connected to the mass spectrometry spray needle through the reducer two-way.

[0045] In one specific embodiment, the sample collection procedure is to use gas pressure to elute the sample from the solid phase extraction column at the end of the gun head into the capillary, the sample passes through the capillary and forms a spray by electrifying the mass spectrometry spray needle, and finally completes the online detection of mass spectrometry.

[0046] An automatic online extraction-mass spectrometry detection method based on integrated microfluidic chip, comprising the following steps:

[0047] Sample preparation: first prepare a 1M capecitabine stock solution, the solvent is DMSO, 500µM capecitabine is diluted from the stock solution with complete medium, and 0.1% formic acid is added to make the PH<4;

[0048] Activate the solid phase extraction column: connect the 100% acetonitrile storage tank to the pressure, make it pass through the sample inlet 1, open the valves ①, ② and ⑦, and close the other valves, so that the channel is filled with acetonitrile, then open ①, ⑦ and ③, and close the other valves, acetonitrile passes through the fluid channel into the gun head loaded with the solid phase extraction column, and the solution stops sampling at 10µL; open ③ and ⑦, and close the other valves, the pressure pushes the solution in the gun head into the fluid channel and through the waste liquid collection port, and the waste liquid is recovered, the above operation is repeated three times;

[0049] Balance the solid phase extraction column: give the 0.1% formic acid storage tank pressure, make it pass through the sample inlet 2, open the valves ⑤, ② and ⑦, and close the other valves, so that the channel is filled with formic acid, then open ⑤, ② and ③, and close the other valves, formic acid passes through the fluid channel into the gun head loaded with the solid phase extraction column, and the solution stops sampling at 10µL; open ③ and ⑦, and close the other valves, the pressure pushes the solution in the gun head into the fluid channel and through the waste liquid collection port, and the waste liquid is recovered, the above operation is repeated three times;

[0050] Load the sample: give the sample storage tank pressure, make it pass through the sample inlet 4, open the valve ④, and close the other valves, the sample passes through the fluid channel into the gun head loaded with the solid phase extraction column, and the solution stops sampling at 10µL; open ③ and ⑦, and close the other valves, the pressure pushes the solution in the gun head into the fluid channel and through the waste liquid collection port, and the waste liquid is recovered, the above operation is repeated 7-10 times;

[0051] Wash the solid phase extraction column: give the 0.1% formic acid storage tank pressure, make it pass through the sample inlet 2, open the valves ⑤, ② and ⑦, and close the other valves, so that the channel is filled with formic acid, then open ⑤, ② and ③, and close the other valves, formic acid passes through the fluid channel into the gun head loaded with the solid phase extraction column, and the solution stops sampling at 10µL; open ③ and ⑦, and close the other valves, the pressure pushes the solution in the gun head into the fluid channel and through the waste liquid collection port, and the waste liquid is recovered, the above operation is repeated three times;

[0052] Sample elution: 1% formic acid / 50% methanol stock solution pool is given pressure to pass through the sample inlet 3, valve ⑥②⑦ is opened, other valves are closed, the channel is filled with eluent, then valve ⑥②③ is opened, other valves are closed, the eluent passes through the fluid channel into the gun head loaded with solid phase extraction column, the solution reaches 10 μL, stop sampling; ③⑦⑧ valve is opened, other valves are closed, the pressure pushes the solution in the gun head into the fluid channel and passes through the mass spectrometry sample inlet, and passes through the two-way into the mass spectrometry spray needle, the mass spectrometry spray needle is electrified to form an electrospray, so as to directly carry out mass spectrometry online detection.

[0053] The embodiment mainly carries out solid phase extraction desalination and mass spectrometry detection on capecitabine diluted by cell culture medium, and the detection result is as shown in Figure 3 After hydrogenation treatment, the molecular ion peak of capecitabine is detected as 360.1560, and capecitabine is better detected.

[0054] The application can realize online sample separation, enrichment and detection, and has wide application prospect in clinical drug detection.

[0055] In the specification, each embodiment is described in a progressive manner, and each embodiment mainly explains the difference from other embodiments, and the same or similar parts of each embodiment can be referred to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the related parts can be referred to the method part.

[0056] The above description of the disclosed embodiments enables a person skilled in the art to implement or use the application. Various modifications to the embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the application. Therefore, the application will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An automated online extraction-mass spectrometry detection system based on an integrated microfluidic chip, characterized in that, include: The system integrates an automated microfluidic chip unit, an online solid-phase extraction desalination unit, an automated control unit, a pressure unit, and an online mass spectrometry detection unit. The automated integrated microfluidic chip unit has multiple PDMS layers, each with different functions, specifically including a fluid layer, a control layer, and a glass encapsulation layer; the fluid layer is provided with a solid phase extraction column interface. The online solid-phase extraction desalination unit inserts a tip with C into the interface of the solid-phase extraction column. 18 The nozzle of the solid phase extraction column is connected to the automated control unit via a rubber tube at the other end. At the same time, the control layer of the automated integrated microfluidic chip unit is connected to the automated control unit. The automated control unit is connected to the pressure unit; The mass spectrometry online detection unit is connected to the automated integrated microfluidic chip unit; Valve ① controls the opening and closing of injection port 1; valve ② controls the opening and closing of the injection-side collection channel and the solid-phase extraction column interface; valve ③ controls the opening and closing of the solid-phase extraction column interface and the waste liquid outlet; valve ④ controls the opening and closing of the sample injection port and the fluid channel; valve ⑤ controls the opening and closing of injection port 2 and the fluid channel; valve ⑥ controls the opening and closing of injection port 3 and the fluid channel; valve ⑦ controls the opening and closing of the fluid channel and the waste liquid outlet; valve ⑧ controls the opening and closing of the fluid channel and the mass spectrometer injection port. An automated online extraction-mass spectrometry detection method based on an integrated microfluidic chip includes the following steps: Sample preparation: First, prepare a 1M capecitabine stock solution in DMSO. Dilute 500µM capecitabine from the stock solution using complete culture medium and add 0.1% formic acid to make the pH < 4. Activating the solid-phase extraction column: Pressurize the 100% acetonitrile reservoir and allow it to enter through inlet 1. Open valves ①②⑦ and close other valves to fill the channel with acetonitrile. Then open valves ①⑦③ and close other valves. Acetonitrile enters the pipette tip containing the solid-phase extraction column through the fluid channel. Stop the injection when the solution reaches 10µL. Open valves ③ and ⑦ and close other valves. The pressure pushes the solution in the pipette tip into the fluid channel and through the waste collection port to recover the waste liquid. Repeat the above operation three times. Equilibrium solid-phase extraction column: 0.1% formic acid reservoir is pressurized and introduced through injection port 2. Valves ⑤②⑦ are opened, and other valves are closed to fill the channel with formic acid. Then, valves ⑤②③ are opened, and other valves are closed. Formic acid enters the pipette tip containing the solid-phase extraction column through the fluid channel. When the solution reaches 10 µL, the injection is stopped. Valves ③ and ⑦ are opened, and other valves are closed. The pressure pushes the solution in the pipette tip into the fluid channel and through the waste collection port to recover the waste liquid. The above operation is repeated three times. Sample loading: Apply pressure to the sample reservoir to allow it to enter through inlet 4. Open valve ④ and close other valves. The sample enters the pipette tip containing the solid-phase extraction column through the fluid channel. Stop the injection when the solution reaches 10 µL. Open valves ③ and ⑦ and close other valves. The pressure pushes the solution in the pipette tip into the fluid channel and through the waste collection port to recover the waste liquid. Repeat the above operation 7-10 times. Cleaning the solid-phase extraction column: Apply pressure to the 0.1% formic acid reservoir and allow it to enter through injection port 2. Open valves ⑤②⑦ and close the other valves to fill the channel with formic acid. Then open valves ⑤②③ and close the other valves. The formic acid enters the pipette tip containing the solid-phase extraction column through the fluid channel. Stop the injection when the solution reaches 10 µL. Open valves ③ and ⑦ and close the other valves. The pressure forces the solution in the pipette tip into the fluid channel and through the waste collection port to recover the waste liquid. Repeat the above operation three times. Sample elution: The 1% formic acid / 50% methanol reservoir is pressurized and allowed to enter through inlet 3. Valves ⑥②⑦ are opened, while other valves are closed, filling the channel with eluent. Then, valves ⑥②③ are opened, while other valves are closed. The eluent enters the pipette tip containing the solid-phase extraction column through the fluid channel. When the solution reaches 10 µL, the injection is stopped. Valves ③⑦⑧ are opened, while other valves are closed. The pressure forces the solution in the pipette tip into the fluid channel and through the mass spectrometer inlet, and then through the two-way valve into the mass spectrometer nozzle. When the mass spectrometer nozzle is energized, an electrospray is formed, allowing for direct online mass spectrometry detection.

2. The automated online extraction-mass spectrometry detection system based on an integrated microfluidic chip according to claim 1, characterized in that, The fluid layer has multiple sample inlets and outlets, as well as a solid-phase extraction column interface. The control layer has multiple valves at different locations, and the valves are connected to an automated control unit.

3. The automated online extraction-mass spectrometry detection system based on an integrated microfluidic chip according to claim 1, characterized in that, The automated integrated microfluidic chip unit has a three-layer structure: an upper fluid layer, a middle control layer, and a lower glass encapsulation layer. The upper and middle layers are made of PDMS, and the lower layer is made of glass. The upper and middle layers are bonded by PDMS in different ratios: 5:1 for the upper layer and 20:1 for the middle layer. The PDMS is cured by heating at 80°C for 20 minutes. The middle and lower layers are bonded by plasma bonding using a plasma cleaner, which involves vacuuming for 150 seconds, introducing air for 50 seconds, and performing a 90-second hydrophilic surface treatment. The surface is cleaned before glass bonding.

4. The automated online extraction-mass spectrometry detection system based on an integrated microfluidic chip according to claim 1, characterized in that, The automated control unit includes a computer, a controller, and a solenoid valve, which are connected in sequence via a circuit. One end of the solenoid valve is connected to the controller, and the other end is connected to the automated integrated microfluidic chip unit via a plastic hose.

5. The automated online extraction-mass spectrometry detection system based on an integrated microfluidic chip according to claim 1, characterized in that, The pressure unit is one of a gas cylinder or a gas pump; it provides pressure to the automated integrated microfluidic chip unit, with a control layer pressure of 20 psi and a fluid layer pressure of 5 psi.

6. The automated online extraction-mass spectrometry detection system based on an integrated microfluidic chip according to claim 1, characterized in that, The online mass spectrometry detection unit includes a capillary with an outer diameter of 669 µm, a spray needle with an outer diameter of 360 µm, a liquid chromatography two-way PEEK variable diameter valve of 1 / 16' to 1 / 32', and a mass spectrometer. The automated integrated microfluidic chip unit is connected to the capillary, and the capillary is connected to the mass spectrometer nozzle via a variable diameter two-way valve.

7. The automated online extraction-mass spectrometry detection system based on an integrated microfluidic chip according to claim 6, characterized in that, The sample collection process involves using gas pressure to elute the sample from the solid-phase extraction column at the end of the nozzle into the capillary. The sample passes through the capillary and is then sprayed by applying electricity to the mass spectrometer nozzle, ultimately completing the online mass spectrometry detection.

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