3D Printing Multifunctional Integrated Microfluidic Chip and Its Preparation Method and Application

Abstract

The present invention discloses a 3D printing multi-functional integrated microfluidic chip and its preparation method and application. A novel microfluidic chip integrating a standardized interface, a cell lysis unit, a monolithic column microextraction unit, and an ICP-MS sample introduction system (direct injection high-efficiency nebulizer) is prepared by 3D printing technology. By directly preparing a three-dimensional mixed lysis unit and a topological structure monolithic column to promote mass transfer, while improving extraction kinetics, the standardized interface and the direct injection high-efficiency nebulizer can effectively reduce the interface dead volume, match the micro-volume injection of the microfluidic chip and the macroscopic determination of ICP-MS, and improve the detection sensitivity. The chip has good preparation reproducibility, low cost, and is easy to expand and popularize. The method established based on this chip has the advantages of low cell consumption, high sensitivity, and low detection limit, and is suitable for the analysis of trace REEs in cell samples, providing strong technical support for the toxicity research of REEs.

Description

Technical Field

[0001] The present invention relates to the technical field of separation and analysis detection, and particularly relates to a 3D printing multi-functional integrated microfluidic chip, a preparation method thereof, and an application thereof. Background Art

[0002] Microfluidics generally refers to a systematic science and technology for processing or operating small-volume (10 -9 ~10 -18 L) liquid samples in channels with a size ranging from several micrometers to several hundred micrometers. It can integrate operation units such as sample preparation, reaction, separation, detection, cell culture, sorting, and lysis involved in fields such as chemistry and biology onto a single chip. A network is formed through microchannels, and a controllable fluid runs through the entire system, which has remarkable characteristics such as fast detection speed, small sample consumption, and high throughput. Pretreatment on the chip unit can effectively improve the efficiency of sample pretreatment. Chip sample pretreatment can efficiently reproduce most conventional operations, mainly including liquid-phase microextraction, membrane extraction, and solid-phase microextraction. As a conventional enrichment extraction method, solid-phase microextraction has a large sample pore volume and a large number of active sites, which can ensure the flow rate and extraction efficiency of samples and has been widely used in the analysis of cell samples. The multi-functional integrated microfluidic chip significantly reduces the cell consumption in ICP-MS analysis, improves the sample throughput and sensitivity, and reduces the pollution and error caused by manual operation. However, with the in-depth research, its shortcomings have gradually emerged. First, currently, the preparation of microfluidic chips usually adopts soft lithography and PDMS molding technology, which generally requires expensive equipment such as a clean laboratory and a lithography machine, as well as experienced operators. It takes a long time and is labor-intensive to adjust the chip structure. Second, the channels prepared by this technology are 2D or 2.5D structures. To construct a multi-functional integrated chip, it is usually necessary to build functional units through multi-layer bonding and alignment, and the preparation reproducibility is poor. In addition, chips with low dead volume, easy disassembly, and compatibility with commercial pipelines and interfaces need to be developed for the micro-macro interface of ICP-MS.

[0003] 3D printing, also known as additive manufacturing, prepares three-dimensional solid models in one step by layer-by-layer printing. It can customize three-dimensional structures according to user needs. Since the structure is created by adding materials and does not require etching or dissolution, the processing environment is friendly and cost-effective. 3D printing breaks the limitations of traditional manufacturing on the minimum feature size, complexity, and functional integration, and will open up a new era of materials science, manufacturing, and engineering applications. There have been a variety of 3D printing technologies developed, including stereolithography (SLA), selective laser sintering (SLS), inkjet and multi-jet printing (i3DP), fused deposition modeling (FDM), laminated object manufacturing (LOM), and direct ink writing (DIW). In contrast, SLA has unique advantages, especially in terms of structural complexity, material diversity, scale span, and functional integration. It can not only achieve the formation of micro-nano scale structures but also the integrated manufacturing of complex three-dimensional spatial structures. It is the most commonly used 3D printing technology for preparing microfluidic chips. The advantages of using 3D printing to prepare microfluidic chips are as follows: (1) It does not require a specific experimental environment such as a clean laboratory and is not restricted by the shape and design of the object; the design, printing, and modification can be completed within a few hours, which is more efficient, inexpensive, and easy to commercialize; (2) It can customize the structure according to user needs and modify the structure with software assistance without reprinting the mask. Compared with the 2.5D PDMS microfluidic device prepared based on soft lithography, 3D printing can conveniently print complex 3D structures (with controllable depth); (3) It can directly print standard commercial interfaces, which are convenient for assembly and easy to combine with detection systems to achieve real-time monitoring. Although the preparation of microfluidic devices by 3D printing is still in its infancy, its revolutionary printing method, unique advantages in preparing true three-dimensional structures, and rich material library for selection have shown great application potential in the field of chip solid-phase microextraction. On the one hand, due to the adsorption characteristics of the existing commercial printing raw materials for analytes, the unique advantage of 3D printing is that it can in-situ manufacture adsorbent materials in the form of particles or meshes inside the chip channels as needed, simultaneously with the printing of the channels. However, the direct use of 3D printing materials for microextraction is limited by the small surface area and insufficient functional groups in the 3D printable commercial materials, and the extraction performance needs to be improved. On the other hand, in order to endow 3D printing materials with more functionality, 3D printing technology is more frequently used to print microextraction channels, and the extraction performance of 3D printing solid-phase microextraction chips for target analytes is improved by integrating commercial materials in the carrier channels, filling functionalized nanomaterials, and in-situ initiating monolithic materials. However, filling functional materials in the channels fails to fully utilize the advantage of the pore structure customization of 3D printing. Surface modification of the customized pore structure is a win-win method for constructing customized microextraction chips. The modification methods mainly include adding functional materials to the raw materials before printing, surface modification after printing, and a combination of pre-printing and post-printing modification. Among them, the surface modification method after printing does not require optimization of the printing material formula and is more simple to operate.However, the current ester group modification based on the surface of photocurable resin involves reaction conditions such as strong bases, organic solvents, or heating, which may cause swelling and damage to the printed microdevices and damage the internal fine structures. Moreover, due to the confidentiality of the components of some commercial materials, the surface functional groups and their distributions are unknown. Therefore, there is a need to develop mild and general modification methods.

[0004] Rare earth elements (REEs) are located in Group IIIB of the periodic table. Their unique physical and chemical properties such as unique electron arrangement characteristics, large ionic radii, and strong metal activities make them irreplaceable in the development of new functional materials. They are widely used in fields such as aerospace, military, electronics, chemical engineering, energy, and biomedicine, becoming the "vitamins" of modern industry. In addition, REEs are also used as crop fertilizers and feed additives. The wide application of REEs increases the risk of human exposure. Research shows that after REEs enter cells, they can cause redox imbalance and chromosome aberration, etc., and their cytotoxicity is affected by the dose and type of REEs. Different REEs may even show completely opposite results. Therefore, developing methods for determining trace REEs in cells is of great significance for studying the cytotoxicity of REEs and developing health-friendly rare earth functional materials. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a 3D printing multi-functional integrated microfluidic chip, its preparation method, and application, which are used for analyzing trace rare earth elements in cells by combining a 3D printing multi-functional integrated microfluidic chip with inductively coupled plasma mass spectrometry.

[0006] To achieve the above purpose, the comprehensive technical solution of the present invention is as follows:

[0007] In the first aspect, the present invention provides a preparation method of a 3D printing multi-functional integrated microfluidic chip, which is characterized by including the following steps:

[0008] S1: The microfluidic system includes a chip interface, a cell lysis unit, and a chip with an integral column;

[0009] Among them, the chip inlet adopts a tubular structure matching a commercial hose and directly fits with the commercial hose; the lysis area is composed of several elliptical units. The ellipse is split from the middle to form a mixing unit; the liquid is split into two streams at the elliptical interface and converges after flowing through the semi-ellipse to accelerate mixing; for the microextraction unit, an integral column composed of several topological repeating units is prepared by simulating the topological structure of metal-organic framework UiO-66. The size of the integral column is flexibly designed to facilitate connection with the commercial hose and reduce the dead volume;

[0010] First, 2mg mL -1Dopamine hydrochloride was dissolved in 10 mM Tris-HCl, pH 8.5 solution and flowed through the monolithic column at a flow rate of 2 μL min -1 for 2 h. After thorough washing with ultrapure water, a 10 mmol L -1 NTA solution was prepared in 10 mM Tris-HCl, pH 8.5. 1 mL of it was taken and flowed through the monolithic column at a flow rate of 2 μL min -1 . After thorough washing with ultrapure water, the above process was repeated several times to obtain an NTA-modified 3D printed monolithic column;

[0011] S2: The direct injection high-efficiency nebulizer consists of a tip nozzle, a bowl-shaped interface directly connected to the torch tube, a triangular prism channel with an inscribed circle diameter of 1.6 mm, a gas inlet, and a PEEK interface. After printing, the capillary was inserted into the PEEK tube, sealed with epoxy resin glue, inserted into the triangular prism of the 3D printed nebulizer, a gasket was stuffed at the interface, and it was tightened with a commercial PEEK head. The three-way gas channels were connected with gas quick connectors and four-way adapters. The carrier gas was evenly divided into three paths and entered the triangular prism, and converged at the nozzle. High-pressure gas was formed at the tip nozzle of the nebulizer to atomize the liquid flowing out of the capillary into an aerosol, which entered the plasma with the carrier gas for ionization;

[0012] S3: The outlet of the 3D printed chip was connected to the six-port valve interface through a commercial hose, and the PEEK tube inlet of the 3D printed nebulizer was connected to the six-port valve interface to form a multifunctional integrated chip integrating standardized interfaces, cell lysis units, monolithic column microextraction units, and sample introduction systems.

[0013] In a second aspect, the present invention provides a 3D printed multifunctional integrated microfluidic chip obtained by the above preparation method.

[0014] In a third aspect, the present invention provides an application of the above 3D printed multifunctional integrated microfluidic chip for analyzing trace rare earth elements in cells, which is characterized in that: a novel microfluidic chip integrating standardized interfaces, cell lysis units, monolithic column microextraction units, and an ICP-MS sample introduction system, i.e., a direct injection high-efficiency nebulizer, was prepared by 3D printing technology. It was on-line coupled with ICP-MS to construct an analysis of trace REEs in a small number of cells when enriching and analyzing 15 rare earth elements in the sample.

[0015] In the specific application of the above technical solution:

[0016] The microfluidic system includes a chip interface, a cell lysis unit, and a chip with an integrated column. Among them, the chip inlet adopts a tubular structure with a length of 3 mm, an inner diameter of 1.5 mm, and an outer diameter of 4 mm, which is matched with a commercial hose (outer diameter 1.5 mm) and directly fits with the commercial hose; the inner diameter of the inlet channel is 150 μm; the lysis zone consists of 5 ellipses with an inner diameter of 150 μm, a major axis of 4 mm, and a minor axis of 1.5 mm. It is designed to split the ellipse in the middle to form a mixing unit, and the distance between two ellipses is 1 mm. The liquid splits into two streams at the ellipse interface and converges after flowing through the semi-ellipse, accelerating mixing. For the microextraction unit, a square integrated column with a length of 1.2 cm, an internal size of 0.7 mm × 0.7 mm × 12 mm, composed of topological repeating units with a rod diameter of 20 μm and a square pore diameter of 80 μm, was prepared by simulating the topological structure of metal-organic framework UiO-66. The outer diameter of the integrated column is 2 mm for easy connection with a commercial hose (2 mm i.d., 3 mm o.d.) to reduce the dead volume. The entire printing process takes 14 h, weighs 0.38 g, and costs 1.0 yuan. First, 2 mg mL -1 of dopamine hydrochloride is dissolved in 10 mM Tris-HCl (pH 8.5) solution and flows through the integrated column at a flow rate of 2 μL min -1 for 2 h. After thorough washing with ultrapure water, a 10 mmol L -1 NTA solution is prepared in 10 mM Tris-HCl (pH 8.5). Take 1 mL and flow it through the integrated column at a flow rate of 2 μL min -1 and wash it thoroughly with ultrapure water. Repeat the above process 4 times to obtain an NTA-modified 3D printed integrated column.

[0017] The direct injection high-efficiency nebulizer consists of a tip nozzle (380 μm i.d.), a bowl-shaped interface directly connected to the torch tube, a triangular prism channel with an inscribed circle diameter of 1.6 mm, a gas inlet, and a PEEK interface. The printing time is 1.6 h, the weight is 8.25 g, and the cost is 1.7 yuan. After printing, a capillary with a length of 3 mm (150 μm i.d., 365 μm o.d.) is inserted into a PEEK tube (0.38 mm i.d., 1.60 mm o.d.), sealed with epoxy resin glue, inserted into the triangular prism of the 3D printed nebulizer, a gasket (1.6 mm i.d., 3.0 mm o.d., thickness 1 mm) is inserted at the interface, and tightened with a commercial PEEK head. The three gas channels are connected by gas quick connectors and a four-way adapter. The carrier gas is evenly divided into three paths and enters the triangular prism, and converges at the nozzle. High-pressure gas is formed at the tip nozzle of the nebulizer to atomize the liquid flowing out of the capillary into an aerosol, which enters the plasma with the carrier gas for ionization.

[0018] Connect the 3D-printed chip outlet to the six-port valve interface through a commercialized hose (2mm i.d., 3mm o.d.), and connect the PEEK tube inlet of the 3D-printed nebulizer to the six-port valve interface to form a multifunctional integrated chip integrating a standardized interface, a cell lysis unit, a monolithic column microextraction unit, and a sample introduction system.

[0019] The technical principle of the present invention is as follows:

[0020] The present invention uses microstereolithography 3D printing technology to prepare a new type of microfluidic chip integrating a standardized interface, a cell lysis unit, a monolithic column microextraction unit, and an ICP-MS sample introduction system (direct injection high-efficiency nebulizer). It is online coupled with ICP-MS to construct a new method for analyzing trace REEs in a small number of cells. The multifunctional integrated microfluidic chip prepared by the emerging 3D printing technology gives full play to the advantages of 3D printing technology in user-customized three-dimensional structures. On the one hand, by directly preparing complex three-dimensional mixed lysis units and monolithic columns with UIO-66 topological structures to promote mass transfer and improve extraction kinetics; on the other hand, by designing standardized interfaces and direct injection high-efficiency nebulizers to reduce interface dead volume, match the micro-volume injection of the microfluidic chip and the macroscopic determination of ICP-MS, and improve detection sensitivity. The prepared direct injection nebulizer has higher sensitivity and the lowest cost than three commercial microfluidic nebulizers. The chip has the advantages of good preparation reproducibility, low cost, easy expansion, and commercial promotion. The method established based on this 3D-printed integrated chip has the advantages of low cell consumption (1200 cells), high sensitivity, and low detection limit, and is suitable for the analysis of trace REEs in cell samples, providing strong technical support for the toxicity research of REEs.

[0021] The beneficial effects and remarkable advantages of the present invention are as follows:

[0022] 1. Compared with traditional soft lithography technology, 3D printing technology can customize the 3D structure of the chip, directly prepare the corresponding functional units in one step, without multiple bonding steps, and the preparation is simple and has good reproducibility.

[0023] 2. The monolithic column prepared based on the 3D topological structure and the mild functionalization reaction mode mediated by polydopamine can effectively improve the liquid mixing efficiency and adsorption efficiency, and has the advantage that the pore structure can be designed as required.

[0024] 3. Directly introducing the chip outlet into the ICP-MS direct injection nebulizer can effectively achieve micro-macro connection, improve the transmission efficiency, and reduce cell consumption. Description of the Drawings

[0025] Figure 1 It is a schematic diagram of the 3D-printed integrated microfluidic system combined with ICP-MS detection of the present invention;

[0026] Figure 2 These are the cross-sections of the monolithic column before and after modification of the present invention and the morphology of its topological structure units: Scanning electron microscope images of the cross-section of the monolithic column based on the 3D-printed chip; where: a and b are before modification; c and d are after modification; e is the topological structure unit of UIO-66.

[0027] Figure 3 This is a schematic diagram of the 3D-printed direct injection high-efficiency atomizer model of the present invention.

[0028] Figure 4 These are the aerosol particle size distributions of the direct injection atomizer of the present invention: (A) Relationship between normalized count percentage and droplet diameter; (B) Relationship between cumulative count percentage and droplet diameter.

[0029] Figure 5 These are the effects of the culture concentration of Ce 3+ in the present invention on cell viability. Detailed implementation manners

[0030] The technical solutions of the present invention will be further elaborated in detail below in conjunction with the embodiments and the drawings.

[0031] A preparation method of a 3D-printed multifunctional integrated microfluidic chip is as follows:

[0032] S1: The microfluidic system includes a chip interface, a cell lysis unit, and a chip with a monolithic column; among them, the chip inlet adopts a tubular structure with a length of 3 mm, an inner diameter of 1.5 mm, and an outer diameter of 4 mm that matches a commercial hose (outer diameter 1.5 mm), and directly fits with the commercial hose; the inner diameter of the inlet channel is 150 μm; the lysis area consists of 5 ellipses with an inner diameter of 150 μm, a major axis of 4 mm, and a minor axis of 1.5 mm. It is designed to split the ellipse in the middle to form a mixing unit, and the distance between two ellipses is 1 mm. The liquid splits into two streams at the ellipse interface and converges after flowing through the semi-ellipse to accelerate mixing. For the microextraction unit, a monolithic column with a length of 1.2 cm and an internal square cross-section of 0.7 mm × 0.7 mm × 12 mm, composed of topological repeating units with a rod diameter of 20 μm and a square pore diameter of 80 μm, is prepared by simulating the topological structure of the metal-organic framework UiO-66. The outer diameter of the monolithic column is 2 mm for easy connection with a commercial hose (2 mm i.d., 3 mm o.d.) to reduce dead volume. The entire printing process takes 14 h, weighs 0.38 g, and costs 1.0 yuan. First, dissolve 2 mg mL -1 dopamine hydrochloride in 10 mM Tris-HCl (pH 8.5) solution, and flow it through the monolithic column at a flow rate of 2 μL min -1 for 2 h. After thorough washing with ultrapure water, prepare 10 mmol L in 10 mM Tris-HCl (pH 8.5)-1 NTA solution, take 1 mL and flow it through the monolithic column at a flow rate of 2 μL min -1 , and wash it thoroughly with ultrapure water. Repeat the above process 4 times to obtain the NTA-modified 3D-printed monolithic column.

[0033] S2: The direct injection high-efficiency atomizer consists of a tip nozzle (380 μm i.d.), a bowl-shaped interface directly connected to the torch tube, a triangular prism channel with an inscribed circle diameter of 1.6 mm, a gas inlet, and a PEEK interface. The printing time is 1.6 h, the weight is 8.25 g, and the cost is 1.7 yuan. After printing, insert a capillary tube (150 μm i.d., 365 μm o.d.) with a length of 3 mm into the PEEK tube (0.38 mm i.d., 1.60 mm o.d.), seal it with epoxy resin glue, insert it into the triangular prism of the 3D-printed atomizer, insert a gasket (1.6 mm i.d., 3.0 mm o.d., thickness 1 mm) at the interface, and tighten it with a commercial PEEK head. The three gas channels are connected by gas quick connectors and a four-way adapter. The carrier gas is evenly divided into three paths and enters the triangular prism, and converges at the nozzle to form high-pressure gas at the tip nozzle of the atomizer, atomize the liquid flowing out of the capillary tube into an aerosol, and enter the plasma with the carrier gas for ionization.

[0034] S3: Connect the 3D-printed chip outlet to the six-way valve interface 6 through a commercial hose (2 mm i.d., 3 mm o.d.), and connect the PEEK tube inlet of the 3D-printed atomizer to the six-way valve interface 3 to form a multifunctional integrated chip integrating standardized interfaces, a cell lysis unit, a monolithic column microextraction unit, and a sample introduction system.

[0035] Example 1

[0036] A schematic diagram of a 3D-printed multifunctional integrated microfluidic chip system, as Figure 1 shown. When the six-way valve is in the Inject state, the cell suspension and the cell lysis solution (0.1% SDS) enter the cell lysis unit from inlets A1 and A3 at a flow rate of 15 μL min -1 respectively. After the cells are lysed and the contents are released, they enter the monolithic column microextraction area. After microextraction by the monolithic column, the waste liquid flows out from the original sample injection port of the six-way valve; then turn the six-way valve to the Load state, and inject 50 μL of 0.4 mol L -1 HNO3 solution into the chip from chip inlet A2 at a flow rate of 50 μLmin -1 for the monolithic column desorption process. The desorbed solution enters the quantitative loop of the six-way valve; then turn it to the Inject state for ICP-MS determination. During the determination, inject the cleaning solution (100 μL ammonium acetate buffer solution) at a flow rate of 100 μLmin -1The flow rate of 1.5 mol / L flows through the monolithic column from the A3 inlet to complete the regeneration of the monolithic column. At this point, the analysis of the cell sample is completed, and the sample throughput is 3 h. -1 .

[0037] A photocuring printer with a resolution of 2 μm was selected. In order to increase the interaction between the solution and the overall column skeleton, a topological repeating unit with a rod diameter of 20 μm and a square aperture of 80 μm was designed to imitate the topological repeating unit structure of UiO-66 with its minimum printable molding size. This topological structure helps to promote the formation of turbulence in the flowing sample solution, increase the contact opportunity between the overall column skeleton and the sample solution, promote extraction kinetics, and improve the extraction rate. Here, the interior of the overall column is a cuboid with a length of 0.75 mm, a width of 0.75 mm, and a height of 12 mm, composed of 2000 topological repeating units (0.15 mm, a width of 0.15 mm, and a height of 0.15 mm), such as Figure 2 shown.

[0038] Considering that the material of the 3D printed integral column is methacrylate material and the specific composition is not disclosed due to commercial secrets, a polydopamine coating-mediated modification method was chosen that has no requirements on the surface properties of the modified material. The surface of the 3D printed integral column skeleton was functionalized with aminocarboxylic acid ligands, which combined with rare earth ions through chelation to adsorb the target rare earth ions.

[0039] Next, the effect of the number of repetitions of the modification process on the adsorption behavior of the monolithic column was investigated. As the number of modifications increased, the adsorption rate gradually increased. After three modifications, the target rare earth ions could be quantitatively adsorbed. After four modifications, the adsorption rate was greater than 95%. The number of modifications was selected as four to prepare the NTA-modified 3D printed monolithic column.

[0040] Example 2

[0041] A 3D printed direct injection high-efficiency atomizer model, such as Figure 3 As shown, considering that the distance from the rectangular tube interface to the outside of the instrument is 10 cm, directly using 3D printing to prepare an atomizer with an inner diameter of micrometers is likely to cause blockage of the channel. On the other hand, in order to prevent the internal sampling tube from shaking under the action of the carrier gas, resulting in unstable signals, a triangular prism-shaped channel with the outer diameter of the internal sampling tube as the inscribed circle is designed to fix the pipeline inserted inside, and a gas interface is designed on the face of the triangular prism to divide the carrier gas into three paths to enter from the gap of the triangular prism, and form high pressure at the tip nozzle to atomize the liquid flowing out of the internal sampling tube into an aerosol and enter the plasma along the rectangular tube for further ionization.

[0042] In the experiment, it was first explored to directly use a PEEK tube with an outer diameter of 1.6 mm and an inner diameter of 50 μm as the internal sampling tube, and the inner diameter of the gas nozzle was 1.62 mm. However, there was almost no signal (less than 1000 CPS) when the tuning solution introduced into the ICP-MS through this nebulizer. It was speculated that it might be because the wall thickness of the PEEK tube was too thick, forming a barrier for gas shearing, resulting in the solution flowing out of the PEEK tube not being fully sheared, forming larger aerosols that could not be transferred to the ICP for further ionization. Therefore, on this basis, the inner diameters of the nozzle and the internal sampling tube were further reduced to form smaller aerosols, improving the transmission efficiency and detection sensitivity. A commercial capillary (150 μm i.d., 360 μm o.d.) was inserted into a PEEK tube (365 μm i.d., 1.6 mm o.d.) as the sampling tube, and at the same time, the inner diameter of the nozzle was reduced to 380 μm.

[0043] Example 3 Aerosol Particle Size Distribution

[0044] In an argon plasma, it is generally considered that aerosols with a diameter less than 8 μm have a favorable contribution to the signal, and droplets larger than 8 μm cannot be completely evaporated in the measurement area, not only making no contribution to the signal intensity but also causing greater plasma perturbation and noise. The aerosol particle size distribution at a distance of 15 mm from the nozzle plane of the nebulizer was measured using a spray laser particle size analyzer, and the results are as Figure 4 shown. The Sauter mean diameter D 3,2 (defined as the volume-surface area ratio) is 5.4 μm, and the percentage of aerosol count with a particle size less than 8 μm is 82.0%.

[0045] The atomization efficiency (solvent transfer efficiency) and transfer efficiency (solute transfer efficiency) of the nebulizer are one of the key factors affecting the detection sensitivity of ICP-MS. For the determination of the atomization efficiency: The nebulizer was connected to a U-tube filled with silica gel (6 - 16 mesh) through a 3D-printed nebulizer-matched interface and a hard silicone tube, and a negative pressure was formed by a vacuum pump to absorb aerosols at the upper end of the nebulizer. High-purity water was atomized continuously at a flow rate of 6 μL min -1 for 3 h, and the atomization efficiency was calculated by the mass difference before and after the U-tube. The atomization efficiency of this nebulizer was measured to be 100%; The direct method was used to measure the transfer efficiency of this nebulizer: Similar to the device for atomization efficiency, the U-tube was replaced with a double-layer glass fiber membrane (diameter 47 mm, pore size 0.3 μm), and an aqueous solution containing Mn 2+ (100 mg L -1 ) was introduced into the nebulizer for atomization at a flow rate of 6 μL min -1 for 1 h. Then the membrane was taken out and placed in 20 mL of 1 mol L -1In nitric acid, heat for 2 h, after ultrasonic treatment for 10 min, take the supernatant and filter it through a filter membrane (pore size 0.22 μm), and then determine the Mn in the supernatant by ICP-MS 2+ The concentration. After calculation, the transmission efficiency of this nebulizer is 81.1%, which is in good agreement with the cumulative counting percentage (82.0%) of the aerosol generated by the nebulizer below 8 μm.

[0046] Under the respective optimal determination conditions of the 3D-printed nebulizer and the commercial Burgener HP microfluidic nebulizer (the applicable flow rate range is 5 - 1500 μL / min -1 ), and under the conditions of an injection flow rate of 6 μL / min -1 , a sampling interval of 100 ms, and an integration time of 100 s, the detection limits and precisions of the two nebulizers were investigated. Among them, the detection limit is defined as the concentration corresponding to 3 times the standard deviation of the blank value (n = 7), and the precision is obtained from the RSD of the signal of the sample solution with a target ion concentration of 10 μg / L -1 within 3 min. The results are listed in Table 1. It can be seen from the table that the 3D-printed detachable total consumption nebulizer has higher sensitivity, which is 4.7 - 6.0 times that of SC-175, the detection limit is equivalent to or even lower than that of SC-175, and it has a lower cost (￥90 vs ￥10,850).

[0047] Table 1 Comparison of the performance of the 3D-printed nebulizer and the commercial nebulizer

[0048]

[0049]

[0050] a : Slope of the working curve

[0051] Example 4

[0052] Examine the uptake of Ce 3+ by cells incubated for 24 h. First, the effect of Ce 3+ on cell viability was investigated by the MTT assay. As 3+ shown, when the incubation concentration of Ce Figure 5 is less than 10 mg / L 3+ , it has no obvious effect on cell viability. Therefore, a Ce -1 incubation concentration of 1 mg / L 3+ was selected, incubated for 24 h, and the uptake of Ce -1 by cells was examined 3+The uptake was investigated, and the cell consumption of this method was examined. The results are listed in Table 2. The results showed that this method still had good accuracy when consuming 500 cells, and the measured cell uptake was 1237 ± 127 fg cell -1 . The total amount of Ce 3+ taken up by cells measured by acid digestion was 1285 ± 20 fg cell -1 , which was in good agreement with the value measured by this method, indicating that in addition to being able to extract Ce 3+ , the prepared chip could also extract other Ce species in cells. This might be because the 4f electrons of lanthanide elements had little effect on coordination bonds, and Ln III complexes had almost ionic properties to a large extent, which was conducive to forming a flexible space-driven coordination geometry with hard carboxylic acid ligands. Coupled with the characteristic of the large ionic radius of Ln III , Ln III (especially light rare earth elements) could form high-coordination-number (8 - 12) complexes, resulting in other binding forms of Ce being able to coordinate with the aminocarboxylic functional groups on the chip monolithic column and thus be extracted.

[0053] Table 2. Analysis results of Ce3+ in MCF-7 cells with different cell numbers (mean ± s.d, n = 3)

[0054]

[0055] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A preparation method of a 3D printing multi-functional integrated microfluidic chip, characterized in that: Including the following steps: S1: The microfluidic system includes a chip interface, a cell lysis unit, and a chip with an integral column; Among them, the chip inlet adopts a tubular structure matching the commercial hose and directly fits with the commercial hose; the lysis zone consists of several elliptical units. The ellipse is split in the middle to form a mixing unit; the liquid is split into two streams at the elliptical interface, converges after flowing through the semi-ellipse, accelerating mixing; for the microextraction unit, an integral column composed of several topological repeating units is prepared by simulating the topological structure of metal-organic framework UiO-66. The size of the integral column is flexibly designed to facilitate connection with the commercial hose, reducing the dead volume; First, dissolve 2 mg / mL -1 dopamine hydrochloride in 10 mM Tris-HCl, pH 8.5 solution, and flow it through the monolithic column at a flow rate of 2 μL / min -1 for 2 h. After thorough washing with ultrapure water, prepare a 10 mmol / L -1 NTA solution in 10 mM Tris-HCl, pH 8.

5. Take 1 mL and flow it through the monolithic column at a flow rate of 2 μL / min -1 for a certain number of times to obtain the NTA-modified 3D printed monolithic column; S2: The direct injection high-efficiency nebulizer consists of a tip nozzle, a bowl-shaped interface directly connected to the torch tube, a triangular prism channel with an inscribed circle diameter of 1.6 mm, a gas inlet, and a PEEK interface; after printing, the capillary is inserted into the PEEK tube, sealed with epoxy resin glue, inserted into the triangular prism of the 3D printed nebulizer, a gasket is inserted at the interface, and tightened with a commercial PEEK head; the three gas channels are connected by gas quick connectors and a four-way adapter, dividing the carrier gas into three equal parts and entering the triangular prism, and converging at the nozzle to form high-pressure gas at the tip nozzle of the nebulizer, atomizing the liquid flowing out of the capillary into an aerosol, and entering the plasma with the carrier gas for ionization; S3: Connect the 3D printed chip outlet to the six-port valve interface through a commercial hose, and connect the PEEK tube inlet of the 3D printed nebulizer to the six-port valve interface to form a multifunctional integrated chip integrating a standardized interface, a cell lysis unit, an integral column microextraction unit, and a sample introduction system.

2. A 3D printed multifunctional integrated microfluidic chip obtained by the preparation method as described in claim 1.

3. Use of the 3D printing multi-functional integrated microfluidic chip as described in claim 2 for analyzing trace rare earth elements in cells, characterized in that: A novel microfluidic chip integrating a standardized interface, a cell lysis unit, an integral column microextraction unit, and an ICP-MS sample introduction system, namely a direct injection high-efficiency nebulizer, is prepared by 3D printing technology. It is online coupled with ICP-MS to construct an analysis of trace REEs in a small amount of cells when enriching and analyzing 15 rare earth elements in the sample.

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