WO2019024182A1 - Microfluidic chip and preparation method and test method therefor - Google Patents

Abstract

A microfluidic chip, and a preparation method and a detection method therefor. The microfluidic chip comprises a microfluidic chip substrate and a microfluidic chip cover covering the microfluidic chip substrate. The microfluidic chip substrate is provided with a reaction cell recessed downwards by a pre-determined depth. A liquid inlet hole and a liquid outlet hole separately communicating with the reaction cell are provided on the periphery of the microfluidic chip substrate. The microfluidic chip is made from a COC resin. The used COC resin has high THz transparency, visible light transparency, high biocompatibility, and is low cost. A surface of the cover is decorated with a GO/Au-NPs nanoparticle complex to capture a target protein, thereby achieving highly sensitive detection. The microfluidic chip substrate and the cover are processed separately and are sealed and packed using a double-sided adhesive or a hot melt adhesive. A square annular spacer can be added between the substrate and the cover to control a distance therebetween, ensuring the invention is affordable and reusable.

Description

Microfluidic chip, preparation method and detection method thereof Technical field

The present invention relates to the field of microfluidic chip technologies, and in particular, to a microfluidic chip, a preparation method thereof and a detection method.

Background technique

Simple, label-free and highly sensitive protein molecular detection methods have been the focus of research in recent years. Terahertz (THz) time-domain spectroscopy is a newly developed coherent detection technique. Studies have shown that many polar macromolecules have vibrational and rotational energy levels in the terahertz band. These molecules have strong absorption of terahertz radiation, so they can be studied by analyzing their characteristic spectra. The use of terahertz technology to study the structure of biological macromolecules, the reaction between molecules, and the interaction between molecules and the environment also have unique advantages, providing fingerprint features for determining the conformation, conformation and environmental effects of molecules.

Protein molecules need to have normal structure and function in the liquid phase environment. Since the terahertz wave can be strongly absorbed by polar substances such as water, the signal of the protein molecules in the liquid phase is greatly attenuated. Most of the existing research objects of terahertz technology are still limited to solid or gaseous state, which hinders the study of biomolecules that need to be active in a liquid environment. The existing protein molecular liquid reaction devices on the market are very expensive and less sensitive.

Therefore, the prior art has yet to be improved and developed.

Summary of the invention

In view of the above deficiencies of the prior art, the object of the present invention is to provide a microfluidic chip, a preparation method thereof and a detection method thereof, aiming at solving the existing loss of THz detection signal for protein molecules in a liquid phase environment, and sensitivity. Insufficient, the existing liquid reaction device is very expensive.

The technical solution of the present invention is as follows:

A microfluidic chip, comprising a microfluidic chip substrate and a microfluidic chip cover sheet covering the microfluidic chip substrate;

The microfluidic chip substrate is provided with a reaction cell recessed downward by a predetermined depth;

The microfluidic chip substrate is provided with a liquid inlet hole and a liquid outlet hole respectively communicating with the reaction pool;

The microfluidic chip is made of COC resin.

The microfluidic chip, wherein the liquid inlet holes are two and are disposed at one end of the microfluidic chip substrate, the liquid outlet hole is one, and the microfluidic chip substrate is disposed on the substrate. One end; the microfluidic chip substrate is further provided with a spare hole around the substrate, and the spare hole is one and disposed between the two liquid inlet holes.

The microfluidic chip, wherein the COC resin is a TOPAS cyclic olefin copolymer; the microfluidic chip has a length of 25-30 mm, a width of 25-30 mm, and a height of 1-3 mm.

The microfluidic chip, wherein the reaction cell is a columnar reaction cell formed by recessing from the middle of the microfluidic chip substrate, and the depth of the reaction cell is downwardly recessed by 0.2-0.25 mm, and the columnar shape The radius of the reaction cell is 8.0-9.0 mm.

The microfluidic chip, wherein a spacer is disposed between the microfluidic chip substrate and the microfluidic chip cover sheet, and the spacer is used for controlling the microfluidic chip substrate and the micro The distance between the flow control chip covers; the microfluidic chip substrate is square, and the spacer is a square annular spacer.

The microfluidic chip, wherein the surface of the microfluidic chip cover sheet is modified with a composite, the composite is a GO/Au-NPs nanoparticle composite, and the GO is an abbreviation for graphene oxide.

A method for preparing a microfluidic chip according to any of the above, comprising:

Step A: preparing a microfluidic chip substrate: forming a reaction cell that is recessed downward by a predetermined depth on the microfluidic chip substrate, and forming a liquid inlet hole and a liquid communicating with the reaction cell around the microfluidic chip substrate Liquid hole

Step B, preparing a microfluidic chip cover sheet;

Step C: covering the microfluidic chip cover sheet on the microfluidic chip substrate.

The method for preparing a microfluidic chip, wherein in the step A, the liquid inlet hole and the liquid outlet hole are formed by a micro injection molding processing technique;

After the step C, the method further comprises: sealing the gap around the microfluidic chip with double-sided tape or hot melt adhesive.

A method for detecting a liquid based on a microfluidic chip according to any of the above, wherein the method comprises the steps of: injecting a liquid into a liquid inlet hole of a microfluidic chip substrate, the liquid entering through the liquid inlet hole a microfluidic chip substrate in a reaction cell;

The reaction cell is placed on a sample holder of a transmissive or reflective terahertz time domain spectrometer and the liquid is then tested.

The microfluidic chip detects a liquid, wherein a liquid is injected into a liquid inlet of a microfluidic chip substrate by using a syringe pump.

Advantageous Effects: Compared with the prior art, the microfluidic chip made of COC resin has the advantages of high THz, visible light transparency, strong biocompatibility and low cost; microfluidic control made of COC resin The surface of the chip cover sheet is modified to achieve protein molecule capture and high sensitivity detection, and overcome the disadvantages of low loss of THz detection signal of liquid protein molecules and expensive detection devices on the market. In addition, the microfluidic chip substrate and the microfluidic chip cover are separated in two parts, which has the advantages of low cost and reusability.

DRAWINGS

1 is a schematic view showing the structure of the structure A in the first embodiment.

2 is a plan view of the structure A in the first embodiment.

Fig. 3 is a side view showing the structure A in the first embodiment.

4 is another side view of the structure A in the first embodiment.

Figure 5 is a graph showing the transmittance of the COC substrate in Example 2 in the range of 0.1 - 2.5 THz.

Fig. 6 is a graph showing the ultraviolet absorption change of GO/Au-NPs complex formed by GO and different addition amounts of HAuCl ₄ in Example 2.

Fig. 7 is a graph showing the relationship between the absorption peak intensity of the GO/Au-NPs complex and the amount of HAuCl ₄ added in Fig. 5.

Fig. 8 is a graph showing the ultraviolet absorption change of the GO and GO/Au-NPs complex in Example 2.

Figures 9a-9c are TEM images of the GO/Au-NPs complex of Example 2.

Figure 10 is an EDX spectrum of the GO/Au-NPs complex of Example 2.

Figure 11 is a graph showing the dynamic light scattering particle size distribution in Example 2.

Detailed ways

The present invention provides a microfluidic chip, a preparation method thereof and a detection method. In order to make the object, technical solution and effect of the present invention more clear and clear, the present invention will be further described in detail below. It is understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

A microfluidic chip of the present invention, comprising a microfluidic chip substrate and covering the microfluidic chip base On-chip microfluidic chip cover. Preferably, the microfluidic chip substrate and the microfluidic chip cover sheet are completely identical in size, and the microfluidic chip cover sheet covers the microfluidic chip substrate to implement packaging. More preferably, after the microfluidic chip cover sheet covers the microfluidic chip substrate, the invention also uses double-sided tape or hot melt adhesive to seal the gap around the microfluidic chip (except the liquid inlet hole) Or outside the liquid outlet) to further improve the sealing effect. In addition, the microfluidic chip substrate and the microfluidic chip cover are separated in two parts, which has the advantages of low cost and reusability.

The microfluidic chip substrate of the present invention is provided with a reaction cell recessed downward by a predetermined depth. Preferably, the reaction cell is a columnar reaction cell formed by recessing from the middle of the microfluidic chip substrate, and the reaction cell is recessed downward to a depth of 0.2-0.25 mm, such as 0.22 mm. Preferably, the columnar reaction cell has a radius of 8.0-9.0 mm, such as 8.5 mm.

The microfluidic chip substrate of the present invention is provided with a liquid inlet hole and a liquid outlet hole respectively communicating with the reaction cell, and the liquid inlet hole is connected with an external syringe pump, and a syringe pump (such as a common manual syringe) is used. The liquid to be tested is injected into the liquid inlet hole, and the liquid passes through the liquid inlet hole to enter the reaction cell for reaction. The present invention can vary the flow rate of the syringe pump to control the progress of the reaction. Preferably, the liquid inlet holes are respectively disposed at one end of the reaction cell, and the liquid outlet holes are disposed at the other end of the reaction cell; the microfluidic chip substrate is further provided with a spare hole around the substrate. The spare holes are one disposed between the two inlet holes. The liquid inlet hole can serve as a vent hole.

A spacer is disposed between the microfluidic chip substrate and the microfluidic chip cover sheet of the present invention, and the spacer is used for controlling between the microfluidic chip substrate and the microfluidic chip cover sheet. distance. The distance between the microfluidic chip substrate and the microfluidic chip cover sheet is controlled by changing the thickness of the spacer. Preferably, the microfluidic chip substrate is square, and the spacer is a square annular spacer. The gasket of the present invention has low cost and can be reused.

The main improvement of the present invention compared to the prior art is that the microfluidic chip is made of COC resin. This is because the COC resin has excellent characteristics of high THz, visible light transparency, high biocompatibility, and low cost. The invention discloses a microfluidic chip made of COC resin, and performs surface modification on the cover sheet, which can realize protein molecule capture and high sensitive detection, and effectively overcomes the loss of THz detection signal of protein molecules in a liquid phase environment, and is on the market. The detection device is expensive and the like. Preferably, the COC resin may be, but not limited to, a TOPAS (COC) cyclic olefin copolymer, for example, may be made of a completely transparent COC5013-L10 material.

Preferably, the microfluidic chip of the present invention has a length of 25-30 mm, a width of 25-30 mm, and a height of 1-3 mm. For example, the microfluidic chip has a length of 26 mm, a width of 26 mm, and a height of 2 mm.

Compared with the prior art, another major improvement of the present invention is that the surface of the microfluidic chip cover sheet is modified with a composite, and the composite is preferably a GO/Au-NPs nanoparticle composite (GO is Graphene oxide). The invention adopts the GO/Au-NPs nanoparticle composite to surface-modify the microfluidic chip cover sheet, and can achieve high sensitive detection of the target protein.

The preparation process of the surface modification of the microfluidic chip cover by GO/Au-NPs nanoparticle composite is as follows: GO and chloroauric acid are continuously stirred at room temperature for about 10 min, then sodium borohydride is added, and the reaction is carried out under continuous mechanical stirring. The gold nanoparticle composite GO/Au-NPs nanoparticle composite was prepared at 15 min.

The prepared microfluidic chip cover sheet was immersed in the prepared GO/Au-NPs nanoparticle composite solution, and allowed to stand at room temperature for 3.5-4.5 h (4 h) to obtain a surface modified GO/Au-NPs nanometer. Microfluidic chip cover sheet for particle composites.

The present invention also provides a method for preparing a microfluidic chip according to any of the above, which comprises:

Step A: preparing a microfluidic chip substrate: forming a reaction cell recessed to a certain depth on the microfluidic chip substrate, and forming a liquid inlet and a liquid outlet communicating with the reaction cell around the microfluidic chip substrate hole. Preferably, the inlet and outlet holes are formed by micro-injection processing techniques.

Step B, preparing a microfluidic chip cover sheet;

Step C: covering the microfluidic chip cover sheet on the microfluidic chip substrate.

In the processing of the present invention, the microfluidic chip substrate and the microfluidic chip cover sheet are separately fabricated. A gasket (such as a square annular gasket) may be disposed between the microfluidic chip substrate and the microfluidic chip cover sheet, and the microfluidic chip cover sheet covers the microfluidic chip On the substrate, the package is realized, and then the gap around the microfluidic chip is sealed by a double-sided tape or a hot melt adhesive (except for the liquid inlet hole or the liquid outlet hole) to further improve the packaging effect.

The present invention also provides a method for detecting a liquid based on a microfluidic chip according to any of the above, wherein the method comprises the steps of:

A liquid is injected into the inlet opening of the microfluidic chip substrate through which the liquid enters the reaction cell of the microfluidic chip substrate. A liquid is injected into the inlet opening of the microfluidic chip substrate using a syringe pump (such as a conventional manual syringe).

Place the reaction cell on a sample holder of a transmissive or reflective terahertz time domain spectrometer and then apply the liquid Detection.

The invention will now be described in detail by way of specific examples.

Example 1

As shown in FIG. 1 to FIG. 4, the main structure of the microfluidic chip of the present embodiment (ie, the microfluidic chip substrate, denoted as A) is as shown in FIG. 1 , wherein the microfluidic chip cover sheet structure is not shown. Out, consists of fully transparent A and B, and a square ring gasket (not shown) between AB. The injection pump of the embodiment adopts a common manual syringe, and the structure is made of a completely transparent COC5013-L10 material. The overall size of the A functional structure is 26 mm long, 26 mm wide, 2 mm high, and a cylindrical reaction tank S2 recessed to a certain depth. The upper surface non-recessed portions S1, S2 have a recessed depth of 0.22 mm and an S2 radius of 8.5 mm. The A structure has a first liquid inlet hole 1, a second liquid inlet hole 2, a third liquid inlet hole 3 and a liquid outlet hole 4. The first liquid inlet hole 1 and the third liquid inlet hole 3 are connected to an external syringe pump, The secondary liquid inlet 2 serves as a spare hole or a vent hole.

The A structure and the B structure are packaged together, where a square annular gasket is placed between the ABs, and the gap around the AB is sealed by hot melt glue (except for the 1, 3, and 4 flow paths), and the B structure is decorated with GO/Au. - NPs nanoparticle composite.

A disposable intravenous infusion needle (or 0.9 mm tetrafluorocapillary capillary) having an outer diameter of 0.9 mm (20 G) is inserted into the first inlet hole 1 and the third inlet port 3, so that the microfluidic chip is connected to the external syringe. The liquid hole 4 is not connected to the syringe for liquid discharge, and the gap is sealed with hot melt adhesive.

The syringes connecting the first inlet hole 1 and the third inlet port 3 are slowly injected with the proteins a and b, respectively, and the proteins a and b are introduced into the S2 for real-time reaction, and the solution flows out from the outlet 4. The THz light is vertically irradiated to the solution in S2, and the solution has a path length of about 0.22 mm for real-time detection.

After the test is completed, A and B are separated, and the functional structure A is retained, and reused after washing.

Example 2

1. Preparation and detection of COC substrate (microfluidic chip cover)

1. Processing of COC substrate

The COC substrate is formed by thermal injection molding.

2. THz transmittance detection of COC substrate

Figure 5 shows the transmittance spectrum of a 2 mm thick COC substrate in the range of 0.1 to 2.5 THz, and the average transmittance is about 90%, which indicates that the COC substrate has high transmission performance for THz light.

2. Preparation and characterization of GO/Au-NPs complexes

1, reagent

1), gold (III) chloride trihydrate, 49.0% gold (Belling); 2), single-layer graphene oxide, 1-20um, pipe company; 3), sodium borohydride (Mclin); 4), (3-mercaptopropyl)trimethoxysilane (Belling); 5), hydrogen peroxide solution (Aladdin); 6), sulfuric acid AR (Shanghai test), 95.0-98.0% national medicine; 7), anhydrous ethanol AR (Shanghai test), ≥99.7% national medicine; 8), deionized water, Merck Millipore Direct-Q3 laboratory reverse osmosis pure water system.

2, equipment

1), VWR agitator; 2), Xinzhi ultrasonic cleaning machine; 3), transmission electron microscope JEM-1230NIPPON TEKNO company; 4), laser micro-confocal Raman spectrometer; 5), nano-particle size detector; 6) , UV-visible spectrophotometer, Agilent Cary 60.

3. Preparation process

1), the choice of reaction and dosage

GO (0.5 mg/mL, 1 mL) and 4 different volume amounts of chloroauric acid (0.1/0.2/0.5/1 ml, 10 mM) were continuously stirred at room temperature for 10 min, then sodium borohydride (3 ml, 10 mM) was added. The reaction was carried out under continuous mechanical stirring for 15 min to form a gold nanoparticle composite composite. During the preparation, it was found that when the volume of chloroauric acid (10 mM) exceeded 0.5 mL, precipitation occurred, so 10 mM 0.5 mL of chloroauric acid was selected for preparation.

2), the characterization of the complex

Table 1, reaction ratio relationship

6 and FIG. 7 are further descriptions of the relationship of the reaction ratios in Table 1, and FIG. 6 is a UV absorption curve of GO/Au-NPs complex formed by GO and different addition amounts of HAuCl ₄ , and FIG. 7 illustrates that at 514 nm. The intensity of the absorption peak appears linearly with the HAuCl ₄ content at the reaction.

As shown in Fig. 8, the ultraviolet absorption chart shows that the wavelength is about 240 nm, which is a characteristic peak of GO, and about 520 nm is a characteristic peak of GO and gold nanoparticles, which indicates that gold nanoparticles and GO are compounded.

9a, 9b, and 9c are TEM images of the composite. As can be seen from the figure, the average particle diameter of the gold nanoparticles is less than 10 nm.

Figure 10 is an EDX elemental analysis of the composite, showing the relationship between the Au and C components in the GO/Au-NPs complex.

Table 2. EDX elemental analysis: the relationship between the composition of Au and C in GO/Au-NPs

element	weight%	atom%
C	59.45	73.40
O	27.65	25.63
Au	12.9	0.97
Total	100.00

Fig. 11 is a graph of DLS (dynamic light scattering particle size distribution), and it can be seen from the figure that the diameter is about 102 nm, and the uniformity is also good (the sharper the peak indicates the more uniform the particles).

3. The GO/Au-NPs complex is transferred to the COC substrate.

1. Processing of COC substrate:

The COC substrate was sealed with a tape on one side, and the COC substrate was repeatedly ultrasonically washed 3 times in absolute ethanol and deionized water, soaked for 15 minutes each time to remove surface grease and the like, and air-dried on the absorbent paper.

2. Transfer process:

1) Preparing a mixed solution of concentrated sulfuric acid and hydrogen peroxide (v1: v2 = 3:1, v1 is the volume of concentrated sulfuric acid, and v2 is the volume of hydrogen peroxide), and the prepared mixed solution is evenly dropped on the surface of the COC substrate, and placed on 80 ° C oven, 30 min; 2) take out the COC substrate, rinse the surface of the COC substrate with deionized water, and dry it on absorbent paper; 3) uniformly drop (3-mercaptopropyl)trimethoxysilane On the surface of the COC substrate, let stand at room temperature for 2 h; 4) take out the COC substrate, rinse the surface of the COC substrate with absolute ethanol and deionized water, and dry it on the absorbent paper; 5) Soak the COC substrate in the configuration. The GO/Au-NPs complex solution was allowed to stand at room temperature for 4 h to complete the preparation of the GO/Au-NPs composite/COC substrate. It was found that the surface of the COC substrate showed lavender, which confirmed the presence of gold nanoparticles.

In summary, the present invention provides a microfluidic chip, a preparation method thereof and a detection method. The invention is made of a microfluidic chip made of COC resin, and the COC resin used has high THz transmittance, visible light transparency, and strong biocompatibility. And the excellent features of low cost, overcome the shortcomings of the THz detection signal loss in the liquid phase environment, and the expensive detection device on the market. In addition, the present invention modifies the GO/Au-NPs nanoparticle composite on the surface of the cover sheet to realize the capture of the protein molecule, and overcomes the THz detection signal of the protein molecule in the liquid phase environment. Weak and ultimately achieve highly sensitive detection. In addition, in the processing, the microfluidic chip substrate and the microfluidic chip cover are separated by two-sided adhesive or hot melt adhesive, and can be packaged in a microfluidic chip substrate and a microfluidic chip cover. The addition of a thin square ring gasket to control the distance between the two parts has the advantage of low cost and reusability. The present invention can also change the flow rate by a syringe pump to control the reaction process.

It is to be understood that the application of the present invention is not limited to the above-described examples, and those skilled in the art can make modifications and changes in accordance with the above description, all of which are within the scope of the appended claims.

Claims (10)

1. A microfluidic chip, comprising: a microfluidic chip substrate and a microfluidic chip cover sheet covering the microfluidic chip substrate;

   The microfluidic chip substrate is provided with a reaction cell recessed downward by a predetermined depth;

   The microfluidic chip substrate is provided with a liquid inlet hole and a liquid outlet hole respectively communicating with the reaction pool;

   The microfluidic chip is made of COC resin.
2. The microfluidic chip according to claim 1, wherein the liquid inlet holes are two and are disposed at one end of the microfluidic chip substrate, and the liquid outlet holes are one and are disposed in the The other end of the microfluidic chip substrate; the microfluidic chip substrate is further provided with a spare hole around the substrate, and the spare hole is one and disposed between the two liquid inlet holes.
3. The microfluidic chip according to claim 1, wherein the COC resin is a TOPAS cyclic olefin copolymer; the microfluidic chip has a length of 25-30 mm, a width of 25-30 mm, and a height of 1- 3mm.
4. The microfluidic chip according to claim 1, wherein the reaction cell is a columnar reaction cell formed by recessing from the middle of the microfluidic chip substrate, and the depth of the reaction cell is downwardly depressed to 0.2- 0.25 mm, the radius of the columnar reaction cell is 8.0-9.0 mm.
5. The microfluidic chip according to claim 1, wherein a spacer is disposed between the microfluidic chip substrate and the microfluidic chip cover sheet, and the spacer is used to control the microfluid The distance between the control chip substrate and the microfluidic chip cover sheet; the microfluidic chip substrate is square, and the spacer is a square annular spacer.
6. The microfluidic chip according to claim 1, wherein the surface of the microfluidic chip cover sheet is modified with a composite, and the composite is a GO/Au-NPs nanoparticle composite.
7. A method for preparing a microfluidic chip according to any one of claims 1 to 6, characterized in that it comprises:

   Step A: preparing a microfluidic chip substrate: forming a reaction cell that is recessed downward by a predetermined depth on the microfluidic chip substrate, and forming a liquid inlet hole and a liquid communicating with the reaction cell around the microfluidic chip substrate Liquid hole

   Step B, preparing a microfluidic chip cover sheet;

   Step C: covering the microfluidic chip cover sheet on the microfluidic chip substrate.
8. The method for preparing a microfluidic chip according to claim 7, wherein in the step A, the liquid inlet hole and the liquid outlet hole are formed by a micro injection molding processing technique;

   After the step C, the method further comprises: sealing the gap around the microfluidic chip with double-sided tape or hot melt adhesive.
9. A method for detecting a liquid based on the microfluidic chip according to any one of claims 1 to 6, wherein Including steps:

   Injecting a liquid into the liquid inlet hole of the microfluidic chip substrate, and the liquid enters the reaction cell of the microfluidic chip substrate through the liquid inlet hole;

   The reaction cell is placed on a sample holder of a transmissive or reflective terahertz time domain spectrometer and the liquid is then tested.
10. A method of detecting a liquid by a microfluidic chip according to claim 9, wherein a liquid is injected into the liquid inlet hole of the microfluidic chip substrate by means of a syringe pump.

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