CN105363505B - A kind of cell capture of three-dimensional structure and release chip and preparation method thereof - Google Patents

Abstract

The invention relates to a cell capture and release chip with a three-dimensional structure, comprising an upper cover and a lower substrate, a microflow channel is etched on the back of the upper cover, and inlets and outlets are respectively set at both ends of the microflow channel of the cover , there is a layer of chitosan film on the substrate, and the cover sheet and the substrate are bonded by an adhesive. This structure can capture and release cells/particles, and pass the liquid to be tested through the three-dimensional structure channel of the microfluidic chip. Through this The height characteristics of this three-dimensional structure are used to capture cells/particles of a specific size, and after the capture is completed, the height of the internal channel of the three-dimensional chip is increased by a specific method, and the cells/particles are finally released. The operation method of capturing and releasing cells is convenient, responds quickly, does not damage cells, and is suitable for capturing and releasing cells and particles in the micron scale. The preparation method of the invention has simple process, and the prepared three-dimensional structure chip has the characteristics of miniaturization, and can be widely used in the field of analysis.

Description

A three-dimensional cell capture and release chip and its preparation method

technical field

The invention relates to a preparation method of a microfluidic chip, in particular to a three-dimensional structure cell capture and release chip, and a method for capturing and releasing cells by using the chip, which is suitable for the capture and release of micron-scale cells and particles.

Background technique

The microfluidic chip is the core of the microanalysis system. The invention and application of the microfluidic chip has provided great convenience to the scientific fields such as chemistry, food, environment, and medicine, and promoted the miniaturization, integration, automation and convenience of some analyzes . It greatly saves the consumption of some chemical reactions on expensive chemical reagents, and also greatly improves the analysis efficiency and reduces the cost.

Usually, microfluidic chips that capture cells mainly use mechanical separation methods, electrical induction separation methods, and magnetic manipulation separation methods. At present, the microfluidic chip produced by mechanical separation method has a capture efficiency of 97%, but it also has some shortcomings. The shortcomings can be summarized as complicated manufacturing process, easy to block the channel, and cannot meet the needs of mass detection.

Currently, microfluidic chips are mainly prepared by photolithography and chemical etching. The main materials used are quartz, glass, and polymers. Microfluidic chips made of polymers are mostly prepared by photolithography. The etched microfluidic chip channels have regular shapes, and the photolithography process is complicated and costly. High, not suitable for promotion and application. The microfluidic chips made of silicon-based media such as glass and quartz are mainly prepared by chemical etching. The microfluidic chips produced by them have good stability and long service life. However, the channels of the microfluidic chip etched by the traditional chemical etching method are very regular, and it is impossible to etch a microfluidic chip with a microfluidic channel of a special shape. Traditional etching methods cannot etch microfluidic chips with special-shaped channels.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art, and provide a three-dimensional cell capture and release chip and its preparation method, which is characterized by simple processing, no clogging, high efficiency, suitable for mass production and detection chips, and has Good chemical stability. Another object is to provide a method for capturing and releasing cells based on a three-dimensional structure chip, which has high capture efficiency and good integrity of released cells.

In order to achieve the above object, the technical solution adopted by the present invention to solve the above technical problems is:

A cell capture and release chip with a three-dimensional structure, including an upper cover and a lower substrate. A microfluidic channel is etched on the back of the upper cover. The two ends of the microfluidic channel of the cover are respectively provided with inlets and outlets. There is a layer of chitosan film, and the cover sheet and base sheet are bonded by adhesive.

The method for preparing the cell capture and release chip with the above three-dimensional structure, the steps are as follows:

(1) Chip preparation:

(a) Selection and cleaning of the substrate: The material of the selected cover and substrate can be hard materials such as silicon wafers, quartz or glass, and at least one of the materials is transparent. Clean the selected cover and substrate, Remove surface oil and other impurities;

(b) Etching mask treatment: Dry the cleaned cover and substrate in an oven at 50°C to 100°C. After drying, adhere a chemical corrosion-resistant mask to the upper and lower surfaces of the cover. Film, by laser cutting, partly cut out the area to be etched, remove the cut anti-corrosion mask, and obtain the area to be etched;

The purpose of the mask treatment is to expose a part of the substrate that needs to be etched, and protect other parts of the substrate that do not need to be etched.

(c) Wet etching: put the processed cover sheet into the etching solution vertically and slowly through the fixture at a rate of 0.01mm/min-1mm/min for etching, because there is a sequence of entering the etching solution , so the etching time is different and the etching depth is different, so the microfluidic channel etched on the cover sheet is a wedge-shaped three-dimensional structure;

(d) Punching: Remove the etching mask on the surface of the cover, so that the entire cover is completely exposed. After the cover is cleaned, the cover is punched. At least two different contours of the cover The area is punched, and the two holes are the entrance hole and the exit hole;

(e) Preparation of chitosan film: use chitosan with a deacetylation degree of 60%-95%, dissolve 0.3g-2.0g chitosan into a 0.1mol/L acetic acid solution with a volume of 10ml, and pass through 1.5-2.5 h stirring to form chitosan colloid; use a pipette to suck 1ml-5ml chitosan gel solution and drop it on the substrate, place the substrate on the homogenizer, and the rotating speed is 800r/min and 1500r/min successively, each rotating 15s, after laying evenly, let it stand until the colloid forms a film;

(f) Combination of the cover sheet and the substrate: After the substrate and the cover sheet are bonded, coat their edges with PDMS (polydimethylsiloxane, polydimethylsiloxane glue), wait for 10-20 minutes and put them into the muffle furnace After heating, the heating temperature range is 200-450° C., and a cell capture and release chip based on a three-dimensional structure is obtained.

A method for capturing and releasing cells based on a chip with a three-dimensional structure, the steps of which are as follows:

(1) Capture of cells:

(a) Inject the liquid sample containing cells to be detected into the fully bonded chip through the syringe pump at a rate of 0.01ul/min-1ul/min from the inlet hole;

(b) Continue to inject phosphate buffer solution (i.e. PBS solution) at the inlet, and inject at a speed of 50-100ul/min for cleaning;

(c) Inject tracer substances (such as: fluorescent dyes such as DAPI or Hoechst dyes that stain the nucleus) or add immune reagents at the entrance to identify target cells;

(d) Inject phosphate buffer solution at the inlet again for cleaning, and place the chip under a microscope to observe the captured target cells;

(2) Release of cells:

(a) The chip captures cells with a specific capture size stuck in the corresponding position, and other cells flow out of the chip through the outlet;

(b) Inject an acetic acid solution with a volume fraction of 3%-5% at the inlet of the chip at a rate of 50ul/min-80ul/min;

(c) Continue to inject phosphate buffer solution (ie PBS solution) at the inlet, inject at a speed of 50-100ul/min for cleaning, the chitosan film in the chip is dissolved, the height inside the chip becomes larger, and the captured cells There were chips flushed out at the exit.

Due to the adoption of the above technical solution, the present invention changes the previous situation where different chips are only made for different cell sizes and blockages often occur in this situation. The preparation method of the present invention has simple processing technology, and the prepared chip for capturing and releasing cells/particles has the characteristics of miniaturized structure, short capturing time and high efficiency, and can be widely used in the field of analysis.

Description of drawings

Figure 1. Top view of the chip.

Figure 2. Cross-sectional view of the chip.

Figure 3. Cross-sectional view of the chip after capturing cells.

Figure 4. The top view of the chip after capturing cells.

Figure 5. Cross-sectional view of chip cell release.

Figure 6. Top view of chip cell release.

Among them: 1-cover sheet, 2-substrate, 3-inlet, 4-microfluidic channel, 5-outlet, 6-chitosan film, 7-ctc circulating tumor cells.

detailed description

The present invention will be further described below in conjunction with implementation examples.

Example 1:

As shown in Figure 1-2, a cell capture and release chip with a three-dimensional structure includes an upper cover 1 and a lower substrate 2, the back of the upper cover 1 is etched with a microfluidic channel 4, and the microfluidic channel 4 of the cover 1 An inlet 3 and an outlet 4 are respectively arranged at both ends of the flow channel 4 , a layer of chitosan film 6 is provided on the substrate 2 , and the cover sheet 1 and the substrate 2 are bonded by an adhesive.

The preparation method of the cell capture and release chip with the above three-dimensional structure, the steps are as follows:

(a) Select glass, silicon wafer or quartz as the material of the cover and substrate, and clean the selected cover and substrate to remove impurities such as oil on the surface;

(b) Dry the cleaned cover and substrate in an oven at 50°C to 100°C. After drying, adhere a chemical-resistant mask to the upper and lower surfaces of the cover, and cut it by laser. Cut out part of the area to be etched, remove the anti-corrosion mask cut out, and obtain the area to be etched;

(c) Put the processed cover sheet into the etchant vertically and slowly at a rate of 0.5mm/min through the fixture for etching. From the cover sheet entering the etching solution to the completion of etching, the total time-consuming is 1 hour, because there is a sequence of entering the etching solution, the etching time is different, resulting in different etching depths, so the microfluidic channel etched on the cover sheet is a wedge-shaped three-dimensional structure;

The etching solution is a typical buffered silicon oxide etching solution, which contains ammonium fluoride (BOE: Buffer OxideEtcher) and deionized water in a mass ratio of 1:10.

(d) Remove the etching mask on the surface of the cover sheet, so that the entire cover sheet and the substrate are completely exposed. After cleaning the cover sheet and the substrate, punch holes in the cover sheet. Drill holes in areas with different contours, with a diameter of 0.6mm, and the two holes are the entrance hole and the exit hole;

(e) Use chitosan with a deacetylation degree of 60%-95%, dissolve 0.3g chitosan into a 0.1mol/L acetic acid solution with a volume of 10ml, and stir for 2 hours to form a chitosan colloid; use a pipette gun Draw 1ml-5ml of chitosan gel solution and drop it on the lower piece, place the lower piece on the glue homogenizer, the rotation speed is 800r/min and 1500r/min, each turn for 15s, lay it evenly and let it stand until the colloid forms a film;

(f) After the substrate and the cover are bonded, coat their edges with PDMS (polydimethylsiloxane, polydimethylsiloxane glue), wait for 15 minutes and put them into the muffle furnace for heating. The heating temperature range is 300°C. , the cell capture and release chip based on the three-dimensional structure was obtained.

Example 2:

A method for preparing a three-dimensional cell capture and release chip, the steps are as follows:

(a) Select the material or glass of the cover and substrate as glass, silicon wafer or quartz, and clean the selected cover and substrate to remove impurities such as oil stains on the surface;

(b) Dry the cleaned cover and substrate in an oven at 50°C to 100°C. After drying, stick a chemical-resistant mask on the upper and lower surfaces of the cover, and cut it by laser. Cut out part of the area to be etched, remove the anti-corrosion mask cut out, and obtain the area to be etched;

(c) Put the processed cover sheet into the etching solution vertically and slowly at the rate of 0.01mm/min or 0.1mm/min or 0.25mm/min or 0.75mm/min or 1mm/min through the fixture for etching Etching, etch for 1 hour, because there is a sequence of entering the etching solution, so the etching time is different, resulting in different etching depths, so the microfluidic channel etched on the cover sheet is a wedge-shaped three-dimensional structure ;

The etching solution is a typical buffered silicon oxide etching solution, which contains ammonium fluoride (BOE: Buffer OxideEtcher) and deionized water in a mass ratio of 1:10.

(d) Remove the etching mask on the surface of the cover sheet, so that the entire cover sheet and the substrate are completely exposed. After cleaning the cover sheet and the substrate, punch holes in the cover sheet. Drill holes in areas with different contours, with a diameter of 0.6mm, and the two holes are the entrance hole and the exit hole;

(e) Using chitosan with a deacetylation degree of 60%-95%, dissolve 0.5g or 0.8g or 1g or 1.5g or 2g of chitosan into a volume of 10ml of 0.1mol/L acetic acid solution, after 1.5 or 2.5 h stirring to form chitosan colloid; use a pipette to suck 1ml-5ml chitosan gel solution and drop it on the lower piece, place the lower piece on the glue homogenizer, the rotating speed is 800r/min and 1500r/min successively, each turn 15s, after laying evenly, let it stand until the colloid forms a film;

(f) After the substrate and the cover are attached, coat their edges with PDMS (polydimethylsiloxane, polydimethylsiloxane glue), wait for 100min or 20min and put them into the muffle furnace for heating. The heating temperature range is 200 °C or 350 °C or 400 °C or 450 °C, the cell capture and release chip based on the three-dimensional structure is obtained.

Example 3:

A method for capturing and releasing cells based on a chip with a three-dimensional structure, the steps of which are as follows:

(1) As shown in Figure 3-4, the capture of cells:

(a) inject the liquid sample containing ctc circulating tumor cells to be detected into the chip of Example 1 through the syringe pump at a rate of 0.51ul/min from the inlet hole;

(b) Continue to inject phosphate buffer solution (i.e. PBS solution) at the inlet, and inject at a speed of 50ul/min for cleaning;

(c) Inject tracer substances (such as: fluorescent dyes such as DAPI or Hoechst dyes that stain the nucleus) at the entrance to identify target cells;

(d) Inject phosphate buffer solution at the inlet again for cleaning, and place the chip under a microscope to observe the captured target cells;

(2) As shown in Figure 5-6, the release of cells:

(a) The chip captures cells with a specific capture size stuck in the corresponding position, and other cells flow out of the chip through the outlet;

(b) Inject an acetic acid solution with a volume fraction of 3% at the inlet of the chip at a rate of 50ul/min;

(c) Continue to inject phosphate buffer solution (i.e. PBS solution) at the inlet, and inject it at a speed of 50ul/min for cleaning. The chitosan film in the chip is dissolved, the height inside the chip becomes larger, and the captured cells have the chip The exit was flushed out.

The capture efficiency can reach up to 96%. By observing the cells captured by the above method through a microscope, it is found that the captured cells have a complete structure and no damage. The cell capture efficiency used was calculated as the ratio of the number of cells that ended up in the chip to the number of cells injected at the beginning of the experiment that were the same size as the captured cells.

Example 4:

A method for capturing and releasing cells based on a chip with a three-dimensional structure, the steps of which are as follows:

(1) Capture of cells:

(a) The liquid sample containing ctc circulating tumor cells to be detected is injected into the key through the inlet hole at a rate of 0.01ul/min or 0.11ul/min or 0.51ul/min or 0.81ul/min or 1ul/min integrated into a complete chip;

(b) Continue to inject phosphate buffer solution (i.e. PBS solution) at the inlet, and inject at a speed of 100ul/min for cleaning;

(c) Inject tracer substances (such as: fluorescent dyes such as DAPI or Hoechst dyes that stain the nucleus) at the entrance to identify target cells;

(d) Inject phosphate buffer solution at the inlet again for cleaning, and place the chip under a microscope to observe the captured target cells;

(2) Release of cells:

(a) The chip captures cells with a specific capture size stuck in the corresponding position, and other cells flow out of the chip through the outlet;

(b) Inject an acetic acid solution with a volume fraction of 4% or 5% at the inlet of the chip at a rate of 80ul/min;

(c) Continue to inject phosphate buffer solution (i.e. PBS solution) at the inlet, and inject it at a speed of 100ul/min for cleaning. The chitosan film in the chip is dissolved, the height inside the chip becomes larger, and the captured cells have the chip The exit was flushed out.

The capture efficiency can reach up to 90%. By observing the cells captured by the above method through a microscope, it is found that the captured cells have a complete structure and no damage. The cell capture efficiency used was calculated as the ratio of the number of cells that ended up in the chip to the number of cells injected at the beginning of the experiment that were the same size as the captured cells.

Claims (2)

1. A method for preparing a three-dimensional cell capture and release chip, the steps of which are as follows: (a) Selection and cleaning of the substrate: select the material of the cover and substrate as silicon wafer, quartz or glass, and clean the selected cover and substrate to remove impurities such as oil on the surface; (b) Etching mask treatment: Dry the cleaned cover and substrate in an oven at 50°C to 100°C. After drying, adhere a chemical corrosion-resistant mask to the upper and lower surfaces of the cover. Film, by laser cutting, partly cut out the area to be etched, remove the cut anti-corrosion mask, and obtain the area to be etched; (c) Wet etching: Put the masked cover sheet into the etching solution vertically and slowly at a rate of 0.01 mm/min-1mm/min through the fixture to etch. The microfluidic channel is a wedge-shaped three-dimensional structure; (d) Punching: Remove the etching mask on the surface of the cover, so that the entire cover is completely exposed. After the cover is cleaned, the cover is punched. At least two different contours of the cover The area is punched, and the two holes are the entrance hole and the exit hole; (e) Preparation of chitosan film: use chitosan with a deacetylation degree of 60%-95%, dissolve 0.3g-2.0g chitosan into 0.1mol/L acetic acid solution with a volume of 10ml, after 1.5h- Stir for 2.5 hours to form chitosan colloid; use a pipette gun to draw 1ml-5ml chitosan gel solution and drop it on the substrate, place the substrate on the homogenizer, the rotation speed is 800r/min and 1500r/min, each Turn for 15 seconds, lay it evenly and let it stand until the colloid forms a film; (f) Combination of the cover sheet and the substrate: after the substrate and the cover sheet are bonded, coat PDMS on their edges, wait for 10-20 minutes and put them into a muffle furnace for heating. The heating temperature range is 200-450°C. The cell capture and release chip based on the three-dimensional structure was obtained; The cell capture and release chip with a three-dimensional structure includes an upper cover (1) and a lower substrate (2), the back of the upper cover (1) is etched with a microfluidic channel (4), the cover (1) The two ends of the microfluidic channel (4) are respectively provided with an inlet (3) and an outlet (4), a layer of chitosan film (6) is on the substrate (2), and the cover sheet (1) and the substrate (2) pass through Adhesive bonding. 2. adopt the described chip of claim 1 to carry out the method for capturing and releasing cells, its steps are as follows: (1) Capture of cells: (a) Inject the liquid sample containing cells to be detected into the fully bonded chip through the syringe pump at a rate of 0.01ul/min-1ul/min from the inlet hole; (b) Continue to inject phosphate buffer solution at the inlet, and inject at a speed of 50ul/min-100ul/min for cleaning; (c) Inject tracer substances or add immune reagents at the entrance to identify target cells; (d) Inject phosphate buffer solution at the inlet again for cleaning, and place the chip under a microscope to observe the captured target cells; (2) Release of cells: (a) The chip captures cells with a specific capture size stuck in the corresponding position, and other cells flow out of the chip through the outlet; (b) Inject an acetic acid solution with a volume fraction of 3%-5% at the inlet of the chip at a rate of 50ul/min-80ul/min; (c) Continue to inject phosphate buffer solution at the inlet, and inject it at a speed of 50ul/min-100ul/min for cleaning. The chitosan film in the chip is dissolved, the height inside the chip becomes larger, and the captured cells have the The exit was flushed out.