An automated concentration gradient generation device for ultra-micro flow rates and its usage method

Abstract

An automated concentration gradient generation device for ultra-microflow rates and its usage method are disclosed, relating to the field of microfluidics. Method: First, a rubber stopper is placed at the outlet of a curved tube. Then, the upper switch is pressed, and the motor controls the spur gear to rotate forward, driving the rack upward. The solution passes through a straight tube, a square tube, and a syringe tube, entering the pump body through the needle at the bottom of the conical nozzle. First, deionized water is drawn in, followed by an equal amount of 5% methylene blue aqueous solution. After the deionized water and methylene blue aqueous solution diffuse naturally, the rubber stopper at the outlet of the curved tube is removed and placed at the outlet of the straight tube. The lower switch is pressed, and the motor controls the spur gear to rotate in reverse, driving the rack downward. The mixed solution passes through the needle at the bottom of the conical nozzle, the syringe tube, and the square tube, finally exiting through the straight tube and the droplets are spotted into a collection device. This invention provides an automated concentration gradient generation device for ultra-microflow rates and its usage method.

Description

Technical Field

[0001] This invention relates to the field of microfluidics, specifically to an automated device for generating concentration gradients at ultra-low flow rates and its usage method. Background Technology

[0002] With the development of science and technology, the generation of ultra-microflow concentration gradients has wide applications in research across multiple fields such as chemistry, physics, and biology. Particularly in fields like cell biology and drug delivery systems, where reagents are expensive, used in extremely low quantities, and not suitable for continuous generation, the automated generation of stable, small-volume, discrete ultra-microflow concentration gradients is crucial for the accuracy and reproducibility of experimental results. Therefore, how to automatically generate stable, small-volume, discrete concentration gradients has become an urgent problem to be solved.

[0003] Free diffusion is a simple and efficient principle for generating concentration gradients. It induces the diffusion of solute molecules due to the concentration difference between two different solutions within a tube, gradually forming a stable concentration gradient. The working principle of an automated concentration gradient generation device based on free diffusion within a small square tube mainly relies on the free diffusion of the solution within the tube. When two solutions of different concentrations come into contact at both ends of the square tube, due to gravity, the higher-density solution spontaneously moves towards the lower-density solution. Solute molecules in the solution also spontaneously move from the high-concentration region to the low-concentration region, thus forming a continuous, three-dimensional wedge-shaped concentration gradient region within the tube. Based on this principle, an automated concentration gradient generation device is designed, which not only simplifies experimental operations but also improves experimental accuracy and controllability. This device, employing the free diffusion principle combined with microfluidic technology, can precisely control the concentration gradient generation process, possessing significant scientific value and broad application prospects.

[0004] While traditional concentration gradient generation devices can generate concentration gradients, these methods suffer from drawbacks such as cumbersome operation, large equipment size, and difficulty in controlling precision, often leading to inconvenience in use. Summary of the Invention

[0005] The purpose of this invention is to solve the above-mentioned technical problems by providing an automated concentration gradient generation device for ultra-micro flow and its usage method.

[0006] An automated concentration gradient generation device with ultra-low flow rate includes an integrated automated pumping system 1 and a free diffusion system 2;

[0007] The integrated automated pumping system 1 includes a circular encapsulation cover 1-1, a spur gear 1-2, a motor 1-3, a rack 1-4, an upper switch 1-5, a lower switch 1-6, a piston 1-7, and a conical nozzle 1-8;

[0008] The free diffusion system 2 includes a square tube 2-2, a needle tube 2-3, a bent tube 2-5, a straight tube 2-6, and a rubber stopper 2-8;

[0009] The integrated automated pumping system 1 has a hollow cylindrical pump body. A conical nozzle 1-8 is located at the bottom of the pump body, and a needle is located at the bottom outlet of the conical nozzle 1-8. A piston 1-7 is located at the bottom of the cylindrical portion of the pump body, and a rack 1-4 is fixedly connected to the upper surface of the piston 1-7. A motor 1-3, a spur gear 1-2, an upper switch 1-5, and a lower switch 1-6 are located on the side of the pump body. The drive shaft of the motor 1-3 is rotatably connected to the center of the spur gear 1-2. The spur gear 1-2 meshes with the rack 1-4 for transmission. Both the upper switch 1-5 and the lower switch 1-6 are electrically connected to the motor 1-3 via wires. A circular encapsulation cover 1-1 is located at the top of the pump body.

[0010] The square tube 2-2 has a through hole at its top end, and one end of the needle tube 2-3 extends into the through hole. The other end of the needle tube 2-3 is connected to the needle at the bottom of the conical nozzle 1-8. The square tube 2-2 has two through holes at its bottom end, and one end of the bent tube 2-5 and the straight tube 2-6 extends into the two through holes respectively. The rubber stopper 2-8 is located at the outlet of the bent tube 2-5 or the straight tube 2-6. The through holes at the top and bottom ends of the square tube 2-2 are sealed.

[0011] The method of using an automated concentration gradient generation device with ultra-low flow rate is as follows:

[0012] Step S1:

[0013] First, use rubber stopper 2-8 to fit at the outlet of bent tube 2-5. Then, press the upper switch 1-5. Motor 1-3 controls spur gear 1-2 to rotate forward, driving rack 1-4 to move upward at a rate of 1~1.5 μL / min. The solution passes through straight tube 2-6, square tube 2-2 and needle tube 2-3 and enters the pump body through the needle at the bottom of conical nozzle 1-8. First, 0.4~0.6 μL of deionized water is drawn, and then 0.4~0.6 μL of 5% methylene blue aqueous solution is drawn.

[0014] Step S2:

[0015] After the deionized water and methylene blue aqueous solution have diffused for 8-12 minutes, remove the rubber stopper 2-8 from the outlet of the bent tube 2-5 and place it on the outlet of the straight tube 2-6. Press the switch 1-6, and the motor 1-3 controls the spur gear 1-2 to reverse, driving the rack 1-4 to move downward at a rate of 0.4-0.6 μL / min. The mixed solution passes through the needle at the bottom of the conical nozzle 1-8, the needle tube 2-3, and the square tube 2-2 in sequence, and is finally discharged through the straight tube 2-6, spotting the droplets into the collection device. The spotting time is 0.8-1.2 s, and the diameter of the droplets is 30-50 nL.

[0016] The working principle of the automated concentration gradient generation device for ultra-microflow mainly relies on the free diffusion phenomenon of the solution within a micromixer. When two solutions of different concentrations come into contact at both ends of a capillary, solute molecules in the solutions spontaneously move from the high-concentration region to the low-concentration region, thereby forming a continuous, three-dimensional wedge-shaped concentration gradient region within the tube. Finally, by controlling the discharge of the solution through an integrated automated pumping system, droplets with a concentration gradient can be generated on the surface of a chip or other applications. Compared with existing technologies, this invention has the following advantages:

[0017] 1. By using an integrated automated pumping system in conjunction with a square tube free diffusion system, it is possible to achieve tiny droplets with concentration gradients without the need for other complex and large equipment (syringe pump, displacement stage), thereby reducing equipment consumption and greatly reducing experimental costs.

[0018] 2. By using a small square tube instead of a capillary tube as a container for free diffusion, the influence of capillary action on the flow of the two solutions is greatly reduced, thereby making full use of free diffusion to achieve efficient and stable generation of concentration gradient, and improving the stability and controllability of concentration gradient generation.

[0019] 3. The use of an integrated automated pumping system in conjunction with a square tube free diffusion system greatly simplifies the cumbersome experimental preparation and time consumption of traditional microfluidic chips, thereby reducing the difficulty of experiments, saving experimental time, and improving the efficiency of concentration gradient generation.

[0020] 4. Depending on the different experimental needs, it can realize the generation of ultra-micro flow concentration gradients. By controlling the flow rate of the liquid outlet of the bent tube and the contact time with the surface of the application chip, it can realize droplets at the level of 30 nL to 100 nL. It has small volume, high adaptability and can be flexibly used in multiple application scenarios.

[0021] 5. Replace the liquid outlet and liquid inlet with separate bent and straight pipes to avoid the influence of residual solution at the same outlet on the experiment.

[0022] 6. Compared with traditional microfluidic chips, the entire device is simpler to prepare and operate, achieving low cost, high automation and high efficiency throughout the process.

[0023] The present invention provides an automated device for generating concentration gradients at ultra-low flow rates and a method for using it. Attached Figure Description

[0024] Figure 1 This diagram illustrates the structure of an automated concentration gradient generation device for ultra-micro flow rates according to the present invention. 1 represents the integrated automated pumping system, and 2 represents the free diffusion system.

[0025] Figure 2 The diagram shows the structure of an integrated automated pumping system. 1-1 represents a circular encapsulation cover, 1-2 represents a spur gear, 1-3 represents a small motor, 1-4 represents a rack, 1-5 represents an upper switch, 1-6 represents a lower switch, 1-7 represents a piston, and 1-8 represents a conical nozzle.

[0026] Figure 3 The diagram shows the structure of a free diffusion system. 2-1 represents the glass substrate, 2-2 represents the square tube, 2-3 represents the needle tube, 2-4 represents the encapsulating adhesive a, 2-5 represents the bent tube, 2-6 represents the straight tube, 2-7 represents the encapsulating adhesive b, and 2-8 represents the rubber stopper.

[0027] Figure 4 A physical diagram showing a free-diffusion system;

[0028] Figure 5 Represents discrete concentration gradient droplets;

[0029] Figure 6 This represents a free diffusion diagram inside the chip.

[0030] Figure 7 This represents a scatter plot of concentration gradients. Detailed Implementation

[0031] Specific Implementation Method 1: This implementation method is an automated concentration gradient generation device with ultra-low flow rate, including an integrated automated pumping system 1 and a free diffusion system 2;

[0032] The integrated automated pumping system 1 includes a circular encapsulation cover 1-1, a spur gear 1-2, a motor 1-3, a rack 1-4, an upper switch 1-5, a lower switch 1-6, a piston 1-7, and a conical nozzle 1-8;

[0033] The free diffusion system 2 includes a square tube 2-2, a needle tube 2-3, a bent tube 2-5, a straight tube 2-6, and a rubber stopper 2-8;

[0034] The integrated automated pumping system 1 has a hollow cylindrical pump body. A conical nozzle 1-8 is located at the bottom of the pump body, and a needle is located at the bottom outlet of the conical nozzle 1-8. A piston 1-7 is located at the bottom of the cylindrical portion of the pump body, and a rack 1-4 is fixedly connected to the upper surface of the piston 1-7. A motor 1-3, a spur gear 1-2, an upper switch 1-5, and a lower switch 1-6 are located on the side of the pump body. The drive shaft of the motor 1-3 is rotatably connected to the center of the spur gear 1-2. The spur gear 1-2 meshes with the rack 1-4 for transmission. Both the upper switch 1-5 and the lower switch 1-6 are electrically connected to the motor 1-3 via wires. A circular encapsulation cover 1-1 is located at the top of the pump body.

[0035] The square tube 2-2 has a through hole at its top end, and one end of the needle tube 2-3 extends into the through hole. The other end of the needle tube 2-3 is connected to the needle at the bottom of the conical nozzle 1-8. The square tube 2-2 has two through holes at its bottom end, and one end of the bent tube 2-5 and the straight tube 2-6 extends into the two through holes respectively. The rubber stopper 2-8 is located at the outlet of the bent tube 2-5 or the straight tube 2-6. The through holes at the top and bottom ends of the square tube 2-2 are sealed.

[0036] Specific Implementation Method Two: The difference between this implementation method and Specific Implementation Method One is that the volume of the pump body is 500~1000 μL.

[0037] The other steps are the same as in Specific Implementation Method 1.

[0038] Specific Implementation Method 3: The difference between this implementation method and Specific Implementation Method 1 or 2 is that the spur gear 1-2 has a module of 0.8 mm, a number of teeth of 17, a pressure angle of 20°, a tooth width of 9~10 mm, an outer diameter of 14~15 mm, and an inner diameter of 3.5~4 mm.

[0039] The other steps are the same as in Specific Implementation Method 1 or 2.

[0040] Specific Implementation Method Four: The difference between this implementation method and Specific Implementation Methods One to Three is that the module of the racks 1-4 is 0.8 mm, the pressure angle is 20°, the tooth width is 9~10 mm, and the length is 28~30 mm.

[0041] The other steps are the same as those in Specific Implementation Methods One to Three.

[0042] Specific Implementation Method 5: The difference between this implementation method and Specific Implementation Methods 1 to 4 is that the inner diameter of the bottom needle of the conical nozzle 1-8 is 0.58~0.61 mm and the outer diameter is 0.91~1.1 mm.

[0043] The other steps are the same as those in Specific Implementation Methods One through Four.

[0044] Specific Implementation Method Six: The difference between this implementation method and Specific Implementation Methods One to Five is that the inner diameter of the needle tube 2-3 is 1.1~1.3 mm and the outer diameter is 1.4~1.51 mm.

[0045] The other steps are the same as those in Specific Implementation Methods 1 to 5.

[0046] Specific Implementation Method Seven: The difference between this implementation method and Specific Implementation Methods One to Six is ​​that the inner diameter of the bent pipe 2-5 and the straight pipe 2-6 is 0.05~0.1 mm and the outer diameter is 0.2~0.3 mm; the length of the bent pipe 2-5 and the straight pipe 2-6 extending into the bottom through hole of the square pipe 2-2 is 2~5 mm; the bending angle of the bent pipe 2-5 is 45~90°.

[0047] The other steps are the same as those in Specific Implementation Methods 1 to 6.

[0048] Specific Implementation Method Eight: The difference between this implementation method and one of the specific implementation methods one to seven is that the square tube 2-2 is disposed on the upper surface of the glass substrate 2-1.

[0049] The other steps are the same as those in Specific Implementation Methods 1 to 7.

[0050] Specific Implementation Method Nine: The difference between this implementation method and Specific Implementation Methods One to Eight is that the through holes at the top and bottom of the square tube 2-2 are sealed with encapsulating adhesive a and encapsulating adhesive b, respectively, and both encapsulating adhesive a and encapsulating adhesive b are AB adhesives.

[0051] The other steps are the same as those in Specific Implementation Methods 1 to 8.

[0052] Specific Implementation Method Ten: This implementation method describes a method for using an automated concentration gradient generation device for ultra-micro flow rates, which is carried out according to the following steps:

[0053] Step S1:

[0054] First, use rubber stopper 2-8 to fit at the outlet of bent tube 2-5. Then, press the upper switch 1-5. Motor 1-3 controls spur gear 1-2 to rotate forward, driving rack 1-4 to move upward at a rate of 1~1.5 μL / min. The solution passes through straight tube 2-6, square tube 2-2 and needle tube 2-3 and enters the pump body through the needle at the bottom of conical nozzle 1-8. First, 0.4~0.6 μL of deionized water is drawn, and then 0.4~0.6 μL of 5% methylene blue aqueous solution is drawn.

[0055] Step S2:

[0056] After the deionized water and methylene blue aqueous solution have diffused for 8-12 minutes, remove the rubber stopper 2-8 from the outlet of the bent tube 2-5 and place it on the outlet of the straight tube 2-6. Press the switch 1-6, and the motor 1-3 controls the spur gear 1-2 to reverse, driving the rack 1-4 to move downward at a rate of 0.4-0.6 μL / min. The mixed solution passes through the needle at the bottom of the conical nozzle 1-8, the needle tube 2-3, and the square tube 2-2 in sequence, and is finally discharged through the straight tube 2-6, spotting the droplets into the collection device. The spotting time is 0.8-1.2 s, and the diameter of the droplets is 30-50 nL.

[0057] Compared to a circular capillary tube, the small square tube 2-2 in this embodiment has a much weaker capillary force. This characteristic can be used to greatly reduce the influence of capillary action on the flow of the two solutions, thereby making full use of gravity to achieve efficient and stable generation of concentration gradient.

[0058] The beneficial effects of the present invention are verified using the following embodiments:

[0059] Example 1:

[0060] This invention uses a concentration gradient generation experiment with nucleic acid aptamers and dipotassium hydrogen phosphate dilution to verify the feasibility of the device and its method.

[0061] like Figure 1 As shown, an automated concentration gradient generation device with ultra-low flow rate includes an integrated automated pumping system 1 (such as...). Figure 2 (as shown) and free diffusion system 2 (as shown) Figure 3 (as shown)

[0062] The integrated automated pumping system 1 includes a circular encapsulation cover 1-1, a spur gear 1-2, a motor 1-3, a rack 1-4, an upper switch 1-5, a lower switch 1-6, a piston 1-7, and a conical nozzle 1-8;

[0063] The free diffusion system 2 includes a square tube 2-2, a needle tube 2-3, a bent tube 2-5, a straight tube 2-6, and a rubber stopper 2-8;

[0064] The integrated automated pumping system 1 has a hollow cylindrical pump body. A conical nozzle 1-8 is located at the bottom of the pump body, and a needle is located at the bottom outlet of the conical nozzle 1-8. A piston 1-7 is located at the bottom of the cylindrical portion of the pump body, and a rack 1-4 is fixedly connected to the upper surface of the piston 1-7. A motor 1-3, a spur gear 1-2, an upper switch 1-5, and a lower switch 1-6 are located on the side of the pump body. The drive shaft of the motor 1-3 is rotatably connected to the center of the spur gear 1-2. The spur gear 1-2 meshes with the rack 1-4 for transmission. Both the upper switch 1-5 and the lower switch 1-6 are electrically connected to the motor 1-3 via wires. A circular encapsulation cover 1-1 is located at the top of the pump body.

[0065] The square tube 2-2 has a through hole at its top end, and one end of the needle tube 2-3 extends into the through hole. The other end of the needle tube 2-3 is connected to the needle at the bottom of the conical nozzle 1-8. The square tube 2-2 has two through holes at its bottom end, and one end of the bent tube 2-5 and the straight tube 2-6 extends into the two through holes respectively. The rubber stopper 2-8 is located at the outlet of the bent tube 2-5 or the straight tube 2-6. The through holes at the top and bottom ends of the square tube 2-2 are sealed.

[0066] The pump body is a miniature pump similar to a syringe, with an overall length of 4.5~6 cm, an effective volume of 500~1000 μL, an outer diameter of 1.4~1.6 cm, a wall thickness of 0.1~0.2 cm, and a circular encapsulation cap 1-1 on top, with a diameter consistent with the outer diameter of the pump body, both being 1.4~1.6 cm, and a thickness of 0.4~0.6 cm.

[0067] The spur gear 1-2 has a module of 0.8 mm, 17 teeth, a pressure angle of 20°, a tooth width of 9~10 mm, an outer diameter of 14~15 mm, and an inner diameter of 3.5~4 mm. It is installed on a small-sized 10mm×10mm×5mm motor 1-3 for operation. The spur gear 1-2 is meshed with a rack 1-4 with a module of 0.8 mm, a pressure angle of 20°, a tooth width of 9~10 mm, and a length of 28~30 mm.

[0068] The upper switch 1-5 and the lower switch 1-6 are used to control the rotation direction of the spur gear 1-2. When the upper switch 1-5 is pressed, the spur gear 1-2 rotates clockwise (forward), driving the rack 1-4 upward to perform the liquid suction function; conversely, when the lower switch 1-6 is pressed, the spur gear 1-2 rotates counterclockwise (reverse), driving the rack 1-4 downward to perform the liquid drainage function.

[0069] Piston 1-7 is the piston connected to the bottom end of rack 1-4. Its diameter is the same as the inner diameter of the pump body, which is 1.2~1.4 cm and its thickness is 0.4~0.6 cm.

[0070] The conical nozzle 1-8 located at the bottom of the pump body has an inner diameter of 0.58~0.61 mm and an outer diameter of 0.91~1.1 mm. It is used to connect to the needle tube 2-3. Its overall schematic diagram and internal structure diagram are shown below. Figure 2-3 As shown.

[0071] The key concentration gradient in this embodiment is mainly implemented in this system, which mainly includes eight parts from 2-1 to 2-8:

[0072] The glass substrate 2-1 is 30-50 mm long, 20-30 mm wide, and 2-3 mm thick.

[0073] The square tube 2-2 has an outer diameter of 1.9~2.0 mm, an inner diameter of 1.5~1.6 mm, and a length of 25~45 mm, and is fixed to the upper surface of the glass substrate 2-1 with UV-curable adhesive.

[0074] Insert a needle tube 2-3 with an inner diameter of 1.1~1.3 mm, an outer diameter of 1.4~1.51 mm, and a length of 10~15 mm into the upper end of the square tube 2-2. The depth of insertion of the front end of the needle tube 2-3 into the square tube 2-2 is most suitable to be between 2~5 mm.

[0075] Two bent tubes, 2-5 and 2-6, with inner diameters of 0.05-0.1 mm, outer diameters of 0.2-0.3 mm, and lengths of 6-8 mm, are inserted into the lower end of square tube 2-2. The lengths of bent tubes 2-5 and straight tubes 2-6 extending into the through hole at the bottom end of square tube 2-2 are both 2-5 mm. Bent tube 2-5 is formed by bending straight tube 2-6 over an alcohol lamp, with a bending angle between 45° and 90° being most suitable. The depth to which bent tubes 2-5 and straight tubes 2-6 are inserted into square tube 2-2 is most suitable at 2-5 mm.

[0076] The through holes at the top and bottom of the square tube 2-2 are sealed with encapsulating adhesive a and encapsulating adhesive b, respectively. Both encapsulating adhesive a and encapsulating adhesive b are AB adhesives, and the airtightness is best after standing for more than 10 hours.

[0077] The rubber stopper 2-8 has an inner diameter of 0.41~0.45 mm and an outer diameter of 0.8~1 mm, and is mainly used to seal the bend 2-5 and the straight pipe 2-6. When the integrated automated pumping system 1 is aspirating liquid, the rubber stopper 2-8 is used to seal the bend 2-5 to achieve the purpose of aspiration; conversely, when the integrated automated pumping system 1 is discharging liquid, the rubber stopper 2-8 is used to seal the straight pipe 2-6 to achieve the purpose of discharging liquid.

[0078] The method of using an automated concentration gradient generation device with ultra-low flow rate is as follows:

[0079] Step S1:

[0080] A 5% methylene blue aqueous solution was prepared as the initial concentration and mixed with deionized water in equal volume. A 24×40 mm ultra-micro flow concentration gradient generation chip and an 8×10 mm micro-column PDMS array chip were fabricated. Each microcolumn had a diameter of 1000 μm, a height of 300 μm, and a distance of 2000 μm between the centers of two adjacent microcolumns.

[0081] Step S2:

[0082] First, use rubber stopper 2-8 to fit at the outlet of bent tube 2-5. Then press the upper switch 1-5. Motor 1-3 controls spur gear 1-2 to rotate forward, driving rack 1-4 to move upward at a rate of 1 μL / min. The solution passes through straight tube 2-6, square tube 2-2 and needle tube 2-3 and enters the pump body through the needle at the bottom of conical nozzle 1-8. First, draw 0.5 μL of deionized water, and then draw 0.5 μL of 5% methylene blue aqueous solution.

[0083] Step S3:

[0084] After the deionized water and methylene blue aqueous solution diffused for 10 minutes, the rubber stopper 2-8 at the outlet of the bent tube 2-5 was removed and placed on the outlet of the straight tube 2-6. The switch 1-6 was pressed, and the motor 1-3 controlled the spur gear 1-2 to reverse, driving the rack 1-4 to move downward at a rate of 0.5 μL / min. The mixed solution passed through the needle at the bottom of the conical nozzle 1-8, the needle tube 2-3, and the square tube 2-2. The mixed solution condensed at the outlet of the bent tube 2-5 was applied to the application surface (chip, glass slide, etc.) by contact spotting for 1 second (at this time, the droplet size is 30~50 nL). This process continued until all the mixed solution with a total volume of 1 μL was applied to the PDMS droplet collection chip with an oil seal on the surface (in this embodiment, the purpose of the oil seal is to prevent the microdroplets from evaporating during subsequent observation).

[0085] Step S4:

[0086] Approximately 20 droplets were captured sequentially and photographed under a microscope for preservation. The grayscale values ​​of the droplets were extracted using ImageJ software, and the difference between the grayscale values ​​of the droplet region and the grayscale values ​​of the blank region was defined as the effective color density for experimental data analysis.

[0087] Depend on Figure 4 It can be seen that when equal amounts of deionized water and methylene blue aqueous solution are successively aspirated and allowed to stand for a certain period of time, allowing them to diffuse freely within the tube, the concentration will spontaneously diffuse from the high-concentration region to the low-concentration region, forming a concentration gradient from bottom to top. The mixed solution is then applied as droplets onto a collecting chip (e.g., ...). Figure 5As shown in the image, droplets of varying colors from dark blue to light blue are formed to represent the concentration gradient. Imagej software is used to extract... Figure 6 The difference between the grayscale values ​​of the blank area and the droplet area is defined as the effective color density, which characterizes the droplet concentration. For example... Figure 6 As shown, the droplets range in color from dark blue to light blue, exhibiting a clear concentration gradient, and the droplets are relatively uniform. This indicates that the device in this embodiment not only generates a concentration gradient but also features stable droplet generation.

[0088] like Figure 7 As shown, as droplets are applied one after another, their grayscale values ​​increase while their relative grayscale values ​​(effective color density) decrease, indicating a decrease in droplet concentration. This is the result of free diffusion mixing. The concentration decreases from high to low, with the high-concentration region representing methylene blue aqueous solution and the low-concentration region representing deionized water. At the bottom of the square tube, methylene blue aqueous solution is predominantly present, with almost no deionized water for mixing and dilution, resulting in a generally high concentration. At the top of the square tube, deionized water is predominantly present, with almost no methylene blue aqueous solution, resulting in a generally low concentration. The middle region is the main area for free diffusion mixing of methylene blue aqueous solution and deionized water, and this mixing gradually diffuses towards both ends over time, forming a concentration gradient.

Claims

1. An automated concentration gradient generation device for ultra-micro flow rates, characterized in that, The ultra-micro flow concentration gradient automatic generation device includes an integrated automatic pumping system (1) and a free diffusion system (2). The integrated automated pumping system (1) includes a circular encapsulation cover (1-1), a spur gear (1-2), a motor (1-3), a rack (1-4), an upper switch (1-5), a lower switch (1-6), a piston (1-7), and a conical nozzle (1-8). The free diffusion system (2) includes a square tube (2-2), a needle tube (2-3), a bent tube (2-5), a straight tube (2-6), and a rubber stopper (2-8). The pump body of the integrated automated pumping system (1) is a hollow cylinder. A conical nozzle (1-8) is provided at the bottom of the pump body, and a needle is provided at the bottom outlet of the conical nozzle (1-8). A piston (1-7) is provided at the bottom of the cylindrical part of the pump body, and a rack (1-4) is fixedly connected to the upper surface of the piston (1-7). A motor (1-3), a spur gear (1-2), an upper switch (1-5), and a lower switch (1-6) are provided on the side of the pump body. The drive shaft of the motor (1-3) is rotatably connected to the center of the spur gear (1-2). The spur gear (1-2) meshes with the rack (1-4). The upper switch (1-5) and the lower switch (1-6) are both electrically connected to the motor (1-3) through wires. A circular encapsulation cover (1-1) is provided on the top of the pump body. The square tube (2-2) has a through hole at its top end, and one end of the needle tube (2-3) extends into the through hole. The other end of the needle tube (2-3) is connected to the needle tip at the bottom of the conical nozzle (1-8). The square tube (2-2) has two through holes at its bottom end, and one end of the bent tube (2-5) and the straight tube (2-6) extends into the two through holes respectively. The rubber stopper (2-8) is placed at the outlet of the bent tube (2-5) or the straight tube (2-6). The through holes at the top and bottom ends of the square tube (2-2) are sealed.

2. The automated concentration gradient generation device for ultra-micro flow rates according to claim 1, characterized in that, The volume of the pump body is 500~1000 μL.

3. The automated concentration gradient generation device for ultra-micro flow rates according to claim 1, characterized in that, The spur gear (1-2) has a module of 0.8 mm, 17 teeth, a pressure angle of 20°, a tooth width of 9~10 mm, an outer diameter of 14~15 mm, and an inner diameter of 3.5~4 mm.

4. The automated concentration gradient generation device for ultra-micro flow rates according to claim 1, characterized in that, The rack (1-4) has a module of 0.8 mm, a pressure angle of 20°, a tooth width of 9~10 mm, and a length of 28~30 mm.

5. The automated concentration gradient generation device for ultra-micro flow rates according to claim 1, characterized in that, The inner diameter of the needle at the bottom of the conical nozzle (1-8) is 0.58~0.61 mm, and the outer diameter is 0.91~1.1 mm.

6. The automated concentration gradient generation device for ultra-micro flow rates according to claim 1, characterized in that, The needle tube (2-3) has an inner diameter of 1.1~1.3 mm and an outer diameter of 1.4~1.51 mm.

7. The automated concentration gradient generation device for ultra-micro flow rates according to claim 1, characterized in that, The inner diameter of the bent pipe (2-5) and the straight pipe (2-6) is 0.05~0.1 mm, and the outer diameter is 0.2~0.3 mm. The length of the bent pipe (2-5) and the straight pipe (2-6) extending into the bottom through hole of the square pipe (2-2) is 2~5 mm. The bending angle of the bent pipe (2-5) is 45~90°.

8. The automated concentration gradient generation device for ultra-micro flow rates according to claim 1, characterized in that, The square tube (2-2) is disposed on the upper surface of the glass substrate (2-1).

9. The automated concentration gradient generation device for ultra-micro flow rates according to claim 1, characterized in that, The through holes at the top and bottom of the square tube (2-2) are sealed with encapsulating adhesive a and encapsulating adhesive b, respectively, and both encapsulating adhesive a and encapsulating adhesive b are AB adhesives.

10. A method of using the automated concentration gradient generation device for ultra-micro flow rates as described in any one of claims 1-9, characterized in that, The usage method is as follows: Step S1: First, use a rubber stopper (2-8) to cover the outlet of the curved tube (2-5), then press the upper switch (1-5). The motor (1-3) controls the spur gear (1-2) to rotate forward, driving the rack (1-4) to move upward at a rate of 1~1.5 μL / min. The solution passes through the straight tube (2-6), square tube (2-2), and syringe (2-3) and enters the pump body through the needle at the bottom of the conical nozzle (1-8). First, 0.4~0.6 μL of deionized water is drawn, and then 0.4~0.6 μL of 5% methylene blue aqueous solution is drawn. Step S2: After the deionized water and methylene blue aqueous solution diffuse for 8-12 minutes, remove the rubber stopper (2-8) from the outlet of the curved tube (2-5) and place it on the outlet of the straight tube (2-6). Press the switch (1-6), and the motor (1-3) controls the spur gear (1-2) to reverse, driving the rack (1-4) to move downward at a rate of 0.4-0.6 μL / min. The mixed solution passes through the needle at the bottom of the conical nozzle (1-8), the syringe (2-3), and the square tube (2-2) in sequence, and is finally discharged through the straight tube (2-6) and the droplets are spotted into the collection device. The spotting time is 0.8-1.2 s, and the diameter of the droplets is 30-50 nL.

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