A microstring biomechanics chip and a preparation method and application thereof

Abstract

The application discloses a kind of microstring biomechanics chip and its preparation method and application, the preparation method includes S1, providing first template with through hole in transverse or longitudinal side face;S2, prepare several through cavities on the non-through hole face of first template and make at least two through holes pass through in the cavity, obtain second template;S3, using strip solid to pass through the through hole of second template, then using sacrificial material to fill the cavity of second template, remove strip solid after solidification, remove second template to obtain third template;S4, using solidifiable material to fill and solidify the third template, then remove third template, obtain microstring biomechanics chip.The application can prepare microstring biomechanics chip with high smoothness, and using it for culture can change the mechanical constraint applied on hydrogel, obtain more diverse and accurate results.

Description

Technical Field

[0001] This invention relates to the field of biochips, and in particular to a microstring biomechanical chip, its fabrication method, and its applications. Background Technology

[0002] With the interdisciplinary integration of mechanics, materials science, engineering, and biomedicine, an increasing number of researchers are dedicated to analyzing and solving biomedical problems from a mechanical perspective, providing new insights for scientific research and disease prevention closely related to human health. Biomechanical chips, as an advanced biomimetic microsystem, have become an important tool for studying biomechanical problems. Among them, engineered stretch tissues based on biomechanical chips provide strong technical support for the mechanobiological study of stretch stimuli. To apply mechanical stimulation to stretch tissues during construction to study the mechanobiological response of cells, the chip structure needs to be designed to anchor the tissue, apply constraints, and exert mechanical stretching. Existing chip anchoring methods are mainly divided into two categories: chemical anchoring and physical anchoring.

[0003] Chemical anchoring fixes the hydrogel by establishing chemical cross-links between the hydrogel and the anchoring material. However, this method has low versatility, and different hydrogel materials and types require different chemical cross-linking methods. Physical anchoring mainly uses clamps at both ends of the chip to fix the hydrogel, or designs the hydrogel into a ring shape similar to a running track, so that two rods can be used to anchor the hydrogel and achieve mechanical stretching. This method has higher versatility but requires high mechanical properties of the hydrogel, is prone to damage during clamping and stretching, and is difficult to obtain small-scale microstructures. With the development of micro-nano fabrication technology, methods based on elastic micropillars or microstrings have received increasing attention, but existing methods still suffer from problems such as high preparation conditions and costs, poor operability, and difficulty in widespread application. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a method for fabricating a micro-string biomechanical chip, comprising the following steps:

[0005] S1. Provide a first template with through holes on its horizontal or vertical sides;

[0006] S2. Prepare several through cavities in the first template and make at least two through holes pass through the cavities to obtain the second template;

[0007] S3. Pass a strip-shaped solid through the through hole of the second template, then fill the cavity of the second template with sacrificial material, remove the strip-shaped solid after curing, and remove the second template to obtain the third template, wherein the volume of the sacrificial material added is greater than the volume of the cavity;

[0008] S4. The third template is filled and cured with a curable material, and then the third template is removed to obtain a micro-string biomechanical chip.

[0009] Further, the first template in step S1 is obtained by curing polydimethylsiloxane and curing agent mixed in a mass ratio of 4 to 40:1 at a curing temperature of 40 to 100°C.

[0010] Furthermore, the sacrificial material in step S3 includes at least one of a gelatin aqueous solution, an alginate aqueous solution, and a hyaluronic acid aqueous solution with a mass concentration of 10% to 40%.

[0011] Furthermore, the curing temperature in step S3 is 0–10°C.

[0012] Furthermore, the curable material in step S4 is a thermosetting material or a photocurable material;

[0013] Furthermore, in step S4, the third template is removed by immersion in water at 50–80°C.

[0014] The present invention also provides a mold used in the above-mentioned preparation method, the mold comprising a lower cover plate, a structural frame, a laying plate and an upper cover plate assembled from bottom to top;

[0015] The structural frame includes a main body with a first storage space in the middle. The main body is provided with micro-grooves that can penetrate the first storage space in the horizontal or vertical direction. The structural frame is used to fill thermosetting materials or sacrificial materials and form through holes.

[0016] The paving board includes a board body with a second storage space in the middle. The bottom areas of the first and second storage spaces are equal, and the bottom areas of the structural frame and the paving board are equal.

[0017] The upper cover plate is provided with an injection hole and an vent hole.

[0018] Furthermore, the diameter of the microgroove is 0.15–0.55 mm, which needs to be slightly larger than the diameter of the strip-shaped solid.

[0019] Furthermore, the through hole can be obtained by passing a strip-shaped solid through a microgroove and then through a mixture of polydimethylsiloxane (PDMS) and a curing agent, and then removing the strip-shaped solid after the mixture has cured; the size of the strip-shaped solid is matched with that of the through hole, and its diameter is 0.1 to 0.5 mm, and the strip-shaped solid includes acupuncture needles, carbon fiber bundles, glass rods, etc.

[0020] The present invention also provides a microstring biomechanical chip, which is prepared using the above-described preparation method.

[0021] The present invention also provides an application of the above-mentioned microstring biomechanical chip in tissue tensile stress detection.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention first prepares a first template with through holes using a mold. Then, a cavity is cut into the first template, with at least two through holes passing through it to obtain a second template. A strip of solid is passed through the second template, and a sacrificial material is filled into the cavity. After curing, the strip of solid is removed, and the second template is removed to obtain a third template, resulting in a sacrificial template. This sacrificial template is then filled with a curable material and removed, yielding a microstring biomechanical chip with controllable microstring dimensions and high smoothness. This invention can prepare highly smooth microstring biomechanical chips, and using them for cultivation can alter the mechanical constraints applied to the hydrogel, resulting in more diverse and precise results. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 A schematic diagram of the mold used in this invention is shown;

[0026] Figure 2 A flowchart illustrating the fabrication method of the micro-string biomechanical chip in Embodiment 1 of the present invention is shown;

[0027] Figure 3a A schematic diagram is shown of a probe connected to a sensor performing a bending test on the microstring of the microstring biomechanical chip of Embodiment 1 of the present invention. Figure 3b The diagram shows the relationship between the mid-span deflection of the microstring and the concentrated load in the microstring biomechanical chip of Example 1.

[0028] Figure 4a Optical microscope images showing the shrinkage and deformation of the hydrogel and the deformation of the microstrings are presented. Figure 4b A schematic diagram of the process for obtaining stretched tissue by culturing collagen hydrogels using a micro-string biomechanical chip;

[0029] Figure 5 The graph shows the relationship between shrinkage force and time during the culture of collagen hydrogels;

[0030] Explanation of reference numerals in the attached figures:

[0031] 1. Lower cover plate; 2. Structural frame; 21. Main body; 22. Micro-groove; 23. First storage space; 3. Paving board; 31. Board body; 32. Second storage space; 4. Upper cover plate; 41. Liquid injection hole; 42. Vent hole. Detailed Implementation

[0032] The endpoints and any values ​​of the ranges disclosed in this invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this invention.

[0033] Given the difficulty in accurately ensuring the formation of microstrings through direct cutting, and the high requirements for the size and smoothness of the microstrings during cutting, this invention first uses PDMS to prepare a substrate with through holes. Then, cavities are formed by cutting on the substrate. A curable and soluble sacrificial material is filled into the cavity to form a sacrificial template. After filling the sacrificial template with PDMS and removing the sacrificial template, a microstring biomechanical chip with controllable microstring size and high smoothness can be obtained.

[0034] To match the fabrication method of the microstring biomechanical chip of the present invention, such as Figure 1 As shown, the mold used in this invention includes a lower cover plate 1, a structural frame 2, a laying plate 3, and an upper cover plate 4 that can be assembled from bottom to top. The structural frame 2 includes a main body 21, with a first storage space 23 in the middle of the main body 21. The main body 21 has a microgroove 22 in its longitudinal direction to accommodate rigid needles and to lead to the first storage space 23. The structural frame 2 is used to fill thermosetting materials or sacrificial materials and to accommodate the passage of rigid needles, and to remove the rigid needles after the thermosetting materials or sacrificial materials have cured, forming a through hole. The laying plate 3 includes a plate body 31, with a second storage space 32 in the middle of the plate body 31. The bottom areas of the first storage space 23 and the second storage space 32 are equal, and the bottom areas of the structural frame 2 and the laying plate 3 are equal. The upper cover plate 4 is provided with an injection hole 41 and an exhaust hole 42.

[0035] More specifically, the bottom surfaces of both the first storage space 23 and the second storage space 32 are rectangles of 30mm × 40mm. Six microgrooves 22, each spaced 3mm apart, are provided to accommodate rigid needles. The injection hole 41 and vent hole 42 of the upper cover plate 4 are both 2mm in diameter. When the mold is assembled, the first storage space 23 of the structural frame 2 corresponds to the second storage space 32 of the cover plate 3. External substances can be injected through the injection hole 41 of the upper cover plate 4, and air in the substances can be expelled through the vent hole 42 of the upper cover plate 4. The structural frame 2 is made of a stable metal, such as aluminum alloy. The lower cover plate 1, cover plate 3, and upper cover plate 4 are all laser-cut stable materials that can withstand temperatures of at least 100°C and do not chemically react with experimental reagents, such as polymethyl methacrylate (PMMA). The thickness of the cover plate 3 can be adjusted to change the thickness of the micro-string chip.

[0036] The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments and accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. The structure of the mold in the embodiments will not be described again.

[0037] Example 1

[0038] A method for fabricating a micro-string biomechanical chip, such as Figure 2 As shown, the steps are as follows:

[0039] Step 1: Prepare PDMS mixture and gelatin solution: Mix PDMS monomer and curing agent evenly at a mass ratio of 10:1. Then, slowly evacuate the vacuum oven until no obvious bubbles rise and burst, to obtain the PDMS mixture. Transfer it to a centrifuge tube and store it at -20℃ in the lower part of the refrigerator for later use. Weigh 12g of gelatin powder and add ultrapure water to 40mL to prepare a 30%wt gelatin solution. Dissolve the gelatin completely at 60℃ and centrifuge at 2000r / min for 2-3min until no obvious bubbles are observed. Store it at 4℃ in the upper part of the refrigerator for later use.

[0040] Step 2: Preparation of PDMS initial template: Assemble the mold in the order of lower cover plate 1, structural frame 2, laying plate 3 and upper cover plate 4, and fix both ends with clamps; pass an acupuncture needle with a diameter of 0.2 mm through the microgroove 22 of the structural frame 2, and inject the PDMS mixture prepared in step 1 through the injection hole 41 of the upper cover plate 4, and then place it at 60℃ for 24 h to allow the PDMS mixture to completely solidify; after solidification, remove the acupuncture needle, take out the PDMS initial template, and laser cut it at a speed of 8 mm / s and a power of 60% to form 18 cavities. Before cutting, use a low-power cutting positioning plate for positioning to ensure that each cavity has two symmetrical through holes;

[0041] Step 3: Preparation of the gelatin template: Preheat the gelatin solution prepared in Step 1 to 40°C until it has sufficient fluidity. Assemble the mold in the order of lower cover plate 1, structural frame 2, laying plate 3, and upper cover plate 4. At this time, the PDMS initial template prepared in Step 2 needs to be assembled together, and an acupuncture needle with a diameter of 0.2 mm is used to pass through the microgroove 22 and the initial template. Before use, clean the PDMS initial template with a plasma cleaner to maintain the activity of PDMS while removing surface dirt. The volume of the injected gelatin solution should be larger than the volume of the cavity of the PDMS initial template to ensure that there is still gelatin solution on the upper part of the PDMS initial template. After fixation, inject the preheated gelatin solution through the injection hole 41 of the upper cover plate 4, and then place it on the upper shelf of the refrigerator (4°C) for 1-2 hours to allow the gelatin to solidify. After solidification, remove the acupuncture needle and carefully separate the initial template to obtain the gelatin template.

[0042] Step 4: Fabrication of the microstring biomechanical chip: Assemble the mold in the order of lower cover plate 1, substrate 3, and upper cover plate 4. Note that the gelatin template prepared in step 3 needs to be assembled together. At this time, the thickness of the microstring chip and whether there is a bottom surface in the cavity can be changed by controlling the thickness of substrate 3. In this embodiment, the thickness of substrate 3 is the same as the total thickness of structural frame 2 and substrate 3 in step 4. After fixing, inject the PDMS mixture prepared in step 1 through the injection hole 41 of the upper cover plate 4. After the PDMS is completely immersed in the microstring cavity formed by the gelatin template, place it at 27°C to allow the PDMS to fully cure for 4 days. After curing, immerse it in water at 60°C to dissolve the gelatin and dry it to obtain the microstring biomechanical chip.

[0043] Example 2

[0044] Application of the micro-string biomechanical chip prepared in Example 1 in tissue tensile stress detection:

[0045] Step 1: Place the probe connected to the sensor vertically in the middle of the microstring and move it up and down at a constant rate. Figure 3a A bending test was performed on the selected microchord, and the collected data was then processed to obtain the force-displacement relationship of the microchord under an approximate concentrated load at mid-span. Figure 3b );

[0046] Step 2: Take 100 μL of 10× phosphate buffer, 33 μL of 0.2 mol / L NaOH aqueous solution, 250 μL of 6 mg / mL collagen, and 417 μL of sterile water, to make a solution with a concentration of 10... 8 200 μL of cell suspension (cells / mL) were thoroughly mixed to obtain a collagen solution with a final concentration of 1.5 mg / mL, in which the cell concentration was 2 × 10⁻⁶ cells / mL. 7 cells / mL, spread evenly in the microstring cavity;

[0047] Step 3: Incubate the hydrogel in a 37℃, 90% RH incubator for 45 minutes to allow the collagen to crosslink into a hydrogel. Continue incubation for 1-2 days and observe the shrinkage and deformation of the hydrogel and the deformation of the microstrings under cell activity. Figure 4a Ultimately, this can result in a contracted stretch tissue. Figure 4b A schematic diagram of this process is shown. The shrinkage force during the deformation of the stretch tissue can be obtained through the deformation of the microstring and its force-displacement relationship curve, and thus its shrinkage force-time relationship can be obtained as follows: Figure 5 As shown.

[0048] This invention first prepares a first template with through holes using a mold. Then, a cavity is cut into the first template, with at least two through holes passing through it to obtain a second template. A strip of solid is passed through the second template, and a sacrificial material is filled into the cavity. After curing, the strip of solid is removed, and the second template is removed to obtain a third template, resulting in a sacrificial template. This sacrificial template is then filled with a curable material and removed, yielding a microstring biomechanical chip with controllable microstring dimensions and high smoothness. This invention can prepare highly smooth microstring biomechanical chips, and using them for cultivation can alter the mechanical constraints applied to the hydrogel, resulting in more diverse and precise results.

[0049] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for fabricating a micro-string biomechanical chip, characterized in that, Includes the following steps, S1. Provide a first template with through holes on its horizontal or vertical sides; S2. Form several through cavities on the non-through-hole surface of the first template and make at least two through holes pass through the cavities to obtain the second template; S3. Pass a strip of solid through the through hole of the second template, then fill the cavity of the second template with sacrificial material, remove the strip of solid after curing, and remove the second template to obtain the third template; S4. The third template is filled and cured with a curable material, and then the third template is removed to obtain a micro-string biomechanical chip.

2. The method for fabricating the microstring biomechanical chip according to claim 1, characterized in that, The first template in step S1 is obtained by curing polydimethylsiloxane and curing agent mixed in a mass ratio of 4~40:1 at a curing temperature of 40~100℃.

3. The method for fabricating the microstring biomechanical chip according to claim 1, characterized in that, The sacrificial material in step S3 includes at least one of the following: a gelatin aqueous solution, an alginate aqueous solution, and a hyaluronic acid aqueous solution with a mass concentration of 10% to 40%.

4. The method for fabricating the microstring biomechanical chip according to claim 1, characterized in that, The curing temperature in step S3 is 0~10℃.

5. The method for fabricating the microstring biomechanical chip according to claim 1, characterized in that, The curable material in step S4 is either a thermosetting material or a photocurable material.

6. The method for fabricating the microstring biomechanical chip according to claim 3, characterized in that, In step S4, the third template is removed by immersion in water at 50~80℃.

7. A mold used in the preparation method according to any one of claims 1 to 6, characterized in that, The mold includes a lower cover plate, a structural frame, a laying plate, and an upper cover plate assembled from bottom to top; the lower cover plate, structural frame, laying plate, and upper cover plate are assembled to prepare a first template and a third template; the lower cover plate, laying plate, and upper cover plate are assembled together with the third template to prepare a micro-string biomechanical chip. The structural frame includes a main body, a first storage space in the middle of the main body, and micro-grooves that can penetrate the first storage space are provided horizontally or vertically in the main body. The through-hole is formed by passing a strip-shaped solid through a microgroove and then through a mixture of polydimethylsiloxane and a curing agent, and then removing the strip-shaped solid after the mixture has cured. The paving board includes a board body with a second storage space in the middle. The bottom areas of the first and second storage spaces are equal, and the bottom areas of the structural frame and the paving board are equal. The upper cover plate is provided with an injection hole and an vent hole.

8. The mold according to claim 7, characterized in that, The diameter of the microgroove is 0.15~0.55mm.

9. A micro-string biomechanical chip, characterized in that, It was prepared using the preparation method described in any one of claims 1 to 6.

10. The application of the microstring biomechanical chip of claim 9 in the detection of tissue tensile stress.

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