A biochip and a method for manufacturing a biochip

Abstract

The application discloses a biochip and a manufacturing method of the biochip, and aims at improving the problem that it is difficult for nano or micron magnetic beads to enter holes or enter holes uniformly. The biochip comprises a first substrate and a plurality of beads. The first substrate has a plurality of recesses, and at least part of the beads is embedded in the recesses. The recesses are provided with first groups on surfaces facing the beads, and the beads are provided with second groups. The first groups of the recesses are connected with the second groups of the beads, so that the beads are fixed in the recesses.

Description

Technical Field

[0001] This invention relates to the field of biochip technology, and more particularly to a biochip and a method for fabricating a biochip. Background Technology

[0002] Gene chips, also known as DNA chips or DNA microarrays, are an important form of biochips. Gene chip technology has developed rapidly since the mid-1990s and has become a crucial technology in molecular biology. Gene chip technology offers numerous advantages, including high throughput, speed, sensitivity, and accuracy. It can simultaneously detect and analyze large numbers of sequences from a sample, thus overcoming the shortcomings of traditional nucleic acid blotting hybridization techniques, such as cumbersome operation, low automation, limited number of sequences processed, and low detection efficiency. Currently, gene chip technology is widely used in medicine, biology, agriculture, environmental protection, and many other fields. In the medical field, gene chip technology can be used for disease diagnosis and treatment, drug research, etc.; in the biological field, it can be used for gene expression detection, DNA sequencing, and the detection of mutants and polymorphisms.

[0003] In related technologies, during the fabrication of biochips, it is necessary to fabricate nano or micro magnetic beads in a substrate with micropores. Existing technologies typically involve introducing nano or micro magnetic beads stored in a preservation solution into the micropores of the substrate through the fluidity of the liquid. However, in this method, due to the small weight of the nano or micro magnetic beads, the preservation solution provides significant resistance to their buoyancy, making it difficult for the nano or micro magnetic beads to enter or uniformly enter the pores. Summary of the Invention

[0004] This invention provides a biochip and a method for fabricating a biochip, thereby improving the difficulty of inserting or uniformly inserting nano or micro magnetic beads into holes.

[0005] This invention provides a biochip, comprising: a first substrate and a plurality of beads; the first substrate has a plurality of recesses, and at least a portion of the beads are embedded in the recesses;

[0006] The recessed portion has a first group on its surface facing the bead, and the bead has a second group; the first group of the recessed portion is connected to the second group of the bead, so that the bead is fixed in the recessed portion.

[0007] In one possible implementation, the first group of the recess is connected to the second group of the bead via covalent bonds and / or hydrogen bonds and / or electrostatic bonds.

[0008] In one possible implementation, the first group comprises -COOH and / or -OH; the second group comprises -NH2, and / or -COOH, and / or -OH.

[0009] In one possible implementation, at least a portion of the outer surface of the bead further has a coating layer comprising the first group.

[0010] In one possible implementation, the coating layer includes at least one of the following:

[0011] paraffin;

[0012] Chitosan;

[0013] Sodium alginate;

[0014] Polycaprolactone diol;

[0015] Cellulose;

[0016] Polylactic acid.

[0017] In one possible implementation, the bead is a magnetic bead, and the size of the bead ranges from 0.1 micrometers to 100 micrometers.

[0018] This disclosure also provides a method for fabricating a biochip, comprising:

[0019] Solid molecularly imprinted polymers are formed by mixing beads with polymers;

[0020] The molecularly imprinted polymer is placed on a sieve plate with multiple sieve holes and ground to obtain a bead precursor with the same sieve hole diameter as the sieve hole diameter. The bead precursor includes: a bead and a coating layer covering the outer surface of the bead.

[0021] The bead precursor is placed on the first substrate having multiple recesses, so that the bead precursor enters the recesses of the first substrate, wherein the diameter of the sieve hole is less than or equal to the diameter of the recess.

[0022] The beads are attracted by a magnet and subjected to a first treatment to remove at least a portion of the coating layer on the surface of the bead precursor.

[0023] In one possible implementation, after obtaining a bead precursor with the same aperture size as the sieve, and before placing the bead precursor on the first substrate having a plurality of recesses, the fabrication method further includes:

[0024] The bead precursor is placed on a second substrate, and a magnet is placed on the other side of the second substrate. The second substrate is then tilted to screen out the beads that are not coated with the polymer.

[0025] In one possible implementation, the solid molecularly imprinted polymer that forms a mixture of beads and polymer comprises: mixing paraffin wax, stearic acid and beads and heating them, then adding a pore-forming agent to form a wax / water emulsion, and adding ethanol to the wax / water emulsion for curing.

[0026] Alternatively, the solid molecularly imprinted polymer that forms a mixture of beads and polymer comprises: mixing chitosan and acetic acid to form a chitosan solution and adding it to the beads, and then dropping the chitosan solution containing the beads into a sodium tripolyphosphate solution to obtain spherical particle media, and drying it.

[0027] Alternatively, the solid molecularly imprinted polymer that forms a mixture of beads and polymer may be further comprising: adding sodium alginate solution to a mixture of CaCl2 and the beads, followed by filtration, washing, and drying.

[0028] Alternatively, the solid molecularly imprinted polymer formed by mixing the beads with the polymer includes: adding the beads to a liquid containing acrylic acid, adding polycaprolactone diol and an initiator to form a gel-like polymer and then drying it.

[0029] Alternatively, the solid molecularly imprinted polymer that forms a mixture of beads and polymer includes: forming a cellulose graft copolymer containing amide groups, reacting it with sodium hydroxide, formaldehyde, and sodium bisulfite to form a modified cellulose graft copolymer, adding the modified cellulose graft copolymer to a solution containing beads, adding a crosslinking agent to react, and then washing and drying.

[0030] Alternatively, the solid molecularly imprinted polymer that forms a mixture of beads and polymer may include: adding an organic phase containing polylactic acid to an aqueous phase containing the beads and an emulsifier to form an emulsion, and performing extraction, evaporation, and extraction processes to evaporate the solvent.

[0031] In one possible implementation, the first treatment to remove at least a portion of the coating layer on the surface of the bead precursor includes: removing at least a portion of the coating layer on the surface of the bead precursor by heating, or acidic liquid treatment, or enzymatic degradation.

[0032] The beneficial effects of the embodiments of the present invention are as follows: In the embodiments of the present invention, the surface of the recessed portion of the first substrate facing the bead has a first group, and the bead has a second group; the first group of the recessed portion is connected to the second group of the bead so that the bead is fixed in the recessed portion. That is, when the bead enters the recessed portion 1 of the first substrate, the bead can be adsorbed into the recessed portion through the interaction between the first group and the second group, which can improve the problem of difficulty in the entry or uniform entry of nano or micro magnetic beads into holes. Attached Figure Description

[0034] Figure 1 This is one of the structural schematic diagrams of the bead and the first substrate provided in an embodiment of the present invention;

[0035] Figure 2 A schematic diagram of the structure of the first substrate is provided for an embodiment of the present invention;

[0036] Figure 3 This is an enlarged schematic diagram of the bead and the first substrate provided in an embodiment of the present invention;

[0037] Figure 4 This is a second schematic diagram of the structure of the bead and the first substrate provided in an embodiment of the present invention;

[0038] Figure 5 One of the schematic diagrams of a bead with a coating layer on its surface provided in an embodiment of the present invention;

[0039] Figure 6 A second schematic diagram showing the surface of a bead covered with a coating layer according to an embodiment of the present invention;

[0040] Figure 7 A schematic diagram illustrating the fabrication process of a biochip provided in an embodiment of the present invention;

[0041] Figure 8 The paraffin molecular formula provided in the embodiments of the present invention;

[0042] Figure 9 The stearic acid structural formula provided in the embodiments of the present invention;

[0043] Figure 10 This is a schematic diagram of the sieve plate provided in an embodiment of the present invention. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of the embodiments of this disclosure clearer, the technical solutions of the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this disclosure. All other embodiments obtained by those skilled in the art based on the described embodiments of this disclosure without creative effort are within the scope of protection of this disclosure.

[0045] Unless otherwise defined, the technical or scientific terms used in this disclosure shall have the ordinary meaning understood by one of ordinary skill in the art to which this disclosure pertains. The terms “first,” “second,” and similar terms used in this disclosure do not indicate any order, quantity, or importance, but are merely used to distinguish different components. Terms such as “comprising” or “including” mean that the element or object preceding the word encompasses the elements or objects listed following the word and their equivalents, without excluding other elements or objects. Terms such as “connected” or “linked” are not limited to physical or mechanical connections, but can include electrical connections, whether direct or indirect. Terms such as “upper,” “lower,” “left,” and “right” are used only to indicate relative positional relationships, and these relative positional relationships may change accordingly when the absolute position of the described objects changes.

[0046] As used herein, “approximately” or “substantially the same” includes the stated value and means within an acceptable range of deviations from the specific value, as determined by a person skilled in the art taking into account the measurement in question and the errors associated with the measurement of the specific quantity (i.e., limitations of the measurement system). For example, “substantially the same” may mean a difference relative to the stated value within one or more standard deviations, or within ±30%, 20%, 10%, or 5%.

[0047] In the accompanying drawings, the thicknesses of layers, films, panels, regions, etc., are enlarged for clarity. Exemplary embodiments are described herein with reference to cross-sectional views that are schematic diagrams of idealized embodiments. Thus, deviations from the shapes shown in the drawings will be expected as a result of, for example, manufacturing techniques and / or tolerances. Therefore, the embodiments described herein should not be construed as limited to the specific shapes of the regions shown herein, but rather include deviations in shape caused, for example, by manufacturing processes. For example, regions illustrated or described as flat may typically have rough and / or non-linear characteristics. Furthermore, sharp corners illustrated may be rounded. Thus, the regions shown in the figures are schematic in nature, and their shapes are not intended to illustrate the precise shapes of the regions, nor are they intended to limit the scope of the claims.

[0048] To keep the following description of the embodiments of this disclosure clear and concise, detailed descriptions of known functions and known components are omitted.

[0049] See Figures 1-4As shown, an embodiment of the present invention provides a biochip, including: a first substrate 1 and a plurality of beads 2; the first substrate 1 has a plurality of recesses 10, and at least a portion of the beads 2 is embedded in the recesses 10; wherein, the surface of the recesses 10 facing the beads 2 has a first group X, and the beads 2 have a second group Y; the first group X of the recesses 10 is connected to the second group Y of the beads 2 so that the beads 2 are fixed in the recesses 10.

[0050] In this embodiment of the invention, the surface of the recessed portion 10 of the first substrate 1 facing the bead 2 has a first group X, and the bead 2 has a second group Y; the first group X of the recessed portion 10 is connected to the second group Y of the bead 2 so that the bead 2 is fixed in the recessed portion 10. That is, when the bead 2 enters the recessed portion 1 of the first substrate 1, the bead 2 can be adsorbed into the recessed portion 10 through the interaction between the first group X and the second group Y, which can improve the problem of difficulty in the entry or uniform entry of nano or micro magnetic beads.

[0051] In one possible implementation, bead 2 can be a magnetic bead, specifically a micron-sized magnetic bead or a nano-sized magnetic bead; the size of bead 2 ranges from 0.1 micrometers to 100 micrometers. Optionally, bead 2 can be a silicon bead.

[0052] Bead array technology, also known as fiber optic microbead chip technology or BeadArray technology, is a gene chip technology based on optical fibers and nanomaterials (such as silicon beads). This technology features high density, high repeatability, high sensitivity, low sample loading, and flexible customization, overcoming several bottlenecks of traditional chip technologies and significantly improving detection and screening speed while reducing research costs. This technology requires the uniform placement of micron-sized magnetic beads within micropores before other operational steps; therefore, ensuring the uniform entry of magnetic beads into the micropores is a prerequisite for its application. In this embodiment of the invention, the biochip can be applied to bead array technology, and the beads can be microbeads within it.

[0053] In one possible implementation, the recesses 10 of the first substrate 1 can be arranged in an array, and correspondingly, the beads 2 can be distributed in an array within the recesses 10.

[0054] In one possible implementation, the first group X of the recess 10 is connected to the second group Y of the bead 2 by covalent bonds and / or hydrogen bonds and / or electrostatic bonds.

[0055] In one possible implementation, the first group X comprises -COOH and / or -OH; the second group Y comprises -NH2, and / or -COOH, and / or -OH. Optionally, the first group X comprises -COOH and the second group Y comprises -NH2; alternatively, the first group X comprises -COOH and the second group Y comprises -OH; alternatively, the first group X comprises -OH and the second group Y comprises -COOH; thus, the interaction force between the first group X and the second group Y is achieved through the mutual attraction between the weak acid and the weak base, thereby enabling the bead 2 to be embedded in the recess 10.

[0056] In one possible implementation, the first group X and the second group Y in this embodiment of the present disclosure may be mutually attracted without undergoing a chemical reaction. For example, the first group X includes -COOH; the second group Y includes -OH. The two may be combined without undergoing a dehydration reaction, but only through the mutual attraction between weak acid and weak base, so that the first group X and the second group Y have an interaction force, thereby embedding the bead 2 in the recess 10. This method is easy to operate and does not require further conditional reactions. In another possible implementation, the first group X and the second group Y in this embodiment of the present disclosure may also be mutually attracted through a chemical reaction. For example, the first group X includes -COOH; the second group Y includes -OH. The two may be combined through a dehydration reaction, thereby achieving a chemical bond bond, so that the first group X and the second group Y have an interaction force, thereby embedding the bead 2 in the recess 10.

[0057] In one possible implementation, at least a portion of the outer surface of the bead 2 further has a coating layer 21, the coating layer including a first group X.

[0058] In this embodiment of the invention, when the bead 2 enters the recess 10 of the first substrate 1, a coating layer 21 can be formed on the outer surface of the bead 2. The coating layer 21 contains a large number of second groups Y. Through the mutual attraction between the second group Y and the first group X in the recess 10, the bead 2 enters the recess 10 of the first substrate 1. Moreover, by forming a coating layer 21 on the outer surface of the bead 2, the method of entering the hole with the preservation liquid in the related technology is changed to direct solid entry into the hole. This avoids the obstruction of liquid buoyancy when the preservation liquid is mixed and entered into the hole. At the same time, the exposed groups of the molecular imprinted layer can be connected with the modified groups in the recess 10 to play a fixing role, thus solving the problem of uniform entry of the bead 2 into the hole.

[0059] In practical implementation, before the bead 2 covered by the coating layer 21 enters the recess 10 of the first substrate 1, the coating layer 21 covers a large portion of the surface of the bead 2, for example, as Figure 5As shown, the coating layer 21 can cover the entire surface of the bead 2. After the bead 2 with the coating layer 21 enters the recess 10 of the first substrate 1, at least a portion of the coating layer 21 on the surface of the bead 2 can be removed by heating, acidic liquid treatment, or enzymatic degradation. In specific implementations, a portion of the coating layer 21 on the surface of the bead 2 may be left. For example, the coating layer 21 on the surface of the bead 2 facing the recess 10 may be left, such as... Figure 6 As shown.

[0060] In one possible implementation, the coating layer 21 can be a molecularly imprinted polymer. Molecular imprinting utilizes the enzyme-substrate or antibody-antigen interaction of molecularly imprinted polymers to specifically recognize imprinted molecules (also called template molecules). A solid medium is obtained by polymerizing the template molecules with a crosslinking agent in a polymer monomer solution. Subsequently, the template molecules in the medium are eluted away by physical or chemical methods, leaving cavities in the polymer that match the spatial structure and binding sites of the template molecules. These cavities have selective recognition properties for template molecules and their analogues. Molecularly imprinted polymers can be prepared using paraffin or similar substances as solvents, porogens, or reaction media, and the rigid backbone of the molecularly imprinted polymer can be destroyed by means of high temperatures or other methods.

[0061] In one possible implementation, the covering layer 21 includes at least one of the following:

[0062] paraffin;

[0063] Chitosan;

[0064] Sodium alginate;

[0065] Polycaprolactone diol;

[0066] Cellulose;

[0067] Polylactic acid.

[0068] Based on the same inventive concept, embodiments of the present invention also provide a method for fabricating a biochip, see [link to relevant documentation]. Figure 7 As shown, the manufacturing method includes:

[0069] Step S100: Forming a solid molecularly imprinted polymer by mixing beads and polymer;

[0070] Step S200: Place the molecularly imprinted polymer on a sieve plate with multiple sieve holes and grind it to obtain a bead precursor with the same sieve hole diameter as the sieve hole diameter. The bead precursor includes: a bead and a coating layer wrapped around the outer surface of the bead.

[0071] Step S300: Place the bead precursor into a first substrate having multiple recesses, so that the bead precursor enters the recesses of the first substrate, wherein the diameter of the sieve hole is less than or equal to the diameter of the recess.

[0072] Step S400: Adsorb the beads with a magnet and perform a first treatment to remove at least part of the coating layer on the surface of the bead precursor.

[0073] Compared to related technologies that involve the beads 2 entering the hole along with the preservation liquid, the present invention directly introduces solids into the hole, avoiding the obstruction of liquid buoyancy when the preservation liquid is mixed into the hole. At the same time, the exposed groups of the molecular imprinted layer (i.e., the coating layer 21) can be connected with the modified groups in the recessed portion 10, playing a fixing role and improving the problem of uniform entry of the beads 2 into the hole.

[0074] In one possible implementation, after step S200 and before step S300, that is, after obtaining a bead precursor with the same aperture as the sieve, and before placing the bead precursor on a first substrate having multiple recesses, the manufacturing method further includes:

[0075] Step S500: Place the bead precursor on the second substrate, place a magnet on the other side of the second substrate, and tilt the second substrate to screen out beads that are not coated with polymer.

[0076] In one possible implementation, step S100, forming a solid molecularly imprinted polymer by mixing beads and polymer, includes: mixing paraffin wax, stearic acid and beads and heating, then adding a pore-forming agent to form a wax / water emulsion, and adding ethanol to the wax / water emulsion for curing.

[0077] Alternatively, a solid molecularly imprinted polymer is formed by mixing beads and polymer, comprising: mixing chitosan and acetic acid to form a chitosan solution and adding beads, and dripping the chitosan solution containing beads into a sodium tripolyphosphate solution to obtain spherical particle media, and then drying them;

[0078] Alternatively, a solid molecularly imprinted polymer is formed by mixing beads with a polymer, including: adding sodium alginate solution to a mixture of CaCl2 and beads, and then filtering, washing, and drying.

[0079] Alternatively, forming a solid molecularly imprinted polymer by mixing beads with a polymer includes: adding beads to a liquid containing acrylic acid, adding polycaprolactone diol and an initiator, forming a gel-like polymer and drying it.

[0080] Alternatively, a solid molecularly imprinted polymer is formed by mixing beads with a polymer, including: forming a cellulose graft copolymer containing amide groups, reacting with sodium hydroxide, formaldehyde, and sodium bisulfite to form a modified cellulose graft copolymer, adding the modified cellulose graft copolymer to a solution containing beads, adding a crosslinking agent to react, and then washing and drying.

[0081] Alternatively, forming a solid molecularly imprinted polymer by mixing beads and polymer, including: adding an organic phase containing polylactic acid to an aqueous phase containing beads and an emulsifier to form an emulsion, and performing extraction, evaporation, and extraction processes to evaporate the solvent.

[0082] In one possible implementation, regarding step S400, a first treatment is performed to remove at least a portion of the coating layer on the surface of the bead precursor, including: removing at least a portion of the coating layer on the surface of the bead precursor by heating, or acidic liquid treatment, or enzymatic degradation.

[0083] To better understand the method for fabricating a biochip provided in the embodiments of the present invention, the following specific examples illustrate the method:

[0084] Example 1

[0085] Step 1: Add a certain amount of paraffin wax, stearic acid (OA), and micron-sized magnetic beads to a glass tube. Place the glass tube in a water bath at 70℃~80℃; after the paraffin wax melts, place the mixture at 70℃ for 5min~10min to form an OA-magnetic bead complex, with a mass ratio of 10:2:1~18:4:1; at a higher temperature, a paraffin wax-stearic acid emulsion is formed; in this emulsion system, OA acts as a "functional monomer" and emulsifier, forming complex and stable imprint droplets. The structural formula of paraffin wax is as follows: Figure 8 As shown, the structural formula of stearic acid is as follows: Figure 9 As shown;

[0086] Step 2: Add a certain amount of 70℃~80℃ deionized water to this tube, and then add a certain amount of n-hexane as a pore-forming agent to the mixture. Let it stand at 70℃~80℃ for 5 minutes; after shaking vigorously by hand, a wax / water emulsion is formed.

[0087] Step 3: Quickly pour the emulsion into a beaker containing a certain amount of ethanol to solidify it, and then vacuum dry it at 30°C for 12 hours to obtain the molecularly imprinted polymer.

[0088] Step 4: Place the obtained molecularly imprinted polymer on a sieve plate and grind it to pass through the sieve plate holes. The diameter of the sieve plate holes is the same as the diameter of the micropores, thus ensuring that the volume of the molecularly imprinted polymer is uniform. The sieve plate can then... Figure 10 As shown, the sieve plate can have multiple sieve holes 3;

[0089] Step 5: The molecularly imprinted polymers after sieving are screened again using a magnet. The molecularly imprinted polymers are placed on a thin plate, and then a magnet is placed under the thin plate. The thin plate is then tilted. The molecularly imprinted polymers coated with micron magnetic beads will be attracted by the magnet, while the molecularly imprinted polymers not coated with micron magnetic beads will slide off, thus achieving the screening.

[0090] Step 6: Spread the screened molecularly imprinted polymer on a microplate. Modify the micropores with -COOH or -OH. The exposed chemical bonds in the paraffin molecularly imprinted coating layer will form hydrogen bonds with the chemical bonds in the micropores (the recessed part 10 of the first substrate 1), thereby fixing it in the micropores. Then remove the excess polymer outside the micropores with deionized water or wind.

[0091] Step 7: Then place the ferromagnet under the well plate and heat the well plate to 70℃~80℃. Due to the properties of paraffin and stearic acid, they will melt and change from solid to liquid. Since the micron magnetic beads are magnetic, they will be firmly attracted to the micropores by the magnet. Then, use deionized water to rinse repeatedly 3 to 5 times to remove the paraffin and stearic acid from the well plate.

[0092] Step 8: Rinse the orifice plate with ethanol and then dry it.

[0093] Example 2: A polymer encapsulating micron-sized magnetic beads was prepared using chitosan to achieve uniform pore placement of the micron-sized magnetic beads;

[0094] Chitosan is a natural polymer material obtained by removing some acetyl groups from the natural polysaccharide chitin. Its main chain contains multiple free amino and hydroxyl groups, and its abundant active functional groups enable it to generate more hydrogen bonds and electrostatic forces, improving its stable binding ability. Chitosan can also be degraded by various methods, including chemical, photodegradation, and enzymatic degradation. Taking advantage of the cationic nature of chitosan in acidic environments, sodium tripolyphosphate is added dropwise to a chitosan solution while stirring. The negatively charged phosphate ions attract the positively charged amino groups on the chitosan molecular chain through electrostatic adsorption, initiating cross-linking between chitosan molecules and ultimately forming microspheres. Based on this, chitosan can be used to achieve uniform pore placement of micron-sized magnetic beads. The specific steps are as follows:

[0095] Step 1: Weigh a certain amount of chitosan and add it to a certain volume of 2% acetic acid solution, stir and swell for 10-20 hours;

[0096] Step 2: Take a certain volume of chitosan solution, add a certain amount of micron magnetic beads to it, mix thoroughly and evenly, sonicate for 2-5 minutes to remove air bubbles, and then use the dropwise addition method to drop the chitosan solution into sodium tripolyphosphate solution to obtain spherical particle media. By controlling the dropwise addition rate, the particle diameter can be controlled to make it approximately the same as the micropore diameter. Then rinse three times with deionized water, dry and set aside for later use.

[0097] Step 3: Use a sieve plate to screen the obtained spherical particles to remove larger particles, and then use a magnet to screen again to obtain particles coated with micron magnetic beads.

[0098] Step 4: Then, the screened particles are spread evenly on the microplate. The abundant active functional groups of chitosan form hydrogen bonds with the chemical bonds in the micropores, thereby fixing them in the micropores. Then, deionized water or wind power is used to remove the excess polymer outside the micropores.

[0099] Step 5: Next, place the magnet below the well plate to fix the micron-sized magnetic beads. Then, repeatedly use weak organic acids such as formic acid to degrade the chitosan, breaking the glycosidic bonds in its molecules and destroying its rigid skeleton, thereby removing the chitosan from the micropores. Alternatively, irradiate the chitosan with ultraviolet, visible, or infrared light to trigger a degradation reaction and remove the chitosan. Experiments have shown that the degradation reaction is more obvious when the wavelength of the irradiated light is less than 360 nm. Alternatively, use chitosanase to specifically degrade the chitosan, breaking its molecular chains into low molecular weight fragments. Then, use an enzyme remover to remove the chitosanase, and then repeatedly wash with deionized water to remove the chitosan and complete the insertion of the micron-sized magnetic beads into the pores.

[0100] Example 3: A polymer encapsulating micron-sized magnetic beads was prepared using sodium alginate to achieve uniform pore placement of the micron-sized magnetic beads;

[0101] Sodium alginate is a natural polysaccharide composed of α-L-mannuronic acid and β-D-guluronic acid linked by 1,4-glycosidic bonds. Sodium alginate can crosslink with Ca2+ to form a gel with high mechanical strength, and thermosetting crosslinking can form microparticles. Sodium alginate is also biodegradable and can be degraded through various methods, thus it can be used to achieve uniform pore formation in micron-sized magnetic beads. The specific steps are as follows:

[0102] Step 1: Add a certain amount of sodium alginate to a certain amount of deionized water, stir thoroughly for 0.5-2 hours in a 30°C water bath, and then let it stand for a period of time.

[0103] Step 2: Weigh a certain amount of CaCl2 and micron-sized magnetic beads, add them to a certain volume of deionized water, stir thoroughly, and then slowly add them to a sodium alginate solution. After the addition is complete, continue stirring for 3-5 hours. The volume of the formed microspheres can be controlled by controlling the dropping rate. Then filter, wash, and transfer to a 60℃ oven to dry and heat-cur for 5-8 hours.

[0104] Step 3: Use a sieve plate to screen the obtained microspheres to remove larger particles, and then use a magnet to screen them again to obtain particles that encapsulate micron magnetic beads.

[0105] Step 4: Then, the screened particles are spread evenly on the microplate. The exposed active functional groups of sodium alginate are used to form hydrogen bonds with the chemical bonds in the micropores, thereby fixing them in the micropores. Then, deionized water or wind is used to remove the excess polymer outside the micropores.

[0106] Step 5: Then, place the ferromagnetic beads under the well plate to fix the micron magnetic beads. Then, use alginate to specifically degrade sodium alginate. Alginate can recognize and cut specific chemical bonds in the sodium alginate molecular chain, thereby decomposing it into low molecular weight compounds and destroying its rigid skeleton. Then, use an enzyme remover to remove the alginate, and then use deionized water to repeatedly wash, thereby removing sodium alginate and completing the insertion of the micron magnetic beads into the well.

[0107] Example 4: Polymers encapsulating micron-sized magnetic beads were prepared using polycaprolactone diol to achieve uniform pore placement of the micron-sized magnetic beads;

[0108] Polycaprolactone diol is a high-molecular-weight organic polymer, mainly produced by ring-opening polymerization of ε-caprolactone monomers under specific catalytic conditions. Polycaprolactone diol belongs to aliphatic polyesters, whose main chains are mostly composed of aliphatic structural units linked by easily hydrolyzable ester bonds. It is readily decomposed and metabolized by various microorganisms or enzymes in plants and animals, exhibiting good biocompatibility and biodegradability. Based on this, polycaprolactone diol can be used to achieve uniform pore formation in micron-sized magnetic beads. The specific steps are as follows:

[0109] Step 1: Dissolve a certain amount of polycaprolactone diol in a certain volume of benzene, add a certain mass of acryloyl chloride and triethylamine, and stir at 80℃ for 1-3 hours. After the reaction is complete, filter the solution and pour the filtrate into a beaker containing a certain volume of n-hexane. Let it stand at 20℃ for 10-15 hours. A white waxy solid precipitate will form at the bottom of the beaker. Dry the solid precipitate under vacuum for 24 hours.

[0110] Step 2: Dissolve a certain amount of the functional monomer acrylic acid in a certain volume of chloroform, then add a certain amount of micron magnetic beads, and let stand at room temperature for 30 minutes to allow the micron magnetic beads to fully interact with the functional monomer.

[0111] Step 3: Subsequently, acrylamide-modified polycaprolactone diol and an initiator were added. The initiator was prepared by dissolving benzoin dimethyl ether in N-vinylpyrrolidone. The mixed solution was irradiated under low-intensity ultraviolet light at a wavelength of 366 nm for 1 hour to obtain a gel-like polymer. After washing with chloroform, it was vacuum dried at 40°C for 24 hours to obtain the molecularly imprinted polymer.

[0112] Step 4: Place the obtained molecularly imprinted polymer on a sieve plate and grind it to pass through the sieve plate holes. The diameter of the sieve plate holes is the same as the diameter of the micropores, so as to ensure that the molecularly imprinted polymer has the same volume.

[0113] Step 5: The molecularly imprinted polymers after sieving are screened again using a magnet. The molecularly imprinted polymers are placed on a thin plate, and then a magnet is placed under the thin plate. The thin plate is then tilted. The molecularly imprinted polymers coated with micron magnetic beads will be attracted by the magnet, while the molecularly imprinted polymers not coated with micron magnetic beads will slide off, thus achieving the screening.

[0114] Step 6: Spread the screened particles evenly on the microplate, and use the active functional groups of polycaprolactone diol to form hydrogen bonds with the chemical bonds in the micropores to fix them in the micropores. Then, use deionized water or wind to remove the excess polymer outside the micropores.

[0115] Step 7: Then, place the magnet under the well plate to fix the micron magnetic beads. Then, use lipase to degrade polycaprolactone diol, breaking the glycosidic bonds in its molecules and destroying its rigid skeleton. At the same time, heating is used to accelerate the degradation process of acrylated polycaprolactone diol. Under high temperature conditions, the molecular chains of PCL will become more active and easier to break. Then, use an enzyme remover to remove the lipase, and then repeatedly wash with deionized water to remove polycaprolactone diol and complete the micron magnetic beads entering the well.

[0116] Example 5: Using cellulose to prepare a polymer that encapsulates micron-sized magnetic beads, achieving uniform pore placement of the micron-sized magnetic beads;

[0117] Cellulose itself possesses advantages such as being non-toxic, highly water-resistant, renewable, and biodegradable. It is a major component of plant cell walls, primarily derived from cotton, hemp, cereals, and other higher plants. Cellulose derivatives, such as cellulose esters, cellulose ethers, and graft copolymers, can be synthesized through chemical modification of cellulose. The cellulose macromolecular chain contains many reactive functional groups (such as -OH, -NH2), which can directly undergo cross-linking reactions with diisocyanates, epichlorohydrin, glutaraldehyde, etc., to obtain the desired molecularly imprinted polymers. Therefore, it can be used to achieve uniform pore formation in micron-sized magnetic beads. The specific steps are as follows:

[0118] Step 1: A certain mass of cotton fiber and ionic liquid 1-butyl-3-methylimidazolium chloride are placed in a reactor and dissolved at 100°C for 4 hours. The solution is then cooled to below 30°C, and a certain mass of chloroacetyl chloride is added under nitrogen purging. The mixture is heated to 50°C and reacted for 4 hours. Nitrogen purging is continued, and then a certain amount of cuprous bromide, 2,2'-bipyridine, ascorbic acid, and acrylamide are added. The living free polymerization reaction is carried out for a predetermined time. The product is then washed with water and ethanol to obtain cellulose graft copolymer containing amide groups.

[0119] Step 2: Add a certain amount of cellulose graft copolymer to a three-necked flask equipped with a stirrer, then add a certain amount of distilled water, adjust the system with sodium hydroxide solution, add a set amount of formaldehyde, heat to the reaction temperature, add a certain amount of sodium bisulfite while stirring, and after reacting for a period of time, the modified cellulose graft copolymer is obtained, filtered through a sieve, washed, and dried.

[0120] Step 3: Weigh a certain mass of the modified cellulose graft copolymer, place it in a certain amount of aqueous solution of micron magnetic beads, add an aqueous solution of glutaraldehyde as a crosslinking agent, and stir the reaction at 50°C for 24 hours. After the reaction is complete, repeatedly wash the product particles, and then dry them in a vacuum oven to obtain the molecularly imprinted polymer;

[0121] Step 4: Place the obtained molecularly imprinted polymer on a sieve plate and grind it to pass through the sieve plate holes. The diameter of the sieve plate holes is the same as the diameter of the micropores, so as to ensure that the molecularly imprinted polymer has the same volume.

[0122] Step 5: The molecularly imprinted polymers after sieving are screened again using a magnet. The molecularly imprinted polymers are placed on a thin plate, and then a magnet is placed under the thin plate. The thin plate is then tilted. The molecularly imprinted polymers coated with micron magnetic beads will be attracted by the magnet, while the molecularly imprinted polymers not coated with micron magnetic beads will slide off, thus achieving the screening.

[0123] Step 6: Spread the screened particles evenly on the microporous plate. Use the active functional groups of cellulose to form hydrogen bonds with the chemical bonds in the micropores to fix them in the micropores. Then use deionized water or wind to remove the excess polymer outside the micropores.

[0124] Step 7: Then place the magnet under the well plate to fix the micron magnetic beads. Then use cellulase to degrade the cellulose and destroy its rigid skeleton. Then use an enzyme remover to remove the cellulase. Then use deionized water to wash repeatedly to remove the cellulose and complete the insertion of the micron magnetic beads into the well.

[0125] Example 6: Polymers encapsulating micron-sized magnetic beads are prepared using polylactic acid to achieve uniform pore placement of the micron-sized magnetic beads;

[0126] Polylactic acid (PLA) is a biodegradable polymer made from lactic acid, also known as polylactide. PLA can break down its ester bonds in vivo or in the natural environment to form oligomers or lactic acid, ultimately degrading into carbon dioxide and water, which are harmless to humans and the environment. PLA can be prepared into biodegradable microspheres using methods such as emulsification solvent evaporation, emulsification solvent coagulation, and spray drying, thus enabling the creation of uniform pores in micron-sized magnetic beads. The specific steps are as follows:

[0127] Step 1: Dissolve polylactic acid in a suitable solvent (such as dichloromethane, trichloromethane, etc.);

[0128] Step 2: Slowly add the organic phase containing polylactic acid to the aqueous phase containing a certain amount of micron magnetic beads and emulsifiers (such as Span 80, Tween 80, etc.), and prepare an emulsion by mechanical stirring or ultrasonic emulsification.

[0129] Step 3: Under slow stirring, the solvent in the internally dispersed phase continuously diffuses to the external phase, transports to the liquid surface, and evaporates into the air. During this process, the extraction-evaporation-extraction process is repeated, causing the polylactic acid carrier material in the internally dispersed phase to precipitate and form a membrane, encapsulating the micron-sized magnetic beads within it;

[0130] Step 4: As the solvent completely evaporates, the microspheres gradually solidify and are collected through steps such as filtration and washing.

[0131] Step 5: Place the obtained molecularly imprinted polymer on a sieve plate and grind it to pass through the sieve plate holes. The diameter of the sieve plate holes is the same as the diameter of the micropores, so as to ensure that the molecularly imprinted polymer has the same volume.

[0132] Step 6: The molecularly imprinted polymers after sieving are screened again using a magnet. The molecularly imprinted polymers are placed on a thin plate, and then a magnet is placed under the thin plate. The thin plate is then tilted. The molecularly imprinted polymers coated with micron magnetic beads will be attracted by the magnet, while the molecularly imprinted polymers not coated with micron magnetic beads will slide off, thus achieving the screening.

[0133] Step 7: Spread the screened molecularly imprinted polymer on a microplate and use the active functional groups of polylactic acid to form hydrogen bonds with the chemical bonds in the micropores to fix it in the micropores. Then, use deionized water or wind to remove the excess polymer outside the micropores.

[0134] Step 8: Then place the magnet under the well plate to fix the micron magnetic beads. Then use lipase or proteinase K to degrade polylactic acid and destroy its rigid skeleton. Then use an enzyme remover to remove the lipase or proteinase K. Then use deionized water to wash repeatedly to remove polylactic acid and complete the micron magnetic beads entering the well.

[0135] Although preferred embodiments of this disclosure have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this disclosure.

[0136] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A biochip, characterized in that, include: A first substrate and a plurality of beads; the first substrate has a plurality of recesses, and at least a portion of the beads are embedded in the recesses. The recessed portion has a first group on its surface facing the bead, and the bead has a second group; the first group of the recessed portion is connected to the second group of the bead, so that the bead is fixed in the recessed portion.

2. The biochip as described in claim 1, characterized in that, The first group of the recessed portion is connected to the second group of the bead through covalent bonds and / or hydrogen bonds and / or electrostatic bonds.

3. The biochip as described in claim 2, characterized in that, The first group includes -COOH and / or -OH; the second group includes -NH2, and / or -COOH, and / or -OH.

4. The biochip as described in claim 1, characterized in that, At least a portion of the outer surface of the bead also has a coating layer, the coating layer comprising the first group.

5. The biochip as described in claim 4, characterized in that, The coating layer includes at least one of the following: paraffin; Chitosan; Sodium alginate; Polycaprolactone diol; Cellulose; Polylactic acid.

6. The biochip as described in claim 1, characterized in that, The bead is a magnetic bead, and the size of the bead ranges from 0.1 micrometers to 100 micrometers.

7. A method for fabricating a biochip, characterized in that, include: Solid molecularly imprinted polymers are formed by mixing beads with polymers; The molecularly imprinted polymer is placed on a sieve plate with multiple sieve holes and ground to obtain a bead precursor with the same sieve hole diameter as the sieve hole diameter. The bead precursor includes: a bead and a coating layer covering the outer surface of the bead. The bead precursor is placed on the first substrate having multiple recesses, so that the bead precursor enters the recesses of the first substrate, wherein the diameter of the sieve hole is less than or equal to the diameter of the recess. The beads are attracted by a magnet and subjected to a first treatment to remove at least a portion of the coating layer on the surface of the bead precursor.

8. The manufacturing method as described in claim 7, characterized in that, After obtaining a bead precursor with the same aperture as the sieve, and before placing the bead precursor on the first substrate having multiple recesses, the manufacturing method further includes: The bead precursor is placed on a second substrate, and a magnet is placed on the other side of the second substrate. The second substrate is then tilted to screen out the beads that are not coated with the polymer.

9. The manufacturing method as described in claim 8, characterized in that, The solid molecularly imprinted polymer that forms a mixture of beads and polymer comprises: mixing paraffin wax, stearic acid and beads and heating them, then adding a pore-forming agent to form a wax / water emulsion, and adding ethanol to the wax / water emulsion for curing. Alternatively, the solid molecularly imprinted polymer that forms a mixture of beads and polymer comprises: mixing chitosan and acetic acid to form a chitosan solution and adding it to the beads, and then dropping the chitosan solution containing the beads into a sodium tripolyphosphate solution to obtain spherical particle media, and drying it. Alternatively, the solid molecularly imprinted polymer that forms a mixture of beads and polymer may be further comprising: adding sodium alginate solution to a mixture of CaCl2 and the beads, followed by filtration, washing, and drying. Alternatively, the solid molecularly imprinted polymer formed by mixing the beads with the polymer includes: adding the beads to a liquid containing acrylic acid, adding polycaprolactone diol and an initiator to form a gel-like polymer and then drying it. Alternatively, the solid molecularly imprinted polymer that forms a mixture of beads and polymer includes: forming a cellulose graft copolymer containing amide groups, reacting it with sodium hydroxide, formaldehyde, and sodium bisulfite to form a modified cellulose graft copolymer, adding the modified cellulose graft copolymer to a solution containing beads, adding a crosslinking agent to react, and then washing and drying. Alternatively, the solid molecularly imprinted polymer that forms a mixture of beads and polymer may include: adding an organic phase containing polylactic acid to an aqueous phase containing the beads and an emulsifier to form an emulsion, and performing extraction, evaporation, and extraction processes to evaporate the solvent.

10. The manufacturing method as described in claim 9, characterized in that, The first treatment, which removes at least a portion of the coating layer on the surface of the bead precursor, includes removing at least a portion of the coating layer on the surface of the bead precursor by heating, or by acidic liquid treatment, or by enzymatic degradation.

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