PDMS (Polydimethylsiloxane) film slide with reactive functional groups as well as preparation method and application of PDMS film slide

Abstract

The invention provides a PDMS (polydimethylsiloxane) membrane slide with reactive functional groups as well as a preparation method and application of the PDMS membrane slide. The PDMS film slide with the reactive functional group prepared by the invention is smooth in surface and uniform in functional group density distribution. The device of the PDMS membrane slide with the reactive functional group, provided by the invention, is simple in structure and convenient to operate, can be used for biochip detection and cell culture, can also be used for microscopic diagnosis, is beneficial to multi-flux detection, reduces the number of experiments and the dosage of reagents, ensures the consistency of the experiments, and improves the efficiency. The method for preparing the PDMS membrane slide with the reactive functional group, provided by the invention, is simple and convenient, does not need to use various reagents, and can introduce the reactive functional group to the surface of the PDMS through a cheap compound, so that the economic benefit is improved, the energy is saved and the environment is protected in the aspects of related surface chemical technologies.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemistry and medicine, and particularly to a multi-flux slide device that can be used in fields such as multi-flux biochips, cell culture, and microscopic diagnosis. Background Art

[0002] Slides are mainly composed of silicon dioxide. The oxide layer protects the silicon from chemical degradation and reactions, and has high mechanical and chemical stability. They are usually used as carriers in the fields of biochips, cell culture, and microscopic diagnosis. For example, they are carriers for biological materials such as nucleic acid fragments, polypeptide molecules, proteins, tissue sections, cells, viruses, bacteria, etc. For the slide substrate, since there are no reactive functional groups on its surface, before applying the slide substrate as a carrier, surface treatment is required to make its surface carry active functional groups so as to link and effectively immobilize various biological molecules.

[0003] Currently, the surface treatment methods before applying slides to the biochip field usually include the following methods: (1) Aldehyde modification: Usually, reagents such as the silane coupling agent APTES and glutaraldehyde are used to modify the glass substrate to make it have aldehyde groups to provide DNA and protein microarrays. (2) Epoxy modification: In addition to silanization, epoxidation is a common method for glass surface modification. Amine-modified DNA and proteins and natural amino groups can be immobilized on the epoxy-modified glass surface to form DNA and protein microarrays respectively. (3) Carboxylation modification: Carboxyl functional groups and amino functional groups can react without any intermediate linking reagents, so it is widely used in the functionalization of carriers. And EDC, NHS, HOBt, TBTU, PAMAM, etc. are commonly used amide coupling reagents for carboxyl and amino groups. (4) Azidation modification: Azide is a stable chemical bond in organic chemistry, and its application in surface chemical modification is profound. To form an azide bond on the glass surface, first, alkylation is carried out using an amino-silane coupling agent, and then the amino functional group forms an azide bond under the action of sodium nitrite. The DNA probe is fixed on the azide surface and can hybridize with PCR products for the detection and identification of pathogens.

[0004] Most of the carriers for cell culture on the traditional market are made of glass. Glass is hydrophilic, so its surface does not require special hydrophilic treatment. However, in actual use, it is prone to adhering to biological molecules such as dust, proteins, bacteria, and viruses, and it is not clean enough, which is likely to contaminate samples. With the rapid development of technology, various polymer materials have gradually replaced glass as the basic processing raw materials for cell culture consumables. Most polymer materials are hydrophobic. To ensure that adherent cells can attach and grow well, the surface of cell culture consumables needs to be specially modified by introducing reactive functional groups on the surface to improve the surface properties to adapt to the growth and reproduction of adherent cells. This treatment is called TC (Tissue Culture) surface treatment. The TC treatment process mainly affects cell attachment and growth by changing the physical and chemical properties of the cell culture dish surface. In the TC treatment process, the methods used include surface physical coating and chemical modification. Commonly used surface coating substances include collagen, fibronectin, laminin, vitronectin, gelatin, poly-L-lysine, polylactic acid, etc. These substances can bind to the receptors on the cell surface and provide the signals and support required for cell attachment. However, due to the lack of reactive functional groups on the surface of conventional cell culture dishes, the surface physical coating is prone to peeling off and has thus been gradually phased out. Currently, the TC treatment of cell culture dishes mainly uses chemical modification. Commonly used chemical modification methods include surface grafting, covalent crosslinking, and ion exchange. Surface grafting is to covalently bind a specific chemical substance to the surface of the cell culture dish to form a cell-affinity surface. Commonly used surface grafting polymers include polyvinyl alcohol, polylactic acid, and polyacrylic acid, etc. These polymers can provide the signals and support required by cells and promote cell attachment and growth. Covalent crosslinking is to crosslink a specific chemical substance on the surface of the cell culture dish through a chemical reaction to form a cell-affinity surface. Commonly used covalent crosslinking agents include glutaraldehyde, ammonium sulfate, and sodium hypochlorite, etc. These covalent crosslinking agents can react with the amino residues on the cell surface to form stable chemical bonds and improve the efficiency of cell attachment and growth. Ion exchange is to introduce specific cations or anions onto the surface of the cell culture dish to form a cell-affinity surface. Commonly used ion exchange agents include polyethyleneimine and polyvinylamine, etc. These ion exchange agents can have charge interactions with anions or cations on the cell surface and improve the cell attachment and growth ability.

[0005] Glass slides have a wide range of applications in the fields of biochemistry and medical technology due to their easy availability, high mechanical properties, and chemical stability. However, the chemical inertness of their surfaces limits their applications in areas such as biochips, cell culture, and microscopic diagnosis. To develop glass slides as carriers for biological materials and broaden their applications in these fields, the glass must be surface-treated to carry active functional groups. The above-mentioned chemical modifications of glass slides first involve using acids, bases, plasma treatment, laser ablation, etc. to expose OH functional groups on the glass slide surface, then using silane coupling agents to make it carry different active functional groups, and finally performing subsequent linking, fixing, and coating of the modification layer. This process is cumbersome, requires a large number of reaction reagents, has harsh reaction conditions, and easily damages the surface morphology and light transmittance of the glass slide, resulting in difficulties in meeting the application requirements for surface modification uniformity and repeatability.

[0006] Polydimethylsiloxane (PDMS), a kind of polymer, with the chemical formula (C2H6OSi) n , has advantages such as good biocompatibility, antioxidant property, thermal stability, good light transmittance, air permeability, low price, and simple processing, and has a wide range of applications in various fields such as biology, chemistry, medicine, daily chemical products, food, and construction. Especially in the biomedical industry, it has received extensive attention. Due to the easy rotation of the Si-O-Si main chain, PDMS presents a highly flexible state, forming a smooth and flat surface. And compared with other polymer materials such as polystyrene (PS), PDMS itself has a low fluorescence background, especially suitable for high-sensitivity fluorescence detection. However, similar to glass substrates, the PDMS membrane has no reactive functional groups on its surface, and its inherent surface hydrophobicity limits its applications in the biomedical field. For the purpose of broadening the application fields of biomedical materials, researchers have conducted a large number of studies, including plasma treatment. However, introducing polar silanol groups through plasma treatment results in a short shelf life of about 6 hours when exposed to ambient air. To extend the lifespan of surface modification, the currently common method is to use acids, bases, plasma treatment, laser ablation, etc. to form silanol groups on the PDMS surface, then use silane coupling agents to make it carry different active functional groups, and finally perform subsequent linking, fixing, and coating of the modification layer. Similar to the surface chemical modification of glass slides, this modification process is cumbersome, requires a large number of reaction reagents, has harsh reaction conditions, and easily damages the surface morphology and light transmittance of the PDMS membrane, resulting in difficulties in meeting the application requirements for surface modification uniformity and repeatability.

[0007] In addition, with the rapid development of the inspection and testing industry, the update speed of testing technologies is also accelerating continuously. With its advantages of multi-flux, low cost, and high speed, multi-flux detection technology has gradually become the development direction of future inspection and testing method development and standard customization, as well as a research hotspot at home and abroad. The so-called multi-flux detection technology refers to the detection of multiple samples or multiple indicators of a single sample at one time, which is relative to the traditional detection technology - only one sample or one indicator is detected at a time. Currently, slide-related products that combine multi-flux detection with slides as carriers have gradually become a research hotspot in the fields of protein chips and cell culture. Summary of the Invention

[0008] The present invention is completed to overcome the limitations of the prior art. Its main purpose is to provide a method for introducing one or more reactive functional groups / oligomer PDMS film layers onto the surface of a slide substrate without going through multiple steps of chemical reactions, without the need for additional chemical reaction organic solvents, and without damaging the surface morphology.

[0009] On the other hand, the present invention provides a method for preparing a PDMS film slide with reactive functional groups. This method can not only easily introduce reactive functional groups, but also introduce functional functional groups onto the surface of the slide substrate through inexpensive small molecule compounds / oligomers, without damaging the surface morphology of the slide, thereby improving economic efficiency in related surface chemistry technologies.

[0010] In one aspect, the present invention provides a PDMS film slide with reactive functional groups prepared by the above method.

[0011] In one aspect, the present invention provides a method for converting existing reactive functional groups into functional groups that are difficult or impossible to introduce after introducing a conventional reactive functional group PDMS film layer onto the surface of a glass substrate, and further reacting with the exposed reactive functional groups of the receptor to fix the receptor on the surface of the PDMS film layer of the glass substrate.

[0012] By using the method provided by the present invention, it is convenient to achieve efficient and stable fixation on the surface of the PDMS film layer of the glass substrate for different exposed reactive functional groups of the receptor, thereby realizing the diversification of the fixation of receptors on the glass substrate.

[0013] Among the above-mentioned receptors, representative ones include biopolymers or organic functional groups used in multiple fields such as protein chips, microfluidics, cell culture, and microscope diagnosis. Not only can proteins such as antibodies or hormones be used, but any substance such as nucleic acids, carbohydrates, cells, etc. can be used as biopolymers. Any form of functional group from carboxyl groups or amino groups to complex biotin or folic acid can be used as an organic functional group.

[0014] In another aspect, the present invention provides an apparatus with a reactive functional group / oligomer PDMS film layer on a glass slide, constructing a reaction tool and a detection tool that can be used for protein chips, cell culture, and microscopy detection, achieving the purpose of high-throughput reaction and detection, and enabling related products to break through the limitations of time and cost.

[0015] Compared with existing methods, the method of introducing reactive functional groups to a glass substrate involved in the present invention is simple, does not require the use of various reagents, can introduce reactive functional groups to the PDMS surface through inexpensive compounds, and does not damage the surface morphology of the glass slide, thereby improving economic benefits in related surface chemistry technologies. In addition, after introducing conventional reactive functional groups to the PDMS surface, existing reactive functional groups can be converted into functional groups that are difficult or impossible to introduce through simple chemical reactions, facilitating efficient and stable immobilization on the PDMS surface for different receptor-exposed reactive functional groups, thereby realizing the diversification of PDMS film-fixed receptors. Moreover, the method is simple and low-cost, and even those without a background in chemistry can easily master it and it is easy to industrialize.

[0016] Furthermore, due to the good biocompatibility of PDMS, introducing the above-mentioned reactive functional group / oligomer PDMS film layer to the surface of the glass substrate not only endows the glass substrate with the function of reacting and immobilizing with various biomolecules, has special functions of oligomers such as polyglycerol, polyethylene glycol, polyvinyl alcohol, poly(N-isopropylacrylamide) (PIPAAm), etc., but also endows the glass substrate with the role of mimicking the natural cell environment. There are multiple elastic values available, covering all elastic ranges from DNA to polypeptides, from proteins to cells, and from soft tissues (e.g., brain or gland) to hard tissues (such as cartilage or bone). It is suitable for the detection and research of biological molecules such as DAN, polypeptides, and proteins; cell culture, cell mechanics research; immunofluorescence staining; live cell imaging of soft surface cell differentiation; transfection, etc.

[0017] In addition, the apparatus with a reactive functional group PDMS film layer on a glass slide constructs a reaction tool and a detection tool that can be used for protein chips, cell culture, and microscopy detection, achieving the purpose of high-throughput reaction and detection, and enabling related products to break through the limitations of time and cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Shows the structural diagram of the apparatus with a reactive functional group film layer on a glass slide;

[0019] Figure 2 Shows the structural diagram of the reactive functional group film layer on a glass slide;

[0020] Figure 3 Shows the preparation method of the reactive functional group film layer on a glass slide;

[0021] Figure 4 Shows the schematic diagram of the conversion of epoxy functional groups into amino functional groups;

[0022] Figure 5 Shows the schematic diagram of the contact angle of the PDMS film layer on the glass substrate, where (a) is the glass substrate of the PDMS film layer without reactive functional groups prepared in Comparative Example 2; (b) is the glass substrate of the epoxy-functionalized PDMS film layer prepared in Example 1; (c) is the glass substrate of the epoxy base-hydrolyzed functional group PDMS film layer prepared in Example 3; (d) is the glass substrate of the epoxy ammonolysis-functionalized PDMS film layer prepared in Example 4; (e) is the glass substrate of the hydroxyl-functionalized PDMS film layer prepared in Example 5; (f) is the glass substrate of the carboxyl-functionalized PDMS film layer prepared in Example 6; (g) is the glass substrate of the hydroxyl-functionalized oligomer PDMS film layer prepared in Example 7;

[0023] Figure 6 Shows the infrared spectra of different functionalized films. Among them, (a) is the glass substrate of the PDMS film layer without functional groups prepared in Comparative Example 2; (b) is the glass substrate of the epoxy-functionalized PDMS film layer prepared in Example 1; (c) is the glass substrate of the epoxy base-hydrolyzed functional group PDMS film layer prepared in Example 2; (d) is the glass substrate of the hydroxyl-functionalized PDMS film layer prepared in Example 3; (e) is the glass substrate of the epoxy ammonolysis-functionalized PDMS film layer prepared in Example 4;

[0024] Figure 7 Shows the schematic diagram of the possible products of the base hydrolysis of the epoxy-functionalized PDMS film layer on the glass substrate;

[0025] Figure 8 Shows the schematic diagram of the possible products of the ammonolysis of the epoxy-functionalized PDMS film; Figure 9 Is an example image of the signal value detection of the mouse IgG antibody immobilized on the functionalized PDMS film layer glass substrate. Detailed implementation manners

[0026] Specifically, the present disclosure provides a method for preparing a glass slide with a reactive functional group PDMS film layer, including the following steps:

[0027] (a) Premixing components A and B of silica gel, and then preforming with the glass slide to obtain a preformed PDMS film layer glass slide;

[0028] (b) Performing plasma surface treatment on the preformed PDMS film layer glass slide;

[0029] (c) Coating the preformed PDMS film layer glass slide obtained in step (b) with a functional group monomer and / or oligomer;

[0030] (d) Fix the functional group monomer and / or oligomer on the surface of the preformed PDMS film slide to obtain a PDMS film slide with reactive functional groups.

[0031] In some embodiments, in step (a), premixing components A and B of silica gel produces polydimethylsiloxane, which is the raw material for preparing PDMS, composed of component A and component B, and contains one or more unsaturated double bonds.

[0032] In some embodiments, in step (a), the mass ratio of component A to component B is about 100 - 1:1. Preferably, the mass ratio of component A to component B is about 15 - 1:1, 10 - 1:1, or 5 - 1:1.

[0033] In some embodiments, in step (a), component A is selected from Sylgard 184 (e.g., Sylgard 184A) produced by Dow Corning Corporation of the United States, poly(methylsiloxane) containing double bonds, and tetra(trimethylsilyloxy)silane. Preferably, component A is Sylgard 184A produced by Dow Corning Corporation of the United States.

[0034] In some embodiments, in step (a), component B is selected from Sylgard 184 (e.g., Sylgard 184B) produced by Dow Corning Corporation of the United States, poly(methylsiloxane) terminated with double bonds, polyfunctional poly(methylsiloxane) containing silicon-hydrogen bonds, and tetramethyltetravinylcyclotetrasiloxane. Preferably, component B is Sylgard 184B produced by Dow Corning Corporation of the United States.

[0035] In some embodiments, in step (a), the preforming methods include, but are not limited to, chemical vapor deposition, room temperature spraying, flame spraying, plasma spraying, electroplating, electroless plating, coating, spin coating, self-leveling, pressing, plasticizing, slip casting, etc. The preforming method can be selected according to the inherent equipment and instruments in the laboratory; the preforming methods with simple operation, simple equipment and instruments, and low price include room temperature spraying, plasma spraying, coating, spin coating, self-leveling, and pressing; preferably, the forming method is spin coating, and the thickness of the coating can be controlled within about 1 μm.

[0036] In some embodiments, in step (a), the preforming method is to mix components A and B evenly, coat them on the surface of the slide, and react at about room temperature - 150 °C for about 5 min - 24 hours.

[0037] Specifically, in step (a), after the pre - forming of component A and component B, they are placed under a temperature condition of about room temperature - 150 °C for about 5 min - 24 hours to control the degree of polymerization of the mixture, so that the prepolymer on the glass surface forms a viscous - semi - solid shape. This viscous - semi - solid prepolymer containing unsaturated double bonds is more likely to form a high - energy state on the surface during the plasma treatment in step b, thus being able to more effectively and uniformly capture and activate the functionalized monomers and / or oligomers containing unsaturated double bonds, and then further undergoing free - radical polymerization under the action of an inherent catalyst in liquid silicone at a certain temperature (for example, room temperature - 150 °C), and then chemically bonding to the surface of the PDMS film layer.

[0038] In some embodiments, in step (b), the surface prepolymer of the pre - formed PDMS film layer glass sheet prepared in step (a) is subjected to plasma surface treatment by a plasma treatment method. The plasma surface treatment achieves the functions of activating the unsaturated double bonds on the prepolymer surface and enhancing the surface wettability.

[0039] In some embodiments, in step (b), the gases used for the plasma surface treatment include: argon, oxygen, nitrogen, ammonia, ethane, hydrogen, or a mixture of two or more of these gases in a volume ratio. Preferably, the gas is a mixed gas of argon and oxygen. In some embodiments, the volume ratio of the gases is about 15 - 1:1, preferably, the volume ratio of the gases is about 5 - 1:1. In some preferred embodiments, the gas is a mixed gas of argon and oxygen with a volume ratio of about 1:1.

[0040] In some embodiments, in step (b), the plasma surface treatment power is selected from 50 - 1000 W, preferably, the power is 500 - 1000 W; more preferably, the plasma surface treatment power is 300 W.

[0041] In some embodiments, in step (b), the treatment time of the plasma surface treatment is about 5 s - 20 minutes, preferably, the time is about 15 s - 20 minutes; more preferably, the treatment time of the plasma surface treatment is about 30 s.

[0042] In some embodiments, in step (c), the functional groups of the functionalized monomers and / or oligomers are selected from one or more of epoxy groups, hydroxyl groups, carboxyl groups, and amino groups.

[0043] In some embodiments, the spin-coated functionalized monomer is an olefin monomer and / or oligomer containing unsaturated double bonds. In some embodiments, the functional groups of the functionalized monomer and / or oligomer include epoxy olefin monomers and / or oligomers containing unsaturated double bonds, hydroxyl olefin monomers and / or oligomers containing unsaturated double bonds, carboxyl olefin monomers and / or oligomers containing unsaturated double bonds, and amino olefin monomers and / or oligomers containing unsaturated double bonds.

[0044] In some embodiments, the functional monomer and / or oligomer is an olefin monomer containing a hydroxyl functional group, which is made from compounds selected from the following group: 6-hepten-1-ol, 7-octene-1,2-diol, 9-decen-1-ol, 10-undecen-1-ol, 5-hexen-1-ol, 3-buten-1-ol, 6-heptanol, 5-hexen-1-ol, 4-penten-1-ol, 2-ene polyethylene glycol (n = 2 - 300), or a combination of any two or more of them. Preferably, the olefin monomer containing a hydroxyl functional group is 10-undecen-1-ol, and the oligomer monomer is 2-ene polyethylene glycol (n = 30).

[0045] In some embodiments, the functional monomer is an olefin monomer containing an epoxy functional group, which is made from compounds selected from the following group: 1,2-epoxy-9-decene, 1,2-epoxy-5-hexene, allyl glycidyl ether, 3,4-epoxy-1-butene, glycidyl methacrylate, or a combination of any two or more of them. Preferably, the olefin monomer containing an epoxy functional group is 1,2-epoxy-9-decene.

[0046] In some embodiments, the functional monomer is an olefin monomer containing a carboxyl functional group, which is made from compounds selected from the following group: 7-octenoic acid, 8-nonenoic acid, 9-decenoic acid, 10-undecenoic acid, 6-heptanoic acid, 5-hexenoic acid, 4-pentenoic acid, or a combination of any two or more of them. Preferably, the olefin monomer containing a carboxyl functional group is 10-undecenoic acid.

[0047] In some embodiments, in step (c), the coating is any one of spin coating, casting, spraying, knife coating, drop coating, or pouring molding. Preferably, the coating is by spin coating, for example, coating with a spin coater or a spin coater.

[0048] Those skilled in the art can select different stage rotation speeds according to the viscosity of the spin-coated monomer and the thickness of the target spin-coated layer. During spin coating, after adding a predetermined amount of monomer, the rotating table holding the glass slide is rotated at a rotation speed faster than the previous stage rotation speed to spread the monomer supplied to the processing surface of the glass slide.

[0049] In some preferred embodiments, in step (c), the rotational speed range of the spin coater is as follows:

[0050] Rotational speed in the first stage: 0 - 100 rpm, time: 3 s - 10 s; preferably, the rotational speed is 100 rpm and the time is 5 s, and / or,

[0051] Rotational speed in the second stage: 200 - 500 rpm, time: 10 - 30 s; preferably, the rotational speed is 300 rpm and the time is 15 s, and / or,

[0052] Rotational speed in the third stage: 500 - 1000 rpm, time: 10 - 30 s; preferably, the rotational speed is 800 rpm and the time is 20 s, and / or,

[0053] Rotational speed in the fourth stage: 1000 - 3000 rpm, time: 10 - 30 s; preferably, the rotational speed is 2000 rpm and the time is 20 s, and / or,

[0054] Rotational speed in the fifth stage: 3000 - 5000 rpm, time: 10 - 30 s; preferably, the rotational speed is 3500 rpm and the time is 30 s.

[0055] In some embodiments, in step (c), the amount of the spin - coated monomer and / or oligomer is in the range of about 50 μL - 5 mL, preferably, the amount is about 100 μL - 1 mL; more preferably, the amount of the spin - coated monomer is about 150 μL and the amount of the oligomer is about 100 μL.

[0056] In some embodiments, in step (d), the functional group monomer and / or oligomer is fixed on the surface of the pre - formed PDMS film - coated glass slide by placing the PDMS film - coated glass slide spin - coated with the functionalized monomer at about room temperature - 150 °C for treatment to obtain a functionalized PDMS film - coated glass slide. In some embodiments, in step (d), the treatment time is about 5 min - 24 hours. Particularly preferably, in step (d), the functional group monomer and / or oligomer is fixed on the surface of the pre - formed PDMS film - coated glass slide by placing the PDMS film - coated glass slide spin - coated with the functionalized monomer and / or oligomer at about room temperature - 150 °C for 5 min - 24 hours to obtain a functionalized PDMS film - coated glass slide.

[0057] In some embodiments, when the functional group monomer is an ethylenic monomer containing an epoxy group functional group, in step (d), after the functional group monomer and / or oligomer are fixed on the surface of the preformed PDMS film-coated glass slide, a ring-opening reaction occurs to obtain a PDMS film-coated glass slide with reactive functional groups / oligomers. The ring-opening reaction is to open the epoxy group of the epoxy group-functionalized PDMS film-coated glass slide through acidolysis, alkalinolysis or aminolysis.

[0058] In some embodiments, the acidolysis is to carry out a ring-opening reaction in an acidic solution to obtain a hydroxyl group-functionalized PDMS film-coated glass slide. The acidic solution is selected from solutions of sulfuric acid, hydrochloric acid, and nitric acid. Preferably, the acidic solution is an aqueous sulfuric acid solution.

[0059] In some embodiments, the alkalinolysis is to carry out a ring-opening reaction in an alkaline solution to obtain a hydroxyl group-functionalized PDMS film-coated glass slide. The alkaline solution is selected from aqueous potassium hydroxide solution, aqueous sodium hydroxide solution, potassium hydroxide methanol solution, sodium hydroxide methanol solution, potassium hydroxide ethanol solution, and sodium hydroxide ethanol solution. Preferably, the alkaline solution is sodium hydroxide methanol solution.

[0060] In some embodiments, the aminolysis is to react a polyamino compound containing an amino group functional group with an epoxy group functional group to obtain a PDMS film-coated glass slide with an amino group functional group on the surface. The compound selected for the aminolysis is selected from the following combinations: hexanediamine, ethylenediamine, triethylenetetramine, tetraethylenepentamine, or a combination thereof, to obtain a PDMS film-coated glass slide with two functional groups of amino / hydroxyl. Preferably, the compound selected for the aminolysis is ethylenediamine.

[0061] Specifically, three functional groups are generated on the surface of the epoxy group-functionalized PDMS film during aminolysis: (1) primary amine: tertiary amine: hydroxyl = 1:1:1; (2) tertiary amine: hydroxyl = 1:1; (3) hydroxyl, see Figure 8 ... Increasing the concentration of ethylenediamine is beneficial to the formation of primary amines. However, at the same time, as the concentration of ethylenediamine increases, the pH value increases. The ethylenediamine chain segment is short, and the primary amino group has strong activity and is likely to attack the adjacent epoxy group, thereby generating a tertiary amine ring and a hydroxyl functional group. This leads to a decrease in the reaction activity of the amino functional group for immobilizing biomolecules; when the concentration of ethylenediamine decreases and the amount of aqueous solution increases, the pH decreases. After the epoxy group is opened, H2O will combine with the epoxy group to form a hydroxyl group. Under the conditions of an aqueous solution of ethylenediamine, these three situations exist simultaneously with different proportions. A schematic diagram of the possible products of the aminolysis of the epoxy group-functionalized PDMS film is as shown in Figure 8 ... To obtain primary amino groups and non-crosslinked PDMS films as much as possible, three ammoniating reagents, namely ammonia water, hexanediamine, and triethyleneamine, can be used to adjust the concentrations of the ammoniating reagent, aqueous solution, and ethylene glycol to obtain the optimal ratio.

[0062] In one aspect, the present invention provides a glass slide with a reactive functional group PDMS film layer prepared by the above method.

[0063] In some embodiments, the film layer thickness of the reactive functional group PDMS film attached to the glass slide with reactive functional groups is about 1 nm - 5 mm.

[0064] As Figure 1 and Figure 2 shown, the present invention provides a glass slide device with a reactive functional group PDMS film layer, comprising a cover body, a well plate, a well plate base and a glass slide;

[0065] The cover body is arranged on the well plate; a plurality of positioning holes are arranged on the well plate, and the well plate is fixedly connected to the well plate base; in a preferred embodiment of the present invention, a protrusion is arranged at the bottom edge position of the well plate; a foldable locking flap corresponding to the position of the protrusion is arranged at the edge position of the well plate base, and the well plate and the well plate base can be fixedly connected by engaging the protrusion with the locking flap.

[0066] The glass slide is arranged between the well plate and the well plate base, gaskets are arranged above and below the glass slide, and a plurality of hole grooves corresponding to the positioning holes are arranged on the gaskets; a reactive functional group PDMS film layer is coated on the glass slide; in a preferred embodiment of the present invention, the PDMS film layer has multifunctionality, with reactive functional groups hanging on the surface, and the reactive functional groups include epoxy groups, amino groups, carboxyl groups, hydroxyl groups, etc. Different reactive functional groups are selected according to the exposed functional groups of different biomolecules; the gasket has a buffering effect to prevent the glass slide from being damaged due to excessive force during collision or assembly of the reaction box; the gasket is designed with two layers of silicone rubber up and down, which can improve the adaptability to the change in the thickness of the glass slide and ensure that there is no leakage between the wells within the range of the glass slide thickness of 1.1 mm - 1.3 mm. The gasket separates a plurality of hole grooves, and the number of the hole grooves is 1 - 1000, preferably, the number of the hole grooves is one, two, four, six, eight, twelve, fourteen, sixteen.

[0067] According to different usage scenarios, the cover body can be optionally equipped with different colors, which can avoid light, isolate air, facilitate observation, prevent dust, particulate impurities and liquid splashing, etc. In a preferred embodiment of the present invention, the cover body is a black cover body, which is used to protect the spotted protein from light and reduce its activity during the chip storage and reaction process. The black cover body can prevent impurities such as dust and liquid splashing during the reaction process. In another preferred embodiment of the present invention, the cover body is a transparent cover body, which is opened when placing cells and adding materials such as culture medium, can isolate external gas, and is convenient for observing the state of cells.

[0068] Similarly, according to different application scenarios, the glass slide needs to be optionally equipped with different colors to improve the signal-to-noise ratio, thereby improving the detection sensitivity, increasing the light transmittance, and facilitating observation, etc. In a preferred embodiment of the present invention, the glass slide is specifically a black glass slide substrate. The glass slide is used for biochips and fluorescence detection. Selecting a black glass slide substrate is beneficial to reducing the background signal, improving the signal-to-noise ratio, and further achieving the purpose of improving the detection sensitivity. In another preferred embodiment of the present invention, the glass slide is used for cell culture and microscope detection. Selecting a glass slide substrate with high light transmittance is beneficial to increasing the light transmittance and facilitating the observation and counting of cell morphology. Based on the glass slide PDMS film layer for this purpose, the highly transparent component is polydimethylsiloxane composed of A / B.

[0069] The device of the glass slide with a reactive functional group PDMS film layer provided by the present invention for invention patents has a simple structure and convenient operation. The preparation of the multifunctional film layer on the glass slide is simple, the required chemical reagents are inexpensive, easy to obtain, do not require the use of additional organic solvents, are environmentally friendly, energy-saving, and easy to industrialize, etc., which determine its broad application prospects. Good biocompatibility, low surface energy, high dielectric properties and mechanical properties, and different reactive functional groups suspended on the surface enable it to be used in biochip detection, cell culture, and also in the field of microscope diagnosis. Moreover, this product is beneficial to multi-flux detection, reduces the number of experiments and the dosage of reagents, ensures the consistency of experiments, improves efficiency, and saves experimental costs.

[0070] Definition

[0071] As used herein, the term "about" modifying a quantity associated with the present invention refers to a variation in the numerical quantity that may occur, for example, through routine testing and processing; through inadvertent error in such testing and processing; through differences in the manufacture, source, or purity of the ingredients used in the present invention, etc. As used herein, "about" a particular value also includes that particular value. For example, about 10% includes 10%. Whether or not modified by the term "about", the claims include equivalent forms of the recited quantity. In one embodiment, the term "about" means within 20%, within 10%, or within 5% of the reported numerical value.

[0072] The term "comprising" and variations thereof do not have a limiting meaning where these terms appear in the specification and claims. Such terms will be understood to imply the inclusion of the stated steps or elements or groups of steps or elements, but not the exclusion of any other steps or elements or groups of steps or elements. The phrase "consisting of" means including and limited to the content following the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory and that no other elements may be present. The phrase "consisting essentially of" means including any elements listed after this phrase and limited to those that do not interfere with or contribute to the activity or action specified for the listed elements in the present disclosure. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but other elements are optional and may or may not be present, depending on whether they substantially affect the activity or action of the listed elements.

[0073] The terms "preferred" and "preferably" refer to embodiments of the present disclosure that may provide certain benefits in certain circumstances. However, in the same or other circumstances, other embodiments may also be preferred. In addition, the recitation of one or more preferred embodiments does not imply that other embodiments are not available and is not intended to exclude other embodiments from the scope of the present disclosure.

[0074] In this application, terms such as "a", "an", and "the" are not intended to refer only to a single entity but include the general category of specific examples that may be used for illustration. The terms "a", "an", "the", and "said" are used interchangeably with the term "at least one". The phrases "at least one of... " and "comprising at least one of... " followed by a list refer to any one of the items in the list and any combination of two or more items in the list.

[0075] The phrases "at least one of... " and "comprising at least one of... " followed by a list refer to any one of the items in the list and any combination of two or more items in the list.

[0076] As used herein, the term "or" is generally used in its ordinary sense, including "and / or", unless the context clearly indicates otherwise.

[0077] The term "and / or" means one or all of the listed elements, or a combination of any two or more of the listed elements.

[0078] Examples

[0079] Compound raw material sources and storage

[0080] For the experimental methods without specific conditions noted in the following examples, they can be carried out according to the conventional conditions of such reactions or according to the conditions recommended by the manufacturer.

[0081] If not otherwise specified, the experimental materials used in the following examples and reagents such as undecenol, undecenoic acid, polyethylene glycol methacrylate, 1,2-epoxy-9-decene, etc. can be obtained from commercial channels.

[0082] The polydimethylsiloxane used in this example is produced by Dow Corning Corporation of the United States, with the model of Sylgard184, and is composed of component A and component B.

[0083] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0084] Example 1 Preparation of epoxy-functionalized PDMS film-coated glass slides

[0085] (1) Accurately weigh 10 g of polydimethylsiloxane component A and 1 g of component B, mix them evenly for 5 min under high-speed stirring (rotation speed: 300 r / min), and use a pipette to suck 100 μL of the AB mixture and drop it onto the glass slide substrate; see Figure 3 .

[0086] (2) Set the spin-coating parameters of the spin coater (the first-stage rotation speed: 500 r / min, time 10 s; the second-stage rotation speed: 1000 r / min, time 20 s; the third-stage rotation speed: 1200 r / min, time 20 s; the fourth-stage rotation speed: 1500 r / min, time 20 s; the fifth-stage rotation speed: 2000 r / min, time 30 s). Spin-coat the AB mixture according to the spin-coater settings.

[0087] (3) Place it at 60 °C for 3 hours. Take out the viscous PDMS film substrate, put it in a vacuum plasma processor, set the plasma processor parameters, the plasma surface treatment power is 300 W, pass in a mixed gas of oxygen / nitrogen (nitrogen / oxygen = 1:1, V / V), start the plasma processor, and treat the viscous PDMS film substrate for 30 s.

[0088] (4) Set the spin coater spin coating parameters (the first stage rotation speed: 100 rpm, time 5 s; the second stage rotation speed: 400 rpm, time 15 s; the third stage rotation speed: 800 rpm, time 20 s; the fourth stage rotation speed: 2000 rpm, time 20 s; the fifth stage rotation speed: 3500 rpm, time 30 s). After setting the spin coater parameters, spin coat 150 μL of 1,2-epoxy-9-decene.

[0089] (5) Place the viscous PDMS film glass substrate spin-coated with 1,2-epoxy-9-decene at 60 °C overnight. Wash it twice with ethanol and three times with water, 30 min each time. Vacuum dry for 24 hours. Preparation of the epoxy-functionalized PDMS film glass substrate in Comparative Example 1 (without the plasma treatment surface process)

[0090] (1) Accurately weigh 10 g of polydimethylsiloxane component A and 1 g of component B, mix them evenly for 5 min under high-speed stirring (rotation speed: 300 rpm), and use a pipette to suck 100 μL of the AB mixture and drop it onto the glass slide substrate;

[0091] (2) Set the spin coater spin coating parameters (the first stage rotation speed: 500 rpm, time 10 s; the second stage rotation speed: 1000 rpm, time 20 s; the third stage rotation speed: 1200 rpm, time 20 s; the fourth stage rotation speed: 1500 rpm, time 20 s; the fifth stage rotation speed: 2000 rpm, time 30 s). Spin coat the AB mixture according to the spin coater settings.

[0092] (3) Set the spin coater spin coating parameters (the first stage rotation speed: 500 rpm, time 10 s; the second stage rotation speed: 1000 rpm, time 20 s; the third stage rotation speed: 1200 rpm, time 20 s; the fourth stage rotation speed: 1500 rpm, time 20 s; the fifth stage rotation speed: 2000 rpm, time 30 s). After setting the spin coater parameters, spin coat 150 μL of 1,2-epoxy-9-decene.

[0093] (4) Place it at 60 °C overnight. Wash it twice with ethanol and three times with water, 30 min each time. Vacuum dry for 24 hours.

[0094] Preparation of Glass Substrate with Non-reactive Functional Group PDMS Film Layer in Comparative Example 2

[0095] (1) Accurately weigh 10 g of polydimethylsiloxane component A and 1 g of component B, mix them evenly for 5 min under high-speed stirring (rotation speed: 300 r / min), and use a pipette to suck 100 μL of the AB mixture and drop it onto the glass slide substrate;

[0096] (2) Set the spin coating parameters of the spin coater (rotation speed in the first stage: 500 r / min, time 10 s; rotation speed in the second stage: 1000 r / min, time 20 s; rotation speed in the third stage: 1200 r / min, time 20 s; rotation speed in the fourth stage: 1500 r / min, time 20 s; rotation speed in the fifth stage: 2000 r / min, time 30 s). Spin coat the AB mixture according to the set parameters of the spin coater.

[0097] (3) Place it at 60 °C and treat it overnight. Wash it twice with ethanol and three times with water, 30 min each time. Dry it in vacuum for 24 hours.

[0098] Contact angle experiments were carried out on the products prepared in Example 1, Comparative Example 1 and Comparative Example 2. Each sample was measured 8 times repeatedly, and the average value and coefficient of variation CV% were calculated. The results are shown in Table 1 and Figure 5 a, Figure 5 b.

[0099] Figure 5 a is the contact angle diagram of the glass substrate with non-reactive functional group PDMS film layer prepared in Comparative Example 2, and the contact angle is 113 °, Figure 5 b is the contact angle diagram of the glass substrate with epoxy-functionalized PDMS film layer prepared in Example 1, and the contact angle is 101 °. Comparing Figure 5 a and Figure 5 b, Figure 5 the contact angle of b is less than Figure 5 a, indicating that the epoxy functional group is coated on the surface of the PDMS film layer, and the hydrophilicity of the epoxy-functionalized film layer surface is stronger than that of the PDMS film layer with non-reactive functional groups.

[0100] Table 1 Contact Angles of Glass Substrates with Non-functionalized, Epoxy-functionalized and Epoxy PDMS Film Layers without Plasma Treatment

[0101]

[0102] As can be seen from the data in Table 1, the contact angle of the epoxy-functionalized PDMS film-coated glass substrate prepared in Example 1 is smaller than that of the non-functionalized PDMS film-coated glass substrate prepared in Comparative Example 2, and the coefficient of variation of the contact angle of the plasma-treated epoxy PDMS film-coated glass substrate prepared in Example 1 is smaller than that of the non-plasma-treated epoxy PDMS film-coated glass substrate prepared in Comparative Example 1.

[0103] The contact angle test results of the plasma-treated epoxy PDMS film-coated glass substrate prepared in Example 1 and the non-plasma-treated epoxy PDMS film-coated glass substrate prepared in Comparative Example 1 in Table 1 show that during the entire preparation process of the functionalized PDMS film-coated glass substrate, plasma surface activation of the viscous PDMS film plays a key role. It is precisely due to the activation of the PDMS film by the plasma that the functionalized monomers can be uniformly coated on the surface of the PDMS after spin coating, providing a prerequisite for the unsaturated double bonds contained in the subsequent functionalized monomers to fully interact with the unsaturated double bonds and catalysts in the PDMS matrix.

[0104] The products prepared in Example 1, Comparative Example 1, and Comparative Example 2 were subjected to infrared testing, and the test results are shown in Figure 6 .

[0105] Figure 6 are the infrared spectra of different functionalized PDMS film-coated glass substrates. Among them, Figure 6 a is the infrared spectrum of the non-functionalized PDMS film-coated glass substrate prepared in Comparative Example 2. Figure 6 b is the infrared spectrum of the epoxy-functionalized PDMS film-coated glass substrate prepared in Example 1. Comparing Figure 6 a and Figure 6 b, it is found that there is an additional characteristic peak of epoxy groups at a wavenumber of 907 cm -1 on the surface of the PDMS film containing epoxy functional groups. This indicates that the epoxy functional groups have been successfully coated onto the surface of the PDMS film-coated glass matrix.

[0106] Acidolysis of the epoxy-functionalized PDMS film in Example 2

[0107] The epoxy PDMS film-coated glass substrate prepared in Example 1 was subjected to ring-opening in a sulfuric acid aqueous solution to prepare a hydroxyl-functionalized PDMS film: The PDMS film-coated glass substrate was placed in 30 mL of sulfuric acid aqueous solution (1 N) and reacted with shaking at 60 °C for 2 h. After the reaction, the PDMS film-coated glass substrate was washed 3 times each in ethanol and water, 30 min each time.

[0108] The product prepared in Example 2 was subjected to infrared testing, and the results are shown in detail in Figure 6 c. Figure 6 c is the infrared spectrum of the hydroxyl-functionalized PDMS film of the PDMS film-coated glass substrate after acidolysis. FromFigure 6 From b and 6c, it can be seen that the characteristic peak of the epoxy group disappears at 907 cm -1 wave number, and a hydroxyl peak appears at 3360 cm -1 It shows that part of the epoxy functional group is ring-opened to form a hydroxyl functional group.

[0109] Example 3 Alkaline hydrolysis of epoxy-functionalized PDMS film layer

[0110] The epoxy-functionalized PDMS film layer on the glass substrate prepared in Example 1 was ring-opened in a sodium hydroxide methanol solution to prepare a hydroxyl-functionalized PDMS film: The epoxy-functionalized PDMS film layer on the glass substrate was placed in 30 mL of a sodium hydroxide methanol solution (10%), and reacted with shaking at 50 °C for 1 h.

[0111] The product prepared in Example 3 was subjected to contact angle measurement and infrared measurement. The results are shown in Figure 5 c and Figure 6 d. Figure 6 d is the infrared spectrum of the hydroxyl-functionalized PDMS film layer formed after the alkaline hydrolysis of the epoxy-functionalized PDMS film layer on the glass substrate. From Figure 6 b and 6d, it can be seen that the characteristic peak of the epoxy group disappears at 907 cm -1 wave number, and new characteristic peaks appear at 904 cm -1 and 3363 cm -1 It shows that the epoxy group of the epoxy-functionalized PDMS film is opened and partially converted into a hydroxyl functional group (3363 cm -1 ); In addition, since the ionization degree of methanol is small, the epoxy group is prone to self-crosslinking after being opened to form a C-O-C chain, and its characteristic peak appears at 904 cm -1 wave number, but the peak height is Figure 6 lower than the amplitude of b. The schematic diagram of the possible product structure after the alkaline hydrolysis of the epoxy-functionalized film is shown in Figure 7 as shown. In addition, the experimental results show that the degree of ring-opening of the epoxy functional group is related to the acidolysis / alkaline hydrolysis time and the strength of acids and bases.

[0112] Figure 5 c is the contact angle diagram of the epoxy-functionalized PDMS film layer on the glass substrate after alkaline hydrolysis, and the contact angle is 78°, Figure 5 b is the contact angle diagram of the epoxy-functionalized PDMS film layer on the glass substrate, and the contact angle is 101°. Comparing Figure 5 c and Figure 5 b, Figure 5 the contact angle of c is less than Figure 5 the contact angle of b by 23°, further indicating that the epoxy functional group is converted into a hydroxyl functional group, and the hydrophilicity of the hydroxyl functional group is greater than that of the epoxy functional group.

[0113] Example 4 Ammonolysis of epoxy-functionalized PDMS film layer

[0114] The epoxy-functionalized PDMS film-coated glass substrate prepared in Example 1 was subjected to ring-opening in a polyamine compound solution to prepare an amino-functionalized PDMS film-coated glass substrate: The epoxy-functionalized film-coated glass substrate was placed in 30 mL of an ethylenediamine / ethanol / aqueous solution (ammonification reagent content 68%, water / ethanol = 4 / 1), and reacted with shaking at 37 °C for 6 h. After the reaction was completed, it was washed three times with ethanol and water, 30 min each time.

[0115] The product prepared in Example 4 was subjected to contact angle measurement and infrared measurement. The results are shown in Figure 5 d and Figure 6 e.

[0116] Figure 6 e is the infrared spectrum of the epoxy-functionalized PDMS film after ammonolysis. From Figure 6 b and 6e, it can be seen that the characteristic peak of the epoxy group at 907 cm -1 wavenumber disappears, indicating that the epoxy functional group has undergone ring-opening to form amino / hydroxy functional groups, and characteristic peaks of hydroxy and amino groups appear at 3366 and 1567 cm -1 .

[0117] Figure 5 d is the contact angle diagram of the epoxy-functionalized PDMS film-coated glass substrate after ammonolysis, and the contact angle is 46°. Figure 5 b is the contact angle diagram of the epoxy-functionalized PDMS film-coated glass substrate, and the contact angle is 101°. Comparing Figure 5 d and Figure 5 b, Figure 5 the contact angle of d is less than Figure 5 the contact angle of b by 55°, further indicating that the epoxy functional group has been converted into amino / hydroxy functional groups, as shown in Figure 4 , and the hydrophilicity of the amino / hydroxy functional group is greater than that of the epoxy functional group.

[0118] Preparation of the hydroxy-functionalized PDMS film-coated glass slide in Example 5

[0119] (1) Accurately weigh 10 g of poly(dimethylsiloxane) component A and 1 g of component B, mix them evenly for 5 min under high-speed stirring (rotation speed: 300 rpm), and use a pipette to aspirate 100 μL of the AB mixture and drop it onto the glass slide substrate;

[0120] (2) Set the spin coating parameters of the spin coater (rotation speed in the first stage: 500 rpm, time 10 s; rotation speed in the second stage: 1000 rpm, time 20 s; rotation speed in the third stage: 1200 rpm, time 20 s; rotation speed in the fourth stage: 1500 rpm, time 20 s; rotation speed in the fifth stage: 2000 rpm, time 30 s). Spin coat the AB mixture according to the parameters set on the spin coater.

[0121] (3) Place it at 60 °C for 3 hours. Take out the viscous PDMS film layer substrate, put it in a vacuum plasma processor, set the plasma treatment parameters, pass in a mixed gas of oxygen / nitrogen (nitrogen / oxygen = 1:1), start the plasma processor, and treat the viscous PDMS film layer substrate.

[0122] (4) Set the spin coating parameters of the spin coater (rotation speed in the first stage: 100 rpm, time 5 s; rotation speed in the second stage: 300 rpm, time 15 s; rotation speed in the third stage 800 rpm, time 15 s; rotation speed in the fourth stage: 2000 rpm, time 15 s; rotation speed in the fifth stage: 4000 rpm, time 30 s). Spin coat 150 μL of undecenol according to the parameters set on the spin coater.

[0123] (5) Place the glass substrate of the viscous PDMS film layer spin-coated with undecenol at 80 °C for 6 hours. Wash it twice with ethanol and three times with water, 30 min each time. Dry it in vacuum for 24 hours.

[0124] Perform a contact angle test on the product prepared in Example 5. The results are shown in Figure 5 e.

[0125] Figure 5 e is the contact angle diagram of the hydroxyl-functionalized PDMS film layer glass substrate, and the contact angle is 88°. Figure 5 a is the contact angle diagram of the non-reactive functionalized PDMS film layer glass substrate, and the contact angle is 113°. Compare Figure 5 e and Figure 5 a. The contact angle of the hydroxyl-functionalized PDMS film layer glass substrate has decreased by 25°, indicating that the hydroxyl-functionalized monomer has been successfully coated on the surface of the PDMS film layer, and the hydrophilicity of the hydroxyl-functionalized PDMS film layer glass substrate is stronger than that of the non-reactive functional group PDMS film layer glass substrate.

[0126] Preparation of carboxyl-functionalized PDMS film layer glass slides in Example 6

[0127] (1) Weigh accurately 10 g of polydimethylsiloxane component A and 1 g of component B, mix them evenly for 5 min under high-speed stirring (rotation speed: 300 r / min), and use a pipette to suck 100 μL of the AB mixture and drop it onto a glass slide substrate.

[0128] (2) Set the spin coating parameters of the spin coater (the first stage rotation speed: 500 r / min, time 10 s; the second stage rotation speed: 1000 r / min, time 20 s; the third stage rotation speed: 1200 r / min, time 20 s; the fourth stage rotation speed: 1500 r / min, time 20 s; the fifth stage rotation speed: 2000 r / min, time 30 s). Spin coat the AB mixture according to the set parameters of the spin coater.

[0129] (3) Place it at 60 °C for 3 hours. Take out the viscous PDMS film layer substrate, put it in a vacuum plasma processor, set the plasma treatment parameters, the plasma surface treatment power is 300 W, pass in a mixed gas of oxygen / nitrogen (nitrogen / oxygen = 1:1), start the plasma processor, and treat the viscous PDMS film layer substrate for 30 s.

[0130] (4) Set the spin coating parameters of the spin coater (the first stage rotation speed: 100 r / min, time 5 s; the second stage rotation speed: 400 r / min, time 10 s; the third stage rotation speed: 900 r / min, time 15 s; the fourth stage rotation speed: 2500 r / min, time 20 s; the fifth stage rotation speed: 4300 r / min, time 30 s). Spin coat 150 μL of undecylenic acid according to the set parameters of the spin coater.

[0131] (5) The glass substrate of the viscous PDMS film layer spin-coated with undecylenic acid is placed at 100 °C for 5 hours. Wash it twice with ethanol and three times with water, 30 min each time. Dry it in vacuum for 24 hours.

[0132] The product prepared in Example 6 was subjected to a contact angle test, and the results are shown in Figure 5 f.

[0133] Figure 5 f is the contact angle diagram of the carboxyl-functionalized PDMS film layer glass substrate, and the contact angle is 68°. Figure 5 a is the contact angle diagram of the non-reactive functionalized PDMS film layer glass substrate, and the contact angle is 113°. Comparing Figure 5 f and Figure 5 a, the contact angle of the carboxyl-functionalized PDMS film layer glass substrate decreased by 35°, indicating that the carboxyl-functionalized monomer was successfully coated on the surface of the PDMS film layer, and the carboxyl-functionalized PDMS film layer glass substrate has stronger hydrophilicity than the non-reactive functional group PDMS film layer glass substrate.

[0134] Preparation of Hydroxyl-functionalized Oligomeric PDMS Film-coated Glass Slide in Example 7

[0135] (1) Accurately weigh 10 g of polydimethylsiloxane component A and 1 g of component B, mix them evenly for 5 min under high-speed stirring (rotation speed: 300 r / min), and use a pipette to aspirate 100 μL of the AB mixture and drop it onto the glass slide substrate;

[0136] (2) Set the spin-coating parameters of the spin coater (the first-stage rotation speed: 500 r / min, time 10 s; the second-stage rotation speed: 1000 r / min, time 20 s; the third-stage rotation speed: 1200 r / min, time 20 s; the fourth-stage rotation speed: 1500 r / min, time 20 s; the fifth-stage rotation speed: 2000 r / min, time 30 s). Spin-coat the AB mixture according to the set parameters of the spin coater.

[0137] (3) Place it at 60 °C for 3 hours. Take out the viscous PDMS film-coated substrate, put it in a vacuum plasma processor, set the plasma treatment parameters, the plasma surface treatment power is 300 W, pass in a mixed gas of oxygen / nitrogen (nitrogen / oxygen = 1:1), start the plasma processor, and treat the viscous PDMS film-coated substrate for 30 s.

[0138] (4) Set the spin-coating parameters of the spin coater (the first-stage rotation speed: 300 r / min, time 10 s; the second-stage rotation speed: 600 r / min, time 15 s; the third-stage rotation speed: 1000 r / min, time 20 s; the fourth-stage rotation speed: 3000 r / min, time 20 s; the fifth-stage rotation speed: 4500 r / min, time 30 s). Spin-coat 150 μL of polyethylene glycol methacrylate (molecular weight 2000) according to the set parameters of the spin coater.

[0139] (5) The viscous PDMS film-coated glass substrate spin-coated with polyethylene glycol methacrylate is placed at 100 °C for 8 hours. Wash it twice with ethanol and three times with water, each time for 30 min. Dry it in vacuum for 24 hours.

[0140] The product prepared in Example 7 was subjected to a contact angle test, and the results are shown in Figure 5 g.

[0141] Figure 5 g is the contact angle diagram of the hydroxyl-functionalized oligomeric PDMS film-coated glass substrate, and the contact angle is 75°. Figure 5 a is the contact angle diagram of the non-reactive functionalized PDMS film-coated glass substrate, and the contact angle is 113°. Comparison Figure 5 g and Figure 5a. The contact angle of the carboxyl-functionalized PDMS film layer on the glass substrate decreased by 38°, indicating that the carboxyl-functionalized monomer was successfully coated on the surface of the PDMS film layer. The hydrophilicity of the carboxyl-functionalized PDMS film layer on the glass substrate is stronger than that of the PDMS film layer on the glass substrate without reactive functional groups.

[0142] Assembly of the device with a reactive functional group / oligomer PDMS film layer glass slide

[0143] Refer to Figure 2 Structural diagram of the device with a reactive functional group / oligomer PDMS film layer glass slide. The device with a reactive functional group / oligomer PDMS film layer glass slide consists of an upper cover 1, a well plate 2, a gasket 3, a glass slide 4, and a lower cover 5.

[0144] The assembly process includes the following steps:

[0145] (1) Remove the lower cover 5;

[0146] (2) Tear off the protective film on the lower side of the gasket, align it with the corresponding hole edges on the lower cover 5, and paste it;

[0147] (3) Tear off the upper protective film of the gasket, then align it with the card slot of the lower cover 5, and paste and fix the glass slide on it;

[0148] (4) Tear off the upper protective film of the gasket, align it with the positioning holes on the well plate 2, and paste the gasket on the well plate;

[0149] (5) Tear off the lower protective film of the gasket, adjust the position of the well plate according to the limit of the lower cover 5, so that the well plate is pasted on the glass slide;

[0150] (6) Fold the locking tab part of the lower cover 5, press the well plate, the glass slide and the lower cover tightly, so that the locking hole on the tab catches the locking protrusion on the well plate and lock it.

[0151] (7) Cover the upper cover, and the multi-channel glass slide is completed for assembly.

[0152] Example 9 Immobilization of epoxy-functionalized PDMS film layer glass with mouse IgG antibody

[0153] This example is the immobilization of the epoxy-functionalized PDMS film layer glass substrate prepared in Example 1 with mouse IgG antibody. It is divided into three immobilizations: 9.1 Epoxy ring-opening immobilization, 9.2 Physical adsorption, and 9.3 Epoxy group conversion to amino-functionalization. The basic immobilization method of the PDMS film layer glass substrate with the epoxy group converted to a hydroxyl group is the same as that of the hydroxyl-functionalized PDMS film layer glass substrate. For details, refer to Example 8.

[0154] 9.1 Epoxy ring-opening immobilization. The method used is the epoxy ring-opening immobilization of the spotting buffer in an alkaline environment. The specific steps are as follows:

[0155] (a) The epoxy-functionalized PDMS film layer glass substrate was adhered to the silicone substrate, and spotting was performed using a Nano-Plotter 2.1 with a spotting array format of 6×6.

[0156] The spotting buffer formulation was: 50 mM pH 9.6 CBS buffer + 5% sucrose; the CBS buffer was a carbonate buffer (1000 mL, pH 9.6): 1.59 g of NaCO3 + 2.93 g of NaHCO3; after spotting was completed, the multi-channel glass slide was assembled according to Example 8.

[0157] (b) It was immobilized at 37°C for 3 hours and then cured at room temperature overnight for 20 hours.

[0158] (c) 200 μL / well of blocking solution was added (the blocking solution formulation was 0.1 M PB buffer + 5% sucrose + 3% BSA (bovine serum albumin)), and blocked at 37°C for 1 h.

[0159] (d) The blocking solution was removed and dried at 37°C for 1 h.

[0160] (e) It was vacuum-sealed and stored at 2 - 8°C.

[0161] 9.2 Physical adsorption immobilization of mouse IgG on the epoxy-functionalized PDMS film layer glass substrate: The specific steps were similar to 9.1, and the main difference was that the spotting buffer used was 25 mM pH 7.4 PB buffer + 5% sucrose. The PB buffer was a phosphate buffer (1250 mL, pH 7.4): 35.5 g of Na2HPO4 + 8.5 g of KH2PO4.

[0162] 9.3 Immobilization of the epoxy group converted to an amino-functionalized PDMS film layer glass substrate and mouse IgG:

[0163] 9.3.1 Using EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) as the activator for the amino functional group, the specific experimental steps were as follows:

[0164] (a) Accurately weigh 10 mg of EDC and dissolve it in 50 mL of 25 mM pH 5.5 MES solution to prepare an activation buffer (prepared and used immediately).

[0165] (b) Take the amino-functionalized PDMS film layer glass substrate and place it in 30 mL of the EDC activation buffer, and activate it at 37°C for 1 hour.

[0166] (c) Discard the activation buffer, paste the EDC-activated PDMS film-coated glass substrate onto the silicone substrate, and use Nano-Plotter 2.1 for spotting. The spotting array is in a 6*6 format. The spotting buffer formulation is as follows: 25 mM pH 5.5 MES buffer: Dissolve 1.3328 g of MES powder in 250 mL of H2O and adjust to pH 5.5 with NaOH; after spotting, assemble the device with the reactive functional group / oligomer PDMS film-coated glass slide according to Example 8.

[0167] The remaining steps are the same as those in 9.1(b), (c), (d), and (e).

[0168] 9.3.2 Physical adsorption and immobilization of the amino-functionalized PDMS film-coated glass substrate with mouse IgG antibody. The specific steps are the same as those for the physical adsorption and immobilization of mouse IgG on the epoxy-functionalized film in 9.2.

[0169] 9.3.3 Use CDI (N,N'-carbonyldiimidazole) as the activator for the amino functional group. The specific experimental steps are similar to those in 9.3.1. The main difference is that the EDC activator is replaced with a CDI activator. The preparation method of the CDI activation buffer is: Accurately weigh 0.5 g of CDI and dissolve it in an anhydrous acetone solution to prepare the activation buffer (prepared and used immediately); the remaining steps are the same as those in 9.3.1.

[0170] 9.3.4 Use glutaraldehyde as the activator for the amino functional group. The specific experimental steps are similar to those in 9.3.1. The main difference is that the EDC activator is replaced with a glutaraldehyde activator. The preparation method of the glutaraldehyde activation buffer is: Accurately weigh 35 mL of glutaraldehyde aqueous solution (25%) and add it to 50 mL of 10 mM pH 7.4 PBS buffer solution to prepare the activation buffer (prepared and used immediately); the remaining steps are the same as those in 9.3.1.

[0171] Example 10 Immobilization of the hydroxy-functionalized PDMS film-coated glass substrate with mouse IgG antibody

[0172] This example is about the immobilization of the prepared hydroxy-functionalized PDMS film-coated glass substrate with mouse IgG antibody in Example 5. The methods for immobilizing mouse IgG antibody on the hydroxy-functionalized PDMS film-coated glass substrate are divided into 10.1 Hydroxy functional group activation and immobilization and 10.2 Physical adsorption and immobilization.

[0173] 10.1 The hydroxy functional group activation and immobilization method uses CDI (N,N'-carbonyldiimidazole) as the activator for the hydroxy functional group. The specific method is the same as that in 9.3.3.

[0174] 10. Physical adsorption and immobilization of the 2-hydroxy functional group PDMS film layer glass substrate and mouse IgG antibody. The specific steps are the same as those for the physical adsorption and immobilization of mouse IgG on the epoxy-functionalized film in 9.2.

[0175] Example 11 Immobilization of the carboxyl-functionalized PDMS film layer glass substrate and mouse IgG antibody

[0176] This example is about the immobilization of the carboxyl-functionalized PDMS film layer glass substrate and mouse IgG antibody prepared in Example 6. The methods for immobilizing mouse IgG antibody on the carboxyl-functionalized PDMS film layer glass substrate are divided into 11.1 and 11.2 carboxyl group functional group activation immobilization (including EDC and CDI as activators) and 11.3 physical adsorption immobilization.

[0177] 11.1 For the carboxyl-functionalized immobilization method, EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) is used as the activator for the carboxyl functional group. The specific method is the same as that in 9.3.1.

[0178] 11.2 For the carboxyl-functionalized immobilization method, CDI (N,N'-carbonyldiimidazole) is used as the activator for the carboxyl functional group. The specific method is the same as that in 9.3.3.

[0179] 11.3 Physical adsorption and immobilization of the carboxyl-functionalized PDMS film layer glass substrate and mouse IgG antibody. The specific steps are the same as those for the physical adsorption and immobilization of mouse IgG on the epoxy-functionalized PDMS film layer glass substrate in 9.2.

[0180] Example 12 Detection of the signal value of mouse IgG antibody immobilized on the functionalized PDMS film layer glass substrate

[0181] This example is about the detection of the signal value of mouse IgG antibody immobilized on the functionalized PDMS film layer glass substrate prepared in Examples 9 - 11.

[0182] The detection principle is to utilize the near-infrared laser confocal technology and adopt the direct method to detect the signal value of IgG antibodies immobilized on the PDMS film-coated glass substrate, and evaluate the immobilization efficiency and uniformity of different PDMS film-coated glass substrates for murine IgG antibodies. Specifically, the biotinylated murine IgG antibody reaction solution is added to the functionalized PDMS film-coated glass substrate immobilized with murine IgG antibodies, incubated to form an antibody-biolabeled antibody complex; the unbound free components are washed away by washing, and then streptavidin fluorescein is added to form a biotin-avidin system, and finally an antibody-biolabeled antibody-avidin fluorescein complex is formed on the PDMS film-coated glass substrate. Then, the signal value of fluorescein on the complex is detected by a biochip scanner. The fluorescence signal value is positively correlated with the concentration of murine IgG immobilized on the PDMS film-coated glass substrate, and the level of the membrane signal value reflects the content of murine IgG immobilized on the membrane and the uniformity of the antibody content immobilized on the PDMS film-coated glass substrate.

[0183] The specific experimental steps are as follows:

[0184] 1. Sampling: Pipette the biotin-labeled murine IgG antibody solution (concentration 0.5 μg / mL) into the glass substrate well plate, 100 μL per well.

[0185] 2. Incubation: Place the glass substrate well plate flat and fix it in a thermostatic shaker, incubate and oscillate at 37 °C and 1000 rpm for 20 min. Discard the liquid in the wells and pat dry.

[0186] 3. Washing: Pipette 200 μL of washing solution into different chip reaction wells, incubate and oscillate at 37 °C and 1000 rpm for 2 min, discard the liquid in the wells and pat dry. Repeat the washing 2 times and pat dry.

[0187] 4. Adding streptavidin fluorescein: Pipette 100 μL of streptavidin fluorescein solution (concentration 0.33 μg / mL) into each well.

[0188] 5. Incubation: Place the chip flat and fix it in a thermostatic shaker, incubate and oscillate at 37 °C and 1000 rpm for 5 min. Discard the liquid in the wells and pat dry.

[0189] 6. Washing: Pipette 200 μL of washing solution into different wells, incubate and oscillate at 37 °C and 1000 rpm for 2 min, discard the liquid in the wells and pat dry. Repeat the washing 2 times and pat dry.

[0190] 7. Adding preservation solution: Pipette 70 μL of preservation solution into each chip well.

[0191] 8. Scanning: Put the reacted protein chip into a biochip scanner (serial number: LCS-01) for scanning and read out the fluorescence intensity.

[0192] Among them,

[0193] Formulation of biotin-labeled mouse IgG antibody solution: Biotin-labeled mouse IgG antibody (0.5 μg / mL) + sucrose (1%) + Tween 80 (5‰) + bovine serum albumin BSA (1%) dissolved in 0.1M PB buffer solution (pH 7.4).

[0194] Formulation of washing solution: Tris (15 mM) + NaCl (0.9%) + Tween 80 (0.1%)

[0195] Formulation of streptavidin fluorescein: Streptavidin fluorescein (0.33 ug / mL) + sucrose (1%) + Tween 80 (5‰) + bovine serum albumin BSA (1%) dissolved in 0.1M PB buffer solution (pH 7.4).

[0196] Formulation of preservation solution: Sucrose (1%) + Tween 80 (5‰) + bovine serum albumin BSA (1%) dissolved in 0.1M PB buffer solution (pH 7.4).

[0197] Figure 9 It is an example image for signal value detection of mouse IgG antibody fixed to the functionalized PDMS film layer glass substrate. It can be seen from the figure that the dots of mouse IgG fixed to the functionalized PDMS film layer glass substrate are uniform and there is no trailing phenomenon.

[0198] The contact angles of the products prepared in Example 1 and Examples 4 - 6 were detected, and the results are shown in Table 2 for details.

[0199] Table 2 Contact angles of the glass substrate with the reactive functional group oligomer PDMS film layer

[0200]

[0201] It can be seen from the data in Table 2 that under the comparison of the same methodology, the signal value of the epoxy group converted to the amino / hydroxyl functionalized glass slide is the highest, the signal value of CDI activation is higher than that of EDC activation, and the signal value of chemical fixation is higher than that of physical adsorption fixation. Among these functionalized membranes and fixation methods, the obtained signal values are all relatively high, greater than 5000, and the coefficient of variation is less than 10%. It shows that the functionalized membrane prepared by this method can be used for the fixation of protein molecules and has good performance.

[0202] It should be understood that the above description can illustrate one or more, but not all, exemplary embodiments of the present invention, and the scope of the present invention should not be limited by any of the above exemplary embodiments.

[0203] All various aspects, embodiments, options, and numerical ranges described herein can be combined in any and all variations.

[0204] The foregoing description of the specific embodiments will so fully reveal the general nature of the present invention that others can, without departing from the general concept of the present invention, readily modify and / or adapt various applications such as these specific embodiments by applying the technical knowledge in the art without undue experimentation. Therefore, such adaptations and modifications are also included within the meaning and scope of the equivalent forms of the disclosed embodiments based on the teachings and guidance presented herein. It should be understood that the words or terms herein are for the purpose of description rather than limitation, and thus the terminology or phrasing of this specification will be interpreted by those skilled in the art in light of the teachings and guidance.

Claims

1. A glass slide device with a PDMS film layer having reactive functional groups, characterized in that, It includes a cover body, an orifice plate, an orifice plate base, and a glass slide; The cover body is arranged on the orifice plate; A number of positioning holes are provided on the orifice plate, and the orifice plate is fixedly connected to the orifice plate base; The glass slide is arranged between the orifice plate and the orifice plate base. Washers are arranged above and below the glass slide, and a number of hole grooves corresponding to the positioning holes are provided on the washers; A PDMS film layer with reactive functional groups is coated on the glass slide.

2. The glass slide device with a PDMS film layer having reactive functional groups as described in claim 1, characterized in that, A protrusion is arranged at the bottom edge position of the orifice plate; a foldable locking fin corresponding to the position of the protrusion is arranged at the edge position of the orifice plate base, and the orifice plate and the orifice plate base can be fixedly connected by engaging the protrusion with the locking fin.

3. The glass slide device with a PDMS film layer having reactive functional groups according to claim 1, characterized in that, The number of the positioning holes is 1 - 1000.

4. The glass slide device with a reactive functional group PDMS film layer as described in claim 1, characterized in that, The cover body is specifically a black cover body.

5. The glass slide device with a reactive functional group PDMS film layer as claimed in claim 1, wherein The cover body is specifically a transparent cover body.

6. The glass slide device with a reactive functional group PDMS film layer as described in claim 1, characterized in that, The glass slide is specifically a glass slide substrate with high light transmittance.

7. The glass slide device with a reactive functional group PDMS film layer as described in claim 1, characterized in that, The glass slide is specifically a black glass slide substrate.

8. A preparation method of a glass slide with a PDMS film layer having reactive functional groups, characterized in that, The method includes the following steps: (a) Premix components A and B of silicone, and then preform with the glass slide to obtain a preformed PDMS film layer glass slide; (b) Perform plasma surface treatment on the preformed PDMS film layer glass slide; (c) Coat the preformed PDMS film layer glass slide obtained in step (b) with a functional group monomer and / or oligomer; (d) Fix the functional group monomer and / or oligomer on the surface of the preformed PDMS film layer glass slide to obtain a PDMS film layer glass slide with reactive functional groups.

9. The preparation method according to claim 8, wherein In step (c), the functional groups of the functional group monomer and / or oligomer are selected from one or more of epoxy group, hydroxyl group, carboxyl group, and amino group.

10. The preparation method according to any one of claims 8 or 9, characterized in that, In step (c), the coating is any one of spin coating method, casting method, spraying method, scraping method, drop coating method, or pouring mold method. Preferably, the coating is carried out by spin coating method, for example, coating with a spin coater or a spin coater.

11. The preparation method according to any one of claims 8-10, characterized in that, In step (c), the amount of the spin-coated monomer and / or oligomer is in the range of about 50 μL - 5 mL. Preferably, the amount is about 100 μL - 1 mL; More preferably, the amount of the spin-coated monomer is about 150 μL, and the amount of the oligomer is about 100 μL.

12. The preparation method according to any one of claims 8-11, characterized in that, In step (d), after the functional group monomer and / or oligomer is fixed on the surface of the preformed PDMS film layer glass slide, a ring-opening reaction occurs.

13. The preparation method according to any one of claims 8-12, characterized in that, In step (d), the fixing of the functional group monomer and / or oligomer on the surface of the preformed PDMS film layer glass slide is to place the PDMS film layer glass slide spin-coated with the functional group monomer and / or oligomer at about room temperature - 150 °C for treatment to obtain a functionalized PDMS film layer glass slide.

14. A PDMS film layer glass slide with reactive functional groups prepared by the method according to any one of claims 8 - 13.

15. The PDMS film-coated glass slide with reactive functional groups as described in claim 14, wherein, The film layer thickness of the reactive functional group PDMS film attached to the PDMS film layer glass slide with reactive functional groups is about 1 nm - 5 mm.

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