A glass slide for blood testing and its preparation method

Abstract

This invention relates to the field of medical device technology, specifically disclosing a glass slide for blood testing and its preparation method. The invention constructs a highly cross-linked multilayer composite coating on the glass slide, enhancing the overall mechanical strength and cohesion of the coating. The alternating adhesion-antibacterial structure overcomes the limitations of traditional single coatings in terms of function and stability. This ensures that cells can be effectively adhered and captured while maximally inhibiting bacterial growth and interference, thereby significantly improving the accuracy of blood testing.

Description

Technical Field

[0001] This invention relates to the field of medical device technology, specifically to a glass slide for blood testing and its preparation method. Background Technology

[0002] Blood test slides are a core consumable in blood morphology analysis, and their surface properties directly determine the accuracy and reliability of the test results. In clinical diagnosis, whether it's routine blood cell differential counting or screening for abnormal cell morphology, parasitic infections, or rare lesions, uniform spreading and firm adhesion of blood cells on the slide surface are required. However, traditional ordinary slides, due to limitations in their surface physicochemical properties, often fail to meet the high requirements of modern precision medicine for detection sensitivity and specificity.

[0003] Specifically, traditional slides suffer from three main problems: insufficient hydrophilicity, poor adhesion, and inadequate antibacterial properties. Insufficient hydrophilicity prevents blood samples from spreading automatically after being added, leading to an "edge effect"—samples clustering at the slide edges, resulting in uneven cell distribution and affecting the accuracy of whole-slide scanning and morphological observation. Furthermore, traditional slides have poor adhesion, especially during subsequent staining, washing, and mounting processes, where cells easily detach from the surface. This is particularly problematic for rare samples such as those from anemic patients or those with minimal blood collection, easily leading to low cell counts or even misdiagnosis. Finally, the lack of antibacterial properties makes traditional slides susceptible to interference from bacteria and their secretions during testing, affecting cell morphology recognition or causing biocontamination. Therefore, developing a novel slide with high hydrophilicity, strong cell adhesion, and antibacterial properties has become an urgent technological direction for improving the quality of blood testing and meeting the needs of accurate clinical diagnosis. Summary of the Invention

[0004] The purpose of this invention is to provide a glass slide for blood testing and its preparation method, which solves the problems of poor hydrophilicity, poor adhesion and insufficient antibacterial properties of traditional glass slides.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution: A method for preparing a glass slide for blood testing, specifically comprising: Step 1: The glass slide is ultrasonically cleaned with acetone and anhydrous ethanol in sequence, then plasma activated, then impregnated with vinyltriethoxysilane solution, and vacuum dried to obtain the pretreated glass slide. Step 2: Using gallic acid and hyaluronic acid as raw materials, N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride were added to the coupling system to synthesize hyaluronic acid-gallic acid copolymer; Step 3: Add ε-polylysine and hyaluronic acid-gallic acid copolymer to PBS buffer, add sodium periodate to obtain polylysine / hyaluronic acid-gallic acid coating solution; Step 4: Using N-acetyl-L-cysteine ​​hydrochloride and chitosan as raw materials, add the coupling system N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to synthesize chitosan-acetylcysteine ​​copolymer; Step 5: Add polyethylene glycol acrylate and chitosan-acetylcysteine ​​copolymer to PBS buffer, add photoinitiator LAP, and obtain polyethylene glycol acrylate / chitosan-acetylcysteine ​​coating solution; Step 6: Alternately spin-coat polylysine / hyaluronic acid-gallic acid coating solution and polyethylene glycol acrylate / chitosan-acetylcysteine ​​coating solution onto the pretreated glass slide and dry and cure to form an alternating deposition composite coating, thus obtaining a glass slide for blood testing.

[0006] As a limitation of the present invention, the preparation method of the hyaluronic acid-gallic acid copolymer is as follows: Gallic acid was added to dimethyl sulfoxide and stirred at 200-300 rpm for 20-30 min to obtain a gallic acid solution. N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride were added to the gallic acid solution, and the mixture was stirred at 400-500 rpm for 1-3 h under light-protected conditions to obtain a GA-NHS solution. Hyaluronic acid was then added to dimethyl sulfoxide and stirred at 200-300 rpm for 20-30 min. N-hydroxysuccinimide and 1-ethyl- 3-(3-Dimethylaminopropyl)carbodiimide hydrochloride was reacted at 25-30℃ and stirred at 400-500 rpm for 3-5 h. Ethylenediamine was added, and the reaction was continued for 20-24 h under nitrogen protection to obtain HA-NH2 solution. HA-NH2 solution was added to GA-NHS solution, and the reaction was carried out under light-protected conditions with stirring at 400-500 rpm for 20-24 h. After the reaction was completed, the mixture was dialyzed sequentially with a mixture of dimethyl sulfoxide and deionized water and deionized water, and then freeze-dried to obtain hyaluronic acid-gallic acid copolymer.

[0007] Gallic acid (GA) and hyaluronic acid (HA) undergo an amidation reaction under the action of a coupling agent system to generate an amide-linked HA-GA copolymer. Specifically, the reaction involves: first, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) in the coupling agent system reacts with the carboxyl group on GA to generate an acylisourea intermediate; then, N-hydroxysuccinimide (NHS) in the system further reacts with the intermediate to generate a stable GA-NHS ester; similarly, HA generates HA-NHS ester under the action of EDC / NHS, and the amino group of the bifunctional molecule ethylenediamine undergoes a substitution reaction with the HA-NHS ester to generate aminated hyaluronic acid (HA-NH2); then, the GA-NHS ester undergoes a substitution reaction with the amino group on HA-NH2 to finally form a stable amide bond, and the GA molecule is linked to the HA-GA copolymer through a covalent bond.

[0008] GA and HA undergo a copolymerization reaction to form an HA-GA copolymer. The GA structural unit contains catechol groups, which are the functional groups responsible for mimicking mussel adhesion. These groups adhere to the pretreated slide and cell surfaces through various non-covalent interactions such as coordination bonds and hydrogen bonds, significantly enhancing the bonding strength between the coating and the substrate, and between coating masks, thereby improving the cell adhesion of the composite coating. HA is a hydrophilic polysaccharide rich in hydroxyl and carboxyl groups, which can bind a large number of water molecules through hydrogen bonds, forming a highly hydrated interface. This gives the coating excellent hydrophilicity, promoting uniform blood spreading on the coating, while also providing good biocompatibility, helping to maintain cell activity and condition.

[0009] As a limitation of the present invention, in the GA-NHS solution, the mass ratio of gallic acid, N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is (5-7):(6-8):(10-12); in the HA-NH2 solution, the mass ratio of hyaluronic acid, ethylenediamine, N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is (10-12):(8-10):(12-15):(22-25).

[0010] As a limitation of the present invention, in the polylysine / hyaluronic acid-gallic acid coating solution, the mass ratio of ε-polylysine, hyaluronic acid-gallic acid copolymer and sodium periodate is (1-1.5):(1-1.5):(0.1-0.15).

[0011] Under the mild oxidation of sodium periodate, the adjacent hydroxyl groups in the unsubstituted sugar ring units of the HA-GA molecular chain are selectively oxidized, resulting in carbon-carbon bond cleavage and ring opening to generate highly reactive aldehyde groups. These newly generated aldehyde groups undergo Schiff base reactions with the amino groups on the ε-polylysine (PLL) molecular chain, dehydrating and condensing to form stable imine bonds (C=N), thus constructing a covalent cross-linked network.

[0012] The HA-GA copolymer and PLL are covalently cross-linked via imine bonds to form a cross-linked network structure, which enhances the cohesive strength and structural stability of the coating, preventing swelling and peeling. The protonated amino groups on the PLL segments of the cross-linked network impart a positive charge to the coating surface, enabling effective attraction and initial anchoring of negatively charged cell membranes through electrostatic attraction. Simultaneously, the catechol groups in HA-GA provide a biomimetic adhesion interface, further synergistically promoting cell spreading and firm adhesion to the coating. The combined effect of these two factors achieves efficient and stable capture of target cells, thereby enhancing the coating's cell adhesion.

[0013] As a limitation of the present invention, the preparation method of the chitosan-acetylcysteine ​​copolymer is as follows: Chitosan was added to a 1 wt% aqueous acetic acid solution and stirred at 200-300 rpm for 20-30 min to obtain a chitosan solution. N-acetyl-L-cysteine ​​hydrochloride was added to deionized water and stirred at 200-300 rpm for 20-30 min to obtain an N-acetyl-L-cysteine ​​hydrochloride solution. N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride were added to deionized water and stirred at 200-300 rpm for 20-30 min to obtain a chitosan solution. Stir at 0 rpm for 20-30 min, then add to N-acetyl-L-cysteine ​​hydrochloride solution. Stir at 25-30℃ and 400-500 rpm for 1-3 h to obtain activated acetylcysteine ​​hydrochloride solution. Add the activated acetylcysteine ​​hydrochloride solution to chitosan solution and react at 25-30℃ and 400-500 rpm for 20-24 h. After the reaction is complete, dialyze with deionized water and freeze dry to obtain chitosan-acetylcysteine ​​copolymer.

[0014] Under the action of the coupling agent system, the carboxyl group in the N-acetyl-L-cysteine ​​(NAC) molecule is first activated to generate a highly reactive intermediate. Subsequently, the intermediate undergoes an amidation reaction with the amino group on the chitosan (CS) molecular chain to form a stable amide bond, thereby covalently grafting cysteine ​​onto chitosan to obtain the CS-NAC copolymer.

[0015] CS and NAC undergo a copolymerization reaction to form a CS-NAC copolymer. The introduction of N-acetyl-L-cysteine ​​introduces an acetylated structure and a thiol group (-SH) into chitosan. The acetylation structure enhances the stability of the thiol group, effectively avoiding the disadvantage of cysteine ​​easily oxidizing to form disulfide bonds during reaction and storage, significantly improving reaction controllability and product reproducibility. The thiol group enhances free radical scavenging ability, improves antioxidant and antibacterial properties, and serves as a reaction site in subsequent polymerization, undergoing a thiol-olefin click reaction with the double bonds of polyethylene glycol acrylate (PEG-DA) to construct a dense and stable three-dimensional cross-linked network, greatly enhancing the mechanical strength and anti-swelling properties of the coating. The inherent positive charge of chitosan and the introduction of N-acetyl-L-cysteine ​​work synergistically to disrupt the integrity of bacterial cell membranes through electrostatic interactions, endowing the coating with excellent broad-spectrum antibacterial properties, effectively inhibiting the growth of common pathogens in blood samples, and ensuring the accuracy of test results.

[0016] As a limitation of the present invention, the mass ratio of chitosan, N-acetyl-L-cysteine ​​hydrochloride, N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is (1-1.5):(0.8-1.2):(0.8-1.0):(0.5-0.7).

[0017] As a limitation of the present invention, in the polyethylene glycol acrylate / chitosan-acetylcysteine ​​coating liquid, the mass ratio of polyethylene glycol acrylate, chitosan-acetylcysteine ​​copolymer and photoinitiator LAP is (1-1.5):(1-1.5):(0.05-0.1).

[0018] The copolymer of polyethylene glycol acrylate (PEG-DA) and CS-NAC achieves covalent cross-linking through a photo-initiated "thiol-ene" click chemistry reaction. Under ultraviolet light irradiation, the photoinitiator LAP decomposes to generate free radicals, which preferentially attack the carbon-carbon double bonds at the ends of PEG-DA to form carbon-center free radicals. Subsequently, the free radicals react with the thiol groups on the CS-NAC molecular chain to generate thioether bonds, forming a cross-linked network structure.

[0019] The PEG-DA copolymer forms a cross-linked network structure, and the PEG-DA in the cross-linked network exhibits good hydrophilicity and antifouling properties. Chitosan and cysteine ​​work synergistically to ensure the cross-linked coating has excellent antibacterial properties.

[0020] As a limitation of the present invention, the plasma activation treatment process conditions for preparing the pretreated glass slide include: argon flow rate of 20-40 sccm, oxygen flow rate of 10-20 sccm, power supply of 80-120W, and treatment time of 2-4 min.

[0021] As a limitation of the present invention, the composite coating consists of alternating polylysine / hyaluronic acid-gallic acid layers and polyethylene glycol acrylate / chitosan-acetylcysteine ​​layers, with the number of layers being 3-7; the thickness of the polylysine / hyaluronic acid-gallic acid layer is 80-100 nm, formed by drying and curing the polylysine / hyaluronic acid-gallic acid coating liquid, under the drying and curing conditions of 30-40℃ for 60-90 min; the thickness of the polyethylene glycol acrylate / chitosan-acetylcysteine ​​layer is 30-50 nm, formed by drying and curing the polyethylene glycol acrylate / chitosan-acetylcysteine ​​coating liquid, under the drying and curing conditions of 365 nm UV irradiation for 2-3 min.

[0022] A glass slide for blood testing, prepared by any of the methods described above.

[0023] Compared with the prior art, the beneficial effects of the present invention are: This invention constructs a highly cross-linked multilayer composite structure by alternately depositing polylysine / hyaluronic acid-gallic acid layers and polyethylene glycol acrylate / chitosan-acetylcysteine ​​layers on a glass slide. Schiff base reaction between PLL and HA-GA, and "thiol-ene" click chemistry between PEG-DA and CS-NAC, form an interpenetrating and stable covalent cross-linked network. This structure not only enhances the overall mechanical strength and cohesion of the coating, effectively preventing detachment due to swelling or erosion in liquid environments, but also greatly strengthens interlayer bonding through multiple interfacial interactions (such as hydrogen bonding and electrostatic interactions), ensuring the coating's durability under long-term use or harsh conditions. The alternating adhesion-antibacterial structure overcomes the limitations and poor stability of traditional single-layer coatings, ensuring effective cell adhesion and capture while maximally inhibiting bacterial growth and interference, thereby significantly improving the accuracy of blood tests. Detailed Implementation

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The dosages recorded in the embodiments are laboratory-scale tests, and can all be scaled up proportionally. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Glass slide (material: borosilicate glass, B content: 13.5 wt%, Si content: 80 wt%, size: 76.2 mm × 25.4 mm, thickness: 1.2 mm), PBS buffer (density: 1.0 g / cm³) 3(pH=7.4), polyethylene glycol acrylate (molecular weight: 700 Da).

[0026] The pretreatment method for the glass slides is as follows: 3g of vinyltriethoxysilane is added to 100mL of toluene and stirred at 200rpm for 20min to obtain a vinyltriethoxysilane toluene solution. The glass slides are then placed in acetone and anhydrous ethanol in sequence, ultrasonically cleaned for 5min each, and vacuum dried at 80℃ for 15min. Subsequently, they are transferred to a plasma device, evacuated to 10Pa, and argon and oxygen are introduced for plasma activation. The argon flow rate is set to 20sccm, the oxygen flow rate to 10sccm, the power supply to 100W, and the treatment time to 2min. The slides are then transferred to the vinyltriethoxysilane toluene solution and immersed at 25-30℃ for 10min. After immersion, the slides are removed, cleaned with toluene and anhydrous ethanol, and vacuum dried at 120℃ for 30min to solidify, thus obtaining the pretreated glass slides.

[0027] Example 1: A method for preparing a glass slide for blood testing, specifically as follows: Step 1: Add 5g gallic acid to 100mL dimethyl sulfoxide and stir at 200rpm for 20min to obtain a gallic acid solution. Add 6g N-hydroxysuccinimide and 10g 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to the gallic acid solution and stir at 400rpm for 2h under light-protected conditions to obtain a GA-NHS solution. Add 10g hyaluronic acid to 100mL dimethyl sulfoxide and stir at 200rpm for 20min. Add 12g N-hydroxysuccinimide and 22g... 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was reacted at 25-30℃ and stirred at 400 rpm for 4 h. 8 g of ethylenediamine was added, and the reaction was continued for 24 h under nitrogen protection to obtain HA-NH2 solution. The HA-NH2 solution was added to GA-NHS solution, and the reaction was carried out at 400 rpm for 24 h under light-protected conditions. After the reaction was completed, the mixture was dialyzed with a mixture of dimethyl sulfoxide and deionized water (v / v=1:1) for 24 h and then dialyzed with deionized water for 48 h to remove unreacted GA, byproducts and solvent. The mixture was then freeze-dried to obtain HA-GA graft copolymer. Step 2: Add 1g of ε-polylysine to 100mL of PBS buffer and stir at 200rpm for 20min to obtain a polylysine solution; add 1g of HA-GA graft copolymer to 100mL of PBS buffer and stir at 200rpm for 20min to obtain an HA-GA graft copolymer solution; mix the polylysine solution and the HA-GA graft copolymer solution, add 0.1g of sodium periodate, and stir until homogeneous to obtain a PLL / HA-GA coating solution; Step 3: Add 1g of chitosan to 100mL of 1wt% acetic acid aqueous solution and stir at 200rpm for 20min to obtain a chitosan solution. Add 0.8g of N-acetyl-L-cysteine ​​hydrochloride to 30mL of deionized water and stir at 200rpm for 20min to obtain an N-acetyl-L-cysteine ​​hydrochloride solution. Add 0.8g of N-hydroxysuccinimide and 0.5g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to 20mL of deionized water and stir at 200rpm for 30min. Add this to the N-acetyl-L-cysteine ​​hydrochloride solution and stir at 25-30℃ and 400rpm for 1h to obtain an activated acetylcysteine ​​hydrochloride solution. Add the activated acetylcysteine ​​hydrochloride solution to the chitosan solution and stir at 25-30℃ and 400rpm for 24h. After the reaction is complete, dialyze with deionized water for 48h and freeze-dry to obtain a chitosan-acetylcysteine ​​copolymer. Step 4: Add 1g of polyethylene glycol acrylate to 100mL of PBS buffer and stir at 200rpm for 20min to obtain a polyethylene glycol acrylate solution. Add 1g of chitosan-acetylcysteine ​​copolymer to 100mL of PBS buffer and stir at 200rpm for 20min to obtain a chitosan-acetylcysteine ​​copolymer solution. Mix the polyethylene glycol acrylate solution and the chitosan-acetylcysteine ​​copolymer solution, add 0.05g of photoinitiator LAP, and stir evenly to obtain the PEG / CS-NAC coating solution. Step 5: Spin-coat the PLL / HA-GA coating solution onto the pretreated glass slide and cure it at 35°C for 60 min to form a 100 nm thick PLL / HA-GA layer. Then spin-coat the PEG / CS-NAC coating solution onto the PLL / HA-GA layer and irradiate it with 365 nm UV light for 3 min to form a 50 nm thick PEG / CS-NAC layer. Deposit another 100 nm thick PLL / HA-GA layer under the same conditions to form a three-layer composite coating, thus obtaining a glass slide for blood testing.

[0028] Example 2: A method for preparing a glass slide for blood testing, specifically as follows: Step 1: Add 6g of gallic acid to 100mL of dimethyl sulfoxide and stir at 200rpm for 20min to obtain a gallic acid solution. Add 6g of N-hydroxysuccinimide and 10g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to the gallic acid solution and stir at 400rpm for 2h under light-protected conditions to obtain a GA-NHS solution. Add 11g of hyaluronic acid to 100mL of dimethyl sulfoxide and stir at 200rpm for 20min. Add 12g of N-hydroxysuccinimide and 22g of... 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was reacted at 25-30℃ and stirred at 400 rpm for 4 h. 9 g of ethylenediamine was added, and the reaction was continued for 24 h under nitrogen protection to obtain HA-NH2 solution. The HA-NH2 solution was added to GA-NHS solution, and the reaction was carried out at 400 rpm for 24 h under light-protected conditions. After the reaction was completed, the mixture was dialyzed with a mixture of dimethyl sulfoxide and deionized water (v / v=1:1) for 24 h and then dialyzed with deionized water for 48 h to remove unreacted GA, byproducts and solvent. The mixture was then freeze-dried to obtain HA-GA graft copolymer. Step 2: Add 1.2g of ε-polylysine to 100mL of PBS buffer and stir at 200rpm for 20min to obtain a polylysine solution; add 1.2g of HA-GA graft copolymer to 100mL of PBS buffer and stir at 200rpm for 20min to obtain an HA-GA graft copolymer solution; mix the polylysine solution and the HA-GA graft copolymer solution, add 0.1g of sodium periodate, and stir until homogeneous to obtain a PLL / HA-GA coating solution; Step 3: Add 1.2g of chitosan to 100mL of 1wt% acetic acid aqueous solution and stir at 200rpm for 20min to obtain a chitosan solution. Add 1g of N-acetyl-L-cysteine ​​hydrochloride to 30mL of deionized water and stir at 200rpm for 20min to obtain an N-acetyl-L-cysteine ​​hydrochloride solution. Add 0.8g of N-hydroxysuccinimide and 0.5g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to 20mL of deionized water and stir at 200rpm for 30min. Add this to the N-acetyl-L-cysteine ​​hydrochloride solution and stir at 25-30℃ and 400rpm for 1h to obtain an activated acetylcysteine ​​hydrochloride solution. Add the activated acetylcysteine ​​hydrochloride solution to the chitosan solution and stir at 25-30℃ and 400rpm for 24h. After the reaction is complete, dialyze with deionized water for 48h and freeze-dry to obtain a chitosan-acetylcysteine ​​copolymer. Step 4: Add 1.2g of polyethylene glycol acrylate to 100mL of PBS buffer and stir at 200rpm for 20min to obtain a polyethylene glycol acrylate solution. Add 1.2g of chitosan-acetylcysteine ​​copolymer to 100mL of PBS buffer and stir at 200rpm for 20min to obtain a chitosan-acetylcysteine ​​copolymer solution. Mix the polyethylene glycol acrylate solution and the chitosan-acetylcysteine ​​copolymer solution, add 0.05g of photoinitiator LAP, and stir evenly to obtain the PEG / CS-NAC coating solution. Step 5: Spin-coat the PLL / HA-GA coating solution onto the pretreated glass slide and cure it at 35°C for 60 min to form a 100 nm thick PLL / HA-GA layer. Then spin-coat the PEG / CS-NAC coating solution onto the PLL / HA-GA layer and irradiate it with 365 nm UV light for 3 min to form a 50 nm thick PEG / CS-NAC layer. Under the same conditions, deposit a 100 nm thick PLL / HA-GA layer, a 50 nm thick PEG / CS-NAC layer, and a 100 nm thick PLL / HA-GA layer again to form a 5-layer composite coating, thus obtaining a glass slide for blood testing.

[0029] Example 3: A method for preparing a glass slide for blood testing, specifically as follows: Step 1: Add 7g gallic acid to 100mL dimethyl sulfoxide and stir at 200rpm for 20min to obtain a gallic acid solution. Add 6g N-hydroxysuccinimide and 10g 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to the gallic acid solution and stir at 400rpm for 2h under light-protected conditions to obtain a GA-NHS solution. Add 12g hyaluronic acid to 100mL dimethyl sulfoxide and stir at 200rpm for 20min. Add 12g N-hydroxysuccinimide and 22g... 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was reacted at 25-30℃ and stirred at 400 rpm for 4 h. 10 g of ethylenediamine was added, and the reaction was continued for 24 h under nitrogen protection to obtain HA-NH2 solution. The HA-NH2 solution was added to GA-NHS solution, and the reaction was carried out at 400 rpm for 24 h under light-protected conditions. After the reaction was completed, the mixture was dialyzed with a mixture of dimethyl sulfoxide and deionized water (v / v=1:1) for 24 h and then dialyzed with deionized water for 48 h to remove unreacted GA, byproducts and solvent. The mixture was then freeze-dried to obtain HA-GA graft copolymer. Step 2: Add 1.5g of ε-polylysine to 100mL of PBS buffer and stir at 200rpm for 20min to obtain a polylysine solution; add 1.5g of HA-GA graft copolymer to 100mL of PBS buffer and stir at 200rpm for 20min to obtain an HA-GA graft copolymer solution; mix the polylysine solution and the HA-GA graft copolymer solution, add 0.1g of sodium periodate, and stir until homogeneous to obtain a PLL / HA-GA coating solution; Step 3: Add 1.5g of chitosan to 100mL of 1wt% acetic acid aqueous solution and stir at 200rpm for 20min to obtain a chitosan solution. Add 1.2g of N-acetyl-L-cysteine ​​hydrochloride to 30mL of deionized water and stir at 200rpm for 20min to obtain an N-acetyl-L-cysteine ​​hydrochloride solution. Add 0.8g of N-hydroxysuccinimide and 0.5g... 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was added to 20 mL of deionized water and stirred at 200 rpm for 30 min. It was then added to N-acetyl-L-cysteine ​​hydrochloride solution and stirred at 25-30 °C and 400 rpm for 1 h to obtain activated acetylcysteine ​​hydrochloride solution. The activated acetylcysteine ​​hydrochloride solution was added to chitosan solution and stirred at 25-30 °C and 400 rpm for 24 h. After the reaction was completed, the mixture was dialyzed with deionized water for 48 h and then freeze-dried to obtain chitosan-acetylcysteine ​​copolymer. Step 4: Add 1.5g of polyethylene glycol acrylate to 100mL of PBS buffer and stir at 200rpm for 20min to obtain a polyethylene glycol acrylate solution. Add 1.5g of chitosan-acetylcysteine ​​copolymer to 100mL of PBS buffer and stir at 200rpm for 20min to obtain a chitosan-acetylcysteine ​​copolymer solution. Mix the polyethylene glycol acrylate solution and the chitosan-acetylcysteine ​​copolymer solution, add 0.05g of photoinitiator LAP, and stir evenly to obtain the PEG / CS-NAC coating solution. Step 5: Spin-coat the PLL / HA-GA coating solution onto the pretreated glass slide and cure it at 35°C for 60 min to form a 100 nm thick PLL / HA-GA layer. Then spin-coat the PEG / CS-NAC coating solution onto the PLL / HA-GA layer and irradiate it with 365 nm UV light for 3 min to form a 50 nm thick PEG / CS-NAC layer. Deposit another 100 nm thick PLL / HA-GA layer under the same conditions to form a three-layer composite coating, thus obtaining a glass slide for blood testing.

[0030] Based on Example 1, the following comparative experiments were conducted, specifically Comparative Example 1, Comparative Example 2, and Comparative Example 3, as described below: Comparative Example 1: This comparative example relates to a method for preparing a glass slide for blood testing. The difference from Example 1 is that gallic acid was not used to modify hyaluronic acid. Specifically: Step 1: Add 1g of ε-polylysine to 100mL of PBS buffer and stir at 200rpm for 20min to obtain a polylysine solution; add 1g of hyaluronic acid to 100mL of PBS buffer and stir at 200rpm for 20min to obtain a hyaluronic acid solution; mix the polylysine solution and the hyaluronic acid solution, add 0.1g of sodium periodate, and stir until homogeneous to obtain the PLL / HA coating solution; Step 2: Add 1g of chitosan to 100mL of 1wt% acetic acid aqueous solution and stir at 200rpm for 20min to obtain a chitosan solution. Add 0.8g of N-acetyl-L-cysteine ​​hydrochloride to 30mL of deionized water and stir at 200rpm for 20min to obtain an N-acetyl-L-cysteine ​​hydrochloride solution. Add 0.8g of N-hydroxysuccinimide and 0.5g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to 20mL of deionized water and stir at 200rpm for 30min. Add this to the N-acetyl-L-cysteine ​​hydrochloride solution and stir at 25-30℃ and 400rpm for 1h to obtain an activated acetylcysteine ​​hydrochloride solution. Add the activated acetylcysteine ​​hydrochloride solution to the chitosan solution and stir at 25-30℃ and 400rpm for 24h. After the reaction is complete, dialyze with deionized water for 48h and freeze-dry to obtain a chitosan-acetylcysteine ​​copolymer. Step 3: Add 1g of polyethylene glycol acrylate to 100mL of PBS buffer and stir at 200rpm for 20min to obtain a polyethylene glycol acrylate solution. Add 1g of chitosan-acetylcysteine ​​copolymer to 100mL of PBS buffer and stir at 200rpm for 20min to obtain a chitosan-acetylcysteine ​​copolymer solution. Mix the polyethylene glycol acrylate solution and the chitosan-acetylcysteine ​​copolymer solution, add 0.05g of photoinitiator LAP, and stir evenly to obtain the PEG / CS-NAC coating solution. Step 5: Spin-coat the PLL / HA coating solution onto the pretreated glass slide and cure it at 35°C for 60 min to form a 100 nm thick PLL / HA layer. Then spin-coat the PEG / CS-NAC coating solution onto the PLL / HA layer and irradiate it with 365 nm UV light for 3 min to form a 50 nm thick PEG / CS-NAC layer. Deposit another 100 nm thick PLL / HA layer under the same conditions to form a three-layer composite coating, thus obtaining a glass slide for blood testing.

[0031] Comparative Example 2: This comparative example relates to a method for preparing a glass slide for blood testing. The difference from Example 1 is that chitosan was not modified with N-acetyl-L-cysteine ​​hydrochloride. Specifically: Step 1: Add 5g gallic acid to 100mL dimethyl sulfoxide and stir at 200rpm for 20min to obtain a gallic acid solution. Add 6g N-hydroxysuccinimide and 10g 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to the gallic acid solution and stir at 400rpm for 2h under light-protected conditions to obtain a GA-NHS solution. Add 10g hyaluronic acid to 100mL dimethyl sulfoxide and stir at 200rpm for 20min. Add 12g N-hydroxysuccinimide and 22g... 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was reacted at 25-30℃ and stirred at 400 rpm for 4 h. 8 g of ethylenediamine was added, and the reaction was continued for 24 h under nitrogen protection to obtain HA-NH2 solution. The HA-NH2 solution was added to GA-NHS solution, and the reaction was carried out at 400 rpm for 24 h under light-protected conditions. After the reaction was completed, the mixture was dialyzed with a mixture of dimethyl sulfoxide and deionized water (v / v=1:1) for 24 h and then dialyzed with deionized water for 48 h to remove unreacted GA, byproducts and solvent. The mixture was then freeze-dried to obtain HA-GA graft copolymer. Step 2: Add 1g of ε-polylysine to 100mL of PBS buffer and stir at 200rpm for 20min to obtain a polylysine solution; add 1g of HA-GA graft copolymer to 100mL of PBS buffer and stir at 200rpm for 20min to obtain an HA-GA graft copolymer solution; mix the polylysine solution and the HA-GA graft copolymer solution, add 0.1g of sodium periodate, and stir until homogeneous to obtain a PLL / HA-GA coating solution; Step 3: Add 1g of polyethylene glycol acrylate to 100mL of PBS buffer and stir at 200rpm for 20min to obtain a polyethylene glycol acrylate solution. Add 1g of chitosan to 100mL of PBS buffer and stir at 200rpm for 20min to obtain a chitosan solution. Mix the polyethylene glycol acrylate solution and the chitosan solution, add 0.05g of photoinitiator LAP, and stir evenly to obtain the PEG / CS coating solution. Step 4: Spin-coat the PLL / HA-GA coating solution onto the pretreated glass slide and cure it at 35°C for 60 min to form a 100 nm thick PLL / HA-GA layer. Then spin-coat the PEG / CS coating solution onto the PLL / HA-GA layer and irradiate it with 365 nm UV light for 3 min to form a 50 nm thick PEG / CS layer. Deposit another 100 nm thick PLL / HA-GA layer under the same conditions to form a three-layer composite coating, thus obtaining a glass slide for blood testing.

[0032] Comparative Example 3: This comparative example relates to a method for preparing a glass slide for blood testing. The difference from Example 1 is that the PLL / HA-GA layer and the PEG / CS-NAC layer in the composite coating are interchanged. Specifically: Step 1: Add 5g gallic acid to 100mL dimethyl sulfoxide and stir at 200rpm for 20min to obtain a gallic acid solution. Add 6g N-hydroxysuccinimide and 10g 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to the gallic acid solution and stir at 400rpm for 2h under light-protected conditions to obtain a GA-NHS solution. Add 10g hyaluronic acid to 100mL dimethyl sulfoxide and stir at 200rpm for 20min. Add 12g N-hydroxysuccinimide and 22g... 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was reacted at 25-30℃ and stirred at 400 rpm for 4 h. 8 g of ethylenediamine was added, and the reaction was continued for 24 h under nitrogen protection to obtain HA-NH2 solution. The HA-NH2 solution was added to GA-NHS solution, and the reaction was carried out at 400 rpm for 24 h under light-protected conditions. After the reaction was completed, the mixture was dialyzed with a mixture of dimethyl sulfoxide and deionized water (v / v=1:1) for 24 h and then dialyzed with deionized water for 48 h to remove unreacted GA, byproducts and solvent. The mixture was then freeze-dried to obtain HA-GA graft copolymer. Step 2: Add 1g of ε-polylysine to 100mL of PBS buffer and stir at 200rpm for 20min to obtain a polylysine solution; add 1g of HA-GA graft copolymer to 100mL of PBS buffer and stir at 200rpm for 20min to obtain an HA-GA graft copolymer solution; mix the polylysine solution and the HA-GA graft copolymer solution, add 0.1g of sodium periodate, and stir until homogeneous to obtain a PLL / HA-GA coating solution; Step 3: Add 1g of chitosan to 100mL of 1wt% acetic acid aqueous solution and stir at 200rpm for 20min to obtain a chitosan solution. Add 0.8g of N-acetyl-L-cysteine ​​hydrochloride to 30mL of deionized water and stir at 200rpm for 20min to obtain an N-acetyl-L-cysteine ​​hydrochloride solution. Add 0.8g of N-hydroxysuccinimide and 0.5g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to 20mL of deionized water and stir at 200rpm for 30min. Add this to the N-acetyl-L-cysteine ​​hydrochloride solution and stir at 25-30℃ and 400rpm for 1h to obtain an activated acetylcysteine ​​hydrochloride solution. Add the activated acetylcysteine ​​hydrochloride solution to the chitosan solution and stir at 25-30℃ and 400rpm for 24h. After the reaction is complete, dialyze with deionized water for 48h and freeze-dry to obtain a chitosan-acetylcysteine ​​copolymer. Step 4: Add 1g of polyethylene glycol acrylate to 100mL of PBS buffer and stir at 200rpm for 20min to obtain a polyethylene glycol acrylate solution. Add 1g of chitosan-acetylcysteine ​​copolymer to 100mL of PBS buffer and stir at 200rpm for 20min to obtain a chitosan-acetylcysteine ​​copolymer solution. Mix the polyethylene glycol acrylate solution and the chitosan-acetylcysteine ​​copolymer solution, add 0.05g of photoinitiator LAP, and stir evenly to obtain the PEG / CS-NAC coating solution. Step 5: Spin-coat the PEG / CS-NAC coating solution onto the pretreated glass slide, irradiate with 365nm UV light for 3 min to form a 50nm thick PEG / CS-NAC layer. Then spin-coat the PEG / CS-NAC layer with PLL / HA-GA coating solution, cure at 35℃ for 60 min to form a 100nm thick PLL / HA-GA layer. Deposit another 50nm thick PEG / CS-NAC layer under the same conditions to form a three-layer composite coating, thus obtaining a glass slide for blood testing.

[0033] Testing experiment: Glass slides for blood testing were processed according to the processes in each embodiment and comparative example, and were used as test samples for the following tests.

[0034] Water contact angle test: The test is conducted in accordance with the "Method for Measurement of Contact Angle of Nanofilms" (GB / T 30447-2013). A 1 μL drop of deionized water is dropped onto the surface of the test sample using a microsyringe. The water contact angle is measured using a contact angle meter. Each test sample is tested 5 times, and the average value is taken.

[0035] Adhesion test: Human skin fibroblasts were selected as test cells. The test cells were seeded on the test sample and cultured in an incubator at 37°C and 5% CO2 concentration for 6 hours. The upper cell culture medium was aspirated, and the non-adhered cells were washed away with PBS buffer. Then, under the dark, FDA staining agent was added and stained for 20 minutes. After removing the FDA staining agent, PBS buffer solution was added and the sample was washed by shaking. The average cell density on the surface of the test sample was counted. Antibacterial activity test: The test was conducted in accordance with the "Determination of Antiviral Activity and Antibacterial Activity of Coatings" (GB / T 21866-2025). Staphylococcus aureus and Escherichia coli were selected as test bacteria, and ordinary glass slides without composite coating were used as the control group. The test bacteria were inoculated onto the test samples and covered with a polyethylene film to spread the bacterial solution evenly on the surface of the test samples. After incubation at 37°C and 90% RH for 24 hours, the bacterial solution was washed off. The supernatant was diluted and incubated in nutrient agar medium for 48 hours. The colony count was performed, and the antibacterial rate of the test samples was calculated.

[0036] Coating adhesion test: The test is conducted in accordance with the "Paints and Varnishes Pull-Off Adhesion Test" (GB / T 5210-2006). Two test samples with the coating side facing each other are bonded together with epoxy adhesive, dried in an oven at 45℃ for 24 hours, and then fixed on the two clamps of a tensile tester. The tensile tester is started, and the tensile speed is set to 1 mm / min until the two glass slides separate. Each test sample is tested 5 times, and the average value of the results is taken.

[0037]

[0038] Conclusion: The test data shows that the slide prepared in Example 1 for blood testing has a lower water contact angle than the comparative examples, and higher average cell density, antibacterial rate, and maximum tensile strength. Compared with Example 1, Comparative Example 1 did not use gallic acid to modify hyaluronic acid, which significantly affected the hydrophilicity and cell adhesion of the slide. Comparative Example 2 did not use cysteine ​​to modify chitosan, which significantly affected the antibacterial properties of the slide. In Comparative Example 3, the bottom and top layers of the composite coating were PEG / CS-NAC layers, resulting in weaker hydrophilicity, cell adhesion, and coating adhesion of the slide compared to Example 1. The slide for blood testing provided by this invention has good hydrophilicity, cell adhesion, and antibacterial properties.

[0039] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A method for preparing a glass slide for blood testing, characterized in that: Specifically: Step 1: The glass slide is ultrasonically cleaned with acetone and anhydrous ethanol in sequence, then plasma activated, then impregnated with vinyltriethoxysilane solution, and vacuum dried to obtain the pretreated glass slide. Step 2: Using gallic acid and hyaluronic acid as raw materials, N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride were added to the coupling system to synthesize hyaluronic acid-gallic acid copolymer; Step 3: Add ε-polylysine and hyaluronic acid-gallic acid copolymer to PBS buffer, add sodium periodate to obtain polylysine / hyaluronic acid-gallic acid coating solution; Step 4: Using N-acetyl-L-cysteine ​​hydrochloride and chitosan as raw materials, add the coupling system N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to synthesize chitosan-acetylcysteine ​​copolymer; Step 5: Add polyethylene glycol acrylate and chitosan-acetylcysteine ​​copolymer to PBS buffer, add photoinitiator LAP, and obtain polyethylene glycol acrylate / chitosan-acetylcysteine ​​coating solution; Step 6: Alternately spin-coat polylysine / hyaluronic acid-gallic acid coating solution and polyethylene glycol acrylate / chitosan-acetylcysteine ​​coating solution onto the pretreated glass slide and dry and cure to form an alternating deposition composite coating, thus obtaining a glass slide for blood testing.

2. The method for preparing a glass slide for blood testing according to claim 1, characterized in that: The preparation method of hyaluronic acid-gallic acid copolymer is as follows: Gallic acid was added to dimethyl sulfoxide and stirred at 200-300 rpm for 20-30 min to obtain a gallic acid solution. N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride were added to the gallic acid solution, and the mixture was stirred at 400-500 rpm for 1-3 h under light-protected conditions to obtain a GA-NHS solution. Hyaluronic acid was then added to dimethyl sulfoxide and stirred at 200-300 rpm for 20-30 min. N-hydroxysuccinimide and 1-ethyl- 3-(3-Dimethylaminopropyl)carbodiimide hydrochloride was reacted at 25-30℃ and stirred at 400-500 rpm for 3-5 h. Ethylenediamine was added, and the reaction was continued for 20-24 h under nitrogen protection to obtain HA-NH2 solution. HA-NH2 solution was added to GA-NHS solution, and the reaction was carried out under light-protected conditions with stirring at 400-500 rpm for 20-24 h. After the reaction was completed, the mixture was dialyzed sequentially with a mixture of dimethyl sulfoxide and deionized water and deionized water, and then freeze-dried to obtain hyaluronic acid-gallic acid copolymer.

3. The method for preparing a glass slide for blood testing according to claim 2, characterized in that: In the GA-NHS solution, the mass ratio of gallic acid, N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is (5-7):(6-8):(10-12); in the HA-NH2 solution, the mass ratio of hyaluronic acid, ethylenediamine, N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is (10-12):(8-10):(12-15):(22-25).

4. The method for preparing a glass slide for blood testing according to claim 1, characterized in that: In the polylysine / hyaluronic acid-gallic acid coating solution, the mass ratio of ε-polylysine, hyaluronic acid-gallic acid copolymer and sodium periodate is (1-1.5):(1-1.5):(0.1-0.15).

5. The method for preparing a glass slide for blood testing according to claim 1, characterized in that: The preparation method of chitosan-acetylcysteine ​​copolymer is as follows: Chitosan was added to a 1 wt% aqueous acetic acid solution and stirred at 200-300 rpm for 20-30 min to obtain a chitosan solution. N-acetyl-L-cysteine ​​hydrochloride was added to deionized water and stirred at 200-300 rpm for 20-30 min to obtain an N-acetyl-L-cysteine ​​hydrochloride solution. N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride were added to deionized water and stirred at 200-300 rpm for 20-30 min to obtain a chitosan solution. Stir at 0 rpm for 20-30 min, then add to N-acetyl-L-cysteine ​​hydrochloride solution. Stir at 25-30℃ and 400-500 rpm for 1-3 h to obtain activated acetylcysteine ​​hydrochloride solution. Add the activated acetylcysteine ​​hydrochloride solution to chitosan solution and react at 25-30℃ and 400-500 rpm for 20-24 h. After the reaction is complete, dialyze with deionized water and freeze dry to obtain chitosan-acetylcysteine ​​copolymer.

6. The method for preparing a glass slide for blood testing according to claim 5, characterized in that: The mass ratio of chitosan, N-acetyl-L-cysteine ​​hydrochloride, N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is (1-1.5):(0.8-1.2):(0.8-1.0):(0.5-0.7).

7. The method for preparing a glass slide for blood testing according to claim 1, characterized in that: In the polyethylene glycol acrylate / chitosan-acetylcysteine ​​coating solution, the mass ratio of polyethylene glycol acrylate, chitosan-acetylcysteine ​​copolymer and photoinitiator LAP is (1-1.5):(1-1.5):(0.05-0.1).

8. The method for preparing a glass slide for blood testing according to claim 1, characterized in that: The plasma activation process conditions for preparing pretreated glass slides include: argon flow rate of 20-40 sccm, oxygen flow rate of 10-20 sccm, power supply of 80-120W, and processing time of 2-4 min.

9. The method for preparing a glass slide for blood testing according to claim 1, characterized in that: The composite coating consists of alternating polylysine / hyaluronic acid-gallic acid layers and polyethylene glycol acrylate / chitosan-acetylcysteine ​​layers, with 3-7 layers in total. The polylysine / hyaluronic acid-gallic acid layer has a thickness of 80-100 nm and is formed by drying and curing the polylysine / hyaluronic acid-gallic acid coating solution under the following conditions: 30-40℃ for 60-90 min. The polyethylene glycol acrylate / chitosan-acetylcysteine ​​layer has a thickness of 30-50 nm and is formed by drying and curing the polyethylene glycol acrylate / chitosan-acetylcysteine ​​coating solution under the following conditions: 365 nm UV irradiation for 2-3 min.

10. A glass slide for blood testing, characterized in that: It is prepared by any one of the preparation methods according to claims 1-9.

Images