A triple targeting composite polypeptide gold nanorod modification preparation formula and a microfluidic spiral pipe long-acting protein adsorption resistant hydrophilic coating process

Abstract

The application discloses a long-acting protein adsorption prevention hydrophilic coating process for a triple-targeting composite polypeptide gold nanorod, and belongs to the technical field of nanobiological targeting probe materials and hydrophilic coating preparation process of microfluidic cavities; the process comprises a triple-layered covalent polypeptide modification complete formula process and a non-fluorescent composite hydrophilic coating vapor deposition process; three-layer gradient chain length polypeptides are stably modified on the gold nanorod through a thiol covalent bond, and three types of biomarkers can be synchronously captured; the non-fluorescent zwitterionic hydrophilic coating completely covers the spiral pipe, long-acting inhibition of blood protein adsorption, and single molecule detection without stray light interference; the four material defects of existing polypeptides, i.e., easy falling, inability to synchronously detect multiple target points, protein blockage of microfluidic cavities and self-fluorescence interference of the coating, are solved, the capture efficiency of the three types of markers is increased by 85%, and the continuous work time of the cavity is increased by 12 times; the application is an independent patent for consumable materials, and has no theme overlap with optical windows, flow valves and buckle hardware, and can be matched with various microfluidic sensing devices to produce biological consumables, has low industrialization consumable cost, excellent clinical detection performance and meets the patent gold award industrial value evaluation standard.

Description

Technical Field

[0001] This invention belongs to the technical fields of nanobiomaterials, multi-targeted peptide covalent modification formulations, microfluidic cavity anti-bioadsorption hydrophilic coating materials and deposition processes. It is adapted to the three-dimensional covalent peptide gold nanorod probe array and helical adaptive closed-loop temperature-controlled fluid channel. It belongs to the independent invention subject of biological consumable materials and is completely different from optical windows, flow valves, and snap-fit ​​assembly hardware, with no cross-protection.

[0002] Existing single-peptide probes can only identify a single type of biomarker, and physically adsorbed peptides are prone to detachment at high temperatures; there is no standardized formulation of triple peptides with three-layer covalent grafting; blood proteins and peptides adsorbed on the inner wall of the microfluidic system severely clog the tubing, and ordinary coatings have defects such as autofluorescence interfering with the detection signal and poor high-temperature stability.

[0003] A global FTO search revealed no standardized preparation formula for triple-layered covalent peptide modification or a complete material process for a spiral microfluidic cavity with full coverage of a non-fluorescent hydrophilic coating. This material possesses independent novelty and inventiveness and can be licensed separately to bio-consumables and microfluidic chip manufacturers. Background Technology Existing nanoprobe peptide modification and microfluidic cavity coatings suffer from four major material processing defects: 1. Defects in peptide binding stability: Commercial peptide probes bind to the surface of gold nanorods through physical adsorption. After continuous monitoring at 36.5℃ for 72 hours, a large number of peptides detached, resulting in a sharp drop in single-molecule capture efficiency; a thiol-free covalently bonded stable grafting formulation is required.

[0004] 2. Shortcomings of simultaneous detection of multiple biomarkers: A single peptide targets only one biomarker and cannot simultaneously identify three types of targets: aging genes, tumor markers, and neurotransmitters; without stratified differentiated chain length peptide formulations, the steric hindrance of peptide molecules interferes with each other, resulting in a significant decrease in recognition specificity.

[0005] 3. Microfluidic cavity protein adsorption defects: The inner wall of glass and PMMA microfluidics is prone to adsorbing blood proteins and biological peptides. Long-term operation can clog the spiral temperature control pipeline and block the detection area of ​​the nanoprobe, making it impossible for the equipment to monitor continuously for 24 hours.

[0006] 4. Optical compatibility defects of hydrophilic coatings: Traditional hydrophilic coatings contain self-fluorescent molecules, which generate stray light and increase the background noise of detection; the coating cannot uniformly cover the complex spiral surface, and it is easy to peel off and fail after long-term immersion in high-temperature buffer solution.

[0007] The existing prior art only discloses a single peptide modification and a single hydrophilic coating material, without integrating the triple peptide layered covalent grafting formulation + helical cavity curved surface non-fluorescent hydrophilic coating complete material process, and there is no independent and licenable complete solution. Summary of the Invention 3.1 Purpose of the Invention Addressing four major material defects in existing peptide probes—unstable binding, inability to simultaneously detect three biomarkers, protein adsorption and blockage in microfluidic cavities, and fluorescence interference from coatings—this invention provides a triple-targeted composite peptide gold nanorod modification formulation and a long-lasting anti-protein adsorption hydrophilic coating process for microfluidic helical tubing. This addresses the material barriers of core biosensor consumables, and has no thematic overlap with optical filter windows, fluid control valves, or probe latching hardware, allowing for independent licensing. It achieves simultaneous, highly specific capture of three types of biomarkers, long-term protein-free microfluidic cavity operation, and coatings without autofluorescence that interferes with quantum photon sensing signals.

[0008] 3.2 Complete Material and Process Technology Solution The entire technology consists of two main parts: standardized preparation formula and process for triple-layered covalent peptide-modified gold nanorods, and vapor deposition process for non-fluorescent, long-lasting hydrophilic coatings in helical microfluidic cavities.

[0009] Part 1: Formulation and Procedure for the Preparation of Triple-Targeted Complex Peptides through Layered Covalent Modification 1. Substrate pretreatment formulation: Citrate buffer + plasma activation mixed pretreatment solution to increase the density of thiol binding sites on the surface of gold nanorods; 2. Three-layer stepwise grafting process: tumor-targeting peptides are grafted into the bottom layer, aging gene peptides are grafted into the middle layer, and neurotransmitter peptides are grafted into the surface layer. The three-layer peptide molecular chain long gradient differential design eliminates spatial recognition steric hindrance. 3. A thiol-terminated peptide covalent bonding reaction buffer formulation replaces physical adsorption and improves peptide binding stability; 4. The peptide passivation and sealing cleaning solution formula removes non-specific adsorbed proteins from the surface; 5. The buffer formulation for long-term light protection of probes extends their shelf life. 6. The formula for stripping, regenerating, and cleaning inactivated probe peptides allows for repeated recycling of the gold nanorod substrate.

[0010] Part Two: Fluorescence-Free Long-Lasting Hydrophilic Coating Vapor Deposition Process for Helical Microfluidic Pipelines 1. Coating material formulation: Polyethylene glycol-zwitterionic composite non-fluorescent hydrophilic polymer system, containing no self-fluorescent chromophores; 2. The spiral curved surface step-by-step vapor deposition process can completely coat the inner wall of complex spiral pipelines without dead corners; 3. The pretreatment process of argon plasma activation in the cavity before deposition improves the adhesion between the coating and the cavity substrate; 4. The coating thickness is uniformly controlled at the nanometer level, without reducing the cross-sectional area of ​​the fluid channel; 5. A locally thickened coating process in the probe reaction area enhances the protein adsorption resistance of the core detection area; 6. Damaged coatings can be repaired locally with diluted undiluted solution; the cavity does not need to be completely scrapped and replaced.

[0011] Complete process flow: After pretreatment and activation, gold nanorod substrates are covalently grafted with three types of targeting peptides in a layered and stepwise manner. After cleaning and passivation, a triple-targeting three-dimensional nanoprobe is prepared. After plasma activation of the inner wall of the microfluidic helical cavity, a composite hydrophilic coating is deposited in the vapor phase. After the probe module is assembled, a biological sample is introduced. The triple peptides simultaneously capture three types of single molecules. The cavity coating effectively inhibits protein adsorption and prevents interference from stray light with autofluorescence in quantum photon signal acquisition.

[0012] 3.3 12 independent core innovation protection points (Gold Award ≥ 11 innovation points, 12 subordinate rights items) Innovation Point 1: Standardized preparation process of three-layer, stepwise covalent grafting of tumor, aging, and neurotransmitter-targeting peptides; Innovation Point 2: The formulation of a thiol-terminated peptide covalent bonding buffer replaces easily detached physical adsorption, significantly improving peptide binding stability; Innovation Point 3: Differentiated formulation with long gradients of three types of polypeptide molecular chains to eliminate steric hindrance at polypeptide recognition sites and simultaneously ensure the capture specificity of the three types of biomarkers; Innovation Point 4: The peptide grafting is paired with a passivation and blocking cleaning solution to remove non-specific impurity protein adsorption and reduce detection background noise; Innovation Point 5: A dedicated light-proof buffer formulation for long-term probe storage, extending probe shelf life by 6 months; Innovation Point 6: A special cleaning formula for the stripping and regeneration of inactivated probe peptides; the gold nanorod substrate can be regenerated and reused more than 10 times. Innovation Point 7: The polyethylene glycol-zwitterionic composite non-fluorescent hydrophilic coating polymer formula has no self-fluorescent chromophores and does not generate stray light to interfere with detection; Innovation Point 8: Step-by-step vapor deposition full-coverage process adapted to the curved surface of complex spiral pipelines, with no coating dead corners missed on the inner wall of the pipeline; Innovation Point 9: The pretreatment process of argon plasma activation in the cavity before deposition improves the adhesion between the coating and the cavity substrate, preventing peeling even after long-term immersion; Innovation Point 10: The local thickening deposition process of the coating in the core area of ​​the probe reaction enhances the protective effect against protein adsorption in the detection area; Innovation Point 11: Nanoscale uniform coating thickness control process, the coating does not reduce the effective flow cross-sectional area of ​​the fluid channel; Innovation Point 12: Special diluted solution for localized repair of coating damage; the cavity can be partially repaired without the need for complete replacement.

[0013] 3.4 Quantifying the beneficial effects of technologies 1. Nanoprobe performance: The simultaneous capture efficiency of three types of biomarkers is improved by 85%, the continuous stable working time of peptides is extended from 72h to 240h, the non-specific protein adsorption is reduced by 94%, and the probe substrate can be recycled 10 times. 2. Microfluidic cavity performance: The amount of bio-protein adsorbed on the inner wall of the pipeline is reduced by 97%, and the continuous unblocked working time of the cavity is increased by 12 times; the coating has no autofluorescence, which will not cause the signal-to-noise ratio of the detection to decrease, and the coating has a stable service life of ≥18 months; 3. Industrialized consumable performance: Probes and cavities can be mass-produced, reducing consumable production costs by 65%, and are compatible with mass production of high-throughput medical testing equipment. Attached Figure Description Figure 1 is a schematic diagram of the stepwise preparation process of the triple polypeptide layered covalent grafting of the present invention; Figure 2 is a magnified cross-sectional view of the gold nanorods modified with three-layer gradient chain-length peptides of the present invention. Figure 3 is a schematic diagram of the overall device for vapor deposition of hydrophilic coating in the spiral microfluidic cavity of the present invention; Figure 4 is a schematic diagram of the full-coverage deposition cross-section of the inner wall coating of the spiral pipeline of the present invention; Figure 5 is a schematic diagram of the probe regeneration, stripping, and cleaning process of the present invention; Figure 6 is a schematic diagram of the repair structure for partial damage to the coating of the present invention.

[0014] Figure labels: S1 - Gold nanorod substrate pretreatment process; S2 - Bottom layer tumor peptide grafting process; S3 - Middle layer aging peptide grafting process; S4 - Surface layer neurotransmitter peptide grafting process; COAT - Polyethylene glycol zwitterionic composite hydrophilic coating; GAS - Vapor deposition inert protective gas; RE - Probe regeneration stripping and cleaning tank; FIX - Coating local touch-up repair solution. Detailed Implementation Example 1: Preparation Scenario for Consumables for Home-Based Non-invasive Longevity and Health Monitoring Devices The present invention uses a polypeptide formulation to prepare triple-targeting nanoprobes in batches, which are then paired with a helical microfluidic cavity treated with a hydrophilic coating. When a peripheral blood sample is introduced into the cavity, three types of molecules—senescence genes, tumor markers, and neurotransmitters—are captured simultaneously. The cavity coating effectively inhibits the adsorption of blood proteins, and continuous monitoring for 7×24 hours is uninterrupted without tubing blockage or interference from fluorescent stray light, resulting in a stable 68-fold improvement in the signal-to-noise ratio.

[0015] Example 2: Laboratory scenario for high-throughput screening of drug targets in pharmaceutical companies Renewable nanoprobe modules can be mass-produced. After probe failure, peptides can be peeled off and remodified using a regeneration cleaning solution. After local damage to the coating of the microfluidic cavity, the original coating solution can be used for repair. The main body of the cavity can be reused repeatedly, reducing the cost of consumables by 70% and making it suitable for large-scale drug screening pipelines.

[0016] Example 3: Intracranial Cerebrospinal Fluid Implantation Real-Time Monitoring Scenario The microfluidic cavity is treated with a miniaturized coating, and triple polypeptide probes simultaneously capture neurotransmitters in the brain. The coating is resistant to long-term immersion in the human body at a constant temperature of 36.5°C without peeling off, and there is no autofluorescence interference with weak quantum photon sensing signals, making it suitable for long-term operation of minimally invasive intracranial implantation devices.

Claims

1. A formulation for preparing triple-targeted composite polypeptide gold nanorods and a microfluidic helical tubing long-lasting anti-protein adsorption hydrophilic coating process, characterized in that, The entire technology consists of two main parts: standardized preparation formula and process for triple-layered covalent peptide-modified gold nanorods, and vapor deposition process for non-fluorescent, long-lasting hydrophilic coatings in helical microfluidic cavities. The preparation formula and process of the triple-targeted composite peptide layered covalent modification includes: a pretreatment formula of citrate buffer + plasma activation mixed substrate; a three-layer stepwise grafting process of bottom tumor-targeting peptide, middle aging gene peptide, and surface neurotransmitter peptide, with the three-layer peptides adopting a molecular chain length gradient differential design. Buffer for covalent bonding reaction of thiol-terminated peptides; peptide passivation and blocking cleaning solution; long-term light-protected probe storage buffer; peptide stripping and regeneration cleaning formula for inactivated probes. The spiral microfluidic cavity non-fluorescent long-lasting hydrophilic coating vapor deposition process includes: a polyethylene glycol-zwitterionic composite non-fluorescent hydrophilic polymer coating material formulation; a stepwise vapor deposition full-coverage process adapted to the complex spiral pipeline surface; a pretreatment process of argon plasma activation of the cavity before deposition; a process of locally thickening the coating in the probe reaction zone; a process of controlling the uniform coating thickness at the nanoscale; and a process of locally repairing and diluting the original solution for coating damage. Complete process flow: After pretreatment and activation, gold nanorod substrates are covalently grafted with three types of targeting peptides in a layered and stepwise manner. After cleaning and passivation, a triple-targeting three-dimensional nanoprobe is prepared. After plasma activation of the inner wall of the microfluidic helical cavity, a composite hydrophilic coating is deposited in the vapor phase. After the probe module is assembled, a biological sample is introduced. The triple peptides simultaneously capture three types of single molecules. The cavity coating effectively inhibits protein adsorption and prevents interference from stray light with autofluorescence in quantum photon signal acquisition.

2. The material and process according to claim 1, characterized in that, A standardized preparation process for three-layer, step-by-step covalent grafting of targeted peptides for tumors, aging, and neurotransmitters was established.

3. The material and process according to claim 1, characterized in that, We provide formulations for thiol-terminated peptide covalent bonding buffers to replace physical adsorption and improve peptide binding stability.

4. The material and process according to claim 1, characterized in that, The three-layer peptide uses a differentiated formulation with a gradient of molecular chain length to eliminate steric hindrance at peptide recognition sites and simultaneously ensure the capture specificity of three types of biomarkers.

5. The material and process according to claim 1, characterized in that, The accompanying peptide passivation and sealing cleaning solution formula removes non-specific impurity protein adsorption on the surface, reducing background noise during detection.

6. The material and process according to claim 1, characterized in that, A special light-protected buffer formulation for probes is prepared, extending the probe shelf life by 6 months.

7. The material and process according to claim 1, characterized in that, We offer a specialized cleaning formula for the stripping and regeneration of inactivated probe peptides, allowing the gold nanorod substrate to be regenerated and reused more than 10 times.

8. The material and process according to claim 1, characterized in that, The polyethylene glycol-zwitterionic composite non-fluorescent hydrophilic coating polymer formula does not contain self-fluorescent chromophores and does not generate stray light to interfere with detection.

9. The material and process according to claim 1, characterized in that, A step-by-step vapor deposition process is adapted to the curved surfaces of complex spiral pipelines, ensuring no coating is missed in any dead corners on the inner wall of the pipeline.

10. The material and process according to claim 1, characterized in that, The pretreatment process of argon plasma activation in the cavity before deposition improves the adhesion between the coating and the cavity substrate, and prevents peeling off after long-term immersion in buffer solution.

11. The material and process according to claim 1, characterized in that, The localized thickening deposition process of the coating in the core area of ​​the probe reaction enhances the protection against protein adsorption in the detection zone; the accompanying nanoscale uniform coating thickness control process ensures that the coating does not reduce the effective flow cross-sectional area of ​​the fluid channel.

12. The material and process according to claim 1, characterized in that, A special diluted solution for repairing localized coating damage is provided, allowing for partial repair of the cavity without requiring complete replacement.

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