SPRi biological anti-fouling sensing detection based on auto-polymerization heterotypic PEG gel coating

Abstract

The invention discloses an SPRi biological anti-pollution sensing detection method based on a self-polymerization heterotype PEG gel coating, and relates to the technical field of clinical molecular biochemical sensing detection. The preparation method specifically comprises the following steps: S1, carrying out functionalization on biotin-polyethylene glycol-amino through tyrosine and a heterobifunctional protein cross-linking agent to obtain biotin-polyethylene glycol-tyrosine and biotin-polyethylene glycol-maleimide; and S2, matching biotin-polyethylene glycol-tyrosine, biotin-polyethylene glycol-maleimide and streptavidin according to a ratio, and carrying out a reaction so as to form the claw-shaped PEG with four branched chains. The double-claw-shaped PEG has excellent anti-fouling performance in complex environment liquid, and the claw-shaped structure at the top end is connected with capture molecules such as an antibody and an aptamer through a reversible disulfide bond, so that the SPRi biosensor can efficiently, quickly, sensitively, specifically and repeatedly carry out direct analysis on a clinical actual sample, and the detection sensitivity is greatly improved. And no sample pretreatment operation is needed.

Description

Technical Field

[0001] The invention relates to the technical field of clinical molecular biochemical sensing detection, and in particular to a self-polymerization heteromorphic PEG gel coating used for SPRi biological anti-fouling sensing detection. Background Art

[0002] Surface plasmon resonance imaging (SPRi) biosensor is a highly sensitive, label-free, rapid optical detection technology. Compared with commonly used detection methods in clinical practice, such as enzyme-linked immunosorbent assay (ELISA) and chemiluminescence, SPRi detection technology has the advantages of simple operation, label-free, real-time monitoring, and high sensitivity. Currently, SPRi has been widely used in the detection of various biomolecular systems, but only a small part of it has been applied to the analysis of clinical patient samples. A key reason why SPRi technology cannot be fully implemented in clinical diagnosis is that SPRi sensors are easily affected by biological contamination in complex biological samples, resulting in poor ability to be directly applied to the analysis of actual clinical samples.

[0003] Biocontamination is believed to be caused by the nonspecific adsorption of macromolecules and biological substances other than the target in complex biological samples. It is an undesirable phenomenon for biosensors working in complex biological media. Sensors operating in complex biological samples are easily affected by various biological contaminants (such as proteins, cells and polysaccharides), resulting in decreased sensitivity and specificity of biosensors, or even failure. In order to avoid biological contamination, SPRi usually detects pretreated biological samples rather than actual samples, and the pretreatment of biological samples is a tedious and time-consuming process. Therefore, in order to enable SPRi biosensors to directly detect actual samples such as whole blood and be widely used in clinical practice, how to avoid various biological contaminations and thus improve detection specificity is a major challenge faced in building simple and efficient SPRi biosensors.

[0004] At present, surface antifouling methods are mainly divided into three categories: physical antifouling, chemical antifouling and biological antifouling. Physical antifouling mainly prevents or slows down the nonspecific adsorption of biological contamination on the sensor interface by size restriction. Currently, commonly used chemical antifouling materials include polyethylene glycol (PEG) and zwitterionic polymers, and biological antifouling materials include DNA and peptides with positive and negative charges.

[0005] Although there are many materials that can be used for surface antifouling, most of them must select an initiator or surface anchoring group based on the substrate in advance so that they can be fixed on the target surface through the method of 'graft from' or 'graft to'. This makes the anchoring group and the substrate a one-to-one correspondence, which greatly limits the application of antifouling coatings. Therefore, it is necessary to develop a universal antifouling coating that can polymerize and adhere to any substrate, so as to make the application of antifouling coatings more extensive.

[0006] Although some new hydrophilic polymers, such as zwitterions and polyacrylamide, have been introduced to prepare antifouling surfaces, polyethylene glycol (PEG) is still the internationally recognized "gold standard" for antifouling and biocompatible polymers and has been approved by the FDA. However, as a linear molecule, PEG has an unstable spatial structure, which makes its antifouling properties vulnerable, thus limiting its application. Therefore, in order to reapply PEG to surface antifouling, it must be modified.

[0007] In recent years, studies have found that block copolymers that form a cyclic conformation have a larger exclusion volume and stronger steric hindrance, so cyclic hydrophilic polymers have been developed to prepare more advanced antifouling coatings. Unlike copolymers that form brushes, these block copolymers have stronger antifouling properties and steric stability, so researchers call it "conformation evolution." Based on this, a team designed an arched block PEG based on dopamine as an antifouling lubricating coating and compared it with a brush-shaped PEG. The experimental results showed that after incubation with bovine serum albumin (BSA) for the same time, the unbound BSA was washed away, and the BSA remaining in the arched PEG was significantly less than that in the brush-shaped PEG, indicating that the arched PEG has better antifouling properties. Therefore, by redesigning the conformation of PEG, it is expected to overcome the shortcomings of traditional brush PEG, thereby making PEG more widely used.

[0008] However, the heteropoly PEG designed in these schemes loses the functionalized tail, which makes it impossible to graft the biorecognition molecules subsequently. Therefore, in order to make the SPR biosensor constructed based on heteropoly PEG have recognition function, it must be improved so that the biorecognition molecules can be grafted. Summary of the invention

[0009] The purpose of the present invention is to solve the shortcomings in the prior art and to propose a self-polymerized heteromorphic PEG gel coating for SPRi bio-antifouling sensing detection.

[0010] In order to achieve the above object, the present invention adopts the following technical solutions:

[0011] The self-polymerized heterogeneous PEG gel coating is used for SPRi bio-antifouling sensing detection, including the following steps:

[0012] S1: functionalizing biotin-PEG-amino groups with tyrosine and heterobifunctional protein cross-linkers to obtain biotin-PEG-tyrosine and biotin-PEG-maleimide;

[0013] S2: Biotin-polyethylene glycol-tyrosine, biotin-polyethylene glycol-maleimide and streptavidin are mixed and reacted in proportion to form a claw-shaped PEG with four branches;

[0014] S3: One chain of the four-branched claw PEG is reacted with the four-arm thiol PEG through Mal to form a double claw PEG;

[0015] S4: The double-clawed PEG is polymerized on the surface of the SPRi chip by tyrosinase to form a reproducible universal double-clawed PEG coating;

[0016] S5: After incubating the antibody on the double-clawed PEG coating surface, the serum is passed into the SPRi sensor to achieve rapid, sensitive and specific detection of the target;

[0017] S6: Finally, TCEP is introduced to cut the disulfide bonds and release the antibodies, so that the coating returns to its original state and can be re-grafted with antibodies.

[0018] Preferably, the biotin-polyethylene glycol-amino group and tyrosine are catalyzed by EDC / NHS at 4° for 24 hours to obtain biotin-polyethylene glycol-tyrosine.

[0019] Further: the heterobifunctional protein cross-linker is set to 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt, and the biotin-polyethylene glycol-amino and 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt are reacted at room temperature for three hours to obtain biotin-polyethylene glycol-maleimide.

[0020] As a preferred embodiment of the present invention, the ratio of biotin-polyethylene glycol-tyrosine, biotin-polyethylene glycol-maleimide and streptavidin is 3:1:1, and the reaction time is one hour at room temperature.

[0021] Based on the above scheme: three chains of the claw-shaped PEG were modified with tyrosine, and the other chain was reacted with four-arm thiol PEG at room temperature for one hour.

[0022] Preferably, the double-clawed PEG is catalyzed by tyrosinase for two hours.

[0023] Furthermore: the bottom and top of the double-clawed PEG are both claw-shaped structures, and the claw-shaped structure at the bottom is polymerized on the surface of the SPRi chip through tyrosine adhesion.

[0024] Based on the above scheme: the claw structure at the top of the double-clawed PEG is connected to the capture molecule through a reversible disulfide bond.

[0025] The beneficial effects of the present invention are:

[0026] This self-polymerized heteromorphic PEG gel coating is used for SPRi biological antifouling sensing detection. The claw structure at the top of the double-claw PEG has excellent antifouling performance in complex environmental liquids, and captures molecules such as antibodies and aptamers through reversible disulfide bonds, enabling the SPRi biosensor to directly analyze actual clinical samples efficiently, quickly, sensitively, specifically and repeatably without any sample pretreatment operations. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is the principle diagram of the present invention;

[0028] Figure 2 This is the linearity graph of the serum sample detection line of the present invention (this scheme vs. traditional scheme);

[0029] Figure 3 This is the linearity graph of the lipemia serum sample detection line of the present invention (this scheme vs. traditional scheme);

[0030] Figure 4 This is the target detection result of this solution of the present invention vs. the ECLIA detection result. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0032] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0033] Based on the self-polymerization heterogeneous PEG gel coating for SPRi bio-antifouling sensing detection, such as Figure 1 As shown, the following steps are included:

[0034] S1: Commercial biotin-PEG-amino was functionalized with tyrosine and a heterobifunctional protein cross-linker to obtain biotin-PEG-tyrosine and biotin-PEG-maleimide (Biotin-PEG-Mal);

[0035] S11: Biotin-PEG-NH2 and tyrosine (Tyr) were catalyzed by EDC / NHS at 4° for 24 hours to obtain biotin-PEG-Tyr;

[0036] EDC is 1-ethyl-(3-dimethylaminopropyl)carbodiimide, an organic compound with the chemical formula C8H 17 N3; NHS is N-hydroxysuccinimide, with the chemical formula C4H5NO3; EDC and NHS are commonly used carboxylic acid activation reagents, which react with carboxylic acids to generate intermediates or activated esters with high reactivity, thereby promoting the coupling reaction between carboxylic acids and groups such as amino groups;

[0037] S12: The heterobifunctional protein cross-linker was set to 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimidyl ester sodium salt (sulfo-SMCC), with the molecular formula C 16 H 17 N2NaO9S; In protein chemical cross-linking, Sulfo-SMCC can react with the sulfhydryl (-SH) on the protein at one end and the amino (-NH2) on the protein at the other end through its bifunctional groups, thereby cross-linking two protein molecules together; Biotin-polyethylene glycol-amino (Biotin-PEG-NH2) and 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt (sulfo-SMCC) react at room temperature for three hours to obtain biotin-polyethylene glycol-maleimide (Biotin-PEG-Mal).

[0038] S2: Biotin-polyethylene glycol-tyrosine, biotin-polyethylene glycol-maleimide and streptavidin are mixed and reacted in proportion to form a claw-shaped PEG with four branches;

[0039] The ratio of biotin-polyethylene glycol-tyrosine (Biotin-PEG-Tyr), biotin-polyethylene glycol-maleimide (Biotin-PEG-Mal) and streptavidin (SA) is 3:1; 1, and the reaction time is one hour at room temperature; streptavidin and biotin can be combined in a ratio of 1:4.

[0040] S3: Three of the four-chain claw-shaped PEG chains were modified with tyrosine, and the other chain was reacted with four-arm thiol PEG through Mal at room temperature for one hour to form a double-clawed PEG.

[0041] S4: The double-clawed PEG was polymerized on the surface of the SPRi chip by tyrosinase for two hours to form a universal double-clawed PEG coating with excellent spatial structure, high hydrophilicity and antifouling properties, and reproducibility;

[0042] The bottom and top of the double-claw PEG are both claw-shaped structures. The claw structure at the bottom is polymerized on the surface of the SPRi chip through tyrosine adhesion, and because of its arched conformation, it can provide excellent anti-fouling performance and spatial stability; the claw structure at the top of the double-claw PEG is connected to the capture molecule (such as antibody or aptamer) through a reversible disulfide bond, thereby specifically identifying and capturing the target, and regenerating the coating.

[0043] S5: After the antibody is incubated on the double-clawed PEG coating surface for one hour, the claw structure at the top of the double-clawed PEG captures the antibody through a reversible disulfide bond, and the serum is passed into the SPRi sensor to achieve rapid, sensitive and specific detection of the target;

[0044] S6: Finally, TCEP is introduced to cut the disulfide bonds and release the antibodies, so that the coating returns to its original state and can be re-grafted with antibodies; TCEP is a very effective thiol reducing agent, widely used as a quantitative reducing agent for disulfide bonds in protein chemistry and proteomics research; the reversible disulfide bond connecting the top of the double-clawed PEG to the captured antibody is broken, the antibody is released, and the double-clawed PEG coating is restored.

[0045] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. Based on the self-polymerization heteromorphic PEG gel coating for SPRi bio-antifouling sensing detection, characterized in that: The steps include: S1: functionalizing biotin-PEG-amino groups with tyrosine and heterobifunctional protein cross-linkers to obtain biotin-PEG-tyrosine and biotin-PEG-maleimide; S2: Biotin-polyethylene glycol-tyrosine, biotin-polyethylene glycol-maleimide and streptavidin are mixed and reacted in proportion to form a claw-shaped PEG with four branches; S3: One chain of the four-branched claw PEG is reacted with the four-arm thiol PEG through Mal to form a double claw PEG; S4: The double-clawed PEG is polymerized on the surface of the SPRi chip by tyrosinase to form a reproducible universal double-clawed PEG coating; S5: After incubating the antibody on the double-clawed PEG coating surface, the serum is passed into the SPRi sensor to achieve rapid, sensitive and specific detection of the target; S6: Finally, TCEP is introduced to cut the disulfide bonds and release the antibodies, so that the coating returns to its original state and can be re-grafted with antibodies.

2. The method according to claim 1, wherein the self-polymerized heteromorphic PEG gel coating is used for SPRi bio-antifouling sensing detection, characterized in that: The biotin-polyethylene glycol-amino and tyrosine are catalyzed by EDC / NHS at 4° for 24 hours to obtain biotin-polyethylene glycol-tyrosine.

3. The self-polymerization heteromorphic PEG gel coating for SPRi bio-antifouling sensing detection according to claim 1, characterized in that: The heterobifunctional protein cross-linker is set to 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonyl succinimide ester sodium salt, and the biotin-polyethylene glycol-amino and 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonyl succinimide ester sodium salt are reacted at room temperature for three hours to obtain biotin-polyethylene glycol-maleimide.

4. The self-polymerized heteromorphic PEG gel coating for SPRi bio-antifouling sensing according to claim 1, characterized in that: The ratio of biotin-polyethylene glycol-tyrosine, biotin-polyethylene glycol-maleimide and streptavidin is 3:1:1, and the reaction time is one hour at room temperature.

5. The self-polymerization heteromorphic PEG gel coating for SPRi bio-antifouling sensing detection according to claim 1, characterized in that: Three chains of the claw-shaped PEG were modified with tyrosine, and the other chain was reacted with four-arm thiol PEG at room temperature for one hour.

6. The self-polymerized heteromorphic PEG gel coating for SPRi bio-antifouling sensing according to claim 1, characterized in that: The catalytic time of the double-clawed PEG by tyrosinase is two hours.

7. The self-polymerization heteromorphic PEG gel coating for SPRi bio-antifouling sensing detection according to claim 1, characterized in that: The bottom and top of the double-clawed PEG are both claw-shaped structures. The claw-shaped structure at the bottom is polymerized on the surface of the SPRi chip through tyrosine adhesion, thereby providing a stable spatial structure and strong hydrophilicity, enabling it to exhibit excellent anti-fouling performance in complex environmental liquids.

8. The self-polymerized heteromorphic PEG gel coating for SPRi bio-antifouling sensing according to claim 1, characterized in that: The claw structure at the top of the double-clawed PEG is connected to the capture molecule through a reversible disulfide bond.

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