WO2020238643A1 - PETx聚合物、制备方法以及三维荆棘状传感器界面 - Google Patents

PETx聚合物、制备方法以及三维荆棘状传感器界面 Download PDF

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WO2020238643A1
WO2020238643A1 PCT/CN2020/090348 CN2020090348W WO2020238643A1 WO 2020238643 A1 WO2020238643 A1 WO 2020238643A1 CN 2020090348 W CN2020090348 W CN 2020090348W WO 2020238643 A1 WO2020238643 A1 WO 2020238643A1
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peg
pll
polymer
functional group
petx
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段学欣
潘文尉
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Tianjin University
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G81/00—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers
    • C08G81/02—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers at least one of the polymers being obtained by reactions involving only carbon-to-carbon unsaturated bonds
    • C08G81/024—Block or graft polymers containing sequences of polymers of C08C or C08F and of polymers of C08G
    • C08G81/028—Block or graft polymers containing sequences of polymers of C08C or C08F and of polymers of C08G containing polyamide sequences
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G81/00—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings

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  • the invention relates to a PETx polymer, a preparation method and a three-dimensional thorn-like sensor interface based on the PETx polymer.
  • Surface-based biosensors often require surface activation methods to enable the surface of the sensor to have specific functions (for example, anti-non-specific adsorption function and specific biological recognition function).
  • surface activation mainly includes two types according to the connection method between the sensor surface and the functionalized material: covalent connection method and non-covalent connection method.
  • Covalent attachment methods have many disadvantages, including: 1) Covalent attachment (such as amino coupling) may interfere with protein structure and affect its binding behavior. 2) The direction and surface density of the protein are difficult to control. 3) The hydrolysis of the silane bond will destroy the long-term stability of the functional group. 4) The covalent bond is irreversible and the sensor can only be used once.
  • PLL poly-L-lysine
  • PLL polypeptide with very clear toxicological significance. It has a high cationic nature at physiological pH, so it is often used as a polymer backbone for the synthesis of polyelectrolytes. This kind of polyelectrolyte with PLL as the skeleton is positively charged due to the amino group on the PLL, which can be modified to the surface of the negatively charged sensor by means of electrostatic action, so that the surface of the sensor has a specific function.
  • Biotin-Streptavidin-System is a new type of biological reaction amplification system developed in the late 1970s, which can almost be combined with various successfully studied markers. Therefore, in the field of biosensing, biotin is often used as a functional group for specific adsorption of streptavidin (SAv).
  • PLL-g-PEG-Biotin is an invented functional material. Its structure diagram is shown in Figure 1. It is a brush-like polymer. The polymer uses PLL as the backbone, the main body of the side chain is PEG (polyethylene glycol), and biotin is used as the functional group at the end of the side chain, to be precise a biological recognition group. It is positively charged at physiological pH and can be modified to the surface of the sensor through electrostatic interaction. The formed sensor interface, because the functional groups are in the same plane, is also called a two-dimensional sensor interface (2D interface). The modified sensor surface has the functions of anti-non-specific adsorption and specific recognition and adsorption of streptavidin (SAv). In addition, by adjusting the pH, PLL-g-PEG-Biotin can fall off the surface of the sensor, thereby realizing the regeneration of the sensor.
  • SAv streptavidin
  • One part of the amino group on the PLL backbone is used to access the side chain, and the other part is used to generate a positive charge. Therefore, in order to ensure that the polyelectrolyte can have sufficient positive charge for electrostatic modification, the grafting rate of the side chain is controlled below 40%, which limits the number of biometric groups biotin and side chain PEG per unit area. , So that the specific biological recognition and anti-non-specific adsorption capabilities of the biosensor surface are limited, thereby limiting the sensing effect of the biosensor.
  • the present invention aims to break the limitations of the traditional 2D interface and develop a new type of polyelectrolyte material, which can form a three-dimensional functional interface (3D interface) on the surface of the sensor, and at the same time make the bio-recognition group and polyethylene glycol in the unit area
  • 3D interface three-dimensional functional interface
  • a PETx polymer is provided.
  • the polymer has a first poly-L-lysine as its main backbone, and a side chain is sequentially connected with a first polyethylene glycol and a second poly-L-lysine.
  • the second poly-L-lysine is saturated with the second polyethylene glycol and the third polyethylene glycol, and there is no excess amino group.
  • the third polyethylene glycol is connected to the end Functional group, wherein the first poly-L-lysine and the second poly-L-lysine have the same or different chain lengths, the first polyethylene glycol, the second The polyethylene glycol and the third polyethylene glycol have the same or different chain lengths; preferably, the PETx polymer is PLL-g- ⁇ PEG k -PLL-g-[(PEG j -functional group) y% (PEG i ) 1-y% ] ⁇ x% , where i, j, and k are all integers greater than or equal to 1, j is not equal to i, and x and y are both in the range greater than 0 and less than 100.
  • the PETx polymer described herein in the present invention refers to a new type of polymer used for polyelectrolyte thin films.
  • the PETx polymer is PLL-g- ⁇ PEG k -PLL-g-[(PEG j -biological recognition group) y% (PEG i ) 1-y% ] ⁇ x% .
  • the functional group is a biological recognition group (or called a biological detection group); the biological recognition group is selected from biotin, Ni-NTA (Ni 2+ chelated nitrilotriacetic acid) or Single-stranded DNA to achieve specific biological detection functions.
  • the biological recognition group is selected from biotin, Ni-NTA (Ni 2+ chelated nitrilotriacetic acid) or Single-stranded DNA to achieve specific biological detection functions.
  • the biological recognition group is biotin.
  • the chain length of PLL and PEG can be arbitrary.
  • the grafting rate on the first poly-L-lysine does not exceed 40%.
  • the molecular weight of the first poly-L-lysine is 15-30 kDa; the molecular weight of the second poly-L-lysine is 3 kDa.
  • the molecular weights of the first poly-L-lysine and the second poly-L-lysine may have other choices according to the chain lengths taken.
  • the general formula of the PETx polymer is as follows: where i, j,
  • k, m, and n are all integers greater than or equal to 1, and x and y are all in the range greater than 0 and less than 100.
  • a method for preparing PETx polymer which includes the following steps: 1) Using the reaction of amino group and active ester, the active ester-polyethylene glycol-functional group (NHS-PEG- Functional group) grafted onto polymer 1 (N 3 -PLL or alkynyl-PLL) to obtain polymer 2 (N 3 -PLL-g-(PEG j -functional group) y% or alkynyl-PLL- g-(PEG j -functional group) y% ) (Reaction formula 1); 2) Excessive active ester-polyethylene glycol-methyl (NHS-PEG-methyl) is added to the reaction to obtain polymer 3 (N 3 -PLL-g-[(PEG j -functional group) y% (PEG i ) 1-y% ] or alkynyl-PLL-g-[(PEG j -functional group) y% (PEG i ) 1-
  • i, j, k, m, and n are all integers greater than or equal to 1, and x and y are all in the range greater than 0 and less than 100.
  • the functional group is a biological recognition group
  • the biological recognition group is selected from biotin, Ni-NTA or single-stranded DNA. More preferably, the biological recognition group is biotin.
  • the click reaction in step 3) adopts the click reaction between poly-L-lysine-azide and alkynyl-polyethylene glycol-active ester to introduce the active ester
  • the click reaction between poly-L-lysine-alkynyl and azide-polyethylene glycol-active ester is used to introduce the active ester.
  • the preparation method of the PETx polymer includes the following steps: 1) Using the reaction between the amino group on the polymer 1 (N 3 -PLL) and the active ester, the active ester-polyethylene Alcohol-biotin (NHS-PEG-biotin) is grafted onto polymer 1 to obtain polymer 2 (reaction formula 1); 2) excess active ester-polyethylene glycol-methyl (NHS-PEG-Methyl) is Add to the reaction to obtain polymer 3 to ensure that there are no remaining amino groups on the poly-L-lysine (reaction formula 2); 3) through the click reaction of the azide group and the alkynyl group, the polyethylene glycol- Active ester (PEG-NHS) is connected to the end of poly-L-lysine in polymer 3 to obtain polymer 4 (reaction formula 3); 4) polymer 4 is grafted at a grafting rate of not more than 40% On the main backbone poly-L-lysine, polymer 5 (PE), Using the reaction between the
  • a three-dimensional thorn-like sensor interface includes a negatively charged sensor surface and a PETx polymer, the PETx polymer is modified to the sensor surface by electrostatic action, Thereby forming a three-dimensional thorn-like sensing interface.
  • the application of the PETx polymer of the present invention or the three-dimensional thorn-like sensor interface of the present invention in biological detection and anti-non-specific adsorption is provided.
  • Figure 1 is a schematic diagram of the structure of the functional material PLL-g-PEG-Biotin that has been invented in the prior art
  • FIG. 2 is a schematic diagram of the structure of a 3D sensing interface formed by PETx whose biometric group is biotin as an example;
  • Figure 3 is the general chemical structure formula of PETx with biotin as an example
  • Figure 4 is a synthesis scheme of PETx with biotin as an example
  • Figure 5 is a hydrogen nuclear magnetic spectrum of polymer 3
  • Figure 6 is a hydrogen nuclear magnetic spectrum of polymer 4.
  • Fig. 7 is the hydrogen nuclear magnetic spectrum of the polymer PETx whose biometric group is biotin as an example
  • Figure 8 is an XPS spectrum of a PETx film on a silica surface
  • Figure 9 is an XPS high-resolution C1s spectrum of a PETx film on a silica surface
  • Figure 10 shows the principle of biodetection of PETx with biotin as an example
  • Fig. 11 is a comparison diagram of real-time biological detection between PETx and 2D interface where the biometric group is biotin as an example;
  • Figure 12 is a comparison diagram of the concentration curves of PETx and 2D interface real-time biological detection in an example where the biological recognition group is biotin;
  • Figure 13 is the anti-non-specific adsorption experiment of PETx and other polyelectrolytes in bovine serum albumin solution with biotin as an example;
  • Figure 15 is the anti-non-specific adsorption experiment of PETx and other polyelectrolytes in 10% serum with biotin as an example;
  • Figure 16 is the anti-non-specific adsorption experiment of PETx and other polyelectrolytes in 50% serum with biotin as an example
  • Fig. 18 is a comparison diagram of the anti-non-specific adsorption effects of PETx and other polyelectrolytes in serum under different dilution conditions with biotin as an example.
  • click chemistry is a synthetic concept proposed by the American chemist Sharpless in 2001.
  • the 1,3-dipolar cycloaddition reaction of azide and alkyne to form 1,2,3-triazole is the most representative one. .
  • the reaction conditions are mild and the yield is high.
  • the application in the immobilization of proteins, especially antibodies has gradually increased.
  • the click reaction between PLL-N 3 and alkynyl-PEG-NHS is used to introduce NHS. It should be noted that the click reaction between PLL-alkynyl and N 3 -PEG-NHS is used The reaction to introduce NHS is equally feasible.
  • reaction formula 1 the reaction between the amino group on the poly-L-lysine and the active ester is used to graft the active ester-polyethylene glycol-biotin to the poly -L-lysine to obtain polymer 2 (reaction formula 1).
  • excess active ester-polyethylene glycol-methyl is added to the reaction to obtain polymer 3 (reaction formula 2) to ensure that there are no remaining amino groups on the polylysine.
  • reaction formula 3 through the reaction of the azide group and the alkynyl group (click chemistry), the polyethylene glycol-active ester is connected to the end of the poly-L-lysine to obtain the polymer 4 (reaction formula 3). Because compound 4 incorporates active ester, it can be grafted to the main backbone poly-L-lysine (15-30kDa) smoothly according to the grafting rate of 40%, to obtain compound 5 (PETx) (reaction formula 4) .
  • Figure 11 shows the real-time detection effect of PETx and PLL-g-PEG-Biotin protein.
  • the ordinate is the thickness of the film formed on the end of the fiber. From the figure, we found that when detecting an excess of streptavidin (200nM), PETx can bind more streptavidin. This is due to the fact that PETx contains more biological recognition groups (biotin). After that, the sensor detected antigens (human immunoglobulin, human IgG, NNCrystal) at different concentrations (1-250 ⁇ g/mL).
  • Figure 15 shows the anti-non-specific adsorption experiment of PETx and other polyelectrolytes in 10% serum with biotin as an example.
  • This program designs and synthesizes a new type of polyelectrolyte PETx, which can form a 3D sensing interface, and has more biotin functional groups and polyethylene glycol content than the traditional 2D PLL-g-PEG-biotin. This makes PETx have a better sensing effect and ability to resist non-specific adsorption.

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Abstract

本发明公开了一种PETx聚合物，其特征在于，该聚合物以第一聚-L-赖氨酸为主骨架，侧链依次连接有第一聚乙二醇和第二聚-L-赖氨酸,在第二聚-L-赖氨酸上饱和地连接有第二聚乙二醇和第三聚乙二醇，而无多余的氨基，在所述第三聚乙二醇的末端连接有功能基团，其中，所述第一聚-L-赖氨酸和所述第二聚-L-赖氨酸具有相同或不同的链长，所述第一聚乙二醇、所述第二聚乙二醇和所述第三聚乙二醇具有相同或不同的链长；优选地，所述PETx聚合物为PLL-g-{PEG k-PLL-g-[(PEG j-生物识别基团) y％(PEG i) 1-y％]} x％，其中，i、j、k、m和n均为大于等于1的整数，j不等于i，x,y均在大于0且小于100的范围内。本发明还涉及该PETx聚合物的制备方法以及基于该PETx聚合物的三维荆棘状传感器界面。本发明可以在传感器表面形成三维的功能化界面，同时使得生物识别基团和聚乙二醇在单位传感器面积上的含量大大增加。

Description

PETx聚合物、制备方法以及三维荆棘状传感器界面 技术领域
本发明涉及一种PETx聚合物、制备方法以及基于该PETx聚合物的三维荆棘状传感器界面。
背景技术
基于表面的生物传感器往往需要通过表面活化的方法，使得传感器的表面具有特定功能(如，抗非特异性吸附功能和特异性生物识别的功能)。这样的表面活化，根据传感器表面与功能化材料的连接方式来分主要包含两种：共价连接方法和非共价连接方法。
共价连接方法有很多的缺点，包括:1)共价结合(如氨基偶联)可能干扰蛋白质结构，影响其结合行为。2)蛋白的方向和表面密度难以控制。3)硅烷键的水解会破坏官能团的长期稳定性。4)共价键是不可逆的，传感器只能使用一次。
对于各种基于表面的生物传感器，大部分传感表面由Au、Si/SiO 2等金属氧化物组成，其带微弱的负电(在生理pH值下)。多聚-L-赖氨酸(PLL)是一种毒理学意义十分明确的多肽，在生理pH值下具有高度的阳离子性质，所以常被作为聚合物的骨架用于聚电解质的合成。这种以PLL为骨架的聚电解质，由于PLL上的氨基而带正电，从而可以通过静电作用的方式修饰到带负电的传感器表面，使得传感器的表面具有特定的功能。
生物素－链霉亲和素系统(Biotin-Streptavidin—System，BAS)是70年代末发展起来的一种新型生物反应放大系统，几乎可与目前研究成功的各种标记物结合。所以在生物传感领域，生物素(Biotin)常被用做链霉亲和素蛋白(Streptavidin，SAv)的特异性吸附的功能基团。
PLL-g-PEG-Biotin是一种已发明的功能化材料。它的结构示意图如图1所示，是一种刷状的聚合物。该聚合物以PLL为骨架，侧链的主体是PEG(聚乙二醇),在侧链的末端以biotin充当功能基团，确切地说生物识别基团。它在生理pH值下带正电荷，从而可以通过静电作用修饰到传感器表面。所形成的传感器界面，由于功能基团处于同一平面，所以又被称作为二维传感器界面(2D界面)。经过修饰的传感器表面同时具有了抗非特异性吸附和特异性识别和吸附链霉亲和素(SAv)的功能。另外，通过调节pH，可以使得PLL-g-PEG-Biotin从传感器表面脱落，从而实现传感器的再生。
PLL骨架上的氨基，一部分用于接入侧链，另一部分用于产生正电荷。所以为了保证该聚电解质可以带有足够的正电荷用以静电修饰，侧链的接枝率被控制在40％以下，这就限制了生物识别基团biotin和侧链PEG在单位面积上的数目，使得该生物传感器表面的特异性生物识别和抗非特异性吸附的能力受到限制，从而限制了该生物传感器的传感效果。
发明内容
本发明旨在打破传统的2D界面的局限，开发一种新型的聚电解材料，可以在传感器表面形成三维的功能化界面(3D界面)，同时使得生物识别基团和聚乙二醇在单位面积上的数目大大增加。
本发明的技术方案是这样实现的：
根据本发明的一个方面，提供了一种PETx聚合物，该聚合物以第一聚-L-赖氨酸为主骨架，侧链依次连接有第一聚乙二醇和第二聚-L-赖氨酸,在第二聚-L-赖氨酸上饱和地连接有第二聚乙二醇和第三聚乙二醇，而无多余的氨基，在所述第三聚乙二醇的末端连接有功能基团，其中，所述第一聚-L-赖氨酸和所述第二聚-L-赖氨酸具有相同或不同的链长，所述第一聚乙二醇、所述第二聚乙二醇和所述第三聚乙二醇具有相同或不同的链长；优选地，所述PETx 聚合物为PLL-g-{PEG k-PLL-g-[(PEG j-功能基团) y％(PEG i) 1-y％]} x％，其中，i、j、k均为大于等于1的整数，j不等于i，x和y均在大于0且小于100的范围内。
本发明这里所述的PETx聚合物，指的是一种新型用于聚电解质薄膜(polyelectrolyte thin films)的聚合物。
优选地，所述PETx聚合物为PLL-g-{PEG k-PLL-g-[(PEG j-生物识别基团) y％(PEG i) 1-y％]} x％。
优选地，所述功能基团是生物识别基团(或称生物检测基团)；所述生物识别基团选自生物素、Ni-NTA(Ni 2+螯合的次氮基三乙酸)或单链DNA，用以实现特定的生物检测功能。
作为本发明的一种实施方式，所述生物识别基团为生物素。
本方案中，PLL、PEG的链长可以为任意。
作为本发明的一种实施方式，所述第一聚-L-赖氨酸上的接枝率不超过40％。
作为本发明的一种实施方式，所述第一聚-L-赖氨酸的分子量为15-30kDa；所述第二聚-L-赖氨酸的分子量为3kDa。所述第一聚-L-赖氨酸和所述第二聚-L-赖氨酸的分子量根据所取的链长不同可有其它的选择。
作为本发明的一种实施方式，所述的PETx聚合物的通式如下：其中，i、j、
Figure PCTCN2020090348-appb-000001
k、m和n均为大于等于1的整数，x,y均在大于0且小于100的范围内。
根据本发明的另一方面，提供了一种PETx聚合物的制备方法，包括以下步骤：1)利用氨基和活性酯的反应，将活性酯-聚乙二醇-功能基团(NHS-PEG-功能基团)接枝到聚合物1(N 3-PLL或炔基-PLL)上得到聚合物2(N 3-PLL-g-(PEG j-功能基团) y％或炔基-PLL-g-(PEG j-功能基团) y％)(反应式1)；2)过量的活性酯-聚乙二醇-甲基(NHS-PEG-甲基)被加入到反应中得到聚合物3(N 3-PLL-g-[(PEG j-功能基团) y％(PEG i) 1-y％]或炔基-PLL-g-[(PEG j-功能基团) y％(PEG i) 1-y％])(反应式2)；3)通过叠氮基团和炔基的点击反应，将聚乙二醇-活性酯(PEG-NHS)接入聚合物中的聚-L-赖氨酸的末端得到聚合物4(NHS-PEG k-PLL-g-[(PEG j-功能基团) y％(PEG i) 1-y％])(反应式3)；4)按照不超过40％的接枝率将聚合物4接枝到主骨架聚-L-赖氨酸上，得到聚合物5(PETx，即PLL-g-{PEG k-PLL-g-[(PEG j-功能基团) y％(PEG i) 1-y％]} x％)(反应式4)，
(1)   N 3-PLL+NHS-PEG j-biotin→N 3-PLL-g-(PEG j-功能基团) y％
或炔基-PLL+NHS-PEG j-功能基团→炔基-PLL-g-(PEG j-功能基团) y％
(2)  N 3-PLL-g-(PEG j-功能基团) y％+NHS-PEG i-甲基→N 3-PLL-g-[(PEG j-功能基团) y％(PEG i) 1-y％]
或
炔基-PLL-g-(PEG j-功能基团) y％+NHS-PEG i-甲基→炔基-PLL-g-[(PEG j-功能基团) y％(PEG i) 1-y％]
(3)  N 3-PLL-g-[(PEG j-功能基团) y％(PEG i) 1-y％]+NHS-PEG k-炔基或→NHS-PEG k-PLL-g-[(PEG j功能基团) y％(PEG i) 1-y％]
炔基-PLL-g-[(PEG j-功能基团) y％(PEG i) 1-y％]+NHS-PEG k-N 3→NHS-PEG k-PLL-g-[(PEG j-功能基团) y％(PEG i) 1-y％]
(4)  PLL+NHS-PEG k-PLL-g-[(PEG j-功能基团) y％(PEG i) 1-y％]→PLL-g-{PEG k-PLL-g-[(PEG j-功能基团) y％(PEG i) 1-y％]} x％
，其中，i、j、k、m和n均为大于等于1的整数，x,y均在大于0且小于100的范围内。
优选地，所述PETx聚合物的制备方法中，所述功能基团是生物识别基团，所述生物识别基团选自生物素、Ni-NTA或单链DNA。更优选地，所述生物识别基团为生物素。
优选地，所述PETx聚合物的制备方法中，步骤3)中的点击反应采用聚-L-赖氨酸-叠氮与炔基-聚乙二醇-活性酯之间的点击反应引入活性酯或者采用聚-L-赖氨酸-炔基与叠氮-聚乙二醇-活性酯之间的点击反应引入活性酯。
作为本发明的一种实施方式，所述PETx聚合物的制备方法，包括以下步骤：1)利用聚合物1(N 3-PLL)上的氨基和活性酯的反应，将活性酯-聚乙二醇-生物素(NHS-PEG-biotin)接枝到聚合物1上得到聚合物2(反应式1)；2)过量的活性酯-聚乙二醇-甲基(NHS-PEG-Methyl)被加入到反应中得到聚合物3，用以确保聚-L-赖氨酸上没有剩余的氨基(反应式2)；3)通过叠氮基团和炔基的点击反应，将聚乙二醇-活性酯(PEG-NHS)接入聚合物3中的聚-L-赖氨酸的末端得到聚合物4(反应式3)；4)按照不超过40％的 接枝率将聚合物4接枝到主骨架聚-L-赖氨酸上，得到聚合物5(PETx，即PLL-g-{PEG k-PLL-g-[(PEG j-biotin) y％(PEG i) 1-y％]} x％)(反应式4)，
Figure PCTCN2020090348-appb-000002
Figure PCTCN2020090348-appb-000003
Figure PCTCN2020090348-appb-000004
Figure PCTCN2020090348-appb-000005
根据本发明的又一方面，提供了一种三维荆棘状传感器界面，所述传感器界面包括带负电的传感器表面和PETx聚合物，所述PETx聚合物通过静电作用的方式修饰到所述传感器表面，从而形成三维的荆棘状的传感界面。
作为本发明的一种实施方式，所述的传感器表面为光纤传感器表面、二氧化硅传感表面、金属传感表面或金属氧化物传感表面。
根据本发明的再一方面，提供了本发明所述的PETx聚合物或本发明所述的三维荆棘状传感器界面在生物检测和抗非特异性吸附方面的应用。
附图说明
图1是现有技术中已发明的功能化材料PLL-g-PEG-Biotin的结构示意图；
图2是生物识别基团为生物素为例的PETx形成的3D传感界面的结构示意图；
图3是生物识别基团为生物素为例的PETx的化学结构通式；
图4是生物识别基团为生物素为例的PETx的合成方案；
图5是聚合物3的核磁氢谱；
图6是聚合物4的核磁氢谱；
图7是生物识别基团为生物素为例的聚合物PETx的核磁氢谱；
图8是二氧化硅表面上的PETx薄膜的XPS谱图；
图9是二氧化硅表面上的PETx薄膜的XPS高分辨率C1s光谱图；
图10是生物识别基团为生物素为例的PETx的生物检测原理；
图11是生物识别基团为生物素为例的PETx和2D界面的实时生物检测对比图；
图12是生物识别基团为生物素为例的PETx和2D界面实时生物检测的浓度曲线对比图；
图13是生物识别基团为生物素为例的PETx和其他聚电解质在牛血清蛋白溶液中的抗非特异性吸附实验；
图14是生物识别基团为生物素为例的PETx和其他聚电解质在牛血清蛋白溶液中的抗非特异性吸附的效果对比图；
图15是生物识别基团为生物素为例的PETx和其他聚电解质在10％血清中的抗非特异性吸附实验；
图16是生物识别基团为生物素为例的PETx和其他聚电解质在50％血清中的抗非特异性吸附实验；
图17是生物识别基团为生物素为例的PETx和其他聚电解质在100％血清中的抗非特异性吸附实验；
图18是生物识别基团为生物素为例的PETx和其他聚电解质在不同稀 释条件下的血清中的抗非特异性吸附的效果对比图。
具体实施方式
下面结合将结合本发明实施例中的附图，对本发明实施例中的技术进行清楚、完整地描述，显然，所描述的实施例仅是本发明的一部分实施例，而不是全部实施例。基于本发明中的实施例，本领域普通技术人员所获得的所有其他实施例，都属于本发明保护的范围。
术语“非特异性吸附”是指蛋白等一些生物分子，由于静电等作用吸附到材料表面，对实验分析引起干扰的现象。
术语“特异性吸附”是指特定的蛋白(或其它生物分子)吸附到特定活性位点的现象。
术语“聚乙二醇(PEG)”具有良好的亲水性和生物相容性，被广泛运用于抗蛋白的非特异性吸附的功能。
术语“点击化学”是2001年由美国化学家Sharpless提出的合成概念，其中以叠氮化物和炔烃的1,3-偶极环加成反应生成1,2,3-三唑最具有代表性。该反应条件温和，产率高，近年来在蛋白尤其是抗体的固定化中应用逐渐增多。在本发明的一种实施方式中采用PLL-N 3与炔基-PEG-NHS之间的点击反应引入NHS，需要注意的是，利用PLL-炔基与N 3-PEG-NHS之间的点击反应来引入NHS，也是同样可行的。
以下实施方式以生物素为功能基团为例。然而本发明不限于此，所述功能基团还可包括用以实现特定的生物检测功能的其它基团，如Ni-NTA(Ni 2+螯合的次氮基三乙酸)或单链DNA等。
实施例1
图2是PETx形成的3D传感界面示意图。PETx由三个部分组成：聚-L-赖氨酸、聚乙二醇和生物素。其化学结构如图3所示，其中，i、j、k、m和n均为大于等于1的整数，x，y均在大于0小于100的范围内。
Figure PCTCN2020090348-appb-000006
接下来，以m＝10、n＝75、j＝12、i＝4、k＝4、x＝40、y＝50为例，介绍PETx的合成方案。
PETx的合成：PETx的合成方案如图4所示。该方案的思路是将传统的2D聚电解质整体当作侧链，接枝到聚-L-赖氨酸的骨架上，形成3D的聚电解质PETx。为了便于引入能和氨基反应的活性酯(NHS)基团，我们选择了末端接有叠氮基团的聚-L-赖氨酸(聚合物1，分子量为3kDa)作为侧链合成过程中的侧骨架。前三步为侧链的合成过程，首先利用聚-L-赖氨酸上的氨基和活性酯的反应，将活性酯-聚乙二醇-生物素按照50％的接枝率接枝到聚-L-赖氨酸上得到聚合物2(反应式1)。之后，过量的活性酯-聚乙二醇-甲基被加入到反应中得到聚合物3(反应式2)，用以确保聚赖氨酸上没有剩余的氨基。接下来就是通过叠氮基团和炔基的反应(点击化学)，将聚乙二醇-活性酯接入聚-L-赖氨酸的末端得到聚合物4(反应式3)。化合物4因为引入了活性酯，所以 可以很顺利地按照40％的接枝率接枝到主骨架聚-L-赖氨酸(15-30kDa)上，得到化合物5(PETx)(反应式4)。
Figure PCTCN2020090348-appb-000007
Figure PCTCN2020090348-appb-000008
Figure PCTCN2020090348-appb-000009
Figure PCTCN2020090348-appb-000010
图5是聚合物3的核磁氢谱：( 1H NMR in D 2O，δ[ppm]＝1.34-1.43(lysineγ-CH 2),(biotin,β-CH 2-),1.47-1.87(lysineβ,δ-CH 2),(biotin,γ-CH 2),2.15(biotin,-CH 2C(O)NH-),2.51(coupled PEG,-CH 2–C(O)–N),2.68(lysine,(-CH 2-C(O)-N)),2.82–2.89(lysine,-CH 2-NH-C(O)),(PEG,-CH 2-NH-C(O)),3.07(biotin,-S-CH 2-),3.21(biotin,-S-CH-),3.27(free lysine,-N-CH 2),3.38(PEG,-O-CH3),3.56(PEG,CH 2-O-),4.13(lysine,N-CH-C(O)-),4.27and 4.45(biotin,2 bridge head CH))。
图6是聚合物4的核磁氢谱：( 1H NMR in D 2O，δ[ppm]＝1.32-1.41(lysineγ-CH 2),(biotin,β-CH 2-),1.48-1.89(lysineβ,δ-CH 2),(biotin,γ-CH 2),2.16(biotin,-CH 2C(O)NH-),2.52(coupled PEG,-CH 2–C(O)–N),2.69(lysine,(-CH 2-C(O)-N)),2.83–2.91(lysine,-CH 2-NH-C(O)),(PEG,-CH 2-NH-C(O)), 3.09(biotin,-S-CH 2-),3.23(biotin,-S-CH-),3.26(free lysine,-N-CH 2),3.37(PEG,-O-CH 3),3.55(PEG,CH 2-O-),4.15(lysine,N-CH-C(O)-),4.26and 4.47(biotin,2 bridge head CH))。
图7是聚合物PETx的核磁氢谱：( 1H NMR in D 2O，δ[ppm]＝1.34-1.42(lysineγ-CH 2),(biotin,β-CH 2-),1.44-1.89(lysineβ,δ-CH 2),(biotin,γ-CH 2),2.13(biotin,-CH 2C(O)NH-),2.54(coupled PEG,-CH 2–C(O)–N),2.71(lysine,(-CH 2-C(O)-N)),2.84–2.92(lysine,-CH 2-NH-C(O)),(PEG,-CH 2-NH-C(O)),3.10(biotin,-S-CH 2-),3.23(biotin,-S-CH-),3.25(free lysine,-N-CH 2),3.36(PEG,-O-CH3),3.57(PEG,CH 2-O-),4.13(lysine,N-CH-C(O)-),4.26and 4.46(biotin,2 bridge head CH))。
图8是二氧化硅表面上的PETx薄膜的XPS谱图，显示了PETx的各个元素组成。
图9是二氧化硅表面上的PETx薄膜的XPS高分辨率C1s光谱图，表明了各个碳元素的价态类型。
实施例2
我们利用光干涉法对比了PETx和2D的PLL-g-PEG-biotin的生物检测效果。本发明产品的工作原理如图10所示，首先将PETx修饰到带负电的光纤端面上，然后该PETx可以特异性吸附链霉亲和素，之后链霉亲和素又可以特异性吸附生物素化的抗人IgG抗体(biotinylated anti-human IgG，NNCrystal公司)，最后抗体可以特异性对抗原进行特异性的检测。
图11展示了PETx和PLL-g-PEG-Biotin的蛋白实时检测效果，纵坐标为该光纤端面上形成的薄膜的厚度。从图中我们发现，当对过量的链霉亲和素(200nM)进行检测时，PETx可以结合更多的链霉亲和素。这得益于PETx中含有更多的生物识别基团(生物素)。之后传感器又对不同浓度(1-250μg/mL) 的抗原(人免疫球蛋白，human IgG，NNCrystal公司)进行了检测。
图12是两种传感界面对抗原的检测的浓度曲线图，曲线的斜率反映了传感器的灵敏度。图中可以发现，经PETx修饰过的传感器的灵敏度要远高于传统的2D界面传感器(PLL-g-PEG-biotin)。
实施例3
我们利用光干涉法对比了PETx和其他聚电解质(PLL-g-PEG-biotin和PLL)的抗非特异性吸附能力。我们先将PETx修饰到带负电的光纤端面上，然后检测该光纤端面在牛血清蛋白(BSA)溶液和不同稀释倍数的血清中的抗非特异性吸附能力。
图13展示了生物识别基团为生物素为例的PETx和其他聚电解质在BSA溶液(15μM,10mM HEPES,pH 7.4)中的抗非特异性吸附实验，纵坐标为该光纤端面上形成的薄膜的厚度变化。从图中可以看出修饰了PETx的光纤端面在浸入BSA溶液后厚度没有增加。这表明PETx具有完美的抗非特异性吸附的能力。
图14直观的对比PETx和其他聚电解质在BSA溶液中的抗非特异性吸附的效果。
图15展示了功能基团为生物素为例的PETx和其他聚电解质在10％血清中的抗非特异性吸附实验。我们将修饰了PETx的光纤端面先浸入HEPES缓冲液中测得基线。之后，将该光纤端面浸入10％血清中，5分钟后取出。取出的光纤端面被重新浸入HEPES缓冲液，观察基线变化。如图所示，修饰了PETx的光纤端面基线并没有上升。这表明PETx具有完美的抗非特异性吸附的能力。
图16展示了功能基团为生物素为例的PETx和其他聚电解质在50％血清中的抗非特异性吸附实验。我们将修饰了PETx的光纤端面先浸入HEPES缓冲液中测得基线。之后，将该光纤端面浸入50％血清中，5分钟后取出。取 出的光纤端面被重新浸入HEPES缓冲液，观察基线变化。如图所示，修饰了PETx的光纤端面基线并没有上升。这表明PETx具有完美的抗非特异性吸附的能力。
图17展示了功能基团为生物素为例的PETx和其他聚电解质在100％血清中的抗非特异性吸附实验。我们将修饰了PETx的光纤端面先浸入HEPES缓冲液中测得基线。之后，将该光纤端面浸入100％血清中，5分钟后取出。取出的光纤端面被重新浸入HEPES缓冲液，观察基线变化。如图所示，修饰了PETx的光纤端面基线并没有上升。这表明PETx具有完美的抗非特异性吸附的能力。
图18直观地对比PETx和其他聚电解质在不同稀释倍数的血清中的抗非特异性吸附的效果。图中显示，PETx在不同稀释倍数的血清中均表现出了优异的抗非特异性吸附的能力。
该方案设计并合成了新型的聚电解质PETx，能够形成3D的传感界面，并且相比于传统2D的PLL-g-PEG-biotin拥有更多的生物素功能基团和聚乙二醇含量,这使得PETx具有更好的传感效果和抗非特异性吸附的能力。
以上所述仅为本发明的较佳实施例而已，并不用于限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种PETx聚合物，其特征在于，该聚合物以第一聚-L-赖氨酸为主骨架，侧链依次连接有第一聚乙二醇和第二聚-L-赖氨酸,在第二聚-L-赖氨酸上饱和地连接有第二聚乙二醇和第三聚乙二醇，而无多余的氨基，在所述第三聚乙二醇的末端连接有功能基团，其中，所述第一聚-L-赖氨酸和所述第二聚-L-赖氨酸具有相同或不同的链长，所述第一聚乙二醇、所述第二聚乙二醇和所述第三聚乙二醇具有相同或不同的链长；优选地，所述PETx聚合物为PLL-g-{PEG k-PLL-g-[(PEG j-功能基团) y％(PEG i) 1-y％]} x％，其中，i、j、k均为大于等于1的整数，j不等于i，x和y均在大于0且小于100的范围内。
  2. 根据权利要求1所述的PETx聚合物，其特征在于，所述功能基团是生物识别基团，所述生物识别基团选自生物素、Ni-NTA或单链DNA。
  3. 根据权利要求1所述的PETx聚合物，其特征在于，所述生物识别基团为生物素。
  4. 根据权利要求1所述的PETx聚合物，其特征在于，所述第一聚-L-赖氨酸上的接枝率不超过40％。
  5. 根据权利要求1所述的PETx聚合物，其特征在于，所述第一聚-L-赖氨酸的分子量为15-30kDa；所述第二聚-L-赖氨酸的分子量为3kDa。
  6. 根据权利要求1所述的PETx聚合物，其特征在于，所述的PETx聚合物的通式如下：
    Figure PCTCN2020090348-appb-100001
    其中，i、j、k、m和n均为大于等于1的整数，x和y均在大于0且小于100的范围内。
  7. 一种PETx聚合物的制备方法，其特征在于，1)利用氨基和活性酯的反应，将活性酯-聚乙二醇-功能基团(NHS-PEG-功能基团)接枝到聚合物1(N 3-PLL或炔基-PLL)上得到聚合物2(N 3-PLL-g-(PEG j-功能基团) y％或炔基-PLL-g-(PEG j-功能基团) y％)(反应式1)；2)过量的活性酯-聚乙二醇-甲基(NHS-PEG-甲基)被加入到反应中得到聚合物3(N 3-PLL-g-[(PEG j-功能基团) y％(PEG i) 1-y％]或炔基-PLL-g-[(PEG j-功能基团) y％(PEG i) 1-y％])(反应式2)；3)通过叠氮基团和炔基的点击反应，将聚乙二醇-活性酯(PEG-NHS)接入聚合物3中的聚-L-赖氨酸的末端得到聚合物4(NHS-PEG k-PLL-g-[(PEG j-功能基团) y％(PEG i) 1-y％])(反应式3)；4)按照不超过40％的接枝率将聚合物4接枝到主骨架聚-L-赖氨酸上，得到聚合物5(PETx，即PLL-g-{PEG k-PLL-g-[(PEG j-功能基团) y％(PEG i) 1-y％]} x％)(反应式4)，
    (1)N 3-PLL+NHS-PEG j-biotin→N 3-PLL-g-(PEG j-功能基团) y％
    或炔基-PLL+NHS-PEG j-功能基团→炔基-PLL-g-(PEG j-功能基团) y％
    (2)N 3-PLL-g-(PEG j-功能基团) y％+NHS-PEG i-甲基→N 3-PLL-g-[(PEG j-功能基团) y％(PEG i) 1-y％]
    或
    炔基-PLL-g-(PEG j-功能基团) y％+NHS-PEG i-甲基→炔基-PLL-g-[(PEG j-功能基团) y％(PEG i) 1-y％]
    (3)N 3-PLL-g-[(PEG j-功能基团) y％(PEG i) 1-y％]+NHS-PEG k-炔基→NHS-PEG k-PLL-g-[(PEG j功能基团) y％(PEG i) 1-y％]
    或
    炔基-PLL-g-[(PEG j-功能基团) y％(PEG i) 1-y％]+NHS-PEG k-N 3→NHS-PEG k-PLL-g-[(PEG j-功能基团) y％(PEG i) 1-y％]
    (4)PLL+NHS-PEG k-PLL-g-[(PEG j-功能基团) y％(PEG i) 1-y％]→PLL-g-{PEG k-PLL-g-[(PEG j-功能基团) y％(PEG i) 1-y％]} x％
    其中，i、j、k、m和n均为大于等于1的整数，x,y均在大于0且小于100的范围内；
    优选地，所述功能基团是生物识别基团。
  8. 一种三维荆棘状传感器界面，其特征在于，所述传感器界面包括带负 电的传感器表面和权利要求1-6任一项所述的PETx聚合物，所述PETx聚合物通过静电作用的方式修饰到所述传感器表面，从而形成三维的荆棘状的传感界面。
  9. 根据权利要求8所述的三维荆棘状传感器界面，其特征在于，所述的传感器表面为光纤传感器表面、二氧化硅传感表面、金属传感表面或金属氧化物传感表面。
  10. 权利要求1-6任一项所述的PETx聚合物或权利要求8-9任一项所述的三维荆棘状传感器界面在制备生物传感器方面的应用。
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