PETx polymer, preparation method and three-dimensional thorn-like sensor interface

Abstract

A PETx polymer including a main backbone which is a first poly-L-lysine, and a side chain which is sequentially connected with a first polyethylene glycol and a second poly-L-lysine, where the second poly-L-lysine is saturately connected with a second polyethylene glycol and a third polyethylene glycol with no remaining amino groups, and the third polyethylene glycol is connected with a functional group at its end, where the first poly-L-lysine and the second poly-L-lysine have the same or different chain lengths, and the first polyethylene glycol, the second polyethylene glycol and the third polyethylene glycol have the same or different chain lengths. Preferably, the PETx polymer is PLL-g-{PEGk-PLL-g-[(PEGj-biological recognition group)y %(PEGi)1-y %]}x %, where i, j, k, m, and n are all integers greater than or equal to 1, j is not equal to i, and x and y are all greater than 0 and less than 100.

Claims

1. A PETx polymer comprising a main backbone which is a first poly-L-lysine, and a side chain which is sequentially connected with a first polyethylene glycol and a second poly-L-lysine, the second poly-L-lysine is saturately connected with a second polyethylene glycol and a third polyethylene glycol without remaining amino groups, and the third polyethylene glycol is connected with a functional group at its end, 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 polyethylene glycol and the third polyethylene glycol have the same or different chain lengths, and wherein the PETx polymer is PLL-g-{PEG.sub.k-PLL-g-[(PEG.sub.j-functional group).sub.y %(PEG.sub.i).sub.1-y %]}.sub.x %, and the PETx polymer has the general formula: ##STR00005## where i, j, k, m and n are all integers greater than or equal to 1, and x and y are in the range greater than 0 and less than 100.

2. The PETx polymer according to claim 1, wherein the functional group is selected from biotin, Ni-NTA or single-stranded DNA.

3. The PETx polymer according to claim 1, wherein the functional group is biotin.

4. The PETx polymer according to claim 1, wherein the first poly-L-lysine has a grafting ratio which does not exceed 40%.

5. The PETx polymer according to claim 1, wherein the first poly-L-lysine has a molecular weight of 15-30 kDa; and the second poly-L-lysine has a molecular weight of 3 kDa.

6. A method for preparing a PETx polymer, comprising: 1) by using a reaction between amino groups of polymer 1 (N3-PLL or alkynyl-PLL) and active ester, grafting an active ester-polyethylene glycol-functional group (NHS-PEG-functional group) onto a polymer 1 (N.sub.3-PLL or alkynyl-PLL) to obtain a polymer 2 (N.sub.3-PLL-g-(PEG.sub.j-functional group).sub.y % or alkynyl-PLL-g-(PEG.sub.j-functional group).sub.y %) (reaction 1); 2) adding excessive active ester-polyethylene glycol-methyl (NHS-PEG-methyl) to the reaction to obtain a polymer 3 (N.sub.3-PLL-g-[(PEG.sub.j-functional group).sub.y %(PEG.sub.i).sub.1-y %] or alkynyl-PLL-g-[(PEG.sub.j-functional group).sub.y %)(PEG.sub.i).sub.1-y %] (reaction 2); 3) by using a click reaction between azide groups and alkynyl groups, grafting the polyethylene glycol-active ester (PEG-NHS) to the end of the poly-L-lysine in the polymer 3 to obtain a polymer 4 (NHS-PEG.sub.k-PLL-g-[(PEG.sub.j-functional group).sub.y %(PEG.sub.i).sub.1-y %]) (reaction 3); 4) grafting the polymer 4 to the main backbone poly-L-lysine with a grafting ratio not exceeding 40% to obtain polymer 5 (PETx, i.e. PLL-g-{PEG.sub.k-PLL-g-[(PEG.sub.j-functional group).sub.y %(PEG.sub.i).sub.1-y %]}.sub.x %) (reaction 4),
N.sub.3-PLL+NHS-PEG.sub.j-biotin.fwdarw.N.sub.3-PLL-g-(PEG.sub.j-functional group).sub.y %
or alkynyl-PLL+NHS-PEG.sub.j-functional group.fwdarw.alkynyl-PLL-g-(PEG.sub.j-functional group).sub.y %  (1)
N.sub.3-PLL-g-(PEG.sub.j-functional group).sub.y %+NHS-PEG.sub.i-methyl.fwdarw.N.sub.3-PLL-g-[(PEG.sub.j-functional group).sub.y %(PEG.sub.i).sub.1-y %]
or
alkynyl-PLL-g-(PEG.sub.j-functional group).sub.y %NHS-PEG.sub.i-methyl.fwdarw.alkynyl-PLL-g-[(PEG.sub.j-functional group).sub.y %(PEG.sub.i).sub.1-y %]  (2)
N.sub.3-PLL-g-[(PEG.sub.j-functional group).sub.y %(PEG.sub.i).sub.1-y %]+NHS-PEG.sub.k-alkynyl.fwdarw.NHS-PEG.sub.k-PLL-g-[(PEG.sub.j-functional group).sub.y %(PEG.sub.i).sub.1-y %]
or
alkynyl-PLL-g-[(PEG.sub.j-functional group).sub.y %(PEG.sub.i).sub.1-y %]NHS-PEG.sub.k-N.sub.3.fwdarw.NHS-PEG.sub.k-PLL-g-[(PEG.sub.j-functional group).sub.y %(PEG.sub.i).sub.1-y %]  (3)
PLL+NHS-PEG.sub.k-PLL-g-[(PEG.sub.j-functional group).sub.y %(PEG.sub.i).sub.1-y %].fwdarw.PLL-g-{PEG.sub.k-PLL-g-[(PEG.sub.j-functional group).sub.y %(PEG.sub.i).sub.1-y %]}.sub.x %  (4) where i, j, k, m, and n are all integers greater than or equal to 1, and x and y are both in the range greater than 0 and less than 100.

7. A three-dimensional thorn-like sensor interface comprising a negatively charged sensor surface and the PETx polymer according to claim 1, wherein the PETx polymer is modified to the sensor surface by electrostatic action, thereby forming the three-dimensional thorn-like sensing interface.

8. The three-dimensional thorn-like sensor interface according to claim 7, wherein the sensor surface is an optical fiber sensor surface, a silica sensor surface, a metal sensor surface, or a metal oxide sensor surface.

9. The three-dimensional thorn-like sensor interface according to claim 7 further comprising using the three-dimensional thorn-like sensor interface.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic diagram of the structure of the functional material PLL-g-PEG-Biotin that has been invented in the prior art.

(2) FIG. 2 is a schematic diagram of the structure of a 3D sensing interface formed by PETx whose biological recognition group is exemplified by biotin.

(3) FIG. 3 is the general formula of chemical structure of PETx whose biological recognition group is exemplified by biotin.

(4) FIG. 4 is a synthesis scheme of PETx whose biological recognition group is exemplified by biotin.

(5) FIG. 5 is an NMR spectrum of polymer 3.

(6) FIG. 6 is an NMR spectrum of polymer 4.

(7) FIG. 7 is an NMR spectrum of a polymer PETx whose biological recognition group is exemplified by biotin.

(8) FIG. 8 is an XPS spectrum of a PETx film on a silica surface.

(9) FIG. 9 is a high-resolution C1s XPS spectrum of a PETx film on a silica surface.

(10) FIG. 10 shows the principle of biodetection of PETx whose biological recognition group is exemplified by biotin.

(11) FIG. 11 is a comparison diagram of real-time biodetection between PETx whose biological recognition group is exemplified by biotin and 2D interface.

(12) FIG. 12 is a comparison diagram of the concentration curves of real-time biodetection of PETx whose biological recognition group is exemplified by biotin and 2D interface.

(13) FIG. 13 shows an anti-nonspecific adsorption experiment of PETx whose biological recognition group is exemplified by biotin and other polyelectrolytes in bovine serum albumin solution.

(14) FIG. 14 is a comparison diagram of the anti-nonspecific adsorption effects of PETx whose biological recognition group is exemplified by biotin and other polyelectrolytes in bovine serum albumin solution.

(15) FIG. 15 shows an anti-nonspecific adsorption experiment of PETx whose biological recognition group is exemplified by biotin and other polyelectrolytes in 10% serum.

(16) FIG. 16 shows an anti-nonspecific adsorption experiment of PETx whose biological recognition group is exemplified by biotin and other polyelectrolytes in 50% serum.

(17) FIG. 17 is an anti-nonspecific adsorption experiment of PETx whose biological recognition group is exemplified by biotin and other polyelectrolytes in 100% serum.

(18) FIG. 18 is a comparison diagram of anti-nonspecific adsorption effects of PETx whose biological recognition group is exemplified by biotin and other polyelectrolytes in serums under different dilution conditions.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(19) The following describes clearly and completely the technology in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, but not all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by a person skilled in the art fall within the protection scope of the present invention.

(20) The term “nonspecific adsorption” refers to a phenomenon that some biological molecules such as proteins are adsorbed to the surface of a material due to the effect of static electricity, etc., which cause interference to the experimental analysis.

(21) The term “specific adsorption” refers to a phenomenon that a specific protein (or other biological molecule) is adsorbed to a specific active site.

(22) The term “polyethylene glycol (PEG)” has good hydrophilicity and biocompatibility, and is widely used to resist nonspecific adsorption of proteins.

(23) The term “click chemistry” is a concept of chemical synthesis proposed by the American chemist Karl Barry Sharpless in 2001, wherein a 1,3-dipolar cycloaddition reaction between an azide and an alkyne to give 1,2,3-triazole is the most representative. The reaction has mild conditions and provides high yield. In recent years, the applications of this reaction have gradually increased in the immobilization of proteins, especially antibodies. In one embodiment of the present invention, the click reaction between PLL-N.sub.3 and alkynyl-PEG-NHS is used to introduce NHS. It should be noted that it is equally feasible to introduce NHS via the click reaction between PLL-alkynyl and N.sub.3-PEG-NHS.

(24) The following embodiments take biotin as a functional group as an example. However, the present invention is not limited to this. The functional group may also include other groups for achieving specific biological detection functions, such as Ni-NTA (Ni.sup.2+ chelated nitrilotriacetic acid) or single-stranded DNA.

Example 1

(25) FIG. 2 is a schematic diagram of a 3D sensing interface formed by PETx. PETx is composed of three parts: poly-L-lysine, polyethylene glycol and biotin. Its chemical structure is shown in FIG. 3, 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.

(26) ##STR00003##

(27) Next, the synthesis scheme of PETx is introduced by taking m=10, n=75, j=12, i=4, k=4, x=40, y=50 as an example.

(28) Synthesis of PETx: The synthesis scheme of PETx is shown in FIG. 3. The idea of this scheme is to take the traditional 2D polyelectrolyte as a whole as a side chain and graft it onto the backbone of poly-L-lysine to form a 3D polyelectrolyte PETx. In order to facilitate the introduction of active ester (NHS) groups that can react with amino groups, we chose poly-L-lysine with an azide group at the end (polymer 1, with a molecular weight of 3 kDa) as a side backbone in a procedure for synthesis of a side chain. The first three steps belong to the procedure for synthesis of the side chain. Firstly, the reaction between the amino groups on the poly-L-lysine and the active ester is used to graft the active ester-polyethylene glycol-biotin to the poly-L-lysine at a grafting ratio of 50% to obtain polymer 2 (Formula 1). Afterwards, an excess of active ester-polyethylene glycol-methyl is added to the reaction to obtain polymer 3 (Formula 2) to ensure that there are no remaining amino groups on the poly-L-lysine. The next step is to connect the polyethylene glycol-active ester to the end of poly-L-lysine through the reaction between the azide group and the alkynyl group (click chemistry) to obtain polymer 4 (Formula 3). Because polymer 4 is introduced with active ester, it can be grafted to the main backbone poly-L-lysine (15-30 kDa) smoothly according to a grafting ratio of 40% to obtain polymer 5 (PETx) (Formula 4).

(29) ##STR00004##

(30) FIG. 5 is an NMR spectrum of polymer 3: (.sup.1H NMR in D.sub.2O δ [ppm]=1.34-1.43 (lysine γ-CH.sub.2), (biotin, β-CH.sub.2—), 1.47-1.87 (lysine β, δ-CH.sub.2), (biotin, γ-CH.sub.2), 2.15 (biotin, —CH.sub.2C(O)NH—), 2.51 (coupled PEG, —CH.sub.2—C(O)—N), 2.68 (lysine, (—CH.sub.2—C(O)—N)), 2.82-2.89 (lysine, —CH.sub.2—NH—C(O)), (PEG, —CH.sub.2—NH—C(O)), 3.07 (biotin, —S—CH.sub.2—), 3.21 (biotin, —S—CH—), 3.27 (free lysine, —N—CH.sub.2), 3.38 (PEG, —O—CH3), 3.56 (PEG, CH.sub.2—O—), 4.13 (lysine, N—CH—C(O)—), 4.27 and 4.45 (biotin, 2 bridge head CH)).

(31) FIG. 6 is an NMR spectrum of polymer 4: (.sup.1H NMR in D.sub.2O, δ [ppm]=1.32-1.41 (lysine γ-CH.sub.2), (biotin, β-CH.sub.2—), 1.48-1.89 (lysine β, δ-CH.sub.2), (biotin, γ-CH.sub.2), 2.16 (biotin, —CH.sub.2C(O)NH—), 2.52 (coupled PEG, —CH.sub.2—C(O)—N), 2.69 (lysine, (—CH.sub.2—C(O)—N)), 2.83-2.91 (lysine, —CH.sub.2—NH—C(O)), (PEG, —CH.sub.2—NH—C(O)), 3.09 (biotin, —S—CH.sub.2—), 3.23 (biotin, —S—CH—), 3.26 (free lysine, —N—CH.sub.2), 3.37 (PEG, —O—CH.sub.3), 3.55 (PEG, CH.sub.2—O—), 4.15 (lysine, N—CH—C(O)—), 4.26 and 4.47 (biotin, 2 bridge head CH)).

(32) FIG. 7 is an NMR spectrum of the polymer PETx: (.sup.1H NMR in D.sub.2O, δ [ppm]=1.34-1.42 (lysine γ-CH.sub.2), (biotin, β-CH.sub.2—), 1.44-1.89 (lysine β, δ-CH.sub.2), (biotin, γ-CH.sub.2), 2.13 (biotin, —CH.sub.2C(O)NH—), 2.54 (coupled PEG, —CH.sub.2—C(O)—N), 2.71 (lysine, (—CH.sub.2—C(O)—N)), 2.84-2.92 (lysine, —CH.sub.2—NH—C(O)), (PEG, —CH.sub.2—NH—C(O)), 3.10 (biotin, —S—CH.sub.2—), 3.23 (biotin, —S—CH—), 3.25 (free lysine, —N—CH.sub.2), 3.36 (PEG, —O—CH3), 3.57 (PEG, CH.sub.2—O—), 4.13 (lysine, N—CH—C(O)—), 4.26 and 4.46 (biotin, 2 bridge head CH)).

(33) FIG. 8 is an XPS spectrum of a PETx film on a silica surface, showing the composition of various elements of PETx.

(34) FIG. 9 is a high-resolution C1s XPS spectrum of a PETx film on a silica surface, indicating the valence type of each carbon element.

Example 2

(35) We compared the biodetection effects of PETx and 2D PLL-g-PEG-biotin by optical interferometry. The working principle of the product of the present invention is shown in FIG. 10. First, PETx is modified on the end face of the negatively charged optical fiber, and then the PETx can specifically adsorb streptavidin, and then streptavidin can specifically adsorb biotinylated anti-human IgG antibody (NNCrystal company), and finally the antibody can specifically detect an antigen.

(36) FIG. 11 shows real-time detection effects of PETx and PLL-g-PEG-Biotin protein. The ordinate is the thickness of the film formed on the end face of the fiber. From the figure, we found that when detecting an excess of streptavidin (200 nM), 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 μg/mL).

(37) FIG. 12 is a graph of the concentration curves for detection of antigen by two types of sensing interfaces, and the slopes of the curves reflect the sensitivity of the sensor. It can be found in the figure that the sensitivity of the sensor modified by PETx is much higher than that of the traditional 2D interface sensor (PLL-g-PEG-biotin).

Example 3

(38) We compared the anti-nonspecific adsorption ability of PETx and other polyelectrolytes (PLL-g-PEG-biotin and PLL) by optical interferometry. We first modified PETx on the end face of the negatively charged optical fiber, and then tested the anti-nonspecific adsorption abilities of the end face of the optical fiber in the bovine serum albumin (BSA) solution and serums of different dilutions.

(39) FIG. 13 shows an anti-nonspecific adsorption experiment of PETx whose biological recognition group is exemplified by biotin and other polyelectrolytes in BSA solution (15 μM, 10 mM HEPES, pH 7.4). The ordinate is the thickness change of the film formed on the end face of the optical fiber. It can be seen from the figure that the thickness of the end face of the optical fiber modified with PETx did not increase after being immersed in the BSA solution. This shows that PETx has a perfect anti-nonspecific adsorption ability.

(40) FIG. 14 intuitively compares anti-nonspecific adsorption effects of PETx and other polyelectrolytes in BSA solution.

(41) FIG. 15 shows an anti-nonspecific adsorption experiment of PETx whose biological recognition group is exemplified by biotin and other polyelectrolytes in 10% serum. We first immersed the PETx-modified end face of the optical fiber in HEPES buffer to measure the baseline. After that, the end face of the optical fiber was immersed in 10% serum and taken out after 5 minutes. The end face of the optical fiber taken out was immersed in HEPES buffer again, and the change of the baseline was observed. As shown in the figure, the baseline of the PETx-modified end face of the optical fiber did not rise. This shows that PETx has a perfect anti-nonspecific adsorption ability.

(42) FIG. 16 shows an anti-nonspecific adsorption experiment of PETx whose biological recognition group is exemplified by biotin and other polyelectrolytes in 50% serum. We first immersed the PETx-modified end face of the optical fiber into HEPES buffer to measure the baseline. After that, the end face of the optical fiber was immersed in 50% serum and taken out after 5 minutes. The end face of the optical fiber taken out was immersed in HEPES buffer again, and the change of the baseline was observed. As shown in the figure, the baseline of the PETx-modified end face of the optical fiber did not rise. This shows that PETx has a perfect anti-nonspecific adsorption ability.

(43) FIG. 17 shows an anti-nonspecific adsorption experiment of PETx whose biological recognition group is exemplified by biotin and other polyelectrolytes in 100% serum. We first immersed the PETx-modified end face of the optical fiber into HEPES buffer to measure the baseline. After that, the end face of the optical fiber was immersed in 100% serum and taken out after 5 minutes. The end face of the optical fiber taken out was immersed in HEPES buffer again, and the change of the baseline was observed. As shown in the figure, the baseline of the PETx-modified end face of the optical fiber did not rise. This shows that PETx has a perfect anti-nonspecific adsorption ability.

(44) FIG. 18 intuitively compares anti-nonspecific adsorption effects of PETx and other polyelectrolytes in serums of different dilutions. The figure shows that PETx has an excellent anti-nonspecific adsorption ability in serums of different dilutions.

(45) This scheme designs and synthesizes a new type of polyelectrolyte PETx, which can form a 3D sensing interface, and has more biotin functional groups and greater content of polyethylene glycol than the traditional 2D PLL-g-PEG-biotin. This makes PETx have a better sensing effect and a better ability to resist nonspecific adsorption.

(46) The above descriptions are only the preferred embodiments of the present invention, and are not used to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the scope of the present disclosure.