CN117126398A - Polylysine grafted polymer, preparation method thereof, polymer modified micro-fluidic chip and detection device comprising polymer modified micro-fluidic chip - Google Patents

Polylysine grafted polymer, preparation method thereof, polymer modified micro-fluidic chip and detection device comprising polymer modified micro-fluidic chip Download PDF

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CN117126398A
CN117126398A CN202210557345.1A CN202210557345A CN117126398A CN 117126398 A CN117126398 A CN 117126398A CN 202210557345 A CN202210557345 A CN 202210557345A CN 117126398 A CN117126398 A CN 117126398A
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景海荣
丁丁
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Abstract

The application provides a lysine graft polymer. The polymer comprises a main chain formed by polymerization of lysine monomers, a side chain formed by grafting lysine, and a side chain containing one or more reactive functional groups for coupling reaction with molecules containing alkoxy units to obtain hydrophilic side chain tail chains. When the lysine graft polymer is used for modifying a microfluidic chip, as the main chain of the graft polymer is a hydrophobic peptide bond and the side chain is a hydrophilic group, the lysine graft polymer can be arranged at the interface of the chip in a regular spiral structure, and the even distribution of hydrophilic units can greatly reduce the defect of the interface of the chip, so that the nonspecific adsorption of nucleic acid, protein and cells on the interface of the chip is further reduced, and the sensitivity and the practicability of the chip in biological detection are improved. The application also provides a preparation method of the lysine graft polymer, a microfluidic chip modified by the lysine graft polymer and a monitoring device comprising the microfluidic chip.

Description

Polylysine grafted polymer, preparation method thereof, polymer modified micro-fluidic chip and detection device comprising polymer modified micro-fluidic chip
Technical Field
The application belongs to the technical field of biomedicine, and particularly relates to a polylysine grafted polymer, a micro-fluidic chip modified by the polylysine grafted polymer and a detection device comprising the polylysine grafted polymer.
Background
The micro-fluidic Chip (Microfluidic Chip), also called Lab-on-a-Chip or biochip, is characterized in that a micro-electromechanical processing technology is used to miniaturize a large laboratory system on a glass or plastic substrate, thereby duplicating complex biological and chemical reaction whole process, and rapidly and automatically completing experiments.
The microfluidic chip generally comprises a substrate and a structure, wherein the substrate is usually made of glass and silicon wafer materials, and the structure is usually made of glass or a micron-sized fluid channel, namely a micro-channel, built by high polymer materials such as PDMS, PMMA, SU8 and the like. The nature of the microchannel surface determines to a large extent the function and application area of the microfluidic chip. However, the surface of the microfluidic chip made of many materials including glass and high polymers is charged or has strong hydrophobicity, so that the sample injection, the flow and the adsorption of solutes of the fluid and the related electrohydrodynamic effects are seriously affected, the range of the analyte is greatly limited, and the sensitivity and the practicability of the microfluidic chip are reduced.
In order to solve the above technical problems, it is generally necessary to perform appropriate molecular modification on the surface of the microfluidic chip to improve the chip performance and increase the sensitivity. At present, the interface modification method for the glass-based microfluidic chip mainly comprises covalent modification of polyethylene glycol, wherein the molecules are considered to be capable of strongly binding water molecules, so that a physical or energy barrier is formed, and proteins, nucleic acids, platelets, cells and the like are far away from the surface of a substrate; in addition, polyethylene glycol molecules have very good biocompatibility, so that the polyethylene glycol molecules are greatly applied to solution antibiosis, surface antifouling, drug delivery and the like. Nevertheless, polyethylene glycol has many defects such as difficulty in functional modification, very slow biodegradation, uneven thickness of a film formed on the surface of glass, and the like, which greatly restrict further improvement of chip performance.
Based on the above, a new molecular modifier or polyethylene glycol derivative is developed for modifying the chip interface, so that the nonspecific zero adsorption of biological analytes is realized, the application of the microfluidic technology in biological medicine and diagnosis research is crucial, and the method is one of the problems to be solved in the field of interface chemistry at present.
Disclosure of Invention
In order to solve the problems of low sensitivity, poor practicability and the like of biological analysis and detection caused by nonspecific adsorption of a microfluidic chip interface in the prior art, the application develops a lysine graft polymer, a microfluidic chip modified by the same and a detection device comprising the microfluidic chip by combining the property that lysine molecules are easy to branch to form multi-reaction functional groups and the property that alkoxy functional groups can inhibit nonspecific adsorption of biomolecules.
In a first aspect, the present application provides a lysine graft polymer having a structure represented by formula (I):
wherein n is more than or equal to 10 and less than or equal to 50, R' is a tail chain group and can be bonded with a chip interface, R 1 Has a structure shown in any one of formulas (II) - (IV),
wherein R is 2 Having the formula (V) - (VI)) The structure shown in any one of the above,
wherein m is more than or equal to 2 and less than or equal to 10. In the structural formula of the applicationIndicated are the ligation sites.
As a specific embodiment of the present application, in the formula (I), 15.ltoreq.n.ltoreq.25 is preferable. As n increases, so too does the dispersity of the polymer, and too much dispersity is detrimental to the modification of the chip interface by the polymer. When n is more than or equal to 15 and less than or equal to 25, the modification effect of the polymer on the chip interface is better; further preferably, n in formula (I) is 15, 18, 20, 22 or 25.
As a specific embodiment of the present application, when the polymer is bonded to the chip interface by hydrophobic interaction, it is preferred that R' is an alkyl carbon chain group; further preferably, R' has a structure represented by any one of formulas (VII) to (VIII),
wherein z is more than or equal to 5 and less than or equal to 20; y is more than or equal to 2 and less than or equal to 8.
As a specific embodiment of the present application, when the polymer is bonded to the chip interface by covalent bond, it is preferable that the main chain or side chain terminal of R ' should contain a siloxane group or a silane group, more preferably, the main chain or side chain of R ' contains an oligoethylene glycol linking chain, still more preferably, R ' has a structure represented by any one of formulas (IX) to (X),
wherein z is more than or equal to 5 and less than or equal to 20.
As a specific embodiment of the present application, in the formula (V), 3.ltoreq.m.ltoreq.6 is preferable. For the graft polymer provided by the disclosure, from the aspect of the synthetic angle, m is more than or equal to 2 and less than or equal to 10, but the larger m is, the yield of the corresponding polymer is obviously reduced due to steric effect, and after m exceeds 10, almost no product exists; from the aspect of the modification effect on the chip surface, the effect of m=2 is close to that of the oligomeric ethylene glycol, and when m is more than or equal to 3 and less than or equal to 6, the effect is better than that of the oligomeric ethylene glycol. Considering comprehensively, m is more than or equal to 3 and less than or equal to 6 is preferable, so that higher synthesis yield can be ensured, and excellent modification performance can be obtained; m in the formula (V) is further preferably 3, 4, 5 or 6.
As a specific embodiment of the present application, the sugar ring structure in formula VI is preferably an oligosaccharide structure, and more preferably, the oligosaccharide structure is a mannose structure, a di-mannose structure or a tri-mannose structure.
The preparation method of the lysine graft polymer is not particularly limited, and the lysine graft polymer can be prepared by a lysine-N-carboxyl cyclic anhydride ring-opening polymerization method and a lysine alpha-amino dehydration condensation method respectively; the former method can be divided into epsilon-amino branching modification before polymerization and epsilon-amino branching modification after polymerization; accordingly, the dehydration condensation method includes two schemes similar to this. The specific steps may be prepared according to methods well known to those skilled in the art, preferably with reference to the following methods:
(1) Epsilon-amino protection: the epsilon-amino group of the lysine-N-carboxycyclic anhydride is protected, such as t-butoxycarbonylation (t-Butyloxy carbonyl, boc) or benzyloxycarbonylation (Cbz).
(2) Polymerization reaction: ring-opening polymerization is carried out on lysine-N-carboxyl cyclic anhydride (molecule 1) protected by epsilon-amino under the action of an initiator, and then protecting groups are removed under the action of a catalyst such as strong organic acid (such as TFA, p-toluenesulfonic acid) and the like to obtain alpha-polylysine (molecule 2);
wherein, the tail end of the initiator is an alkyl carbon chain or an ethoxy chain containing amino, and can contain an aromatic ring structure and a heteroatom group. When the initiator is combined with the chip interface through covalent bonds, the main chain or the side chain terminal of the initiator needs to contain a siloxane group or a silane group; the terminal end of the initiator includes, for example, but is not limited to, the following structure:
wherein z is more than or equal to 5 and less than or equal to 20; y is more than or equal to 2 and less than or equal to 8.
Wherein the catalyst is trifluoroacetic acid (TFA), p-toluenesulfonic acid and other strong organic acids.
(3) Grafting reaction: the alpha-polylysine and 2, 6-di-tert-butyloxycarbonyl aminocaproic acid (molecule 3) undergo condensation reaction, and the protecting group is removed under the action of a catalyst such as strong organic acid (such as TFA, p-toluenesulfonic acid) to obtain a molecule 4 with a side chain terminal of diamino; molecule 4 and tert-butyloxycarbonyl aminooxy acetic acid pentafluorophenyl ester (molecule 5) undergo nucleophilic substitution reaction, and the protecting group is removed again to obtain molecule 6 containing an active amino group at the end; the amino at the end of the molecule 6 and the hydroxyl at the end of the oligoethylene glycol or the oligosaccharide molecule are subjected to substitution reaction to obtain the target product 7.
More preferably prepared with reference to the following synthetic route:
(1) Under the action of an initiator, ring-opening polymerization is carried out on lysine-N-carboxyl cyclic anhydride molecule 1, and protective groups are removed under the action of trifluoroacetic acid to obtain alpha-polylysine, namely molecule 2.
(2) The molecule 2 and the molecule 3 are subjected to dehydration condensation reaction, and the protecting group is removed to obtain the molecule 4 with the side chain terminal of the double amino groups.
(3) Molecule 4 and molecule 5 undergo nucleophilic substitution reaction and the protecting group is removed to obtain molecule 6 with an active amino group at the end.
(4) The amino at the end of the molecule 6 and the hydroxyl at the end of the oligoethylene glycol or the oligosaccharide molecule are subjected to substitution reaction to obtain the target product 7.
The above-mentioned raw materials in the present application are all self-made or commercially available, and the present application is not particularly limited thereto.
As can be seen from the above examples of the synthesis method, the lysine graft polymer disclosed by the application is simple to synthesize, and derivatives with different side chains can be obtained by only a few consecutive identical reactions.
In a second aspect, the application provides a preparation method of the polylysine graft polymer, wherein the polylysine graft polymer is prepared by a method (1) of lysine-N-carboxyl cyclic anhydride ring-opening polymerization or a method (2) of lysine alpha-amino dehydration condensation; preferably, the method comprises the steps of,
the method (1) is an epsilon-amino branching modification method before polymerization of the method (1-1) or an epsilon-amino branching modification method after polymerization of the method (1-2);
the method (2) is an epsilon-amino branching modification method before dehydration condensation of the method (2-1) or an epsilon-amino branching modification method after dehydration condensation of the method (1-2).
As a specific embodiment of the present application, preferably, the preparation method includes the steps of:
(1) Epsilon-amino protection: protecting epsilon-amino of lysine-N-carboxyl ring anhydride by t-butyloxycarbonyl or benzyloxy carbonyl to obtain the lysine-N-carboxyl ring anhydride with epsilon-amino protection;
(2) Polymerization reaction: ring-opening polymerization is carried out on lysine-N-carboxyl cyclic anhydride protected by epsilon-amino under the action of an initiator, and then protecting groups are removed under the action of a strong organic acid catalyst to obtain alpha-polylysine; further preferably, the initiator is terminated with an amino-containing alkyl carbon chain or an ethoxy chain, still further preferably, the initiator is terminated with a structure according to any one of formulas (IX) - (X),
in the formula (IX) and the formula (X), z is more than or equal to 5 and less than or equal to 20;
or the terminal of the initiator is an alkyl carbon chain, and more preferably, the terminal of the initiator has a structure shown in any one of formulas (VII-VIII),
in the formula (VII) and the formula (VIII), z is more than or equal to 5 and less than or equal to 20; y is more than or equal to 2 and less than or equal to 8;
(3) Grafting reaction: the method comprises the steps of (1) carrying out condensation reaction on alpha-polylysine and 2, 6-di-tert-butoxycarbonyl amino caproic acid, removing a protecting group under the action of a strong organic acid catalyst, carrying out nucleophilic substitution reaction on the alpha-polylysine and tert-butoxycarbonyl amino oxy acetic acid pentafluorophenyl ester, removing the protecting group again to obtain active amino, and carrying out substitution reaction on the amino and hydroxyl at the tail end of an oligomeric ethylene glycol or oligosaccharide molecule to obtain a target product;
preferably, the strong organic acid catalyst in step (2) and step (3) is at least one selected from trifluoroacetic acid and p-toluenesulfonic acid.
In a third aspect, the present application provides a microfluidic chip having a part or all of its interfaces modified by the lysine graft polymer described above.
As a specific embodiment of the present application, preferably, the substrate of the microfluidic chip is glass or a silicon wafer material.
In a fourth aspect, the present application provides a detection device comprising a microfluidic chip as described above.
As a specific embodiment of the present application, the detection device can be used for separation of blood dissociation DNA (cell free DNA), immunodetection, and the like.
The beneficial effects of the application are as follows:
the present disclosure provides graft polymers comprising a backbone formed by polymerization of lysine monomers, side chains formed by grafting of lysine, the side chains comprising one or more reactive functional groups for coupling reactions with molecules containing alkoxy units to form side chain tail chains containing alkoxy units. This structure has the following advantages:
(1) Because the main chain is a hydrophobic peptide bond, and the tail chain of the side chain is a hydrophilic group, the polymer can be arranged on the chip interface in a regular spiral structure, and simultaneously, the alkoxy groups of the tail chain of the side chain can be uniformly and compactly distributed on the chip interface, so that the defect of the chip interface is greatly reduced, and the nonspecific adsorption of nucleic acid, protein and cells by the chip interface is reduced.
(2) Because the lysine side chain is in a branched structure, a plurality of functional side chain tail chains can be derived, so that a plurality of alkoxy tail chains are uniformly distributed around the spiral structure, and a complete physical barrier is formed on the surface of the substrate to prevent nonspecific adsorption of biomolecules on the surface of the substrate.
In conclusion, the polymer is used for interface modification of the microfluidic chip, can obviously reduce nonspecific adsorption of nucleic acid, protein and cells, and is beneficial to improving the sensitivity and practicality of the microfluidic chip in biological detection.
Drawings
FIG. 1 shows the preparation of P (Lys-EG) according to example 4 of the present application 1 ) Schematic structural diagram of the modified glass chip surface;
FIG. 2 is a schematic diagram of the structure of a glass-based chip channel in the detection device according to embodiment 5;
1. microfluidic channels, 11, sample injection channels, 12, reaction channels, 13 and sample discharge channels,
2. a color development area, 21, a sample capture color development area, 22, a sample capture color development area.
Detailed Description
The application is further illustrated below in connection with specific examples, which are not to be construed as limiting the application in any way.
The specific raw materials used in the examples of the present application are self-made or commercially available, and the present application is not particularly limited thereto.
The lysine graft polymer provided by the present application is preferably prepared with reference to the following synthetic scheme 1:
(1) Under the action of an initiator, ring-opening polymerization is carried out on lysine-N-carboxyl cyclic anhydride molecule 1, and protective groups are removed under the action of trifluoroacetic acid to obtain alpha-polylysine, namely molecule 2.
(2) The molecule 2 and the molecule 3 are subjected to dehydration condensation reaction, and the protecting group is removed to obtain the molecule 4 with the side chain terminal of the double amino groups.
(3) Molecule 4 and molecule 5 undergo nucleophilic substitution reaction and the protecting group is removed to obtain molecule 6 with an active amino group at the end.
(4) The amino at the end of the molecule 6 and the hydroxyl at the end of the oligoethylene glycol or the oligosaccharide molecule are subjected to substitution reaction to obtain the target product 7.
Preparation of lysine graft Polymer
Example 1
Branched Polymer P (Lys-EG) with lysine side chain containing an oligoethylene glycol molecule 1 ) Is synthesized by (a)
(1) Referring to scheme 1, molecule 2 is obtained from the ring-opening polymerization of molecule 1. In anhydrous tetrahydrofuran, anhydrous N, N-dimethylformamide or N-methylpyrrolidone solvent (1.0 mL), tert-butoxycarbonyl (Boc) -protected lysine-N-carboxycyclic anhydride (0.15 mmol) was stirred at room temperature (27 ℃ C.) for overnight (15 hours) under the action of dodecylamine (0.0075 mmol) as an initiator and then ring-opening polymerized, followed by reaction in a mixed solvent of 2mL trifluoroacetic acid (TFA) and dichloromethane (v: v=1:1) at room temperature (27 ℃ C.) for 0.5 hour to remove the Boc protecting group, to give the α -polylysine represented by molecule 2.
(2) A solution of t-butoxycarbonylaminooxyacetic acid (15.5 mmol) and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (18.6 mmol) in methylene chloride (50 mL) was cooled to zero on ice, after which a solution of pentafluorophenol (17.1 mmol) in methylene chloride (5 mL) was added and gradually returned to room temperature (27 ℃ C.) and stirred for 5 hours to give the tert-butyl oxycarbonylaminooxyacetate, pentafluorobenzene, molecule 5.
(3) Preparation of molecules of formula 6-1 from the reaction of molecules 2 and 5 (similar to molecule 6 in scheme 1 in structure, with a different degree of branching)
Molecule 2 (1.30 mmol) and N, N-Diisopropylethylamine (DIPEA) (3.88 mmol) were dissolved in 1mL DMSO and molecule 5 (25.0 mmol) was added; the resulting reaction mixture was stirred at room temperature for 1 hour, 2mL of methanol was added, and the mixture was precipitated with diethyl ether. Then, 2mL of trifluoroacetic acid (TFA) was added to the precipitated solid, stirred at room temperature for 0.5 hour, the excess TFA was distilled off under reduced pressure, 2mL of methanol was added to the residue, and the mixture was precipitated with diethyl ether to obtain a molecule of formula 6-1.
(4) Dissolving a molecule having the structure of formula 6-1, oligoethylene glycol and aniline in HAc/NaAc buffer (ph=4.2); reacting at 37 ℃ overnight; dialyzing with distilled water, and lyophilizing the obtained solution to obtain branched polymer P (Lys-EG) 1 ). Wherein the molar ratio of the molecule of formula 6-1 to the oligoethylene glycol to the aniline is 0.80:1.0:0.50.
Example 2
Branched Polymer P (Lys-EG) with lysine side chain containing two oligoethylene glycol molecules 2 ) Is synthesized by (a)
Branched Polymer P (Lys-EG) 2 ) Synthesis of (C) and P (Lys-EG) in example 1 1 ) The synthesis of (2) differs only in the preparation of molecule 4, the remaining steps being identical. Molecule 4 was prepared by putting molecule 2 and molecule 3 in a mixed solvent of dichloromethane and hydroxybenzotriazole and catalyzed condensation by EDC to give molecule 4. The method specifically comprises the following steps:
Boc-Lys (Boc) -OH (molecule 3, 20.1 mmol), EDC (23.0 mmol), hydroxybenzotriazole (23.0 mmol) were dissolved in dichloromethane (50 mL) and cooled to zero on ice; n, N-diisopropylethylamine (26.0 mmol) and molecule 2 (1.30 mmol) were then slowly added thereto in this order, followed by recovery to room temperature (27 ℃ C.) and reaction overnight (15 hours). After the reaction is finished, the mixture is washed once by 2M (mol/L) hydrochloric acid, and then is separated by silica gel column chromatography.
The final branched polymer P (Lys-EG) 2 ) The structural formula of the (C) is shown as follows,
example 3
Branched Polymer P (Lys-EG) with four oligoethylene glycol molecules in the lysine side chain 4 ) Is synthesized by (a)
Branched polymersP(Lys-EG 4 ) With the branched Polymer P (Lys-EG) of example 2 2 ) The synthesis differs only in that molecule 4-3 is further prepared on the basis of molecule 4 (similar in structure to molecule 4 in scheme 1, with a different degree of branching), the remainder of the procedure being identical.
Preparation of molecule 4-3: catalytic condensation of molecule 4 (1.30 mmol) with molecule 3 (Boc-Lys (Boc) -OH,38.0 mmol) in a mixed solvent of dichloromethane (50 mL) and hydroxybenzotriazole (23.0 mmol) with EDC (23.0 mmol) gives molecule 4-3; the specific experimental procedure was the same as for the preparation of molecule 4 in example 2.
The final branched polymer P (Lys-EG) 4 ) The structural formula of the (C) is shown as follows,
preparation and performance of (II) lysine grafted polymer modified microfluidic chip
Example 4
P(Lys-EG 1 ) Modifying glass-based surface and its adsorption performance to protein and nucleic acid
1. The silicon oxide on the chip surface is converted into silicon hydroxyl.
Placing the glass chip with the silicon oxide on the surface into piranha washing liquid (piranha solution), and soaking for 10 hours at the temperature of 85-90 ℃; the piranha solution consists of 98wt% of concentrated sulfuric acid and 30wt% of hydrogen peroxide, wherein the volume ratio of the concentrated sulfuric acid to the 30% of hydrogen peroxide is 1:3.
The chip was then rinsed twice with deionized water. And (3) carrying out ultrasonic cleaning treatment on the washed chip in deionized water, and drying with nitrogen for later use.
2. And (3) hydrophobically modifying the surface of the glass chip.
The glass chip with the silicon hydroxyl on the surface is put into 0.5% (v/v) toluene solution of dodecyl trimethoxy silane, and is hermetically soaked for 30 hours at 35 ℃. After the reaction is finished, pouring out the reaction liquid, flushing the chip twice by adopting deionized water, and ultrasonically cleaning the chip by using the deionized water for 5 minutes to remove impurities stained on the chip; the chip was then blow-dried with nitrogen.
3、P(Lys-EG 1 ) Modified glass chip
Branched polylysine P (Lys-EG) prepared using example 1 1 ) A solution having a concentration of 5.0mg/mL was prepared with PBS buffer (pH 7.4). The washed glass chip was then immersed in the above polymer solution and incubated at room temperature for 24 hours on a shaker. Finally, the coated glass-based chip was rinsed thoroughly with ultrapure water and dried with a nitrogen stream.
Branched polylysine P (Lys-EG) prepared in example 2 2 ) And branched polylysine P (Lys-EG) prepared in example 3 4 ) The procedure for modifying the glass chip is the same as described above.
The preparation method of the PEG coated glass chip is the same as the steps, and the PEG is cetyl polyoxyethylene ether.
4. Protein and nucleic acid adsorption experiments
Polymer-free Glass chips (Bare Glass), glass chips with grafted polymer (P (Lys-EG) 1 )、P(Lys-EG 2 )、P(Lys-EG 4 ) Coated glass chips and glass chips with PEG coating. Freshly prepared protein solutions (BSA, 1.0 mg/mL) or oligonucleotide solutions (21 nt-dsDNA,1.5 mg/mL) were passed through the chip at 25℃for 15 minutes at a flow rate of 50. Mu.L/min, respectively, and the chip surface was rinsed with PBS buffer for 15 minutes. The amount of the protein and the oligonucleotide adsorbed on the glass surface was determined by measuring the fluorescent groups modified on the above molecules, and the measurement results are shown in Table 1.
TABLE 1
21nt dsDNA BSA
Bare Glass 100％ 100％
P(Lys-EG 1 ) 3.2％ 4.0％
P(Lys-EG 2 ) 3.4％ 3.7％
P(Lys-E 4 ) 2.7％ 2.9％
PEG 10.2％ 13.0％
As can be seen from Table 1, the lysine graft polymer of the present disclosure significantly reduced the nonspecific adsorption of oligonucleotides and proteins after coating the glass interface, compared to PEG, and was prepared from P (Lys-EG 1 ) To P (Lys-EG) 2 ) To P (Lys-EG) 4 ) The more the number of branched side chain terminal oligoethylene glycol increases, the more obvious the nonspecific "zero" adsorption effect is.
(III) detection device
Example 5
This example provides a pharmaceutical composition comprising the preparation of example 4 having P (Lys-EG 1 ) CoatingThe detection device of the glass chip of the layer is used for realizing nonspecific zero adsorption of analytes and improving the signal to noise ratio of detection parts, and is used for biological detection, the structure of a glass-based chip channel is shown in fig. 2, wherein the microfluidic channel 1 comprises a sample inlet channel 11, a reaction channel 12 and a sample outlet channel 13; the color development region 2 includes a sample capturing color development region 21 provided between the sample introduction flow path 11 and the reaction flow path 12, and a sample capturing color development region 22 provided between the reaction flow path 12 and the sample discharge flow path 13.
In order to avoid non-specific adsorption of the sample on the glass-based surface during the flow process and to reduce the sensitivity of the experiment, the embodiment performs P (Lys-EG 1 ) And (3) coating. The reaction occurring in the sample capture colour zone 21, 22 may be a hybridization capture reaction of an oligonucleotide single strand or an antigen-antibody specific recognition reaction, and may be designed according to specific experimental details without undue limitation.
Of course, in other embodiments, it is also possible to coat all or part of the color-developing region 2 simultaneously, leaving only the region to be reacted.
In summary, the present disclosure provides graft polymers comprising a backbone formed by polymerization of lysine monomers, side chains formed by grafting of lysine, the side chains comprising one or more reactive functional groups for coupling with molecules comprising alkoxy units to form side chain tail chains comprising alkoxy units. The polymer is used for interface modification of the microfluidic chip, can obviously reduce nonspecific adsorption of nucleic acid, protein and cells, and is beneficial to improving the sensitivity and practicality of the microfluidic chip in biological detection. The structure of the polymer has the following advantages:
(1) Because the main chain is a hydrophobic peptide bond, and the tail chain of the side chain is a hydrophilic group, the polymer can be arranged on the chip interface in a regular spiral structure, and simultaneously, the alkoxy groups of the tail chain of the side chain can be uniformly and compactly distributed on the chip interface, so that the defect of the chip interface is greatly reduced, and the nonspecific adsorption of nucleic acid, protein and cells by the chip interface is reduced.
(2) Because the lysine side chain is in a branched structure, a plurality of functional side chain tail chains can be derived, so that a plurality of alkoxy tail chains are uniformly distributed around the spiral structure, and a complete physical barrier is formed on the surface of the substrate to prevent nonspecific adsorption of biomolecules on the surface of the substrate.
Any numerical value recited in this disclosure includes all values incremented by one unit from the lowest value to the highest value if there is only a two unit interval between any lowest value and any highest value. For example, if the amount of one component, or the value of a process variable such as temperature, pressure, time, etc., is stated to be 50-90, it is meant in this specification that values such as 51-89, 52-88 … …, and 69-71, and 70-71 are specifically recited. For non-integer values, 0.1, 0.01, 0.001 or 0.0001 units may be considered as appropriate. This is only a few examples of the specific designations. In a similar manner, all possible combinations of values between the lowest value and the highest value enumerated are to be considered to be disclosed.
It should be noted that the above-described embodiments are only for explaining the present application and do not constitute any limitation of the present application. The application has been described with reference to exemplary embodiments, but it is understood that the words which have been used are words of description and illustration, rather than words of limitation. Modifications may be made to the application as defined in the appended claims, and the application may be modified without departing from the scope and spirit of the application. Although the application is described herein with reference to particular means, materials and embodiments, the application is not intended to be limited to the particulars disclosed herein, as the application extends to all other means and applications which perform the same function.

Claims (10)

1. A polylysine graft polymer is characterized by having a structure shown in formula (I):
wherein n is more than or equal to 10 and less than or equal to 50, R' is a tail chain group, R 1 Has a structure shown in any one of formulas (II) - (IV),
wherein R is 2 Has a structure shown in any one of formulas (V) - (VI),
wherein m is more than or equal to 2 and less than or equal to 10.
2. The polymer according to claim 1, wherein 15.ltoreq.n.ltoreq.25, preferably 15, 18, 20, 22 or 25.
3. The polymer according to claim 1 or 2, wherein R 'is an alkyl carbon chain group, preferably R' has a structure according to any one of formulas (VII) to (VIII),
wherein z is more than or equal to 5 and less than or equal to 20; y is more than or equal to 2 and less than or equal to 8.
4. A polymer according to claim 1 or 2, wherein the backbone or side chain ends of R 'contain a siloxy or silane group, preferably the backbone or side chain of R' contains an oligoethylene glycol linking chain; more preferably, R' has a structure represented by any one of formulas (IX) to (X),
wherein z is more than or equal to 5 and less than or equal to 20.
5. The polymer according to any of claims 1 to 4, wherein 3.ltoreq.m.ltoreq.6, preferably 3, 4, 5 or 6.
6. The polymer of any one of claims 1 to 5, wherein the sugar ring structure in formula VI is an oligosaccharide structure, preferably the oligosaccharide structure is a mannose structure, a di-mannose structure or a tri-mannose structure.
7. A method for preparing a polylysine graft polymer according to any one of claims 1-6 wherein polylysine graft polymer is prepared by a method (1) of ring-opening polymerization of lysine-N-carboxycyclic anhydride or a method (2) of dehydration condensation of lysine alpha-amino; preferably, the method comprises the steps of,
the method (1) is an epsilon-amino branching modification method before polymerization of the method (1-1) or an epsilon-amino branching modification method after polymerization of the method (1-2);
the method (2) is an epsilon-amino branching modification method before dehydration condensation of the method (2-1) or an epsilon-amino branching modification method after dehydration condensation of the method (1-2).
8. The preparation method according to claim 7, characterized in that the preparation method comprises the steps of:
(1) Epsilon-amino protection: protecting epsilon-amino of lysine-N-carboxyl ring anhydride by t-butyloxycarbonyl or benzyloxy carbonyl to obtain the lysine-N-carboxyl ring anhydride with epsilon-amino protection;
(2) Polymerization reaction: ring-opening polymerization is carried out on lysine-N-carboxyl cyclic anhydride protected by epsilon-amino under the action of an initiator, and then protecting groups are removed under the action of a strong organic acid catalyst to obtain alpha-polylysine;
preferably, the initiator is terminated with an amino-containing alkyl carbon chain or an ethoxy chain, further preferably, the initiator is terminated with a structure as shown in any one of formulas (IX) - (X),
in the formula (IX) and the formula (X), z is more than or equal to 5 and less than or equal to 20;
or the terminal of the initiator is an alkyl carbon chain, further preferably, the terminal of the initiator has a structure shown in any one of formulas (VII-VIII),
in the formula (VII) and the formula (VIII), z is more than or equal to 5 and less than or equal to 20; y is more than or equal to 2 and less than or equal to 8;
(3) Grafting reaction: the method comprises the steps of (1) carrying out condensation reaction on alpha-polylysine and 2, 6-di-tert-butoxycarbonyl amino caproic acid, removing a protecting group under the action of a strong organic acid catalyst, carrying out nucleophilic substitution reaction on the alpha-polylysine and tert-butoxycarbonyl amino oxy acetic acid pentafluorophenyl ester, removing the protecting group again to obtain active amino, and carrying out substitution reaction on the amino and hydroxyl at the tail end of an oligomeric ethylene glycol or oligosaccharide molecule to obtain a target product;
preferably, the strong organic acid catalyst in step (2) and step (3) is at least one selected from trifluoroacetic acid and p-toluenesulfonic acid.
9. A microfluidic chip, characterized in that part or all of the interface is modified by the polylysine graft polymer according to any one of claims 1-6 or by the polylysine graft polymer made by the preparation method according to any one of claims 7-8, preferably the substrate of the microfluidic chip is glass or silicon wafer material.
10. A detection device comprising the microfluidic chip of claim 9.
CN202210557345.1A 2022-05-20 2022-05-20 Polylysine grafted polymer, preparation method thereof, polymer modified micro-fluidic chip and detection device comprising polymer modified micro-fluidic chip Pending CN117126398A (en)

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