A coated substrate for in situ synthesis and bio-detection of high density polypeptide arrays by one-step acid hydrolysis and uses thereof

Abstract

The application discloses a coating matrix for realizing in-situ synthesis and biological detection of high-density polypeptide arrays through one-step acid hydrolysis and application thereof, and relates to the technical field of biotechnology.The technical scheme points of the coating matrix are as follows: the structural composition of the coating matrix comprises a hydrophilic hydrogel matrix, and side-chain-protected polyamino acids are synthesized on the hydrophilic hydrogel matrix; the polyamino acids are one of hydroxyl group-protected serine [O'-X]S or hydroxyl group-protected threonine [O'-X]T, so that the coating matrix is modified by polymers with the structure of [O'-X]S)n or [O'-X]T)n; the coating matrix has low wettability to aprotic polar solvents used in polypeptide array synthesis, and the contact angle θ of the coating matrix is greater than 25°; the coating matrix has an amino group at the end, and can be chemically reacted with an electrophilic reagent; and the coating matrix can be treated by trifluoroacetic acid to remove the protecting group X, so as to be converted into a coating matrix with high wettability, and the contact angle θ of the coating matrix is less than 5°.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, more particularly, it relates to a coating matrix for in situ synthesis and bioassay of high-density polypeptide array by one-step acid hydrolysis and its application. BACKGROUND

[0002] Synthesis of biomolecules into high-density arrays and the ability to produce them at high yield, and to apply the arrays directly to biochemistry or cell biology screening, can provide a powerful tool for the fields of biological materials, chemical biology and pharmacy. There are two major challenges for in situ synthesis of biomolecule arrays: 1) in situ multi-step combinatorial chemical synthesis on a plane with small enough feature size and high yield and efficiency; 2) not only a coating surface compatible with chemical reagents, but also a surface compatible with subsequent protein or cell-based screening analysis.

[0003] There are two major in situ array synthesis methods, each with its own advantages and disadvantages. The first is the SPOT method, which includes the use of standard Fmoc-based solid-phase peptide synthesis to synthesize peptides on cellulose membranes step by step. The high porosity of cellulose makes the matrix an ideal matrix for solid-phase synthesis, which can absorb the reactants in the matrix and be easy to clean. However, due to the high porosity, it is difficult to achieve small feature size and high-density arrays on cellulose membranes, and its array is only 25 / cm 2 . Larger feature size and porous matrix also lead to higher protein consumption. In order to image the cells adhered to the surface, the strong light scattering of cellulose fibers interferes with fluorescence-based imaging and data analysis. The second method is to generate more than 100,000 / cm 2 high-density peptide arrays on glass by printing polymer particles containing pre-activated compound monomers or using light-labile protecting groups, but it also has the following disadvantages: first, most methods require special chemicals or commercial instruments, such as: amino acid carrier ink, laser-based transfer technology, scanning probe lithography technology, maskless lithography or pre-patterning of surface wettability, etc.; second, compared with the SPOT method, it is relatively more difficult to achieve automation.

[0004] It is well known that oligonucleotide arrays for DNA and RNA analysis represent one of the most important applications of array technology. However, in the field of drug screening and discovery, one-well-one-analysis remains the most common approach. Most cell-material interaction studies are also performed in the form of reaction wells, and only a few reports have been published on the screening of biological materials in array format. As an alternative to addressable arrays, one-bead-one-compound synthesis has been developed for the preparation of large libraries of polypeptides and peptoids. Compounds on selected beads can be identified by polypeptide sequencing and mass spectrometry. However, synthesis of addressable arrays or screening of compounds on beads is more complicated than the equal distribution of solutions in different wells. In addition, like other screening technologies, most array-based screening is also limited to pre-synthesized collections of compounds.

[0005] Aprotic polar solvents such as DMF and DMSO cannot form droplets on glass or aminofunctionalized glass surfaces. Their high wettability to the substrate results in a small contact angle (0 « 10°), leading to the spreading of droplets into irregular larger areas. This problem is difficult to solve by adjusting the contact angle through changing the surface of the support or the solvent. A weakening of the attractive forces between the solvent and the surface can lead to unstable movement of the droplets, a common but undesirable phenomenon in droplet deposition. Therefore, a surface with optimal wettability is important for in situ synthesis of arrays. In addition to chemical synthesis, the compatibility of the generated molecular array with various biochemical assays and cell adhesion is also important. The lipophilic polymers such as polystyrene used in traditional solid-phase polypeptide synthesis are hydrophobic, which can lead to excessive non-specific interactions with many proteins.

[0006] In our previous work, we developed a lipid-grafted amphiphilic coating on glass surfaces, on which small droplets of organic solvents can be deposited with a relatively large contact angle and inhibited movement, allowing multiple rounds of combinatorial synthesis of small molecule compounds and polypeptides. By removing the lipid modification, the amphiphilic surface can be converted to a hydrophilic surface, forming a high-density array suitable for protein and cell screening. However, in the preparation of the coating, the reaction operation for introducing hydrophobic groups on the hydroxyl group is complex, the yield is unstable, and in the final side chain deprotection in polypeptide synthesis and the regulation of the wettability of the support surface, respectively, in the TFA acid treatment and the saponification in ammonia solution two reaction steps. Compared with side chain deprotection, saponification is not complete, leaving a small amount of lipid modification in the prepared coating matrix, which can interfere with subsequent screening experiments. In addition, long-term use of ammonia solution treatment can damage some sensitive amino acids in polypeptide array synthesis, making it difficult to well control the synthesis quality of the polypeptide array, thereby affecting the result output in the subsequent biological screening detection. SUMMARY

[0007] In view of the deficiencies of the prior art, the purpose of the present application is to provide a coating matrix for realizing in-situ synthesis and biological detection of high-density polypeptide array through one-step acid hydrolysis, and application thereof, wherein a poly-(O-tert-butyl-serine) / poly-(O-tert-butyl-threonine) linker is used to replace the lipid modification in the previous work to obtain the characteristics of the surface with amphiphilicity, and the final side chain deprotection in polypeptide synthesis and the switching of the wettability of the carrier surface can be realized through one-step acid treatment reaction, thereby avoiding the damage to the lipid bond-containing peptide compounds caused by the ammonia treatment, and simplifying the steps while synthesizing the array with higher density.

[0008] To achieve the above-mentioned purpose, the present application provides the following technical scheme: a coating matrix for realizing in-situ synthesis and biological detection of high-density polypeptide array through one-step acid hydrolysis, wherein the structure of the coating matrix comprises a hydrophilic hydrogel matrix, and side chain-protected polyamino acids are synthesized on the hydrophilic hydrogel matrix, the polyamino acids are one of serine [O'-X]S protected by an X group or threonine [O'-X]T protected by an X group, so that the coating matrix is modified by a polymer with the structure of [O'-X]S)n or [O'-X]T)n.

[0009] The coating matrix has low wettability to the aprotic polar solvent used in the synthesis of the polypeptide array, and the contact angle θ is greater than 25°.

[0010] The coating matrix has an amino group at the end, which can be chemically reacted with an electrophilic reagent.

[0011] The coating matrix can remove the protecting group X by trifluoroacetic acid treatment, so as to be converted into a polymer with high wettability, and the contact angle θ is less than 5°.

[0012] Further, the hydrophilic hydrogel matrix is an oligoamino polysaccharide.

[0013] Further, the side chain-protected polyamino acid is connected to the amino group of the hydrophilic hydrogel matrix through the C-terminal, and 3≤n<20.

[0014] Further, the connection is a direct connection through an amide bond, or a connection composed of one of single or multiple glycine, -alanine or 8-amino-3,6-dioxaoctanoic acid, wherein the length of the connection is a 3-18 carbon atom chain.

[0015] Further, the amino group reacted with the electrophilic reagent is the N-terminal amino group of the [O'-X]S)n or [O'-X]T)n polymer, or an amino group connected to the N-terminal amino group of the [O'-X]S)n or [O'-X]T)n polymer.

[0016] Furthermore, the linker is composed of a single or multiple glycine, β-alanine or 8-amino-3,6-dioxaoctanoic acid, and the linker length is a carbon atom chain of 3-60.

[0017] Furthermore, the side chain protecting group is a hydrophobic group.

[0018] Furthermore, the side chain protecting group is a tert-butyl group.

[0019] A solid phase carrier comprises a flat surface coated with any of the above coating matrices. The solid phase carrier is one of glass, quartz, silicon wafer, steel wafer, ceramic wafer or plastic wafer with activated groups on the surface.

[0020] An application of the above solid phase carrier, on which a matrix of polypeptide combinations containing different sequences synthesized by solid phase polypeptides is used for detecting and / or identifying protein binding compounds, biological materials, enzyme substrates or enzyme inhibitors.

[0021] In summary, the present invention has the following beneficial effects: The present invention utilizes a poly(O-tert-butyl-serine) / poly(O-tert-butyl-threonine) linker to form a coating surface with amphiphilic properties. Final side chain deprotection and control of support surface wettability during peptide synthesis are both achieved in a single acid treatment step. This novel coating matrix not only improves the quality of in situ synthesis reactions and simplifies the peptide array synthesis process, but also increases the resolution of array synthesis to 10,000 features / cm. 2 , which has important application prospects in the field of biology. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Shown are the synthetic steps for coating the inventive material on a glass surface.

[0023] Figure 2 Shown are the contact angle measurements of DMSO on G4 surfaces without TFA treatment (A), and the droplet sizes of DMSO on G4 surfaces with and without TFA treatment (B).

[0024] Figure 3 Shown are the contact angle measurements of H2O on the G4 surface without TFA treatment (A) and the contact angle measurements of the G4 surface after TFA treatment (B).

[0025] Figure 4 Shown are arrays of biotin- and fluorescein-labeled peptides synthesized directly on the slide surface.

[0026] Figure 5 Shown is the binding of neutravidin to a biotinylated peptide synthesized directly on the slide surface.

[0027] Figure 6 Epitope map of monoclonal anti-Flag antibody is shown. DETAILED DESCRIPTION

[0028] The application is further illustrated in conjunction with the following examples.

[0029] Example 1 Covalent coating of a material of the application on a glass surface

[0030] Step 1 : Amino-functionalization of the surface of the glass slide with 3- aminopropyltriethoxysilane.

[0031] Step 2: Treatment of the amino-silane functionalized glass slide with a solution of 0.1 M succinic anhydride and 0.3 M N-methylmorpholine in N,N- dimethylformamide for 2 hours, converting the surface amino groups to carboxyl groups.

[0032] Step 3-I: Treatment of the surface carboxyl groups with a solution of 0.1 M N,N'-diisopropylcarbodiimide and 1 M N-hydroxysuccinimide in N,N- dimethylformamide for 30 minutes, converting the surface carboxyl groups to N- hydroxysuccinimide esters.

[0033] Step 3-II: Immersion of the glass slide in a solution of chitosan (1% chitosan dissolved in 1% acetic acid) overnight, immobilizing the chitosan on the surface via amide bond formation.

[0034] Step 3-III: Coupling of the remaining amino groups on the surface chitosan with Fmoc-Ala-OH by treatment with freshly mixed equal volumes of 0.5 M Fmoc-Ala-OH, 0.5 M HATU, 1.5 M N-methylmorpholine in N,N-dimethylformamide for 1 hour.

[0035] Steps 3-I to 3-III were repeated twice to form a chitosan hydrogel on the surface of the glass slide.

[0036] Step 3-IV: Removal of the Fmoc protecting group by treatment of the surface with 20% piperidine in N,N-dimethylformamide for 30 minutes.

[0037] Step 4-I: Treatment of the surface with freshly mixed equal volumes of 0.5 M Fmoc-Ser(tBu)-OH, 0.5 M HATU, 1.5 M N-methylmorpholine in N,N- dimethylformamide for 1 hour.

[0038] Step 4-II: Fmoc deprotection with 20% piperidine in N,N-dimethylformamide and washing to couple the free amino groups with Fmoc-Ser(tBu)-OH.

[0039] Step 4-I and 4-II are repeated 2, 5 or 8 times to produce 3, 6 or 9 repeats of polyserine on the surface.

[0040] Step 4-III: The free amino groups are coupled to Fmoc-O2Oc-OH by treating the surface with freshly mixed equal volumes of 0.5 M Fmoc-O2Oc-OH, 0.5 M HATU, 1.5 M N-methylmorpholine solution (solvents are both N,N-dimethylformamide) in DMF for 1 hour, followed by repeating Step 4-II.

[0041] Step 4-III is repeated once.

[0042] The final G4 surface, on which organic solvents can form stable droplets in situ, is ready for the synthesis of a polypeptide array. After the synthesis of the polypeptide array is completed, the side chains of the polyserine are also deprotected when the side chains of the polypeptide amino acids are deprotected with TFA.

[0043] Example 2 Measurement of the contact angle of dimethyl sulfoxide on G4 surfaces that have not been treated with TFA and measurement of the droplet size of dimethyl sulfoxide on G4 surfaces that have and have not been treated with TFA.

[0044] A 10 microliter droplet of dimethyl sulfoxide is added to the G4 surface that has not been treated with TFA using a motorized syringe pump and the droplet on the surface is imaged using a digital camera to measure the contact angle as shown in Figure 2 (A), which increases as the number of repeats of the side chain tert-butyl protected serine increases.

[0045] A 5 microliter droplet of dimethyl sulfoxide is added to the G4 surface that has and has not been treated with TFA using a motorized syringe pump and the droplet on the surface is imaged using a digital camera as shown in Figure 2 (B), which spreads out on the G4 surface after treatment with TFA and forms very small droplets on the G4 surface without TFA treatment.

[0046] Example 3 Measurement of the contact angle of water on G4 surfaces that have and have not been treated with TFA.

[0047] A 10 microliter droplet of dimethyl sulfoxide is added to the G4 surface that has not been treated with TFA using a motorized syringe pump and the droplet on the surface is imaged using a digital camera to measure the contact angle as shown in Figure 3 (A), which increases as the number of repeats of the side chain tert-butyl protected serine increases; and Figure 3 (B), which decreases to the same value after treatment with TFA regardless of the number of repeats of the serine.

[0048] Example 4. Synthesis and imaging of biotinylated polypeptides (biotin-G7O2Oc) and fluorescein labeled polypeptides (F-G7O2Oc).

[0049] The polypeptide array was synthesized directly on the G4 surface by Fmoc solid phase synthesis method, and the side chains of the amino acids were deprotected by TFA. The synthesized polypeptide array was incubated with 10 micromolar Dylight633 labeled Neutravidin solution. After washing, the slide was imaged by fluorescence microscope. As shown in Figure 4 , the fluorescence signals of Dylight633 labeled Neutravidin and fluorescein did not merge when the array density reached 10,000 per square centimeter, indicating that each peptide on the array was synthesized individually at this density.

[0050] Example 5. Dot array imaging of various biotinylated polypeptides.

[0051] The polypeptide array (polypeptides a-k) was incubated with 10 micromolar Dylight633 labeled Neutravidin solution, washed, and the polypeptides on the slide were imaged by fluorescence microscope. After each synthesis reaction, the amino groups on the G4 surface that did not react were blocked with acetic anhydride, so only the full-length polypeptides could be labeled with biotin. As shown in Figure 5 , all polypeptides had fluorescence signals, indicating that all polypeptides were successfully synthesized.

[0052] Example 6. Imaging of epitope mapping of anti-FLAG tag antibody.

[0053] The FLAG tag peptide array with different mutations was incubated with 141 nM Dylight633 labeled anti-FLAG tag M2 antibody solution. After washing, the slide was imaged by fluorescence microscope. As shown in Figure 6 , the anti-Flag tag antibody can specifically bind to the FLAG tag peptide, Y2 and K3 are the key to recognition, which is consistent with the published results; the original sequence NH2-DYKDDDDK-COOH is marked with a white circle.

[0054] From the above examples, it can be seen that the polyserine (hydroxyl side chain protected by tert-butanol) can realize the final side chain deprotection in polypeptide synthesis and the switching of surface wettability by one-step acid treatment. The new coating matrix simplifies array synthesis and improves array density, which can be as high as 10,000 features / cm 2 ; the surface properties switched by acid treatment are better, the deprotection is more gentle and complete, and it will not damage the peptide compounds with lipid bonds, etc., and will not affect subsequent protein binding experiments, etc.

[0055] The embodiments are only used for explaining the present application, and are not used for limiting the present application, and the person skilled in the art can make the modification of the embodiments without the creative contribution according to the need after reading the description, and as long as the modification is within the scope of the claims of the present application, it is protected by the patent law.

Claims

1. A coating matrix for in situ synthesis and biodetection of high-density peptide arrays by one-step acid hydrolysis, characterized in that: The coating matrix comprises a hydrophilic hydrogel matrix, on which a side chain-protected polyamino acid is synthesized, wherein the side chain-protected polyamino acid is connected to an amino group of the hydrophilic hydrogel matrix via its C-terminus, wherein 3≤n<20, and the polyamino acid is one of serine [O'-X]S whose hydroxyl group is protected by an X group or threonine [O'-X]T whose hydroxyl group is protected by an X group, so that the coating matrix has a polymer modification having a [O'-X]S)n or [O'-X]T)n structure; The coating matrix has low wettability to the aprotic polar solvent used in the synthesis of the polypeptide array, and its contact angle θ is greater than 25°; The coating matrix has an amino group at the end, which can react chemically with an electrophilic reagent; The coating matrix can be treated with trifluoroacetic acid to remove the protecting group X, thereby converting it into a matrix with high wettability, with a contact angle θ of <5°.

2. The coating matrix according to claim 1, characterized in that The hydrophilic hydrogel matrix is ​​oligoaminopolysaccharide.

3. The coating matrix according to claim 1, characterized in that The linkage is one of direct linkage via an amide bond or linkage consisting of single or multiple glycine, β-alanine or 8-amino-3,6-dioxaoctanoic acid, wherein the linkage length is a 3-18 carbon atom chain.

4. The coating matrix according to claim 1, characterized in that The amino group that reacts with the electrophilic reagent is the N-terminal amino group of the [O'-X]S)n or [O'-X]T)n polymer, or the amino group connected to the N-terminal amino group of the [O'-X]S)n or [O'-X]T)n polymer.

5. The coating matrix according to claim 4, characterized in that The linker is composed of a single or multiple glycine, β-alanine or 8-amino-3,6-dioxaoctanoic acid, and the linker length is a carbon atom chain of 3-60.

6. The coating matrix according to claim 1, characterized in that The side chain protecting group is a hydrophobic group.

7. The coating matrix according to claim 1 or 6, characterized in that The side chain protecting group is tert-butyl.

8. A solid phase carrier, characterized in that The invention comprises a flat surface coated with a coating matrix according to any one of claims 1 to 7, wherein the solid phase carrier is one of glass, quartz, silicon wafer, steel wafer, ceramic wafer or plastic wafer with activation groups on the surface.

9. A use of the solid phase carrier according to claim 8, characterized in that: A matrix of polypeptide combinations containing different sequences synthesized using solid phase polypeptides is used to detect and / or identify protein-binding compounds, biological materials, enzyme substrates or enzyme inhibitors.

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