Amino blocking method for chip microspheres and application thereof

Abstract

The application provides a chip microsphere amino blocking method and application. After activation of the chip microsphere and before coupling of an oligonucleotide chain, a group containing an acyl group or a benzyl group is used to block the excess amino group. The specific steps include three processing steps of microsphere activation, amino blocking and coupling of the oligonucleotide chain. Effect verification results show that the microsphere obtained by the application has significantly reduced non-specificity in the hybridization process and the specific hybridization is not affected, and the decoding result accuracy based on hybridization can be greatly improved. At the same time, the application uses acetic anhydride, fluorine-substituted acetic anhydride, succinic anhydride, glutaric anhydride or benzyl halogen substitutes as the amino blocking reagent, the reagent is easy to obtain, the blocking reaction condition is mild, and the blocking reaction is easy to realize.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of chip preparation, and relates to a chip microsphere amino blocking method, a chip microsphere prepared by the method and a chip containing the microsphere. BACKGROUND

[0002] Single nucleotide polymorphism (SNP) refers to a single nucleotide variation existing at a specific position of a genome, including base conversion, transversion, insertion and deletion, etc. SNPs exist universally in human and animal genomes, and there is an average of about 1000 bases in the human genome for one SNP. As the third generation of genetic diagnosis markers, SNPs can directly affect the susceptibility of individuals to diseases, metabolic differences of exogenous substances and adverse drug reactions. Chip detection, as a detection method with low cost, large throughput and high accuracy, has become a powerful detection tool for large-scale SNP analysis, and provides strong technical support for genetic research analysis, individualized medical treatment and agricultural breeding research.

[0003] In a chip composed of a glass substrate and microbeads, the microbeads are the core part of the chip, and each microbead surface is coupled with an oligonucleotide fragment of a sequence. The glass substrate is etched to form many small holes arranged in an orderly manner, and the size of the small holes matches the microbeads, with one small hole accommodating one microbead. In the production of the chip, hundreds of thousands of microbeads for the chip are mixed according to a certain proportion and scattered on the glass substrate. The microbeads are randomly dropped into the small holes on the substrate, and then hybridization decoding is used to determine which microbead is in each small hole.

[0004] The microbeads are obtained by modification, modification, coupling and other steps of silica microspheres. Because a part of the amino groups may not be completely reacted during the modification process, when the microbeads are directly used for hybridization decoding, the excess amino groups will cause a certain degree of non-specific adsorption, which will greatly reduce the accuracy of the hybridization decoding. Therefore, it is very important to reduce this non-specific adsorption in the preparation process of the microbeads.

[0005] In order to solve the above technical problems, the prior art has made many beneficial explorations, such as the Chinese invention patent with the patent number CN106596961A discloses a construction method of a high-sensitivity microstructure biological chip. After the glass sheet is sequentially etched, epoxy group reaction and amination reaction, the amination glass surface on the unmarked amination glass sheet is subjected to a blocking reaction by using an ethanolamine blocking solution, and then anhydrous ethanol is used for elution to obtain the blocked glass sheet. SUMMARY

[0006] The present application is based on the foregoing research, and several amino blocking reagents in the chip preparation process are screened out. By blocking the excess amino groups on the activated microspheres, the influence of the excess amino groups on the subsequent hybridization process is reduced and eliminated, so as to reduce the hybridization non-specificity. The first object of the present application is to provide a microsphere modification method, which blocks the excess amino groups of the chip microspheres by using the screened amino blocking reagent. The oligonucleotide modified microspheres obtained by the method have low hybridization non-specificity. The second object of the present application is to provide the chip microspheres modified by the above method. The third object of the present application is to provide a chip comprising the above microspheres, in particular, an SNP chip.

[0007] In a first aspect of the present application, a chip microsphere amino blocking method is provided, in which a R group is used to block the excess amino groups after the activation of the chip microspheres and before the coupling of the oligonucleotide chains, wherein the R group is an acyl group or a benzyl group.

[0008] The technical solution adopted is as follows:

[0009]

[0010] In the above formula 1 to formula 4, R represents any one of acetyl, trifluoroacetyl, 4-carboxylpropionyl, 5-carboxybutyryl, and benzyl.

[0011] The specific steps are as follows:

[0012] A. Microsphere activation

[0013] The amino modified microspheres represented by formula 1 are suspended in dry acetonitrile, and N, N-diisopropyl ethylamine and cyanuric chloride are sequentially added. The particle size of the microspheres is 0.5-5 μm, the concentration of the microspheres is 50-150 mg / mL, the amount of N, N-diisopropyl ethylamine used is 150-250 μL per gram of microspheres, and the amount of cyanuric chloride used is 50-150 mg per gram of microspheres. The reaction is carried out at room temperature for 0.5-3 hours, and then the microspheres are sequentially washed with dry acetonitrile for 3-5 times, washed with sodium borate solution for 3-5 times, and then washed with the solvent to be used in the next step for 3-5 times, to obtain the compound of formula 2.

[0014] B. Amino blocking

[0015] The activated amino microspheres in step A are added to a reaction solvent, and a blocking reagent is added at room temperature and reacted for 30 minutes. Then, the reaction is carried out at 25-50 °C for 0.5-2 hours, the supernatant is removed by centrifugation, and the compound of formula 3 is obtained by washing with sodium borate solution for 3-5 times, which is directly used in the next step.

[0016] The reaction solvent is any one or combination of acetonitrile, DMF, N-methyl pyrrolidone, or any one of a mixed solution of the above three solvents and a sodium borate solution; and the blocking reagent is any one of acetic anhydride, fluorine-substituted acetic anhydride, succinic anhydride, glutaric anhydride or benzyl halogen substituent.

[0017] C, coupling oligonucleotide chain

[0018] The activated microspheres with blocked excess amino groups obtained in step B are suspended in a sodium borate solution, and a sodium chloride solution of the 5' amino-modified oligonucleotide chain is added at room temperature, wherein the microsphere concentration is 25-100 mg / mL, the oligonucleotide chain concentration is 80-120 nanomoles / mL, and the sodium chloride concentration is 0.5-1 mol / L. The reaction is carried out at room temperature for 3-8 hours, and then the compound of formula 4 is obtained after 3-5 times of ultrapure water washing and storage.

[0019] In a second aspect of the present application, a chip microsphere prepared by the above-mentioned amino blocking method of chip microspheres is provided.

[0020] In a third aspect of the present application, a chip containing the above-mentioned microspheres is provided.

[0021] The oligonucleotide-modified microspheres are laid into a chip matched with the particle size of the microspheres, and hybridization is carried out with the target 1 and the target 2 at the same time (the target 1 is an oligonucleotide chain modified with FAM at the end and complementary to the oligonucleotide chain on the microspheres, and the target 2 is an oligonucleotide chain modified with Cy3 at the end and non-complementary to the oligonucleotide chain on the microspheres), and after hybridization, the FAM and Cy3 channels of the chip scanner are scanned respectively. It can be seen from the comparison that the hybridization signal of the microspheres treated with the amino blocking reagent in the modification process does not decrease, and the non-specific signal significantly decreases.

[0022] Compared with the prior art, the technical effects of the present application are as follows:

[0023] In terms of function, the microspheres obtained by the present application have significantly reduced non-specificity in the hybridization process and do not affect specific hybridization, which can greatly improve the correctness of the decoding result based on hybridization.

[0024] In terms of preparation, the present application uses acetic anhydride, fluorine-substituted acetic anhydride, succinic anhydride, glutaric anhydride or benzyl halogen substituent as the amino blocking reagent, which is easy to obtain, and the blocking reaction conditions are mild and easy to realize. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 is a scanning graph of the mixture of the oligonucleotide-modified microspheres without blocking amino groups and the target 1 and the target 2 after hybridization in the FAM channel.

[0026] Figure 2is the scanning graph of the mixture of the oligonucleotide modified microspheres with blocked amino groups and the target 1 and the target 2 under the Cy3 channel after hybridization.

[0027] Figure 3 is the scanning graph of the mixture of the oligonucleotide modified microspheres with blocked amino groups and the target 1 and the target 2 under the FAM channel after hybridization.

[0028] Figure 4 is the scanning graph of the mixture of the oligonucleotide modified microspheres with blocked amino groups and the target 1 and the target 2 under the Cy3 channel after hybridization. DETAILED DESCRIPTION

[0029] The application will be described in detail below with reference to the embodiments and the accompanying drawings. However, the following embodiments should not be regarded as limiting the scope of the application.

[0030] Example 1 acyl blocked microspheres

[0031] Taking the preparation of the microspheres with the linked oligonucleotide chain as shown in the following formula 5 as an example, the specific preparation method is as follows:

[0032]

[0033] 1. Preparation of activated microspheres

[0034] Into a 50 mL reaction bottle, 1 g of amino microspheres with a particle size of 3 μm, 20 mL of anhydrous acetonitrile, 0.3 mL of N, N-diisopropyl ethylamine and 0.15 g of cyanuric chloride were sequentially added. The reaction was carried out at room temperature for 1 hour, sequentially washed with dry acetonitrile for 3-5 times, washed with sodium borate solution for 3-5 times, and then washed with dry acetonitrile for 3-5 times, to obtain the activated microspheres with the structural formula as shown in formula 6, which was directly used in the next step reaction.

[0035]

[0036] 2. Amino blocking

[0037] Into a 50 mL reaction bottle, 1 g of the activated microspheres in step 1, 20 mL of anhydrous acetonitrile, and 0.1 mL of acetic anhydride were sequentially added, and the reaction was carried out at room temperature for 30 minutes, and then heated to 35 °C for 1 hour. The supernatant was removed by centrifugation, and the microspheres were washed with sodium borate solution for 3-5 times, to obtain the microspheres with the structural formula as shown in formula 7, which was directly used in the next step reaction.

[0038]

[0039] 3. Coupling of oligonucleotides

[0040] Into a 100 mL reaction bottle, 1 gram of microspheres from Step 2 was added and suspended in 7.5 mL of a sodium borate solution (pH 8.5, concentration 0.05 mol / L). A 5' amino-modified oligonucleotide chain was dissolved in 2.5 mL of a sodium chloride solution (concentration 2 mol / L), and the reaction was carried out at room temperature for 5 hours. After washing 3-5 times with ultrapure water, the product having the structure shown in Formula 8 was obtained and stored for later use.

[0041]

[0042] Example 2: Benzyl-protected microspheres

[0043] For example, to prepare a microsphere of a linked oligonucleotide chain having the structure shown in Formula 9, the following specific preparation method was used:

[0044]

[0045] 1. Preparation of activated microspheres

[0046] Into a 50 mL reaction bottle, 1 gram of amino microspheres having a particle size of 3 μm was added, followed by 20 mL of anhydrous acetonitrile, 0.3 mL of N,N-diisopropyl ethylamine, and 0.15 grams of cyanuric chloride. The reaction was carried out at room temperature for 1 hour. The product was washed 3-5 times with dry acetonitrile, 3-5 times with a sodium borate solution, and 3-5 times with dry acetonitrile to obtain activated microspheres having the structure shown in Formula 10, which were directly used in the next step.

[0047]

[0048] 2. Amino group protection

[0049] Into a 50 mL reaction bottle, 1 gram of activated microspheres from Step 1 was added, followed by 20 mL of anhydrous DMF, 0.15 mL of N,N-diisopropyl ethylamine, and 0.1 mL of benzyl bromide, which was slowly added dropwise under ice bath cooling. The reaction was carried out at this temperature for 30 minutes, and then at 35°C for 2 hours. The supernatant was removed by centrifugation, and the product was washed 3-5 times with a sodium borate solution to obtain microspheres having the structure shown in Formula 11, which were directly used in the next step.

[0050]

[0051] 3. Coupling of an oligonucleotide

[0052] Into a 100 mL reaction bottle, 1 gram of microspheres from Step 2 was added and suspended in 7.5 mL of a sodium borate solution (pH 8.5, concentration 0.05 mol / L). A 5' amino-modified oligonucleotide chain was dissolved in 2.5 mL of a sodium chloride solution (concentration 2 mol / L), and the reaction was carried out at room temperature for 5 hours. After washing 3-5 times with ultrapure water, the product having the structure shown in Formula 8 was obtained and stored for later use.

[0053]

[0054] Comparative Example 1

[0055] The specific preparation method of the modified oligonucleotide chain microspheres with the structure shown in formula 13 without blocking the excess amino group is as follows:

[0056]

[0057] 1. Preparation of activated microspheres

[0058] Into a 50 mL reaction bottle, 1 gram of amino microspheres with a particle size of 3 μm, 20 mL of anhydrous acetonitrile, 0.3 mL of N, N-diisopropyl ethylamine and 0.15 g of cyanuric chloride were sequentially added. The reaction was carried out at room temperature for 1 hour, sequentially washed with dry acetonitrile for 3-5 times, washed with a sodium borate solution for 3-5 times, and then washed with dry acetonitrile for 3-5 times, to obtain the activated microspheres with the structure shown in formula 14, which was directly used in the next step reaction.

[0059]

[0060] 2. Coupling of oligonucleotides

[0061] Into a 100 mL reaction bottle, 1 gram of the microspheres in step 1 was added and suspended in 7.5 mL of a sodium borate solution (pH 8.5, concentration 0.05 mol / L), 5' amino modified oligonucleotide chain was dissolved in 2.5 mL of a sodium chloride solution (concentration 2 mol / L), and the reaction was carried out at room temperature for 5 hours. After washing with ultrapure water for 3-5 times, the product with the structure shown in formula 15 was obtained and stored for use.

[0062]

[0063] Effect comparison

[0064] The specific method for comparing the non-specificity of the oligonucleotide modified microspheres without blocking the amino group and the oligonucleotide modified microspheres with blocked amino group is as follows:

[0065] 2 mg (modified oligonucleotide chain structure is the same) of the modified microspheres in comparative example 1 and example 1 were taken and laid in the same chip, a mixed solution of target 1 and target 2 was added, and incubated at 20 ℃ lower than the Tm value for 30 minutes. After removing the mixed solution, scanning was carried out by using the FAM channel and the Cy3 channel of a scanner.

[0066] Comparison Figure 1 and Figure 3 It can be known that the hybridization signal of the microspheres treated by using the amino blocking reagent in the modification process of the application does not decrease. Figure 2 and Figure 4 It can be known that the non-specific signal of the microspheres of the application is obviously decreased.

[0067] The above description of the embodiments is only for the purpose of illustrating the present application, facilitating the understanding and use of the present application by those skilled in the art, and is not intended to limit the present application. The present application is not limited to the above-described embodiments, and improvements and modifications made by those skilled in the art based on the present application without departing from the scope of the present application are within the scope of the present application.

Claims

1. A method for blocking amino groups of chip microspheres, characterized in that, R group is selected from any one of acetyl, trifluoroacetyl, 4-carboxypropionyl, 5-carboxybutyryl or benzyl, The amino blocking method is as follows: the activated amino microspheres are added into a reaction solvent, a blocking reagent is added at room temperature and reacted for 30 minutes, then reacted at 25-50°C for 0.5-2 hours, the supernatant is removed by centrifugation, and the microspheres are washed with sodium borate solution for 3-5 times to obtain the activated microspheres after blocking the excess amino groups, which are directly used in the next step, The blocking reagent is selected from any one of acetic anhydride, fluorine-substituted acetic anhydride, succinic anhydride, glutaric anhydride or benzyl bromide.

2. The amino blocking method of the chip microspheres according to claim 1, wherein: The microsphere activation method is as follows: the amino-modified microspheres are suspended in dry solvent, N, N-diisopropyl ethylamine and cyanuric chloride are sequentially added, reacted at room temperature, sequentially washed with dry acetonitrile, sodium borate solution for multiple times, and then washed with the solvent to be used in the next step to obtain the activated microspheres; The coupling method of the oligonucleotide chain is as follows: the obtained activated microspheres after blocking the excess amino groups are suspended in sodium borate solution, a sodium chloride solution of 5' amino-modified oligonucleotide chain is added at room temperature, reacted at room temperature for 3-8 hours, washed with ultrapure water for 3-5 times to obtain the chip microspheres coupled with the oligonucleotide chain, which are stored for use.

3. The amino blocking method of the chip microspheres according to claim 2, wherein: In the microsphere activation method, the dry solvent is dry acetonitrile; The particle size of the microspheres is 0.5-5 μm, the microsphere concentration is 50-150 mg / mL, the amount of N, N-diisopropyl ethylamine is 150-250 μL per gram of microspheres, and the amount of cyanuric chloride is 50-150 mg per gram of microspheres; wherein After reacting at room temperature for 0.5-3 hours, the microspheres are sequentially washed with dry acetonitrile for 3-5 times, sodium borate solution for 3-5 times, and the solvent to be used in the next step for 3-5 times.

4. The amino blocking method of the chip microspheres according to claim 1, wherein: In the amino blocking method, the reaction solvent is any one or a combination of acetonitrile, DMF, N-methyl pyrrolidone, or a mixed solution with sodium borate solution.

5. The amino blocking method of the chip microspheres according to claim 2, wherein: wherein In the coupling method of the oligonucleotide chain, the microsphere concentration is 25-100 mg / mL, the oligonucleotide chain concentration is 80-120 nanomole / mL, and the sodium chloride concentration is 0.5-1 mol / L.

6. The chip microspheres prepared by the amino blocking method of the chip microspheres according to claim 2. wherein A microsphere is arranged thereon, and the microsphere is the chip microsphere according to claim 6. ​ 7. A SNP chip, characterized by, ​

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