CN103881999B - Low-residual inorganic-organic hybridization integrated substrate immobilized enzyme reactor and preparation thereof - Google Patents

Abstract

The present invention relates to a kind of low-residual inorganic-organic hybrid silica gel integrated substrate immobilized enzyme reactor, with tetramethoxy-silicane, vinyltrimethoxy silane, methacrylic acid is as monomer, with Polyethylene Glycol as perforating agent, in-situ polymerization under ethanol ammonia mixed system, obtains a kind of hydridization rigid matrix with loose structure.By introducing hydrophilic active reactive group on the substrate surface, make protein or protease molecule to be firmly bonded on integrated substrate, thus prepare highly active low-residual immobilized enzyme reactor.The advantages such as it is high that the method has stability, simple, quick.Compared with traditional protein immobilization method, on the integrated substrate prepared, not only there is highly active reactive group, the molecule can with protein etc. rapidly with primary amine groups reacts, simultaneously because the introducing of stromal surface hydrophilic group, significantly improve the response rate of protein or enzymatic hydrolysate.

Description

Low-residual inorganic-organic hybridization integrated substrate immobilized enzyme reactor and preparation thereof

Technical field

The present invention relates to a kind of low-residual inorganic-organic hybridization integrated substrate immobilized enzyme reactor, available Low-residual, rapid enzymolysis in protein.

Background technology

Proteomic analytical methods based on checking order by peptide fragment dactylogram and second order ms peptide fragment In, protein digestion is very important sample pretreatment step.But, conventional free solution enzyme Solution method has enzymolysis time length, enzyme can not reuse, easily from shortcomings such as degradeds.In recent years, In order to overcome these problems, protease is fixed in different substrate by people, is prepared as immobilized enzyme Reactor (IMER, immobilized enzymatic reactor), thus improve the enzymolysis effect of protein Rate, it is to avoid the manual operations of protein example, decreases the contaminated probability of protein example.Mesh Before, multiple material is all applied to enzyme immobilizatio, such as nano material (Qiao, L., et.al Chem.Eur.J. 2008,14,151-157), membrane material (Cooper, J.W., et.al Anal.Chem.2003,75, 1067-1074.) and integral material (Ma, J.F., et.al.Anal.Chem.2008,80,2949-2956.) Deng.

In above-mentioned material, the IMER of hybridisation silica gel integral post is because its specific surface area is big, mass transfer velocity Hurry up, and the surface easily plurality of advantages such as modified, it is preferable immobilization proteinase carrier.But, although For comparing monolithic silica column, the silicone hydroxyl on hybridisation silica gel integral post surface obtains a certain degree of suppression, But the non-specific adsorption of protein still cannot be avoided by completely.Therefore, for tradition hybrid silicon The problem that glue integrated substrate enzyme reactor exists, it is whole that we have developed a kind of low-residual inorganic-organic hybridization Body substrate immobilized enzyme reactor.This enzyme reactor is possible not only to realize high-throughout protein digestion, But also the protein response rate in processing procedure can be improved, have in proteomics research Well application prospect.

Summary of the invention

In order to solve the problems referred to above, it is an object of the invention to provide a kind of low-residual inorganic-organic hybridization Silica gel integrated substrate immobilized enzyme reactor.This enzyme reactor is possible not only to realize high-throughout protein Enzymolysis, but also the protein response rate in processing procedure can be improved.In order to realize this purpose, The technical scheme is that

1) tetramethoxy-silicane, vinyltrimethoxy silane, Polyethylene Glycol mix in an acidic solution, 0 DEG C of stirring prepares reaction solution A;

Wherein the addition of tetramethoxy-silicane accounts for the mass percent scope of reaction solution A and is 10%-30%, the addition of vinyltrimethoxy silane accounts for the mass percent scope of reaction solution A For 3%-10%, the addition of Polyethylene Glycol accounts for the mass percent scope of reaction solution A and is 5%-20%, it is 40%-80% that the addition of acid solution accounts for the mass percent scope of reaction solution A；

2) in reaction solution A, methacrylic acid is added, in dehydrated alcohol-ammonia mixed system mixing, Add initiator, degasification, carry out adding heat polymerization no less than 12 hours at 35-60 DEG C；Had There is the hydridization rigid matrix of loose structure

Wherein the addition of methacrylic acid is the 5%-20% of reaction solution A mass number；Added Dehydrated alcohol is the 40%-80% of reaction solution A mass number, and the ammonia added is reaction solution A matter 1-5 times of amount number,

The initiator added is Ammonium persulfate. (APS) or azo isobutyronitrile (AIBN), and its addition is Tetramethoxy-silicane, vinyltrimethoxy silane and three kinds of monomers of methacrylic acid add gross mass 0.5%-2%；

3) hydridization rigid matrix surface introduces hydrophilic active reactive group, specifically comprises the following steps that

Use 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides (EDC) and N-hydroxysuccinimidyl acyl sub- Amine (NHS) activated substrate surface carboxyl groups in buffer, adds hydrophilic polymine (PEI), the carboxyl of activation is little no less than 4 with hydrophilic polymine (PEI) room temperature reaction again Time, obtain hydrophilic immobilized substrate；

Wherein the quality of EDC/NHS is than scope 1/1-1/10；EDC is concentration in buffer 1-20mg/mL

The final concentration scope using PEI is 1-15mg/mL；

4) hybrid matrix surface fixing protein enzyme, specifically comprises the following steps that

Use EDC and NHS carboxyl of activated protein enzyme in buffer, obtain mixed solution, will Mixed solution is passed through in hydrophilic immobilized substrate, mixed solution and the primary amine groups on hydrophilic matrix material Room temperature reaction is no less than 6 hours, makes protease molecule to be firmly bonded on integrated substrate；

In mixed solution, the quality of EDC/NHS is than scope 1/1-1/10；EDC is concentration in mixed solution 1-20mg/mL, the protease concentration scope being suitable in mixed solution is 1-10mg/mL.

Present invention have the advantage that

1, porous hybrid silica gel integrated substrate preparation condition is gentle, is easily controlled, prepares reproducible；

2, using PEI is modifying agent, not only increases the hydrophilic of stromal surface, but also can cover miscellaneous The silicone hydroxyl that SiClx glue integral material unreacted is complete；

3, use EDC and NHS activating surface group, the modification time can be shortened, improve and make flux；

4, the low-residual hybridisation silica gel integrated substrate enzyme reactor of preparation, is possible not only to realize high-throughout albumen Matter enzymolysis, but also the protein response rate in processing procedure can be improved.

Accompanying drawing explanation

Fig. 1, the hydrophilic evaluation of low-residual hybrid matrix material；The standard of (a) bovine serum albumin (BSA) Curve；The standard curve of (b) polypeptide (DRVYVHPHL)；C () is with 0.1mg/mL BSA and 0.1mg/mL Polypeptide is sample, is passed through in hybrid matrix material with 1 μ L/min, collects the chromatographic fractionation figure of component, Wherein 1 is BSA, and 2 is polypeptide.

Fig. 2, low-residual immobilized enzyme reactor continuous enzymolysis bovine serum albumin (a) and Myoglobin (b) Mass spectral analysis figure.

Detailed description of the invention

Embodiment 1

1, the preparation of hybridisation silica gel integrated substrate material: weigh PEG-10000 540mg, use 5mL0.01M Acetic acid is abundant, then adds 1.8mL tetramethoxy-silicane, 0.6mL vinyl trimethoxy Silane, 0 DEG C of stirring 1h, makes reaction solution A.Weigh Ammonium persulfate. 2mg, with 200 μ L reactions Solution A is dissolved, and sequentially adds 200 μ L dehydrated alcohol, 10 μ L methacrylic acids, oscillating ultrasonic 5min.Add 50 μ L water, 50 μ L0.5M ammonia after cooling, pour into after vibration in capillary tube, Put into 40 DEG C of water-bath reaction 24h.

2, stromal surface is modified: after taking out integral post, first with water by perforating agents such as PEG-10000 with other not The little molecule of reaction is rinsed well.Weigh each 10mg and 20mg of EDC, NHS, use 1mL50mM The phosphate-buffered salt of pH5.0 dissolves, and then passes to integral post reaction 4h, then uses phosphate-buffered salt to incite somebody to action After complete EDC and NHS of unreacted rinses well, it is passed through 10mg/mL PEI, reacts 4h, then use PEI complete for unreacted is rinsed well by phosphate-buffered salt.

3, enzyme immobilizatio: weigh each 10mg and 20mg of EDC, NHS, trypsin Trypsin) 3mg, benzene first narrows 32mg, and after dissolving with the phosphate-buffered salt of 50mM pH5.0, shaken at room temperature reacts 1h, then passes to react in the hybridisation silica gel integral post that PEI modifies 6h, i.e. prepares that can to obtain low-residual miscellaneous SiClx glue immobilized enzyme reactor.

Embodiment 2

The hybridisation silica gel integral material substrate of the modification PEI investigation to the proteins and peptides response rate: preparation is many Peptide (DRVYHPHL) and each 1mg/mL of BSA, be respectively adopted 50mM ammonium hydrogen carbonate (pH8.0) Dilution, and mix with 1:1 ratio, mixed solution concentration is respectively 50 μ g/mL, 100 μ g/mL, 200 μ g/mL, 300 μ g/mL, 500 μ g/mL, and draw BSA (2a) and the standard of polypeptide (2b) Curve, separation condition is as follows: chromatographic column: Proteonavi post (4.6mm i.d × 150mm, 5 μ,), Flowing phase: A:0.1%TFA+H₂O;B:0.1%TFA+ACN;Gradient: 0-5min, 5%B, 5-35min, 5%B-80%B;Flow: 0.5mL/min, detection: 214nm;Sampling volume: 20 μ L.In order to Investigate the substrate absorption to sample, intercept the integral post of one section of 5cm length, be passed through with 1 μ L/min 100 μ g/mL biased samples, collect 100min, then sample introduction 20 μ L, chromatogram as illustrated in figure 1 c, After calculating peak area, shown that by regression equation calculation the response rate of BSA and polypeptide is respectively 106% ± 0.1% and 105% ± 0.1%.

Embodiment 3

The evaluation of low-residual hybridisation silica gel integrated substrate enzyme reactor: successively by 0.1mg/mL BSA and 0.05mg/mL Myoglobin (Myo) is passed through immobilized enzyme reactor continuously with 1 μ L/min In (0.25mm i.d × 50mm), collect component, and detected by MALDI-TOF MS, through number According to library searching, the sequential covering rate of BSA is 75%(such as Fig. 2 a), the sequential covering rate of Myoglobin is 98%(such as Fig. 2 b).

Claims (3)

1. a low-residual inorganic-organic hybridization integrated substrate immobilized enzyme reactor, it is characterised in that: Using tetramethoxy-silicane, vinyltrimethoxy silane, methacrylic acid as monomer, with poly-second two Alcohol is perforating agent, carries out in-situ polymerization in ethanol-ammonia mixed system, obtains one and has porous knot The hydridization rigid matrix of structure；By introducing hydrophilic active reactive group on the substrate surface, make protein Or protease molecule can be firmly bonded on integrated substrate, thus prepare highly active low-residual Immobilized enzyme reactor.

2. low-residual inorganic-organic hybridization integrated substrate immobilized enzyme reactor described in a claim 1 Preparation method, it is characterised in that:

1) tetramethoxy-silicane, vinyltrimethoxy silane, Polyethylene Glycol mix in an acidic solution, 0 DEG C of stirring prepares reaction solution A；

Wherein the addition of tetramethoxy-silicane accounts for the mass percent scope of reaction solution A and is 10%-30%, the addition of vinyltrimethoxy silane accounts for the mass percent scope of reaction solution A For 3%-10%, the addition of Polyethylene Glycol accounts for the mass percent scope of reaction solution A and is 5%-20%, it is 40%-80% that the addition of acid solution accounts for the mass percent scope of reaction solution A；

2) in reaction solution A, methacrylic acid is added, in dehydrated alcohol-ammonia mixed system mixing, Add initiator, degasification, carry out adding heat polymerization no less than 12 hours at 35-60 DEG C；Had There is the hydridization rigid matrix of loose structure；

Wherein the addition of methacrylic acid is the 5%-20% of reaction solution A mass number；Added Dehydrated alcohol is the 40%-80% of reaction solution A mass number, and the ammonia added is reaction solution A matter 1-5 times of amount number,

Described initiator is Ammonium persulfate. (APS) or azo isobutyronitrile (AIBN), and its addition is Tetramethoxy-silicane, vinyltrimethoxy silane and three kinds of monomers of methacrylic acid add gross mass 0.5%-2%；

3) hydridization rigid matrix surface introduces hydrophilic active reactive group, specifically comprises the following steps that

A. 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides (EDC) and N-hydroxysuccinimidyl are used Acid imide (NHS) activated substrate surface carboxyl groups in buffer, adds hydrophilic polymine (PEI), the carboxyl of activation is little no less than 4 with hydrophilic polymine (PEI) room temperature reaction again Time, obtain hydrophilic immobilized substrate；

Wherein the quality of EDC/NHS is than scope 1/1-1/10；EDC is concentration in buffer 1-20mg/mL

The final concentration scope using PEI is 1-15mg/mL；

B. use EDC and NHS carboxyl of activated protein enzyme in buffer, obtain mixed solution, Mixed solution is passed through in hydrophilic immobilized substrate, mixed solution and the primary amine on hydrophilic matrix material Base room temperature reaction is no less than 6 hours, makes protease molecule to be firmly bonded on integrated substrate；

In mixed solution, the quality of EDC/NHS is than scope 1/1-1/10；EDC is concentration in mixed solution 1-20mg/mL, the protease concentration scope being suitable in mixed solution is 1-10mg/mL.

3. according to the preparation method described in claim 2, it is characterised in that:

Acid solution is acetic acid, formic acid, and concentration is 0.01-0.1M, and buffer is phosphate, borate, Concentration is 10mM-100mM.