CN114768774B

CN114768774B - Acetylated molecularly imprinted polymer for constructing microfluidic chip integrated platform

Info

Publication number: CN114768774B
Application number: CN202210364653.2A
Authority: CN
Inventors: 刘照胜; 张雪; 黄艳萍
Original assignee: Tianjin Medical University
Current assignee: Tianjin Medical University
Priority date: 2022-04-08
Filing date: 2022-04-08
Publication date: 2023-09-15
Anticipated expiration: 2042-04-08
Also published as: CN114768774A

Abstract

The invention relates to a mesoporous molecular sieve doped acetylated molecularly imprinted polymer and a construction of a microfluidic chip platform. The imprinted polymer is used as an adsorbent for solid phase extraction and is used for analyzing the acetylated modified peptide. According to the method, the SBA-15 mesoporous molecular sieve is doped into the acetylated molecularly imprinted polymer, so that the imprinting effect of the template peptide can be obviously improved. Meanwhile, polyethylene glycol dimethacrylate is added during preparation, and the polyethylene glycol dimethacrylate and ethylene glycol dimethacrylate are crosslinked together, so that the hydrophilicity of the polymer can be increased, the nonspecific adsorption can be effectively reduced, and the obvious imprinting effect (imprinting factor 3.2) can be obtained. And then integrating a plurality of functional areas for protein fractionation, denaturation, enzymolysis and acetylated peptide enrichment to construct a microfluidic chip platform, which can be used for online enrichment of the acetylated peptide in the biological sample.

Description

Acetylated molecularly imprinted polymer for constructing microfluidic chip integrated platform

Technical Field

The invention relates to an acetylated molecularly imprinted polymer for constructing a microfluidic chip integrated platform, in particular to a mesoporous molecular sieve doped acetylated molecularly imprinted polymer and construction of a microfluidic chip platform.

Background

Lysine acetylation (Lysine acetylation, kac) is a widely dynamic and reversible Post-protein modification (Post-Translational Modification, PTM), and is widely involved in multiple cellular processes such as gene transcription, protein degradation, cellular metabolism, stress reaction, and the like, playing an important regulatory role in metabolic-related diseases and neurodegenerative diseases. However, since the content of the acetylated modified proteins is low, signals of the acetylated modified proteins are interfered by a large amount of non-acetylated proteins in the identification process, which brings great challenges to the identification and functional research of the acetylated modified proteins, and in order to better research the acetylated modified proteins, development of efficient and specific lysine acetylation enrichment strategies is needed. Currently, the commonly used Kac peptide enrichment methods include agarose enrichment, isoelectric focusing, strong cation exchange, immunoaffinity methods and the like. Among them, immunoaffinity methods are the most common and effective enrichment methods, but are costly and environmentally sensitive. Meanwhile, due to the strong substrate specificity of the biological antibody, various affinity reagents need to be tested to achieve the ideal enrichment performance. Therefore, developing a material which has strong specificity, good stability, low cost and easy preparation has good practical significance for enriching Kac peptide.

The molecular imprinting technology (Molecular Imprinting Technique, MIT) is a technology for simulating the interaction principle of antigen/antibody to synthesize a preselected stationary phase, and is widely applied to the fields of material synthesis, separation and purification, drug delivery systems and the like. By virtue of the advantages of selectivity and the like of the tailoring, the method has great application potential in the field of targeted proteomics. The molecularly imprinted polymer (Molecularly Imprinted Polymer, MIP) prepared on the basis of MIT has the characteristics of molecular shape and spatial structure selectivity. MIP has better thermal, physical and chemical stability than antibodies that also rely on molecular recognition. In recent years, short peptide fragments common in different peptide fragments are used as a single template for immobilization, and the MIP can realize enrichment of various modified peptides at specific positions. In addition, dong Xiang successfully achieved specific enrichment of acetylated peptides in histone and HeLa cell lysates using MIPs prepared by epitope blotting towards the subject group. The application of the technology not only ensures good specificity and selectivity of the target peptide, but also has the advantages of low cost and high economic value, and has great application potential for realizing the selective enrichment of the target peptide in complex protein biological samples. Although MIPs have many attractive properties, MIPs have limited ability to capture templates from complex samples and have low selectivity and/or affinity for MIPs. In order to improve the imprinting performance of MIP, the introduction of nanomaterials is becoming more important.

The SBA-15 mesoporous molecular sieve material has uniform hexagonal channels, high surface area, thicker pore walls (3.1-6.4 nm) and large pore diameterAnd narrow size distribution. The characteristics lead the nanometer-sized porous ceramic to have great potential application value in the aspects of adsorption, catalysis, biological separation, nanometer assembly and the like. MIP research based on mesoporous silica materials is prepared by combining MIT with a spatial structure and a binding site which are completely matched with template molecules, and is widely applied to the fields of adsorption separation, microreactors, drug delivery, shape selective catalysis and the like at present. Meanwhile, mesoporous silica materials have also been used for the treatment of polypeptides in proteolytic liquidsEnriching. Highly ordered pore structures, high molecular weight species can be removed by size exclusion to ensure high selectivity of the target, large surface area enabling high capacity enrichment of smaller peptides that can pass through the pores. If interfering peptides enter the pores, the mixture can be further separated by hydrophilic/hydrophobic interactions of different functional groups with the surface groups. However, there are few reports on the application of MIP based on SBA-15 mesoporous molecular sieve in polypeptide separation and enrichment. The molecular imprinting polymer is prepared by utilizing the high efficiency and the aperture effect of the SBA-15 mesoporous molecular sieve, so that the imprinting site embedding phenomenon in the traditional MIP is hopeful to be solved, and the specific recognition of the polypeptide in the field of proteomics is realized. Chinese patent CN113304708A discloses that a molecular sieve-based surface-oriented imprinted polymer (SBA-15@mip) is prepared by using an SBA-15 mesoporous molecular sieve as a carrier, 2, 3-difluoro-4-formylphenylboronic acid as a monomer, and N-acetylneuraminic acid as a fragment template. Further, an SBA-15@MIP glycoprotein microreactor was prepared for selective enrichment of N-glycopeptides of protein samples.

Microfluidic technology is a technology for integrating functions of an analysis laboratory of micro fluid on a chip, has the characteristics of high analysis speed, low cost, high throughput, multifunctional integration and the like, is widely applied to the fields of biochemistry, medicine, environment detection and the like, and has become one of the leading edge analysis technologies which are paid attention to in recent years. The technology can realize integration of a series of processes such as sample processing, separation, analysis and the like, is an emerging technology capable of rapidly and accurately completing the analysis process, and relates to the field of multiple disciplines such as surface chemistry, electrochemistry, micromachining and the like. Along with the continuous progress of micro-processing technology, the integration of multifunctional components in the micro-fluidic chip better reflects the characteristics of high efficiency, simple operation, bionics and the like, so that the micro-fluidic chip has outstanding advantages in aspects of cell culture research, sample analysis, and simulation of tissue structures, microenvironments and mechanical forces of organs in a body to realize organoid functions.

The microfluidic chip integrates basic operation units such as sample preparation, biological and chemical reactions, separation and detection and the like in the fields such as biology, chemistry and the like on a small operation platform, and is widely applied to protein separation analysis. The multidimensional separation system built on the basis of the microfluidic chip is already applied to the field of solid phase extraction, realizes the on-line automatic separation and enrichment of samples, and solves the problems that the multidimensional system has complicated steps, incompatible running conditions among the dimensions and difficulty in realizing on-line sample processing. At present, a sample concentration and enrichment technology based on a microfluidic chip analysis platform is widely applied, and is an effective method for improving the pre-enrichment analysis efficiency and sensitivity of low-abundance proteins. CN106362810a reports a molecular engram polymer film modification-two-electrode electrochemistry micro-fluidic chip and a preparation method thereof, wherein the molecular engram polymer is modified on a metal electrode. By introducing a molecular imprinting technology into the preparation of a microfluidic chip, a novel microfluidic electrochemical sensor platform is constructed.

The method is used for doping the SBA-15 mesoporous molecular sieve for the first time and adopts a hydrophilic glycol dimethacrylate cross-linking agent to develop a novel acetylated molecularly imprinted material. And the protein is integrated with a protein grading column, a chip denaturation region and an enzyme reactor on line, so that a novel protein sample pretreatment platform is successfully constructed, and the efficient enrichment of protein on-line grading, denaturation, enzymolysis and acetylation modified peptides is realized.

Disclosure of Invention

The invention aims to provide an SBA-15 mesoporous molecular sieve doped acetylated molecularly imprinted polymer and a construction of a microfluidic chip platform. The SBA-15 mesoporous molecular sieve is doped into the acetylated molecularly imprinted polymer, so that the imprinting effect of the template peptide can be obviously improved. Meanwhile, polyethylene glycol dimethacrylate (PEGDA) is added during preparation, and the polyethylene glycol dimethacrylate (EDMA) is crosslinked together, so that the hydrophilicity of the polymer can be increased, the adsorption quantity of NIP is effectively reduced, and a more remarkable imprinting effect is achieved.

The SBA-15 mesoporous molecular sieve doped acetylated molecularly imprinted polymer comprises the following raw materials in mass ratio:

the sum of the mass compositions of the raw materials is 100 percent.

The invention provides a preparation method of an SBA-15 mesoporous molecular sieve doped acetylated molecularly imprinted polymer, which specifically comprises the following steps:

1) According to the measurement, the functional monomer zinc acrylate, a template KacQLAT, a doping agent SBA-15 mesoporous molecular sieve, an initiator azo-diisobutyronitrile, a crosslinking agent PEGDA and EDMA are dissolved in a mixed pore-foaming agent solution of methanol and N, N-dimethylformamide; dissolving with ultrasonic (power 150W) for 10-15min, dissolving, clarifying, introducing nitrogen to remove oxygen in the liquid, sealing, and reacting in water bath at 60deg.C for 12 hr.

2) The obtained acetylated molecularly imprinted polymer is placed in a mortar for grinding, and is washed by acetonitrile to remove unreacted residual impurities.

The synthesis of the acetylated molecularly imprinted polymer free of SBA-15 mesoporous molecular sieve is the same as the rest steps except that the SBA-15 mesoporous molecular sieve is removed.

Synthesis of molecularly imprinted polymers without PEGDA the procedure was as above except that the amount of PEGDA was replaced with the crosslinker EDMA.

Synthesis of non-imprinted Polymer (NIP) the procedure was followed except that the template molecule KacQLAT was not added.

The preparation method of the electrochemical sensor of the SBA-15 mesoporous molecular sieve doped acetylated molecularly imprinted polymer provided by the invention comprises the following steps:

(1) Polishing a Glassy Carbon Electrode (GCE) on a polishing cloth by using GCE alumina water slurry, and then cleaning by using sulfuric acid solution, deionized water and ethanol respectively;

(2) Adding an SBA-15 mesoporous molecular sieve doped acetylated molecularly imprinted polymer into a chitosan solution, and adding a multi-walled carbon nanotube DMF solution to increase conductivity to obtain an SBA-15@MIP mixture solution; and (2) dripping the SBA-15@MIP mixture solution on the surface of the GCE in the step (1), drying a glassy carbon electrode at 50 ℃ for 15min, immersing the glassy carbon electrode in acetonitrile/acetic acid/water (5/5/90, v/v/v) to remove a template KacQLAT, and drying at room temperature to obtain the SBA-15@MIP/GCE of the SBA-15 mesoporous molecular sieve doped acetylated molecularly imprinted polymer electrochemical sensor.

(3) Electrochemical sensor detection: selecting K containing KCl ₃ [Fe(CN) ₆ ]As an electrolyte solution; template SBA-15@MIP/GCE and SBA-15@NIP/GCE are removed and placed in a 50mM Tris-HCl (pH 8.0) solution containing a template for incubation; in order to remove nonspecific adsorption on the surface of the electrode film, the adsorbed electric sensor is put into Tris-HCl buffer salt; finally, placing the electric sensor after adsorption in electrolyte, and measuring the current response of the electric sensor through DPV after balancing; all tests were measured in triplicate; the adsorption performance of the electric sensor is characterized by the change of current, and the calculation of the current change quantity is according to the formula (1)

ΔI＝I _o -I _e (1)

Wherein I is _o And I _e (μA) represents the initial current and the post-sink current, respectively.

The blotting factor (IF) was used to evaluate the blotting performance of MIPs:

wherein DeltaI _MIP And DeltaI _NIP (μA) represents the current change values of SBA-15/MIP/GCE and SBA-15/NIP/GCE in the electrolyte after adsorption of KacQLAT, respectively.

The invention provides a multifunctional micro-fluidic chip integrated platform (device) for enriching acetylated peptides, which mainly comprises a protein grading column unit, a denaturation chip unit, an enzyme reactor unit, an SBA-15 mesoporous molecular sieve doped acetylated molecularly imprinted monolithic column unit, a micro digital injection pump and an FEP connecting pipeline; the front of the denatured chip unit is connected with a protein grading unit, the rear of the denatured chip unit is sequentially connected with an enzyme reactor unit and an SBA-15 mesoporous molecular sieve doped acetylated molecularly imprinted monolithic column unit, the four units are connected in series by an FEP tube, and then the chip is fixed by a clamp; the FEP tube at the channel inlet and the inlet D, E are connected with a micro digital injection pump for adjusting the flow rate, and the final outlet end of the liquid is connected with a small centrifuge tube; the SBA-15 mesoporous molecular sieve doped acetylated molecular imprinting monolithic column unit is finally connected with a liquid chromatography tandem mass spectrometry device for monitoring; the two ends of the protein classification column are provided with an inlet A and an outlet B which are connected; the denaturation chip is divided into a denaturation I region and a denaturation II region which are connected with each other, the front end of the denaturation I region is provided with an inlet C and an inlet D of a reducing agent, the front end of the denaturation II region is provided with an inlet E for an alkylating agent, and the rear end of the denaturation II region is provided with an outlet F, wherein each region comprises a plurality of groups of snakelike channels capable of randomly regulating the total length of the channel; the enzyme reactor unit is composed of an enzyme reactor with an outlet and an inlet; the acetylated molecularly imprinted monolithic column unit is formed by an SBA-15 mesoporous molecular sieve doped acetylated molecularly imprinted monolithic column with an outlet and an inlet. Each channel of the denaturation column is 2cm long, 250 μm wide and 100 μm deep.

The invention provides an application of a multifunctional microfluidic chip integrated platform in the aspect of enriching acetylated peptides, which comprises the following specific application method steps:

1) The protein was prepared to a concentration of 1.5mg/mL with ultrapure water, and 50. Mu.L was injected from inlet A and discharged from outlet B at a flow rate of 0.3. Mu.L/min using a digital syringe pump. Simultaneously, 20mM DTT and 30mM IAA in 50mM Tris-HCl buffer (pH 8.0) were injected from inlets D and E into the chip at a flow rate of 0.3mL/min, respectively. Reduction of the protein is accomplished in a first serpentine channel and alkylation of the protein is accomplished in a second serpentine channel. And then, the denatured protein solution sequentially enters IMER for enzymolysis, and the acetylated peptides in the enzymolysis solution are enriched by an SBA-15 mesoporous molecular sieve doped acetylated molecularly imprinted monolithic column. Then, 50. Mu.L of 50mM Tris-HCl buffer (pH 8.0) and 100. Mu.LACN/HAC/H were performed in an off-line manner at a flow rate of 1.0. Mu.L/min ₂ O (5/5/90, v/v/v) respectively eluting and eluting the SBA-15 mesoporous molecular sieve doped acetylated molecularly imprinted monolithic column. The final effluent and eluate were collected.

2) The column was rinsed with ultra pure water (0.3. Mu.L/min, 20. Mu.L) and the rinse was discarded. Then, the solution at inlet A is changed to H ₂ O (pH 12.0), the protein adsorbed on the column was eluted in an elution volume of 50. Mu.L. The protein eluate from outlet B is mixed with DTT and IAA at the inlets of D and E. Subsequent operationsStep "I" is treated identically. And collecting the final effluent and the eluent of the SBA-15 mesoporous molecular sieve doped acetylated molecularly imprinted monolithic column. Freeze-drying and desalting untreated serum proteins and all collected samplesZipTips C18 column (Merck Millipore Led, ireland) was identified by LC-MS/MS.

The invention provides a preparation method of an acetylated molecularly imprinted polymer containing an SBA-15 mesoporous molecular sieve, and the optimal doping amount of the SBA-15 mesoporous molecular sieve in the synthesized acetylated molecularly imprinted polymer is found through experimental design, and the doping agent type (compared with a titanium nanotube TNTs) is optimized, so that the molecularly imprinted polymer with good imprinting effect and high selectivity on a template is prepared. PEGDA is a hydrophilic crosslinking monomer that contains short oligomeric (ethylene glycol) side chains that have been incorporated into polymers to increase the hydrophilicity of the polymeric material, which can effectively reduce the non-specific adsorption of non-hydrophilic peptides. In addition, the addition of the molecular engram polymer can increase the hydrophilicity of the engram cavity and improve the selectivity of the molecular engram polymer to hydrophilic template molecules. Therefore, the addition of the hydrophilic cross-linking agent PEGDA can significantly improve the blotting effect of the molecularly imprinted polymer on KacQLAT (if=3.2). The integrated platform of the micro-fluidic chip which can be used for enriching the acetylated peptide is built by the integrated platform and other functional units (protein grading, denaturation and enzymolysis functional units). Therefore, the invention can be used for efficient separation and specific enrichment of acetylated peptides.

Drawings

FIG. 1 is a scanning electron microscope image of MIP (a), NIP (b) and SBA-15 mesoporous molecular sieves (c) of the invention.

FIG. 2 is a graph showing the comparison of the imprinting effect and the adsorption effect of the dopant type on the template for the acetylated molecularly imprinted polymer prepared by the invention.

FIG. 3 is a cyclic voltammogram of different electrodes of the present invention (after extraction of the template molecules of a.SBA-15@MIP/GCE; before extraction of the template molecules of b.SBA-15@MIP/GCE; c.SBA-15@NIP/GCE; d.GCE).

Fig. 4 is a schematic diagram of a multifunctional microfluidic chip integration platform constructed by the invention.

Detailed Description

The invention will be further illustrated in detail with reference to specific examples. The experimental methods for which specific conditions are not specified in the examples are generally as described in conventional conditions and handbooks, or as suggested by the manufacturer; the general equipment, materials, reagents, etc. used, unless otherwise indicated, are all commercially available.

Example 1

The preparation condition of the molecular imprinting polymer is optimized to improve the specificity of the molecular imprinting polymer to template molecules, so that the imprinting effect is more remarkable. In order to examine the effect of the functional monomer/crosslinker ratio, dopant type on the imprinting and adsorption effects of the templates. The specific operation steps are as follows:

the preparation method of the SBA-15 mesoporous molecular sieve doped acetylated molecularly imprinted polymer comprises the following steps:

0.50% of SBA-15 mesoporous molecular sieve (8-12 nm), 1.44% of template KacQLAT (95% of Shanghai blaze biotechnology Co., ltd., product No. 04010047537), 3.97% of functional monomer zinc acrylate, 15.81% of cross-linking agent PEGDA and 9.50% of EDMA, 0.46% of initiator azodiisobutyronitrile and the mixture solution of 36.05% of pore-forming agent methanol and 32.27% of N, N-dimethylformamide; dissolving with ultrasonic (power 150W) for 15min, dissolving, clarifying, introducing nitrogen, removing oxygen from the prepolymerization solution, sealing the prepolymerization solution, and reacting in a water bath at 60deg.C for 12 hr.

Acetylated lysine-glutamine-leucine-alanine-threonine, named reference tanata 2020,224:121810 Wei et al. Specifically, lysine-glutamine-leucine-alanine-threonine (KacQLAT) was acetylated. Reference is made to: "Wei Z H, zhang X, zhao X, jiao Y J, huang Y P, liu Z S.construction of a microfluidic platformintegrating online protein fractionation, registration, digenesis, and peptide construction Talanta," abstract "means" a-lysine-glycine-glycine (KGG) imprinted monolith ".

b. The Molecularly Imprinted Polymer (MIP) product obtained above was washed with acetonitrile for 3 times to remove unreacted residual impurities, dried at 50 ℃ for 2 hours, and placed in a mortar for grinding to obtain a molecularly imprinted polymer with a size of about 5-10 μm.

The result shows that compared with NIP, MIP has more pore structure, pore size is 100nm-250nm, and particle surface is rough; SBA-15 mesoporous molecular sieves exhibit an ordered mesoporous structure (see FIG. 1). Comparison of different dopant types shows that the acetylated molecularly imprinted polymer doped with SBA-15 mesoporous molecular sieve has the highest imprinting factor (see FIG. 2).

When the content of the SBA-15 mesoporous molecular sieve is 0.00%, the synthesis of the molecularly imprinted polymer without the SBA-15 mesoporous molecular sieve is shown. The effect of different dopants on the imprinting effect of the molecularly imprinted polymer was obtained in comparison with the molecularly imprinted polymer doped with the SBA-15 mesoporous molecular sieve of example 1, which provides support.

Example 2

The prepared electrochemical sensor based on the molecularly imprinted electrode technology is applied to detection of a template KacQLAT. The method comprises the following specific steps:

preparation of SBA-15@MIP/GCE electric sensor:

SBA-15@MIP/GCE electrical sensors were prepared using a drop coating method. First, the Glassy Carbon Electrode (GCE) was polished on a polishing cloth with 1.0, 0.3 and 0.05. Mu.M aqueous GCE alumina slurries, respectively, and then rinsed with sulfuric acid solution (1M), deionized water and ethanol, respectively, for 10min. Thereafter, 2mg of the imprinted polymer was added to 1mL of chitosan solution (0.5% wt, containing 20. Mu.L of acetic acid), and 50. Mu.L (1 mg/mL) of multi-walled carbon nanotube DMF solution was added to increase conductivity. To uniformly disperse the polymer and the multi-walled carbon nanotubes, the prepared mixture was sonicated and mixed for 5min. Finally, 10. Mu.L of SBA-15@MIP mixture was applied dropwise to the surface of the GCE and dried at 50℃for 15min. And (3) soaking the dried glassy carbon electrode in acetonitrile/acetic acid/water (5/5/90, v/v/v) to remove template peptide KacQLAT, and drying at room temperature to obtain an electrochemical sensor SBA-15@MIP/GCE of the SBA-15 mesoporous molecular sieve doped acetylated molecularly imprinted polymer.

b. Electrochemical sensor detection:

the detection is completed in a traditional three-electrode system, a platinum wire electrode is used as a counter electrode, a silver/silver chloride electrode is used as a reference electrode, the prepared electrochemical sensor based on the molecularly imprinted electrode technology is used as a working electrode, and 5 multiplied by 10 of 0.02mol/L KCl is selected ^-3 mol/LK ₃ [Fe(CN) ₆ ]As electrolyte solution. The glassy carbon electrode is firstly subjected to grinding and polishing treatment on alpha-alumina polishing powder with the particle size of 50nm, and then is sequentially subjected to ultrasonic washing by deionized water and absolute ethyl alcohol.

Electrochemical measurement procedure we first incubated the template-removed SBA-15@MIP/GCE with SBA-15@NIP/GCE in template solution (pH 8.0, 50mM Tris-HCl,20 mL). Thereafter, to remove non-specific adsorption on the electrode membrane surface, the drug-loaded electrical sensor was placed in Tris-HCl buffer salt (pH 8.0, 50 mM) for 5min. Finally, we placed the adsorbed electrical sensor in electrolyte and measured the current response of the electrical sensor by DPV after 15min of equilibration. All tests in this experiment were measured in triplicate. The adsorption performance of the electric sensor is characterized by the change of current, and the calculation of the current change quantity is according to the formula (1)

ΔI＝I _o -I _e (1)

The blotting factor (IF) was used to evaluate the blotting performance of MIPs, IF calculation was as per formula (2):

FIG. 3 shows the cyclic voltammetry curve of an electrochemical sensor based on molecularly imprinted electrode technology for detection of KacQLAT in 50mM Tris-HCl buffer pH 8.0.

Example 3

In order to reduce sample consumption and loss, shorten analysis time and cost, realize automation and integration targets, a multifunctional microfluidic chip integration platform is built. The method comprises the following specific steps:

a. sample online treatment:

the protein classification is connected in front of the denaturation chip, the enzyme reactor and the acetylation molecular imprinting monolithic column are connected at the back, the four units are connected in series on line by a thin connecting pipe, and the length requirement and the size requirement of the denaturation channel and the position distribution of each liquid storage tank are comprehensively considered. The online denaturation column is divided into a denaturation zone I and a denaturation zone II, each zone comprises a plurality of groups of snake-shaped channels, the length of each channel is 2cm, the total length of the channels can be adjusted at will, and all channels are 250 mu m wide and 100 mu m deep. The prepared chip is placed on a clamp, a Fluorinated Ethylene Propylene (FEP) heat-shrinkable tube is inserted into an inlet and an outlet (shown as an inlet A, C, D, E and an outlet B, F in fig. 4) at the tail end of each channel, the prepared chip is screwed and fixed by nuts, the FEP tube at the inlet of the channel is connected with a microinjection pump, and the outlet is connected with a small centrifuge tube. Regulating the flow speed of the microinjection pump, and examining the accurate control of the volume and the flow of the passage; the functional areas are connected by FEP tubing, and the microinjection pump at the main inlet is turned on.

b. Preparation of protein fractionation column:

1.20% of template titanium dioxide nanotube with mass fraction, 0.15% of initiator azodiisobutyronitrile, 16.02% of functional monomer glycidyl methacrylate and 7.21% of ethylene glycol dimethacrylate are dissolved in a mixed solution of pore-forming agent n-dodecanol 47.28% and cyclohexanol 28.14%; dissolving with ultrasonic wave (power 150W) for 15min, dissolving and clarifying, introducing nitrogen, removing oxygen in the prepolymerization solution, injecting the prepolymerization solution into a clean capillary (with inner diameter=250μm), sealing two ends with rubber, reacting for 3.5h in a constant temperature water bath at 60 ℃, flushing the synthesized monolithic column with acetonitrile to remove unreacted monomer and pore-forming agent, injecting 4.5mol/L ammonia water into the synthesized capillary, grafting amino on the surface of the polymer in water bath at 60 ℃ for 2h, and flushing with acetonitrile to remove unreacted monomer and pore-forming agent;

c. preparation of the enzyme reactor:

24.12% of TRIM (trimethoxypropyl trimethyl acrylate) serving as a functional monomer and 0.22% of azodiisobutyronitrile serving as an initiator are dissolved in a mixed solution of 20.34% of toluene serving as a pore-forming agent and 55.32% of isooctane; dissolving with ultrasonic wave (power 150W) for 15min, clarifying, introducing nitrogen gas, removing oxygen in the prepolymerization solution, injecting the prepolymerization solution into a clean capillary tube, sealing two ends with rubber, injecting into the capillary tube (inner diameter=250μm), placing in a water bath at 50deg.C for reaction for 24h, and washing unreacted monomer and pore-forming agent with acetonitrile after the reaction is finished.

10mg/mL of trypsin and 1mg/mL of tris (2-formylethyl) phosphine hydrochloride (TCEP) solution were prepared with 20mmol/LTris-HCl solution (pH 8.0), the trypsin solution was mixed with the TCEP solution at 10:1 (v/v), reacted at 25℃for 3 hours, and then centrifuged at 10000r/min at 4℃for 10 minutes, after standing to room temperature, the supernatant was taken, and ammonium persulfate and N, N-methylenebisacrylamide were added in amounts such that the concentrations were 1mg/mL and 2mg/mL, respectively. And (3) injecting the enzyme derivative liquid into the synthesized functional area monolithic column, sealing two ends of the channel by using a rubber plug, and reacting for 5 hours at 25 ℃. After the reaction, the mixture was rinsed with 20mmol/LTris-HCl solution (pH 8.0) and stored under refrigeration at 4 ℃.

Preparation of SBA-15 mesoporous molecular sieve doped acetylated molecularly imprinted monolithic column:

the preparation of the pre-polymerized liquid is the same as that of the acetylated molecularly imprinted polymer doped with the SBA-15 mesoporous molecular sieve, the pre-polymerized liquid is injected into a clean capillary (with the inner diameter of=250 mu m), two ends are sealed by rubber, the reaction is carried out for 12 hours in a constant-temperature water bath kettle at 60 ℃, the unreacted materials at the position are washed by acetonitrile, and then the template is washed by acetonitrile/acetic acid/water (5/5/90, v/v/v).

The synthesis of the non-imprinted monolithic column was performed in the same manner as above except that the template molecule KacQLAT was not added.

Fig. 4 shows the on-line denaturation technology operating route of the microfluidic chip platform integrating protein fractionation-denaturation-enzymolysis-acetylated peptide enrichment multifunctional.

Example 4

The built microfluidic chip platform is used for enriching the acetylated peptides of serum extracted proteins of patients with macular degeneration

Extraction of serum proteins: taking 5mL of whole blood of a patient suffering from macular degeneration, and placing the whole blood in an anticoagulation tube; then centrifuging, and regulating the rotational speed to 3500-4000 r/min for 5min; sucking the uppermost serum layer into a blank test tube by using a prepared suction tube after centrifuging; 100 mu L of serum is taken, 200 mu L of serum protein extraction kit (Shanghai Bei Bo Biotechnology Co., ltd., product No. BB-3137-1) is added, the mixture is refrigerated and precipitated for 0.5h, the mixture is centrifuged at 1000r/min for 10min, the supernatant is taken, and the supernatant is dialyzed and freeze-dried to obtain the serum protein extract.

1) Serum-extracted protein was prepared with ultrapure water to a concentration of 1.5mg/mL, and 50. Mu.L was injected from inlet A and discharged from outlet B at a flow rate of 0.3. Mu.L/min using a digital syringe pump. Simultaneously, 20mM DTT (dithiothreitol) and 30mM IAA (iodoacetamide) in 50mM Tris-HCl buffer (pH 8.0) were injected from inlets D and E at a flow rate of 0.3mL/min into the chip, respectively, at 50. Mu.L. Reduction of the protein is accomplished in a first serpentine channel and alkylation of the protein is accomplished in a second serpentine channel. And then, the denatured protein solution sequentially enters IMER for enzymolysis, and the acetylated peptides in the enzymolysis solution are enriched by an SBA-15 mesoporous molecular sieve doped acetylated molecularly imprinted monolithic column. Then, 50. Mu.L of 50mM Tris-HCl buffer (pH 8.0) and 100. Mu.L of ACN/HAC/H were performed in an off-line manner at a flow rate of 1.0. Mu.L/min ₂ O (5/5/90, v/v/v) respectively eluting and eluting the SBA-15 mesoporous molecular sieve doped acetylated molecularly imprinted monolithic column. The final effluent and eluate were collected.

2) The column was rinsed with ultra pure water (0.3. Mu.L/min, 20. Mu.L) and the rinse was discarded. Then, the solution at inlet A is changed to H ₂ O (pH 12.0), the protein adsorbed on the column was eluted in an elution volume of 50. Mu.L. The protein eluate from outlet B is mixed with DTT and IAA at the inlets of D and E. The subsequent operations are the same as the process of step 1). And collecting the final effluent and the eluent of the SBA-15 mesoporous molecular sieve doped acetylated molecularly imprinted monolithic column.

Subjecting untreated serum proteins, and all samples collected after the platform treatment, to lyophilization and desaltingZipTips C18 column (Merck Millipore Led, ireland) was identified by LC-MS/MS (liquid chromatography-tandem mass spectrometry).

After the microfluidic chip integrated platform is applied to pretreatment of human serum protein samples, the mass spectrum identification result shows that the identification capacity of Kac peptide and Kac protein is improved by 1.6 times (44 vs 71) and 2.0 times (22 vs 43) after the platform is treated. The processing process of the protein sample of the platform comprises protein fractionation, denaturation, enzymolysis and enrichment of acetylated peptides, and the processing process of all protein samples is completed, and the platform only needs 9.1h. However, if the off-line method is adopted, the sample treatment needs at least 22.4 hours (denaturation for 1.3 hours, enzymolysis for 12 hours and sample treatment for 9.1 hours), mainly because the denaturation and enzymolysis in the traditional method are not finished on line, and the consumed time is 1.3 hours and 12 hours respectively. As can be seen, the present platform shortens the processing time of protein samples by about 2.5-fold (from 22.4h to 9.1 h). In addition, the sample consumption of the solution enzymolysis is 10mg, and the sample consumption of the microfluidic chip integrated platform is 0.075mg, which is reduced by 133 times compared with the traditional method. Therefore, the micro-fluidic chip platform can obviously improve the identification effect of the acetylation modification and the pretreatment efficiency of the protein sample.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims

1. The SBA-15 mesoporous molecular sieve doped acetylated molecularly imprinted polymer is characterized by comprising the following raw materials in parts by mass:

the preparation method comprises the following steps: adding SBA-15 mesoporous molecular sieve, template KacQLAT, functional monomer zinc acrylate, cross-linking agent PEGDA, EDMA and initiator azodiisobutyronitrile into mixed solution of methanol and N, N-dimethylformamide, ultrasonically dissolving, clarifying, introducing nitrogen to remove oxygen in the prepolymerization solution, sealing the prepolymerization solution, and reacting for 10-12h at 55-60 ℃; the unreacted material was removed by washing with acetonitrile and the product was dried.

2. The method for preparing the SBA-15 mesoporous molecular sieve doped acetylated molecularly imprinted polymer of claim 1, which is characterized by comprising the following steps:

1) According to the measurement, dissolving SBA-15 mesoporous molecular sieve, template KacQLAT, functional monomer zinc acrylate, cross-linking agents PEGDA and EDMA, initiator azo diisobutyronitrile in a mixed solution of pore-forming agent methanol and N, N-dimethylformamide; ultrasonic dissolving for 15min, ultrasonic power of 150W to dissolve and clarify, then introducing nitrogen to remove oxygen in the prepolymerization liquid, sealing the prepolymerization liquid, and reacting in a constant temperature water bath kettle at 55-60 ℃ for 10-12h;

2) And washing the obtained Molecularly Imprinted Polymer (MIP) with acetonitrile to remove unreacted residual impurities, drying in vacuum, and grinding in a mortar to obtain the SBA-15 mesoporous molecular sieve doped acetylated molecularly imprinted polymer.

3. A method for preparing an electrochemical sensor comprising the SBA-15 mesoporous molecular sieve doped acetylated molecularly imprinted polymer of claim 1, comprising the steps of:

1) Polishing a Glassy Carbon Electrode (GCE) on a polishing cloth by using GCE alumina water slurry, and then cleaning by using sulfuric acid solution, deionized water and ethanol respectively;

2) Adding an SBA-15 mesoporous molecular sieve doped acetylated molecularly imprinted polymer into a chitosan solution, and adding a multi-walled carbon nanotube DMF solution to increase conductivity to obtain an SBA-15@MIP mixture solution; dripping SBA-15@MIP mixture solution on the surface of the GCE in the step 1), drying a glassy carbon electrode at 50 ℃ for 15min, soaking the glassy carbon electrode in acetonitrile/acetic acid/water solution with the volume ratio of 5/5/90 to remove a template KacQLAT, and drying at room temperature to obtain an electrochemical sensor SBA-15@MIP/GCE of an SBA-15 mesoporous molecular sieve doped acetylated molecularly imprinted polymer;

3) Electrochemical sensor detection: selecting K containing KCl ₃ [Fe(CN) ₆ ]As an electrolyte solution; removing the template SBA-15@MIP/GCE and SBA-15@NIP/GCE, and placing the template and the SBA-15@MIP/GCE into a 50mM Tris-HCl solution with pH of 8.0 for incubation; in order to remove nonspecific adsorption on the surface of the electrode film, the adsorbed electric sensor is put into Tris-HCl buffer salt; finally, placing the electric sensor after adsorption in electrolyte, and measuring the current response of the electric sensor through DPV after balancing; all tests were measured in triplicate; the adsorption performance of the electric sensor is characterized by the change of current, and the calculation of the current change quantity is as follows in the formula (1):

ΔI＝Io-Ie (1)，

wherein Io and Ie represent initial current and current after adsorption respectively, and the unit is μA;

the blotting factor (IF) was used to evaluate the blotting performance of MIPs:

wherein DeltaI _MIP And DeltaI _NIP The current change values of SBA-15/MIP/GCE and SBA-15/NIP/GCE in the electrolyte after KacQLAT adsorption are expressed in mu A.

4. A method of manufacturing an electrochemical sensor according to claim 3, characterized in that:

the concentration of the GCE alumina slurry in the step 1) is respectively 1.0, 0.3 and 0.05 mu M, and the sulfuric acid solution is 1M;

the mass of the imprinted polymer in the step 2) is 2mg; the volume of the chitosan solution is 1mL, the mass percentage is 0.5 percent and the chitosan solution contains 20 mu L of acetic acid; the drop volume of the SBA-15@MIP mixture is 10 mu L; the volume of the DMF solution of the wall carbon nano tube is 50 mu L, and the concentration of the solution is 1mg/mL; the volume ratio of acetonitrile/acetic acid/water is 5/5/90;

step 3) the concentration of KCl solution is 0.02mol/L; k (K) ₃ [Fe(CN) ₆ ]The concentration of the solution was 5X 10 ^-3 mol/L; the template solution is 50mM Tris-HCl, the pH of the solution is 8.0, and the volume of the solution is 20mL; tris-HCl buffer salt at pH8.0 at a concentration of 50mM; the adsorption time is 20min; the equilibration time was 10min.

5. The multifunctional microfluidic chip integrated platform for enriching the acetylated peptides is characterized by mainly comprising a protein grading column unit, a denaturation chip unit, an enzyme reactor unit, an SBA-15 mesoporous molecular sieve doped acetylated molecularly imprinted monolithic column unit, a micro digital injection pump and an FEP connecting pipeline; the front of the denatured chip unit is connected with a protein grading unit, the rear of the denatured chip unit is sequentially connected with an enzyme reactor unit and an SBA-15 mesoporous molecular sieve doped acetylated molecularly imprinted monolithic column unit, the four units are connected in series by an FEP tube, and then the chip is fixed by a clamp; the FEP tube at the channel inlet and the inlet D, E are connected with a micro digital injection pump for adjusting the flow rate, and the final outlet end of the effluent liquid is connected with a small centrifuge tube;

the protein classifying column is provided with an inlet A and an outlet B which are connected;

the denaturation chip is divided into a denaturation I region and a denaturation II region which are connected with each other, the front end of the denaturation I region is provided with an inlet C and an inlet D of a reducing agent, the front end of the denaturation II region is provided with an inlet E for an alkylating agent, and the rear end of the denaturation II region is provided with an outlet F, wherein each region comprises a plurality of groups of snakelike channels capable of randomly regulating the total length of the channel; the enzyme reactor unit is composed of an enzyme reactor with an outlet and an inlet; the acetylated molecularly imprinted monolithic column unit is formed by an SBA-15 mesoporous molecular sieve doped acetylated molecularly imprinted monolithic column with an outlet and an inlet;

the preparation method of the SBA-15 mesoporous molecular sieve doped acetylated molecularly imprinted monolithic column comprises the following steps: the preparation of the pre-polymerized liquid is the same as that of the SBA-15 mesoporous molecular sieve doped acetylated molecularly imprinted polymer in claim 1, the pre-polymerized liquid is injected into a clean capillary, the inner diameter of the capillary is=250 mu m, two ends of the capillary are sealed by rubber, the reaction is carried out for 12 hours in a constant-temperature water bath kettle at 60 ℃, unreacted substances at the positions are washed by acetonitrile, a template is washed by acetonitrile/acetic acid/water, and the volume ratio of acetonitrile/acetic acid/water is 5/5/90.

6. A method for constructing a multifunctional microfluidic chip integration platform for enriching acetylated peptides according to claim 5, characterized in that: the method comprises the following steps:

1) Sample on-line processing unit construction: the front of the modified chip is connected with a protein grading column, the rear is connected with an enzyme reactor and an SBA-15 mesoporous molecular sieve doped acetylated molecular imprinting monolithic column, so that a microfluidic chip integrated platform comprising four functional units of the protein grading column, the modified chip, the enzyme reactor and the molecular imprinting monolithic column is constructed, and the four units are connected in series on line by a Fluorinated Ethylene Propylene (FEP) heat shrink tube, wherein the four units comprise interfaces A-F; placing the chip in a matched fixture, dividing the denatured chip into a denatured I area and a denatured II area, and inserting an FEP heat-shrinkable tube into a fixture interface to be connected with other functional units; the FEP pipes at the channel inlets A, D and E are connected with a microinjection pump, and the effluent outlet is connected with a small centrifuge tube;

2) Preparation of protein fractionation column: dissolving titanium dioxide nanotubes, azodiisobutyronitrile, glycidyl methacrylate and ethylene glycol dimethacrylate in pore-forming agents n-dodecanol and cyclohexanol; ultrasonic dissolution and one-step polymerization; washing the synthesized monolithic column with acetonitrile to remove unreacted monomers and a pore-forming agent, then injecting ammonia water into the synthesized capillary, heating in a water bath to graft amino groups on the surface of the polymer, and washing with deionized water to neutrality;

3) Preparation of the enzyme reactor: dissolving TRIM and azodiisobutyronitrile in toluene and isooctane; ultrasonic dissolving, injecting the pre-polymerized liquid into a clean capillary tube, placing the capillary tube in a water bath at 50 ℃ for reaction for 24 hours to obtain a TRIM column, and flushing unreacted monomers and a pore-forming agent by acetonitrile;

preparing trypsin and TCEP solution by using Tris-HCl solution, mixing the trypsin solution and the TCEP solution, centrifuging, standing to room temperature, taking supernatant, and adding ammonium persulfate and N, N-methylene bisacrylamide; injecting the enzymolysis liquid into the synthesized TRIM monolithic column, and sealing the two ends of the channel by using a rubber plug; after the reaction is finished, flushing with Tris-HCl solution, and refrigerating at 4 ℃;

4) Preparation of SBA-15 mesoporous molecular sieve doped acetylated molecularly imprinted monolithic column:

the preparation of the pre-polymerized liquid is the same as that of the acetylated molecularly imprinted polymer doped with the SBA-15 mesoporous molecular sieve, the pre-polymerized liquid is injected into a clean capillary, two ends are sealed by rubber, the water bath heating reaction is carried out, the unreacted substance at the washing position of acetonitrile is used, and then the acetonitrile/acetic acid/water is used for washing out the template.

7. The method according to claim 6, wherein: the denaturation I area and the denaturation II area in the step 1), wherein each area comprises a plurality of groups of snake-shaped channels, and each channel is 2cm long; the total length of the channels can be adjusted at will, and all channels in the chip have the width of 250 mu m and the depth of 100 mu m;

the mass fractions of the titanium dioxide nanotube, the azodiisobutyronitrile, the glycidyl methacrylate, the ethylene glycol dimethacrylate, the n-dodecanol and the cyclohexanol in the step 2) are respectively 1.20%, 0.15%, 16.02%, 7.21%, 47.28% and 28.14%; the concentration of the ammonia water is 4.5mol/L; the polymerization time for synthesizing the capillary base column by a one-step method is 3.5h; the reaction time of the ammonia water in the synthesized capillary tube of the base column is 2.5 hours;

the mass fractions of TRIM, azodiisobutyronitrile, toluene and isooctane in the step 3) are 24.12%, 0.22%, 20.34% and 55.32% respectively; the concentration of the Tris-HCl solution is 20mmol/L, and the pH of the solution is 8.0; trypsin concentration is 10mg/mL; the concentration of the TCEP solution is 1mg/mL; the volume ratio of the protease solution to the TCEP solution is 10:1; the concentrations of ammonium persulfate and N, N-methylene bisacrylamide are respectively 1mg/mL and 2mg/mL; the mixing time of the trypsin solution and the TCEP solution is 2 hours; injecting the enzyme derivative liquid into the synthesized TRIM monolithic column, wherein the reaction time is 5 hours;

the water bath heating temperature in the step 4) is 60 ℃, and the reaction time is 12 hours; the volume ratio of acetonitrile/acetic acid/water was 5/5/90.

8. The application method of the multifunctional microfluidic chip integrated platform constructed by the construction method of claim 6 in enriching acetylated peptides is characterized by comprising the following steps:

1) The protein is prepared into a certain concentration by ultra-pure water, and flows out from an inlet A to an outlet B,

meanwhile, DTT and IAA prepared by Tris-HCl buffer solution are respectively injected into the chip from inlets D and E; reduction of the protein is accomplished in a first serpentine channel, alkylation of the protein is accomplished in a second serpentine channel,

then, the denatured protein solution sequentially enters IMER for enzymolysis, and the acetylated peptides in the enzymolysis solution are enriched by an acetylated molecular imprinting monolithic column; then, in an off-line manner, tris-HCl buffer and ACN/HAC/H, respectively ₂ O leaching and eluting the acetylated molecularly imprinted monolithic column; collecting the final effluent and the eluent of the acetylated molecularly imprinted monolithic column;

2) Eluting the protein classification column with ultrapure water, discarding the eluent,

then, the solution at inlet A is changed to H ₂ O, eluting the proteins adsorbed on the classification column; the protein eluent flowing out from the outlet B is mixed with DTT and IAA of the inlets of D and E; the subsequent operation is the same as the treatment of the step 1); and collecting the final effluent and the eluent of the acetylated molecularly imprinted monolithic column.

9. The application method according to claim 8, wherein: the protein concentration in the step 1) is 1.5mg/mL, the concentration of the Tris-HCl buffer solution is 50mM, and the pH is 8.0;

the concentration of DTT and IAA are respectively 20mM and 30mM, the flow rate of the digital injection pump is 0.3 mu L/min, and the volume is 50 mu L; off-line rinsing and eluting volumes of the acetylated molecularly imprinted monolithic column are 50 mu L and 100 mu L respectively, the flow rate is 1.0 mu L/min, and the ACN/HAC/H is high ₂ The volume ratio of O is 5/5/90;

the rinsing volume of the protein classification column in the step 2) is 20 mu L, the flow rate is 0.3 mu L/min, and H ₂ The pH of O was 12.0.