CN110196324B

CN110196324B - Magnetic mesoporous silica nanoparticle probe and preparation method and application thereof

Info

Publication number: CN110196324B
Application number: CN201910475019.4A
Authority: CN
Inventors: 周昕; 李真真
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2019-05-31
Filing date: 2019-05-31
Publication date: 2020-09-29
Anticipated expiration: 2039-05-31
Also published as: CN110196324A

Abstract

The invention discloses a magnetic mesoporous silica nanoparticle probe, which is prepared by loading magnetic mesoporous silica nanoparticles with peroxidase to obtain M-MSN/POD, wrapping the M-MSN/POD with polyethyleneimine to encapsulate the peroxidase to obtain M-MSN/POD/PEI nanoparticles, and mixing the M-MSN/POD/PEI nanoparticles with an antibody to form the magnetic mesoporous silica nanoparticle probe. The invention also discloses a preparation method of the magnetic mesoporous silica nanoparticle probe. Compared with the prior art, the product prepared by the invention has the characteristics of high specificity, good stability, wide application field and the like, and has simple and convenient process and lower cost, thereby having good popularization prospect.

Description

Magnetic mesoporous silica nanoparticle probe and preparation method and application thereof

Technical Field

The invention belongs to the technical field of mesoporous silica probe preparation, and particularly relates to a magnetic mesoporous silica nanoparticle probe and a preparation method and application thereof.

Background

Since a series of materials of a novel mesoporous molecular sieve was first reported by Kresge et al in 1992, mesoporous silica materials have attracted extensive attention from researchers. Due to simple synthesis and low cost, the mesoporous silicon dioxide has a porous structure with large specific surface area, adjustable size and structure and internal and external surfaces which are easy to be chemically modified, so that the mesoporous silicon dioxide becomes an excellent carrier for loading small molecules.

In the past, a series of researches on the application of mesoporous silica materials in adsorption, imaging, drug delivery, biosensors and the like have been carried out. The mesoporous silica has porous performance and is generally used for carrying other small molecular substances such as drugs, proteins, fluorescent substances and the like in the research process. Has better loading capacity and biodegradability. The method has great application potential in the field of biological diagnosis and analysis, and obtains fruitful research results. The mesoporous shell layer endows the M-MSNs with two surfaces, namely the whole particle surface and the mesoporous inner surface. Therefore, the pore channel of the mesoporous material can not only act on the surface of the material with the biomolecules with the same size, but also penetrate through the whole orderly arranged mesoscopic restricted space of the material, thereby promoting the controllable release of the guest molecules. And secondly, the mesoporous shell layer can effectively protect the internal magnetic nanoparticles, so that the defects of instability, easy agglomeration and oxidation of the magnetic nanoparticles are overcome, and the M-MSNs have the advantage of more favorable composite synergy. And thirdly, from the viewpoint of multifunctional compounding, the functional modification can be flexibly and simply realized on the pore wall of the mesoporous silicon oxide layer, so that the interaction between the loading substance and the carrier can be optimized, and the surface charge, the dispersion stability and the targeting property can be controlled.

Most of clinical means for detecting virus diseases are complex, and laboratory detection has the defects of high operation requirement, long time consumption, expensive instruments and the like, and needs operation of professionals. The most typical test of signal amplification effect frequently used in laboratory detection is an enzyme-linked immunosorbent assay, and signal amplification can be realized by using a chemical means, but the operation process of the enzyme-linked immunosorbent assay is complex and needs to be operated by professional personnel, and the operation process is easy to cause result deviation due to errors.

Disclosure of Invention

The purpose of the invention is as follows: in order to solve the problems in the prior art, the invention aims to utilize the signal amplification principle of ELISA, use magnetic mesoporous silica nanoparticles to carry peroxidase, and utilize the principle that positive and negative charges can be assembled layer by layer to form a nanoprobe, so that virus particles in a sample can be directly detected. The high catalytic efficiency of the enzyme is utilized, the reaction effect can be greatly amplified, so that the determination method achieves high sensitivity, the use of expensive ELISA instruments can be reduced, and the method has obvious advantages in clinical detection.

The technical scheme is as follows: in order to solve the technical problems, the technical scheme adopted by the invention is as follows: the magnetic mesoporous silica nanoparticle probe is prepared by loading magnetic mesoporous silica nanoparticles with peroxidase to obtain M-MSN/POD, wrapping the M-MSN/POD with polyethyleneimine to encapsulate the peroxidase to obtain M-MSN/POD/PEI nanoparticles, and mixing the M-MSN/POD/PEI nanoparticles with an antibody to form the magnetic mesoporous silica nanoparticle probe.

Wherein, the magnetic mesoporous silicon dioxide nano-particles are modified by amino groups, the size is 60-65nm, and the mesoporous aperture is 5-6 nm.

Wherein, the polyethyleneimine is linear and has a molecular weight of 25 KDa.

The invention also discloses a preparation method of the magnetic mesoporous silica nanoparticle probe, which comprises the following steps:

1) loading of peroxidase: mixing peroxidase and M-MSN nanoparticles to obtain POD-loaded M-MSN nanoparticles, namely M-MSN/POD;

2) PEI coated M-MSN-/POD: mixing PEI with M-MSN/POD to obtain magnetic mesoporous silicon nanoparticles (M-MSN/POD/PEI nanoparticles) wrapped and loaded by polyethyleneimine;

3) coupling of virus antibodies: and mixing the M-MSN/POD/PEI nano particles with the antibody, and mixing the obtained product to obtain the magnetic mesoporous silica nano particle probe.

Wherein, the mass ratio of the peroxidase to the M-MSN nanoparticles in the step 1) is 1: 1.

Wherein, the mass ratio of the PEI and the M-MSN/POD in the step 2) is 1: 10.

Wherein the mixing time in the step 2) is 8-12 h.

Wherein, the mass ratio of the M-MSN/POD/PEI nanoparticle to the virus antibody in the step 3) is 10: 1.

The antibody of step 3) includes but is not limited to newcastle disease virus antibody, and other antibodies can be suitable.

The virus includes, but is not limited to, Newcastle Disease (NDV) virus, and other viruses may be suitable.

The invention also discloses the application of the magnetic mesoporous silica nanoparticle probe in the preparation of the test strip for detecting viruses.

Has the advantages that: compared with the prior art, the invention has the advantages that:

1) the invention utilizes the carrying characteristic of a porous structure, carries the common chemical color reagent peroxidase, utilizes the mesoporous characteristic of mesoporous silica to load the peroxidase into the mesoporous structure, and uses high molecular polymer polyethyleneimine to temporarily 'plug' the pore opening of the mesoporous, thereby ensuring the enzymatic activity of the peroxidase in the mesoporous. The preparation method combines the signal amplification principle of the traditional enzyme-linked immunosorbent assay, and finally can indicate the detection result in a color development mode; compared with the prior art, the novel nano probe prepared by the method has the characteristics of high specificity, good stability, wide application field and the like, and has simple and convenient process and lower cost, thereby having good popularization prospect.

2) The invention has mild reaction conditions, does not influence the activity of protein, has good dispersity of the probe in the solution, and provides a new technical route and a new research idea for treating diseases such as cancer and the like and detecting the specificity of pathogens.

Drawings

FIG. 1 TEM representation of magnetic mesoporous silica;

FIG. 2 is a SEM representation of magnetic mesoporous silica;

FIG. 3 is a SDS-PAGE graph of peroxidase-loaded magnetic mesoporous silica; wherein 1 is a pre-dyed protein marker, 2 is probe magnetic rack supernatant, 3 is magnetic mesoporous silica loaded peroxidase, 4 is magnetic mesoporous silica, and 5 is simple peroxidase 20 μ g;

FIG. 4 is a demonstration chart of probe preparation to verify SDS-PAGE patterns of mesoporous silicon nanoparticles bound to antibodies; wherein M is a pre-dyed protein marker, 1 is peroxidase of 2 mug, 2 is antibody of 2 mug, and 3 is a magnetic dielectric silicon loaded peroxidase and antibody linked nanoprobe;

FIG. 5 shows a nucleic acid gel electrophoresis of the PCR amplification product of example 3, marker2000 in lane M and NDV virus in lane I; lane II is POD/M-MSN/PEI/Ab/NDV;

FIG. 6 shows the PCR amplification product of example 4 subjected to nucleic acid gel electrophoresis, marker2000 in lane M, and INDV virus in lane; lane II NDV: POD/M-MSN/PEI/Ab ═ 1: 1; lane III is NDV: POD/M-MSN/PEI/Ab 1/2; lane IV is NDV: POD/M-MSN/PEI/Ab 1/4; lane V is NDV: POD/M-MSN/PEI/Ab 1/6;

FIG. 7 TEM binding image of magnetic mesoporous silica nanoprobe binding NDV virus;

FIG. 8 shows the PCR amplification product of example 6 subjected to nucleic acid gel electrophoresis, marker2000 in lane M and H9N2 virus in lane I; lane II is POD/M-MSN/PEI/Ab/H9N 2;

fig. 9 TEM image in example 7.

Detailed Description

The present invention is further illustrated by the following specific examples, it should be noted that, for those skilled in the art, variations and modifications can be made without departing from the principle of the present invention, and these should also be construed as falling within the scope of the present invention. The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. In the quantitative experiments in the following examples, three replicates were set up and the results averaged.

The magnetic mesoporous silica nanoparticles are purchased from Shanghai Sophia biological company, and have the following goods numbers: M-MSNs-50; peroxidase was purchased from beijing solibao bio-inc; the natural immunity signal, immunity and infection research laboratory of the Newcastle disease virus (strain: lasota) Jiangsu province Yangzhou university college of veterinary medicine is a laboratory for storing strains; the Newcastle disease virus antibody is Newcastle disease virus antibody |8H2(BIO-RAD, MCA 2822); magnetic stand machines are available from Eppendorf corporation, germany; PBS (10X) was purchased from Jiangsu Biyuntian Biotech limited; both the clean bench and the mixer were purchased from Thermo Fisher; the RNA virus extraction kit is purchased from Beijing Probiotics, Inc., Hao Biotech; the RNA reverse transcription kit is purchased from Beijing kang, a century Biotechnology Co., Ltd; 2 × Taq Master Mix (p111-w3) was purchased from Nanjing Novowed Biotech, Inc.; primer synthesis comes from the company of Biotechnology engineering (Shanghai) and influenza virus H9N2, and the natural immune signal, immune and infection research laboratory of Yangzhou university college of Yongsu province stores strains for the laboratory. All the following examples used PBS diluted 1 x PBS (pH 7.2-7.4).

Example 1 construction of a magnetic mesoporous silica nanoparticle Probe Using a Newcastle disease Virus detection Probe as an example

1. Characterization of M-MSN nanoparticles: the M-MSN nano particles are diluted to be 1 mug/muL, then 10 muL of sample application copper mesh is taken, and then the size and the aperture of the nano particles are observed by a transmission electron microscope and a scanning electron microscope, and the result is shown in figure 1 and figure 2.

2. Loading of Peroxidase (POD): mixing commercial peroxidase and the diluted M-MSN nanoparticles obtained in the step 1 according to the mass ratio of 1: 1 (namely 100 mu g of M-MSN: 100 mu g of POD), and incubating the mixed solution on the mixure at 4 ℃ for 8-12 h; then separating and washing the unbound peroxidase by a magnetic rack to obtain a supernatant and a precipitate, wherein the obtained precipitate is POD/M-MSN; dissolving the precipitate in 100 μ L PBS; simultaneously respectively dissolving 20 mu g of M-MSN nano particles into 100 mu L of PBS, and dissolving 20 mu g of POD into 20 mu L of PBS for later use;

20 mu L of supernatant, 20 mu L of precipitate dissolved in PBS, 20 mu L of M-MSN nano-particles dissolved in PBS and 20 mu L of POD dissolved in PBS are respectively taken, 5 mu L of 5 × loading buffer is added, the sample is boiled at 100 ℃ for 5 minutes, and then protein gel electrophoresis is carried out, the voltage is 120V, and the current is 90 mA. The time is 1h, then Coomassie brilliant blue is used for dyeing for 25min, and the decolorization solution is used for decolorizing for 24h, and the result is shown in figure 3. As can be seen from FIG. 3, the POD/M-MSN does load the original POD into the mesopores of the M-MSN.

3. Mixing the M-MSN/POD constructed in the step 2 with 5% polyethyleneimine according to the mass ratio of 10: 1, and incubating for 1h at 4 ℃; and separating the supernatant and the precipitate by a magnetic frame to obtain M-MSN/POD/PEI nano particles for later use.

4. Coupling of newcastle disease virus antibodies: mixing the M-MSN/POD/PEI nano particles obtained in the step 3 and Newcastle Disease Virus antibody |8H2(BIO-RAD, MCA2822) according to the ratio of 10: 1, and incubating for 8H at 4 ℃; obtaining a magnetic mesoporous silica nanoparticle probe POD/M-MSN/PEI/Ab, and dissolving the POD/M-MSN/PEI/Ab in 20 mu L of PBS for later use; at the same time, 2. mu.g of antibody was dissolved in 20. mu.L of PBS; mu.g POD in 20. mu.L PBS; and (5) standby.

mu.L of POD in PBS, 20. mu.L of Newcastle disease virus antibody in PBS, and 20. mu.L of POD/M-MSN/PEI/Ab in PBS were added with 5. mu.L of 5 × loading buffer, respectively. Cooking the sample at 100 deg.C for 5 min. Then protein electrophoresis is carried out, the voltage is 120V, and the current is 90 mA. The time is 1h, then Coomassie brilliant blue is used for dyeing for 25min, and the decoloration solution is used for decoloring for 24 h. The photograph was taken and the results are shown in FIG. 4, in which it can be seen that the newcastle disease virus antibody has been successfully linked to POD/M-MSN/PEI.

Example 2 detection of peroxidase Activity

In order to detect the activity of peroxidase in the magnetic mesoporous silica, TEM is selected as a substrate, the TEM reacts with peroxidase in different states for color development, the TEM is specifically divided into a pure Peroxidase (POD), a peroxidase (POD/M-MSN) wrapped by the magnetic mesoporous silica, a magnetic mesoporous silica (POD/M-MSN/PEI) wrapped by polyethyleneimine and loaded with peroxidase, and a constructed nanoprobe (POD/M-MSN/PEI/Ab), and then a microplate reader is used to read the value of OD 450. As a result of the examination, it was found that the enzymatic activity of peroxidase was not changed in any state after loading the peroxidase into the magnetic mesoporous silica. The specific detection results are as follows:

content of POD	OD450
		POD 0.2μg	3.9267
POD/M-MSN 0.2μg	3.9389
		POD/M-MSN/PEI 0.2μg	3.9165
POD/M-MSN/PEI/Ab 0.2μg	3.7982

Example 3 detection of Newcastle disease Virus by magnetic mesoporous silica nanoparticle Probe

1. The magnetic mesoporous silica nanoparticle probe (POD/M-MSN/PEI/Ab) prepared in example 1 was bound to a Newcastle disease virus: the combination ratio of the magnetic mesoporous silica nanoparticle probe to the Newcastle disease virus is 1: 1, the reaction is carried out for 1 hour at room temperature, after 1 hour, a magnetic frame is used for separating supernatant and precipitate, and the supernatant and the precipitate are washed for 3 times by 1 XPBS. And precipitating for later use.

2. Extracting virus RNA and reverse transcription: and (3) extracting RNA from the precipitation product obtained in the step (1) by using an RNA extraction kit, and then reversely transcribing the extracted RNA into cDNA by using a reverse transcription kit. The reverse transcription system is as follows:

the reverse transcription process was 37 deg.C, 15min, 85 deg.C, 5 s.

3. And (3) detecting the binding efficiency: and (3) carrying out PCR identification on the reverse transcription product obtained in the step (2) and detecting the combination efficiency of the magnetic mesoporous silica nanoprobe. Based on the whole gene sequence of newcastle disease virus (GenBank accession No.: AE001363), a primer specific to the gene was designed using Primier 5.0 software, and the upstream primer (F): 5'-ctatccgggttgcgctggta-3' (SEQ ID No. 1); downstream primer (R): 5'-ttccaagtaggtggcacgca-3' (SEQ ID No.2), the length of the amplification product is 866 bp. The reaction system identified by PCR is as follows:

preparing a PCR reaction system with a total volume of 25 mu L

PCR reaction procedure: 5min at 95 ℃, 5s at 95 ℃, 30s at 57 ℃, 45s at 72 ℃ and 10min at 72 ℃ for 25 cycles in total, and 50min at 13 ℃.

And (3) carrying out agarose gel electrophoresis on the PCR amplification product: preparing 1.5% agarose gel, with electrophoresis parameter of 120V and time of 40 min. Referring to fig. 5, the electrophoresis result shows that the efficiency of combining the mesoporous silicon nanoprobe with newcastle disease virus is high, and newcastle disease virus is not detected in the supernatant after separation by the mesoporous silicon nanoprobe.

Example 4 sensitivity test of magnetic mesoporous silica nanoparticle probes for detecting Newcastle disease Virus

1. Combining the magnetic mesoporous silica nanoparticle probe (POD/M-MSN/PEI/Ab) prepared in example 1 with Newcastle disease viruses with different proportions, and reducing the amount of the viruses under the condition of keeping the amount of the magnetic mesoporous silica nanoparticle probe (POD/M-MSN/PEI/Ab) unchanged; the specific selection ratios are as follows: NDV, POD/M-MSN/PEI/Ab is 1: 1; NDV: POD/M-MSN/PEI/Ab 1/2; NDV: POD/M-MSN/PEI/Ab 1/4; NDV: POD/M-MSN/PEI/Ab ═ 1/6, was reacted at room temperature for 1 hour, and after 1 hour, the supernatant and the precipitate were separated using a magnetic holder and washed 3 times with 1 XPBS. And precipitating for later use.

the reverse transcription process was 37 deg.C, 15min, 85 deg.C, 5 s.

preparing a PCR reaction system with a total volume of 25 mu L

And (3) carrying out agarose gel electrophoresis on the PCR amplification product: preparing 1.5% agarose gel, with electrophoresis parameter of 120V and time of 40 min. Electrophoresis results referring to fig. 6, the results show that the mesoporous silicon nanoprobe has high binding efficiency to newcastle disease virus, and can still capture newcastle disease virus when the amount of virus is reduced to 1/6.

Example 5 detection of Newcastle disease Virus by magnetic mesoporous silica nanoparticle Probe

The probe POD/M-MSN/PEI/Ab was bound to the Newcastle disease virus and reacted at room temperature for 1 hour, then unbound virus in the supernatant was separated using a magnetic frame, and the probe was washed 3 times using PBS. Then, a product obtained by combining the mesoporous silica nanoprobe with the newcastle disease virus is spotted on a copper mesh, the copper mesh is dried for 10 minutes at room temperature, then the copper mesh is negatively stained with 2% phosphotungstic acid for 5 minutes, the copper mesh is placed under an electron microscope for observation, and as shown in figure 7, the magnetic mesoporous silica nanoprobe is combined with the newcastle disease virus.

Example 6

To test the specificity of the probe, the POD/M-MSN/PEI/Ab nanoprobe constructed in example 3 was combined with influenza virus H9N2, the reaction time was 1 hour at room temperature, after the reaction was finished, the magnetic frame was used for separation, PBS was used for washing three times, then the obtained product was subjected to RNA extraction, and the obtained RNA was subjected to reverse transcription into cDNA, wherein the reverse transcription system is as follows:

the reverse transcription process was 37 deg.C, 15min, 85 deg.C, 5 s.

The binding efficiency was then detected by PCR.

Preparing a PCR reaction system with a total volume of 25 mu L

And (3) carrying out agarose gel electrophoresis on the PCR amplification product: preparing 1.5% agarose gel, with electrophoresis parameter of 120V and time of 40 min. The results are shown in FIG. 8, without any banding. The probe can be proved to have specificity, and the possibility of non-specific adsorption is eliminated.

Example 7

In order to detect the specificity of the probe, the POD/M-MSN/PEI/Ab nanoprobe in example 3 is combined with influenza virus H9N2, the reaction time at room temperature is also 1 hour, after the reaction is finished, a magnetic frame is used for separation, and PBS is used for washing three times; and then, taking a product obtained by combining the mesoporous silica nanoprobe with the virus H9N2, spotting the product on a copper net, drying the product at room temperature for 10 minutes, then carrying out negative staining by using 2% phosphotungstic acid for 5 minutes, and placing the copper net under an electron microscope for observation, wherein the specific result is shown in figure 9, and an electron microscope photo of the combination of the mesoporous silica nanoprobe and the virus is not found.

In conclusion, the mesoporous silica nanoprobe prepared by the invention has good specificity and great development potential in the later period, and can be applied to other detection fields such as detection of test strips and detection chips.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Variations or modifications in other variations may occur to those skilled in the art based upon the foregoing description. Not all embodiments need be illustrated or described herein. And obvious variations or modifications of this embodiment may be made without departing from the spirit or scope of the invention.

Sequence listing

<110> Yangzhou university

<120> magnetic mesoporous silica nanoparticle probe and preparation method and application thereof

<160>2

<170>SIPOSequenceListing 1.0

<210>1

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

ctatccgggt tgcgctggta 20

<210>2

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

ttccaagtag gtggcacgca 20

Claims

1. The magnetic mesoporous silica nanoparticle probe is characterized in that magnetic mesoporous silica nanoparticles are loaded with peroxidase to obtain M-MSN/POD, polyethyleneimine wraps the M-MSN/POD to encapsulate the peroxidase to obtain M-MSN/POD/PEI nanoparticles, and the M-MSN/POD/PEI nanoparticles are mixed with an antibody to form the magnetic mesoporous silica nanoparticle probe, wherein the magnetic mesoporous silica nanoparticles are amino-modified, have the size of 60-65nm and the mesoporous aperture of 5-6nm, and the polyethyleneimine is linear and has the molecular weight of 25 KDa.

2. The preparation method of the magnetic mesoporous silica nanoparticle probe of claim 1, comprising the following steps:

3) coupling of the antibody: mixing the M-MSN/POD/PEI nano particles with the antibody, and mixing the obtained product to obtain a magnetic mesoporous silica nano particle probe;

the mass ratio of the peroxidase to the M-MSN nanoparticles in the step 1) is 1: 1, the mass ratio of the PEI to the M-MSN/POD in the step 2) is 1: 10, the mixing time of the step 2) is 8-12h, and the mass ratio of the M-MSN/POD/PEI nanoparticle to the antibody of the step 3) is 10: 1, the antibody in the step 3) is a Newcastle disease virus antibody.

3. The use of the magnetic mesoporous silica nanoparticle probe of claim 1 in the preparation of a test strip for detecting viruses.