CN1164607C

CN1164607C - Application of silicon amide shell type nano granular material in nucleic acid enriching and nano sensing process

Info

Publication number: CN1164607C
Application number: CNB021141584A
Authority: CN
Inventors: 王柯敏; 何晓晓; 李杜; 谭蔚泓
Original assignee: Hunan University
Current assignee: Hunan University
Priority date: 2002-05-28
Filing date: 2002-05-28
Publication date: 2004-09-01
Anticipated expiration: 2022-05-28
Also published as: CN1385439A

Abstract

The present invention relates to the application of amination silicon shell nanometer particle materials in ribonucleic acid enrichment and nanometer sensing. In the present invention, core materials with special biochemistry properties, sound properties, light properties, electricity properties, heat properties, magnetic properties, etc. are filled in the silicon dioxide network molecule shell layers of amination silicon shell nanometer particle materials; the positive value electrokinetic potential of nanometer particles in neutral environment is used for separating, enriching, extracting and reprinting ribonucleic acid from biological samples; amino groups on the surfaces of the nanometer particles directly react with biological macromolecules or indirectly react with biological macromolecules through organic molecules for causing the surfaces of particles and the biological macromolecules to be mutually decorated; the decorated nanometer particles are used as sensors for measuring single cell reification indexes. The present invention slickly combines nanometer particles and molecule biology/medical science, provides a fire-new chance for the prominent development of molecule biology/medical science and has a wide market prospect and social benefit.

Description

The application of silicon amide shell nano particle material in nucleic acid enriching and nanosensor

Technical field: the present invention relates to the nanobiology field, relate in particular to the application and development of nano particle at biomedical sector.

Background technology: contemporary medical science, biology have been deep into molecular level, to biomacromolecule or its segment is separated efficiently, enrichment, extraction, reprinting etc. have become the technical issues that need to address day by day urgently, and utilize dimensionally corresponding of nano particle and biomacromolecule to finish above-mentioned biology/medical research or actually operating, be an important approach; But the nano particle of existing other kinds, or be not used because of physicochemical property is unstable, or because of produce difficulty, the raw material costliness is difficult to popularization; And existing nano SiO 2 particle surface tissue is single, electrokinetic potential in neutral solution is a negative value, do not possess required biochemical characteristic and be difficult to and modify, bioactive molecules and segment thereof being separated, meeting difficulty in the operation such as enrichment, extraction, reprinting, be difficult in molecular biosciences/medical science, be applied.

Above-mentioned predicament at prior art, the inventor has adopted multiple silylating reagent to prepare a kind of silicon amide shell nano particle material with the processing method of one-step hydrolysis, described particle surface is a silicon-dioxide network molecule shell, the branch subshell is submitted and is associated with end of the chain amino, zeta potential value in neutral solution is higher than+10mv, and waiting an electricity is about pH9.6.

Summary of the invention: for this reason, technical solution of the present invention is the application of silicon amide shell nano particle material in nucleic acid enriching and nanosensor;

Described application is the inner nuclear material that enriches character such as special biochemistry, sound, light, electricity, heat, magnetic in the silicon-dioxide network molecule shell of silicon amide shell nano particle material.

Described application be utilize nano particle in the neutral environment on the occasion of electrokinetic potential separation from biological sample, enrichment, extraction, reprinting nucleic acid.

Described application be make its surface amino directly or by organic molecule indirectly and biomacromolecule react, particle surface and biomacromolecule are modified each other.

Described application is to utilize modified nano particle as transmitter, and single celled materialization index is measured.

Above-mentioned advantages of application is:

Described nano particle, in acidity and neutral medium, the silica sphere of uncrosslinked amino still keeps materialization stability widely, and can take place protonated at the crosslinked amino of its particle surface, produce clean positive electricity effectively, in pH neutral normal temperature medium, the electrokinetic potential of described silicon amide shell nano-particle material is up to 35mv, thereby can be easily and electronegative DNA, bioactive moleculess such as RNA form " nano particle-DNA " by electrostatic interaction, the mixture of " nano particle-RNA " etc., with DNA, the binding ability of biomacromolecules such as RNA is very strong, and the bonded biomacromolecule had obvious provide protection, for to DNA, RNA etc. separate, enrichment, extract, condition has been created in reprinting;

The amino of nano grain surface has reactive behavior, be easy to direct or indirect and other biological macromole active group reaction by the chemical cross-linking agent of tool double-functional group, modify each other realizing, reach certain biochemistry operation or testing goal then as structure biological nano transmitter etc.;

Nano particle forms shell structure, can enrich with functional kernels such as biochemistry, sound, light, electricity, heat, magnetic in shell inside, and these functional kernels are externally under the exciting of electromagnetic field etc., can implement specific function as: orientation movement, excite monochromatic light, specific change or reaction etc., thereby realize the biochemistry operation of expection efficiently or detect;

The present invention combines nano particle dexterously with molecular biosciences/medical science, for the breakthrough of molecular biosciences/medical science provides brand-new opportunity, has wide market outlook and social benefit.

Description of drawings: now in conjunction with the accompanying drawings the present invention is explained.

Fig. 1 is the structural representation of silicon amide core-shell nanoparticles of the present invention.

The electrokinetic potential that Fig. 2 compares with other nano particles for nano particle of the present invention and the graphic representation of pH value.

Fig. 3 is the detected through gel electrophoresis image of DNaseI digested plasmid DNA.

Fig. 4 is the image of COS-7 cell under simple microscope of amination nano SiO 2 particle-pIRGFP mixture transfection.

Fig. 5 is the image of COS-7 cell under fluorescent microscope of amination nano SiO 2 particle-pIRGFP mixture transfection.

Fig. 6 is the fluorescence spectrum figure of amination nano SiO 2 particle to the enrichment experiment of fluorescently-labeled dna segment.

Silicon amide shell nano particle material as shown in Figure 1, its particle surface are silicon-dioxide network molecular structure shell 2, submit at silicon-dioxide network molecule shell 2 that to be associated with carbon value be C ₃～C ₁₀The diamine alkyl; Silicon-dioxide network molecule shell 2 includes inner nuclear material 1 and comprises: functional inner nuclear material such as silicon-dioxide or special biochemistry, sound, light, electricity, heat, magnetic is as dyestuff, fluorescent substance, magnetic substance, quantum dot, metal, medicine etc.; Further, end of the chain amino crosslinked on the shell 2 can react with biomacromolecule active group 3 (comprising: RNA, DNA, antibody, hormone, somatomedin, lectin, enzyme molecule etc.) directly or indirectly, realizes the particle surface modification;

As shown in Figure 2: the electrokinetic potential measurement shows: in pH neutral normal temperature medium, silicon amide shell nano particle 4 of the present invention (comprising: magnetic, fluorescence etc.) surface amino can produce clean positive electricity effectively, and electrokinetic potential is higher than 35mv, only when pH is higher than iso-electric point about 9.6, the apparent deprotonation of amino generation, electrokinetic potential reaches null value and negative trend; Comparatively speaking, the electrokinetic potential of ferroferric oxide gel nano particle 5, silica magnetic nano particle 6, simple nano SiO 2 particle 7 etc. just presents negative value as far back as pH in 7 sour environment, all far short of what is expected than the correlated performance of silicon amide shell nano particle 4.

Embodiment:

Embodiment one (preparation method): A) in the oil phase of hexanaphthene/n-hexyl alcohol, add entry and TritonX100 and dispersion formation water-in-oil microemulsion, B) adding functional nuclear matters such as biochemistry, sound, light, electricity, heat, magnetic goes in the microemulsion, adding tetraethoxy and N-(β-aminoethyl)-γ-aminopropyl-triethoxyl silane then simultaneously goes in the microemulsion, C) adding catalyst ammonia water goes in the microemulsion, react and finished synchronous hydrolysis reaction in 24 hours, generate silicon amide shell nano-particle material as shown in Figure 1.

Embodiment two (nano particle is to the enrichment of fluorescently-labeled dna segment): with amidized nano SiO 2 particle with by fluorescently-labeled incomplementarity single strain oligonucleotide segment (3 '-CAT-AGT-GCT-CCG-GGA-TAC-GC-5 '-TMAR) cultivates, detect by fluorophotometer and agarose gel electrophoresis to show (referring to the fluorescence spectrum figure of Fig. 6): the fluorescence intensity that TMAR produces in the solution when nano particle drops into as yet maximum intensity do not occur shown in curve a; Dropping into amidized nano SiO 2 particle enters in the solution, the nano particle effect of fluorescently-labeled DNA and input and with solution separating after, there has not been fluorescence in the supernatant liquor, illustrate that DNA all is combined in nano grain surface, the fluorescence intensity intensity shown in curve b in the supernatant solution is tending towards null value at this moment; Change conditions such as ionic strength or potential of hydrogen, nano particle combining and can dissociate with DNA's, and the TMAR after dissociating gets back to the fluorescence intensity that produces in the solution shown in curve c: almost return to initial high fluorescent.

Embodiment three (magnetic nanoparticle is to the extraction of the total DNA of animal tissues): in the silicon amide shell nano particle preparation process, synchronously magneticsubstance is enriched and enter in the silicon shell, the silicon amide shell magnetic nanoparticles that generates has superparamagnetism, with the biological tissue samples liquid (blood after this nano particle and the cracking, marrow, tissue etc.) one work, positively charged magnetic nanoparticle and electronegative DNA or its segment form the mixture of " nano particle-DNA " in the electrostatic attraction mode, externally under the guide effect in magnetic field, its extraction procedure and time obviously shorten; Neither need again nano particle to be modified, also do not need the phenol-chloroform extracting in the ordinary method; In the agarose gel electrophoresis experiment, total DNA that this method is extracted compares with ordinary method, and the characteristic of DNA is constant.

Embodiment four (nano particle of enrichment DNA is as novel non-viral genophore): with silicon amide core-shell nanoparticles and plasmid DNA (green fluorescence protein expression carrier pIRGFP) with certain mass than mixing, the agarose gel electrophoresis qualitative detection is the result show, increase along with the plasmid DNA amount, the electrokinetic potential of " nano particle-plasmid DNA " mixture reduces gradually, when the amount that adds DNA reaches 0.0972 (w/w) is saturated, and " nano particle-plasmid DNA " mixture reaches iso-electric point.Then in the standard (its gel band (1) in referring to Fig. 3) of plasmid DNA, reach the DNaseI that all adds equivalent in " nano particle-plasmid DNA " mixture (referring to the gel band among Fig. 3 (3)), water-bath digestion is after 1 hour in the time of 37 ℃, carry out detected through gel electrophoresis again, found that not had DNA (being gel band (2) completely dissolve among Fig. 3) in the standard of DNA; And " nano particle-plasmid DNA " mixture still has DNA to have (seeing the gel band (4) among Fig. 3) in the point sample hole, relatively gel band (3), (4) are as seen, the formation of " nano particle-DNA " mixture has obvious provide protection to DNA, and is just low slightly in the undigested composite sample of postdigestive concentration ratio; Be further developed into thus the non-viral gene transfection method as: with mixture plasmid DNA is brought in the America green monkey kidney cell (COS-7 clone), cell after the transfection place common and fluorescent microscope under, obtain the

image

4,5 of COS-7 cell under common and fluorescent microscope of amination nano SiO 2 particle-pIRGFP

mixture transfection.Image

4,5 shows that nano particle can effectively shift the pIRGFP plasmid and enter the COS-7 cell, and can produce high-level GFP expression, and transfection efficiency reaches 60-70%.

Embodiment five (the biochemical molecular group is modified in the amino indirect reaction of particle surface): on silicon amide shell fluorescent nano particle surface, make amino and glutaraldehyde (2%) room temperature reaction 1 hour, glutaraldehyde is attached at nano grain surface, to fix some biomacromolecule as glucose oxidase, Streptavidin etc.

Embodiment six (the amino direct reaction of particle surface is modified the biochemical molecular group): with bipyridyl ruthenium title complex Ru (BPY) ₃Be the silicon amide shell fluorescent nano particle surface of kernel, available fluorescein isothiocyanate (FITC), rhodamine isothiocyanate etc. are directly modified.

Embodiment seven (nano grain surface of fluorescence kernel directly modify be used for pH transmitter): with bipyridyl ruthenium title complex Ru (BPY) ₃Silicon amide shell fluorescent nano particle surface for kernel, directly modify with fluorescent reagent fluorescein isothiocyanate (FITC) the pH sensitivity, make it tangible response be arranged, thereby be used as the pH nano-sensor, realize the measurement of unicellular interior pH by the endocytosis of cell pH.

Claims

1, the application of silicon amide shell nano particle material in nucleic acid enriching and nanometer pH sensing, described particle surface is a silicon-dioxide network molecule shell, the branch subshell is submitted and is associated with end of the chain amino, enriches the inner nuclear material of biochemistry, sound, light, electricity, heat, magnetic property in the shell.

2, the application of silicon amide shell nano particle material as claimed in claim 1 in nucleic acid enriching and nanometer pH sensing is characterized in that: described application is to enrich bipyridyl ruthenium title complex or magnetic substance as inner nuclear material in the silicon-dioxide network molecule shell of silicon amide shell nano particle material.

3, the application of silicon amide shell nano particle material as claimed in claim 1 or 2 in nucleic acid enriching and nanometer pH sensing is characterized in that: described application be utilize nano particle in the pH neutral environment on the occasion of electrokinetic potential separation from biological sample, enrichment, extraction, reprinting nucleic acid.

4, the application of silicon amide shell nano particle material as claimed in claim 1 or 2 in nucleic acid enriching and nanometer pH sensing, it is characterized in that: described application be make its surface amino directly or by glutaraldehyde indirectly and glucose oxidase, Streptavidin or rhodamine isothiocyanate react, particle surface and glucose oxidase, Streptavidin or rhodamine isothiocyanate are modified each other.

5, the application of silicon amide shell nano particle material as claimed in claim 4 in nucleic acid enriching and nanometer pH sensing is characterized in that: described application is to utilize modified nano particle as transmitter, and single celled pH value is measured.