CN111996186A - Preparation method of probe positioning modified soluble nano core cap particles - Google Patents
Preparation method of probe positioning modified soluble nano core cap particles Download PDFInfo
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- CN111996186A CN111996186A CN202010794367.0A CN202010794367A CN111996186A CN 111996186 A CN111996186 A CN 111996186A CN 202010794367 A CN202010794367 A CN 202010794367A CN 111996186 A CN111996186 A CN 111996186A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
Abstract
The invention discloses a preparation method of probe positioning modified soluble nano core cap particles, which is characterized in that by controlling the growth of the mounting position, the number and the posture of a seed probe, the seed probe molecules are self-positioned and only modified in the cap top central area of a three-dimensional space curved surface of a core cap, and the rest part of the outer surface of the cap is modified with a system supporting molecules to stabilize the posture of the probe molecules, so that the nano core cap particles can be freely dispersed in an aqueous solution.
Description
Technical Field
The invention belongs to the field of nanotechnology and biotechnology, relates to probe positioning modification of an aspheric symmetrical three-dimensional space curved surface, and particularly relates to a preparation method of probe positioning modified soluble nanometer core cap particles.
Background
The core cap nanometer structure is characterized in that a layer of noble metal shell with nanometer thickness is wrapped on the outer surface of the spherical core, different cap depths are formed according to the proportion of the shell layer covering the surface of the spherical core, and the cap depths can be smaller than, equal to or larger than half shells. When the shell covers 100% of the spherical core surface, the core cap nanostructure evolves into a core-shell structure. The two nano structures increasingly show good commercial value in the fields of biosensing, molecular probe imaging, nano optoelectronic devices and the like in recent years. The applications mainly derive from the unique optical, electric, magnetic and other physical properties of the noble metal nano structure.
The intrinsic Local Surface Plasmon Resonance (LSPR) effect of the noble metal nano-structured particles enables the noble metal nano-structured particles to have strong extinction characteristics in a visible-near infrared band, generally show different colors and positions of photo-heat, and are developed into novel multi-modal and multifunctional molecular probe markers. Of the numerous noble metal nanostructures, core-shell structures have found a great deal of use because of the broad spectral tuning range that can be obtained by small changes in structural parameters (e.g., core-shell ratio). Compared with a core-shell structure, the core-cap structure also has broadband adjustment characteristics, and parameters which can be manually adjusted are richer, particularly after the spherical symmetry is broken, the LSPR mode of the core-cap structure has strong dependence on the electric field vector of exciting light: electric field vectors exhibit galvanic and magnetic dipole behavior, respectively, when parallel and perpendicular to their symmetry axes, each exhibiting different optical properties. The spherical symmetry break creates new patterns, however it also presents a greater challenge to the surface molecular modification sites, since such patterns, which are related to the orientation of the symmetry axis, require that the molecule be located at a characteristic position to facilitate manual manipulation, where the central region where the symmetry axis intersects the outer surface of the core cap is particularly important in forming dimers based on core cap nanostructures (patent application: 2018112376362).
The self-directed modification of biomolecules to non-spherical symmetric three-dimensional surfaces is a great challenge, and patent application 2020100382957, based on the core-shell structure, proposes a method for preparing directionally assembled core-cap heterodimers by attaching another particle to the apex of the core cap via a PVP molecule located in the central region of the cap apex. The method realizes the site-specific modification of molecules to the surface of the core cap, but the structure modified by the method is usually limited in PDMS (polydimethylsiloxane) and does not pay attention to the degree of freedom (namely free suspension in solution) required in the application of molecular probes, and the chemical reagents used in the process of releasing PDMS to release core cap particles cause the probe molecules to fall off or inactivate, so that the probe core cap particles stably dispersed in the aqueous solution are difficult to obtain. Therefore, the application provides a preparation method of the probe positioning modified soluble nano core cap, so that nano core cap particles modified with probe molecules only in the central region of the top of the cap can be freely dispersed in aqueous solution.
Disclosure of Invention
The invention aims to solve the technical problem of providing a preparation method of probe positioning modified soluble nano core cap particles, which enables seed probe molecules to be modified only in the central region of the outer surface of the cap top, and the rest part of the outer surface of the cap modifies system support molecules to stabilize the posture of the probe molecules and the system balance of the nano core cap particles in aqueous solution.
The technical scheme of the invention is as follows:
a method for preparing soluble nanometer core cap particles modified by probe positioning is characterized in that single-stranded DNA/RNA molecules are used as seed probes, the seed probes are only modified in the central area of the outer surface of the top of a nanometer core cap structure, and the rest parts of the outer surface of the cap are uniformly modified with system supporting molecules so as to stabilize the posture of the seed probes and balance the dispersion of the nanometer core cap particles in aqueous solution; the preparation method is characterized in that the method for controlling the installation position, the number and the posture of the seed probe comprises the following steps:
1) the single-stranded DNA/RNA seed probe is connected to the substrate through base pair complementation and DNA/RNA hybridization which has uniformly grown on the substrate, and the free end of the single-stranded DNA/RNA seed probe modified with sulfydryl is used for capturing precious metal nano particles;
2) immersing the substrate obtained in the step 1) into a solution dispersed with the nano core-shell particles, wherein the nano core-shell particles are captured and connected to the substrate through the sulfydryl of the seed probe;
3) after the substrate is cleaned and dried, the substrate is subjected to dry etching along the reverse direction of the external normal of the substrate, and part of metal shells of the nano core shell particles are removed;
4) immersing the etched substrate into an aqueous solution containing sulfhydryl molecules, wherein the sulfhydryl molecules are selected according to the following principle: the probe is connected to the outer surface of the nano core cap structure through sulfydryl, supports the posture of a probe molecule through carried charges, and stabilizes the balance of nano core cap particles in an aqueous solution;
5) the DNA/RNA double strand uncoils, releases the nano core cap particles into the solution, and obtains the nano core cap particles which are freely suspended in the solution and only decorated in the central area of the outer surface of the cap top by the seed probe;
according to the preparation method of the probe positioning modified soluble nano core cap particle, the area ratio of the central region of the outer surface of the probe modified cap top depends on the number (or chain length) of basic groups of a seed probe;
according to the preparation method of the probe positioning modified soluble nano core cap particles, the nano core shell particles are of a spherical symmetrical structure, and the outer surface of the spherical symmetrical structure is 100% covered with a noble metal material;
according to the preparation method of the probe-positioned modified soluble nano core cap particle, the seed probe can be firstly hybridized with intermediate DNA/RNA, and then the intermediate DNA/RNA is hybridized with target DNA/RNA to form a sandwich structure so as to adapt to a detection target.
The invention has the beneficial effects that: the seed probe molecules are self-positioned and only modified in the central area of the cap top of the three-dimensional space curved surface of the core cap, the rest part of the outer surface of the cap is modified by a system to support the molecules so as to stabilize the posture of the probe molecules and enable the nano core cap particles to be freely dispersed in the aqueous solution, and the prepared probe positioning modified soluble nano core cap particles can be used for high-specificity molecule detection, imaging and identification.
Drawings
FIG. 1 is a schematic diagram of a method for preparing a probe-location-modified soluble nano core cap particle.
Detailed Description
The embodiments are described in detail below with reference to the accompanying drawings and technical solutions.
Examples
A preparation method of probe positioning modified soluble nano core cap particles is shown in figure 1, and the nano core cap particles are characterized in that single-stranded DNA molecules are used as seed probes and are only modified in the central area of the outer surface of the top of a nano core cap structure, and the rest part of the outer surface of the cap is uniformly modified with system supporting molecules to stabilize the posture of the seed probes and the dispersion balance of the nano core cap particles in aqueous solution; the preparation method is characterized in that the method for controlling the installation position, the number and the posture of the seed probe comprises the following key steps:
1) DNA A (5' -NH)2-TTTTTTTTTTCGTCATCGTGCTG-3') is fixed on the base plate by the following steps: a. cleaningThe glass slide is immersed in 3-aminopropyl trimethoxy silane (APS) with the volume fraction of 0.5 percent, and is soaked in acetone after 2min, 5min each time (fresh acetone is replaced each time), and 10 times in total; oven drying for 45 min; soaking 1, 4-benzene diisothiocyanate (PDC) with the mass concentration of 1g/L for 45 min; soaking in methanol for 5 min; soaking in acetone for 5 min; drying at room temperature to obtain an isothiocyanate modified slide; b. uniformly spotting 2 mu M of DNA A fragment on the surface of the isothiocyanate modified slide by using a micropipette, wherein the spotting volume of each spot is 0.5 mu L; placing the glass slide in a constant temperature and humidity box with 37 ℃ and 90% humidity for heat preservation overnight, so that the DNA A and the glass slide are fully crosslinked; c. sealing the slide, washing with ammonia water with the volume fraction of 1% for 5min, washing with double distilled water and drying, and finally, covalently bonding the DNA A with the isothiocyanic acid group on the surface of the slide through the 5' end amino group of the DNA A and fixing the DNA A on the slide; after obtaining the slide glass on which the above reaction was completed, single-stranded DNA B (5 '-SH-CAGCACGATGACGAA-FAM-3') as a seed probe was hybridized with single-stranded DNA A uniformly grown on the substrate by base pair complementation to be ligated to the substrate, as follows: dripping a DNA B solution on the glass slide, and hybridizing for 6h at 37 ℃; washing with washing solution (2 × SSPE, 1% SDS) for 3 times, 5min each time, washing with water and drying; the free end of the DNA B modified with sulfydryl is used for capturing the noble metal nanoparticles;
2) immersing the hybridized slide glass into a 20mM Tris solution (pH7.4), and then adding a 0.1mM TCEP solution to activate the 5' terminal sulfhydryl of the seed probe DNAB; adding dispersed SiO2@ Au core-shell structure particle solution, overnight; then, dripping a NaCl and SDS mixed solution until the salt ion concentration is 0.1M, standing for 5 hours, then adding the NaCl and SDS mixed solution until the salt ion concentration is 0.2M, standing for 5 hours, then adding the NaCl and SDS mixed solution until the salt ion concentration is 0.3M, and standing for 5 hours; SiO 22The @ Au core-shell structure particles and mercapto groups of the seed probe molecules form Au-S covalent bonds through the surfaces of the gold shells and are fixed on the glass slide;
3) blowing N onto the substrate2Blow-drying, and dry etching along the reverse direction of the substrate external normal to remove SiO250% of gold shell of the @ Au core-shell structure particles to obtain SiO2@ Au core cap structured particles;
4) immersing the etched substrate into a hydrosulfuryl succinic acid aqueous solution, standing for 10h, wherein as the central area of the cap top of the core cap structure particles is modified with the seed probe, under the influence of steric hindrance factors, the other areas except the outer surface of the cap of the central area of the cap top are modified with the hydrosulfuryl succinic acid, so that probe molecules are ensured not to lie on the particle surface and the system balance of the particles in the aqueous solution;
5) passing the substrate through N2And drying, immersing the substrate in 1M NaCl buffer solution, heating in water bath to 70 ℃, uncoiling DNA double strands, and enabling a connector of the seed probe and the core cap structure particles to fall into the solution from the substrate, thereby obtaining the soluble nano core cap particles with the central area of the cap top positioned and modified.
It should be noted that: the above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (5)
1. A preparation method of probe positioning modified soluble nanometer core cap particles is characterized in that single-stranded DNA/RNA molecules are adopted as seed probes for the soluble nanometer core cap particles, the seed probes are only modified in the central area of the outer surface of the top of a nanometer core cap structure, and the rest parts of the outer surface of the cap are uniformly modified with system supporting molecules so as to stabilize the posture of the seed probes and balance the dispersion of the nanometer core cap particles in aqueous solution; the preparation method is characterized in that the method for controlling the installation position, the number and the posture of the seed probe comprises the following steps:
1) the single-stranded DNA/RNA seed probe is connected to the substrate through base pair complementation and DNA/RNA hybridization which has uniformly grown on the substrate, and the free end of the single-stranded DNA/RNA seed probe modified with sulfydryl is used for capturing precious metal nano particles;
2) immersing the substrate obtained in the step 1) into a solution dispersed with the nano core-shell particles, wherein the nano core-shell particles are captured and connected to the substrate through the sulfydryl of the seed probe;
3) after the substrate is cleaned and dried, the substrate is subjected to dry etching along the reverse direction of the external normal of the substrate, and part of the metal shell of the nano core shell particles is removed;
4) immersing the etched substrate into an aqueous solution containing sulfhydryl molecules, wherein the sulfhydryl molecules are selected according to the following principle: the probe is connected to the outer surface of the nano core cap structure through sulfydryl, supports the posture of a probe molecule through carried charges, and stabilizes the balance of nano core cap particles in an aqueous solution;
5) and (3) unwinding the DNA/RNA double strand, releasing the nano core cap particles into the solution, and obtaining the freely suspended nano core cap structure particles of which the seed probe only modifies the central area of the outer surface of the cap top in the solution.
2. The method for preparing the probe-localization-modified soluble nano core cap particle according to claim 1, wherein the area ratio of the central region of the outer surface of the top of the probe-modified cap depends on the number of bases or the chain length of the seed probe.
3. The method for preparing the probe location modified soluble nano core cap particle according to claim 1 or 2, wherein the nano core shell particle is a spherical symmetric structure with 100% of the outer surface covered with noble metal material.
4. The method as claimed in claim 1 or 2, wherein the seed probe is first hybridized with the intermediate DNA/RNA, and then hybridized with the target DNA/RNA to form a sandwich structure to adapt to the target.
5. The method as claimed in claim 3, wherein the seed probe is first hybridized with the intermediate DNA/RNA, and then hybridized with the target DNA/RNA to form a sandwich structure to adapt to the target.
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Citations (5)
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| CN2560946Y (en) * | 2002-06-11 | 2003-07-16 | 刘全俊 | Nano gold babel silver hepatitis-infected detection-type gene chip |
| CN101382491A (en) * | 2008-09-25 | 2009-03-11 | 湖南大学 | Long life luminous nanometer bio probe for detecting pathogenic microorganism and preparing and detecting method thereof |
| US20110159291A1 (en) * | 2009-12-24 | 2011-06-30 | Yi Sun | Solution stable and chemically reactive metallic nanoparticles |
| CN110412291A (en) * | 2019-07-30 | 2019-11-05 | 福建师范大学 | A method of building SERS spectra probe in detecting markers for breast cancer EGFR phosphorylated tyrosine |
| CN111204705A (en) * | 2020-01-14 | 2020-05-29 | 大连理工大学 | Preparation method of directionally assembled core-cap heterogeneous dimer structure |
-
2020
- 2020-08-10 CN CN202010794367.0A patent/CN111996186A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2560946Y (en) * | 2002-06-11 | 2003-07-16 | 刘全俊 | Nano gold babel silver hepatitis-infected detection-type gene chip |
| CN101382491A (en) * | 2008-09-25 | 2009-03-11 | 湖南大学 | Long life luminous nanometer bio probe for detecting pathogenic microorganism and preparing and detecting method thereof |
| US20110159291A1 (en) * | 2009-12-24 | 2011-06-30 | Yi Sun | Solution stable and chemically reactive metallic nanoparticles |
| CN110412291A (en) * | 2019-07-30 | 2019-11-05 | 福建师范大学 | A method of building SERS spectra probe in detecting markers for breast cancer EGFR phosphorylated tyrosine |
| CN111204705A (en) * | 2020-01-14 | 2020-05-29 | 大连理工大学 | Preparation method of directionally assembled core-cap heterogeneous dimer structure |
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| SARIT S. AGASTI等: "Nanoparticles for Detection and Diagnosis", 《ADV DRUG DELIV REV.》 * |
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