CN111392775A - RNA nucleic acid biological probe and preparation method thereof - Google Patents

RNA nucleic acid biological probe and preparation method thereof Download PDF

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CN111392775A
CN111392775A CN202010208327.3A CN202010208327A CN111392775A CN 111392775 A CN111392775 A CN 111392775A CN 202010208327 A CN202010208327 A CN 202010208327A CN 111392775 A CN111392775 A CN 111392775A
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蒋连福
董文英
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Nanjing Beige Electronic Technology Co ltd
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Abstract

The invention discloses an RNA nucleic acid biological probe and a preparation method thereof, belonging to the field of biological probes. The invention, by H2O2Preparation of orange MoO by oxidizing high-purity molybdenum powder3Adding a certain amount of absolute ethyl alcohol into the precursor solution, and then heating for 48 hours at 180 ℃ to obtain black MoO2‑MoO3Filtering, washing and drying the suspension to obtain high-purity one-dimensional MoO2‑MoO3A nanomaterial; last one-dimensional structure MoO2‑MoO3And modifying the nano material and coupling the modified nano material with the probe RNA. According to the invention, the substrate plate is introduced to accelerate the crystal growth speed and fineness of the precursor solution on the surface of the substrate plate in the vertical direction, so that the biological probe with high toughness and high sensitivity is obtained.

Description

RNA nucleic acid biological probe and preparation method thereof
The invention is a divisional application, and the original application information is as follows, the name: a preparation method of molybdenum oxide with a one-dimensional nano structure and application thereof are disclosed in the application number: 2019104703530, filing date: 2019.05.31.
Technical Field
The invention belongs to the field of biological probes, and particularly relates to an RNA nucleic acid biological probe and a preparation method thereof.
Background
MoO, a potential application of molybdenum oxide with performance increased by controlling and excavating morphology of molybdenum oxide through a plurality of research subjects in recent years2Is an excellent semi-metallic semiconductor material, although the research on the application base is far inferior to that of MoO3The method has wide application range, but has wider application effect in the aspect of building functional microelectronic devices; and due to MoO2Has huge surface area, which has obvious advantages in nano-scale interfacial chemistry and nano-adsorption.
The development of biosensors has been over 30 years, the existing biological probes generally select porous silicon sensitive materials as substrate materials, and the porous silicon sensitive nano materials have small particle size and large specific surface area, so that the high specific surface area promotes the combination of bioactive probes in porous silicon and detected molecules, and the biological sensitivity has many advantages. Meanwhile, due to the fact that the porous silicon is made of the non-metallic semiconductor material, on one hand, the toughness of the porous silicon is poor, a few of incompletely-formed substrates are easy to break in a long-term use process, the service life of the biosensor is greatly shortened, and on the other hand, due to the poor conductivity of the porous silicon, when the porous silicon is used as a biological probe substrate, metal nano particles need to be filled, and therefore the sensitivity of the biosensor is low.
Disclosure of Invention
The purpose of the invention is as follows: provides a preparation method of RNA nucleic acid biological probe to solve the problems in the background technology.
The technical scheme is as follows: the substrate of RNA nucleic acid biological probe includes the following steps:
the method comprises the following steps: the synthesized MoO2-MoO3Soaking the one-dimensional nano material in 0.01 mol/L isobutyl triethoxy silicon solution for 2h, washing with ionized water, drying, then continuing soaking in 2.5% glutaraldehyde aqueous solution for 2h, washing with phosphate buffer solution,Drying;
step two: with functionalized MoO2-MoO3One-dimensional nano material is used as a substrate, probe RNA with the concentration of 40 umol/L is dripped into MoO by a micropipette2-MoO3Standing the surface of the one-dimensional nano material for 2 hours at the constant temperature of 36.5 ℃, and then adding MoO2-MoO3The one-dimensional nano material is soaked in 3 mol/L Ethanolamine (EA) solution, placed for 2 hours at the constant temperature of 36.5 ℃, and finally washed by PBS to obtain unconjugated probe RNA.
In further embodiments, the MoO2-MoO3The preparation process of the one-dimensional nano material comprises the following steps:
s1, slowly adding 4g of high-purity molybdenum powder into 30-40 g of H for 4-8 times2O2And 58-68 g of purified water, and fully stirring for 3-5 h at normal temperature to form an orange precursor solution;
s2, adding absolute ethyl alcohol with the mass being 5-15% of that of the precursor solution into the orange precursor solution, and stirring for 2-3 h at constant temperature;
s3, adding the precursor mixed solution into a sealed reaction kettle, putting the reaction kettle into a constant-temperature heating box, heating for 48-96 hours at the temperature of 100-200 ℃, and obtaining black MoO after the reaction is finished2- MoO3Suspending liquid;
s4, filtering, washing and drying to obtain MoO2-MoO3A one-dimensional nanomaterial.
In further embodiments, the H2O2Is an oxidation source and has the mass fraction of 30 to 100 percent.
In further implementation, in the step S1, the molybdenum powder is mixed with H2O2The molar concentration ratio of (1): (25-30).
In the further implementation process, the reaction kettle shell in the steps S1 and S3 is made of high-temperature and high-pressure resistant sealed stainless steel, and a layer of sealed polytetrafluoroethylene material is embedded in the inner layer to form a protective layer.
In a further implementation process, in the step S1, a temperature measuring step is further included, in which a temperature measuring device is disposed in the reaction vessel for measuring the temperature of the reaction system; and detecting the absorbance in the orange solution by using a spectrophotometric agent, and calculating the concentration of the precursor solution.
In a further implementation, the step of S2 may further be: diluting the orange solution to a water solution with the mass concentration of 0.05g/ml, adding the diluted orange solution into a transparent glass container, then thoroughly cleaning and drying the surface of the substrate plate, stably placing the substrate plate on the liquid surface of the diluted solution, sealing the glass container, finally placing the sealed glass container into an oven, and heating for 6-24 hours at the temperature of 40-60 ℃.
In a further embodiment, the substrate plate has an area of 1 × 1cm2Hollow simple substance silicon is used as a main body, and the average density of the whole body is 1.1 g/ml; and a 600-900 nm silicon dioxide coating is plated on the surface of the simple substance silicon.
In a further implementation process, in the step S3, the filtering, washing, and drying processes specifically include:
the method comprises the following steps: solid MoO on filter paper2- MoO3The one-dimensional nano material is transferred into a beaker, and absolute ethyl alcohol washing liquid is sprayed to the solid MoO coagulated into blocks from a spray head at a high speed2- MoO3Scattering the one-dimensional nano material into particles;
step two: slightly stirring for 10-15 min to enable solid MoO2- MoO3One-dimensional nano material and absolute ethyl alcohol washing liquid form uniformly distributed heterogeneous dispersion liquid, and then the dispersion liquid is filtered and separated to obtain solid MoO2- MoO3A one-dimensional nanomaterial filter cake;
step three: repeating the first step and the second step for 1-2 times; replacing the absolute ethyl alcohol washing liquid with distilled water, and continuously repeating the first step and the second step for 2-3 times;
step four: drying solid MoO by natural air drying2- MoO3And (3) a one-dimensional nano material filter cake.
On the other hand, the application of the molybdenum oxide with the one-dimensional nano structure can be used as a substrate for manufacturing various biological probes.
In a further implementation, as
Has the advantages that: the invention relates to a preparation method of an RNA nucleic acid biological probe, which is prepared by H2O2Preparation of orange MoO by oxidizing high-purity molybdenum powder3Adding a certain amount of absolute ethyl alcohol into the precursor solution, and then heating for 48 hours at 180 ℃ to obtain black MoO2-MoO3Finally filtering, washing and drying the suspension to obtain high-purity MoO with a one-dimensional structure2-MoO3A nanomaterial; by introducing the substrate plate, the crystal growth speed and fineness of the precursor solution on the surface of the substrate plate in the vertical direction are accelerated. Provides a preparation method of a substrate material of a biological probe with high toughness and high sensitivity.
Drawings
FIG. 1 shows X-ray diffraction (XRD) data of molybdenum oxide samples obtained in examples 1 to 4.
FIG. 2 shows the red shift of the reflectance spectra for different RNA concentrations obtained in example 1.
FIG. 3 shows the RNA detection limit and sensitivity of the RNA nucleic acid bioprobe samples obtained in examples 1 and 4 to 6.
Detailed Description
In the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present invention. It will be apparent, however, to one skilled in the art, that the present invention may be practiced without one or more of these specific details. In other instances, well-known features have not been described in order to avoid obscuring the invention.
In order to solve the problems in the prior art, after the applicant intensively studies the preparation process of the existing biological probe-supporting material, the following findings are obtained: 1. most of the existing biological probes adopt porous silicon nano materials as substrates; the porous silicon nano material is mostly prepared by adopting an electrochemical carving method in the preparation process, and belongs to a top-down process for cutting and refining macroscopic substances by adopting a physical or chemical method to obtain the nano-scale substances. One dimension thus preparedThe purity, nano-fineness and one-dimensional structural integrity of the nano-materials are generally low, thus affecting the use effect and service life of the biological probe. And are not discussed in detail herein. 2. MoO in a conventional one-dimensional structure3Adding a certain amount of absolute ethyl alcohol, and in the hydrothermal reaction crystallization process, under the reduction action of the ethyl alcohol, MoO3Re-dissolving and re-crystallizing nano wire to obtain MoO2In a one-dimensional structure of MoO3A layer of rough MoO is formed on the surface2Layer, MoO of one-dimensional structure during long-term reaction3Can be completely converted into coarse MoO2. 3. When the substrate plate is placed on the surface of the solution, the substrate plate can float on the liquid surface due to the density of the substrate plate is close to that of the diluted solution and the tension between the substrate plate and the liquid surface, and because the lattice matching degree between the coating and molybdenum oxide is poor, the substrate plate can only perform crystal growth on the surface of the substrate plate and cannot grow into the coating, a layer of compact molybdenum oxide film is formed on the substrate plate, molybdenum oxide particles gradually crystallize downwards along the film under the action of gravity, and MoO with a one-dimensional structure is formed in the vertical direction3Array of wherein MoO3The surface is reduced by ethanol to generate MoO2Form MoO2-MoO3An array; by introducing a substrate plate, MoO is accelerated3And the precursor solution is subjected to crystallization growth speed and fineness in the vertical direction on the surface of the substrate plate.
In order to facilitate understanding of the technical scheme and the design idea, the applicant designs the following scheme for verification and explanation.
Example 1
A preparation method of an RNA nucleic acid biological probe comprises the following steps:
s1, 4g of high-purity molybdenum powder is slowly added into 30g of H by 4 times2O2And 68g of purified water, and fully stirring for 3 hours at normal temperature to form an orange precursor solution;
s2, adding absolute ethyl alcohol accounting for 5% of the mass of the precursor solution into the orange precursor solution, and stirring at constant temperature for 2 hours;
s3, adding the precursor mixed solution into a sealed reaction kettle at the temperature of 200 DEG CThen, heating for 48h to obtain black MoO after the reaction is finished2- MoO3Suspending liquid;
s4, filtering, washing and drying to obtain MoO2-MoO3A one-dimensional nanomaterial.
Wherein, the reaction kettle shell in the steps S1 and S3 is made of high temperature and high pressure resistant closed stainless steel, and the inner layer is embedded with a layer of closed polytetrafluoroethylene material to make a protective layer. In the step S1, a temperature measuring step is further included, in which a temperature measuring device is provided in the reaction vessel for measuring the temperature of the reaction system; after adding the molybdenum powder once, sampling once, and detecting the absorbance of the orange solution by using a spectrophotometric agent.
The specific steps of filtering, washing and drying are as follows:
step S401: solid MoO on filter paper2- MoO3The one-dimensional nano material is transferred into a beaker, and absolute ethyl alcohol washing liquid is sprayed to the solid MoO coagulated into blocks from a spray head at a high speed2- MoO3Scattering the one-dimensional nano material into particles;
step S402: slightly stirring for 15min to make solid MoO2- MoO3One-dimensional nano material and absolute ethyl alcohol washing liquid form uniformly distributed heterogeneous dispersion liquid, and then the dispersion liquid is filtered and separated to obtain solid MoO2- MoO3A one-dimensional nanomaterial filter cake;
step S403: repeating the first step and the second step 2 times; replacing the absolute ethyl alcohol washing solution with distilled water, and continuously repeating the first step and the second step for 3 times;
step S404: dry solid MoO2- MoO3A one-dimensional nanomaterial filter cake; and (5) adopting natural air drying.
The RNA nucleic acid biological probe is prepared by adopting the one-dimensional nano material, and the specific synthetic steps are as follows:
the method comprises the following steps: the synthesized MoO2-MoO3Soaking the one-dimensional nano material in 0.01 mol/L isobutyl triethoxy silicon solution for 2h, washing with ionized water, drying, and further soaking in 2.5% glutaraldehyde aqueous solution for 2hWashing with phosphate buffer solution and drying;
step two: with functionalized MoO2-MoO3One-dimensional nano material is used as a substrate, probe RNA with the concentration of 40 umol/L is dripped into MoO by a micropipette2-MoO3Standing the surface of the one-dimensional nano material for 2 hours at the constant temperature of 36.5 ℃, and then adding MoO2-MoO3The one-dimensional nano material is soaked in 3 mol/L Ethanolamine (EA) solution, placed for 2 hours at the constant temperature of 36.5 ℃, and finally washed by PBS to obtain unconjugated probe RNA.
Example 2
The present embodiment is different from embodiment 1 in that step S3: heating at 200 deg.C for 120 h. The rest of the procedure was the same as in example 1.
Example 3
The present embodiment is different from embodiment 1 in that step S2: adding absolute ethyl alcohol accounting for 15% of the mass of the precursor solution into the orange precursor solution, and stirring for 2 hours at constant temperature; the rest of the procedure was the same as in example 1.
Example 4
The present embodiment differs from embodiment 1 in that step S2 is not performed; the rest of the procedure was the same as in example 1.
Example 5
The difference between this example and example 1 is that in step S3, an orange-yellow precursor solution is diluted/purified to an aqueous solution with a mass concentration of 0.5 mol/L, then the surfaces of a plurality of substrate plates are thoroughly cleaned and dried, and the substrate plates are stably placed on the liquid level of the diluted solution, and finally the temperature of the reaction kettle is adjusted and heated at 40 ℃ for 24 hours, wherein the area of each substrate plate is 1 × 1cm2Hollow simple substance silicon is used as a main body, and the average density of the whole body is 1 g/ml; a600 nm copper plating layer is plated on the surface of the simple substance silicon. The rest of the procedure was the same as in example 1.
Example 6
The difference between this example and example 1 is that an RNA nucleic acid biological probe is prepared by using a porous silicon nanomaterial, and the specific steps are as follows:
step one, soaking the synthesized porous silicon nano material in isobutyl triethoxy silicon solution of 0.01 mol/L for 2h, washing with ionized water, drying, then continuing soaking in 2.5% glutaraldehyde aqueous solution for 2h, washing with phosphate buffer solution, and drying;
step two, taking the functionalized porous silicon nano material as a substrate, dropwise adding probe RNA with the concentration of 40 umol/L on the surface of the porous silicon nano material by using a micropipette, standing for 2 hours at the constant temperature of 36.5 ℃, then soaking the porous silicon nano material in 3 mol/L Ethanolamine (EA) solution, standing for 2 hours at the constant temperature of 36.5 ℃, and finally washing the unconjugated probe RNA by using PBS.
40u L complementary peptide chain with the concentration from 0.20 to 70.00 umol/L is dripped on the RNA biological probe by a micropipette, then the sample is placed in a 37 ℃ incubator for 1h to generate sufficient specific reaction, after the reaction is completed, the unreacted complementary DNA is removed by cleaning with physiological saline and is dried in nitrogen, then the reflection spectrum of each sample between 800nm and 2000nm is tested, the reflection spectrum is analyzed, and the corresponding red shift is calculated to obtain the accompanying figures 2-3. from the accompanying figure 2, the red shift of the reflection spectrum is linearly related to the concentration of the complementary DNA, wherein the slope of the linear equation is the sensitivity of the RNA biological probe, and the detection limit formula is that the detection limit = instrument minimum resolution/sensor sensitivity, wherein, the minimum resolution of the ultraviolet spectrophotometer adopted in the embodiment is 0.01 nm.
In the present invention, from the X-ray diffraction (XRD) data of the molybdenum oxide sample, it can be seen that: molybdenum oxide obtained in example 1 contains MoO as a main component2-MoO3Composition having an inner layer structure of MoO3The surface layer is MoO2(ii) a The molybdenum oxide obtained in examples 2 to 3 contained MoO as a main component2One-dimensional, linear structures have been destroyed and cannot become substrates for biological probes; molybdenum oxide obtained in example 4 contains MoO as a main component3. The data of RNA detection limit and sensitivity of the RNA nucleic acid bioprobe samples obtained in comparative example 1 and examples 4 to 6 are preferably the data of example 5.
It should be noted that the various features described in the above embodiments may be combined in any suitable manner without departing from the scope of the invention. The invention is not described in detail in order to avoid unnecessary repetition.

Claims (9)

1. A preparation method of an RNA nucleic acid biological probe is characterized by comprising the following steps:
the method comprises the following steps: the synthesized MoO2-MoO3Soaking the one-dimensional nano material in an isobutyl triethoxy silicon solution of 0.01 mol/L for 2h, washing with ionized water, drying, then continuously soaking in a 2.5% glutaraldehyde aqueous solution for 2h, washing with a phosphate buffer solution, and drying;
step two: with functionalized MoO2-MoO3One-dimensional nano material is used as a substrate, probe RNA with the concentration of 40 umol/L is dripped into MoO by a micropipette2-MoO3Standing the surface of the one-dimensional nano material for 2 hours at the constant temperature of 36.5 ℃, and then adding MoO2-MoO3The one-dimensional nano material is soaked in 3 mol/L Ethanolamine (EA) solution, placed for 2 hours at the constant temperature of 36.5 ℃, and finally washed by PBS to obtain unconjugated probe RNA.
2. The method for preparing RNA nucleic acid bioprobe according to claim 1, wherein the MoO is2-MoO3The preparation process of the one-dimensional nano material comprises the following steps:
s1, slowly adding 4g of high-purity molybdenum powder into 30-40 g of H for 4-8 times2O2Fully stirring the solution and 58-68 g of purified water at normal temperature for 3-5 h to form an orange precursor solution;
s2, diluting the orange solution to a water solution with the mass concentration of 0.05g/ml, adding the orange solution into a transparent glass container, then thoroughly cleaning and drying the surface of the substrate plate, stably placing the substrate plate on the liquid level of the diluted solution, sealing the glass container, finally placing the sealed glass container into an oven, and heating for 6-24 hours at the temperature of 40-60 ℃;
s3, adding the precursor mixed solution into a sealed reaction kettle, putting the reaction kettle into a constant-temperature heating box, heating for 48-96 hours at the temperature of 100-200 ℃, and obtaining black MoO after the reaction is finished2- MoO3Suspending liquid;
s4, filtering, washing and drying to obtain MoO2-MoO3A one-dimensional nanomaterial.
3. The method for preparing an RNA nucleic acid bioprobe according to claim 2, wherein the H is2O2The solution is an oxidation source and the mass fraction of the solution is 30-100%.
4. The method for preparing RNA nucleic acid bioprobe according to claim 2, wherein in the step S1, the molybdenum powder and H2O2The molar concentration ratio of (1): (25-30).
5. The method of claim 2, wherein the outer shell of the reaction vessel is made of high temperature and high pressure resistant stainless steel, and the inner layer is embedded with a protective layer made of a sealed polytetrafluoroethylene material in steps S1 and S3.
6. The method for preparing an RNA nucleic acid bioprobe according to claim 2, further comprising a temperature measuring step of setting a temperature measuring device in the reaction vessel for measuring the temperature of the reaction system in step S1; and detecting the absorbance in the orange solution by using a spectrophotometric agent, and calculating the concentration of the precursor solution.
7. The method for preparing RNA nucleic acid bioprobe according to claim 2, wherein the area of the substrate plate is 1 × 1cm2Hollow simple substance silicon is used as a main body, and the average density of the whole body is 1.1 g/ml; and a 600-900 nm silicon dioxide coating is plated on the surface of the simple substance silicon.
8. The method for preparing the RNA nucleic acid bioprobe according to claim 2, wherein the step S3 comprises the following steps:
the method comprises the following steps: solid MoO on filter paper2-MoO3The one-dimensional nano material is transferred into a beaker, and absolute ethyl alcohol washing liquid is sprayed to the solid MoO coagulated into blocks from a spray head at a high speed2-MoO3Scattering the one-dimensional nano material into particles;
step two: slightly stirring for 10-15 min to enable solid MoO2-MoO3One-dimensional nano material and absolute ethyl alcohol washing liquid form uniformly distributed heterogeneous dispersion liquid, and then the dispersion liquid is filtered and separated to obtain solid MoO2-MoO3A one-dimensional nanomaterial filter cake;
step three: repeating the first step and the second step for 1-2 times; replacing the absolute ethyl alcohol washing liquid with distilled water, and continuously repeating the first step and the second step for 2-3 times;
step four: drying solid MoO by natural air drying2-MoO3And (3) a one-dimensional nano material filter cake.
9. An RNA nucleic acid biological probe obtained by the preparation method according to any one of claims 1 to 8.
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Application publication date: 20200710