CN118050342A - Charge transfer Raman enhanced substrate material based on plasma resonance enhancement, preparation and application thereof - Google Patents

Charge transfer Raman enhanced substrate material based on plasma resonance enhancement, preparation and application thereof Download PDF

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CN118050342A
CN118050342A CN202211423242.2A CN202211423242A CN118050342A CN 118050342 A CN118050342 A CN 118050342A CN 202211423242 A CN202211423242 A CN 202211423242A CN 118050342 A CN118050342 A CN 118050342A
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nano
raman
dna
array
mir
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田阳
郑婷婷
冯恩铎
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East China Normal University
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East China Normal University
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Priority to PCT/CN2023/127688 priority patent/WO2024104104A1/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering

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  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
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  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

The invention discloses a charge transfer Raman enhanced substrate material based on plasma resonance enhancement and a preparation method thereof. Firstly, a novel Surface Enhanced Raman Spectrum (SERS) substrate with a Raman enhancement factor as high as 3.19 multiplied by 10 10, namely a Cu 2 O nano array, is prepared by taking a Cu 2 O nano wire as a basic unit through a self-assembly mode; secondly, the Cu 2 O nano array spacing is successfully controlled in a large range by the surface modification and the nano material co-assembly; finally, by utilizing the extremely high SERS sensitivity and selectivity of the Cu 2 O nano array to the signal molecule PTCDA and taking a specific nucleic acid sequence as a specific recognition unit of miR-34c, a miR-34c Raman analysis chip based on the Cu 2 O nano array is constructed, and the quick analysis and detection of the representative miR-34c are realized.

Description

Charge transfer Raman enhanced substrate material based on plasma resonance enhancement, preparation and application thereof
Technical Field
The invention discloses a charge transfer Raman enhanced substrate material based on plasma resonance enhancement, a preparation method and application thereof, and belongs to the technical fields of material synthesis and biosensing.
Background
Mirnas are an important class of signaling substances in organisms that play an important role in a variety of disease metabolic processes, such as neurodegenerative diseases, cancer, and many other chronic diseases. Although the concentration content of miRNA in body fluid is low and is only fM-nM, the tiny concentration change of miRNA often indicates the occurrence of serious diseases, so that an ultra-high-sensitivity analysis and detection means is constructed, and the high-sensitivity rapid analysis and detection of miRNA in body fluid is of great significance to screening and rapid diagnosis of human diseases. Surface Enhanced Raman Spectroscopy (SERS) is a spectroscopic analysis technique that is capable of increasing the raman signal of molecules adsorbed on the surface of a material by a factor of about 10 6. Thus, SERS-based analytical techniques generally have extremely high sensitivity, enabling analytical detection of trace species in a variety of environments. However, the conventional SERS enhancement substrate mainly uses precious metal nano materials such as Au, ag, cu, and the like, and has shortcomings in analysis selectivity, signal stability, and the like.
In contrast, the semiconductor nano SERS substrate represented by TiO 2、Cu2 O exhibits more excellent raman molecular selectivity and signal stability. But is limited by its poor SERS enhancement effect, such semiconductor SERS substrate materials have poor analytical sensitivity, and are very challenging to analyze and detect for trace species. Therefore, the development of semiconductor nanomaterials with high enhancement factors is an important solution to achieve high selectivity, high sensitivity, high accuracy surface enhanced raman spectroscopy. Currently, a variety of semiconductor nanomaterials, including CuTe, WO 3-x, have been used as SERS substrates and achieve raman enhancement effects of about 10 6. However, it is still difficult to meet the sensitivity requirements of miRNA detection.
Disclosure of Invention
The invention aims to provide a charge transfer Raman enhancement substrate material-Cu 2 O nano-array based on plasma resonance enhancement, wherein the Cu 2 O nano-array is used as a brand new charge transfer Raman enhancement substrate material based on plasma resonance enhancement, an important basis is provided for the preparation of a miR-34c nucleic acid sequence Raman analysis chip, and the miR-34c nucleic acid sequence Raman analysis chip prepared by the invention has the advantages of high analysis speed, high molecular selectivity, high detection sensitivity, low price, simplicity and convenience in operation, easiness in storage and the like.
The invention provides a method for synthesizing Cu 2 O nanowires, which is characterized in that a hydrothermal method is adopted to take copper acetate as a raw material, O-methoxy aniline as a reducing agent and a surfactant, and the preparation of the Cu 2 O nanowires is realized under a relatively mild condition, and comprises the following steps:
(1) The Cu 2 O nanowire is prepared by placing a solution containing copper acetate (CuAc 2), ultrapure water and O-methoxy aniline into a hydrothermal reaction kettle through a hydrothermal method and performing high-temperature treatment.
(2) And (3) centrifugally washing the Cu 2 O nanowire obtained by the hydrothermal method in the step (1) for multiple times to remove impurities, and drying to obtain a pure Cu 2 O nanowire product.
In the step (1), the hydrothermal reaction kettle is preferably respectively pre-ultrasonically cleaned in ultrapure water and absolute ethyl alcohol for 15 minutes, and is preferably used after being dried by nitrogen.
In the step (1), the dosage ratio of the CuAc 2, the ultrapure water and the o-methoxy aniline is 0.05g-0.2g:30-50mL:0.05-0.2mol/L; preferably 0.1g:50mL:0.1mol/L.
In the step (1), the mixing time of the solution is 15-45 min; preferably 30min.
In the step (1), the volume of the hydrothermal reaction kettle is 50mL or 100mL; preferably 50mL.
In the step (1), the temperature of the hydrothermal reaction is 160-200 ℃; preferably 180 ℃.
In the step (1), the time of the hydrothermal reaction is 8-15 h; preferably 10h.
In the step (2), the centrifugal rotating speed is 3000rpm-8000rpm; preferably 5000rpm.
In the step (2), the centrifugation time is 3-10 min; preferably, it is 5min.
In the step (2), the washing solvent is one or two of ultrapure water, absolute methanol, absolute ethanol and acetone; preferably ultrapure water and absolute ethanol.
In the step (2), the washing times of the solvent are 2-5 times; preferably 5 times.
In the step (2), the temperature of the drying process is 40-70 ℃; preferably 60 ℃.
In the step (2), the time of the drying process is 8-12 h; preferably 10h.
In a specific embodiment, the step of synthesizing the Cu 2 O nanowire comprises: 0.1g of CuAc 2 was dissolved in 50mL of ultrapure water, o-methoxyaniline was added thereto at a concentration of 0.1mol/L, and the mixture was sufficiently stirred for 30 minutes, transferred to a 50mL hydrothermal reaction vessel, and further, the temperature was kept at 180℃in an oven for 10 hours. Then, the hydrothermal reaction kettle is naturally cooled to room temperature, the dispersion liquid is transferred into a centrifuge tube, and the precipitate is obtained by centrifugation at 5000 rpm. The product was further washed 5 times with ultrapure water and absolute ethanol as washing solvents, respectively. Finally, the obtained product is dried for 10 hours at 60 ℃ to obtain the final product.
The invention also provides the Cu 2 O nanowire prepared by the method.
The invention also provides application of the Cu 2 O nanowire in preparation of a Cu 2 O nano array, pitch regulation and control, surface enhanced Raman spectrum, miRNA analysis and detection and the like.
The invention also provides a preparation method of the Cu 2 O nano-array, which is characterized in that the surface of the Cu 2 O nano-wire is modified by adopting a surfactant, and the regular self-assembly of the Cu 2 O nano-wire is realized by utilizing an oil-water interface, so that the Cu 2 O nano-array is constructed, and the specific steps are as follows:
Carrying out surface modification on the Cu 2 O nanowire, and obtaining surface modified Cu 2 O through centrifugal separation; based on Langmuir-Blodgett (LB) method, self-assembly of modified Cu 2 O nanowires is carried out at an oil/water interface, and the modified Cu 2 O nanowires are placed in an oven for drying to obtain a final product, namely a Cu 2 O nanoarray.
Wherein the surface modifying active agent of the Cu 2 O nanowire is one of polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), polystyrene (PS), cetyl Trimethyl Ammonium Chloride (CTAC) and the like; preferably Polystyrene (PS).
Wherein the molecular weight of the surface modification active agent used for assembling the Cu 2 O nano-array is one of 100-1000000; preferably 100000.
Wherein the surface modifying active agent modifying group is sulfhydryl (-SH), amino (-NH 2) and carboxyl (-COOH); preferably, it is a sulfhydryl group (-SH).
Wherein the polystyrene concentration is 1mM-50mM; preferably 10mM.
Wherein the dosage of the Cu 2 O nanowire is 10-100 mug; preferably 30 μg.
Wherein the solvent used for modifying the surface of the Cu 2 O nanowire is one of ultrapure water, absolute ethyl alcohol and absolute methyl alcohol; preferably ultrapure water.
Wherein the time for the surface modification process of the Cu 2 O nanowire is 8-15h; preferably 12h.
Wherein the temperature used in the surface modification process of the Cu 2 O nanowire is 20-40 ℃; preferably 30 ℃.
Wherein the centrifugal speed of the separation of the modified Cu 2 O nanowire is 3000rpm-8000rpm; preferably 8000rpm.
Wherein the separation and centrifugation time of the modified Cu 2 O nanowire is 3-10min; preferably, it is 5min.
Wherein the oil-water interface used in the LB assembling process is one of water/chloroform, water/cyclohexane and water/dichloromethane; preferably, water/cyclohexane.
Wherein the ratio of the oil-water interface is 1:0.5-1:3; preferably 1:1.
Wherein the total volume of the oil-water dispersion solvent is 0.5-2mL; preferably 1mL.
Wherein the concentration rate of the oil-water interface is 10-30cm 2/min; preferably 20cm 2/min.
Wherein the drying temperature is 40-80 ℃; preferably 60 ℃.
The invention also provides the Cu 2 O nano-array prepared by the method, a surface structure modification and coupling method of the Cu 2 O nano-array, and application of the Cu 2 O nano-array in constructing a Raman analysis chip.
The invention also provides a method for regulating and controlling the spacing between nanowires of the Cu 2 O nano-array and application of the Cu 2 O nano-array in surface enhanced Raman spectroscopy and miRNA analysis.
The preparation of the Cu 2 O nano-array refers to the assembly of the synthesized Cu 2 O nano-wire with a disordered structure into a nano-assembly body with a uniform structure and a unique fixed distance, namely the Cu 2 O nano-array, and the main technical points of the preparation are factors such as solvent selection, evaporation rate and the like used in the self-assembly process of the Cu 2 O nano-wire. On the basis of Cu 2 O nano-arrays, in the further experiments of the invention, a mode for regulating and controlling the spacing of the Cu 2 O nano-arrays is invented, and the technical points are mainly focused on the selection of the types and the molecular weights of the surfactants.
The invention also provides a method for regulating and controlling the spacing of the Cu 2 O nano-array, which comprises the following specific steps: the Cu 2 O nano array (2-20 nm) with adjustable space is obtained after drying according to the preparation method of the Cu 2 O nano array by adjusting the molecular weight and the type of the Cu 2 O nano wire surfactant.
Because the types and the molecular weights of the surfactants have important influence on the spacing regulation of the Cu 2 O nano-arrays in the invention, the key technical difficulties of the spacing regulation of the Cu 2 O nano-arrays in the invention are mainly that: firstly, how to realize the specific modification of different surfactants on the surface of a nano array element Cu 2 O nanowire; secondly, selecting a proper surfactant as a surface modifying active agent of the Cu 2 O nanowire according to factors such as molecular structures, molecular electrical properties, interaction relations and the like of different types of surfactants; thirdly, selecting proper molecular weight of the surfactant according to the nature of the given surfactant, and realizing continuous regulation of the pitch of the nano array.
The distance regulating and controlling part of the Cu 2 O nano-array relates to two main variables, namely the type of the surfactant and the molecular weight of the surfactant. The types of the surfactants are the basis of Cu 2 O nanowire assembly, and as the molecular structures and intermolecular interaction relations of different surfactants are different, the selection of the surfactants with proper physical and chemical properties (such as surface charge, hydrophilicity and hydrophobicity, modification groups and the like) is a key for completing Cu 2 O nanowire assembly. The pitch of the nano-arrays produced by different interactions among different surfactant molecules is also different, so that the regulation and control of the material pitch can be realized by changing the types of the surfactants. On the other hand, for a specific surfactant, by changing the molecular weight of the surfactant, the interactions among the surfactant molecules are also obviously different, so that the pitch regulation of the Cu 2 O nano-array can be realized.
Wherein, the surface modifying active agent of the Cu 2 O nanowire is one of polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), polystyrene (PS), cetyltrimethylammonium chloride (CTAC) and cetyltrimethylammonium bromide (CTAB); preferably polyvinylpyrrolidone (PVP).
Wherein the molecular weight of the surface modifying active agent used for assembling the Cu 2 O nano-array is adjusted to be one of 300-300000 according to different nano-array pitches; preferably 10000 (pitch 2 nm), 100000 (pitch 10 nm), 200000 (pitch 20 nm).
Wherein, the surface modification active modification group of the Cu 2 O nano-array is mercapto (-SH), amino (-NH 2) and carboxyl (-COOH); preferably, it is a sulfhydryl group (-SH).
Wherein the concentration of polyvinylpyrrolidone (PVP) is 1mM-50mM; preferably 10mM.
In one specific embodiment, the preparation method of the Cu 2 O line nano-array comprises the following steps: the Cu 2 O nanowire prepared as described above was taken as a basic assembly unit, and polystyrene (SH-PS) having a thiol-modified molecular weight of 50000 was dissolved in 5mL of ultrapure water to prepare a 10mM SH-PS solution, to which 30. Mu.g of Cu 2 O nanowire powder was added, and stirring was continued at 30℃for 12 hours. And centrifuging at 8000rpm for 5min, and collecting precipitate to obtain PS modified Cu 2 O nanowire. Subsequently, the resulting nanowires were well dispersed in 500. Mu.L cyclohexane and transferred to the surface of a 2cm 2 single crystal silicon substrate. Then 500. Mu.L of ultrapure water is dripped on the surface of the monocrystalline silicon substrate, and after the interface is stabilized, the sample is concentrated at a speed of 20cm 2/min in a dry environment. And after the sample is completely dried, cleaning the surface by absolute ethyl alcohol, and finally drying the sample in an oven at 60 ℃ overnight to obtain the Cu 2 O nano-array. Likewise, the continuous regulation and control of the spacing of the Cu 2 O nano array between 2nm and 20nm can be realized by adjusting the types and the molecular weights of the Cu 2 O nano-wire surfactant. Specifically, when the surface active group is changed into mercapto-modified polyvinylpyrrolidone (SH-PVP) with molecular weight of 10000, cu 2 O nano-arrays with interval of 2nm can be prepared and obtained. When the molecular weight of SH-PS used was adjusted to 100000 or 200000, cu 2 O nanoarrays with pitches of 10nm and 20nm, respectively, could be obtained.
The invention also provides the Cu 2 O nano array with adjustable space, which is prepared by the method.
The invention also provides application of the Cu 2 O nano-array with adjustable spacing in surface enhanced Raman spectroscopy and miRNA analysis.
The invention also tests the performance of the Cu 2 O nano-array as a novel Surface Enhanced Raman Spectroscopy (SERS) substrate and analyzes the enhancement mechanism.
And (3) taking the Cu 2 O nano-array as a SERS substrate material, selecting small organic signal molecules as Raman signal molecules, and testing the SERS enhancement effect of the Cu 2 O nano-array. The test shows that the Cu 2 O nano array with adjustable space produces obvious Raman enhancement effect on small organic signal molecules, and the Raman enhancement factor can reach 3.19 multiplied by 10 10 through calculation.
Wherein, the supporting substrate used by the Cu 2 O nano array is one of a silicon wafer, a cover glass, a glass slide and a quartz plate; preferably a silicon wafer.
Wherein the size of the silicon wafer is one of 0.5-2cm 2; preferably 1cm 2.
Wherein, the spacing of the nanowires in the Cu 2 O nano array is one of 2-20 nm; preferably 5nm.
Wherein the organic signal small molecule is one of perylene tetracarboxylic dianhydride (PTCDA), rhodamine 6G (R6G), rhodamine B (RhB), cyanine and derivatives thereof, tetracyanoquinodimethane (TCNQ) and derivatives thereof, bathocuproine (BCP) and derivatives thereof, phenylacetylene and derivatives thereof; preferably perylene tetracarboxylic dianhydride (PTCDA).
Wherein the concentration of the organic signal small molecule solution is 1nM-100 mu M; preferably 100nM.
Wherein the volume of the organic signal small molecule solution is 10 mu L-200 mu L; preferably 100 μl.
Wherein the laser wavelength of the Raman analysis detection is one of 532nm, 633nm and 785 nm; preferably 633nm.
Wherein the laser power used in the Raman spectrum test is 0.5-5mW; preferably 1mW.
Wherein the test exposure time used for the Raman spectrum test is 0.5-2s; preferably 1s.
Wherein the test exposure time used for the Raman spectrum test is 0.5-2s; preferably 1s.
The Cu 2 O nano array is used as a base material, small organic molecules are selected as representative molecules, a Cu 2 O nano array-molecule composite is constructed, and a transient absorption spectrum is adopted to characterize a Raman enhancement mechanism of the composite.
Wherein, the supporting substrate used by the Cu 2 O nano array is one of a silicon wafer, a cover glass, a glass slide and a quartz plate; preferably a quartz plate.
Wherein the size of the quartz plate is one of 2-5cm 2; preferably 4cm 2.
Wherein, the spacing of the nanowires in the Cu 2 O nano array is one of 2-20 nm; preferably 5nm.
Wherein the organic signal small molecule is one of perylene tetracarboxylic dianhydride (PTCDA), rhodamine 6G (R6G), rhodamine B (RhB), cyanine and derivatives thereof, tetracyanoquinodimethane (TCNQ) and derivatives thereof, bathocuproine (BCP) and derivatives thereof, phenylacetylene and derivatives thereof; preferably perylene tetracarboxylic dianhydride (PTCDA).
Wherein the concentration of the organic signal small molecule solution is 1nM-100 mu M; preferably 100nM.
Wherein the volume of the organic signal small molecule solution is 10 mu L-200 mu L; preferably 100 μl.
The laser wavelength of the transient absorption spectrum test is one of 325nm, 630nm and 530 nm; preferably 630nm.
Wherein the laser power used in the Raman spectrum test is 0.5-5mW; preferably 1mW.
The Raman spectrum test uses a test time range of 100fs-1ns; preferably 100fs-500ps.
In a specific embodiment, a silicon wafer is used as a Cu 2 O nano array assembly substrate, a Cu 2 O nano array SERS substrate with a nanowire spacing of 5nm is prepared, 100nM (nanometer-wavelength-scanning complementary metal oxide semiconductor) and 100 mu L PTCDA (pulse length division multiple access) are used as Raman signal molecules, 633nm is used as an excitation wavelength, and analysis and detection are carried out on the SERS performance of the Cu 2 O nano array, wherein the laser power is 1mW and the exposure time is 1s. Then, a Cu 2 O nano-array was prepared using a quartz plate as an assembly substrate, and a transient absorption spectrum test was performed using 100nM,100 μL PTCDA as a signal molecule. Wherein the excitation light wavelength used was 630nm. Scanning wavelength ranges from 100fs to 500ps, and the Cu 2 O nano-array surface enhanced Raman spectrum mechanism is verified.
The invention also provides a preparation method of the Raman analysis chip of the miR-34c nucleic acid sequence, and the surface enhanced Raman spectrum performance test of the Cu 2 O nano array with adjustable spacing shows that the Cu 2 O nano array with the spacing of 5nm can generate the optimal surface enhanced Raman effect. In order to obtain higher sensitivity, the Raman chip is constructed on the basis of a Cu 2 O nano-array with a 5nm pitch, and the method comprises the following steps:
(1) First, a PBS solution containing thiol-modified DNA capture chains (SH-DNA) was dropped onto the prepared surface of Cu 2 O nanoarrays with a pitch of 5nm, and incubated, to prepare SH-DNA modified Cu 2 O nanoarrays (SH-DNA-Cu 2 ONAs).
(2) Adding Raman signal molecules, N-hydroxysuccinimide (NHS) and amino-modified DNA signal chains (DNAPRIMER) into a solution containing 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), reacting for a period of time, adding hydrochloric acid into the solution, repeatedly washing and filtering to obtain a solid product which is a DNA signal probe.
(3) And (3) dropwise adding a reference small molecule on the surface of the SH-DNA modified Cu 2 O nano array, and drying to prepare SH-DNA-Cu 2 O NAs with a reference signal, thereby obtaining the miR-34c Raman analysis chip based on the Cu 2 O nano array.
In step (1), the nucleic acid sequence of the DNA capture strand (SH-DNA) is:
5’-SH-GCCACTGTGCAGCTAACTACACTGCCTGCAGACT-3’(SEQ ID NO.1)。
in step (1), the nucleic acid sequence of the DNA signal strand (DNA-primer) is:
5’-CCCGCAATC-NH2-3’。
in the step (1), the SH-DNA concentration in PBS solution is 10-50 mu M; preferably 20. Mu.M.
In the step (1), the volume of the PBS solution of SH-DNA dripped on the surface of the Cu 2 O nano-array is 100 mu L-1mL; preferably 500. Mu.L.
In the step (1), the incubation temperature of the SH-DNA and the Cu 2 O nano array is 20-50 ℃; preferably 37 ℃.
In the step (1), the incubation time of the SH-DNA and the Cu 2 O nano array is 3-5 h; preferably 3h.
In the step (2), the raman signal molecule is one of perylene tetracarboxylic dianhydride (PTCDA), rhodamine 6G (R6G), rhodamine B (RhB), cyanine and derivatives thereof; preferably perylene tetracarboxylic dianhydride (PTCDA).
In the step (2), the dosage of the Raman signal molecules is 100 mu M-1000 mu M; preferably 500 μm.
In the step (2), the dosage of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) is 2mg-20mg; preferably 10mg.
In the step (2), the dosage of the N-hydroxysuccinimide (NHS) is 1mg-10mg; preferably 5mg.
In the step (2), the total volume of the solution containing 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) is 1mL-20mL; preferably 10mL.
In the step (2), the amount of the amino-modified DNA signal chain (DNAPRIMER) is 10 mu M-100 mu M; preferably 20. Mu.M.
In the step (2), the reaction time for modifying the DNA-primer by the Raman signal molecule is 10-20 h; preferably 16h.
In the step (2), the reaction temperature used by the Raman signal molecule for modifying the DNA-primer is 60-90 ℃; preferably 85 ℃.
In the step (2), the concentration of the hydrochloric acid is 0.5M-2M; preferably 1M.
In the step (2), the volume of the hydrochloric acid is 50-200 mL; preferably 100mL.
In the step (2), the repeated times of the hydrochloric acid washing process are 2-5 times; preferably 3 times.
In the step (3), the reference small molecule is one of Tetracyanoquinodimethane (TCNQ) and derivatives thereof, bathocuproine (BCP) and derivatives thereof, phenylacetylene and derivatives thereof; preferably, it is Tetracyanoquinodimethane (TCNQ).
In the step (3), the concentration of the reference small molecule solution is 100 mu M-2mM; preferably 1mM.
In the step (3), the volume of the reference small molecule solution is 100-1000 mu L; preferably 500. Mu.L.
In the step (3), the drying time of the reference small molecules is 30-60 min; preferably 30min.
In a specific embodiment, the preparation steps of the miR-34c Raman analysis chip comprise: mu.L of a 20. Mu.M SH-DNA solution was dropped onto the surface of the Cu 2 O nanoarray prepared in the step (2) at a pitch of 5nm, and incubated at a constant temperature for 3 hours at 37 ℃. Then 500 mu L of 1mM of reference small molecule TCNQ is dripped on the surface of the nano array, further incubation is carried out for 30min at 37 ℃, and then RNase-free water is used for cleaning the surface, so that the miR-34c Raman analysis chip based on the Cu 2 O nano array is obtained. Meanwhile, to 1mL of an aqueous solution containing 10mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 5mg of N-hydroxysuccinimide (NHS), DNA-primer was added to a concentration of 20. Mu.M, perylene tetracarboxylic dianhydride (PTCDA) was then added thereto to 500. Mu.M, the above reaction system was further reacted at a constant temperature of 85℃for 16 hours, 100mL of a washing product of 1M hydrochloric acid was further reused three times, and the precipitate was filtered and kept, and dried in an oven at 40℃for 15 hours to obtain a DNA signaling probe.
The invention also provides a Raman analysis chip of the miR-34c nucleic acid sequence prepared by the method.
The invention also provides application of the Raman analysis chip of the miR-34c nucleic acid sequence in miR-34c high-sensitivity analysis and detection.
The invention also provides a miR-34c rapid sensitive analysis and detection method based on the Raman analysis chip and a DNA signal probe, which comprises the following steps:
(1) And dripping an RNase-free mixed system containing miR-34c gene sequences with different concentrations and DNA signal probes onto the surface of the Raman analysis chip for mixed incubation.
(2) And (3) cleaning the surface of the Raman analysis chip by RNase-free water and quantitatively analyzing by adopting a Raman spectrometer.
In the step (1), the volume of the RNase-free mixed system is 200-800 mu L; preferably 500. Mu.L.
In the step (1), the concentration of the DNA signaling probe is 0.5-2 mu M; preferably 1 μm.
In the step (1), the temperature of mixed incubation is 20-50 ℃; preferably 37 ℃.
In the step (1), the mixed incubation time is 5-20min; preferably 15min.
In the step (2), the amount of RNase-free water is 1mL-2mL; preferably 1mL.
In the step (2), the times of washing the RNase-free water are 1 to 3 times; preferably 2 times.
In the step (2), the laser wavelength used in the raman spectrum test is one of 532nm, 633nm and 785 nm; preferably 633nm.
In the step (2), the laser power used in the Raman spectrum test is 0.5-5mW; preferably 1mW.
In the step (2), the test exposure time used in the Raman spectrum test is 0.5-2s; preferably 1s.
In a specific embodiment, the method of detecting miR-34c comprises: mu.L of RNase-free solution containing miR-34c at different concentrations and 1. Mu.M DNA signaling probe was added dropwise to the surface of the Raman analysis chip, and incubated at 37℃for 15min. After washing twice with 1mL of RNase-free water, detection was performed by a Raman spectrometer using a laser wavelength of 633nm with a laser power of 1mW and an exposure time of 1s.
According to the invention, the specific analysis and detection of miR-34c sequences in a sample can be realized by measuring the Raman enhancement effects of different degrees of signal molecules PTCDA and reference molecules TCNQ on a Cu 2 O nano-array and by using the ratio of PTCDA Raman signal intensity to TCNQ Raman signal intensity.
In one specific embodiment of the invention, the construction method of the Cu 2 O nano-array SERS substrate and the detection method for miR-34c sequence based on the Cu 2 O nano-array comprise the following steps:
(1) Preparation of Cu 2 O nanowire
Synthesis of Cu 2 O nanowires: 0.1g of CuAc 2 was dissolved in 50mL of ultrapure water, o-methoxyaniline was added thereto at a concentration of 0.1mol/L, and the mixture was sufficiently stirred for 30 minutes, transferred to a 50mL hydrothermal reaction vessel, and further, the temperature was kept at 180℃in an oven for 10 hours. Then, the hydrothermal reaction kettle is naturally cooled to room temperature, the dispersion liquid is transferred into a centrifuge tube, and the precipitate is obtained by centrifugation at 5000 rpm. The product was further washed 5 times with ultrapure water and absolute ethanol as washing solvents, respectively. Finally, the obtained product is dried for 10 hours at 60 ℃ to obtain the final product.
(2) Preparation of Cu 2 O line nano array
The Cu 2 O nanowire prepared as described above was taken as a basic assembly unit, and polystyrene (SH-PS) having a thiol-modified molecular weight of 50000 was dissolved in 5mL of ultrapure water to prepare a 10mM SH-PS solution, to which 30. Mu.g of Cu 2 O nanowire powder was added, and stirring was continued at 30℃for 12 hours. And centrifuging at 8000rpm for 5min, and collecting precipitate to obtain PS modified Cu 2 O nanowire. Subsequently, the resulting nanowires were well dispersed in 500. Mu.L cyclohexane and transferred to the surface of a 2cm 2 single crystal silicon substrate. Then 500. Mu.L of ultrapure water is dripped on the surface of the monocrystalline silicon substrate, and after the interface is stabilized, the sample is concentrated at a speed of 20cm 2/min in a dry environment. And after the sample is completely dried, cleaning the surface by absolute ethyl alcohol, and finally drying the sample in an oven at 60 ℃ overnight to obtain the Cu 2 O nano-array. Likewise, the continuous regulation and control of the spacing of the Cu 2 O nano array between 2nm and 20nm can be realized by adjusting the types and the molecular weights of the Cu 2 O nano-wire surfactant. Specifically, when the surface active group is changed into mercapto-modified polyvinylpyrrolidone (SH-PVP) with molecular weight of 10000, cu 2 O nano-arrays with interval of 2nm can be prepared and obtained. When the molecular weight of SH-PS used was adjusted to 100000 or 200000, cu 2 O nanoarrays with pitches of 10nm and 20nm, respectively, could be obtained.
(3) SERS performance characterization and Raman enhancement mechanism analysis of Cu 2 O nano-array
The method comprises the steps of taking a silicon wafer as a Cu 2 O nano array assembly substrate, preparing a Cu 2 O nano array SERS substrate with a nanowire spacing of 5nm, taking 100nM and 100 mu L of PTCDA as Raman signal molecules, and taking 633nm as excitation wavelength, and analyzing and detecting the SERS performance of the Cu 2 O nano array, wherein the laser power is 1mW and the exposure time is 1s. Then, a Cu 2 O nano-array was prepared using a quartz plate as an assembly substrate, and a transient absorption spectrum test was performed using 100nM,100 μL PTCDA as a signal molecule. Wherein the excitation light wavelength used was 630nm. Scanning wavelength ranges from 100fs to 500ps, and the Cu 2 O nano-array surface enhanced Raman spectrum mechanism is verified.
(4) Preparation of miR-34c Raman analysis chip
Mu.L of a 20. Mu.M SH-DNA solution was dropped onto the surface of the Cu 2 O nanoarray prepared in the step (2) at a pitch of 5nm, and incubated at a constant temperature for 3 hours at 37 ℃. Then 500 mu L of 1mM of reference small molecule TCNQ is dripped on the surface of the nano array, further incubation is carried out for 30min at 37 ℃, and then RNase-free water is used for cleaning the surface, so that the miR-34c Raman analysis chip based on the Cu 2 O nano array is obtained. Meanwhile, to 1mL of an aqueous solution containing 10mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 5mg of N-hydroxysuccinimide (NHS), DNA-primer was added to a concentration of 20. Mu.M, then perylene tetracarboxylic dianhydride (PTCDA) was added thereto to 500. Mu.M, the above reaction system was continuously reacted at a constant temperature of 85℃for 16 hours, 100mL of a washing product of 1M hydrochloric acid was further reused three times, and the precipitate was filtered and kept, and dried in an oven at 40℃for 15 hours to obtain a DNA signaling probe.
(5) Detection of miR-34 c: miR-34c analog sample analysis
Mu.L of RNase-free solution containing miR-34c at different concentrations and 1. Mu.M DNA signaling probe was added dropwise to the surface of the Raman analysis chip, and incubated at 37℃for 15min. After washing twice with 1mLRNase-free water, detection was performed by a raman spectrometer using a laser wavelength of 633nm, a laser power of 1mW, and an exposure time of 1s.
The method has the advantages that the Cu 2 O nanowire is synthesized by a hydrothermal method at one step, the Langmuir-Blodgett method is adopted for the first time, the self-assembly of the Cu 2 O nanowire is realized at an oil/water interface through surface modification, and the Cu 2 O nanowire array with controllable nanowire spacing (spacing is 2-20 nm) is obtained for the first time. By utilizing the Local Surface Plasmon Resonance (LSPR) coupling effect of the Cu 2 O nanowire, the LSPR effect of the semiconductor nanomaterial Cu 2 O in the visible light region is realized, and the ultra-high sensitivity and high selectivity Raman enhancement effect on Raman signal molecules PTCDA, TCNQ and the like is realized based on an electromagnetic field mechanism and a charge transfer mechanism. The enhancement factor reaches 3.19×10 10. More importantly, through the characterization of transient absorption spectrum, the invention verifies a charge transfer mechanism based on plasma induction, provides a brand-new surface enhanced Raman spectrum mechanism, and provides a high-sensitivity, high-selection and high-stability biological analysis platform for realizing the analysis of various trace biomarkers in a biological system. Further, based on the SERS analysis platform, the invention designs a brand new miR-34c specific and high-sensitivity Raman analysis chip. PTCDA is used as a response signal molecule, TCNQ is used as a signal reference molecule, and based on the specific identification of a specific DNA sequence in a Raman analysis chip to miR-34c, the high-sensitivity and high-selectivity miR-34c rapid quantitative analysis without amplification and pretreatment links within 15min is realized by combining mobile handheld Raman spectrum analysis equipment, and the detection limit reaches 0.06fM. The Raman analysis chip not only can realize the nucleic acid specific identification of single-base mismatch, but also has the advantages of high sensitivity, high accuracy, simplicity and convenience in operation and portability, and provides a high-quality platform for rapid screening and accurate detection of samples based on miRNA.
Drawings
FIG. 1 is an SEM image of Cu 2 O nanowires synthesized in example 1 of the present invention.
FIG. 2 is an HR-TEM image of Cu 2 O nanowires synthesized in example 1 of the present invention.
FIG. 3 is an XRD spectrum of Cu 2 O nanowires synthesized in example 1 of the present invention.
FIG. 4 is an ultraviolet-visible absorption spectrum of Cu 2 O nanowire synthesized in example 1 of the present invention.
FIG. 5 is an SEM image of a Cu 2 O nanoarray synthesized in example 2 of the present invention, where A is a Cu 2 O nanoarray at a pitch of 2nm, B is a Cu 2 O nanoarray at a pitch of 5nm, C is a Cu 2 O nanoarray at a pitch of 10nm, and D is a Cu 2 O nanoarray at a pitch of 20 nm.
FIG. 6 is an ultraviolet-visible absorption spectrum of the Cu 2 O nanoarray synthesized in example 2 of the present invention.
FIG. 7 is a graph of the surface enhanced Raman spectrum and enhancement factor statistics of PTCDA, TCNQ, BCP molecules of a Cu 2 O nanoarray with a spacing of 5nm synthesized in example 3 of the present invention, wherein A is a Raman spectrum of the three molecules on the surface of the Cu 2 O nanoarray, and B is a graph of enhancement factor data statistics of the three molecules on the surface of the Cu 2 O nanoarray.
FIG. 8 is an ultraviolet-visible absorption spectrum of the DNA signaling probe prepared in example 4 of the present invention.
Fig. 9 is a linear relationship between a raman analysis spectrum of a prepared miR-34c raman analysis chip and a raman analysis spectrum of a solution of miR-34c with different concentrations in embodiment 5, wherein a is a raman analysis spectrum of the miR-34c raman analysis chip and the miR-34c with different concentrations, and B is a linear raman analysis graph of the miR-34c raman analysis chip and the miR-34c with different concentrations.
Fig. 10 is a graph of a comparison of raman responses of different interferents in an organism tested using the prepared miR-34c raman analysis chip in example 5 of the present invention.
FIG. 11 is a response time curve of miR-34c in an organism using a prepared miR-34c Raman analysis chip in example 5 of this invention.
FIG. 12 is a graph of the response intensity of miR-34c to miR-34c, prepared in example 5 of this invention, after storage for different times.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and drawings. The procedures, conditions, experimental methods, etc. for carrying out the present invention are common knowledge and common knowledge in the art, except for the following specific references, and the present invention is not particularly limited.
Example 1 preparation of Cu 2 O nanowires
Synthesis of Cu 2 O nanowires: 0.1g of CuAc 2 was dissolved in 50mL of ultrapure water, o-methoxyaniline was added thereto at a concentration of 0.1mol/L, and the mixture was sufficiently stirred for 30 minutes, transferred to a 50mL hydrothermal reaction vessel, and kept at 180℃for 10 hours in an oven. Then, the hydrothermal reaction kettle is naturally cooled to room temperature, the dispersion liquid is transferred into a centrifuge tube, and the precipitate is obtained by centrifugation at 5000 rpm. The product was further washed 5 times with ultrapure water and absolute ethanol as washing solvents, respectively. Finally, the obtained product is dried for 10 hours at 60 ℃ to obtain the final product. Similarly, the temperature of the reaction kettle is further raised to 250 ℃ and kept for 5 hours, and the Cu 2 O nanowire with similar size and good dispersibility can be obtained.
FIG. 1 shows that Cu 2 O nanowires were successfully prepared by hydrothermal method, with a diameter of about 20nm and a length of 2-3 μm. FIG. 2 is a high resolution transmission electron microscope (HR-TEM) image of Cu 2 O nanowires, from which it can be seen that the Cu 2 O nanowire has a surface interplanar spacing of 0.297nm, attributed to the (110) crystal plane of the Cu 2 O crystal. Fig. 3 shows an X-ray diffraction pattern (XRD) of Cu 2 O nanowires, and XRD diffraction peaks ascribed to the (110), (111), (200) and (131) crystal planes of Cu 2 O can be observed, which proves that the crystallinity of Cu 2 O nanowires is good. FIG. 4 is an ultraviolet visible absorption spectrum of Cu 2 O nanowire, with the absorption peak at 476nm attributed to band gap absorption of the material.
Example 2 preparation of Cu 2 O line nanoarrays and pitch control
The Cu 2 O nanowire prepared as described above was taken as a basic assembly unit, and polystyrene (SH-PS) having a thiol-modified molecular weight of 50000 was dissolved in 5mL of ultrapure water to prepare a 10mM SH-PS solution, to which 30. Mu.g of Cu 2 O nanowire powder was added, and stirring was continued at 30℃for 12 hours. And centrifuging at 8000rpm for 5min, and collecting precipitate to obtain PS modified Cu 2 O nanowire. Subsequently, the resulting nanowires were well dispersed in 500. Mu.L cyclohexane and transferred to the surface of a 2cm 2 single crystal silicon substrate. Then 500. Mu.L of ultrapure water is dripped on the surface of the monocrystalline silicon substrate, and after the interface is stabilized, the sample is concentrated at a speed of 20cm 2/min in a dry environment. And after the sample is completely dried, cleaning the surface by absolute ethyl alcohol, and finally drying the sample in an oven at 60 ℃ overnight to obtain the Cu 2 O nano-array. Likewise, the continuous regulation and control of the spacing of the Cu 2 O nano array between 2nm and 20nm can be realized by adjusting the types and the molecular weights of the Cu 2 O nano-wire surfactant. Specifically, when the surface active group is changed into mercapto-modified polyvinylpyrrolidone (SH-PVP) with molecular weight of 10000, cu 2 O nano-arrays with interval of 2nm can be prepared and obtained. When the molecular weight of SH-PS used was adjusted to 100000 or 200000, cu 2 O nanoarrays with pitches of 10nm and 20nm, respectively, could be obtained.
Fig. 5 is a Scanning Electron Microscope (SEM) image of Cu 2 O nanoarrays with different pitches, from which it can be seen that the Cu 2 O nanoarrays are uniformly aligned with the nanowire pitches being continuously varied among 2nm (fig. 5A), 5nm (fig. 5B), 10nm (fig. 5C) and 20nm (fig. 5D). FIG. 6 is an ultraviolet-visible absorption spectrum of a Cu 2 O nanoarray, showing that for a Cu 2 O nanoarray with a 5nm spacing, a new ultraviolet-visible absorption peak appears at 687nm, in addition to the absorption peak at 476nm being attributed to the band gap absorption of the material, indicating that a Cu 2 O nanoarray appears as LSPR absorption in the visible spectrum due to coupling between Cu 2 O nanoarrays. Accordingly, for a 2nm pitch Cu 2 O nanoarray, a corresponding LSPR absorption signal occurs at 519 nm. In the Cu 2 O nano-array with the distance of 10nm and 20nm, the weak coupling effect between the nano-wires is not shown to be obvious in the visible light range.
Example 3 surface enhanced Raman Spectroscopy characterization and enhanced mechanism resolution of Cu 2 O nanoarrays
The method comprises the steps of taking a silicon wafer as a Cu 2 O nano array assembly substrate, taking the silicon wafer as a Cu 2 O nano array assembly substrate, preparing a Cu 2 O nano array SERS substrate with a nanowire spacing of 5nm, taking 100nM and 100 mu L of PTCDA as Raman signal molecules, taking 633nm as excitation wavelength, and analyzing and detecting the SERS performance of the Cu 2 O nano array, wherein the laser power is 1mW and the exposure time is 1s. Then, a Cu 2 O nano array is prepared by taking a quartz plate as an assembly substrate, 100nM and 100 mu L of PTCDA, TCNQ and BCP are respectively taken as signal molecules, and transient absorption spectrum test is carried out. Wherein the excitation light wavelength used was 630nm. Scanning wavelength ranges from 100fs to 500ps, and the Cu 2 O nano-array surface enhanced Raman spectrum mechanism is verified.
Fig. 7 shows the SERS spectrum of PTCDA, TCNQ, BCP molecules on the Cu 2 O nanowire surface, and it can be seen from the figure that the raman signals of the three PTCDA, TCNQ, BCP molecules obtain a significant enhancement effect on the Cu 2 O nanoarray surface compared to the single molecules, wherein the enhancement factor of the PTCDA molecule is highest, reaching 3.19×10 10. Through experiments, the raman enhancement of the Cu 2 O nano-array is verified to be generated by the synergistic effect of three parts, namely the enhancement of an LSPR induced electromagnetic field, the enhancement of photoinduced charge transfer and the LSPR induced charge transfer.
Example 4 miR-34c preparation of Raman analysis chip
Mu.L of a 20. Mu.M SH-DNA solution was dropped onto the surface of the Cu 2 O nanoarray prepared in the step (2) at a pitch of 5nm, and incubated at a constant temperature for 3 hours at 37 ℃. Then 500 mu L of 1mM of reference small molecule TCNQ is dripped on the surface of the nano array, further incubation is carried out for 30min at 37 ℃, and then RNase-free water is used for cleaning the surface, so that the miR-34c Raman analysis chip based on the Cu 2 O nano array is obtained. Meanwhile, to 1mL of an aqueous solution containing 10mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 5mg of N-hydroxysuccinimide (NHS), DNA-primer was added to a concentration of 20. Mu.M, then perylene tetracarboxylic dianhydride (PTCDA) was added thereto to 500. Mu.M, the above reaction system was continuously reacted at a constant temperature of 85℃for 16 hours, 100mL of a washing product of 1M hydrochloric acid was further reused three times, and the precipitate was filtered and kept, and dried in an oven at 40℃for 15 hours to obtain a DNA signaling probe.
FIG. 8 is a graph showing the ultraviolet-visible absorption spectrum of the prepared DNA signaling probe, wherein three absorption peak signals attributed to the absorption of PTCDA molecules appear between 400nm and 600nm, and signals attributed to the absorption of DNA molecules appear around 230nm to 260nm, which proves that the PTCDA molecules are successfully modified by the DNA structure, and further proves that the DNA signaling probe is successfully prepared. Provides a research basis for the next Raman analysis.
Examples 5 miR-34c assays
MiR-34c simulation sample analysis: mu.L of RNase-free solution containing miR-34c at different concentrations and 1. Mu.M DNA signaling probe was added dropwise to the surface of the Raman analysis chip, and incubated at 37℃for 15min. After washing twice with 1mL of RNase-free water, detection was performed by a Raman spectrometer using a laser wavelength of 633nm with a laser power of 1mW and an exposure time of 1s.
FIG. 9A is a graph of the Raman analysis detection spectrum of miR-34c at different concentrations in RNase-free solution based on the miR-34c Raman analysis chip prepared in example 3. In the graph, the Raman spectrum peak intensity (I 1381) of the Raman spectrum peak at 1381cm -1、1565cm-1, which belongs to PTCDA molecules, is gradually enhanced along with the increase of the concentration of miR-34c sequences, while the Raman spectrum signal (I 2225) at 2225cm -1, which belongs to TCNQ, is kept unchanged, so that the SERS analysis platform can rapidly and accurately realize sensitive analysis and detection of miR-34 c. FIG. 9B is a graph showing the relationship between the concentration of the Raman ratio type signal I 1381/I2225, and shows that the signal intensity of I 1381/I2225 and the concentration of miR-34c are in a linear relationship in the range of 0.1fM-0.1nM, and the detection limit is as low as 0.06fM. The Raman analysis chip constructed in the invention can be used for detecting miR-34 c. FIG. 10 is a statistical graph of the Raman signal response of the prepared miR-34c Raman analysis chip in different interfering substance solutions (metal ions Cu 2+、Fe3+、Ca2+、K+、Na+, non-specific proteins Bovine Serum Albumin (BSA), pepsin (pepsin), immunoglobulin (IgG), thrombin (Thrombin), trypsin (Trypsin), base mismatch nucleic acid sequences Mis-1, mis-2, mis-3, mis-4 and Mis-5). The graph shows that the miR-34c Raman analysis platform has excellent selectivity on miR-34c sequences, and is an ideal choice for achieving miR-34c analysis. Fig. 11 is a graph of response time kinetics of the raman analysis chip to miR-34c, and as can be seen from the graph, the response time of the raman analysis chip is only 5min. FIG. 12 is a graph of Raman enhancement signals from a 300nM miR-34c assay using a Raman analysis chip stored for various times. As can be seen from the graph, the Raman analysis chip constructed by the invention has long-term stability, and the activity of the Raman analysis chip can still be maintained to be more than 97% in normal-temperature storage for more than 200 days.
The method has the advantages that the Cu 2 O nanowire is synthesized by a hydrothermal method at one step, the Langmuir-Blodgett method is adopted for the first time, the self-assembly of the Cu 2 O nanowire is realized at an oil/water interface through surface modification, and the Cu 2 O nanowire array with controllable nanowire spacing (spacing is 2-20 nm) is obtained for the first time. By utilizing the Local Surface Plasmon Resonance (LSPR) coupling effect of the Cu 2 O nanowire, the LSPR effect of the semiconductor nanomaterial Cu 2 O in the visible light region is realized, and the ultra-high sensitivity and high selectivity Raman enhancement effect on Raman signal molecules PTCDA, TCNQ and the like is realized based on an electromagnetic field mechanism and a charge transfer mechanism. The enhancement factor reaches 3.19×10 10. More importantly, a charge transfer mechanism based on plasma induction is verified, a novel surface enhanced Raman spectrum mechanism is provided, and a high-sensitivity, high-selection and high-stability biological analysis platform is provided for analysis of various trace biomarkers in a biological system. Further, based on the SERS analysis platform, the invention designs a brand new miR-34c specific and high-sensitivity Raman analysis chip. PTCDA is used as a response signal molecule, TCNQ is used as a signal reference molecule, and based on the specific identification of a specific DNA sequence in a Raman analysis chip to miR-34c, the high-sensitivity and high-selectivity miR-34c rapid quantitative analysis without amplification and pretreatment links within 15min is realized by combining mobile handheld Raman spectrum analysis equipment, and the detection limit reaches 0.06fM. The Raman analysis chip not only can realize the specific identification of nucleic acid with single base mismatch, but also has the advantages of high sensitivity, high accuracy, simple and convenient operation and portability, and provides a high-quality platform for rapid screening and accurate detection of diseases based on nucleic acid.
The protection of the present invention is not limited to the above embodiments. Variations and advantages that would occur to one skilled in the art are included in the invention without departing from the spirit and scope of the inventive concept, and the scope of the invention is defined by the appended claims.

Claims (12)

1. The preparation method of the charge transfer Raman enhanced substrate material based on the plasma resonance enhancement is characterized by comprising the following steps of:
(1) Preparation of Cu 2 O nanowires: the Cu 2 O nanowire is prepared by placing a solution containing copper acetate CuAc 2, ultrapure water and O-methoxy aniline into a hydrothermal reaction kettle through a hydrothermal method and performing high-temperature treatment;
(2) Preparation and space regulation of Cu 2 O nano-arrays: carrying out surface modification on the Cu 2 O nanowire, and obtaining surface modified Cu 2 O through centrifugal separation; based on Langmuir-Blodgett method, carrying out self-assembly of modified Cu 2 O nano-wires at an oil/water interface, and drying in an oven to obtain a final product Cu 2 O nano-array;
Or the self-assembly is carried out by changing the molecular weight and the type of the Cu 2 O nanowire surfactant, so that the spacing regulation of the Cu 2 O nano array is realized.
2. The method according to claim 1, wherein in the step (1), the ratio of the amount of CuAc 2, ultrapure water and o-methoxyaniline is 0.05g to 0.2g:30-50mL:0.05-0.2mol/L; the mixing time of the solution is 15min-45min; the volume of the hydrothermal reaction kettle is 50mL or 100mL; the temperature of the hydrothermal reaction is 160-200 ℃; the hydrothermal reaction time is 8-15 h.
3. The method according to claim 1, wherein in the step (2), the surface modifying active agent of the Cu 2 O nanowire is one of polyvinylpyrrolidone, polyethylene glycol, polystyrene, cetyltrimethylammonium chloride; the molecular weight of the surface modification active agent used for assembling the Cu 2 O nano-array is one of 100-1000000; the surface modifying active agent modifying group is sulfhydryl, amino and carboxyl; the time for the surface modification process of the Cu 2 O nanowire is 8-15 hours; the temperature used in the surface modification process of the Cu 2 O nanowire is 20-40 ℃; the rotational speed of the centrifugation is 3000rpm-8000rpm; the oil-water interface is one of water/chloroform, water/cyclohexane and water/dichloromethane; the ratio of the oil-water interface is 1:0.5-1:3; the total volume of the oil-water dispersion solvent is 0.5-2mL; the concentration rate of the oil-water interface is 10-30cm 2/min.
4. A Cu 2 O nanowire or a Cu 2 O nanoarray prepared according to the method of claim 1.
5. A preparation method of a Raman analysis chip of miR-34c nucleic acid sequence is characterized by comprising the following steps:
(1) Dropwise adding PBS solution containing mercapto modified DNA capture chain SH-DNA to the surface of the Cu 2 O nano array with the interval of 5nm for incubation, so as to prepare an SH-DNA modified Cu 2 O nano array;
(2) Adding a Raman signal molecule, N-hydroxysuccinimide and an amino-modified DNA signal chain into a solution containing 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, reacting for a period of time, adding hydrochloric acid into the solution, repeatedly washing and filtering to obtain a solid product which is a DNA signal probe;
(3) And (3) dropwise adding a reference small molecule on the surface of the SH-DNA modified Cu 2 O nano-array, and drying to prepare SH-DNA-Cu 2 O NAs with a reference signal, thereby obtaining the Raman analysis chip of miR-34c nucleic acid sequence based on the Cu 2 O nano-array.
6. The method according to claim 5, wherein in the step (1), the SH-DNA is 5'-SH-GCCACTGTGCAGCTAACTACACTGCCTGCAGACT-3'; the sequence of the DNA-primer is 5'-CCCGCAATC-NH 2 -3'; the concentration of SH-DNA in PBS solution is 10-50 mu M; the volume of the PBS solution of SH-DNA dripped on the surface of the Cu 2 O nano-array is 100 mu L-1mL; the incubation temperature of the SH-DNA and the Cu 2 O nano array is 20-50 ℃; the incubation time of the SH-DNA and the Cu 2 O nano array is 3-5 h.
7. The method according to claim 5, wherein in the step (2), the raman signal molecule is one of perylene tetracarboxylic dianhydride, rhodamine 6G, rhodamine B, cyanine and derivatives thereof; the dosage of the Raman signal molecules is 100 mu M-1000 mu M; the dosage of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 2mg-20mg; the dosage of the N-hydroxysuccinimide is 1mg-10mg; the total volume of the solution containing 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 1mL-20mL; the dosage of the amino modified DNA signal chain is 10 mu M-100 mu M; the reaction time for modifying the DNA-primer by the Raman signal molecule is 10-20 h; the reaction temperature used by the Raman signal molecule for modifying the DNA-primer is 60-90 ℃; the concentration of the hydrochloric acid is 0.5M-2M; the volume of the hydrochloric acid is 50mL-200mL.
8. The method of claim 5, wherein in step (3), the reference small molecule is one of tetracyanoquinodimethane and its derivatives, bathocuproine and its derivatives, phenylacetylene and its derivatives; the concentration of the reference small molecule solution is 100 mu M-2mM; the volume of the reference small molecule solution is 100 mu L-1000 mu L; the drying time of the reference small molecule is 30-60 min.
9. A raman analysis chip of miR-34c nucleic acid sequences prepared according to the method of any one of claims 5-8.
10. The use of the Cu 2 O nanowires of claim 4 in the preparation of Cu 2 O wire nanoarrays and pitch control, and in surface enhanced raman spectroscopy and miRNA analytical detection.
11. The Cu 2 O nanoarray according to claim 4, use in nanowire pitch modulation methods and in surface enhanced raman spectroscopy, miRNA analysis.
12. The use of the raman analysis chip of the miR-34c nucleic acid sequence according to claim 9 in miR-34c high-sensitivity analysis detection.
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