CN113667720A - Biosensor for detecting miRNA-182 and preparation method and application thereof - Google Patents
Biosensor for detecting miRNA-182 and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a biosensor for detecting miRNA-182, a preparation method and application thereof, and belongs to the field of biosensors. The preparation method of the biosensor comprises the following steps: preparation of aminated Fe3O4Magnetic nanospheres; reacting 2,4, 6-trihydroxybenzene-1, 3, 5-trimethyl aldehyde monomer with the aminated magnetic nanospheres to prepare core-shell MCOF nanospheres wrapped by COF layers; and (3) incubating the hairpin DNA probe 1 and the probe 2 with miRNA-182 at room temperature, and then carrying out quenching reaction with the core-shell MCOF nanospheres to construct a miRNA-182 biosensor with amplified fluorescence signals. The biosensor can quantitatively measure miRNA-182 in serum of a brain tumor patient, has the characteristics of low detection limit, wide linear range and high stability, can realize miRNA detection visualization by combining the biosensor with a capillary system, and is beneficial to non-invasive simple and rapid diagnosis and prognosis monitoring of the brain tumor.
Description
Technical Field
The invention relates to the field of biosensors, in particular to a biosensor for detecting miRNA-182 and a preparation method and application thereof.
Background
Brain tumors have a poor prognosis and are difficult to find in time due to special positions, so that most brain tumor patients miss the optimal treatment period after diagnosis. Therefore, timely, early detection and accurate diagnosis of brain tumors are critical to effective clinical treatment. Currently, Magnetic Resonance Imaging (MRI) and Computed Tomography (CT) imaging methods are generally used for clinical diagnosis of brain tumors. However, these methods do not distinguish benign lesions from malignant lesions, especially early stage small size lesions. Although tissue biopsy can improve accuracy, it is invasive and thus not conducive to the diagnosis of brain tumors located in the central nervous system. Liquid biopsies based on tumor markers (including miRNA, ctDNA, proteins, exosomes and CTCs) in biological fluids have great application prospects in cancer diagnosis because of their simplicity, rapidity and non-invasiveness. Among them, miRNA is used as non-coding small RNA for targeting specific messenger RNA to down-regulate some gene expression, and shows high specificity in the process of tumorigenesis and development. miRNA-182 plays an important regulating role in the proliferation and invasion of brain tumor cells, and research reports that miRNA-182 can indicate the occurrence and development of brain tumors with high specificity, so that miRNA-182 can be used as a biomarker for simple and rapid diagnosis and prognosis monitoring of brain tumors. However, the content of miRNA-182 in human blood is very low and the resolution of homologous miRNA is not high, so that the above problems need to be solved to realize sensitive detection of miRNA.
As a simple and effective isothermal signal amplification technique, the hybrid chain reaction has many applications in biosensing, bioimaging and biomedicine, and shows advantages in the detection of low-abundance miRNA. However, the hybrid chain reaction requires a suitable signal amplification sensing platform. Traditional fluorescence quenchers such as graphene, molybdenum disulfide and Black Phosphorus (BP) nanosheets can achieve fluorescence sensing detection through pi-pi interaction with nucleic acid, but a fluorescence signal amplification is rarely utilized to enhance the sensitivity of miRNA detection in cooperation with a sensing platform. The two-dimensional covalent organic framework nanosheet can be combined with a hybrid chain reaction, and DNA sensing based on fluorescence signal amplification is realized through sensitive interaction between a crystalline Covalent Organic Framework (COF) and a special DNA chain. However, most COF nanoplatelets aggregate severely, have low crystallinity and are susceptible to degradation (instability), which greatly reduces the biosensing performance of the nanoplatelets. Furthermore, after the COF organic nano material is doped into a complex detection system (blood), the COF organic nano material is difficult to separate in the detection process. These all reduce the sensitivity and stability of COF-based fluorescent biosensors. Therefore, there is an urgent need to prepare magnetic COF nanospheres with high crystallinity to realize the interaction between the magnetic COF nanospheres and nucleic acid molecules, and simultaneously realize simple and convenient magnetic separation for sensitive miRNA detection and rapid brain tumor diagnosis and prognosis monitoring.
Disclosure of Invention
The invention aims to provide a biosensor for detecting miRNA-182, a preparation method and an application thereof, aiming at solving the problems in the prior art, the miRNA-182 in the serum of a brain tumor patient can be quantitatively measured by the miRNA biosensor, the miRNA biosensor has the characteristics of low detection limit, wide linear range and high stability, the visualization of miRNA detection can be realized by combining the biosensor and a capillary system, and the noninvasive simple and rapid diagnosis and prognosis monitoring of the brain tumor are facilitated.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a preparation method of a biosensor for detecting miRNA-182, which comprises the following steps:
step 1: by adding Fe3O4Adding tetraethyl orthosilicate TEOS and (3-aminopropyl) triethoxysilane APTES into the nanospheres to prepare aminated magnetic nanospheres;
step 2: reacting monomer 2,4, 6-trihydroxybenzene-1, 3, 5-trimethyl aldehyde with the aminated magnetic nanospheres to prepare 2,4, 6-trihydroxybenzene-1, 3, 5-trimethyl aldehyde functionalized magnetic nanospheres;
then carrying out COF (chip on film) mediated in-situ growth on the 2,4, 6-trihydroxybenzene-1, 3, 5-trimethylaldehyde functionalized magnetic nanosphere to prepare a core-shell MCOF nanosphere;
and step 3: and (3) incubating the hairpin DNA probe 1 and the hairpin DNA probe 2 with miRNA-182 at room temperature, and then carrying out quenching reaction with the core-shell MCOF nanospheres to construct a miRNA-182 biosensor with amplified fluorescence signals.
Preferably, in step 2, monomer 2,4, 6-trihydroxybenzene-1, 3, 5-trimethyl aldehyde is grafted to the surface of the aminated nanosphere through Schiff base reaction, and then the magnetic nanosphere functionalized by 2,4, 6-trihydroxybenzene-1, 3, 5-trimethyl aldehyde and monomer 2,4, 6-trihydroxybenzene-1, 3, 5-trimethyl aldehyde and benzidine undergo in-situ growth of COF at high temperature to prepare the core-shell MCOF nanosphere.
Preferably, the high temperature conditions are: reacting for 10-72 h at 60-240 ℃.
Preferably, in step 3, the hairpin DNA probe 1 and the hairpin DNA probe 2 are two sequences having a length of 18 to 32 bases and capable of undergoing a hybridization chain reaction.
Preferably, in step 3, the final concentration of the hairpin DNA probe 1 and the hairpin DNA probe 2 is 1nM to 1. mu.M; the final concentration of the core-shell MCOF nanosphere is 0.02mg/ml to 1 mg/ml.
Preferably, in step 3, the incubation time is: 30 min-24 h; the quenching time is 5 min-2 h.
Preferably, the size distribution of the core-shell MCOF nanosphere is 300-600 nm, and the Zeta potential is-50.0 mV.
The invention also provides a biosensor for detecting miRNA-182, which is prepared by the preparation method.
The invention also provides application of the biosensor in preparation of a miRNA-182 detection reagent.
Preferably, after the biosensor is constructed, the supernatant is separated under the action of a magnetic field, and the supernatant is taken for fluorescence measurement to detect the content of miRNA-182 in the sample to be detected.
The invention discloses the following technical effects:
the invention prepares dispersed and high-crystallization magnetic Covalent Organic Framework (COF) wrapped Fe by a monomer-mediated in-situ interface growth strategy3O4Magnetic nanospheres (MCOF). By utilizing the unique interaction between the MCOF and the hairpin DNA molecules, the miRNA biosensor with amplified fluorescence signal is constructed. By means of the special magnetic COF nanosphere and the unique sensing system, the miRNA biosensor can realize sensitive detection of miRNA-182 in different matrixes. Through experimental verification, the biosensor can quantitatively determine miRNA-182 in serum of a brain tumor patient, detection limit, linear range and regression coefficient (R)2) Respectively reaches 20fM, 0.1pM to 10pM and 0.991, and has the characteristics of low detection limit, wide linear range and high stability. In addition, the biosensor has good stability and accuracyAccuracy and precision. On the basis, a strategy for realizing visual detection of miRNA-182 in a micro sample by combining the biosensor and a capillary chip system is provided. The method provides a reliable candidate method for non-invasive simple and rapid diagnosis and prognosis monitoring of brain tumors, and has great application potential.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a structural representation of a core-shell MCOF nanosphere; a: magnetic Fe3O4TEM images and ED images of nanospheres (inset); b: TEM images and ED images of multiple core-shell MCOF nanospheres; c: TEM images of single core-shell MCOF nanospheres; d: HRTEM image of COF shell; e: magnetic Fe3O4HRTEM images of nuclei; f: STEM image and EDS element mapping of MCOF; g: XRD pattern of MCOF; h: monomer (TT: 2,4, 6-trihydroxybenzene-1, 3, 5-triformal; benzidine), intermediate (Fe)3O4、Fe3O4@SiO2) And FT-IR spectra of MCOF nanospheres;
FIG. 2 is a structural analysis of core-shell MCOF nanospheres; a: XPS total spectrum; b: c1s, C corresponding to the total spectrum: n1s, d corresponding to the total spectrum: o1s corresponding to the total spectrum; e: an Fe 2p spectrum corresponding to the total spectrum; f: nitrogen adsorption-desorption isotherms and pore size distribution curves;
FIG. 3 is an optimization of a fluorescence signal amplification biosensor; a-b fluorescence signal amplification biosensor fluorescence spectra for miRNA biosensing in the absence of H2 and in the presence of both H1 and H2, respectively; c-f respectively amplifying the relative fluorescence intensity of the biosensor by using a fluorescence signal for miRNA biosensing at different hybridization strand reaction temperatures, different concentrations of H1 and H2 probes, with or without magnetic separation and in different media;
FIG. 4 is a standard curve for miRNA detection in real blood samples;
FIG. 5 is a graph of relative fluorescence intensity of MCOF-based biosensors at different target sequences; t: a target miRNA; SM: single base mismatched mirnas; FM: a four base mismatched miRNA; r: a random miRNA;
FIG. 6 is a graph of miRNA-182 concentration in serum samples from 12 healthy donor sera, 12 glioma patients pre-and post-surgery via MCOF-based biosensors; GP: patients with preoperative glioma; AS: after the operation; HD: a healthy donor;
fig. 7 is a fluorescence image and schematic of an MCOF-based capillary-assisted visualization sensing system at different miRNA concentrations;
FIG. 8 is a TGA curve of MCOF;
FIG. 9 is an image of MCOF solution before and after magnetic adsorption;
FIG. 10 is a schematic diagram of the structures of the H1 sequence and the H2 sequence.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Example 1A biosensor for detecting miRNA-182
(1)150mg Fe3O4The nanospheres were dispersed in 100ml ethanol, then 25ml pure water and 1.2ml ammonia water were added and mixed well. 100 μ l TEOS ethanol solution was added and reacted for 9 h. The particles were obtained by high speed centrifugation and washed. Dispersing the product into 120ml isopropanol, adding 0.5ml APTES, mechanically stirring for 9h, centrifuging to separate the product, washing, and freeze-drying to obtain the aminated magnetic nanosphere.
Dispersing the obtained aminated magnetic nanospheres into 10ml of dioxane, adding 10mg of monomer 2,4, 6-trihydroxybenzene-1, 3, 5-trimethylaldehyde (TT) and 150 mu l of pure acetic acid, uniformly mixing, transferring into a reaction kettle, heating at 120 ℃ for 1h, centrifuging at a high speed to separate a product, washing with DMF (dimethyl formamide), dioxane and mesitylene in sequence, and freeze-drying to obtain the TT functionalized magnetic nanospheres. And then dispersing the product with 21mg of monomer TT and 27.6mg of benzidine in 5ml of mesitylene/dioxane (1:1), uniformly mixing, transferring the mixture into a reaction kettle, and reacting at 60 ℃ for 48 hours to perform in-situ growth of COF to prepare the core-shell MCOF nanosphere.
(2) Total RNA is extracted from a serum sample according to an extraction step by using a total RNA rapid extraction kit. Hairpin DNA probe 1(H1) (50 nM final concentration) and hairpin DNA probe 2(H2) (50 nM final concentration) were added to PBS with miRNA-182, mixed well and incubated at room temperature for 1H. Then, core-shell MCOF nanosphere solution (final concentration 0.2mg/ml) was added to the above mixture, quenched for 15min, separated by magnetic field, and the supernatant was subjected to fluorescence measurement.
Wherein, H1 sequence (5 '-3'):
AGTGTGAGTTCTACCATTGCCAAACAAAGTTTTGGCAA
h2 sequence (5 '-3'):
TTTGGCAATGGTAGAACTCACACTAGTTCTACCATTGCCAAAACTTTG
for two strands designed for a target sequence, the sequence H1 can be complementary with the target sequence and a part of the sequence H2, the 5' end of the sequence H1 is modified with Texas Red fluorophore, and the sequences H1 and H2 can continue to self-complement and extend under the condition that the target sequence (corresponding to a primer) exists to realize a cascade reaction (as shown in FIG. 10).
Example 2A biosensor for detecting miRNA-182
(1)150mg Fe3O4The nanospheres were dispersed in 100ml ethanol, then 25ml pure water and 1.2ml ammonia water were added and mixed well. 100 μ l TEOS ethanol solution was added and reacted for 9 h. The particles were obtained by high speed centrifugation and washed. Dispersing the product into 120ml isopropanol, adding 0.5ml APTES, mechanically stirring for 9h, centrifuging to separate the product, washing, and freeze-drying to obtain the aminated magnetic nanosphere.
Dispersing the obtained aminated magnetic nanospheres into 10ml of dioxane, adding 10mg of monomer 2,4, 6-trihydroxybenzene-1, 3, 5-trimethylaldehyde (TT) and 150 mu l of pure acetic acid, uniformly mixing, transferring into a reaction kettle, heating at 120 ℃ for 1h, centrifuging at a high speed to separate a product, washing with DMF (dimethyl formamide), dioxane and mesitylene in sequence, and freeze-drying to obtain the TT functionalized magnetic nanospheres. And then dispersing the product with 21mg of monomer TT and 27.6mg of benzidine in 5ml of mesitylene/dioxane (1:1), uniformly mixing, transferring the mixture into a reaction kettle, and reacting at 120 ℃ for 48 hours to perform in-situ growth of COF (chip-on-film) to prepare the core-shell MCOF nanosphere.
(2) Total RNA is extracted from a serum sample according to an extraction step by using a total RNA rapid extraction kit. Hairpin DNA probe 1(H1) (50 nM final concentration) and hairpin DNA probe 2(H2) (50 nM final concentration) were added to PBS with miRNA-182, mixed well and incubated at room temperature for 1H. Then, core-shell MCOF nanosphere solution (final concentration 0.2mg/ml) was added to the above mixture, quenched for 15min, separated by magnetic field, and the supernatant was subjected to fluorescence measurement.
Example 3A biosensor for detecting miRNA-182
The difference from the embodiment 1 is that: the "subsequent dispersion of the above product of example 1 with 21mg monomer TT, 27.6mg benzidine in 5ml mesitylene/dioxane (1:1), after mixing, transfer to a reaction kettle for 48h reaction at 60 ℃ for COF in situ growth, and the 60 ℃ in the preparation of core-shell MCOF nanospheres was replaced by 180 ℃ without changing other conditions and steps.
Example 4A biosensor for detecting miRNA-182
The difference from the embodiment 1 is that: the "product from example 1 followed by dispersion of the above product with 21mg monomer TT, 27.6mg benzidine in 5ml mesitylene/dioxane (1:1), mixing well and transferring to a reaction kettle for 48h reaction at 60 ℃ for COF in situ growth, and the 60 ℃ in the preparation of core-shell MCOF nanospheres was replaced by 240 ℃ without changing other conditions and steps.
Example 5A biosensor for detecting miRNA-182
The difference from the embodiment 2 is that: "reaction 48 h" in preparation of core-shell MCOF nanosphere "by dispersing the product of example 2" subsequently above with 21mg of monomer TT, 27.6mg of benzidine in 5ml of mesitylene/dioxane (1:1), mixing uniformly and transferring to a reaction kettle for reaction at 60 ℃ for 48h for COF in-situ growth, and "reaction 10 h" without changing other conditions and steps.
Example 6A biosensor for detecting miRNA-182
The difference from the embodiment 2 is that: "reaction 48 h" in preparation of core-shell MCOF nanosphere "by dispersing the product of example 2" subsequently above with 21mg of monomer TT, 27.6mg of benzidine in 5ml of mesitylene/dioxane (1:1), mixing uniformly and transferring to a reaction kettle for reaction at 60 ℃ for 48h for COF in-situ growth, and "reaction 24 h" without changing other conditions and steps.
Example 7A biosensor for detecting miRNA-182
The difference from the embodiment 2 is that: "reaction 48 h" in preparation of core-shell MCOF nanosphere "by dispersing the product of example 2" subsequently above with 21mg of monomer TT, 27.6mg of benzidine in 5ml of mesitylene/dioxane (1:1), mixing uniformly and transferring to a reaction kettle for reaction at 60 ℃ for 48h for COF in-situ growth, and "reaction 72 h" without changing other conditions and steps.
Example 8A biosensor for detecting miRNA-182
The difference from the embodiment 2 is that: h1 at a final concentration of 1nM and H2 at a final concentration of 1 nM; other conditions and steps were unchanged.
Example 9A biosensor for detecting miRNA-182
The difference from the embodiment 2 is that: a final concentration of 5nM H1 and a final concentration of 5nM H2; other conditions and steps were unchanged.
Example 10A biosensor for detecting miRNA-182
The difference from the embodiment 2 is that: h1 at a final concentration of 10nM and H2 at a final concentration of 10 nM; other conditions and steps were unchanged.
Example 11A biosensor for detecting miRNA-182
The difference from the embodiment 2 is that: a final concentration of H1 of 20nM and H2 of 20 nM; other conditions and steps were unchanged.
Example 12A biosensor for detecting miRNA-182
The difference from the embodiment 2 is that: a final concentration of 100nM H1 and a final concentration of 100nM H2; other conditions and steps were unchanged.
Example 13A biosensor for detecting miRNA-182
The difference from the embodiment 2 is that: a final concentration of H1 of 500nM and H2 of 500 nM; other conditions and steps were unchanged.
Example 14A biosensor for detecting miRNA-182
The difference from the embodiment 2 is that: h1 at a final concentration of 1 μ M and H2 at a final concentration of 1 μ M; other conditions and steps were unchanged.
Example 15A biosensor for detecting miRNA-182
The difference from the embodiment 2 is that: (2) in the specification, the 'room temperature incubation for 1 h' is replaced by 'room temperature incubation for 30 min'; other conditions and steps were unchanged.
Example 16A biosensor for detecting miRNA-182
The difference from the embodiment 2 is that: (2) in the specification, the 'room temperature incubation 1 h' is replaced by 'room temperature incubation 2 h'; other conditions and steps were unchanged.
Example 17A biosensor for detecting miRNA-182
The difference from the embodiment 2 is that: (2) in the specification, the 'room temperature incubation 1 h' is replaced by 'room temperature incubation 10 h'; other conditions and steps were unchanged.
Example 18A biosensor for detecting miRNA-182
The difference from the embodiment 2 is that: (2) in the specification, the 'room temperature incubation 1 h' is replaced by 'room temperature incubation 24 h'; other conditions and steps were unchanged.
Example 19A biosensor for detecting miRNA-182
The difference from the embodiment 2 is that: (2) in the above description, the "core-shell MCOF nanosphere solution (final concentration 0.2 mg/ml)" is replaced with "core-shell MCOF nanosphere solution (final concentration 0.02 mg/ml)"; other conditions and steps were unchanged.
Example 20A biosensor for detecting miRNA-182
The difference from the embodiment 2 is that: (2) in the above description, the "core-shell MCOF nanosphere solution (final concentration 0.2 mg/ml)" is replaced with "core-shell MCOF nanosphere solution (final concentration 0.05 mg/ml)"; other conditions and steps were unchanged.
Example 21A biosensor for detecting miRNA-182
The difference from the embodiment 2 is that: (2) in the above description, the "core-shell MCOF nanosphere solution (final concentration 0.2 mg/ml)" is replaced with "core-shell MCOF nanosphere solution (final concentration 0.1 mg/ml)"; other conditions and steps were unchanged.
Example 22A biosensor for detecting miRNA-182
The difference from the embodiment 2 is that: (2) in the above description, the "core-shell MCOF nanosphere solution (final concentration 0.2 mg/ml)" is replaced with "core-shell MCOF nanosphere solution (final concentration 0.5 mg/ml)"; other conditions and steps were unchanged.
Example 23A biosensor for detecting miRNA-182
The difference from the embodiment 2 is that: (2) in the above description, the "core-shell MCOF nanosphere solution (final concentration: 0.2 mg/ml)" is replaced with "core-shell MCOF nanosphere solution (final concentration: 1 mg/ml)"; other conditions and steps were unchanged.
Example 24A biosensor for detecting miRNA-182
The difference from the embodiment 2 is that: (2) in the above step, "quench for 15 min" is replaced with "quench for 5 min"; other conditions and steps were unchanged.
Example 25A biosensor for detecting miRNA-182
The difference from the embodiment 2 is that: (2) in the above step, "quench for 15 min" was replaced with "quench for 10 min"; other conditions and steps were unchanged.
Example 26A biosensor for detecting miRNA-182
The difference from the embodiment 2 is that: (2) in the above step, "quench for 15 min" was replaced with "quench for 30 min"; other conditions and steps were unchanged.
Example 27A biosensor for detecting miRNA-182
The difference from the embodiment 2 is that: (2) in the above step, the "quenching time of 15 min" is replaced by the "quenching time of 2 h"; other conditions and steps were unchanged.
Example 28
The morphology, the crystal lattice and the elemental composition of the core-shell MCOF nanospheres prepared in example 2 were observed by a field emission transmission electron microscope, the powder X-ray diffraction of the core-shell MCOF nanospheres was recorded by a Bruker D8 diffractometer, and the infrared spectrum was obtained by an infrared spectrometer.
As shown in FIG. 1, the results show that core-shell MCOF nanospheres are made of uniform Fe3O4Nanospheres are the core and highly crystalline Covalent Organic Frameworks (COFs) are the shell.
Example 29
The molecular structure, element content, specific surface area and pore size distribution of the core-shell MCOF nanospheres prepared in example 2 were analyzed by X-ray photoelectron spectroscopy and specific surface and pore size analyzer.
As shown in fig. 2, the results showed C, O and N element distributions and confirmed the crystalline COF framework, and the absence of significant Fe signal in XPS confirmed Fe3O4Completely wrapped by a COF shell. The BET surface area and the pore volume of MCOF respectively reach 224m2And 0.32 cc/g.
Example 30
The influence of probe synergy, hybridization strand reaction temperature, probe concentration, magnetic field effect and different media on the detection of the biosensor prepared in example 2 was analyzed by a fluorescence spectrometer.
H1 and H2 (or no H2) and miRNA-182 (or no miRNA-182) are added into PBS, and after being mixed well, the mixture is incubated for 1H at room temperature. And then adding the core-shell MCOF nanosphere solution (or not adding the MCOF nanosphere solution) into the mixture, quenching for 15min, separating under the action of a magnetic field, taking the supernatant, performing fluorescence measurement, and inspecting the synergistic effect of the probe.
By adding H1 (final concentration 50nM) and H2 (final concentration 50nM) and miRNA-182 to PBS, after mixing well, incubation was performed at room temperature (or 4 deg.C, 37 deg.C) for 1H. Then adding core-shell MCOF nanosphere solution (final concentration is 0.2mg/ml) into the mixture, quenching for 15min, separating under the action of magnetic field, taking supernatant, performing fluorescence measurement, and examining the influence of hybridization chain reaction temperature.
The effect of different probe concentrations was examined by example 8-example 14.
By adding H1 (final concentration 50nM) and H2 (final concentration 50nM) and miRNA-182 to PBS, after mixing well, incubation was performed at room temperature for 1H. Then adding core-shell MCOF nanosphere solution (final concentration of 0.2mg/ml) into the mixture, quenching for 15min, separating under magnetic field action (or without magnetic field action), collecting supernatant, and performing fluorescence measurement to examine the influence of magnetic field action.
By adding H1 (final concentration 50nM) and H2 (final concentration 50nM) and miRNA-182 to PBS (or rat plasma, serum, whole blood), mixing well, and incubating at room temperature for 1H. Then adding a core-shell MCOF nanosphere solution (with a final concentration of 0.2mg/ml) into the mixture, quenching for 15min, separating under the action of a magnetic field, taking the supernatant, performing fluorescence measurement, and inspecting the influence of different media.
As shown in fig. 3, the results show that dsDNA cannot be formed without H2, MCOF quenches the fluorescence of H1 by specific adsorption, and in the presence of both H1 and H2, this quenching is significantly blocked due to miRNA hybridizing with H1 and H2 to non-adsorbable dsDNA. The result shows that the detection temperature is 25 ℃, the concentration of the H1+ H2 probe is 50nM, and the probe has the best detection performance under the condition of synergistic effect with magnetic separation.
Example 31
The linearity and specificity of the detection method of the biosensor prepared in example 2 in the serum matrix were analyzed by fluorescence spectroscopy.
The miRNA-182 (with the final concentration gradient of 10 fM-1 nM) and H1 (with the final concentration of 50nM) and H2 (with the final concentration of 50nM) were added to PBS, mixed well and incubated at room temperature for 1H. Then, a core-shell MCOF nanosphere solution (final concentration of 0.2mg/ml) was added to the mixture, quenched for 15min, separated by magnetic field, and the supernatant was subjected to fluorescence measurement to examine linearity.
By adding H1 (final concentration 50nM) and H2 (final concentration 50nM) and miRNA-182 (or single base mismatch sequence, four base mismatch sequence, random sequence) into PBS, mixing well, and incubating at room temperature for 1H. Then adding core-shell MCOF nanosphere solution (final concentration is 0.2mg/ml) into the mixture, quenching for 15min, separating under the action of magnetic field, taking supernatant, performing fluorescence measurement, and examining specificity.
As shown in fig. 4 and 5, the results show that the biosensor has high specificity and good linearity for miRNA in serum.
Example 32
Pre/post operative sera of 12 glioma patients and sera of 12 healthy subjects were examined by the biosensor prepared in example 2.
As shown in FIG. 6, the results showed that the mean concentration of miRNA-182 in pre-operative serum of glioma patients was significantly higher than that of healthy persons. After 1 week of surgery, the values dropped significantly to near normal levels.
Example 33
H1 (final concentration 50nM) and H2 (final concentration 50nM) were added to the PBS with miRNA-182 and after thorough mixing, incubated at room temperature for 1H. Then, the core-shell MCOF nanosphere solution (final concentration 0.2mg/ml) prepared in example 2 was added to the above mixture, quenched for 15min, separated by magnetic field, and the supernatant was taken from the capillary tube, and the feedback of the capillary chip biosensor system was observed by laser scanning confocal microscope under different miRNA concentrations, and the results showed that the red fluorescence in the capillary tube was gradually enhanced with the increase of miRNA concentration. This particular and sensitive biosensing performance is in good agreement with the fluorescence spectroscopy measurements described above, as shown in FIG. 7.
Example 34
The thermal stability of the MCOF material is characterized by thermogravimetric analysis, and the magnetic separation capability of the MCOF material is observed by applying a magnetic field, so that the MCOF material is shown to have good thermal stability and magnetic separation capability, as shown in figures 8 and 9.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (10)
1. A preparation method of a biosensor for detecting miRNA-182 is characterized by comprising the following steps:
step 1: by adding Fe3O4Adding tetraethyl orthosilicate TEOS and (3-aminopropyl) triethoxysilane APTES into the nanospheres to prepare aminated magnetic nanospheres;
step 2: reacting monomer 2,4, 6-trihydroxybenzene-1, 3, 5-trimethyl aldehyde with the aminated magnetic nanospheres to prepare 2,4, 6-trihydroxybenzene-1, 3, 5-trimethyl aldehyde functionalized magnetic nanospheres;
then carrying out COF (chip on film) mediated in-situ growth on the 2,4, 6-trihydroxybenzene-1, 3, 5-trimethylaldehyde functionalized magnetic nanosphere to prepare a core-shell MCOF nanosphere;
and step 3: and (3) incubating the hairpin DNA probe 1 and the hairpin DNA probe 2 with miRNA-182 at room temperature, and then carrying out quenching reaction with the core-shell MCOF nanospheres to construct a miRNA-182 biosensor with amplified fluorescence signals.
2. The preparation method of claim 1, wherein in step 2, the monomer 2,4, 6-trihydroxybenzene-1, 3, 5-triformal is grafted to the surface of the aminated nanosphere through Schiff base reaction, and then the 2,4, 6-trihydroxybenzene-1, 3, 5-triformal functionalized magnetic nanosphere and the monomer 2,4, 6-trihydroxybenzene-1, 3, 5-triformal and benzidine are subjected to in-situ growth of COF at high temperature to prepare the core-shell type MCOF nanosphere.
3. The method of claim 2, wherein the elevated temperature conditions are: reacting for 10-72 h at 60-240 ℃.
4. The method according to claim 1, wherein in step 3, the hairpin DNA probe 1 and the hairpin DNA probe 2 have two sequences of 18 to 32 bases in length that can undergo a hybridization reaction.
5. The method according to claim 1, wherein in step 3, the final concentration of the hairpin DNA probe 1 and the hairpin DNA probe 2 is 1nM to 1. mu.M; the final concentration of the core-shell MCOF nanosphere is 0.02mg/ml to 1 mg/ml.
6. The method according to claim 1, wherein in step 3, the incubation time is: 30 min-24 h; the quenching time is 5 min-2 h.
7. The preparation method of claim 1, wherein the size distribution of the core-shell MCOF nanospheres is 300nm to 600nm, and the Zeta potential is-50.0 mV to 50.0 mV.
8. A biosensor for detecting miRNA-182, prepared by the method of any one of claims 1-7.
9. Use of the biosensor of claim 8 in the preparation of a reagent for detecting miRNA-182.
10. The use according to claim 9, wherein after the biosensor according to claim 8 is constructed, the supernatant is separated by the action of a magnetic field, and the supernatant is subjected to fluorescence measurement to detect the content of miRNA-182 in the sample to be detected.
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