CN111007128A

CN111007128A - Iron oxyhydroxide-MoS2Nanometer hybrid material and preparation method thereof, electrode for aptamer sensor and aptamer sensor

Info

Publication number: CN111007128A
Application number: CN201911379603.6A
Authority: CN
Inventors: 张治红; 胡彬; 黄顺江; 何领好; 王明花; 崔静; 宋英攀; 贾巧娟
Original assignee: Zhengzhou University of Light Industry
Current assignee: Zhengzhou University of Light Industry
Priority date: 2019-12-27
Filing date: 2019-12-27
Publication date: 2020-04-14
Anticipated expiration: 2039-12-27
Also published as: CN111007128B

Abstract

The invention belongs to the technical field of nano hybrid materials, and particularly relates to iron oxyhydroxide-MoS₂A nano hybrid material, a preparation method thereof, an electrode for an aptamer sensor and the aptamer sensor. The iron oxyhydroxide-MoS₂The nano hybrid material comprises a hydroxyl ferric oxide nano rod and MoS grown on the surface of the hydroxyl ferric oxide nano rod₂Nanosheets, said MoS₂The nanosheet is obtained by carrying out solvothermal reaction on phosphomolybdic acid hydrate and thioacetamide. Iron oxyhydroxide-MoS of the invention₂In the nano-hybrid material, MoS₂The nano-sheet vertically grows on a rodlike β -FeOOH substrate to form a nano-hybrid multi-component structure with uniform components, and the miRNA-21 biosensor obtained based on the nano-hybrid material has excellent sensing propertyHigh sensitivity, excellent stability and reproducibility, high selectivity and feasibility.

Description

Iron oxyhydroxide-MoS2Nanometer hybrid material and preparation method thereof, electrode for aptamer sensor and aptamer sensor

Technical Field

The invention belongs to the technical field of nano hybrid materials, and particularly relates to iron oxyhydroxide-MoS₂A nano hybrid material, a preparation method thereof, an electrode for an aptamer sensor and the aptamer sensor.

Background

Early screening and detection of cancer is crucial for its effective treatment and improvement of patient survival, and development of rapid, low-cost sensing systems is a focus of attention. Micrornas (mirnas), which are endogenous and non-coding short-chain RNAs, are considered oncogenes and tumor suppressor factors due to their overexpression in cancer cells. Among them, miRNA-21 has been of great interest as a potent biomarker because it is involved in different cellular and biological functions. However, micrornas often exhibit characteristics such as short chain, sequence similarity, and low abundance, which pose significant challenges to their clinical diagnosis.

Currently, scientists invented various analysis methods and biosensors aiming at the sensitive detection of miRNA in biology, such as fluorescence spectroscopy, chemiluminescence spectroscopy, inductively coupled plasma mass spectrometry, raman spectroscopy, photoelectrochemical analysis, electrochemiluminescence analysis, electrochemical techniques, and the like. Among them, the electrochemical technique is considered to be one of the most effective methods due to its advantages of low cost, high detection sensitivity, good specificity, simple instrument, etc. However, miRNA is present in plasma or serum in low levels of femtomolar (fM), nanomolar (nM), and enhancing the detection signal is a major problem for miRNA sensors. Although many methods have been attempted to develop various sensors for detecting mirnas, the relatively low detection sensitivity limits their practical applications. Therefore, it is urgent to develop an ultra-sensitive and cost-effective biosensor for detecting miRNA.

A typical DNA electrochemical biosensor consists of a gold/glassy carbon electrode with an anchored single stranded DNA or RNA probe. Through the hybridization reaction of the single-stranded DNA or RNA probe aptamer and a target detection object, the electrochemical behavior of the interface of the electrode and the electrolyte is changed, so that the electrochemical signal is changed. At present, people construct different electrochemical biosensors based on miRNA and complementary targets thereof in different ways, such as bispecific and nuclease-assisted targeted cyclic signal amplification, two-dimensional DNA nanoprobes and enzyme-free target cyclic amplification, glucose/O-based₂A paper self-powered system of a biofuel cell, a self-assembled pH-sensitive continuous circular DNA nano switch, a homogeneous phase electrochemical method based on a nucleic acid functionalized metal-organic framework, an exonuclease-assisted target circulation method and the like. However, the electrochemical biosensor has problems of complicated preparation, complicated DNA sequence design, or complicated signal amplification/indicator development design, and the like, which limits its application.

The nano material, such as conductive polymer, carbon nano material, metal organic framework or covalent organic framework material, can be used as a scaffold modified electrode for fixing probe molecules due to large specific surface area and high conductivity. In addition, the nano material can effectively fix the biological recognition molecules and accelerate the electron transfer of the biosensor. Therefore, the method for preparing the miRNA detection sensitive biosensor by optimizing the electrode material by a feasible method has important significance.

MoS₂Nanosheets (NSs) have received a great deal of attention in recent years due to their abundant and unique structural properties and have found wide application in a variety of fields. However, MoS₂The practical application of the electrochemical catalyst is limited by low electrochemical activity and easy agglomeration. To overcome the above drawbacks, many efforts have been made, one of the most important of which is to integrate it with some conductive materials and use it for the construction of MoS₂The base complex detects different targets, such as miRNA, alpha-fetoprotein or 5 hmC. However, the above MoS₂The electrochemical activity and biocompatibility of the base composite electrode modified material are limited, and the detection sensitivity of the constructed electrochemical biosensor is limitedTo be improved.

Disclosure of Invention

The invention aims to provide iron oxyhydroxide-MoS₂The nanometer hybrid material has excellent electrochemical activity and biocompatibility, and can be used as an electrode modification material of an aptamer sensor for detecting miRNA-21 to improve detection sensitivity.

Another object of the present invention is to provide an iron oxyhydroxide-MoS₂Method for preparing nano hybrid material to improve existing MoS₂The electrochemical activity and biocompatibility of the base composite electrode modified material.

The third purpose of the invention is to provide an electrode for an aptamer sensor, which has excellent biocompatibility, high electrochemical activity and strong biological affinity to miRNA-21 complementary DNA, and can be used for constructing the aptamer sensor so as to improve the miRNA-21 detection sensitivity.

The fourth purpose of the invention is to provide an aptamer sensor, so as to improve the detection sensitivity of miRNA-21.

To achieve the above object, the iron oxyhydroxide-MoS of the present invention₂The specific technical scheme of the nano hybrid material is as follows:

iron oxyhydroxide-MoS₂Nano hybrid material comprising iron oxyhydroxide nanorods and MoS grown on the surface thereof₂Nanosheets, said MoS₂The nanosheet is obtained by carrying out solvothermal reaction on phosphomolybdic acid hydrate and thioacetamide.

Iron oxyhydroxide-MoS of the invention₂Nano hybrid material, abbreviated as pd-MoS₂@ β -FeOOH nano hybrid material based on existing conductive material and MoS₂Composite MoS₂When the base compound is used as an electrode modification material, the electrochemical activity and biocompatibility cannot meet the requirement of low-content miRNA-21 detection, and MoS is obtained by designing and synthesizing phosphomolybdic acid hydrate and thioacetamide through hydrothermal reaction₂Nanosheet (pd-MoS)₂NSs) and hybridized with the hydroxyl ferric oxide nano-rod with a large tunnel type structure to obtain a nano hybrid material with high electrochemical activity and excellent biocompatibility to promote electron transfer andthe sensing sensitivity of the material is improved.

Specifically, the phosphomolybdate hydrate simultaneously contains oxygen, phosphorus and transition metal, has changeable chemical structure and excellent electronic universality, can be combined with various electrons, and has higher electrochemical activity; the hydroxyl ferric oxide has a large tunnel structure, and the firmly combined Fe atom can convert H into H₂O is firmly anchored in tunnels and is commonly used in fields such as oxidation catalysts, batteries, semiconductor electronics, magnetic storage media, and bioimaging. MoS derived from phosphomolybdic acid hydrate₂The nanosheet-combined iron oxyhydroxide nanorod as an electrode modification material of the aptamer sensor can promote electron transfer and improve the sensing sensitivity of the sensor.

Iron oxyhydroxide-MoS of the invention₂pd-MoS in nano hybrid material₂The nano-sheets grow around the iron oxyhydroxide nano-rods to form a nano-hybrid multi-component structure with uniform components, and the hybrid material contains Mo⁴⁺/Mo⁵⁺And Fe⁰/Fe²⁺/Fe³⁺The product has chemical valence, rich oxygen vacancy and Fe-O bond. The miRNA-21 biosensor obtained based on the nano hybrid material has excellent sensing performance, high sensitivity, excellent stability and reproducibility, high selectivity and feasibility.

Iron oxyhydroxide-MoS of the invention₂The specific technical scheme of the preparation method of the nano hybrid material is as follows:

iron oxyhydroxide-MoS₂The preparation method of the nano hybrid material comprises the following steps: carrying out solvothermal reaction on a mixed solution consisting of the iron oxyhydroxide nanorod, the phosphomolybdic acid hydrate, the thioacetamide and the solvent to obtain the nano-composite material.

Iron oxyhydroxide-MoS of the invention₂The preparation method of nano hybrid material uses phosphomolybdic acid hydrate as molybdenum source and thioacetamide as sulfur source, and grows MoS on the iron oxyhydroxide nano-rod₂Nanosheets to give iron oxyhydroxide-MoS₂The preparation method of the nano hybrid material is simple, and the obtained nano hybrid material has good electrochemical activity and biocompatibility.

In order to further optimize the electrochemical activity and biocompatibility of the obtained nano hybrid material, the mass ratio of the iron oxyhydroxide nanorod, the phosphomolybdate hydrate and the thioacetamide is (1-4): 1: 1.

In order to improve the reaction rate, the reaction temperature is 180-220 ℃, and in order to ensure full reaction, the reaction time is 8-16 h.

The mixed solution is obtained by mixing the iron oxyhydroxide nanorod, the phosphomolybdic acid hydrate aqueous solution and the thioacetamide methanol solution.

The iron oxyhydroxide nanorod can be obtained by carrying out solvothermal reaction on ferric chloride and glucose, and is marked as β -FeOOH nanorod (β -FeOOH NRs), further, the reaction temperature is 150-180 ℃, and the reaction time is 5-8 h.

The specific technical scheme of the electrode for the aptamer sensor comprises the following steps:

the electrode for the aptamer sensor comprises an electrode substrate and an electrode modification material on the surface of the electrode, wherein the electrode modification material is the iron oxyhydroxide-MoS₂A nano hybrid material.

The electrode has excellent biocompatibility, high electrochemical activity and strong biological affinity to miRNA-21 complementary DNA, and can be used for constructing aptamer sensors to improve the detection sensitivity of nucleic acid aptamers.

The aptamer sensor has the specific technical scheme that:

an aptamer sensor comprises an electrode substrate, an electrode modification material on the surface of an electrode and a nucleic acid aptamer fixed on the electrode modification material, wherein the electrode modification material is the iron oxyhydroxide-MoS₂A nano hybrid material.

The aptamer sensor can improve the detection sensitivity of the aptamer, and specifically comprises the following steps: pd-MoS₂@ β -FeOOH nano hybrid material and its use as electrode material to construct electrochemical biosensor for detecting miRNA, pd-MoS₂@ β -FeOOH not only can be used as a platform to anchor the cDNA chain of miRNA-21, but also can effectively conduct electrochemical signalsIn pd-MoS₂@ β -FeOOH has good biocompatibility and high electrochemical activity, and a large number of cDNA strands are fixed on pd-MoS through van der Waals force between nucleobases and interaction between β -FeOOH/DNA₂@ β -FeOOH surface when miRNA-21 is present, the miRNA and cDNA form a helical duplex by hybridization, resulting in a conformational change in the cDNA, a change at each step in the sensor construction process, such as pd-MoS₂The electrode surface changes such as @ β -FeOOH modified electrode, cDNA adsorption and DNA chain conformation changes and the like can be expressed by electrochemical technologies such as EIS, CV and the like.

Furthermore, the aptamer is complementary DNA of miRNA-21, and a sensor constructed by the aptamer can improve the detection sensitivity of miRNA-21 and is beneficial to early screening and detection of cancers.

Drawings

FIG. 1 shows iron oxyhydroxide-MoS according to the invention₂SEM image of nano-hybrid;

FIG. 2 shows the iron oxyhydroxide-MoS of the present invention₂High resolution TEM images of the nano-hybrid;

FIG. 3 shows the iron oxyhydroxide-MoS of the present invention₂A high resolution TEM image of the nano-hybrid, wherein (d) corresponds to element Mo, (e) corresponds to element Fe, (f) corresponds to element O, (g) corresponds to element C;

FIG. 4 is an SEM image of β -FeOOH nanorods of the comparative example of the present invention;

FIG. 5 is a high resolution TEM image of β -FeOOH nanorods of the comparative example of the present invention;

FIG. 6 shows pd-MoS of comparative example of the invention₂SEM images of the nanoplatelets;

FIG. 7 shows pd-MoS of comparative example of the invention₂High resolution TEM images of the nanoplates;

FIG. 8 shows a schematic representation of the iron oxyhydroxide-MoS of the present invention₂Nano hybrid material, β -FeOOH nanorod and pd-MoS nanorod of comparative example₂XRD spectra of the nanosheets;

FIG. 9 shows a schematic representation of the iron oxyhydroxide-MoS of the present invention₂Nano hybrid material, β -FeOOH nanorod and pd-MoS nanorod of comparative example₂FT-IR spectra of the nanoplatelets;

FIG. 10 shows a schematic representation of the iron oxyhydroxide-MoS of the present invention₂Nano hybrid material, β -FeOOH nanorod and pd-MoS nanorod of comparative example₂XPS spectra of the nanoplates;

FIG. 11 shows a schematic representation of the iron oxyhydroxide-MoS of the present invention₂High resolution XPS spectra of (h) Mo 2p, (i) S2 p and (j) Fe2p in the nano hybrid material;

FIG. 12 shows a schematic representation of the iron oxyhydroxide-MoS of the present invention₂C1s high resolution XPS spectrum of the nano hybrid material;

FIG. 13 shows a schematic representation of the iron oxyhydroxide-MoS of the present invention₂High-resolution XPS spectrum of O1s of the nano hybrid material;

FIG. 14 shows a schematic representation of the iron oxyhydroxide-MoS of the present invention₂Nano hybrid material and β -FeOOH nano rod, pd-MoS nano rod of comparative example₂Influence of the nanosheets on the viability of the L929 cells;

FIG. 15 shows iron oxyhydroxide-MoS according to the present invention₂Nano hybrid material and β -FeOOH nano rod, pd-MoS nano rod of comparative example₂The effect of the nanosheets on the viability of the MCF-7 cells;

FIG. 16 is an EIS curve of each step in the process of detecting miRNA-21 by using an aptamer sensor constructed by β -FeOOH nanorods of a comparative example;

FIG. 17 is a CV curve of each step in the process of detecting miRNA-21 of an aptamer sensor constructed by using β -FeOOH nanorods of a comparative example;

FIG. 18 shows pd-MoS using comparative example₂An EIS curve of each step of the aptamer sensor constructed by the nanosheets in the miRNA-21 detection process;

FIG. 19 shows pd-MoS using comparative example₂The CV curve of each step of the aptamer sensor constructed by the nanosheets in the miRNA-21 detection process;

FIG. 20 shows a schematic representation of iron oxyhydroxide-MoS using the present invention₂An aptamer sensor constructed by the nano hybrid material detects an EIS curve of each step in the miRNA-21 detection process;

FIG. 21 shows a schematic representation of iron oxyhydroxide-MoS using the present invention₂The CV curve of each step of an aptamer sensor constructed by the nano hybrid material in the process of detecting miRNA-21;

FIG. 22 shows the use of the present inventionMing iron oxyhydroxide-MoS₂Nano hybrid material and β -FeOOH nano rod, pd-MoS nano rod of comparative example₂Aptamer sensor constructed by nanosheet modified electrode and used for detecting delta R of each stage in preparation process of miRNA-21_ctA value;

FIG. 23 is a.DELTA.R of each step in detecting miRNA-21 using the aptamer sensor of the present invention_ctAnd pd-MoS₂The relationship of dosage of @ β -FeOOH nano hybrid material;

FIG. 24 is a graph of the effect of aptamer concentration on the detection of miRNA-21 by the aptamer sensor of the invention;

FIG. 25 is a graph showing the Δ R obtained from the aptamer sensor of the invention with miRNA-21 at different action times_ctA value;

FIG. 26 is an EIS plot of miRNA-21 at different concentrations detected using the aptamer sensor of the invention;

FIG. 27 shows Δ R_ctAnd miRNA-21 concentration;

FIG. 28 shows Δ R_ctThe concentration logarithmic relation with miRNA-21 and a fitted curve, wherein an error line is a standard deviation when n is 3;

FIG. 29 is a diagram showing the specificity of miRNA-21 detection by the aptamer sensor of the invention;

FIG. 30 is a graph showing the reproducibility of the detection of miRNA-21 by the aptamer sensor of the invention;

FIG. 31 is a graph showing the detection of Δ R for miRNA-21 for 15 consecutive days_ctA value;

FIG. 32 shows R obtained by 10 detections of the aptamer sensor of the invention_ctA value;

FIG. 33 is a diagram showing detection of R of miRNA-21 contained in L929 cells and MCF-7 cells by the aptamer sensor of the present invention_ctA change in value.

Detailed Description

The present invention will be further described with reference to the following specific examples.

All chemicals were analytical reagent grade and no further purification was required.

All solutions were prepared with Milli-Q ultra pure water (. gtoreq.18.2 M.OMEGA.cm).

In the examples, the sources of the raw materials are as follows:

thioacetamide (TAA) was purchased from Komiou Chemical Reagent co., Ltd. (tianjin, china);

ferric chloride hexahydrate (FeCl)₃·6H₂O) purchased from national pharmaceutical group chemical agents, Inc.;

glucose and phosphomolybdic acid hydrate (H)₃P(Mo₃O₁₀)₄·xH₂O) purchased from Maclean biochemical technology co., Ltd. (shanghai, china);

cDNA, miRNA-21, miRNA-141, miRNA-155, miRNA-126, single base mismatched miRNA-21(MM1) and three base mismatched miRNA-21(MM3) were purchased from Qingke Biological Technology Co., Ltd (Beijing, China), wherein:

cDNA:5′-TTTT CAA CAT CAG TCT GAT AAG CTA TTT-3′

miRNA-21:5′-UAG CUU AUC AGA CUG AUG UUG A-3′；

MM1:5′-UAG CUU AUA AGA CUG AUG UUG A-3′

MM3:5′-UAG CUU AUA ACC CUG AUG UUG A-3′

miRNA-141:5′-UAA CAC UGU CUG GUA AAG AUG G-3′

miRNA-155:5′-UUA AUG CUA AUC GUG AUA GGG G-3′

miRNA-126:5′-UCG UAC CGU GAG UAA UAA UGC G-3′

KCl，NaCl，KH₂PO₄，Na₂HPO₄，K₃[Fe(CN)₆]·H₂o is purchased from national drug-controlled chemical reagents ltd (beijing, china);

the concentration of the PBS buffer solution is 0.1M, the pH value is 7.4, and the specific preparation method comprises the following steps: mixing 1.44gNa₂HPO₄、0.24gKH₂PO₄8g of NaCl and 0.2g of KCl in 1000mL of deionized water.

Human breast cancer cells (MCF-7) and mouse fibroblasts (L929) were obtained from the Chinese academy of sciences cell line (Shanghai, China).

Mono, hydroxy ferric oxide-MoS₂Examples of Nanohybridization materials

Example 1

pd-MoS of this example₂@ β -FeOOH nano hybrid material, comprising iron oxyhydroxide nano rod and MoS grown on the surface thereof₂Nanosheets, said MoS₂The nanosheet consists of H₃P(Mo₃O₁₀)₄·xH₂Carrying out solvothermal reaction on O and thioacetamide to obtain the compound.

Di, hydroxy iron oxide-MoS₂Examples of the preparation of Nanohydrated materials

Example 2

pd-MoS of this example₂Preparation method of @ β -FeOOH nano hybrid material, for pd-MoS in example 1₂The preparation of the @ β -FeOOH nano hybrid material is explained, and the specific steps are as follows:

(1) mixing 25mgH₃P(Mo₃O₁₀)₄·xH₂O dissolved in 20mL H₂Obtaining solution A in O, dissolving 25mg of TAA in 30mL of methanol to form solution B, dropwise adding the solution A into the solution B under magnetic stirring, and simultaneously adding 50mg of β -FeOOH nano rod into the mixture in the system;

(2) transferring the obtained mixture into a 50mL autoclave, and reacting for 12h at 200 ℃;

(3) after the reaction is finished, naturally cooling to room temperature, washing the product with ethanol, and drying at 60 ℃ for 12h to obtain pd-MoS₂@ β -FeOOH nano hybrid material.

The β -FeOOH nano rod is synthesized by adopting the existing solvothermal method, and specifically comprises the following steps:

(1) 4.5g FeCl₃·6H₂O and 70mg of glucose were dissolved in 35mL of ultrapure water;

(2) transferring the clear solution into a 50mL sealed autoclave, and reacting for 6 hours at 163 ℃;

(3) after the reaction was completed, it was cooled to room temperature, centrifuged and washed with ethanol, and then dried at 100 ℃ for 3 hours to obtain β -FeOOH yellow powder.

Example 3

(1) 50mg of H₃P(Mo₃O₁₀)₄·xH₂O dissolved in 20mL H₂Obtaining solution A in O, dissolving 50mg of TAA in 30mL of methanol to form solution B, dropwise adding the solution A into the solution B under magnetic stirring, and simultaneously adding 50mg of β -FeOOH nano-rod into the mixture in the system;

(2) transferring the obtained mixture into a 50mL autoclave, and reacting for 12h at 220 ℃;

(2) transferring the clear solution into a 50mL sealed autoclave, and reacting for 5 hours at 180 ℃;

Example 4

(1) adding 25mg of H₃P(Mo₃O₁₀)₄·xH₂O dissolved in 20mL H₂Obtaining solution A in O, dissolving 25mg of TAA in 30mL of methanol to form solution B, dropwise adding the solution A into the solution B under magnetic stirring, and simultaneously adding 75mg of β -FeOOH nano rod into the mixture in the system;

(2) transferring the obtained mixture into a 50mL autoclave, and reacting for 15h at 200 ℃;

(1) 4.5g of FeCl₃·6H₂O and 70mg of glucose were dissolved in 35mL of ultrapure water;

(2) transferring the clear solution into a 50mL sealed autoclave, and reacting for 8 hours at 150 ℃;

Embodiments of electrodes for aptamer Sensors

Example 3

The electrode for the aptamer sensor comprises an electrode substrate and an electrode modification material on the surface of the electrode substrate, wherein the electrode modification material is the iron oxyhydroxide-MoS of the embodiment 1₂A nano hybrid material.

Fourth, embodiments of aptamer sensor

Example 4

The aptamer sensor comprises an electrode modification material and a nucleic acid aptamer, wherein the electrode modification material is an electrode substrate decorated on the surface of an electrode, and the nucleic acid aptamer is fixed on the electrode modification material, and the electrode modification material is pd-MoS in example 1₂@ β -FeOOH nano hybrid material, nucleic acid aptamer is complementary DNA of miRNA-21.

(1) 1, 2, 5, 10 and 20mg of pd-MoS₂@ β -FeOOH powder was dispersed in 10mL of Milli-Q water, respectively, and sonicated for 10 minutes to give concentrations of 0.1, 0.2, 0.5, 1 and 2 mg-mL, respectively^-1pd-MoS of₂@ β -FeOOH dispersion solution;

(2) the above pd-MoS₂@ β -FeOOH dispersion (5.0. mu.L) was dropped onto pretreated bare gold electrode (AE) at room temperature₂Drying to obtain pd-MoS₂@ β -FeOOH modified electrode material in pd-MoS₂@ β -FeOOH/AE, the above pretreatment process is specifically to physically polish the bare gold electrode on a chamois leather with 0.5 μm alumina powder, and then put the bare gold electrode in piranha solution (H)₂SO₄：H₂O₂7:3(v/v)) for 15min, and then dividingRespectively performing ultrasonic treatment with anhydrous ethanol and deionized water for 5 min; then taking out the bare gold electrode for drying, and adopting Cyclic Voltammetry (CV) at H of 0.5M₂SO₄Activating treatment is carried out in (-0.2-1.6V), then washing is carried out by Milli-Q water, and drying is carried out under nitrogen gas, thus obtaining the pretreated gold electrode;

(3) pd-MoS₂@ β -FeOOH/AE were reacted with cDNA solutions of different concentrations (10, 20, 50, 100 and 200nM) for 2h, washed with PBS and N₂Drying to obtain cDNA fixed electrode material as cDNA/pd-MoS₂@ β -FeOOH/AE.

The sensor was stored in a refrigerator at 4 ℃ for future use. The use of cDNA materials fixed at different concentrations can be used to evaluate the sensitivity of detecting miRNA-21.

Fifth, comparative example

Comparative example 1

In the aptamer sensor of the comparative example, the electrode modification material is β -FeOOH nanorod, and the concentration of the dispersion solution is 1 mg/mL^-1Otherwise, as in example 4, the obtained β -FeOOH modified electrode material was represented by β -FeOOH/AE, and the obtained cDNA immobilized electrode material was represented by cDNA/β -FeOOH/AE.

Comparative example 2

In the aptamer sensor of the comparative example, the electrode modification material was pd-MoS₂The concentration of the nanosheet in the dispersion solution was 1 mg/mL^-1pd-MoS obtained in the same manner as in example 4₂Modified electrode materials with pd-MoS₂The term ` AE ` used for the cDNA-immobilized electrode material, and the term cDNA/pd-MoS₂and/AE.

Wherein, pd-MoS₂The preparation process of the nano-sheet is as follows:

(1) mixing 25mgH₃PO₄·12MoO₃Dissolved in 20mL of H₂Solution A was obtained in O, and 25mg of TAA was dissolved in 30mL of methanol to obtain solution B. Dropwise adding the solution A into the solution B under magnetic stirring;

(3) after the reaction is finished, naturally cooling to room temperature, washing the product with ethanol, and then drying at 60 ℃ for 12 hours to obtainpd-MoS₂Nanosheets.

Sixth, Experimental example

(one) pd-MoS₂@ β -FeOOH nano hybrid material, β -FeOOH NRs, pd-MoS₂Characterization of NSs

Experimental example 1: topography testing

The synthesized pd-MoS₂The surface morphology and the microstructure of the @ β -FeOOH nano hybrid material are observed by SEM and TEM, the SEM images of the three materials are respectively shown in figures 1, 4 and 6, and the TEM images of the three materials are respectively shown in figures 2, 5 and 7.

pd-MoS₂SEM of NSs showed a sphere assembled from multiple nanoplates with a uniform size of 500nm, consistent with TEM images. High resolution TEM images show irregular lattice spacing of 0.62nm, corresponding to pd-MoS₂The (002) crystal face of (A) is consistent with the XRD result, indicating that H₃PO₄·12MoO₃Reaction with TAA to MoS₂NSs。

Pure β -FeOOH exhibits a bullet-like nanorod structure about 500nm in length, consistent with TEM images, a lattice spacing of d 0.524nm is observed in TEM, due to the (111) lattice plane of β -FeOOH.

pd-MoS₂SEM of @ β -FeOOH nano hybrid material shows pd-MoS₂NSs are grown vertically on the surface of β -FeOOH NRs, as further confirmed by TEM images high resolution TEM images show two interplanar spacings of d 0.26 and 0.62nm, corresponding to (012) and MoS, respectively, for β -FeOOH₂And can be seen from the element distribution diagram of the high-resolution TEM image, see FIG. 3, pd-MoS₂The elements in the @ β -FeOOH nano hybrid material are uniformly distributed, namely the nano hybrid material has uniform chemical composition, and compared with the pure β -FeOOH material, pd-MoS₂The structure of the @ β -FeOOH nano hybrid material is loose, which is beneficial to electron transfer and the fixation of aptamer and target.

Experimental example 2: characterization of the Crystal Structure

pd-MoS₂@ β -FeOOH nano hybrid material, β -FeOOH NRs, pd-MoS₂The crystal structure of NSs was characterized by XRD and the results are shown in figure 8. As can be seen from the figure, pd-MoS₂NSs is 9.1 ° at 2 θ,diffraction peaks at 14.6 °, 32.1 ° and 56.6 ° due to MoS₂Consistent with high resolution TEM results, XRD of β -FeOOH NRs showed a characteristic peak of β -FeOOH (JCPDS No.78-1594)₂No MoS is observed in XRD pattern of @ β -FeOOH nano hybrid material₂The characteristic peak of (2) is only the diffraction peak of β -FeOOH, probably due to the fact that the pd-MoS is prepared₂In the process of (3), β -FeOOH is added to influence the MoS₂The crystallinity of (4) is reduced. At the same time, due to a small amount of pd-MoS₂NSs cover β -FeOOH, resulting in a lower peak density of β -FeOOH.

Experimental example 3: chemical Structure characterization

pd-MoS₂@ β -FeOOH nano hybrid material, β -FeOOH NRs, pd-MoS₂The chemical structure of NSs was characterized by fourier transform infrared (FT-IR) and XPS spectra, as shown in fig. 9, 10, 11, respectively.

In FIG. 9, pd-MoS₂FT-IR spectra of NSs vibrate at 3432cm due to O-H stretching^-1Shows a strong adsorption peak at 1630cm^-1The absorption peak at position corresponds to C ═ O stretching vibration and is located at 1395cm^-1And 1079cm^-1The absorption peaks at (A) correspond to C-OH and C-O-C, 558cm^-1And 774cm^-1The absorption peak at (a) is due to Mo-S and Mo-O stretching vibration at 1114cm^-1The absorption peak at (A) corresponds to the asymmetric stretching vibration of S ═ O. β -FeOOH NRs is located at 3427cm in the FT-IR spectrum^-1The peak at (a) corresponds to the stretching mode of the O-H bond, at 1623cm^-1The peak at (A) is due to C ═ O stretching vibration at 643 and 865cm^-1Characteristic absorption of the corresponding OH. Under pd-MoS₂The infrared spectrum of @ β -FeOOH can be simultaneously observed to be pd-MoS₂And β characteristic peaks of FeOOH, such as 556cm^-1Due to pd-MoS₂Characteristic peak sum of Mo-S in NSs 864cm^-1The position corresponds to a characteristic peak of β -OH in FeOOH.

In FIG. 10, for pd-MoS₂NSs, Mo 3d (227.6eV), S2 p (162.3eV), C1S (284.6eV), N1S (400eV) and O1S (531.5eV) signals were observed, and for β -FeOOH NRs, Fe2p (711.3eV), O1S (531.5eV) and C1S (284.6eV) signals, pd-MoS, appeared₂The XPS spectrum of the @ β -FeOOH nano hybrid material contains C1s, O1s and N1s signals, and is derived from pd-MoS₂Mo 3d (229.8 eV) and S2 p (162.3eV) of (9) and β -FeOOH of Fe2 p.

pd-MoS₂The chemical compositions and environments of elements in the @ β -FeOOH nano hybrid material are fitted with a high-resolution XPS spectrum by XPSPEAK1 software, and the XPS spectrum is shown in FIG. 11(h) and a plurality of peaks with Binding Energy (BEs) of 228.2 eV and 231.4eV respectively correspond to MoS₂Mo of⁴⁺3d_5/2And Mo ⁴⁺3d_3/2And (4) components. The peak at 229.6eV is attributed to Mo⁶⁺And (4) components. pd-MoS₂Mo in @ β -FeOOH nano hybrid material⁴⁺State to Mo⁶⁺The edges are slightly oxidized during the transition of the states. This finding can be found by corresponding to Mo-O_xThe 235.7eV peak of the bond is further demonstrated. The peak with binding energy at 225.9eV is attributed to pd-MoS₂S2S in the composition, high resolution S2 p XPS spectrogram, see FIG. 11(i), can be divided into two main peaks 161.4eV and 162.2eV, respectively attributed to S^2-S2 p of_3/2And S2 p_1/2. The binding energies at 163.2eV and 164.4eV are attributed to S₂ ^2-S2 p of_3/2And S2 p_1/2. Peaks for BE at 166.3 and 168.6eV are attributed to S⁶⁺S2 p of_3/2And S2 p_1/2. The BE peak with a peak value of 166.8eV is attributed to SO₄ ^2-A key. XPS spectra of high resolution Fe2p, FIG. 11(j), can be decomposed into Fe2p_3/2Fe (b) of⁰(707.1eV)，Fe²⁺(708.7eV) and Fe³⁺(711.85eV) and Fe²⁺Peak of (717.85eV) and Fe2p_1/2Fe (b) of³⁺(721.2 eV). The peaks at 714.9 and 723.9eV are Fe2p, respectively_3/2And Fe2p_1/2And (4) accompanying peaks.

pd-MoS₂The C1s XPS spectrum of @ β -FeOOH, see FIG. 12, can BE divided into 284.2, 285.7 and 288.5eV, corresponding to the groups C-C, C-O and COO, respectively, As shown by the O1s XPS spectrum, see FIG. 13, four peaks are obtained at BE due to M-O (M stands for metal), O vacancies, C ═ O and C-O groups, 529.9, 530.3, 531.2 and 532.5eV, respectively, M-O bonds can BE Fe-O and Mo-O, and O vacancies can significantly enhance electron transfer.

pd-MoS₂@ β -FeOOH Fe in nano hybrid material⁰、Fe²⁺、Fe³⁺The coexistence of the nano-hybrid material and oxygen vacancy can greatly promote electron transfer, thereby improving the sensing performance of the nano-hybrid material.

(II) pd-MoS₂@ β -FeOOH nano hybrid material, β -FeOOH NRs, pd-MoS₂Cytotoxicity assays for NSs

Experimental example 4:

because the aptamer sensor is mainly used for detecting human serum, the pd-MoS is firstly aligned before the aptamer sensor is constructed₂The biocompatibility of @ β -FeOOH nano-hybrid materials was tested, as shown in FIGS. 14 and 15, pd-MoS₂NSs, &lTtT translation = β "&gTt β &lTt/T &gTt-FeOOH NRs and pd-MoS₂The cytotoxicity of the @ β -FeOOH nano hybrid material is verified by a typical MTT test on L929 normal cells and MCF-7 cancer cells, and the results show that the cell survival rate is changed from 10 to 200 mu g/mL along with the concentration after the L929 and MCF-7 cells are cultured for 24 hours by using three nano materials^-1And increased by an increase. Wherein pd-MoS is adopted₂The survival rate of the cultured cells is the highest, indicating good biocompatibility. When the concentration is as high as 200 mug. mL^-1When, pd-MoS₂The survival rates of L929 cells and MCF-7 cells cultured by the @ β -FeOOH nano hybrid material are 67.1 percent and 70 percent, which indicates that pd-MoS₂The presence of NSs can enhance pd-MoS₂The biocompatibility of the @ β -FeOOH nano hybrid material.

Serum samples used in the experiment were collected after informed consent was obtained from the first subsidiary hospital of zhengzhou university. The study protocol met the ethical standards of Helsinki declaration, and its amendments made in 1964, and was approved by the ethical Committee of the first subsidiary hospital of Zhengzhou university. To evaluate pd-MoS₂The applicability of the @ β -FeOOH-based sensor to real samples is characterized in that miRNA-21 with different concentrations is added into collected human serum samples by a standard adding method, the sensor constructed by the invention is used for testing, the concentration of the miRNA-21 is detected according to a calibration curve and compared with an actual value, and in addition, the applicability of the sensor is verified by detecting miRNA-21 contained in L929 cells and breast cancer cells (MCF-7).

(III) detection experiment of aptamer sensor on miRNA-21

Experimental example 5: EIS and CV experiments

pd-MoS₂The sensing performance of the @ β -FeOOH nano hybrid material for detecting miRNA-21 adopts two electrochemical technologies, namely Electrochemical Impedance Spectroscopy (EIS) and Cyclic Voltammetry (CV) research, and the EIS method is used for detecting pd-MoS₂Aptamer sensor with @ β -FeOOH as electrode modification material (hereinafter referred to as pd-MoS)₂@ β -FeOOH-based aptamer sensor, similarly, with pd-MoS₂β -FeOOH is used as an aptamer sensor of electrode modification materials and is respectively abbreviated as pd-MoS₂Aptamer-based sensors, β -FeOOH-based aptamer sensors) electrode interface performance at each step in the construction process was typically analyzed using Zview2 software.

In order to optimize the performance of the constructed aptamer sensor, the pd-MoS is subjected to pre-detection₂The parameters of @ β -FeOOH dosage, cDNA concentration, miRNA-21 action time and the like are optimized, and the cDNA/pd-MoS is used for measuring the parameters under the optimal measurement parameter condition₂@ β -FeOOH/AE detects miRNA with different concentrations to obtain pd-MoS₂@ β -lowest limit of detection (LOD) for FeOOH-based aptamer sensors.

pd-MoS₂Construction of @ β -FeOOH-based aptamer sensor and EIS curve in the detection of miRNA-21 are shown in FIG. 20 for comparison, for β -FeOOH and pd-MoS₂The performance of the aptamer sensors was also compared using EIS and the results are shown in fig. 16, fig. 18. In FIG. 20, the bare gold electrode AE shows fast electron transfer with the electrolyte, thus resulting in a lower R_ctA value of 97. omega. with pd-MoS₂After electrode AE is modified by @ β -FeOOH nano hybrid material, pd-MoS₂R of @ β -FeOOH/AE_ctThe value increased to 445.2 Ω because of pd-MoS₂The electrochemical activity of @ β -FeOOH was weak, hindering electron transfer at the interface of the electrode and electrolyte solution anchoring of the cDNA aptamer at pd-MoS₂@ β -FeOOH nano hybrid material, cDNA/pd-MoS₂R of @ β -FeOOH/AE_ctThe value increased to 1.1 k.OMEGA.due to the large amount of negatively charged phosphate groups caused by the cDNA strand on the electrode surface, effectively hindering [ Fe (CN)₆]^3-/4-Oxidation by oxygenReducing electron transport between the probe and the electrode. When the constructed aptamer sensor detects miRNA-21, miRNA-21/cDNA/pd-MoS₂R of @ β -FeOOH/AE_ctThe value further increased to 2.2 k.OMEGA.indicating that hybridization between cDNA and miRNA-21 further prevented electron transfer at the solid/liquid interface. pd-MoS₂And β -FeOOH-based aptamer sensors gave similar results.

Table 1 summarizes β -FeOOH, pd-MoS for the different electrodes₂And pd-MoS₂R of @ β -FeOOH group aptamer sensor in different steps_ctThe value is obtained. pd-MoS₂Bare gold electrode AE, pd-MoS in basal aptamer sensor₂/AE、cDNA/pd-MoS₂AE and miRNA-21/cDNA/pd-MoS₂R caused by/AE_ctValues of 59.4 omega, 192 omega, 488 omega and 724 omega respectively β -FeOOH group R in aptamer sensor caused by naked gold electrode AE, β -FeOOH/AE, cDNA/β -FeOOH/AE and miRNA-21/cDNA/β -FeOOH/AE_ctThe values were 80 Ω, 399.3 Ω, 611.7 Ω and 892.5 Ω, respectively.

TABLE 1R of three aptamer sensors at each step in the detection of miRNA-21_ctValue of

However, it is difficult to directly analyze and compare the sensing efficiency by means of the EIS curve for detecting miRNA-21 by means of these aptamer sensors alone. pd-MoS₂@ β -FeOOH nano material modification, cDNA fixation and detection of R around miRNA-21_ctThe difference represents the adsorption amount of the adsorption layer. The Δ R for each step during miRNA-21 detection for the different aptamer sensors is summarized in FIG. 22_ctValue, wherein, pd-MoS₂Delta R of/AE_ctThe value was the smallest, 132.6 Ω, indicating excellent electrochemical activity. pd-MoS₂Δ R of @ β -FeOOH/AE_ctThe value (348.1. omega.) was slightly higher than β -FeOOH/AE (319.3. omega.), indicating pd-MoS₂The presence of NSs may facilitate electron transfer of the electrode nanomaterial. Immobilization of cDNA in pd-MoS₂post-R on/AE and β -FeOOH/AE_ctThe increase in value was negligible, 280.8 and 236 Ω, respectively. In contrast, pd-MoS₂@β-The FeOOH nano hybrid material and cDNA show stronger combination effect, resulting in higher Delta R_ct(643.8 Ω). Thus, pd-MoS₂The @ β -FeOOH-based aptamer sensor shows excellent detection performance on miRNA-21 pd-MoS₂And β -FeOOH-based aptamer sensor shows relatively small R when detecting miRNA-21_ctTherefore, the β -FeOOH and pd-MoS are combined₂The electrochemical activity of the electrode material can be enhanced, the cDNA fixing capacity can be improved, and the reaction efficiency between the cDNA and the miRNA-21 can be enhanced. Thus, the present invention selects pd-MoS₂The @ β -FeOOH nano hybrid material is used as an anchoring cDNA chain platform and is used for further detecting miRNA-21.

pd-MoS₂Similar results were shown in CV curves for the @ β -FeOOH-based aptamer sensor, see FIG. 21, pd-MoS₂CV curve of @ β -FeOOH-based aptamer sensor at 1mM [ Fe (CN)₆]^3-/4-Test in Redox electrolyte for comparison, β -FeOOH group and pd-MoS under the same conditions₂CV curves of the aptamer-based sensors are shown in fig. 17 and 19.

For the β -FeOOH based aptamer sensor, as shown in FIG. 17, the naked AE showed a pair of distinct redox peaks with an inter-peak amplitude (Δ Ep) of 0.275V, in contrast, the CV curve of β -FeOOH/AE showed a lower current density and a wider Δ Ep of 0.325V, indicating that the β -FeOOH layer hindered electron transfer, consistent with the EIS results, when the cDNA was anchored and used to detect miRNA-21, the current density was further decreased and the peak Δ Ep of the electrode was gradually broadened to 0.36V and 0.42V, respectively, due to the formation of double strands by hybridization of the adsorbed single stranded cDNA oligonucleotides with miRNA-21 at the electrode surface, hindering electron transfer at the interface.

For pd-MoS₂Aptamer-based sensor, pd-MoS as shown in FIG. 19₂The CV curve for/AE became plateau, indicating a nonlinear, slow diffusion in the electrolyte, indicating a pd-MoS of the electrolyte₂There is a complex mass transfer process between NSs. cDNA/pd-MoS₂AE and miRNA-21/cDNA/pd-MoS₂the/AE maintains the CV shape, but the CV area is continuously reducedThis indicates that the fixation of the cDNA aptamer and the hybridization reaction with miRNA-21 hinder electron transfer at the solid/liquid interface.

For pd-MoS₂@ β -FeOOH-based aptamer sensor, see FIG. 21, cDNA/pd-MoS₂@ β -FeOOH/AE and miRNA-21/cDNA/pd-MoS₂The Δ Ep for @ β -FeOOH/AE was 0.461V and 0.523V, respectively, for the β -FeOOH-based aptamer sensor cDNA/β -FeOOH/AE and miRNA-21/cDNA/β -FeOOH/AE was 0.360V and 0.420V, respectively, based on pd-MoS₂cDNA/pd-MoS of basal adapted sensor₂AE and miRNA-21/cDNA/pd-MoS₂Δ Ep for/AE was 0.616V and 0.783V, respectively, indicating pd-MoS₂The change of the @ β -FeOOH aptamer sensor is more obvious, namely the sensing performance is more excellent and is consistent with the EIS result.

To realize pd-MoS₂The @ β -FeOOH-based aptamer sensor achieves the best sensing performance when detecting miRNA-21, and before constructing the aptamer sensor, a series of experimental parameters such as aptamer concentration, pd-MoS₂The dosage of @ β -FeOOH and the action time of miRNA-21 are optimized.

As shown in FIG. 23, EIS signal (. DELTA.Rct) and pd-MoS obtained at each step of miRNA-21 detection were evaluated₂The relationship between the dosage of @ β -FeOOH nano hybrid material and pd-MoS₂@ β -FeOOH concentration increase, pd-MoS₂The delta Rct values of @ β -FeOOH modified AE, immobilized aptamer and miRNA-21 detection are gradually increased and are in pd-MoS₂@ β -FeOOH concentration 4 mg. mL^-1Equilibrium is reached. This is due to the thicker pd-MoS₂The @ β -FeOOH layer blocks electron transfer, causing R_ctThe value increases, the more and more aptamer chains are anchored on the electrode surface, resulting in strong repulsion with redox ions increasing R_ctValue resulting in R of miRNA-21_ctThe value increases. When pd-MoS₂The concentration of @ β -FeOOH is more than 1 mg/mL^-1When the electrode is used, the nanometer material with excessive thickness falls off from the surface of the electrode. Therefore, the concentration selected in this experiment was 1.0 mg/mL^-1pd-MoS of₂The surface of an AE electrode is modified by @ β -FeOOH.

FIG. 24 shows pd-MoS₂Effect of aptamer concentration in the @ β -FeOOH-based aptamer sensor on miRNA-21 detection As seen from the figure, aptamer anchoringAnd R in the miRNA-21 detection process_ctValues increased with increasing aptamer concentration, reaching an equilibrium stage when aptamer concentration was higher than 100nM, indicating saturation of aptamer strand binding to the electrode at this concentration. Therefore, the optimal aptamer concentration for constructing a sensor for detecting miRNA-21 in the invention is 100 nM.

FIG. 25 is a graph showing the Δ R obtained from the aptamer sensor of the invention with miRNA-21 at different action times_ctValues, obtained from fig. 24. Δ R when aptamer strand interacts with miRNA_ctThe values increase gradually with time of action. Δ R when the binding time is longer than 60 minutes_ctThe value is basically unchanged, which indicates that the hybridization reaction of the aptamer chain and miRNA-21 reaches saturation. Dissociation of miRNA-21 hybridization reaction due to aptamer and dissociation from pd-MoS when washed with PBS (0.1M)₂@ β -FeOOH material sloughs off, resulting in Δ R_ctThe value decreases slightly. Therefore, the action time of the aptamer and the miRNA in 60 minutes is the pd-MoS constructed by the invention₂@ β -optimal time of action for FeOOH-based aptamer sensors.

Experimental example 6: sensitivity test

Sensitivity is one of the important factors in evaluating the sensing performance of electrochemical biosensors and is usually expressed by the limit of detection (LOD) obtained in the concentration titration experiment of miRNA-21. pd-MoS constructed by the invention₂The LOD of the @ β -FeOOH aptamer sensor is determined by miRNA-21 with different concentrations under the optimized optimal experimental parameter conditions to obtain an EIS diagram shown in FIG. 26, and the Δ R_ctThe variation with miRNA-21 concentration is shown in FIG. 27. As can be seen from the figure, Δ R increases with the miRNA-21 concentration from 0.001pM to 5000pM_ctThe value increased significantly and then tended to be unchanged, indicating that miRNA-21 is in equilibrium at this time. FIG. 28 shows a linear relationship between miRNA-21 concentration in the range of 0.001pM to 5000pM and Δ Rct values versus the logarithm of miRNA-21 concentration, a linear regression equation was obtained by fitting: Δ R_ct(Ω)＝0.34C_miRNA-21(pM) +1.20, correlation coefficient 0.9925. According to the simulation method of the International Union of pure and applied chemistry, the LOD of the miRNA-21 detected by the sensor is 0.11fM based on three times of signal to noise ratio. Compared with other reported aptamer sensors for detecting miRNA-21 (Table 2), the pd-MoS₂The @ β -FeOOH group sensor has higher sensitivity and lower LOD₂@ β -FeOOH as platform, existing pd-MoS₂NSs has excellent electrochemical activity, large specific surface area and good biocompatibility, and has good sensing performance on miRNA-21. And pd-MoS₂The combination of NSs and β -FeOOH NRs further has a synergistic effect, producing Mo⁴⁺/Mo⁵⁺And Fe⁰/Fe²⁺/Fe³⁺The abundant oxygen vacancies and Fe-O bonds can significantly enhance the immobilization of cDNA. Thus, with pd-MoS₂The sensor constructed by adopting the @ β -FeOOH platform can stabilize the double chains generated by the reaction of the cDNA and the miRNA-21, thereby improving the sensing performance of the sensor.

TABLE 2 comparison of the sensor constructed according to the present invention with other reported miRNA-21 detection techniques

Reference documents:

[1]Peng,L.,Yuan,Y.,Fu,X.,Fu,A.,Zhang,P.,Chai,Y.,Gan,X.,Yuan,R.,2019.Anal.Chem.91(5),3239-3245.

[2]He,C.,Wang,M.,Sun,X.,Zhu,Y.,Zhou,X.,Xiao,S.,Zhang,Q.,Liu,F.,Yu,Y.,Liang,H.,Zou,G.,2019.Biosens.Bioelectron.129,50-57.

[3]Tian,L.,Qi,J.,Ma,X.,Wang,X.,Yao,C.,Song,W.,Wang,Y.,2018.Biosens.Bioelectron.122,43-50.

[4]Salahandish,R.,Ghaffarinejad,A.,Omidinia,E.,Zargartalebi,H.,Majidzadeh-A,K.,Naghib,S.M.,Sanati-Nezhad,A.,2018.Biosens.Bioelectron.120,129-136.

experimental example 7: experiment of specificity

To evaluate pd-MoS₂The specificity of the @ β -FeOOH aptamer sensor takes the possible interferents (with the concentration of 1pM) such as MM1, MM3, miRNA 141, miRNA 155 and miRNA 166 in a biological system as targetsOver pd-MoS₂The @ β -FeOOH aptamer sensor detects the interferent, and the obtained electrochemical signal is compared with the electrochemical signal obtained after diluting 1000 times of miRNA-21, so that the specificity of the sensor to miRNA is obtained.

Using MM1, MM3 and 3 TMM strands, hybridization reactions with cDNA were performed under the same conditions, respectively, to obtain a series of EIS results, and Δ Rct values, as shown in FIG. 29. For the three TMM chains, only negligible Δ R was observed_ctThe value: hybridization of aptamer strands to MM1miRNA-21 resulted in smaller Δ Rct (44 Ω), and hybridization of aptamers to MM3 miRNA-21 resulted in lower Δ Rct values (32 Ω). Indicating that the hybridization reaction between the cDNA and the interfering molecule is weak. In contrast, the larger Δ R produced when the interaction between cDNA and miRNA-21 was obtained_ctValues, indicating successful binding. When cDNA is acted with mixed solution of MM1miRNA-21, MM3 miRNA-21 and miRNA-21, the obtained Delta R_ctThe value is compared with the value of Delta R when miRNA-21 exists alone_ctSubstantially identical. From the above results, pd-MoS₂The @ β -FeOOH-based aptamer sensor shows good specificity in detecting miRNA-21 in a complex environment.

Experimental example 8: stability and repeatability

pd-MoS of the invention₂Stability and repeatability of the @ β -FeOOH-based aptamer sensor by preparing five identical pd-MoS₂The @ β -FeOOH-based aptamer sensors each detected 1pM miRNA-21 to evaluate the reproducibility between the sensors, and the results are shown in FIG. 30, with the Relative Standard Deviation (RSD) of the five sensors being only 6.14%.

Selecting a pd-MoS₂The @ β -FeOOH-based aptamer sensor continuously detects 1pM miRNA-21 every day for 15 days to obtain a 15-day delta R_ctThe value is shown in FIG. 31, and the RSD is as low as 7.44%. The sensor is stored for 15 days and then subjected to detection on R obtained by detecting 1pM miRNA-21_ctThe value was about 121.9% of the initial value. The above results show that the pd-MoS constructed by the invention₂The @ β -FeOOH-based electrochemical biosensor has good stability and excellent repeatability.

Experimental example 9: reproducibility

pd-MoS constructed by the invention₂@ β -FeOOH-based aptamer sensorThe washed aptamer sensor was re-detected for miRNA-21(1 pm. mL) by soaking in 0.05M NaOH and washing 3 times with Milli-Q water^-1) The EIS electrochemical signal obtained was compared to that obtained without washing. pd-MoS constructed by the invention₂Reproducibility of the @ β -FeOOH-based aptamer sensor by repeated measurements, R was found to be obtained by 10 measurements_ctThe value was slightly decreased as shown in fig. 32, indicating that it has excellent reproducibility.

Experimental example 10: detection of aptamer sensor in human serum

pd-MoS constructed by the invention₂The practical applicability of the @ β -FeOOH-based aptamer sensor in miRNA-21 detection is verified in a human serum sample, firstly, miRNA-21 with different concentrations is doped into human serum, an EIS method is adopted for detection, the theoretical concentration of the miRNA-21 is deduced according to a calibration curve of figure 27 and is shown in table 3, and the theoretical concentration is compared with the practical concentration of the miRNA, so that the miRNA-21 sensor has better consistency.

TABLE 3 use of pd-MoS₂Results of @ β -FeOOH-based aptamer sensor for detecting miRNA-21(n ═ 3) in human serum

Added amounts(pM)	Found amounts(pM)	Recovery(％)	RSD(％)
				0.001	0.00104	104	2.08
0.01	0.00966	96.6	4.63
				0.1	0.103	103	3.65
1.0	0.918	91.8	4.10
				10	10.64	106.4	3.17
100	99.53	99.5	2.73

Note: in table 3, "n-3" indicates that each sample was measured 3 times.

In addition, the utility of the aptamer sensor of the present invention was further verified by detecting L929 cells and MCF-7 cells, which are breast cancer cells. As shown in FIG. 33, when the cell concentration was 100,000cell mL^-1The concentration of miRNA-21 in MCF-7 cells was more than 50 times higher than that in L929 cells. The above results show that the pd-MoS of the present invention₂The @ β -FeOOH-based sensor shows excellent applicability.

The EIS, CV, cytotoxicity and other tests show that the pd-MoS of the invention₂The @ β -FeOOH nano hybrid material has good electrochemical activity and biocompatibility, and the novel electrochemical aptamer sensor constructed by the nano hybrid material is prepared byThe hybridization mechanism of the cDNA and the target miRNA-21 detects the miRNA-21, and the sensing sensitivity is higher. The invention relates to pd-MoS obtained by a solvothermal synthesis method₂NSs grow vertically and closely on β -FeOOH nano-rod to obtain pd-MoS₂@ β -FeOOH nano hybrid material due to the multi-scale nano structure having Mo⁴⁺/Mo⁵⁺And Fe⁰/Fe²⁺/Fe³⁺Mixed chemical valence, rich oxygen vacancies and Fe-O bonds with pd-MoS₂NSs compared to β -FeOOH NRs, pd-MoS₂The @ β -FeOOH nano hybrid material shows good electrochemical activity and strong cDNA anchoring capability, can carry out hybridization reaction with miRNA-21 to the maximum extent, the constructed aptamer sensor has higher sensitivity in the linear detection range of miRNA-21 concentration of 1fM-5nM, the LOD of the aptamer sensor is 0.11fM, and the aptamer sensor has high selectivity, good stability, excellent reproducibility and applicability₂The @ β -FeOOH-based aptamer sensor provides a new idea for developing a biological detection analysis method with excellent sensing performance.

Claims

1. Iron oxyhydroxide-MoS₂The nano hybrid material is characterized by comprising iron oxyhydroxide nanorods and MoS grown on the surfaces of the iron oxyhydroxide nanorods₂Nanosheets, said MoS₂The nanosheet is obtained by carrying out solvothermal reaction on phosphomolybdic acid hydrate and thioacetamide.

2. The iron oxyhydroxide nanorod-MoS of claim 1₂The preparation method of the nano hybrid material is characterized by comprising the following steps: carrying out solvothermal reaction on a mixed solution consisting of the iron oxyhydroxide nanorod, the phosphomolybdic acid hydrate, the thioacetamide and the solvent to obtain the nano-composite material.

3. The iron oxyhydroxide nanorod-MoS of claim 2₂The preparation method of the nano-sheet nano hybrid material is characterized in that the mass ratio of the iron oxyhydroxide nano-rod, the phosphomolybdate hydrate and the thioacetamide is (1-4) to 1: 1.

4. The iron oxyhydroxide nanorod-MoS of claim 2₂The preparation method of the nano hybrid material is characterized in that the reaction temperature is 180-220 ℃.

5. The iron oxyhydroxide nanorod-MoS of claim 2₂The preparation method of the nano hybrid material is characterized in that the reaction time is 8-16 h.

6. The iron oxyhydroxide nanorod-MoS of claim 2₂The preparation method of the nano-sheet nano hybrid material is characterized in that the mixed solution is obtained by mixing the hydroxyl iron oxide nano-rods, the aqueous solution of phosphomolybdic acid hydrate and the methanol solution of thioacetamide.

7. The iron oxyhydroxide nanorod-MoS of any one of claims 1-6₂The preparation method of the nano-sheet nano hybrid material is characterized in that the iron oxyhydroxide nano-rod is obtained by carrying out solvothermal reaction on ferric chloride and glucose, wherein the reaction temperature is 150-180 ℃, and the reaction time is 5-8 h.

8. An electrode for an aptamer sensor, which comprises an electrode substrate and an electrode modification material on the surface of the electrode substrate, wherein the electrode modification material is the iron oxyhydroxide-MoS according to claim 1₂A nano hybrid material.

9. An aptamer sensor, which is characterized by comprising an electrode substrate, an electrode modification material on the surface of an electrode and a nucleic acid aptamer fixed on the electrode modification material, wherein the electrode modification material is the iron oxyhydroxide-MoS according to claim 1₂A nano hybrid material.

10. The aptamer sensor of claim 9, wherein the nucleic acid aptamer is a complementary DNA of miRNA-21.