CN113219032A - Electrochemical sensor for detecting hepatitis B exosome miRNA and preparation and application thereof - Google Patents
Electrochemical sensor for detecting hepatitis B exosome miRNA and preparation and application thereof Download PDFInfo
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Abstract
The invention discloses an electrochemical sensor for detecting hepatitis B exosome miRNA and preparation and application thereof. The sensor includes a working electrode: the surface is fixed with a DNA nano-bracket marked by a capture probe sulfydryl; target substance T chain induced cascade strand displacement reaction (L-TEDCR) system: a cascade T-shaped structure compound Ts and a cascade reaction auxiliary chain Fs, wherein the Ts consists of a DNA single-chain P chain, an R chain, an L chain and an L chain0The Fs consist of a single DNA strand, the F strand and the L strand0Chain composition; and MOF/DNA cascade enzyme amplification detection system. After the exosome miRNA is added, the L-TEDCR is rapidly triggered to generate a large number of DNA nano-scaffolds; the resulting DNA nanoscaffold was immobilized on an electrode and bound to MOF/DNA cascades, generating a significantly amplified electrochemical signal for the detection of exosome mirnas. The sensor of the invention has high sensitivityThe detection speed is high, the specificity is good, and the like.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an electrochemical sensor for detecting hepatitis B exosome miRNA and preparation and application thereof.
Background
Hepatitis B Virus (HBV) infection seriously threatens human health, and HBV infection markers are numerous, including hepatitis B surface antigen (HBsAg), hepatitis B antigen (HBeAg), hepatitis B virus quantification (HBV DNA), and recently novel serological markers including hepatitis B core associated antigen (hbcrhbag), HBV pregenomic RNA (pgRNA), and the like, which are used more frequently in clinical practice. Although the existing detection means for the peripheral blood virological marker has advantages in diagnosis, efficacy monitoring and the like, the existing detection means cannot well replace the detection of covalently closed circular DNA (cccDNA) of liver tissue, so more research is needed to search for better serological indexes.
Exosomes are first found in sheep reticulocytes in 1983, have the size of about 30-150nm, are mainly derived from multivesicular bodies formed by invagination of lysosome microparticles in cells, are released into extracellular matrixes after being fused with cell membranes through multivesicular body outer membranes, and certain exosomes are detected as serum markers of different liver diseases. Researches show that HBV can encode HBV related microRNA, the HBV related microRNA is named as HBV miR21, HBV miR21 is highly expressed in a liver cancer cell line, and the HBV miR21 is positively correlated with HBV DNA in a hepatitis active patient. Currently, the common method for detecting exosome mirnas is quantitative reverse transcription polymerase chain reaction (qRT-PCR). However, this method is time consuming and laborious, thus prompting researchers to develop a new method for accurately measuring exosome mirnas with rapidity and high sensitivity.
Recently, enzyme-free signal amplification methods such as entropy-driven cycling reaction (EDCR), Catalytic Hairpin Assembly (CHA), and hybrid strand reaction (HCR) have been explored for the amplification detection of miRNA. In order to significantly increase the reaction speed, diffusible reactants participating in the signal amplification sensing system are assembled in a compact space. For example, Ju et al designed a localized HCR, i.e., a hairpin probe that binds alternately to a long DNA strand produced by Rolling Circle Amplification (RCA) for mRNA detection, exhibiting an extremely rapid reaction rate compared to conventional HCRs. Similarly, Wei et al developed a localized CHA for miRNA imaging, in which the CHA reactants were immobilized on assembled DNA nanowires by multiple single strand hybridization. Although localized reaction substrates in the sensing system can greatly enhance the reactionSpeed, but these strategies have the following drawbacks: i) the construction of a local substrate requires multiple assembly steps, resulting in time and labor; ii) the hybridization efficiency of diffusible reactants with DNA nanowires may be low under physical conditions, directly leading to incomplete localization structures; iii) inherent characteristics of CHA and HCR, such as unacceptable cycle leakage and stringent reaction conditions, i.e., divalent metal ions (e.g., Mg)2+) Invariably adversely affects the performance of the assay, limiting their further use in early tumor diagnosis. In addition, leakage may be more severe because the increased number of DNA hairpins in a compact space is more susceptible to non-specific interactions.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide an electrochemical sensor for detecting hepatitis B exosome miRNA and preparation and application thereof.
In order to achieve the above and other related objects, a first aspect of the present invention provides an electrochemical sensor for detecting miRNA of exosome of hepatitis b, including a working electrode, a target T-strand induced cascade strand displacement reaction (L-TEDCR) system, and an MOF/DNA cascade enzyme amplification detection system, where the working electrode is a substrate electrode with a capture probe fixed on the surface, the capture probe is a DNA nano-scaffold labeled with thiol, and is designed for the amplification product of miRNA of exosome; the cascade strand displacement reaction system comprises a cascade T-shaped structure compound Ts and a cascade reaction auxiliary strand Fs, and the MOF/DNA cascade enzyme amplification detection system comprises MOF/DNA cascade enzyme; the Ts consists of a DNA single-chain P chain, an R chain, an L chain and an L chain0The Fs consist of a single DNA strand, the F strand and the L strand0Chain composition; two foothold points T1 and T2 are arranged at the tail end of the Ts, and T1 and T2 are both positioned on an L chain; the target substance can bind to the first foothold T1 and replace the P chain by a foothold mediated strand displacement reaction, exposing the second foothold T2 located in the L chain; fs hybridizes with T2, the target substance and R chain are displaced by base-pairing extension, and DNA nano-scaffolds are generated and displacedThe target substance of (a) binds to the adjacent foothold T1, triggering the next cycle reaction, producing a large number of DNA nanoscaffolds; the DNA nano-scaffold is fixed on the surface of the electrode, is specifically hybridized with the MOF/DNA cascade enzyme to capture the MOF/DNA cascade enzyme to form a three-layer composite material, generates an electrochemical signal, and realizes the detection of the exosome miRNA through detecting the electrochemical signal.
Further, the nucleotide sequence of the P chain is as follows:
5′-TTTTTCCCTTAGCTTATCAGACTGA-3′(SEQ ID NO.1)。
further, the nucleotide sequence of the R chain is as follows:
5′-CCTACGTCTCCAACTAACTTACGG-3′(SEQ ID NO.2)。
further, the nucleotide sequence of the L chain is:
5′-GTAGAATGCAGTGTAGTAGAATGCAGTGTAGTAGAATGCAGTGTAGTAGAATGCAGTGTAGTAGAATGCAGTGTA-3′(SEQ ID NO.3)。
further, L is0The nucleotide sequence of the strand is:
5′-TCAACATCAGTCTGATAAGCTAAGGGCCGTAAGTTAGTTGGAGACGTAGGTTTACACTGCATTCTAC-3′(SEQ ID NO.4)。
further, the nucleotide sequence of the F chain is as follows:
5′-CCTACGTCTCCAACTAACTTACGGCCCTTAGCTTATCAGACTGA-3′(SEQ ID NO.5)。
further, the nucleotide sequence of the target substance T chain (target sequence T) is:
5′-TAGCTTATCAGACTGATGTTGA-3′(SEQ ID NO.6)。
further, T1 is a sequence of red portion on L chain, the sequence base is TCAACA, T2 is a sequence of green portion on L chain, the sequence base is AGGG.
Furthermore, the MOF/DNA cascade enzyme is formed by combining AuNPs/MOF with glucose oxidase activity and hemin/G-quadruplex DNAzyme (G4-hemin DNAzyme) with peroxidase-like activity, the AuNPs/MOF is formed by combining gold nanoparticle AuNPs and metal organic framework material MOF, and the hemin/G-quadruplex DNAzyme (G4-hemin DNAzyme, G4-hemin deoxyribozyme) is a compound formed by G-quadruplex (G-4) and hemin (hemin), and has good peroxidase-like activity.
Further, the base electrode is a gold electrode.
Further, the electrochemical sensor further comprises a reference electrode and a counter electrode.
Optionally, the reference electrode is selected from any one of a saturated calomel electrode or a silver chloride electrode (Ag/AgCl); preferably, the reference electrode is a calomel electrode.
Optionally, the counter electrode is a platinum wire electrode.
Further, the electrochemical sensor also comprises a substrate solution, and the substrate solution is PBS buffer solution.
In a second aspect, the present invention provides a method for preparing an electrochemical sensor for detecting exosome miRNA according to the first aspect, including the following steps:
(1) preparing a cascade strand displacement reaction system: preparing a cascade T-shaped structure compound Ts by adopting a DNA single-chain P chain, an R chain, an L chain and an L0 chain through a one-step annealing method, and preparing a cascade reaction auxiliary chain Fs by adopting a DNA single-chain F chain and an L0 chain through a one-step annealing method; mixing Ts, Fs, a target substance T chain and a DNA hybridization solution, and incubating to obtain a cascade chain displacement reaction system;
(2) preparation of MOF/DNA Cascade enzymes: combining gold nanoparticles AuNPs and metal organic framework material MOF to prepare AuNPs/MOF, and then combining the AuNPs/MOF and hemin/G-quadruplex DNAzyme to prepare MOF/DNA cascade enzyme;
(3) dropwise adding the cascade chain displacement reaction system obtained in the step (1) to the surface of a working electrode fixed with a capture probe, and incubating;
(4) and (3) mixing the MOF/DNA cascade enzyme obtained in the step (2) with a DNA hybridization solution for reaction, dripping the obtained reaction solution onto the working electrode treated in the step (3), and incubating to obtain the MOF/DNA cascade enzyme amplification detection system.
Further, in the step (1), P chain, R chain, L chain and L chain0The molar ratio of the chains is (4-6): (1), preferably (4.5-5): 1, more preferably 5: 1.
Further, in the step (1), P chain, R chain, L chain and L chain are firstly carried out0The strands were dissolved in the DNA hybridization solution, and mixed and incubated.
Further, in the step (1), the F chain and the L chain0The molar ratio of the chains is (4-6) to 1, preferably (4.5-5) to 1, more preferably 5: 1.
Further, in the step (1), the molar ratio of Ts to Fs is 1: 1.
Further, in the step (1), the final concentration of the target substance T chain in the reaction solution is not less than 100 aM.
Further, in the step (1), the incubation temperature is 37 ℃, and the incubation time is more than or equal to 30 min.
Further, in the step (1), the DNA hybridization solution includes: 10mM Tris buffer, 480mM NaCl, 5mM MgCl2。
Further, in the step (2), the preparation method of the metal organic framework material MOF comprises the following steps: FeCl is added3·6H2O、H2BDC (2-amino terephthalic acid) is dissolved in an organic solvent, then the mixture is placed in a high-pressure reaction kettle for heating reaction, after the reaction is finished, reaction liquid is centrifuged to obtain a product, and the product is washed and dried to obtain the MOF.
Alternatively, in the preparation method of the metal organic framework material MOF, FeCl3·6H2O and H2The molar ratio of BDC (2-aminoterephthalic acid) is (1.5-2) to 1, preferably 2: 1.
Alternatively, in the method for preparing a metal organic framework material MOF, the organic solvent is selected from DMF. Optionally, in the preparation method of the metal organic framework material MOF, the reaction temperature is 110-140 ℃, and preferably 120 ℃; the reaction time is 3-5h, preferably 4 h.
Alternatively, in the preparation method of the metal organic framework material MOF, C is adopted2H5The centrifuged product was washed with OH and DMF.
Optionally, in the preparation method of the metal organic framework material MOF, the drying temperature is 50-70 ℃, preferably 60 ℃; the drying time is 5-8h, preferably 6 h.
Further, in the step (2), the AuNPs goldThe preparation method of the nano particles comprises the following steps: adding sodium citrate to the boiled HAuCl4And in the solution, after the color of the solution is observed to change from light yellow to deep red, stopping heating, stirring the solution to cool to room temperature (23-25 ℃), and continuing stirring for 20-30 minutes to obtain the solution containing the AuNPs gold nanoparticles.
Optionally, in the preparation method of the AuNPs gold nanoparticles, sodium citrate and HAuCl are used4The molar ratio of (3.0-4.0) to 1, preferably (3.8-4.0) to 1.
Further, in the step (2), the gold nanoparticles are spherical, and the diameter average size is 9-11 nm.
Further, in the step (2), the reaction temperature for preparing AuNPs/MOF by combining the gold nanoparticles AuNPs and the metal organic framework material MOF is 37 ℃, and the time is 10-15h, preferably 12 h; the reaction temperature for preparing MOF/DNA cascade enzyme by combining AuNPs/MOF and hemin/G-four-chain DNAzyme is 37 ℃, and the reaction time is 0.5-2h, preferably 1 h.
Further, in the step (3), the incubation temperature is 37 ℃ and the incubation time is 1-1.5h, preferably 1 h.
Further, in the step (4), the DNA hybridization solution includes: 10mM Tris buffer, 480mM NaCl, 5mM MgCl2。
Further, in the step (4), the temperature of the MOF/DNA cascade enzyme and the DNA hybridization solution is 4 ℃, and the time is 1-2h, preferably 1 h.
Further, in the step (4), the incubation temperature is 4 ℃ and the incubation time is 1-2h, preferably 1 h.
Further, in the step (3), the working electrode immobilized with the capture probe is prepared according to the following steps:
surface treatment of an electrode: polishing the surface of the substrate electrode to make the surface smooth; then using piranha solution (H)2SO4∶H2O2Treating the electrode at a ratio of 3: 1), cleaning the electrode and drying;
fixing a capture probe: taking a DNA nano-stent marked by sulfydryl as a capture probe, dropwise adding the capture probe on the surface of a cleanly treated electrode, and incubating;
sealing the electrode: and (3) blocking the non-specific adsorption sites by using 6-mercapto-1-ethanol (MCH) to obtain the working electrode on which the capture probe is immobilized.
Optionally, in the step (i), polishing is performed on the substrate electrode by using alumina powder.
Optionally, in the step II, the concentration of the capture probe is 100-1000 nmol/L.
Optionally, in the second step, the incubation temperature is 4 ℃ and the incubation time is 8-12 hours.
Optionally, in the step (c), the sealing time is 0.5 h.
The third aspect of the invention provides a method for detecting hepatitis B exosome miRNA, which adopts the electrochemical sensor of the first aspect and/or the electrochemical sensor prepared by the preparation method of the second aspect.
Further, the detection method comprises the following steps:
the working electrode, the reference electrode and the counter electrode are correctly connected to an electrochemical workstation, PBS buffer solution is used as a reaction substrate solution, and the electrochemical signal is measured by Differential Pulse Voltammetry (DPV).
As mentioned above, the electrochemical sensor for detecting the hepatitis B exosome miRNA and the preparation and the application thereof have the following beneficial effects:
the invention constructs an electrochemical sensor based on a localized cascade structure strand displacement reaction (L-TEDCR) system and an MOF/DNA cascade enzyme amplification detection system, and develops a stable and high-sensitivity enzyme-free hepatitis B exosome miRNA detection biosensing strategy based on the electrochemical sensor. The invention combines MOF/DNA cascade enzyme and cascade strand displacement structure for the first time and is used for detecting exosome miRNA. Wherein, the L-TEDCR reaction substrates (Ts and Fs) are assembled only by a simple one-step annealing method so as to ensure the sufficient hybridization of a plurality of short single chains; once Exo-miRNA is added, L-TEDCR is rapidly triggered, resulting in the generation of large amounts of DNA nano-scaffolds; the resulting DNA nanoscaffold was then immobilized on a working electrode, binding to the MOF/DNA cascade, thereby generating a significantly amplified electrochemical signal for detection of Exo-miRNA.
Compared with the classical EDCR, the design of the localized cascade structure strand displacement reaction in the invention increases the detection sensitivity to the target substance mutation and improves the detection speed. The sensor of the invention shows the advantages of high sensitivity, strong stability, good reproducibility, fast reaction speed and the like for the determination of exosome miRNA, is expected to be applied to the determination of actual samples and clinical specimens, and is developed into a sensor with clinical application value.
Drawings
FIG. 1 shows a schematic diagram of the detection of the method of the present invention.
Fig. 2 shows a TEM characterization result of gold nanoparticles in example 1.
FIG. 3 is a graph showing the results of PAGE electrophoretic characterization of the cascade strand displacement reaction (L-TEDCR) of the present invention in example 2.
FIG. 4 is a graph showing the TEM characterization results of the MOF/DNA cascades in example 3.
FIG. 5 is a graph showing the results of characterization of the L-TEDCR fluorescence feasibility spectrum in example 4.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
The specific implementation process of the invention is as follows:
example 1
Preparation of electrochemical sensor and detection of exosome miRNA
1. Material
6-mercapto-1-hexanol (MCH) was obtained from Sigma-Aldrich (St Louis, MO, USA). The HPLC purified oligonucleotides were synthesized from Shanghai. Hemin G-four-chain DNAzyme (solution) was purchased from Biotechnology, Inc. (Shanghai).
2. Detection instrument
Shanghai Chenghua CHI660D electrochemical workstation, the detection system is a three-electrode system, and the three-electrode system comprises a reference electrode which is an Ag/AgCl electrode, a counter electrode which is a platinum wire electrode, and a gold electrode of which the working electrode diameter is 3 mm.
3. Principle of detection
As shown in figure 1, the invention constructs an enzyme-free and label-free electrochemical sensor based on a cascade strand displacement reaction (L-TEDCR) and MOF/DNA cascade enzyme, which is used for detecting exosome miRNA, and the detection principle is as follows: the cascade strand displacement reaction system consists of a cascade T-shaped structure compound (Ts) and an auxiliary strand (Fs). Ts, Fs are assembled by one-pot thermal annealing, wherein Ts is composed of a P chain, an R chain, an L chain and an L chain0Chains, Fs consisting of F chain, L0Chain composition. Two footholds T1 and T2 are arranged at the tail end of Ts, T1 is a sequence of a red part on an L chain, a sequence base is TCAACA, T2 is a sequence of a green part on the L chain, and a sequence base is AGGG. . When added to a biosensing system, the target can bind to the first foothold on the L chain (red region) and displace the P chain by a foothold-mediated strand displacement reaction, exposing a blocked foothold T2 located in the L chain (green region). Subsequently, the Fs strand hybridizes to the exposed foothold domain, and the target strand (target substance T strand) and the R strand are displaced as the base pairs are extended. The displaced target material then rapidly binds to the adjacent foothold T1 to trigger the next cycling reaction, resulting in the replacement of a large number of P strands while producing a large number of DNA nanoscaffolds. The generated large amount of displaced DNA nano-scaffolds are fixed on the surface of the electrode, and are specifically hybridized with MOF/DNA cascade enzyme assembly to further capture the constructed MOF/DNA cascade enzyme. Successfully capturing MOF/DNA cascade enzyme to form a three-layer composite material for catalyzing glucose to generate H2O2And generating an electrochemical signal. The content of the target substance can be obtained by detecting the electrochemical signal, and the detection of the exosome miRNA is realized.
In addition, after the target substance is added, the target substance substitutes for the P chain and binds to the L chain, while the auxiliary chain Fs is used to substitute for the target substance and the R chain, thereby recycling the target substance; l is0The strands regularly arrange the strand displacement reaction substrates (PRL complex, i.e., P strand + R strand + L strand) in a compact arrangementIn space. The nucleotide sequence designed by the invention is shown as follows:
a P chain: 5'-TTTTTCCCTTAGCTTATCAGACTGA-3' (SEQ ID NO. 1).
Chain R: 5'-CCTACGTCTCCAACTAACTTACGG-3' (SEQ ID NO. 2).
L chain:
5′-GTAGAATGCAGTGTAGTAGAATGCAGTGTAGTAGAATGCAGTGTAGTAGAATGCAGTGTAGTAGAATGCAGTGTA-3′(SEQ ID NO.3)。
L0chain:
5′-TCAACATCAGTCTGATAAGCTAAGGGCCGTAAGTTAGTTGGAGACGTAGGTTTACACTGCATTCTAC-3′(SEQ ID NO.4)。
chain F: 5'-CCTACGTCTCCAACTAACTTACGGCCCTTAGCTTATCAGACTGA-3' (SEQ ID NO. 5).
Target substance T chain (target sequence T):
5′-TAGCTTATCAGACTGATGTTGA-3′(SEQ ID NO.6)。
4. preparation process
(1) Preparing a cascade strand displacement reaction system:
firstly, DNA single-chain P chain, R chain, L chain and L chain0The chain is prepared into a cascade T-shaped structure compound Ts by a one-step annealing method according to a molar ratio of 5: 1: firstly, P chain, R chain, L chain and L chain0The strands were dissolved in DNA hybridization solutions (10mM Tris buffer, 480mM NaCl, 5mM MgCl)2) Then 25. mu.L of 18.79. mu.M P chain, 30.6. mu.L of 15.12. mu.M R chain, 33. mu.L of 15.18. mu.M L chain and 6.8. mu.L of 14.58. mu.M L chain0The single strand DNA was dissolved in 4.6. mu.L of a DNA hybridization solution (10mM Tris buffer, 480mM NaCl, 5mM MgCl)2) Then mixed and incubated at 37 ℃ for 30 min.
② DNA single strands F and L0Preparing cascade reaction auxiliary chain Fs by a one-step annealing method according to the molar ratio of 5: 1: first 27.7. mu.L of 18. mu. M F strand and 7.7. mu.L of 13. mu. M L strand0Each strand was dissolved in 64.6. mu.L (volume) of a DNA hybridization solution (10mM Tris buffer, 480mM NaCl, 5mM MgCl)2) Then mixed and incubated at 37 ℃ for 30 min.
③ taking Ts (9.6 muL 0.5 muM), Fs (9.6 muL 0.5 muM) and a target substance T chain (exosome miRNA with different concentrations),97.98 vol DNA hybridization solution (10mM Tris buffer, 480mM NaCl, 5mM MgCl) was added2) Stirring and mixing, and incubating for 1h at 37 ℃ to obtain a cascade strand displacement reaction system.
(2) Preparation of MOF/DNA Cascade enzymes:
the AuNPs/MOF is prepared by combining the gold nanoparticles AuNPs and the metal organic framework material MOF, and then the MOF/DNA cascade enzyme is prepared by combining the AuNPs/MOF and the hemin/G-four-chain DNAzyme.
Preparation of MOF: firstly 0.187g FeCl3And 0.126g H2BDC (2-aminoterephthalic acid) was dissolved in 10mL of DMF, which was then subjected to ultrasonic mixing treatment for 10min, and the resulting homogeneous yellowish green solution was transferred to a 20mL autoclave lined with polytetrafluoroethylene and reacted with heating at 120 ℃ for 4 hours. After the solution was cooled to room temperature, the resulting suspension was centrifuged at 8000rpm for 10min to separate MOF nanoplates, then washed 3 times with anhydrous ethanol, and the synthesized nanomaterial was purified by centrifugation (8000rpm, 10 min). Finally, the resulting MOF material was resuspended in absolute ethanol and stored in a 4 ℃ freezer for further use.
② preparing AuNPs gold nano particles: sodium citrate (10.1mL, 34mM) was added rapidly to HAuCl4In a boiling solution (88.2mL, 1mM), it was observed that the solution changed color from pale yellow to deep red within 1 min. The heating system was then turned off, stirred and allowed to cool to room temperature (23-25 ℃) and then after another 20min stirring it was stored at 4 ℃ for subsequent use.
As shown in fig. 2, the gold nanoparticles are spherical and have a uniform distribution of particle sizes, with a diameter average size of about 10 nm.
Thirdly, mixing 1mL of the AuNPs solution of the gold nanoparticles obtained in the third step with 1mL of the MOF solution of the metal organic framework material obtained in the first step, stirring overnight at 37 ℃ to prepare the AuNPs/MOF solution, and then stirring 1mL of the AuNPs/MOF solution and 100 mu L of hemin chloride/G-quadruplex DNAzyme at 37 ℃ for 1h to prepare the solution containing MOF/DNA cascade enzyme.
(3) Preparing a working electrode:
surface treatment of a gold electrode:
using 0.05 μm aluminum powder pairsPolishing the gold electrode to be in a mirror surface shape, and ultrasonically cleaning the gold electrode for 3 times for 3min by using deionized water; then, using piranha solution (H)2SO4∶H2O2Treating the gold electrode for 10min at a ratio of 3: 1), washing with deionized water, and drying the surface of the electrode by using an ear washing ball.
Fixing a capture probe: and (3) dropwise adding the sulfydryl-labeled DNA nano-stent capture probe on the surface of the treated electrode, and standing at 4 ℃ overnight.
Adopting MCH to seal the electrode: the surface of the capture probe-assembled electrode was washed three times with 1 XPBS buffer and blocked for 1h by dropping 10. mu.L of 1mM MCH. And repeatedly washing the electrode to obtain the working electrode modified by the probe for later use.
(4) And (3) dropwise adding 10 mu L of the cascade strand displacement reaction system obtained in the step (1) to the surface of a working electrode (MCH/CP/GE) on which the capture probe is immobilized after the treatment in the step (3), incubating for 1h at 37 ℃, and then washing with a Tris-HCl buffer solution.
(5) Taking 100 μ L of the MOF/DNA cascade enzyme-containing solution obtained in step (2), adding 100 μ L of LDNA hybridization solution (10mM Tris buffer, 480mM NaCl, 5mM MgCl)2) And (4) stirring and mixing, incubating at 4 ℃ for 60min, dripping the obtained reaction solution onto the working electrode treated in the step (4), and incubating at 4 ℃ for 1 h.
(6) And (3) taking a PBS buffer solution as a substrate solution for electrochemical measurement, placing the electrode to be measured processed in the step (5) in the PBS buffer solution, taking an Ag/AgCl electrode as a reference electrode, taking a platinum wire electrode as a counter electrode, and measuring an electrochemical signal by using a Differential Pulse Voltammetry (DPV) at room temperature.
Example 2
Feasibility of verifying and detecting exosome miRNA electrochemical sensor
Whether Exo-miRNA triggered L-TEDCR was determined by PAGE experiments, the results are shown in figure 3.
FIG. 3 shows the results of PAGE electrophoretic characterization of the cascade strand displacement reaction (L-TEDCR) of the present invention. Wherein, Lane M is a DNA gradient marker, lanes 1-5 are respectively a P chain, an R chain, an L chain, and an L chain0Fs, Lane 6 is the Ts complex, Lane 7 is Ts + Fs, Lane 8 is Ts + Fs, Lane 9 isTs + helper strand Fs + target substance.
From FIG. 3, it can be seen that in the absence of Exo-miRNA, almost no P-strand was produced (lanes 7, 8), indicating that the constructed L-TEDCR strategy has negligible background leakage. In the presence of different concentrations of Exo-miRNA, the Fs helper strand decreased, while a new late band appeared in lane 9, consistent with the position in lane 1, indicating that a large number of P strands were produced (lane 9), demonstrating that L-TEDCR performed successfully as expected with signal amplification.
Example 3
Taking the MOF nanosheet prepared in example 1, the synthesis of MOF was verified by TEM.
Fig. 4 shows a TEM image of MOF nanosheets, and as can be seen from fig. 4, the MOFs synthesized by the present invention exhibit a distinct lamellar structure with a lateral dimension of about 2 μm, indicating that the MOF nanosheets have a large specific surface area, which facilitates loading of more biomolecules. Indicating that the MOFs we constructed were successfully synthesized.
Example 4
The feasibility of L-TEDCR fluorescence was verified in the presence and absence of target material.
The cascade T-type structural complex Ts (16. mu.L) and the auxiliary strand Fs (16. mu.L) obtained in example 1 were reacted with 0.28. mu.L of the target substance (Exo-miRNA 21) and 87.72. mu.L of deionized water at 37 ℃ for 1 hour, and the fluorescence signal thereof was detected. The fluorescence signal intensity of the constructed L-TEDCR system with and without the target substance is shown in FIG. 2.
As can be seen from fig. 5, the difference in fluorescence intensity is significant between the presence of the target substance (red curve) and the absence of the target substance (black curve), and the fluorescence signal intensity is more significant in the presence of the target substance (red curve), indicating that the L-TEDCR reaction constructed according to the present invention was successfully performed.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
SEQUENCE LISTING
<110> Chongqing university of medical science
<120> electrochemical sensor for detecting hepatitis B exosome miRNA, preparation and application thereof
<130> PCQYK2110476-HZ
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 25
<212> DNA
<213> Artificial
<220>
<223> P chain
<400> 1
tttttccctt agcttatcag actga 25
<210> 2
<211> 24
<212> DNA
<213> Artificial
<220>
<223> R chain
<400> 2
cctacgtctc caactaactt acgg 24
<210> 3
<211> 75
<212> DNA
<213> Artificial
<220>
<223> L chain
<400> 3
gtagaatgca gtgtagtaga atgcagtgta gtagaatgca gtgtagtaga atgcagtgta 60
gtagaatgca gtgta 75
<210> 4
<211> 67
<212> DNA
<213> Artificial
<220>
<223> L0 chain
<400> 4
tcaacatcag tctgataagc taagggccgt aagttagttg gagacgtagg tttacactgc 60
attctac 67
<210> 5
<211> 44
<212> DNA
<213> Artificial
<220>
<223> F chain
<400> 5
cctacgtctc caactaactt acggccctta gcttatcaga ctga 44
<210> 6
<211> 22
<212> DNA
<213> Artificial
<220>
<223> target sequence T
<400> 6
tagcttatca gactgatgtt ga 22
Claims (10)
1. An electrochemical sensor for detecting hepatitis B exosome miRNA is characterized by comprising a working electrode, a target substance T chain induced cascade strand displacement reaction system and an MOF/DNA cascade enzyme amplification detection system, wherein the working electrode is a substrate electrode of which the surface is fixed with a capture probe, the capture probe is a DNA nano-scaffold marked by sulfydryl, and the electrochemical sensor is designed for exosome miRNA amplification products; the cascade strand displacement reaction system comprises a cascadeA T-shaped structure compound Ts and a cascade reaction auxiliary chain Fs, wherein the MOF/DNA cascade enzyme amplification detection system comprises MOF/DNA cascade enzyme; the Ts consists of a DNA single-chain P chain, an R chain, an L chain and an L chain0The Fs consist of a single DNA strand, the F strand and the L strand0Chain composition; two footholds T1 and T2 are arranged at the tail end of the Ts, and a target substance can be combined with the first foothold T1 and replace a P chain through a foothold mediated chain replacement reaction to expose a second foothold T2 in an L chain; fs is hybridized with T2, the target substance and the R chain are displaced through base pairing extension, DNA nano-scaffolds are generated simultaneously, the displaced target substance is combined to the adjacent foothold T1, the next cycle reaction is triggered, and a large number of DNA nano-scaffolds are generated; the DNA nano-scaffold is fixed on the surface of the electrode, is specifically hybridized with the MOF/DNA cascade enzyme to capture the MOF/DNA cascade enzyme to form a three-layer composite material, generates an electrochemical signal, and realizes the detection of the exosome miRNA through detecting the electrochemical signal.
2. The electrochemical sensor of claim 1, wherein: the nucleotide sequence of the P chain is as follows:
5′-TTTTTCCCTTAGCTTATCAGACTGA-3′(SEQ ID NO.1)；
the nucleotide sequence of the R chain is as follows:
5′-CCTACGTCTCCAACTAACTTACGG-3′(SEQ ID NO.2)；
the nucleotide sequence of the L chain is as follows:
5′-GTAGAATGCAGTGTAGTAGAATGCAGTGTAGTAGAATGCAGTGTAGTAGAATGCAGTGTAGTAGAATGCAGTGTA-3′(SEQ ID NO.3)；
said L0The nucleotide sequence of the strand is:
5′-TCAACATCAGTCTGATAAGCTAAGGGCCGTAAGTTAGTTGGAGACGTAGGTTTACACTGCATTCTAC-3′(SEQ ID NO.4)；
the nucleotide sequence of the F chain is as follows:
5′-CCTACGTCTCCAACTAACTTACGGCCCTTAGCTTATCAGACTGA-3′(SEQ ID NO.5)；
the nucleotide sequence of the target substance T chain (target sequence T) is:
5′-TAGCTTATCAGACTGATGTTGA-3′(SEQ ID NO.6)。
3. the electrochemical sensor of claim 1, wherein: the MOF/DNA cascade enzyme is formed by combining AuNPs/MOF with glucose oxidase-like activity and hemin/G-quadruplex DNAzyme with peroxidase-like activity, the AuNPs/MOF is formed by combining gold nanoparticle AuNPs and metal organic framework material MOF, and the hemin/G-quadruplex DNAzyme is a compound formed by a G-quadruplex and hemin.
4. The electrochemical sensor of claim 1, wherein: the substrate electrode is a gold electrode; the electrochemical sensor further includes a reference electrode and a counter electrode.
5. A method of manufacturing an electrochemical sensor according to any one of claims 1 to 4, comprising the steps of:
(1) preparing a cascade strand displacement reaction system: preparing a cascade T-shaped structure compound Ts by adopting a DNA single-chain P chain, an R chain, an L chain and an L0 chain through a one-step annealing method, and preparing a cascade reaction auxiliary chain Fs by adopting a DNA single-chain F chain and an L0 chain through a one-step annealing method; mixing Ts, Fs, a target substance T chain and a DNA hybridization solution, and incubating to obtain a cascade chain displacement reaction system;
(2) preparation of MOF/DNA Cascade enzymes: combining gold nanoparticles AuNPs and metal organic framework material MOF to prepare AuNPs/MOF, and then combining the AuNPs/MOF and hemin/G-quadruplex DNAzyme to prepare MOF/DNA cascade enzyme;
(3) dropwise adding the cascade chain displacement reaction system obtained in the step (1) to the surface of a working electrode fixed with a capture probe, and incubating;
(4) and (3) mixing the MOF/DNA cascade enzyme obtained in the step (2) with a DNA hybridization solution for reaction, dripping the obtained reaction solution onto the working electrode treated in the step (3), and incubating to obtain the MOF/DNA cascade enzyme amplification detection system.
6. The method of claim 5, wherein: in the step (1), the molar ratio of the P chain, the R chain, the L chain and the L0 chain is (4-6) to 1;
and/or, in the step (1), the P chain, the R chain, the L chain and the L chain are firstly connected0Dissolving the strands in DNA hybridization solution respectively, and mixing and incubating;
and/or, in said step (1), the F chain and the L chain0The molar ratio of the chains is (4-6) to 1;
and/or, in the step (1), the molar ratio of Ts to Fs is 1: 1;
and/or, in the step (1), the incubation temperature is 37 ℃, and the incubation time is more than or equal to 30 min;
and/or, in the step (1), the DNA hybridization solution comprises: 10mM Tris buffer, 480mM NaCl, 5mM MgCl2。
7. The method of claim 5, wherein: in the step (2), the preparation method of the metal organic framework material MOF comprises the following steps: FeCl is added3·6H2O、H2BDC (2-amino terephthalic acid) is dissolved in an organic solvent, then the mixture is placed in a high-pressure reaction kettle for heating reaction, after the reaction is finished, reaction liquid is centrifuged to obtain a product, and the product is washed and dried to obtain MOF;
in the step (2), the preparation method of the AuNPs gold nanoparticles comprises the following steps: adding sodium citrate to the boiled HAuCl4In the solution, after the color of the solution is observed to change from light yellow to deep red, stopping heating, stirring the solution to cool to room temperature (23-25 ℃), and continuing stirring for 20-30 minutes to obtain a solution containing AuNPs gold nanoparticles;
in the step (2), the reaction temperature for preparing AuNPs/MOF by combining the gold nanoparticles AuNPs and the metal organic framework material MOF is 37 ℃, and the reaction time is 10-15 h; the reaction temperature for preparing MOF/DNA cascade enzyme by combining AuNPs/MOF and hemin/G-four-chain DNAzyme is 37 ℃, and the reaction time is 0.5-2 h.
8. The method of claim 7, wherein: in the preparation method of metal organic framework material MOF, FeCl3·6H2O and H2Molar ratio of BDC (2-aminoterephthalic acid)1 to 1 in proportion of (1.5-2);
and/or in the preparation method of the metal organic framework material MOF, the reaction temperature is 110-140 ℃, and the reaction time is 3-5 h;
and/or, in the preparation method of metal organic framework material MOF, C is adopted2H5OH and DMF wash the centrifuged product;
and/or in the preparation method of the metal organic framework material MOF, the drying temperature is 50-70 ℃, and the drying time is 5-8 h;
and/or, in the preparation method of the AuNPs gold nanoparticles, sodium citrate and HAuCl4The molar ratio of (3.0-4.0) to 1.
9. The method of claim 5, wherein:
and/or, in the step (3), the incubation temperature is 37 ℃ and the incubation time is 1-1.5 h;
and/or, in the step (4), the DNA hybridization solution comprises: 10mM Tris buffer, 480mM NaCl, 5mM MgCl2；
And/or in the step (4), the MOF/DNA cascade enzyme and the DNA hybridization solution are mixed and reacted at the temperature of 4 ℃ for 1-2 h;
and/or, in the step (4), the incubation temperature is 4 ℃ and the incubation time is 1-2 h;
and/or, in the step (3), the working electrode immobilized with the capture probe is prepared according to the following steps:
surface treatment of an electrode: polishing the surface of the substrate electrode to make the surface smooth; then, treating the electrode with piranha solution, cleaning the electrode and drying;
fixing a capture probe: taking a DNA nano-stent marked by sulfydryl as a capture probe, dropwise adding the capture probe on the surface of a cleanly treated electrode, and incubating;
sealing the electrode: and (3) blocking the nonspecific adsorption sites by using 6-mercapto-1-ethanol to obtain the working electrode on which the capture probe is immobilized.
10. A method for detecting hepatitis b exosome miRNA, using the electrochemical sensor according to any one of claims 1-4 and/or the electrochemical sensor prepared according to any one of claims 5-9.
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