CN113624815B - Double-signal miRNA-21 detection method based on three-dimensional DNA Walker and Turnbull's blue - Google Patents
Double-signal miRNA-21 detection method based on three-dimensional DNA Walker and Turnbull's blue Download PDFInfo
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Abstract
The invention discloses a double-signal miRNA-21 detection method for generating Turnbull's blue by inducing based on three-dimensional DNA Walker and MOF-Fe (II). The method is to combine the affinity modified MB with the biotin modified hairpin chain H 1 After mixed incubation, the mixture is mixed with hairpin chain H 2 Mixing and incubating an-Au NP-GOx probe and a miRNA-21 solution to obtain a DNA Walker product; adding a DNA Walker product into a glucose solution for catalytic reaction, and performing magnetic separation to obtain a solution containing hydrogen peroxide; and then reacting the MOF-Fe (II) with a solution containing hydrogen peroxide and inducing the generation of TB at an electrode to realize electrochemical detection, or inducing the generation of TB in the solution by using the MOF-Fe (II) solution containing hydrogen peroxide to realize photothermal detection, so that the method can be used for the dual-signal detection of miRNA, and has low detection lower limit and high selectivity on miRNA target.
Description
Technical Field
The invention relates to a miRNA-21 detection method, in particular to a double-signal miRNA-21 detection method for inducing the immediate generation of Turnbull's blue based on three-dimensional DNA Walker and MOF-Fe (II), and belongs to the field of electrochemistry and photothermal bioanalysis.
Background
Micrornas (mirnas) are a class of short, non-coding sequences, 18-25 nucleotides in length, that play a crucial role in various biological processes, such as: gene expression, transcription, cell proliferation, differentiation, apoptosis, and hematopoiesis. In addition, aberrant expression of mirnas will lead to the formation, invasion and metastasis of cancer. In recent years, many of the reported analysis methods combined with bioanalysis have been successfully used for rapid quantitative or semi-quantitative analysis of miRNA, including electrochemical, fluorescent, colorimetric, surface plasmon resonance, and electrochemiluminescence-based bioanalysis. Compared with other methods, the electrochemical and temperature-based sensing platform has the obvious advantages of simplicity, low cost, high sensitivity and the like, and can realize sensitive detection of miRNA, but a method for simultaneously realizing electrochemical and photothermal detection of miRNA-21 by inducing Turnbull's blue through three-dimensional DNA Walker and MOF-Fe (II) in the prior art is not reported.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a double-signal miRNA-21 detection method for inducing generation of Turnbull's blue based on three-dimensional DNA Walker and MOF-Fe (II), wherein the method is based on miRNA-21 and hairpin chain H 1 The hybridization reaction skillfully designs the DNA Walker signal amplification and the generation of H by catalyzing glucose with GOx 2 O 2 And a signal transduction strategy for generating Turnbull's Blue (TB) in situ by means of self-sacrifice of MOF-Fe (II) so as to realize high-sensitivity and high-selectivity dual-signal detection of miRNA-21.
In order to realize the technical purpose, the invention provides a double-signal miRNA-21 detection method based on the generation of three-dimensional DNA Walker and MOF-Fe (II) induced Turnbull's blue, which can be realized by an electrochemical detection method or a photothermal detection method;
the electrochemical detection method comprises the following steps:
1) The avidin-modified MB is linked to the biotin-modified hairpin chain H 1 After mixed incubation, the resulting product is combined with hairpin chain H 2 Mixing and incubating an-Au NP-GOx probe and a miRNA-21 solution to obtain a DNA Walker product; adding the DNA Walker product into a glucose solution to perform catalytic reaction, and magnetically separating the DNA Walker product to obtain a solution containing hydrogen peroxide;
2) Dripping MOF-Fe (II) dispersion liquid on the surface of an electrode, drying, dripping the solution containing hydrogen peroxide on the surface of the electrode, incubating, dripping potassium ferricyanide solution on the surface of the electrode, reacting, and placing the electrode in a buffer solution for electrochemical detection after the reaction is finished to obtain a current response value;
3) Replacing the miRNA-21 solution in the step 1) with a series of standard miRNA-21 solutions with different concentrations to perform the steps 1) and 2) to obtain a series of current response values, and constructing a standard curve between the miRNA-21 concentration and the current response values;
4) Replacing the miRNA-21 solution in the step 1) with the miRNA-21 solution to be detected to perform the steps 1) and 2), obtaining corresponding current response values, and calculating the concentration of the miRNA-21 solution to be detected according to a standard curve;
or,
the photothermal detection method comprises the following steps:
i) The avidin-modified MB is linked to the biotin-modified hairpin chain H 1 After mixed incubation, the resulting product is combined with hairpin chain H 2 Mixing and incubating an-Au NP-GOx probe and a miRNA-21 solution to obtain a DNA Walker product; adding the DNA Walker product into a glucose solution to perform catalytic reaction, and magnetically separating the DNA Walker product to obtain a solution containing hydrogen peroxide;
II) incubating the solution containing hydrogen peroxide in the MOF-Fe (II) dispersion liquid, adding a potassium ferricyanide solution, reacting, and performing photo-thermal detection after the reaction is finished to obtain a temperature response value;
III) replacing the miRNA-21 solution in the step I) with a series of standard miRNA-21 solutions with different concentrations to perform the step I) and the step 2) to obtain a series of temperature response values, and constructing a standard curve between the miRNA-21 concentration and the temperature response values;
IV) replacing the miRNA-21 solution to be detected with the miRNA-21 solution in the step I) to perform the step I) and the step II) to obtain a corresponding temperature response value, and calculating the concentration of the miRNA-21 solution to be detected according to a standard curve;
as a preferred embodiment, the avidin-modified MB is linked to the biotin-modified hairpin chain H 1 After mixing, incubating for 20-40 m at the temperature of 30-40 DEG Cin, the resulting product and the hairpin chain H 2 Mixing the-Au NP-GOx probe and the miRNA-21 solution, and continuously incubating for 1.0-2 h at the temperature of 30-40 ℃. miRNA-21 can be fully developed into clip chain H through mixed incubation 1 Triggering the DNA Walker mechanism to allow more hairpin chains H 2 -Au NP-GOx Probe and hairpin chain H 1 And the signal amplification effect is achieved by pairing and combining.
As a preferred embodiment, the avidin-modified MB is linked to the biotin-modified hairpin chain H 1 The proportion of (A) is 4-6 g: 1-3 mu mol. Further preferred is the case of the biotin-modified hairpin chain H 1 The concentration was 2. Mu.M and the concentration of avidin-modified MB was 5mg mL -1 。
The DNA Walker product of the invention mainly catalyzes glucose reaction, and GOx (glucose oxidase) is utilized to catalyze the conversion of glucose into gluconic acid and hydrogen peroxide (H) 2 O 2 )。
As a preferred scheme, the MOF-Fe (II) is prepared by the following method: adding ferrous acetate water solution into DMF solution of 2-amino terephthalic acid, mixing uniformly, transferring into a high-pressure kettle, and carrying out solvent thermal reaction. The MOF-Fe (II) prepared by the method has a nano-scale, uniform-particle rice-shaped structure morphology, a large surface area and rich Fe 2+ Has high reaction activity, and can be hydrogen peroxide and K 3 [Fe(CN) 6 ]The reaction produces the Turnbull's blue with strong electric activity.
As a preferable scheme, the molar ratio of the ferrous acetate to the 2-amino terephthalic acid is 1.
As a preferred scheme, the conditions of the solvothermal reaction are as follows: reacting for 0.5-1.5 hours at the temperature of 40-60 ℃.
As a preferable scheme, the time for adding the DNA Walker product into the glucose solution to carry out the catalytic reaction is 20-40 min.
In a preferable embodiment, in step 2), a solution containing hydrogen peroxide is dropped on the surface of the electrode, and incubation is performed for 0.5 to 1.5min (Fe (II) can be oxidized into Fe (III)), and a potassium ferricyanide solution is dropped on the surface of the electrode, and reaction is performed for 20 to 40min (in situ generation of TB).
As a preferable scheme, in the step II), a solution containing hydrogen peroxide in the MOF-Fe (II) dispersion liquid is incubated for 2 to 8min, and then a potassium ferricyanide solution is added for reaction for 10 to 20min.
Hairpin chain H 2 the-Au NP-GOx probe was synthesized by the following method: adopting Na as gold colloid 2 CO 3 Adjusting the pH value of the solution to 9.0-9.5 to form a stable gold colloid solution, and then adding a hair clip chain H 2 Adding the mixture of GOx and the mixture into a gold colloid solution, incubating overnight, and centrifuging to obtain hairpin chain H 2 Au NP-GOx probe. The more specific preparation method comprises the following steps: using Na 2 CO 3 The pH value of the gold colloid solution is adjusted to 9.0-9.5 by the aqueous solution to prevent the gold colloid solution from coagulating; under slight shaking, will contain hairpin chain H 2 And GOx (both 1.0mg mL) -1 ) After the mixture of (4 ℃) was added to the gold colloid solution (0.2 mM), and incubated overnight at 4 ℃, the mixture was centrifuged at 14000rpm for 20 minutes to separate the unbound hairpin chain H 2 And GOx, and precipitating the resulting precipitate (i.e., hairpin chain H 2 Au NP-GOx) was redispersed in 0.1M PBS (pH 7.4) buffer solution containing 1.0wt% BSA and stored at 4 ℃ for use.
The invention relates to a three-dimensional DNA Walker product synthesis process which comprises the following steps: first, miRNA-21 and hairpin chain H are utilized 1 So that the hairpin chain H on the Magnetic Bead (MB) is hybridized 1 Is opened; at the same time, a hairpin chain H is added 1 Base complementary pairing generation composite probe hairpin chain H 2 And (2) Au NP-GOx, enabling the target miRNA-21 to be released competitively, participating in the next cycle, repeating the steps continuously, finally forming a plurality of DNA duplexes on the magnetic beads, and after the reaction is finished, carrying out magnetic washing separation to remove redundant probes.
The DNA Walker product related by the invention catalyzes the reaction of glucose solution to convert into H 2 O 2 The process of (2): mixing and incubating the DNA Walker product with a glucose solution for about 30min, allowing GOx to play a catalytic role to generate H 2 O 2 And gluconic acid, magnetic separation to obtainA solution containing hydrogen peroxide. Further preferably, the concentration of the glucose solution is 4mM.
The MOF-Fe (II) related to the invention is prepared by the following method: mixing ferrous acetate and 2-amino terephthalic acid (BDC-NH) 2 ) Synthesizing the rice grain-shaped MOF-Fe (II) through a hydrothermal reaction. More specifically, 10mL of 0.57M FeAc was prepared first 2 An aqueous solution; subsequently, 0.9g of BDC-NH was accurately weighed 2 Adding the mixture into 30mL of DMF, and carrying out ultrasonic treatment for about 10min to fully dissolve the mixture; under stirring, feAc is added 2 The aqueous solution is added to BDC-NH 2 Stirring the solution in DMF for 15min, transferring the solution into a high-pressure kettle, and reacting the solution for 1 hour at 50 ℃; after cooling to ambient temperature, the product was collected by centrifugation and washed 3 times with DMF and absolute ethanol, respectively; finally, the reddish brown product obtained was dried in a vacuum oven overnight.
The invention relates to a DNA Walker mediated TB in situ generation method, which comprises the following steps: (a) Ultrasonically dispersing MOF-Fe (II) into the solution, dropwise adding the solution onto the surface of a GCE electrode, and drying at room temperature; (b) Dripping the DNA Walker product solution on MOF-Fe (II)/GCE, and incubating for several minutes; (c) After washing, K is washed 3 [Fe(CN) 6 ]Dropping on the electrode surface, and reacting at room temperature. Further preferably, the concentration of the MOF-Fe (II) dispersion is 0.5mg mL -1 . The reaction time of the DNA Walker product solution and MOF-Fe (II)/GCE is 1min. K 3 [Fe(CN) 6 ]The concentration was 2mM. Most preferred method for the in situ generation of DNA Walker-mediated TB: (a) Ultrasonically dispersing 1mg of MOF-Fe (II) in 2mL of methanol, dropwise adding 4.5 mu L of the mixture to the surface of a GCE electrode, and drying at room temperature; (b) Dripping a product solution obtained by the DNA Walker reaction on MOF-Fe (II)/GCE, and incubating for 1min; (c) After washing, the concentration of K was 2mM 3 [Fe(CN)]Dropping on the surface of the electrode, and reacting at room temperature to generate TB in situ.
In the electrochemical detection process, phosphate Buffer Solution (PBS) containing KCl is used as electrolyte, and a three-electrode system is used for detecting a current value, namely: GCE is the working electrode; the platinum wire is used as a counter electrode; ag/AgCl is used as a reference electrode. The PBS was at a concentration of 0.01M and pH 6.0. In the photo-thermal detection, the MOF-Fe (I) is usedI) Increase in amount with H 2 O 2 The reaction time of (2) is prolonged to 5min, the laser wavelength is 800nm, and the power density is 2.63W cm -2 The irradiation time was 2min.
The synthesized metal organic framework MOF-Fe (II) has a typical rice grain structure, has a large specific surface area and contains a large amount of Fe 2+ Provides abundant raw materials for the generation of TB. When no target is present, MOF-Fe (II) is reacted with K [ Fe (CN)]The reaction generates a large amount of TB with a strong background signal. The presence of the target can cause H to be generated in the DNA reaction process 2 O 2 So that Fe (II) in the MOF-Fe (II) is oxidized into Fe (III), the generation of TB is inhibited, and the electrochemical signal is reduced.
The detection principle of the double-signal miRNA-21 detection method based on the generation of the three-dimensional DNA Walker and MOF-Fe (II) induced Turnbull's blue is as follows: first, when the target is present, a large number of hairpin chains H are bound to MB 1 Is opened; subsequently, when a hairpin chain H is added 2 when-Au NP-GOx Probe, it can react with H 1 Competitive binding, GOx loading on MB and miRNA-21 releasing, the released target can participate in the next cycle, after a period of reaction, the magnetic beads pass through hairpin chain H 1 With hairpin chain H 2 The base pair of (2) is complementary to load more GOx, so as to achieve the effect of signal amplification. When glucose is added into the magnetic compound, GOx in the magnetic compound can catalyze the glucose to generate gluconic acid and hydrogen peroxide (H) 2 O 2 ). After the reaction is finished, the catalyst contains H 2 O 2 The supernatant is dripped into a MOF-Fe (II) modified Glassy Carbon Electrode (GCE), so that Fe (II) is oxidized into Fe (III), the formation of TB on the MOFs surface is inhibited, and the electrochemical signal is reduced. Likewise, since the amount of TB generated is reduced, the photothermal effect is reduced, the temperature difference is small, and dual signal detection of the target object is achieved. The signal response values of the two detection methods are in linear correlation with the miRNA-21 concentration in a certain range. Therefore, the biosensing platform with excellent detection performance and excellent feasibility provides a new way for detecting miRNA-21.
The invention provides a double-signal miRNA-21 detection method for generating Turnbull's blue by inducing based on three-dimensional DNA Walker and MOF-Fe (II), which comprises the following steps:
(1) Construction of the electrochemical biosensor: mu.L of avidin-modified MB (Strep-MB, 5mg mL) -1 ) With 10. Mu.L of biotin-modified hairpin chain H 1 (2. Mu.M) and incubated at 37 ℃ for 30min. Magnetic separation, washing the magnetic product three times with Tris-HCl buffer (5 mM, pH 7.4), adding 30. Mu.L of hairpin chain H 2 Au NP-GOx Probe (among them, hairpin chain H) 2 The concentration is about 1.8 mu M, and hairpin chain H in the mixed solution 2 With hairpin chain H 1 In a molar ratio of about 2.5) to a target miRNA-21 at a concentration of 0.001 to 100pM, and containing MgCl 2 Tris-HCl (10 mM, pH 7.4) buffer (50 mM) and KCl (1 mM) to 50. Mu.L; mixing and incubating for 90min at 37 ℃ to fully develop the clip chain H of the miRNA-21 1 Triggering the DNA Walker mechanism, resulting in more hairpin chain H 2 -AuNP-GOx Probe and hairpin chain H 1 The signal amplification effect is achieved through pairing and combination; subsequently, after magnetic washing, 50. Mu.L of a 4mM glucose solution (0.5 mM, prepared in PBS at pH 7.0) was added to continue the reaction for 30min, allowing GOx to catalyze glucose to generate gluconic acid and hydrogen peroxide (H) 2 O 2 ) And H is 2 O 2 Fe (II) in the MOF-Fe (II) can be oxidized into Fe (III) to inhibit the generation of TB;
the three-dimensional DNA Walker reaction in the step (1) to form double-stranded DNA connected with a large amount of GOx is carried out by the following steps:
first, miRNA-21 and hairpin chain H are utilized 1 The hybridization reaction of (2), so that the hairpin chain H on the magnetic bead 1 Is opened; at the same time, a hairpin chain H is added 1 Base complementary pairing generation composite probe hairpin chain H 2 Au NP-GOx, so that the target miRNA-21 is competitively released to participate in the next cycle, and the process is repeated continuously, and finally a plurality of DNA duplexes are formed on the magnetic beads; after the reaction is finished, the reaction solution is magnetically washed and separated, and redundant probes are removed.
H in the step (1) 2 O 2 The formation process of (a) and the reaction thereof with MOF-Fe (II) are carried out by the following steps:
to the above productsAdding glucose solution with concentration of 4mM into the substance, mixing and incubating for about 30min, allowing GOx to play a catalytic role to generate H 2 O 2 And gluconic acid; after magnetic separation, the supernatant is dripped on the surface of the electrode modified with MOF-Fe (II), and incubation is continued for 1min, so that Fe (II) in the MOF-Fe (II) is oxidized into Fe (III).
The preparation method of MOF-Fe (II) and the in-situ generation of TB in the step (1) are prepared by the following steps:
(a) First, 10mL of 0.57M FeAc was prepared 2 An aqueous solution; subsequently, 0.9g of BDC-NH was accurately weighed 2 Adding the mixture into 30mL of DMF, and carrying out ultrasonic treatment for about 10min to fully dissolve the mixture; under stirring, feAc is added 2 The aqueous solution is added to BDC-NH 2 Stirring the solution in DMF for 15min, transferring the solution to a high-pressure reaction kettle, and reacting the solution for 1 hour at the temperature of 50 ℃; after cooling to ambient temperature, the product was collected by centrifugation and washed 3 times with DMF and absolute ethanol, respectively; finally, the reddish brown product obtained was dried in a vacuum oven overnight.
(b) Ultrasonically dispersing 1mg of MOF-Fe (II) in 2mL of methanol, dropwise adding 4.5 mu L of the mixture to the surface of a GCE electrode, and drying at room temperature; dripping a product solution obtained by the DNA Walker reaction on MOF-Fe (II)/GCE, and incubating for 1min; after washing, K [ Fe (CN) ] was dropped to the above electrode surface at a concentration of 2mM, and reacted at room temperature to generate TB in situ.
(2) Electrochemical detection of miRNA-21: 4.5 μ L of MOF-Fe (II) (0.5 mg/mL) was added dropwise to the GCE and dried at room temperature. After that, 10. Mu.L of the supernatant obtained by the DNA Walker reaction was applied to an MOF-Fe (II)/GCE electrode, incubated at room temperature for 1min, and then the electrode surface was gently rinsed with ultrapure water. Finally, 10. Mu.L of 2mM K [ Fe (CN) ]]Dropping on the surface of the electrode, and reacting at room temperature for 30min to remove H 2 O 2 Oxidized MOF-Fe (II) can be reacted with K [ Fe (CN)]Sufficient reaction to generate Turnbull's Blue (TB) to generate electrochemical signal. The electrochemical response was measured using Differential Pulse Voltammetry (DPV) using a three-electrode system with 0.01M PBS as the electrolyte. Since the concentration of miRNA-21 added in step (1) is different, the amount of DNA duplexes generated on the magnetic beads is different, i.e., contains different amountsGOx, resulting in H 2 O 2 The amount of MOF-Fe (II) oxidized Fe (II) also varied, and the amount of TB produced varied, so the magnitude of the decrease in electrochemical response varied. Repeating the above operations, and drawing a standard curve according to the logarithm of the current response value to the standard sample concentration; and replacing the miRNA-21 standard solution with the solution to be detected for the detection, and obtaining a concentration result through a standard curve.
(3) And (3) miRNA-21 photothermal detection: the DNA reaction was carried out in the same manner as in step (2) at a concentration of 100 mg L in a medium containing 40. Mu.L of DNA -1 Adding 50. Mu.L of MOF-Fe (II) containing H 2 O 2 The supernatant of the DNA reaction of (1) was incubated for 5min. Subsequently, 10. Mu.L of K [ Fe (CN) at a concentration of 2mM was added]And (3) solution. After the reaction was completed, the mixed solution was continuously irradiated with a laser having a wavelength of 808nm for 2min. Recording the temperature difference before and after irradiation, namely drawing a standard curve according to the logarithm of the temperature difference to the concentration of the standard sample; and replacing the miRNA-21 standard solution with the solution to be detected for detection, and obtaining a concentration result through a standard curve.
Compared with the prior art, the technical scheme of the invention has the beneficial technical effects that:
1) The invention adopts a self-sacrifice method, utilizes MOF-Fe (II) and K [ Fe (CN) ] to react to generate the Turnbull's blue with strong electric activity and photothermal effect to generate signals, can realize double-signal detection on a target object, and greatly embodies the versatility of the biosensor.
2) The MOF-Fe (II) is synthesized by one step of hydrothermal reaction, the synthesis method is simple, the cost is low, and the obtained MOF-Fe (II) has the shape of a nano-grade rice grain, has a large surface area and contains rich Fe 2+ The reaction activity is higher.
3) The invention improves the detection sensitivity by an amplification strategy based on biomolecules, the three-dimensional DNA Walker signal amplification reaction is triggered by a target object, and the released target object can be circularly amplified, so that a great amount of GOx-modified H can be obtained 2 And H 1 Hybridization to produce a DNA duplex containing a significant amount of GOx. After adding glucose, a large amount of H can be catalytically generated 2 O 2 To oxidize MOF-Fe 2 + The Fe (II) in (C) is,the generation of TB is inhibited, and the purpose of signal amplification is achieved;
4) The signal reduction type dual-signal sensing strategy for miRNA-21 detection, which is constructed by the invention, has a good linear relation between current and temperature difference and target object concentration logarithm in a concentration range of 1fM-100pM, and has the advantages of wide linear range and low detection line.
Drawings
FIG. 1 is a schematic diagram of a double-signal MicroRNA-21 detection method based on the instant generation of Tengtze blue induced by three-dimensional DNA Walker and MOF-Fe (II).
FIGS. 2 (A) and (C) are a scanning electron micrograph and a transmission electron micrograph of rice grain-shaped MOF-Fe (II), respectively; FIGS. 2 (B) and (D) are a scanning electron micrograph and a transmission electron micrograph of TB.
FIG. 3 shows the results of the detection of the standard sample in example 1, A: example 1 test target concentration versus corresponding electrochemical response plot; b: example 1 electrochemical response values are plotted against log-linear concentration of the detection target; c: example 1 detecting a target concentration and a corresponding thermal imaging profile; d: example 1 temperature difference versus log linear concentration of the target.
FIG. 4 shows the results of selectivity and reproducibility studies of example 1.
In FIG. 5, A and B are the influence of the reaction time and incubation temperature on the amplification degree of DNA Walker in the process of synthesizing DNA Walker products, respectively; c and D are respectively the influence of the time of producing hydrogen peroxide by catalyzing glucose by the DNA Walker product and the formation time of TB on the electrochemical signal intensity.
Detailed Description
The technical solution of the present invention is further illustrated by the following specific implementation examples, but the scope of the present invention is not limited thereby.
Example 1
(1) First, 10mL of 0.57M FeAc was prepared 2 An aqueous solution; subsequently, 0.9g of BDC-NH was accurately weighed 2 Adding the mixture into 30mL of DMF, and carrying out ultrasonic treatment for about 10min to fully dissolve the mixture; under stirring, feAc is added 2 The aqueous solution is added to BDC-NH 2 Stirring for 15min, transferring into an autoclave, and allowing to stand in DMFReacting for 1 hour at 50 ℃; after cooling to ambient temperature, the product was collected by centrifugation and washed 3 times with DMF and absolute ethanol, respectively; finally, the reddish brown product obtained was dried in a vacuum oven overnight.
(2) Ultrasonically dispersing 1mg of MOF-Fe (II) powder prepared in the step (1) in 2mL of methanol, and dropwise adding 4.5L of the powder onto the surface of GCE. Drying at room temperature to obtain the MOF-Fe (II)/GCE electrode.
(3) mu.L of avidin-modified MB (Strep-MB, 5mg mL) -1 ) With 10. Mu.L of biotin-modified hairpin chain H 1 (2. Mu.M) and incubated at 37 ℃ for 30min. Magnetic separation, washing the magnetic product with Tris-HCl buffer (5 mM, pH 7.4) for three times, and adding 30. Mu.L of hairpin chain H 2 Au NP-GOx Probe (among them, hairpin chain H) 2 The concentration is about 1.8 mu M, and the hairpin chain H in the mixture 2 With hairpin chain H 1 About 2.5 molar ratio 2 Tris-HCl (10 mM, pH 7.4) buffer (50 mM) and KCl (1 mM) to 50. Mu.L; mixing and incubating for 90min at 37 ℃ to fully develop the clip chain H of the miRNA-21 1 Triggering the DNA Walker mechanism, resulting in more hairpin chain H 2 -AuNP-GOx Probe and hairpin chain H 1 The signal amplification effect is achieved through pairing and combination;
(4) After the DNA Walker product obtained in the step (3) is magnetically washed, 50 mu L of glucose solution with the concentration of 4mM (0.5 mM, PBS preparation with pH 7.0) is added for continuing the reaction for 30min, so that GOx catalyzes glucose to generate gluconic acid and hydrogen peroxide (H) 2 O 2 ) (ii) a Magnetic separation, and storage of the resulting supernatant at 4 ℃.
(5) Dropwise adding 10L of the product solution obtained in the step (4) on the electrode in the step (2), and incubating at room temperature for 1min to oxidize Fe (II) in the MOF-Fe (II) modified on the surface of the electrode; k [ Fe (CN) ] was dropped to the above electrode surface at a concentration of 2mM and reacted at room temperature to generate TB in situ. After washing, the electrochemical response was measured using Differential Pulse Voltammetry (DPV) with 0.01M PBS (pH 6.0) as the electrolyte. Similarly, the mixed solution was subjected to photothermal detection by continuous irradiation with a laser beam having a wavelength of 808nm for 2min, and the temperature difference between before and after the irradiation was recorded. Due to the signal amplification effect of the DNAwalker, the current response value and the temperature difference are remarkably reduced, and the current response value and the temperature difference have a determined relation with the concentration of the miRNA-21, so that the miRNA-21 can be sensitively detected; and replacing the miRNA-21 standard solution with the solution to be detected for the detection, and obtaining a concentration result through a standard curve.
The DNA sequence used in example 1 is as follows:
FIG. 1 is a schematic diagram of the principle and process of a double-signal miRNA-21 detection method based on the generation of Turnbull's blue induced by three-dimensional DNA Walker and MOF-Fe (II) according to the present invention. FIG. 2 is a scanning electron micrograph and a transmission electron micrograph of MOF-Fe (II) and in situ generated TB. The scanning electron micrograph shows that the MOF-Fe (II) is in a typical rice grain shape and has the diameter of between 250 and 300 nm. After the TB is generated, small particles are generated on the surface of the MOF-Fe (II) rice grains, the original appearance is partially broken, but the distribution is more uniform, and the generated TB is proved to have better dispersibility. FIG. 3 shows the results of the standard samples tested in example 1, wherein the current and temperature both decrease with the increase of miRNA-21 concentration. As shown in FIGS. 3B and 3D, there was a good linear relationship between the current response and the temperature difference as a function of the logarithm of the target concentration over the miRNA-21 concentration range of 1fM-100 pM. In addition, in order to prove the practical applicability of the invention in life, the selectivity and the reproducibility of the method on miRNA-21 are also examined. The experimental results show (fig. 4A) that the current value of the high concentration interferent is similar to the current value of the target alone. In addition, the Relative Standard Deviation (RSD) of the results of the five parallel sensors was less than 5%, with good reproducibility (fig. 4B). Therefore, the method for detecting miRNA-21 has good selectivity and reproducibility.
Condition optimization experiment:
1. optimization of conditions for synthesis of DNA Walker products (including reaction time and incubation temperature).
In order to realize the sensitive detection of the electrochemical biosensing platform on the miRNA-21, the relevant real objects are detected by using differential pulse voltammetryThe test conditions and parameters are optimized. First, the influence of the reaction conditions for synthesizing three-dimensional DNA Walker on the sensing platform was investigated in a PBS (0.01M, pH 6.0) solution containing 0.1M KCl. As shown in fig. 5 (a), the DPV signal decreases as the reaction time increases from 20min to 90min, and reaches a plateau when the reaction time is further extended to 110 min. Therefore, the optimal reaction time for synthesizing the three-dimensional DNA Walker is 90min. Further, as shown in fig. 5 (B), the DPV signal gradually decreased as the incubation temperature gradually increased to 37 ℃, while the signal gradually increased as the incubation temperature continued to increase from 37 ℃ to 47 ℃. This phenomenon indicates that when the temperature exceeds 37 ℃, the degree of DNA Walker amplification is affected, only part of H1 and H2 hybridize, and a small amount of H is generated 2 O 2 Resulting in a reduced amplitude of signal degradation. Therefore, the optimal incubation temperature for the DNA Walker reaction is 37 ℃.
2. Optimizing the time for producing hydrogen peroxide by catalyzing glucose with the DNA Walker product and the TB formation time:
the catalytic time of GOx on glucose and the formation time of TB are also critical to the detection effect of the sensor. FIG. 5 (C) shows that the DPV response current value decreases with the increase of the catalytic time of GOx to glucose, the current value is lowest at 30min, and the current value gradually stabilizes after 30min, i.e. glucose can be oxidized into gluconic acid and H by GOx within a certain time 2 O 2 So that the length of the catalytic time directly determines the production of H 2 O 2 The amount of (c). Therefore, 30min was chosen as the optimal catalysis time for GOx on glucose. For the duration of TB formation, K [ Fe (CN) increases with time]Compete for more Fe in MOF-Fe (II) 2+ I.e. more and more TB is generated at the electrode surface, resulting in a stronger electrochemical signal. As can be seen from FIG. 5 (D), when the reaction time was 30min, the DPV signal reached a minimum and remained stable, indicating that the electrode surface K [ Fe (CN) ]]With MOF-Fe 2+ The reaction of (2) reaches a saturation state. Therefore, 30min was chosen as the optimal reaction time for TB production.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made according to the claims of the present invention should be covered by the present invention.
Claims (8)
1. A double-signal miRNA-21 detection method based on generation of Teng blue induced by three-dimensional DNA Walker and MOF-Fe (II) is characterized in that:
comprises the following steps:
1) The avidin-modified MB is coupled to the biotin-modified hairpin chain H 1 After mixed incubation, the resulting product is combined with hairpin chain H 2 Mixing and incubating an-Au NP-GOx probe and a miRNA-21 solution to obtain a DNA Walker product; adding the DNA Walker product into a glucose solution to perform catalytic reaction, and magnetically separating the DNA Walker product to obtain a solution containing hydrogen peroxide;
2) Dripping MOF-Fe (II) dispersion liquid on the surface of an electrode, drying, dripping the solution containing hydrogen peroxide on the surface of the electrode, incubating, dripping potassium ferricyanide solution on the surface of the electrode, reacting, and placing the electrode in a buffer solution for electrochemical detection after the reaction is finished to obtain a current response value;
3) Replacing the miRNA-21 solution in the step 1) with a series of standard miRNA-21 solutions with different concentrations to perform the step 1) and the step 2) to obtain a series of current response values, and constructing a standard curve between the miRNA-21 concentration and the current response values;
4) Replacing the miRNA-21 solution in the step 1) with the miRNA-21 solution to be detected to perform the steps 1) and 2), obtaining a corresponding current response value, and calculating the concentration of the miRNA-21 solution to be detected according to a standard curve;
or,
comprises the following steps:
i) The avidin-modified MB is linked to the biotin-modified hairpin chain H 1 After mixed incubation, the resulting product is combined with hairpin chain H 2 Mixing and incubating an-Au NP-GOx probe and a miRNA-21 solution to obtain a DNA Walker product; adding the DNA Walker product into a glucose solution to perform catalytic reaction, and magnetically separating the DNA Walker product to obtain a solution containing hydrogen peroxide;
II) incubating the solution containing hydrogen peroxide in the MOF-Fe (II) dispersion liquid, adding a potassium ferricyanide solution, reacting, and performing photo-thermal detection after the reaction is finished to obtain a temperature response value;
III) replacing the miRNA-21 solution in the step I) with a series of standard miRNA-21 solutions with different concentrations to perform the step I) and the step 2) to obtain a series of temperature response values, and constructing a standard curve between the miRNA-21 concentration and the temperature response values;
IV) replacing the miRNA-21 solution in the step I) with the miRNA-21 solution to be detected to perform the step I) and the step II), obtaining a corresponding temperature response value, and calculating the concentration of the miRNA-21 solution to be detected according to a standard curve.
2. The method for detecting the dual-signal miRNA-21 based on the induction of Turnbull's blue generation by the three-dimensional DNA Walker and the MOF-Fe (II) as claimed in claim 1, wherein the method comprises the following steps: avidin-modified MB and biotin-modified hairpin chain H 1 Mixing, incubating at 30-40 deg.C for 20-40 min, and mixing the obtained product with hairpin chain H 2 Mixing the-Au NP-GOx probe and the miRNA-21 solution, and continuously incubating for 1.0-2 h at the temperature of 30-40 ℃.
3. The method for detecting the dual-signal miRNA-21 based on the induction of Turnbull's blue generation by the three-dimensional DNA Walker and the MOF-Fe (II) as claimed in claim 1, wherein the method comprises the following steps: avidin-modified MB and biotin-modified hairpin chain H 1 The mixing proportion is 4-6 g.
4. The method for detecting the dual-signal miRNA-21 based on the induction of Turnbull's blue generation by the three-dimensional DNA Walker and the MOF-Fe (II) as claimed in claim 1, wherein the method comprises the following steps: the MOF-Fe (II) is prepared by the following method: adding the ferrous acetate aqueous solution into the DMF solution of 2-amino terephthalic acid, uniformly mixing, transferring into a high-pressure kettle, and carrying out solvothermal reaction.
5. The method for detecting the dual-signal miRNA-21 based on the induction of Turnbull's blue generation by the three-dimensional DNA Walker and the MOF-Fe (II) as claimed in claim 4, wherein the method comprises the following steps:
the molar ratio of the ferrous acetate to the 2-amino terephthalic acid is 1-1.2;
the conditions of the solvothermal reaction are as follows: reacting for 0.5-1.5 hours at the temperature of 40-60 ℃.
6. The method for detecting the dual-signal miRNA-21 based on the induction of Turnbull's blue generation by the three-dimensional DNA Walker and the MOF-Fe (II) as claimed in claim 4, wherein the method comprises the following steps: the time for adding the DNA Walker product into the glucose solution to carry out catalytic reaction is 20-40 min.
7. The method for detecting the dual-signal miRNA-21 based on the induction of Turnbull's blue generation by the three-dimensional DNA Walker and the MOF-Fe (II) as claimed in claim 1, wherein the method comprises the following steps: in the step 2), a solution containing hydrogen peroxide is dripped on the surface of the electrode for incubation for 0.5-1.5 min, and a potassium ferricyanide solution is dripped on the surface of the electrode for reaction for 20-40 min.
8. The method for detecting the dual-signal miRNA-21 based on the induction of Turnbull's blue generation by the three-dimensional DNA Walker and the MOF-Fe (II) as claimed in claim 1, wherein the method comprises the following steps: in the step II), incubating the solution containing hydrogen peroxide in the MOF-Fe (II) dispersion liquid for 2-8 min, adding a potassium ferricyanide solution, and reacting for 10-20 min.
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