阅读说明：本技术 一种基于三维DNA Walker和滕氏蓝的双信号miRNA-21检测方法 (Double-signal miRNA-21 detection method based on three-dimensional DNA Walker and Turnbull's blue ) 是由 汤娟 程宏丽 刘丽萍 高珊 于 2021-06-30 设计创作，主要内容包括：本发明公开了一种基于三维DNA Walker和MOF-Fe(II)诱导滕氏蓝生成的双信号miRNA-21检测方法。该方法是将亲和素修饰的MB与生物素修饰的发夹链H-(1)混合孵育后,再与发夹链H-(2)-Au NP-GOx探针及miRNA-21溶液混合孵育,得到DNA Walker产物；在葡萄糖溶液中加入DNA Walker产物进行催化反应,磁性分离,得到含过氧化氢的溶液；再利用MOF-Fe(II)与含过氧化氢的溶液反应并在电极诱导TB生成,以实现电化学检测,或者利用MOF-Fe(II)含过氧化氢的溶液并在溶液中诱导TB生成,以实现光热检测,从而可以用于miRNA的双信号检测,且对miRNA目标物检测下限低、选择性高。(The invention discloses a double-signal miRNA-21 detection method for inducing generation of Turnbull's blue based on three-dimensional DNA Walker and MOF-Fe (II). The method is to combine the MB modified by the avidin and the hairpin chain H modified by the biotin 1 After mixed incubation, the mixture is further 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; then MOF-Fe (II) is used for reacting with a solution containing hydrogen peroxide and inducing TB generation at an electrode to realize electrochemical detection, or MOF-Fe (II) solution containing hydrogen peroxide is used for inducing TB generation in the solution to realize photothermal detection, so that the method can be used for double-signal detection of miRNA, has low detection lower limit on miRNA target substances, and is selectedThe performance is high.)

1. A double-signal miRNA-21 detection method based on three-dimensional DNA Walker and MOF-Fe (II) induction of generation of Turnbull's blue is characterized in that:

comprises the following steps:

1) the avidin-modified MB is linked to the biotin-modified hairpin chain H₁After mixed incubation, the resulting product is combined with hairpin chain H₂Mixing 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 a corresponding current response value, and calculating the concentration of the miRNA-21 solution to be detected according to a standard curve;

alternatively, the first and second electrodes may be,

comprises the following steps:

I) the avidin-modified MB is linked to the biotin-modified hairpin chain H₁After mixed incubation, the resulting product is combined with hairpin chain H₂Mixing 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 three-dimensional DNA Walker and the MOF-Fe (II) for inducing the generation of the Turnbull's blue as claimed in claim 1, wherein the method comprises the following steps: avidin-modified MB and biotin-modified hairpin chain H₁Mixing, incubating for 20-40 min at 30-40 ℃, and mixing the obtained product with the hairpin chain H₂Mixing the-Au NP-GOx probe and the miRNA-21 solution, and continuously incubating for 1.0-2 h at 30-40 ℃.

3. The method for detecting the dual-signal miRNA-21 based on the three-dimensional DNA Walker and the MOF-Fe (II) for inducing the generation of the Turnbull's blue as claimed in claim 1, wherein the method comprises the following steps: avidin-modified MB and biotin-modified hairpin chain H₁The mixing ratio is 4-6 g: 1-3 μmol.

4. The method for detecting the dual-signal miRNA-21 based on the three-dimensional DNA Walker and the MOF-Fe (II) for inducing the generation of the Turnbull's blue 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 three-dimensional DNA Walker and the MOF-Fe (II) for inducing the generation of the Turnbull's blue 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: 1-1.2;

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 three-dimensional DNA Walker and the MOF-Fe (II) for inducing the generation of the Turnbull's blue as claimed in claim 4, wherein the method comprises the following steps: and adding the DNA Walker product into the glucose solution for catalytic reaction for 20-40 min.

7. The method for detecting the dual-signal miRNA-21 based on the three-dimensional DNA Walker and the MOF-Fe (II) for inducing the generation of the Turnbull's blue as claimed in claim 1, wherein the method comprises the following steps: and in the step 2), dropwise adding a solution containing hydrogen peroxide on the surface of the electrode, incubating for 0.5-1.5 min, dropwise adding a potassium ferricyanide solution on the surface of the electrode, and reacting for 20-40 min.

8. The method for detecting the dual-signal miRNA-21 based on the three-dimensional DNA Walker and the MOF-Fe (II) for inducing the generation of the Turnbull's blue 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.

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

MicroRNA (miRNA) is a short non-coding sequence with a length of 18-25 nucleotides, and plays 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 in the prior art, a method for inducing generation of Turnbull's blue by using three-dimensional DNA Walker and MOF-Fe (II) and realizing electrochemical and photothermal detection of miRNA-21 is not reported.

Disclosure of Invention

In order to overcome the defects and shortcomings 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 H₁The hybridization reaction skillfully designs the amplification of DNA Walker signals and the generation of H by catalyzing glucose by GOx₂O₂And a signal transduction strategy of self-sacrifice in-situ generation of Turnbull's Blue (TB) by means of MOF-Fe (II) to realize high-sensitivity and high-selectivity dual-signal detection of miRNA-21.

In order to achieve the technical purpose, the invention provides a double-signal miRNA-21 detection method for inducing generation of Turnbull's blue based on three-dimensional DNA Walker and MOF-Fe (II), 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 H₁After mixed incubation, the resulting product is combined with hairpin chain H₂Mixing 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 a corresponding current response value, and calculating the concentration of the miRNA-21 solution to be detected according to a standard curve;

alternatively, the first and second electrodes may be,

the photothermal detection method comprises the following steps:

I) the avidin-modified MB is linked to the biotin-modified hairpin chain H₁After mixed incubation, the resulting product is combined with hairpin chain H₂Mixing 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;

as a preferred embodiment, the avidin-modified MB is linked to the biotin-modified hairpin chain H₁After mixing, incubating at a temperature of 30-40 ℃ for 20 to c40min, and the obtained product and hairpin chain H₂Mixing the-Au NP-GOx probe and the miRNA-21 solution, and continuously incubating for 1.0-2 h at 30-40 ℃. miRNA-21 can be fully developed into clip chain H through mixed incubation₁Triggering the DNA Walker mechanism to allow more hairpin chains H₂-Au NP-GOx Probe and hairpin chain H₁And 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 H₁The ratio of (A) to (B) is 4-6 g: 1-3. mu. mol. Further preferred is the case of the biotin-modified hairpin chain H₁The 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)₂O₂)。

As a preferred embodiment, 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. The MOF-Fe (II) prepared by the method has a nano-scale and uniform-particle rice-grain-shaped structure appearance, has a large surface area and contains rich Fe²⁺Has high reaction activity, and can be hydrogen peroxide and K₃[Fe(CN)₆]The reaction produces the Teng's blue with strong electric activity.

Preferably, the molar ratio of the ferrous acetate to the 2-amino terephthalic acid is 1: 1-1.2.

As a preferred embodiment, the solvothermal reaction conditions are: 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 catalytic reaction is 20-40 min.

Preferably, in step 2), a solution containing hydrogen peroxide is dropped on the surface of the electrode, incubation is performed for 0.5-1.5 min (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 20-40 min (in-situ generation of TB).

As a preferable scheme, in the step II), a solution containing hydrogen peroxide in the MOF-fe (II) dispersion is incubated for 2-8 min, and then a potassium ferricyanide solution is added to react for 10-20 min.

Hairpin chain H₂the-Au NP-GOx probe was synthesized by the following method: adopting Na as gold colloid₂CO₃Adjusting 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 H₂Adding the mixture of GOx and the mixture into a gold colloid solution, incubating overnight, and centrifuging to obtain hairpin chain H₂-Au NP-GOx probe. The more specific preparation method comprises the following steps: using Na₂CO₃Adjusting the pH value of the gold colloid solution to 9.0-9.5 by using the aqueous solution to prevent the gold colloid solution from coagulating; under slight shaking, will contain hairpin chain H₂And GOx (all 1.0mg mL)^-1) After the mixture of (1) was added to the gold colloid solution (0.2mM) and incubated overnight at 4 ℃, the mixture was centrifuged at 14000rpm for 20 minutes to separate the unbound hairpin chain H₂And GOx, and precipitating the resulting precipitate (i.e., hairpin chain H₂-Au NP-GOx) was redispersed in 0.1M PBS (pH 7.4) buffer containing 1.0 wt% 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 utilized₁So that the hairpin chain H on the Magnetic Bead (MB) is hybridized₁Is opened; at the same time, a hairpin chain H is added₁Base complementary pairing generation composite probe hairpin chain H₂And (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 H₂O₂The process of (2): mixing and incubating the DNA Walker product with a glucose solution for about 30min, and allowing GOx to play a catalytic role to generate H₂O₂And gluconic acid, magnetic separation to obtainTo a solution containing hydrogen peroxide. Further preferably, the concentration of the glucose solution is 4 mM.

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 rice grain-shaped MOF-Fe (II) by a hydrothermal reaction. More specifically, 10mL of 0.57M FeAc was prepared₂An aqueous solution; subsequently, 0.9g of BDC-NH was accurately weighed₂Adding the mixture into 30mL of DMF, and carrying out ultrasonic treatment for about 10min to fully dissolve the mixture; under stirring, FeAc is added₂The aqueous solution is added to BDC-NH₂Stirring the solution in DMF for 15min, transferring the solution into a high-pressure 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.

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 to 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 washed₃[Fe(CN)₆]Dropped onto the surface of the electrode and reacted 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 1 min. K₃[Fe(CN)₆]The concentration was 2 mM. 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 1 min; (c) after washing, the K was added at a concentration of 2mM₃[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 the 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 concentration was 0.01M and pH was 6.0. In the case of photothermal detection, the MOF-Fe increase in the amount of (II) with H₂O₂The reaction time of (2) is prolonged to 5min, the laser wavelength is 800nm, and the power density is 2.63W cm^-2The irradiation time was 2 min.

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 Fe²⁺Provides abundant raw materials for the generation of TB. When no target is present, MOF-Fe (II) and K [ Fe (CN)]The reaction produces 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 process₂O₂So 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 three-dimensional DNA Walker and the induction of the MOF-Fe (II) to the generation of the Turnbull's blue is as follows: first, when the target is present, a large number of hairpin chains H are bound to MB₁Is opened; subsequently, when a hairpin chain H is added₂when-Au NP-GOx Probe, it can react with H₁Competitive binding, GOx loading on MB and miRNA-21 releasing, the released target can participate in the next cycle, and after a period of reaction, the magnetic beads pass through hairpin chain H₁With hairpin chain H₂The 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)₂O₂). After the reaction is finished, the catalyst contains H₂O₂The supernatant of (a) is dripped into an MOF-Fe (II) modified Glassy Carbon Electrode (GCE), so that Fe (II) is oxidized into Fe (III), the formation of TB on the surface of MOFs is inhibited, and the electrochemical signal is reduced. Likewise, since the amount of TB generated is reduced, the photothermal effect is reduced and the temperature difference is small, thereby achieving dual signal detection of the target. 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 inducing generation of Turnbull's blue 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 H₁(2. mu.M) and incubated at 37 ℃ for 30 min. Magnetic separation, washing the magnetic product with Tris-HCl buffer (5mM, pH 7.4) for three times, and adding 30. mu.L of hairpin chain H₂Au NP-GOx Probe (among others, hairpin chain H)₂The concentration is about 1.8 mu M, and the hairpin chain H in the mixture₂With hairpin chain H₁In a molar ratio of about 2.5:1) and a target miRNA-21 at a concentration of 0.001-100 pM, and MgCl₂Tris-HCl (10mM, pH 7.4) buffer (50mM) and KCl (1mM) to 50. mu.L; mixing and incubating for 90min at 37 ℃ to fully develop the clip chain H of the miRNA-21₁Triggering the DNA Walker mechanism to allow more hairpin chains H₂-AuNP-GOx Probe and hairpin chain H₁The signal amplification effect is achieved through pairing and combination; subsequently, after magnetic washing, 50. mu.L of a 4mM glucose solution (0.5mM, prepared in PBS pH 7.0) was added to continue the reaction for 30min, allowing GOx to catalyze glucose to generate gluconic acid and hydrogen peroxide (H)₂O₂) And H is₂O₂Fe (II) in the MOF-Fe (II) can be oxidized into Fe (III), so that the generation of TB is inhibited;

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 utilized₁The hybridization reaction of (2) so that the hairpin chain H on the magnetic bead₁Is opened; at the same time, a hairpin chain H is added₁Base complementary pairing generation composite probe hairpin chain H₂Au 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)₂O₂The formation process and the reaction with MOF-Fe (II) are carried out by the following steps:

to the aboveAdding glucose solution with concentration of 4mM into the product, mixing and incubating for about 30min, and allowing GOx to play a catalytic role to generate H₂O₂And 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) firstly preparing 10mL of 0.57M FeAc₂An aqueous solution; subsequently, 0.9g of BDC-NH was accurately weighed₂Adding the mixture into 30mL of DMF, and carrying out ultrasonic treatment for about 10min to fully dissolve the mixture; under stirring, FeAc is added₂The aqueous solution is added to BDC-NH₂Stirring 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 1 min; after washing, K [ Fe (CN) ] was dropped to the surface of the above electrode 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) (0.5mg/mL) was added dropwise to GCE and dried at room temperature. Then, 10. mu.L of the supernatant obtained from 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 H₂O₂Oxidized MOF-Fe (II) with K [ Fe (CN)]The reaction is complete to generate Turnbull's Blue (TB), which generates an 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 of (2) resulting in H being generated₂O₂The amount of MOF-fe (ii) and hence the amount of oxidized fe (ii) also varied, resulting in a different amount of TB produced and therefore a different magnitude of reduction in electrochemical response. 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^-1Adding 50 μ L of MOF-Fe (II) containing H₂O₂The supernatant of the DNA reaction of (1) was incubated for 5 min. Subsequently, 10. mu.L of 2mM K [ Fe (CN) ]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 2 min. 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 the 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 of a target object, and greatly embodies the multifunctionality 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 nano-scale rice grains, has larger surface area and contains rich Fe²⁺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 obtained₂And H₁Hybridization to produce a DNA duplex containing a significant amount of GOx. After adding glucose, a large amount of H can be catalytically generated₂O₂To oxidize MOF-Fe^{2 +}Fe (I) in (1)I) 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 three-dimensional DNA Walker and MOF-Fe (II) for inducing immediate generation of Turnbull's blue.

FIGS. 2(A) and (C) are respectively a scanning electron micrograph and a transmission electron micrograph of rice grain MOF-Fe (II); FIGS. 2(B) and (D) are a scanning electron micrograph and a transmission electron micrograph, respectively, 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 the selectivity and reproducibility test 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 influence of the TB formation time 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) Firstly preparing 10mL of 0.57M FeAc₂An aqueous solution; subsequently, 0.9g of BDC-NH was accurately weighed₂Adding the mixture into 30mL of DMF, and carrying out ultrasonic treatment for about 10min to fully dissolve the mixture; under stirring, FeAc is added₂The aqueous solution is added to BDC-NH₂Stirring for 15min, transferring into autoclave, and allowing to standReacting at 50 ℃ for 1 hour; 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) 1mg of MOF-Fe (II) powder prepared in step (1) was ultrasonically dispersed in 2mL of methanol, and 4.5L was added dropwise to the surface of GCE. After drying at room temperature, the MOF-Fe (II)/GCE electrode is obtained.

(3) mu.L of avidin-modified MB (Strep-MB, 5mg mL)^-1) With 10. mu.L of biotin-modified hairpin chain H₁(2. mu.M) and incubated at 37 ℃ for 30 min. Magnetic separation, washing the magnetic product with Tris-HCl buffer (5mM, pH 7.4) for three times, and adding 30. mu.L of hairpin chain H₂Au NP-GOx Probe (among others, hairpin chain H)₂The concentration is about 1.8 mu M, and the hairpin chain H in the mixture₂With hairpin chain H₁At a molar ratio of about 2.5:1) and a target miRNA-21 concentration of 1pM, and containing MgCl₂Tris-HCl (10mM, pH 7.4) buffer (50mM) and KCl (1mM) to 50. mu.L; mixing and incubating for 90min at 37 ℃ to fully develop the clip chain H of the miRNA-21₁Triggering the DNA Walker mechanism to allow more hairpin chains H₂-AuNP-GOx Probe and hairpin chain H₁The 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 (0.5mM, PBS preparation with pH of 7.0) is added for continuing the reaction for 30min, so that GOx catalyzes glucose to generate gluconic acid and hydrogen peroxide (H)₂O₂) (ii) a Magnetic separation, and storage of the resulting supernatant at 4 ℃.

(5) Dripping 10L of the product solution obtained in the step (4) on the electrode in the step (2), and incubating for 1min at room temperature to oxidize Fe (II) in the MOF-Fe (II) modified on the surface of the electrode; k [ Fe (CN) ] was dropped to the surface of the electrode 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 for inducing generation of Turnbull's blue based on 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 microscope image shows that the MOF-Fe (II) has a typical rice-grain shape and the diameter is about 250-300 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 be better in dispersity. 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 differential pulse voltammetry is used for carrying out the related detectionThe experimental conditions and parameters were optimized. First, the effect of the reaction conditions for the synthesis of three-dimensional DNA Walker on the sensing platform was explored 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 90 min. 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, and only parts of H1 and H2 hybridize to produce a small amount of H₂O₂Resulting 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 time₂O₂Therefore, the length of the catalytic time directly determines the generation of H₂O₂The amount of (c). Therefore, 30min was chosen as the optimal catalysis time for GOx on glucose. For the duration of TB formation, with increasing time, K [ Fe (CN)]Will 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 the lowest value and remained stable, indicating that the electrode surface K [ Fe (CN) ]]With MOF-Fe²⁺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 for the best mode of the invention, and all equivalent changes and modifications made in accordance with the claims of the invention should be covered by the present invention.