阅读说明：本技术 一种基于钒酸铋的光电化学检测miRNA-21含量的方法 (Bismuth vanadate-based method for photoelectrochemical detection of miRNA-21 content ) 是由 王光丽 刘田利 顾萌萌 孙冬雪 于 2020-06-15 设计创作，主要内容包括：本发明公开了一种基于钒酸铋的光电化学检测miRNA-21含量的方法,属于分析检测领域。本发明方法结合miRNA-21介导的滚环扩增、内切酶特异性识别水解反应与亚甲基蓝或耐尔蓝对双链DNA的特异性嵌入所造成的BiVO<Sub>4</Sub>修饰电极的光电信号激发,构建信号“增强型”检测。本发明方法在0.005-10000pM浓度范围内可实现高灵敏检测miRNA-21,检测限低至0.3fM。与传统方法相比,本发明所提出的无标记光电化学检测方法具有成本低,操作简便灵活,试剂用量小,灵敏度高,背景信号小等优点,有望成为有效检测miRNA-21的方法之一。(The invention discloses a bismuth vanadate-based method for photoelectrochemical detection of miRNA-21 content, and belongs to the field of analysis and detection. The method combines miRNA-21 mediated rolling circle amplification, endonuclease specific recognition hydrolysis reaction and BiVO (BiVO) caused by specific embedding of methylene blue or Nerlan to double-stranded DNA (deoxyribonucleic acid) 4 And (3) exciting a photoelectric signal of the modified electrode to construct signal 'enhanced' detection. The method can realize high-sensitivity detection of miRNA-21 within the concentration range of 0.005-10000pM, and the detection limit is as low as 0.3 fM. Compared with the traditional method, the label-free photoelectrochemical detection method provided by the invention has the advantages of low cost, simple and flexible operation, small reagent dosage, high sensitivity, small background signal and the like, and is expected to become one of the methods for effectively detecting miRNA-21.)

1. A method for measuring miRNA-21 content by photoelectrochemistry is characterized by comprising the following steps:

(1)BiVO₄preparation of ITO electrode: BiVO (bismuth oxide) is added₄Dispersing in water to form BiVO₄Suspending liquid; then dropping BiVO on the surface of the ITO electrode₄The suspension is dried to obtain BiVO₄An ITO electrode;

(2) biological recognition and signal amplification reaction: respectively carrying out amplification reaction on miRNA-21 systems with different concentrations, and then adding endonuclease to carry out enzyme digestion reaction to obtain lysate containing more single-stranded DNA;

(3) electrode surface hybridization recognition reaction and photocurrent measurement: immobilization of amine-functionalized Probe DNA to BiVO by glutaraldehyde crosslinking reaction₄On the ITO electrode; adding the lysate and buffer solution system obtained in the step (2) to the surface of an electrode, performing hybridization reaction, adding EXO I to continue reaction, and adding a signal molecule solution to perform incubation; after incubation is finished, taking the electrode as a working electrode, and adopting a three-electrode system to perform photocurrent measurement;

(4) constructing a linear model: calculating by using the photocurrent in the step (3) to obtain photocurrent difference values under different concentrations, and then constructing by using the photocurrent difference and the concentration of miRNA-21 to obtain a linear model; wherein the photocurrent difference is the difference between the photocurrent values of different miRNA-21 concentrations and the photocurrent value of miRNA-21 concentration of 0.

2. The method of claim 1, wherein the signal molecule in step (3) is methylene blue and/or Nerlan blue.

3. The method of claim 1, wherein BiVO is obtained in step (1)₄The concentration of the suspension was 1 mg/mL.

4. The method of claim 1, wherein BiVO is obtained in step (1)₄The preparation method comprises the following steps:

mixing a bismuth salt solution and a vanadate solution, wherein the mass concentration ratio of the two solutions is 1:1, uniformly mixing, and transferring into a high-pressure reaction kettle for reaction; after the reaction is finished, cooling, washing and drying to obtain powdery BiVO₄And (3) obtaining the product.

5. The method of claim 4, wherein the vanadate is one of ammonium metavanadate or potassium vanadate; the bismuth salt is bismuth nitrate.

6. The method as claimed in claim 4, wherein the reaction temperature is 100 ℃ and 200 ℃ and the reaction time is 8-20 h.

7. The method according to any one of claims 1 to 6, wherein in the step (3), the lysis solution and the buffer system are dripped on the surface of the electrode, and the hybridization reaction is carried out at 37 +/-0.5 ℃; introducing EXO I, and continuing to react at 37 +/-0.5 ℃; finally, 10. mu.M signal molecule solution was incubated at 37. + -. 0.5 ℃ on the electrode surface.

8. The method according to any one of claims 1 to 7, wherein the photocurrent measurement in step (3) is carried out using a three-electrode system by a current-time technique using the prepared electrode as a working electrode, in Tris-HCl at pH 7, at a voltage of 0V relative to an Ag/AgCl reference electrode.

9. The method of any one of claims 1 to 8, wherein the miRNA-21 base sequence used in the biological recognition and signal amplification reaction is 5'-UAG CUU AUC AGA CUG AUG UUG A-3';the base sequence of the padlock probe is 5'-TGA TAA GCT ACC ACC TCC GCG CGA AGT CTC AAC ATC AGT C-3'; the base sequence of the probe DNA on the fixed electrode is 5' -NH₂–C₆CCA CCT CCG CGC GAA GTC TCA A–3'。

10. The method according to any one of claims 1-9, further comprising: processing a sample to be detected according to the step (2) to obtain corresponding lysate; then measuring the light current value of the sample to be measured through the step (3); and finally, calculating to obtain the concentration content of the miRNA-21 in the sample to be detected through the linear model obtained in the step (4).

Technical Field

The invention belongs to the field of analysis and detection, and relates to a method for photoelectrochemical detection of miRNA-21 content based on bismuth vanadate.

Background

MicroRNA (miRNA) is an endogenous, evolutionarily conserved, non-coding single-stranded RNA [ He L, Hannon G.J.Nat.Rev.Genet.2004,5: 522-containing 531 ], which plays a crucial role in regulating transcription and in a variety of biological processes (such as cell proliferation, differentiation, apoptosis and hematopoiesis) [ Wang Y, Li Z H, Weber TJ, Hu D H, Lin C T, Li J H, Lin Y H.anal.chem.2013,85: 6775-containing 6782; wang Y, Tang L H, Li Z H, Lin Y H, Li J H. nat. Protoc.2014,9: 1944-. Studies have shown that expression of mirnas may be closely related to many human diseases [ essula-Kerscher a, Slack F j. nat. rev. cancer 2006,6: 259-269; gupta A, Gartner J J, Sethupathy P, Hatzigeorgiou A G, Fraser NW.Nature 2008,451: 600-. The traditional miRNA detection method has the disadvantages of time-consuming sample pretreatment, complex experimental process and high experimental cost, and severely limits the practical application of the traditional miRNA detection method [ Jia H, Li Z, Liu C, ChengY.Angew.chem.int.Ed.2010,49: 5498-. Therefore, the development of a high-sensitivity detection method for detecting miRNA is of great significance.

Disclosure of Invention

Photoelectrochemical (PEC) analysis is a novel analytical method established on the basis of direct or indirect interaction between a photoactive material under photoexcitation and an analyte. In the detection process, the electric signals generated by the external excitation light source can be effectively distinguished, so that the background signals can be obviously reduced, and the performance of the sensor is higher. In recent years, photo-electrochemical biosensors have been rapidly developed and widely used in many fields.

The photoelectric material adopted by the invention is a green and environment-friendly bismuth-based compound BiVO₄[Li H,SunH.Curr.Opin.Chem.Biol.2012,16:74-83.]. The bismuth-based compound is composed of (Bi)₂O₂)²⁺The layered structure formed by the host layer and other guest ion layers is beneficial to the migration of photo-generated electrons between the layers, effectively reduces the recombination of the photo-generated electrons and holes and further improves the utilization efficiency of photo-generated carriers. BiVO₄Under the excitation of the LED light source, electrons on the valence band transition to the conduction band, and meanwhile, holes are left in the valence band, and the conduction band is transmitted to the electrode to generate anode photocurrent. Electronic power supplyBiVO can be effectively consumed in the presence of body₄The holes on the valence band reduce the recombination efficiency of hole electrons, so that the photocurrent is greatly improved. The sensor constructed by the invention initiates a rolling circle amplification reaction in the presence of a target molecule miRNA, and generates a plurality of single-stranded DNAs with the assistance of endonuclease; and can hybridize with DNA fixed on the electrode to form double-stranded DNA, and embed signal molecule into the double-stranded DNA on the electrode to excite BiVO₄The generated photocurrent signal variation difference value is related to the target concentration, thereby achieving the purpose of detecting miRNA. The biological probes used in the method do not need complicated and expensive marks, so that the method greatly saves the test cost, improves the convenience of detection, and has high sensitivity and good selectivity.

The invention aims to provide a rapid and efficient miRNA photoelectrochemical detection method which has the advantages of wide linear range, low detection limit (high sensitivity), strong specificity, convenience in use, low cost and the like.

Based on the purpose, the design idea of the invention is as follows: RCA reaction, endonuclease digestion reaction and DNA hybridization reaction on the surface of the electrode, which are initiated in the presence of a target, lead to that a large amount of signal molecules are fixed on the surface of the electrode through double-stranded DNA, and high photocurrent signals can be measured; when no target exists, the reaction is inhibited, only single-stranded DNA exists on the surface of the electrode, and EXO I is introduced to digest and hydrolyze the single-stranded DNA, so that the background signal is successfully reduced, and the measured photocurrent signal is lower.

The invention provides a method for photoelectrochemical detection of miRNA-21 content, which comprises the following steps:

(1)BiVO₄preparation of ITO electrode: BiVO (bismuth oxide) is added₄Dispersing in water to form BiVO₄Suspending liquid; then, the surface of the ITO electrode is coated with BiVO₄The suspension is dried to obtain BiVO₄An ITO electrode;

(2) biological recognition and signal amplification reaction: respectively carrying out amplification reaction on samples with different miRNA-21 concentrations, and then adding endonuclease to carry out enzyme digestion reaction to obtain lysate containing more single-stranded DNA;

(3) electrode surface hybridization recognition reaction andphotocurrent measurement: immobilization of amine-functionalized Probe DNA to BiVO by glutaraldehyde crosslinking reaction₄On the ITO electrode; adding the lysate and buffer solution system obtained in the step (2) to the surface of an electrode, performing hybridization reaction, adding EXO I to continue reaction, and adding a signal molecule solution to perform incubation; after incubation is finished, taking the electrode as a working electrode, and adopting a three-electrode system to perform photocurrent measurement to obtain photocurrent values corresponding to miRNA-21 systems with different known concentrations;

(4) constructing a linear model: calculating the photocurrent values of the samples with different miRNA-21 concentrations and the sample with miRNA-21 concentration of 0 obtained in the step (3) to obtain photocurrent difference values delta I (delta I is the difference obtained by subtracting the photocurrents of the samples with miRNA-21 or without miRNA-21); and then constructing a linear model by using the photocurrent difference and the concentration of miRNA-21.

In one embodiment of the present invention, BiVO in the step (1)₄The concentration of the suspension was 1 mg/mL.

In one embodiment of the present invention, BiVO in the step (1)₄Can be prepared by the following method:

mixing a bismuth salt solution and a vanadate solution, wherein the mass concentration ratio of the two solutions is 1:1, uniformly mixing, transferring into a high-pressure reaction kettle, and reacting for a certain time at a certain temperature; and naturally cooling to room temperature, alternately washing the product by using absolute ethyl alcohol and distilled water, and drying in a drying oven overnight to obtain a powdery product.

In one embodiment of the invention, BiVO is prepared₄The vanadate is one of ammonium metavanadate or potassium vanadate; the bismuth salt is bismuth nitrate.

In one embodiment of the invention, BiVO is prepared₄The reaction temperature is 100-200 ℃, and the reaction time is 8-20 h.

In one embodiment of the present invention, the amplification reaction in step (2) comprises the following processes:

in the presence of 10mM MgCl₂At a pH of 7.540mM Tris-HCl buffer solution, mixing padlock probes with different concentrations of miRNA-21 at 37Hybridization incubation is carried out for 1 hour at +/-0.5 ℃; then, T4DNA ligase was added and 10mM MgCl was added₂0.5mM ATP, 7.540mM Tris-HCl buffer, incubated at 22. + -. 0.5 ℃ for 1 hour; next, phi29DNA polymerase, deoxyribonucleoside triphosphates, and a solution containing 10.0mM MgCl₂、50mM KCl、5mM(NH₄)₂SO₄At a pH of 7.540.0mM in Tris-HCl buffer solution, and the amplification reaction was carried out at 30. + -. 0.5 ℃ for 2 hours.

In an embodiment of the present invention, the enzyme digestion reaction in step (2) includes the following processes:

adding endonuclease and the mixture containing 50mM NaCl and 5mM MgCl₂The digestion reaction was carried out at 55. + -. 0.5 ℃ for 1 hour in 0.5mM dithiothreitol pH 7.925mM Tris-HCl buffer solution.

In one embodiment of the present invention, in the step (3), specifically, the amine-functionalized probe DNA is immobilized to BiVO by glutaraldehyde crosslinking reaction₄On the ITO electrode, the mixture is incubated overnight at 4 +/-0.5 ℃ in a humid environment.

In one embodiment of the present invention, the buffer liquid in step (3) contains 0.1M NaCl, 1mM MgCl₂pH 7.410 mM Tris-HCl buffer solution.

In one embodiment of the invention, in the step (3), the lysis solution and the buffer system are dripped on the surface of the electrode, and the hybridization reaction is carried out at 37 +/-0.5 ℃ for 1 hour; introducing EXO I, and reacting for 30 minutes at 37 +/-0.5 ℃; finally, 10. mu.M signal molecule solution was incubated for 15 minutes at 37. + -. 0.5 ℃ on the electrode surface.

In one embodiment of the present invention, the photocurrent measurement in step (3) is performed by using a three-electrode system and a current-time technique, using the prepared electrode as a working electrode, in Tris-HCl at pH 7, at a voltage of 0V with respect to an Ag/AgCl reference electrode.

In one embodiment of the invention, the miRNA-21 base sequence used in the biological recognition and signal amplification reaction is 5'-UAG CUU AUC AGA CUG AUG UUG A-3'; the base sequence of the padlock probe is 5' -TGATAA GCTACC ACC TCC GCG CGA AGT CTC AAC ATC AGT C-3'; the base sequence of the probe DNA on the fixed electrode is 5' -NH₂–C₆CCA CCT CCG CGC GAA GTC TCA A–3'。

In one embodiment of the present invention, the signal molecule in step (3) is Methylene Blue (MB) and/or Nai Blue (NB).

In one embodiment of the invention, the method further comprises: processing the sample to be detected according to the step (2) to obtain corresponding lysate, and then measuring the photocurrent value of the sample to be detected through the step (3); and (5) calculating to obtain the concentration of miRNA-21 in the sample to be detected through the linear model obtained in the step (4).

In an embodiment of the present invention, the method specifically includes the following steps:

a、BiVO₄the preparation of (1): mixing 20mL of bismuth nitrate solution and 20mL of vanadate solution, wherein the mass concentration ratio of the two solutions is 1:1, stirring for 30 minutes, transferring into a high-pressure reaction kettle, and reacting for a certain time at a certain temperature; naturally cooling to room temperature, alternately washing the product with absolute ethyl alcohol and distilled water, and drying in a drying oven overnight to obtain a powdery product;

b、BiVO₄preparing a modified ITO electrode: weighing the prepared BiVO₄Adding the powder into water, and performing ultrasonic treatment to obtain a suspension of 1 mg/mL; 30 mu L of BiVO is dripped on the surface of the ITO electrode which is cleaned in advance₄Drying the suspension at 40 deg.C for 2 hr;

c. biological recognition and signal amplification reaction: in the presence of 10mM MgCl₂In 7.540mM Tris-HCl buffer solution, mixing padlock probes with miRNA-21 of different concentrations, and hybridizing and incubating at 37 ℃ for 1 hour; then, T4DNA ligase was added and 10mM MgCl was included₂0.5mM ATP, 7.540mM Tris-HCl buffer solution, incubated at 22 ℃ for 1 hour; next, phi29DNA polymerase, deoxyribonucleoside triphosphates, and a solution containing 10.0mM MgCl were added₂、50mM KCl、5mM(NH₄)₂SO₄At a pH of 7.540.0mM in Tris-HCl buffer solution, and carrying out an amplification reaction at 30 ℃ for 2 hours; finally, addEndonucleases and DNA primers containing 50mM NaCl, 5mM MgCl₂The digestion reaction was carried out at 55 ℃ for 1 hour in 0.5mM dithiothreitol pH 7.925mM Tris-HCl buffer solution;

d. and (3) carrying out DNA hybridization recognition reaction on the surface of the electrode and measuring photocurrent: immobilization of amine-functionalized Probe DNA to BiVO by glutaraldehyde crosslinking reaction₄C, incubating overnight at 4 ℃ in a humid environment on the ITO electrode, and taking the final lysate in the step c and the lysate containing 0.1M NaCl and 1mM MgCl₂Is added dropwise to the electrode surface, and hybridization reaction is carried out at 37 ℃ for 1 hour; EXO I is introduced and reacted for 30 minutes at 37 ℃; finally, 10 μ M signal molecule solution is incubated for 15 minutes at 37 ℃ on the electrode surface; and d, adopting a three-electrode system, using the electrode prepared in the step d as a working electrode through a current-time technology, placing the working electrode in Tris-HCl with the pH value of 7, and measuring the photocurrent at the voltage of 0V relative to the reference electrode of Ag/AgCl.

In one embodiment of the invention, when target miRNA-21 exists in a sample to be detected, miRNA-21 is combined with a padlock probe to initiate constant-temperature rolling circle amplification, nucleic acid is exponentially amplified in a short time to generate a long single-stranded DNA, endonuclease is introduced to digest and hydrolyze RCA to generate the single-stranded DNA into short cDNA, the cDNA is hybridized with probe DNA fixed on an electrode to generate the double-stranded DNA, signal molecules can be effectively embedded, and the unhybridized probe DNA is introduced into EXO I to digest and hydrolyze, so that the background is effectively reduced; using signal molecules with BiVO₄The photocurrent signal generated between the two electrodes achieves the detection purpose.

The invention has the beneficial effects that:

the detection method is rapid and efficient, has the advantages of wide linear range (0.005-10000pM), low detection limit to 0.3fM (high sensitivity), strong specificity, convenient use, low cost and the like, and has very wide application prospect.

Drawings

FIG. 1 is BiVO₄Photocurrent profiles before (a) and after (b) addition of MB to the modified electrode.

FIG. 2A) different concentrations of MB (a to i in the order of 0,0.005,0.01,0.05,0.1,0.5,1,5, 10. mu. moL/L) BiVO prepared in example 1₄Photocurrent effects; B) the photocurrent variation difference was plotted linearly against the logarithm of MB concentration.

FIG. 3 is BiVO prepared by practicing example 1₄Response profiles to different concentrations of miRNA-21(a to k are sequentially 0,0.005,0.01,0.05,0.1,0.5,1,10,100,1000,10000 pM).

FIG. 4 is a linear plot of photocurrent variation versus log of miRNA-21 concentration obtained in practicing the method of example 1.

Detailed Description