CN111638255A - Bismuth vanadate-based method for photoelectrochemical detection of miRNA-21 content - Google Patents
Bismuth vanadate-based method for photoelectrochemical detection of miRNA-21 content Download PDFInfo
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Abstract
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)4And (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.
Description
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
Example 1
a、BiVO4The preparation of (1): weighing 2.0g of bismuth nitrate, dissolving the bismuth nitrate in 20mL of ethylene glycol, and magnetically stirring until the bismuth nitrate is dissolved to obtain a solution A; weighing 0.5g of ammonium metavanadate, and adding 20mL of 80 ℃ deionized water, and magnetically stirring until the ammonium metavanadate is dissolved to obtain a solution B; dropwise adding the solution B into the solution A, stirring for 30 minutes, transferring into an autoclave, and heating at 100 ℃ for 15 hours; naturally cooling to room temperature, alternately washing with anhydrous ethanol and distilled water until the supernatant is neutral, and oven drying in a drying oven at 60 deg.C overnight;
b、BiVO4preparing a modified ITO electrode: weighing the prepared BiVO4Adding the powder into deionized water, and performing ultrasonic treatment to obtain 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: mu.L of a 1. mu.M padlock probe was mixed with 10. mu.L of miRNA-21 at various concentrations in 50. mu.L of a mixture containing 10mM MgCl2At 37 ℃ for 1 hour in 7.540mM Tris-HCl buffer solution; then, 40U T4DNA ligase and 50. mu.L of MgCl containing 10mM were added20.5mM ATP, 7.540mM Tris-HCl buffer solution at 22 ℃ for 1 hour; next, 10U phi29DNA polymerase, 4.4. mu.L, 10mM deoxyribonucleoside triphosphate and 50. mu.L MgCl containing 10.0mM2、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; BstNB I nucleic acid, 20U, was addedEndonuclease and 20. mu.L of a DNA containing 50mM NaCl, 5mM MgCl20.5mM dithiothreitol in 25mM Tris-HCl buffer solution at pH 7.9, and cleaving the enzyme at 55 ℃ for 1 hour;
d. and (3) carrying out DNA hybridization recognition reaction on the surface of the electrode and measuring photocurrent: mu.L, 2. mu.M of amino-functionalized probe DNA was immobilized to BiVO by glutaraldehyde cross-linking reaction4On an ITO electrode, incubating overnight at 4 ℃; then, 15. mu.L of monoethanolamine was taken out at 37 ℃ in a concentration of 1mM, and the electrode was subjected to blocking reaction for 1 hour in the absence of light; then, 15. mu.L of the final lysate from step c and 15. mu.L of a lysate containing 0.1M NaCl, 1mM MgCl2After mixing with Tris-HCl hybridization buffer (pH 7.410 mM), the mixture was dropped on the surface of an electrode to perform hybridization reaction at 37 ℃ for 1 hour; then, 3U of EXO I and 18. mu.L of a solution containing 67mM glycine-KOH (pH 9.5), 6.7mM MgCl were introduced210mM dithiothreitol (pH 7.410 mM Tris-HCl buffer solution), and digesting and hydrolyzing at 37 ℃ for 30 minutes to remove non-hybridized single-stranded DNA on the surface of the electrode; finally, 28. mu.L of 10. mu.M MB solution was 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.
The photocurrent measurement uses a platinum wire electrode as a counter electrode, a 500W xenon lamp as an excitation light source (provided with a 400nm cut-off filter), and the photocurrent of the working electrode is recorded through an electrochemical workstation.
The results are shown in fig. 3-4, where the method has a sensitive response to miRNA, with a linear equation of Δ i (na) 145.5log [ CmiRNA-21(pM)]+2127.7, linear range 0.005 to 10000pM, linear correlation coefficient R20.992 with a detection limit of 0.3 fM.
Example 2
a、BiVO4The preparation of (1): weighing 2.0g of bismuth nitrate, dissolving the bismuth nitrate in 20mL of glycerol aqueous solution (50 v/v%), and magnetically stirring until the bismuth nitrate is dissolved to obtain solution A; weighing 0.8g of potassium vanadate in 20mL of deionized water, and magnetically stirring until the potassium vanadate is dissolved to obtain a solution B; the solution B was added dropwise to the solution A, and the pH of the mixed solution was adjusted to 3 with nitric acid.0, stirring for 30 minutes, transferring into an autoclave, and heating for 8 hours at 180 ℃; naturally cooling to room temperature, alternately washing with anhydrous ethanol and distilled water until the supernatant is neutral, and oven drying at 80 deg.C in a drying oven overnight;
b、BiVO4preparing a modified ITO electrode: weighing the prepared BiVO4Adding the powder into deionized water, and performing ultrasonic treatment to obtain 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 ℃ for 2 hours for later use;
c. biological recognition and signal amplification reaction: mu.L of a 1. mu.M padlock probe was mixed with 10. mu.L of miRNA-21 at various concentrations in 50. mu.L of a mixture containing 10mM MgCl2At 37 ℃ for 1 hour in 7.540mM Tris-HCl buffer solution; then, 40U T4DNA ligase and 50. mu.L of MgCl containing 10mM were added20.5mM ATP, 7.540mM Tris-HCl buffer solution at 22 ℃ for 1 hour; next, 10U phi29DNA polymerase, 4.4. mu.L, 10mM deoxyribonucleoside triphosphate and 50. mu.L containing 10mM MgCl were added2、50mM KCl、5mM(NH4)2SO4At a pH of 7.540mM in Tris-HCl buffer solution, and carrying out an amplification reaction at 30 ℃ for 2 hours; finally, 20U of Nt.BstNB I endonuclease and 20. mu.L of DNA containing 50mM NaCl, 5mM MgCl were added20.5mM dithiothreitol in 25mM Tris-HCl buffer solution at pH 7.9, and cleaving the enzyme at 55 ℃ for 1 hour;
d. DNA hybridization recognition reaction and photocurrent measurement on the surface of the electrode: mu.L, 2. mu.M of amino-functionalized probe DNA was immobilized to BiVO by glutaraldehyde cross-linking reaction4On an ITO electrode, incubating overnight at 4 ℃; then, 15. mu.L of monoethanolamine was taken out at 37 ℃ in a concentration of 1mM, and the electrode was subjected to blocking reaction for 1 hour in the absence of light; then, 15. mu.L of the final lysate from step c and 15. mu.L of a lysate containing 0.1M NaCl, 1mM MgCl2After mixing with Tris-HCl hybridization buffer (pH 7.410 mM), the mixture was dropped on the surface of an electrode to perform hybridization reaction at 37 ℃ for 1 hour; then, 3U of EXO I and 18. mu.L of MgCl containing 67mM glycine-KOH (pH 9.5) and 6.7mM were introduced210mM dithiothreitol (pH 7.410 mM Tris-HCl buffer solution) at 37 deg.CDigesting and hydrolyzing for 30 minutes to remove non-hybridized single-stranded DNA on the surface of the electrode; finally, 28. mu.L of 10. mu.M NB solution was 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.
Claims (10)
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).
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| CN112525971A (en) * | 2020-12-16 | 2021-03-19 | 江南大学 | Method for photoelectrochemical detection of chloramphenicol based on bismuth tungstate |
| CN114199970A (en) * | 2021-12-15 | 2022-03-18 | 江南大学 | Cathode photoelectrochemical detection model of T4 polynucleotide kinase and application thereof |
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| CN114199970A (en) * | 2021-12-15 | 2022-03-18 | 江南大学 | Cathode photoelectrochemical detection model of T4 polynucleotide kinase and application thereof |
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