CN112378965B

CN112378965B - Electrochemical detection method for hypersensitive microRNA of endopeptidase-driven polypod DNA molecular machine

Info

Publication number: CN112378965B
Application number: CN202011252668.7A
Authority: CN
Inventors: 许永杰; 罗洁; 达静静; 郑翔; 李学英; 张志敏; 闫旭东
Original assignee: Guizhou Provincial Peoples Hospital
Current assignee: Guizhou Provincial Peoples Hospital
Priority date: 2020-11-11
Filing date: 2020-11-11
Publication date: 2023-01-31
Anticipated expiration: 2040-11-11
Also published as: CN112378965A

Abstract

The invention relates to the technical field of molecular diagnosis, and discloses a hypersensitive microRNA electrochemical detection method of an endonuclease driven polypod DNA molecular machine, which comprises the following steps: aiming at target microRNA, detection probes 1, 2 and 3 (probes 1, 2 and 3) are designed and synthesized in a programmed mode to serve as recognition elements, a sulfhydrylation modified probe4 (probe 4) is designed and synthesized to serve as a report element, the sulfhydrylation probe4 is dripped on the surface of a gold electrode, and the gold electrode is incubated overnight in a refrigerator at 4 ℃ to promote the report probe to be anchored on the surface of the gold electrode, so that a molecular track is prepared, a target sequence and the detection probes are recognized and carry out cascade programming reaction to form a three-fork DNA nano structure, each end of the structure is designed with a short piece of nucleic acid restriction endonuclease site serving as a foot of a molecular machine, and then the endonuclease-driven multi-foot molecular machine can be formed.

Description

Electrochemical detection method for hypersensitive microRNA of endopeptidase driven polypod DNA molecular machine

Technical Field

The invention relates to the technical field of molecular diagnosis, in particular to a hypersensitive microRNA electrochemical detection method of an endonuclease driven polypod DNA molecular machine.

Background

The subcloned population of tumor cells evolves continuously with various selective pressures, promoting their proliferation and dissemination, and reducing therapeutic susceptibility. Accurate diagnosis and monitoring are realized, ineffective treatment is avoided, and a molecular detection technology for continuously probing dynamic molecular evolution information of tumor cells is urgently needed. Circulating microRNA (18-24 nucleotides) plays a key role in tumor proliferation, differentiation and apoptosis, and can be used as a potential biomarker for treatment monitoring, drug resistance evaluation and quantification of minimal residual diseases. Meanwhile, the detection of the circulating microRNA in the blood has the advantages of simple and almost non-invasive molecular dynamic landscape for real-time reaction of the tumor, thereby showing wide application prospect.

Nucleic acid detection techniques based on Polymerase Chain Reaction (PCR) have high sensitivity, but are often limited by specialized laboratories, expensive instruments, and complex primer designs. The method for exploring a simple and high-performance microRNA quantitative new method has important research value.

Disclosure of Invention

Technical problem to be solved

Aiming at the defects of the prior art, the invention provides a hypersensitive microRNA electrochemical detection method of an endonuclease driven polypod DNA molecular machine, which has the advantages of no need of a PCR instrument with accurate temperature control, amplification and amplification under an isothermal condition, simplicity and convenience, high efficiency of the DNA molecular machine, single-molecule controllability and detection and analysis performance improvement, provides the advantages of a simple, convenient and sensitive candidate technology with application prospect for cyclic microRNA detection by using an electrochemical sensing technology of the gene polypod molecular machine, and solves the problem that the nucleic acid detection technology based on polymerase chain reaction has high sensitivity but is often limited by professional laboratories, expensive instruments, complex primer designs and the like.

(II) technical scheme

In order to realize the PCR instrument without accurate temperature control, amplification can be realized under the isothermal condition, the PCR instrument has the advantages of simplicity and convenience, the DNA molecular machine has high efficiency and single-molecule controllability, and the detection and analysis performance is improved, the electrochemical sensing technology of the gene multi-foot molecular machine provides a simple, convenient and sensitive candidate technology with application prospect for circulating microRNA detection, and the invention provides the following technical scheme: a hypersensitive microRNA electrochemical detection method of an endonuclease driven polypod DNA molecular machine is characterized by comprising the following steps:

aiming at target microRNA, detecting

probes

1, 2 and 3 (

probes

1, 2 and 3) are designed and synthesized in a programming way to be used as recognition elements, and a thiol-modified probe4 (probe 4) is designed and synthesized to be used as a report element;

dropwise adding the thiolated probe4 on the surface of the gold electrode, and incubating overnight in a refrigerator at 4 ℃ to promote the report probe to be anchored on the surface of the gold electrode, thereby preparing a molecular rail;

through the identification of the target sequence and the detection probe and the occurrence of cascade programming reaction, the three-fork DNA nano structure is formed. Each segment of the structure is designed with a short piece of restriction endonuclease site as a foot of a molecular machine, so that a multi-foot molecular machine driven by the endonuclease can be formed;

wherein the sequence of Probe1 (Probe 1) is CCTCAGCTTCAACATCAGTCTGATAAGCTAGATGTTGTAGCTTATCAGACTG, the sequence of Probe2 (Probe 2) is CCTCAGCTCAGTCTGATAAGCTACAACATCTTCAGACTGATGTTGTAGCTTAT, and the sequence of Probe3 (Probe 3) is CCTCAGCTATAAGCTACAACATCAGTCTGATAGCTTATCAGACTGTTGTTGT.

Preferably, when the target microRNA exists, the target microRNA triggers three detection probes (

probes

1, 2 and 3) to perform a programmed self-assembly reaction, and the three detection probes are assembled to form a three-fork type DNA nano-structure.

Preferably, each 5' end of the trifurcate structure extends out of a short single-stranded nucleic acid fragment, so as to form a multi-footed DNA molecular machine, and each foot of the multi-footed DNA molecular machine is recognized and hybridized with a reporter probe (probe 4) fixed on the surface of a gold electrode to form a DNA double strand, so as to be captured on the surface of the electrode.

Preferably, the double strand is designed with a restriction enzyme site, the restriction enzyme recognizes and cleaves the double strand to promote the cleavage of the reporter from the probe, and after the cleavage, the restriction enzyme site is sufficient to recognize and hybridize with the next reporter probe, and then enter the next round of the endonuclease reaction.

Preferably, the hybridization and cutting process is repeatedly identified, and then the polypod molecular machine is driven to continuously walk on the reporting probe track on the surface of the gold electrode.

Preferably, each time the single chain extending from each end of the trifurcated structure is combined with and cut on the surface of the electrode, the reporter group is cut off from the probe, so that the electric signal is weakened, and the amplified output of the signal is realized.

Preferably, when the target microRNA does not exist, the programmed reaction cannot be triggered, the detection probes (

probe

1, 2 and 3) exist independently, a short single strand extending from the tail end of the detection probe cannot form a double strand due to weak binding force with the electrode surface probe, the report probe cannot be cut by the restriction enzyme, and the electrochemical signal is unchanged.

(III) advantageous effects

Compared with the prior art, the invention provides a hypersensitive microRNA electrochemical detection method of an endonuclease driven polypod DNA molecular machine, which has the following beneficial effects:

1. according to the electrochemical detection method for the hypersensitive microRNA of the endopeptidase-driven polypod DNA molecular machine, the polypod DNA walking molecular machine is formed by

programming detection probes

1, 2 and 3 (

probes

1, 2 and 3), and the sulfhydrylation modification probe4 (probe 4) is designed to serve as a report probe track on the surface of a gold electrode, the DNA molecular machine walks on the report track on the surface of the gold electrode under the drive of the restriction endonuclease, so that the amplification output of signals is realized, and further, a novel technology for the electrochemical sensing detection of the cycling microRNA based on the polypod molecular machine is developed, the novel technology does not need a PCR instrument with accurate temperature control, the amplification can be realized under the isothermal condition, and the electrochemical detection method has the advantages of simplicity and convenience.

2. According to the electrochemical detection method of the hypersensitivity microRNA of the endonuclease driven polypod DNA molecular machine, the DNA molecular machine has high efficiency and single molecule controllability, the detection and analysis performance is improved, and the electrochemical sensing technology of the polypod molecular machine provides a simple, convenient and sensitive candidate technology with application prospect for circulating microRNA detection.

Drawings

FIG. 1 is a schematic diagram of electrochemical sensing detection based on a triplex DNA walking machine in example 1 of the present invention;

FIG. 2 is a diagram showing the electrophoretic verification of the assembly of the polypod molecular machine in example 2 of the present invention;

FIG. 3 is a graph showing the verification of the electrochemical signals of the multi-legged molecular machine in example 2 of the present invention;

FIG. 4 is a graph of impedance characterization of the electrode preparation process in example 2 of the present invention;

FIG. 5 is a graph illustrating the correlation analysis between the logarithm of microRNA concentration and a detection signal in example 2 of the present invention;

FIG. 6 is a graph showing the current response of target microRNAs with different concentrations in example 2 of the present invention;

FIG. 7 is a graph of the current response of different target sequences in example 2 of the present invention;

FIG. 8 is a bar graph of signal values of different target sequences in example 2 of the present invention;

FIG. 9 is a graph showing the recovery analysis of a simulated sample of an actual sample in example 2 of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

Referring to fig. 1-9, in one aspect of the embodiments of the present invention, a method for electrochemical detection of hypersensitive microRNA by an endonuclease driven polypod DNA molecular machine is provided, which includes:

for a target microRNA, programming and synthesizing

detection probes

1, 2 and 3 (

probes

1, 2 and 3) as recognition elements, and designing and synthesizing a thiol-modified probe4 (probe 4) as a report element;

dripping the sulfhydrylation probe4 on the surface of the gold electrode, and incubating overnight in a refrigerator at 4 ℃ to promote the report probe to be anchored on the surface of the gold electrode, thereby preparing a molecular orbit;

through the recognition of the target sequence and the detection probe and the generation of cascade programming reaction, the three-fork type DNA nano structure is formed. Each segment of the structure is designed with a short piece of restriction endonuclease site as a foot of a molecular machine, so that a multi-foot molecular machine driven by the endonuclease can be formed;

When the target microRNA exists, the target microRNA triggers three detection probes (

probes

1, 2 and 3) to perform programmed self-assembly reaction, and a three-way type DNA nano structure is formed by assembly.

Each 5' end of the trifurcate structure extends out a short single-stranded nucleic acid segment, so that a multi-foot DNA molecular machine is formed, and each foot of the multi-foot DNA molecular machine is identified and hybridized with a report probe (probe 4) fixed on the surface of a gold electrode to form a DNA double strand, so that the DNA double strand is captured on the surface of the electrode.

And designing a restriction enzyme site on the double strand, recognizing and cutting the double strand by the restriction enzyme to promote the reporter group to be cut off from the probe, after cutting, recognizing and hybridizing the restriction enzyme site with the next reporter probe, and then entering the next round of internal reaction.

The process of identifying hybridization and cutting is repeated, and then the multi-foot molecular machine is driven to continuously walk on the surface of the gold electrode by the report probe track.

The combination and the cutting of the single chain extended from each end of the three-fork structure and the surface of the electrode each time promote the cutting and the falling of the report group from the probe, so that the electric signal is weakened, and the amplification and the output of the signal are realized.

When the target microRNA does not exist, a programmed reaction cannot be triggered, the detection probes (

probes

1, 2 and 3) exist independently, a short single chain extending from the tail end of the detection probe cannot form a double chain due to weak binding force with the electrode surface probe, the report probe cannot be cut by restriction enzyme, and the electrochemical signal is unchanged.

Example 1

FIG. 1 is a schematic diagram of the electrochemical sensing detection principle based on the trident-type walking DNA molecular machine in example 1 of the present invention.

(1) The sequence of the biological probe used in the electrochemical detection process of microRNA in the embodiment is shown in Table 1:

table 1: biological probe sequence used in electrochemical detection process of microRNA

(2) Preparing a gold electrode interface by using a biological probe:

the gold electrode is first scanned in 0.5M dilute sulfuric acid for 12 weeks, then polished on the deer skin with 0.3 μ M and 0.05 μ M aluminum powder for 10min, the surface of the gold electrode is cleaned with clear water, and then with piranha solution (H) ₂ SO ₄ :H ₂ O ₂ Treatment 3 times, 5min each time, then washing the electrode surface with clean water, drying the gold-loaded electrode surface with nitrogen gas, dropping a reporter probe (1 μ M,5 μ L) onto the electrode surface, and in a 4-degree refrigerator for 10h to prepare a detection electrode.

(3) And (3) electrophoretic characterization:

0.5 g of agar sugar powder is weighed and then added into 25mL of 0.5 XTBE buffer solution to prepare 2% agarose gel, 6 mu L of reaction system solution is respectively added into gel holes, electrophoresis is carried out for 30min under the constant voltage of 110V, and results are photographed and stored under a gel imaging system.

(4) Electrochemical detection:

the concentration of each of

probes

1, 2 and 3 (

Probe

1, 2 and 3) was 200nM, the concentration of restriction endonuclease (Nb. BbvCI) was 0.05U/. Mu.L, and the concentration of target microRNA was 1X 10 ^-16 、1×10 ^-15 、1×10 ^-14 、1×10 ^-13 、1×10 ^-12 、1×10 ^-11 、1×10 ^-10 、1×10 ^-9 、1×10 ^-8 Adding one liter of mols (mol/L) solution to the surface of a gold electrode for incubation for 1h, respectively washing the surface of the electrode by using PBS solution and PBST solution after reaction, then adding streptavidin-labeled alkaline phosphatase (1.00 mu g/mL) for incubation for 35 min, and respectively washing the surface of the electrode by using PBS solution and PBST solutionThen, the gold electrode was placed in a substrate solution containing alpha-naphthol (1 mg/mL) for 90 seconds, and electrochemical signal detection was carried out at a scanning voltage of 0 to 0.45V.

Example 2

(1) Feasibility verification results of nucleic acid detection:

agarose gel (3%) electrophoresis verified the polypod DNA nanostructure construction, and the results are shown in fig. 2: lane 1 is 500bp DNA marker, lanes 2, 3, 4, and 5 are target microRNA and detection probes (probe 1, 2, and 3), respectively; lane 6 is assembly strand microRNA + probe1; lane 7 is assembly strand microRNA + probe1+ probe2; lane 8 is assembly strand microRNA + probe1+ probe2+ probe3; lane 9 is probe1+ probe2+ probe, the maker lane is 500bp DNA ladder (Takara), lanes 1 to 4 are assembly strands, as an electrophoresis control, and lanes 5 to 7 are DNA assembly process, and compared to the control band, the band is gradually set back because the assembly strands are gradually assembled into a large molecular weight DNA hybrid, thereby causing a slow electrophoretic moving rate, which suggests that: the construction of the multi-foot DNA nano structure is feasible, the responsiveness result of the electrochemical sensing detection system is shown in figure 3, and when no target microRNA exists in the detection system, the signal value is about 15 muA; when the detection system is added with the target microRNA, the current value is remarkably reduced by about 2.1 muA, and the result indicates that: the construction of nucleic acid detection bodies based on nb.

(2) And (3) detecting a characterization result of an electrode preparation process:

the results of the electrode surface preparation process by impedance characterization are shown in fig. 4 (a-d): the preparation steps include that 1, a bare gold electrode is prepared, 2, a report Probe Probe4 is fixed on the surface of the gold electrode, 3, a mercaptoethanol sealed gold electrode surface is prepared, and 4, a fetal bovine serum sealed gold electrode surface is prepared, wherein resistance is gradually increased in the preparation process of the electrode surface (from a to d), and because nucleic acid and sealing molecules on the surface of the electrode gradually block electron transfer on the surface of the gold electrode, impedance is increased, and the preparation of the electrode surface detection Probe is successful. After the electrode surface detection probe is successfully prepared, the molecular reaction process on the electrode surface is further characterized by impedance as shown in fig. 4 (e-f): adding streptavidin-labeled alkaline phosphatase (e), adding Probe1, 2, 3, nb.BbvCI and target microRNA (f) again, wherein the impedance value e of the added streptavidin-labeled alkaline phosphatase is increased compared with the impedance value d in the step 4, because the streptavidin-labeled alkaline phosphatase connected to the surface of the electrode gradually obstructs electron transfer on the surface of the gold electrode, so that the impedance is increased, and when the reaction system (

Probe

1, 2, 3, nb.BbvCI and target microRNA) is added for acting for 60min, the impedance value f is greatly reduced, because the resistance is reduced due to the fact that the reporter Probe Probe4 is sheared off the surface of the electrode, and the result shows that the electrochemical sensing detection system works according to the experimental design.

(3) Sensitivity and linear range results:

as shown in FIG. 5, the results were each at a concentration of 1X 10 ^-16 、1×10 ^-15 、1×10 ^-14 、1×10 ^-13 、1×10 ^-12 、1×10 ^-11 、1×10 ^-10 、1×10 ^-9 、1×10 ^-8 The electrochemical signal spectrum under mol per liter (mol/L) is shown in figure 6, the result is the correlation analysis of the target microRNA concentration and the detection signal, and the correlation analysis is at 1X 10 ^-16 mol/L to 1X 10 ^-8 The logarithm of the concentration value between mol/L is linearly related to the current signal value, and the equation is Y = -8.19-1.3log10C (R) ² = 0.999), according to the principle of the Mean of signal values of 6 blank experiments +3 times standard deviation (Mean) _Blank +3 δ), the lowest detection limit was calculated to be 0.29fM.

(4) The specific result of the novel electrochemical sensing detection technology is as follows:

fig. 7-8 are specific analyses of the new electrochemical sensing detection technology, and the specific analyses (b) are performed through single base mismatching (e), double base pairing (d), triple base mismatching (c) and incomplete mismatching respectively, as shown in fig. 7, current curves b, c, d and e of different target sequences have no significant change in electrochemical signals compared with blank (a), when a target sequence exists, the electrochemical signals are significantly reduced (f) compared with blank (a), when single base mismatching exists, the signals are slightly reduced (e), fig. 8 is a current value histogram corresponding to the current curves of fig. 7, and fig. 8 shows that the newly established new electrochemical sensing detection technology has single base mismatching distinguishing capability and high specificity.

(5) Recovery rate experimental results:

FIG. 9 shows the recovery rate analysis of the new electrochemical sensing detection technology, in which artificially synthesized target microRNA is added to serum of a healthy person to prepare a simulated actual sample for subsequent detection. Specifically, a simulated actual sample containing 10pM of target microRNA and a standard solution containing 10pM of target microRNA are respectively added into a new detection system, a current signal is detected after reaction, the current signal (a) is a signal of the simulated actual sample containing microRNA, the current signal (b) is a signal of the standard solution containing the target microRNA, the corresponding concentration of the detected current signal (b) is about 9.85pM, the recovery rate is 98.50 percent, and the recovery rate shows that the constructed new electrochemical sensing detection technology still has good analysis performance in a complex matrix solution, the performance is not influenced by a complex mechanism, so the new detection system has a good prospect of being applied to the detection and analysis of the concentration of the microRNA in an actual sample.

It should be noted that, in this document, relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrases "comprising one of 8230; \8230;" 8230; "does not exclude the presence of additional like elements in a process, method, article, or apparatus that comprises the element.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

1. A hypersensitive microRNA electrochemical detection method of an endonuclease driven polypod DNA molecular machine is characterized by comprising the following steps:

aiming at target microRNA, detecting probes 1, 2 and 3 (probes 1, 2 and 3) are designed and synthesized in a programming way to be used as recognition elements, and a thiol-modified probe4 (probe 4) is designed and synthesized to be used as a report element;

through the recognition of a target sequence and a detection probe and the occurrence of cascade programming reaction, a three-fork DNA nano structure is formed, each section of the structure is designed with a short piece of nucleic acid restriction endonuclease site as a foot of a molecular machine, and further a multi-foot molecular machine driven by the nucleic acid restriction endonuclease can be formed;

wherein the sequence of Probe1 (Probe 1) is CCTCAGCTTCAACATCAGTCTGATAAGCTAGATGTTG TAGCTTATCAGACTG, the sequence of Probe2 (Probe 2) is CCTCAGCTCAGTCTGATAAGCTACAACA TCTCAGACTGATGTTGTAGCTTAT, and the sequence of Probe3 (Probe 3) is CCTCAGCTATAAGCTACAAA CATCAGTCTGATAGCTTATCAGGAACTGTTGTTGT;

the binding and cutting of the single chain extending from each end of the trident structure with the surface of the electrode each time promotes the cutting and falling of the reporter group from the probe, so that the electric signal is weakened, the amplification output of the signal is realized, and the target microRNA is detected;

when the target microRNA does not exist, the programmed reaction cannot be triggered, the detection probes (probes 1, 2 and 3) exist independently, a short single chain extending from the tail end of the detection probe cannot form a double chain due to weak binding force with the electrode surface probe, the report probe cannot be cut by the restriction endonuclease, and the electrochemical signal is unchanged.

2. The method according to claim 1, wherein when the target microRNA exists, the target microRNA triggers three detection probes (probe 1, 2 and 3) to perform a programmed self-assembly reaction, and the three detection probes are assembled to form a ternary DNA nanostructure.

3. The method of claim 2, wherein a short single-stranded nucleic acid fragment extends from each 5' end of the trifurcated structure, thereby forming a polypod DNA molecular machine, each foot of the polypod DNA molecular machine recognizing and hybridizing with a reporter probe (probe 4) immobilized on the surface of the gold electrode to form a DNA double strand, thereby being captured on the surface of the electrode.

4. The method of claim 3, wherein the double strand is designed with a restriction enzyme site, the restriction enzyme recognizes and cleaves the double strand to facilitate cleavage of the reporter from the probe, and after cleavage, the restriction enzyme site is sufficient to recognize and hybridize with the next reporter probe for the next round of the cleavage reaction.

5. The method of claim 4, wherein the hybridization and cleavage process is identified and repeated, and the polypod molecular machine is driven to continuously walk on the report probe track on the surface of the gold electrode.