CN110988345A - Method for detecting thrombin by using nanopore - Google Patents
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Abstract
The invention discloses a method for detecting thrombin by using a nanopore, belonging to the technical field of biochemical analysis. The method is based on a double-stranded DNA probe. The probe consists of an aptamer, cDNA-16 and a capture arm. The double-stranded DNA probe is fixed on the surface of a streptavidin magnetic bead through the action of biotin-streptavidin, and a formed double-stranded DNA-magnetic bead compound is used as the probe. The aptamer is attached to a magnetic bead, and in the presence of thrombin, the aptamer binds specifically to the thrombin, which is then magnetically separated, releasing the hybridization product (output DNA) of the capture arm to the cDNA. The released DNA is subjected to nanopore detection, which generates a characteristic blocking current signal. The higher the concentration of thrombin, the more DNA is released, the more current blocking signals are present in the nanopore, and the frequency of the signals is proportional to the concentration of thrombin. Therefore, quantitative detection of proteins can be achieved by analyzing the signal frequency.
Description
Technical Field
The invention relates to a method for detecting thrombin by using a nanopore, belonging to the technical field of biochemical analysis.
Background
Cancer is one of the major diseases that seriously threaten human health and life. Statistical data show that the number of new cancer cases in China in 2015 is 429.2 ten thousand, which is equivalent to 1.2 ten thousand new cases per day on average; there are 281.4 ten thousand cancer deaths, equivalent to an average of 7700 deaths per day from cancer. Studies have suggested that the higher mortality rate of cancer is mainly associated with low sensitivity and low diagnosis rate for early detection of cancer. It has been reported that, according to the current medical level, 5-year survival rates after treatment of early cancer patients can exceed 80%, while 5-year survival rates after treatment of late cancer patients are still less than 10%. Therefore, early detection of cancer is of great importance for the treatment of cancer. At present, the clinical diagnosis method of cancer is still mainly based on imaging examination, and has low resolution, thus being difficult to meet the requirement of early diagnosis of cancer. The tumor marker is used as an important index for diagnosing tumors, can realize early screening of cancers by detecting active substances in malignant tumor cells, and plays a very important role in prognosis. Therefore, a new tumor marker detection method with good specificity and high sensitivity is established, and the method has important scientific significance and clinical value for early diagnosis and treatment of cancer. Thrombin is a multifunctional serine protease, can recognize various macromolecular substrates, and has procoagulant and anticoagulant functions. Early clinical studies found that the coagulation system is activated in most patients with malignant tumors and that thrombin promotes the growth, invasion and spontaneous metastasis of tumor cells and also stimulates angiogenesis of tumors. Thrombin, an important participant in many physiopathological processes, is a marker for the diagnosis of some diseases such as cancer and alzheimer's disease. The detection of thrombin is therefore of medical importance.
In recent years, nanopore sensing technology has been widely used in many research fields such as chemistry and biology due to its advantages of rapidness, low cost, no need of fluorescent labeling, and the like, and has developed into a novel and unique single molecule analysis means. The working principle is that under the drive of an external electric field force, a single molecule to be detected is limited in a nano-sized channel, and due to the physical occupation effect and the like of the single molecule in a nanopore, the resistance of the channel is changed, so that the ion current flowing through the nanopore is changed, and a blocking current signal is formed. The blocking degree and blocking time of each signal can reflect the information of the composition, structural characteristics and the like of the molecules, and the signal frequency can reflect the concentration of the molecules. The nanopore sensing technology is applied to sensitive detection of biomarkers, and has important scientific significance and clinical value for diagnosis and screening of diseases and guidance and treatment.
How to design an effective strategy to realize simple, efficient, rapid and sensitive detection of thrombin is a technical problem to be solved urgently in the field.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for detecting thrombin (thrombin) by using a nanopore.
The invention adopts the following technical scheme:
a method for detecting thrombin by using a nanopore, comprising the steps of:
(1) preparation of double-stranded DNA substrate Probe: respectively taking a proper amount of capture arm Throm-DNA, cDNA-16 and aptamer Throm-apt to be mixed in a buffer solution, reacting for 5 minutes at 95 ℃ after uniform mixing, fully hybridizing, slowly cooling to room temperature to obtain a double-chain reaction solution, mixing the double-chain reaction solution with streptavidin magnetic beads, putting the mixture into a constant temperature oscillator for incubation for 15 minutes at 37 ℃, carrying out magnetic separation after the reaction is finished, and removing supernatant to obtain a double-chain DNA substrate probe combined with the magnetic beads;
(2) mixing thrombin and a substrate probe for incubation reaction, namely adding 100 mu L of thrombin with different concentrations into the double-stranded DNA substrate probe respectively, placing the mixture in a constant temperature oscillator for incubation for half an hour at 37 ℃, specifically identifying and combining the thrombin and an aptamer connected to a magnetic bead, obtaining supernatant through magnetic separation, and taking 100 mu L of the supernatant to carry out α -hemolysin nanopore detection;
(3) α -hemolysin nanopore assembly, which is to uniformly coat phospholipid solution on the inner side and the outer side of a small hole of a 1mL trans detection cell by using a 000 # mink brush, respectively adding 1mL of electrolyte solution after assembling a cis detection cell and the trans detection cell, immersing a pair of Ag/AgCl electrodes into the electrolyte solution, applying +100mV voltage to two ends of the phospholipid bilayer membrane by using a current amplifier probe, and forming the phospholipid bilayer membrane at the small hole of the trans cell by using a pulling method, wherein α -hemolysin is added into the cis detection cell, when α -hemolysin forms a stable nano channel by self-assembly on the phospholipid bilayer membrane, ion flow quantitively steps, and the stable opening current of a single nanopore is 160 +/-10 pA under the condition of +160mV voltage;
(4) the α -hemolysin nanopore is used for detecting and outputting DNA, the supernatant obtained in the step (2) is injected into a cis detection cell, molecules to be detected pass through the α -hemolysin nanopore one by one under the drive of an external electric field to generate blocking signals, the current generated in the experiment is amplified and collected through Axomatch 200B, is converted into digital quantity through a DigitalData1440A digital-to-analog converter and is transmitted to a computer, the nanometer channel single molecule experiment data is observed and recorded in real time through PClamp 10.6 software, and data analysis is carried out by using originLab9.0 to realize the quantitative analysis and detection of thrombin.
In the step (1)
The sequence of cDNA-16 is AACCACACAACCTACC; the sequence of the Throm-DNA is C30GGTAGGTTGTGTGGTTAT(CCC) AGTCACCCCAAC, respectively; the Throm-apt sequence is Biotin-T10AGTCCGTGGTAGGGCAGGTTGGGGTGACT, respectively; wherein C is30Means 30C, T10Refers to 10T, the underline CCC indicates the branch attached to the single stranded DNA.
The concentrations of the Throm-DNA of the capture arm, the cDNA-16 and the aptamer Throm-apt in the step (1) are all 10-6M, the dosage is 20 mu L; the buffer was used in an amount of 20. mu.L.
Preferably, the buffer composition is 150mM NaCl,20mM Tris-HCl, pH 7.9.
The specific method for mixing the double-chain reaction solution and the streptavidin magnetic beads in the step (1) comprises the following steps: first, 50. mu.L of magnetic beads were washed three times with 1mL of 1 XBW buffer, then 25. mu.L of the double-stranded reaction solution, 25. mu.L of ultrapure water and the washed magnetic beads were mixed in 50. mu.L of 2 XBW buffer, vortexed for 15 minutes and then magnetically separated, the supernatant was poured out, and the magnetic beads were retained.
The dosage of the double-stranded DNA substrate probe in the step (2) is 100 mu L,the concentration is 2X 10-7M。
And (3) the phospholipid solution in the step (3) is a phospholipid n-decane solution of 30 mg/mL.
The electrolyte solution cis pool in the step (3) is 1.0M KCl,10mM Tris-HCl,1mM EDTA, pH7.8; the trans pool was 1.0M KCl,10mM Tris-HCl,1mM EDTA, pH 7.8.
The addition amount of α -hemolysin in the step (3) is 1 mu L and 5 mu g/mL.
The principle of the detection method of the present invention is that, as shown in FIG. 1, the method is based on a double-stranded DNA probe composed of an aptamer (Throm-apt), cDNA-16 and a capture arm (Throm-DNA), the double-stranded DNA probe is immobilized on the surface of streptavidin magnetic beads by the biotin-streptavidin effect, and the formed double-stranded DNA-magnetic bead complex serves as a probe, the aptamer is attached to the magnetic beads, the aptamer binds specifically to thrombin in the presence of thrombin, and then is magnetically separated, releasing the hybridization product (export DNA) of the capture arm and cDNA, and the released DNA undergoes nanopore detection, generating a characteristic blocking current signal.
The streptavidin magnetic beads of the present invention are dynabeads myone purchased from Invitrogen (California, u.s.a.)TMStreptavidin T1(10mg/mL, 1.0 μm diameter).
The invention has the beneficial effects that:
(1) the specificity is good: because the method is based on the specific recognition and combination of thrombin and aptamer, the specificity of the reaction is extremely high; moreover, the inventor also carries out comprehensive and fine optimization on specific reaction conditions, so that nonspecific reaction hardly occurs; the resulting products produce highly characteristic blocking signals in the nanopore, which also greatly enhances the specificity of the method.
(2) The sensitivity is high: the nanopore is a very sensitive single-molecule analysis means, and higher voltage of 160mV is used in experimental operation, so that the detection sensitivity is greatly improved, and the lower detection limit of the method can reach 3.11 multiplied by 10-12M。
(3) No labeling and cyclic amplification required: the scheme does not carry out any marking or signal amplification, does not introduce any cyclic amplification, and can realize the sensitive detection of the thrombin only by utilizing the advantage of high sensitivity of the nanopore. This is not possible with other detection methods such as fluorescence, colorimetry.
(4) The application range is wide: high specificity and high sensitivity detection of other proteins, nucleic acids or small molecules can be realized only by changing the aptamer sequence in the substrate probe.
Drawings
FIG. 1 is a schematic diagram of the detection of thrombin using a nanopore.
FIG. 2 is a graph of data for detection of DNA hybridization product H16-fork. Wherein A is a schematic diagram of a current signal, and B is a scatter diagram of statistical analysis of a large number of blocking current events.
FIG. 3 is a graph of TB data from thrombin at various concentrations. Graph a is a plot of the frequency of a characteristic current event caused by detection of TB. Graph B is the correlation of event frequency with TB concentration.
FIG. 4 shows the results of the target protein recovery test in normal human serum. Graph A is a current signal diagram and graph B is a graph of different concentrations (10)-10-10-8M) TB was added to the recovery data after human serum testing.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
Example 1
A method for detecting thrombin by using a nanopore, comprising the steps of:
(1) preparation of double-stranded DNA Probe: the double-stranded DNA probe is prepared by mixing capture arm DNA, cDNA and aptamer (the concentration is 10)-6M) of the three, 20 mu L of the three are mixed in 20 mu L of buffer solution (150mM NaCl,20mM Tris-HCl, pH 7.9), incubated at the temperature of 95 ℃ for 5 minutes and then gradually cooled to room temperature, and finally, a dsDNA-based hybridization double-strand reaction solution is obtained; immobilizing double-stranded DNA probes on the surface of streptavidin Magnetic Beads (MB): magnetic beads (50. mu.L, 10mg/mL) were washed three times with 1mL of 1 XBW buffer (1M NaCl,1mM EDTA,10mM Tris-HCl, pH 7.5), then 25. mu.L of the double-stranded DNA reaction solution, 25. mu.L of ultrapure water and the magnetic beads were mixed in 50. mu.L of 2 XBW buffer, vortexed for 15 minutes, and after the reaction was completed, the supernatant was removed by magnetic separation to obtain double-stranded DNA substrate probes bound to the magnetic beads.
(2) The aptamer binds specifically to thrombin and releases the hybrid (H16-fork). Taking 100 μ L of double-stranded DNA substrate probe, the concentration is 2X 10-7M, then thrombin TB (100. mu.L, 10) was added-8M), incubating for 40 minutes at 37 ℃ in a constant temperature oscillator, occasionally vortexing, and collecting supernatant after magnetic separation to obtain 100 mu L of hybrid DNA for nanopore detection. . Because thrombin can specifically recognize aptamers in the double-stranded DNA substrate, the double-stranded DNA-magnetic bead complex forms two parts after incubation with thrombin, wherein one part is a free Throm-DNA/DNA16 hybrid chain, and the other part is a magnetic bead-single-stranded DNA complex. The free Throm-DNA/DNA16 hybrid strand contained in the supernatant collected after magnetic separation was analyzed further.
(3) α -assembling hemolysin nanopore, evacuating phospholipid chloroform solution, pumping, adding n-decane to obtain 30mgmL solution-1And (3) solution. Uniformly smearing 30mg mL of writing brush with 000 # mink on the inner side and the outer side of a small hole of a 1mL trans detection pool-1Phospholipid n-decane solution. After assembling the cis and trans detection cells, 1mL of electrolyte solution ((cis:1.0M KCl,10mM Tris-HCl,1mM EDTA, pH 7.8; trans:1.0M KCl,10mM Tris-HCl,1mM EDTA, pH 7.8) was added, a pair of Ag/AgCl electrodes were immersed in the electrolyte solution, a voltage of +100mV was applied across the phospholipid bilayer membrane using a current amplifier probeThe pulled method forms a phospholipid bilayer membrane at the small pore of the trans pool. 1 uL of 5. mu.gmL was added to the cis detection cell-1α -hemolysin, when α -hemolysin self-assembles to form a stable nano-channel on the phospholipid bilayer membrane, the ion current is subjected to quantization step change, and under the voltage condition of +160mV, the stable opening current of a single nanopore is 160 +/-10 pA.
(4) Injecting the supernatant obtained in the step (2) into a cis detection cell, driving the cis detection cell by an external electric field, enabling molecules to be detected to pass through α -hemolysin nanopores one by one to generate a blocking signal, amplifying and collecting current generated in an experiment through Axoatch 200B (Axon Instruments, USA), converting the current into digital quantity through a DigitalData1440A digital-to-analog converter (Axon Instruments, USA), transmitting the digital quantity to a computer, observing and recording single-molecule experimental data of a nano channel in real time through PClamp 10.6 software (Axon Instruments, USA), and analyzing the data by using OriginLab 9.0.
Example 2
When thrombin is detected by the method of the invention, the aptamer binds specifically to thrombin and releases the Throm-DNA/DNA16 hybrid (H16-fork), and nanopore analysis is performed on the DNA double stranded substrate Throm-DNA/DNA16 in 160mV, 1M KCl, as shown in FIG. 2. (A) The current signal diagram shows that the hybridization product H16-fork almost completely eliminates pore current. (B) The green dots represent a scatter plot of the signal produced by Throm-DNA/DNA16 through the pore with a blocking time centered between 0.7ms and 30ms and a blocking degree centered between 0.97 and 1 (where I is the pore current that is plugged, I is0Pore current to open).
Example 3
The invention also relates to a target protein thrombin TB (0-10)-7M) is mixed with the probe, the correlation of the characteristic current event frequency of the target protein and the concentration is analyzed, and the characteristic current event frequency caused by TB is separately detected, and the specific result is shown in figure 3. The characteristic current frequency of the target protein gradually increases with increasing target concentration. Wherein the concentration of thrombin is in the range of 5X 10-12M to 10-7M is greater than or equal to the total weight of the composition. These data are at a voltage of +160mV on 1M KClAnd (4) obtaining. Error bars represent standard deviations of three experiments.
Example 4
To further explore the applicability of this method to the analysis of real biological samples, different concentrations (10) were used-10-10-8M) was added to human serum, and then different concentrations of protein were detected by standard methods and the recovery was calculated. The results are shown in FIG. 4. There was a low level of noise-like spike in the current signal of the original serum sample, but the addition of protein resulted in a large number of characteristic current signals that were readily distinguishable (FIG. 4A). The recovery of thrombin from the spiked standard in FIG. 4B was between 90% and 110%. Error bars were collected at 1M KCl +160mV potential for all data representing the standard deviation of three experiments.
Sequence listing
<110> Linyi university
<120> a method for detecting thrombin using nanopore
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<170>SIPOSequenceListing 1.0
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<213> Artificial Sequence (Artificial Sequence)
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aaccacacaa cctacc 16
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tttttttttt agtccgtggt agggcaggtt ggggtgact 39
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<213> Artificial Sequence (Artificial Sequence)
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cccccccccc cccccccccc cccccccccc ggtaggttgt gtggttatcc cagtcacccc 60
aac 63
Claims (9)
1. A method for detecting thrombin by using a nanopore, which is characterized by comprising the following steps:
(1) preparation of double-stranded DNA substrate Probe: respectively taking a proper amount of capture arm Throm-DNA, cDNA-16 and aptamer Throm-apt to be mixed in a buffer solution, reacting for 5 minutes at 95 ℃ after uniform mixing, fully hybridizing, slowly cooling to room temperature to obtain a double-chain reaction solution, mixing the double-chain reaction solution with streptavidin magnetic beads, putting the mixture into a constant temperature oscillator for incubation for 15 minutes at 37 ℃, carrying out magnetic separation after the reaction is finished, and removing supernatant to obtain a double-chain DNA substrate probe combined with the magnetic beads;
(2) mixing thrombin and a substrate probe for incubation reaction, namely adding 100 mu L of thrombin with different concentrations into the double-stranded DNA substrate probe respectively, placing the mixture in a constant temperature oscillator for incubation for half an hour at 37 ℃, specifically identifying and combining the thrombin and an aptamer connected to a magnetic bead, obtaining supernatant through magnetic separation, and taking 100 mu L of the supernatant to carry out α -hemolysin nanopore detection;
(3) α -hemolysin nanopore assembly, which is to uniformly coat phospholipid solution on the inner side and the outer side of a small hole of a 1mL trans detection cell by using a 000 # mink brush, respectively adding 1mL of electrolyte solution after assembling a cis detection cell and the trans detection cell, immersing a pair of Ag/AgCl electrodes into the electrolyte solution, applying +100mV voltage to two ends of the phospholipid bilayer membrane by using a current amplifier probe, and forming the phospholipid bilayer membrane at the small hole of the trans cell by using a pulling method, wherein α -hemolysin is added into the cis detection cell, when α -hemolysin forms a stable nano channel by self-assembly on the phospholipid bilayer membrane, ion flow quantitively steps, and the stable opening current of a single nanopore is 160 +/-10 pA under the condition of +160mV voltage;
(4) the α -hemolysin nanopore is used for detecting output DNA, the supernatant obtained in the step (2) is injected into a cis detection cell, molecules to be detected pass through the α -hemolysin nanopore one by one under the drive of an external electric field to generate blocking signals, the current generated in the experiment is amplified and collected through Axomatch 200B, is converted into digital quantity through a DigitalData1440A digital-to-analog converter and is transmitted to a computer, the nanometer channel single molecule experiment data is observed and recorded in real time through PClamp 10.6 software, and data analysis is carried out by using originLab9.0 to realize the quantitative analysis and detection of thrombin.
2. The method for detecting thrombin according to claim 1, wherein the sequence of cDNA-16 in step (1) is AACCACACAACCTACC; the sequence of the Throm-DNA is C30GGTAGGTTGTGTGGTTAT(CCC) AGTCACCCCAAC, respectively; the Throm-apt sequence is Biotin-T10AGTCCGTGGTAGGGCAGGTTGGGGTGACT, respectively; wherein C is30Means 30C, T10The underlined CCC in the 10T, Throm-DNA sequence indicates the branching attached to the single-stranded DNA.
3. The method for detecting thrombin by using nanopore according to claim 1, wherein the concentrations of the Throm-DNA, cDNA-16 and aptamer Throm-apt in step (1) are all 10-6M, the dosage is 20 mu L; the buffer was used in an amount of 20. mu.L.
4. The method for detecting thrombin according to claim 3, wherein the buffer composition is 150mM NaCl,20mM Tris-HCl, pH 7.9.
5. The method for detecting thrombin by using a nanopore according to claim 1, wherein the specific method for mixing the double-stranded reaction solution and the streptavidin magnetic bead in the step (1) is as follows: first, 50. mu.L of magnetic beads were washed three times with 1mL of 1 XBW buffer, then 25. mu.L of the double-stranded reaction solution, 25. mu.L of ultrapure water and the washed magnetic beads were mixed in 50. mu.L of 2 XBW buffer, vortexed for 15 minutes and then magnetically separated, the supernatant was poured out, and the magnetic beads were retained.
6. The method for detecting thrombin according to claim 1, wherein the amount of the double-stranded DNA substrate probe used in step (2) is 100. mu.L and the concentration is 2X 10-7M。
7. The method for detecting thrombin according to claim 1, wherein the phospholipid solution in the step (3) is a 30mg/mL phospholipid n-decanone solution.
8. The method for detecting thrombin according to claim 1, wherein the electrolyte solution cis pool in the step (3) is 1.0M KCl,10mM Tris-HCl,1mM EDTA, pH 7.8; the trans pool was 1.0MKCl,10mM Tris-HCl,1mM EDTA, pH 7.8.
9. The method for detecting thrombin according to claim 1, wherein the α -hemolysin is added in an amount of 1 μ L and 5 μ g/mL in said step (3).
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