CN110734957B - General method for constructing structure switch type aptamer, general sensor applying same and construction method thereof - Google Patents
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Abstract
The invention relates to a general method for constructing a structure switch type aptamer and a general sensor for detecting various types of targets by applying the general method. A probe (Apt-G4) having a structure switching property is constructed by linking sequences rich in guanine nucleotide (G) to both ends of an aptamer, respectively. Apt-G4 forms a G-tetramer structure (G4) when no target capable of specifically binding to the aptamer is present; when the target is present, the target binds to the aptamer and G4 is destroyed. Apt-G4 design has general applicability to aptamers to different types of targets. The use of G4 can obviously enhance the catalysis of H by chlorhematin 2 O 2 Reacting with colorless 2' -hydrazine-bis-3-ethylbenzthiazoline-6-sulfonic Acid (ABTS) to generate green ABTS +· The colorimetric detection of signal reduction of mercury ions, thrombin and sulfadimethoxine is realized respectively.
Description
Technical Field
The invention relates to a general method for constructing a structural switch type aptamer, a general sensor for detecting various types of targets (metal ions, proteins and organic small molecules) by applying the general method and a construction method thereof. Belongs to the field of biotechnology.
Background
Aptamers are obtained from in vitro synthesized oligonucleotide libraries through multiple rounds of affinity Enrichment and polymerase chain reaction steps by in vitro screening techniques, i.e., exponential Enrichment of Ligands by expression Evolution, SELEX (Nature, 1990,346 (6287), 818-822, science,1990,249 (4968), 505-510), biorecognition molecules with high specificity and affinity for screening targets. Aptamers can recognize many types of targets, including metal ions, proteins, small organic molecules, and the like. Aptamers find wide application in a variety of fields, particularly in the field of biosensors.
Structurally Switched Aptamers (SSA) are a class of aptamers that undergo significant conformational changes upon binding to a target. Because the conformational change induced by the target can be conveniently converted into detectable signals such as electrochemistry, optics and the like, great interest is brought to the field of biosensing. In recent years, SSA-based sensors are diversified, and have the remarkable advantages of simple structure, simplicity in operation, high sensitivity and good specificity. However, the current screening methods for SSA are very few and inefficient (proc. Natl.acad.sci.u.s.a.2010,107, 14053-14058.), require 15-20 screening cycles, and the obtained aptamers have poor selectivity and low affinity. The number of SSA reported to date is very limited, and most aptamers have no or only minor conformational changes, and thus a target bound to the aptamer is not able to induce a change that produces a significant detection signal. Moreover, SSA of different targets often has different structural switch modes, for example, SSA can form different structures such as double strand, Y-type, G-tetramer, etc. when binding with a target, there is a lack of uniformity, and the design of a universal SSA sensor is also limited.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a general method for constructing a structure switch type aptamer and a general sensor for detecting various types of targets (metal ions, proteins and organic small molecules) by using the same.
The invention provides a general method for constructing a structure switch type aptamer, which comprises the following steps: design of aptamers with structural switching properties: the two ends of the aptamer are respectively connected with a sequence rich in guanine nucleotide G to construct a probe Apt-G4 with structure switch performance.
Further, in the above general method for constructing Sub>A structure switch type aptamer, the probe Apt-G4 having Sub>A structure switch property is Sub>A-SULF-Sub>A; A-TBA29-A; and A-T11-A.
In addition, the invention also provides a construction method of the universal sensor, which comprises the following steps: designing a nucleic acid aptamer with structural switch performance: connecting the two ends of the aptamer with sequences rich in guanine nucleotide G respectively to construct a probe Apt-G4 with structure switch performance; step (2), heating Apt-G4 at 95 ℃, and slowly cooling to room temperature; adding substances to be detected with different concentrations and Apt-G4 to incubate to form a mixed solution; adding hemin (hemin) to incubate with the mixed solution; and (5) simultaneously adding hydrogen peroxide and 2' -hydrazine-bis-3-ethylbenzthiazoline-6-sulfonic acid, measuring the ultraviolet absorption at 418nm of a selected time, and drawing a working curve by taking the ultraviolet absorption value as a vertical coordinate and the concentration of the substance to be measured as a horizontal coordinate.
In the method for constructing the general-purpose sensor, in the step (2), the temperature is maintained at 95 ℃ for 6 minutes, and then the temperature is gradually cooled to 25 ℃.
In addition, the method for constructing the general-purpose sensor further comprises the step (6) of a selectivity test: and (5) replacing the substances to be detected with other substances with equal concentrations respectively, and repeating the experiments in the steps (1) to (5).
Further, in the above construction method of the general sensor, the probe Apt-G4 having the structure switching property is prepared as a solution having a concentration of 0.375. Mu.M.
The invention also provides a general sensor for constructing the structure switch type aptamer, which comprises the aptamer with the structure switch performance, and the two ends of the aptamer are respectively connected with a sequence rich in guanine nucleotide G to construct a probe Apt-G4 with the structure switch performance. The probe Apt-G4 with the structural switch performance is A-SULF-A; A-TBA29-A; and A-T11-A. The probe Apt-G4 having the structure switching property was configured into a solution having a concentration of 0.375. Mu.M.
The specific experimental steps of the invention are as follows:
(1) Design of aptamers with structural switching properties: connecting sequences rich in guanine nucleotide (G) to two ends of the aptamer respectively to construct a probe (Apt-G4) with structure switch performance;
(2) Heating Apt-G4 at 95 ℃, and slowly cooling to room temperature;
(3) Adding substances to be detected with different concentrations and incubating with Apt-G4;
(4) Adding hemin (hemin) and incubating with the above mixture;
(5) With addition of hydrogen peroxide (H) 2 O 2 ) With 2' -hydrazine-bis-3-ethylbenzothiazoleMeasuring ultraviolet absorption at 418nm at a selected time by using quinoline-6-sulfonic Acid (ABTS), and drawing a working curve by using the ultraviolet absorption value as a vertical coordinate and the concentration of a substance to be measured as a horizontal coordinate;
(6) And (3) selective test: and replacing the substances to be detected with other substances with equal concentrations respectively, and repeating the experiment in 1-5 steps.
The method and the sensor thereof have the following advantages:
1) Any aptamer can be designed using the SSA design method of the present invention as an aptamer with significant structural switching properties, i.e., SSA;
2) The SSA design method has good universality, not only can convert non-SSA into SSA, but also can convert SSA with different structural switch performances into SSA with uniform conformational change, namely SSA with G-tetramer structural switch performance;
3) The SSA designed by the invention can be used for constructing a sensor with universality and can be used for detecting various different types of targets.
Drawings
FIG. 1 is a schematic diagram of the colorimetric detection of a target using SSA constructed by the methods of the present invention.
Fig. 2 is an ultraviolet-visible absorption spectrum (A) and a working curve (B) of sulfadoxine detected by the method of the invention.
FIG. 3 is a graph of the results of a selectivity test of a sulfadoxine Xin Bise sensor constructed in accordance with the method of the present invention.
FIG. 4 shows a UV-visible absorption spectrum (A) and a working curve (B) of thrombin detected according to the method of the present invention.
FIG. 5 is a graph showing the results of a selectivity test of a thrombin colorimetric sensor constructed according to the method of the present invention.
Fig. 6 is a uv-vis absorption spectrum (a) and a working curve (B) for detecting mercury ions according to the method of the present invention.
FIG. 7 is a graph of the results of a selectivity test for a mercury ion colorimetric sensor constructed according to the method of the present invention.
FIG. 8 is Sub>A circular dichroism spectrum of three SSAs constructed according to the methods of the present invention (A: A-SULF-A; B: A-TBA29-A; and C: A-T11-A) before and after addition of heme and target (A: sulfadoxine; B: thrombin; C: mercuric ions).
Detailed Description
Table 1: sequence information of the DNA used in the present invention.
| Name of probe | Sequence (5 '-3') |
| A-SULF-A | AGGGACGGGACTAGAGAGGGCAACGAGTGTTTATAGAAGGGACGGGA |
| A-TBA29-A | AGGGACGGGAAGTCCGTGGTAGGGCAGGTTGGGGTGACTAGGGACGGGA |
| A-T11-A | AGGGACGGGATTTTTTTTTTTAGGGACGGGA |
FIG. 1 is a schematic diagram of the colorimetric detection of a target using SSA constructed by the method of the present invention. The two ends of any aptamer are respectively connected with a sequence rich in guanine nucleotide (G) to construct a probe (Apt-G4) with structure switch performance; when no target exists, apt-G4 forms a G-tetramer structure which can enhance heme catalysis ABTS oxidation to form a colored product ABTS +· Forming; when the target exists, the aptamer segment in Apt-G4 is combined with the target, the G-tetramer structure is destroyed, and the formation of a colored product ABT by the oxidation of heme catalytic ABTS (ethylene-based phosphotriethylation transferase) cannot be enhancedS +· Is performed.
Fig. 2 is an ultraviolet-visible absorption spectrum (A) and a working curve (B) of sulfadoxine detected by the method of the invention. In FIG. 2, the left graph A is a graph of the ultraviolet-visible absorption spectrum, and the ordinate of the right graph B is the absorbance value at 418 nanometers (nm) of the test sample at the eighth minute. The SSA used was A-SULF-A (Table 1).
FIG. 3 is a graph of the results of a selectivity test of a sulfadoxine Xin Bise sensor constructed in accordance with the method of the present invention. The final concentrations of sulfadoxine, ampicillin, kanamycin A, kanamycin B, tetracycline were all 1 millimole per liter (mM). The values on the ordinate were calculated from the absorbance values at 418nm at the eighth minute for each sample and blank. The difference between the absorbance of the blank experiment and the absorbance of the sample containing sulfadoxine is one hundred percent.
FIG. 4 shows a UV-visible absorption spectrum (A) and a working curve (B) of thrombin detected according to the method of the present invention. In FIG. 4, the left side A is a graph of the UV-visible absorption spectrum, and the ordinate of the right side B is the absorbance value at 418nm of the test sample at the eighth minute. The SSA used was A-TBA29-A (Table 1).
FIG. 5 is a graph showing the results of a selectivity test of a thrombin colorimetric sensor constructed according to the method of the present invention. The final concentrations of thrombin and bovine serum albumin were 20 micromoles per liter (. Mu.M). The values on the ordinate were calculated from the absorbance values at 418nm at the eighth minute for each sample and blank. The difference between the absorbance of the blank and the absorbance of the thrombin-containing sample was one hundred percent.
Fig. 6 is a uv-vis absorption spectrum (a) and a working curve (B) for detecting mercury ions according to the method of the present invention. In FIG. 6, the left graph A is a graph of the UV-visible absorption spectrum, and the ordinate of the right graph B is the absorbance value of the test sample at 418nm at the eighth minute. The SSA used was A-T11-A (Table 1).
FIG. 7 is a graph of the results of a selectivity test for a mercury ion colorimetric sensor constructed according to the method of the present invention. Hg 2+ 、Pb 2 + 、Fe 2+ 、Fe 3+ 、Mg 2+ 、Ca 2+ 、Cd 2+ 、Co 2+ 、Zn 2+ 、Ni 2+ All concentrations of (2) were 2. Mu.M. The values on the ordinate were calculated from the absorbance value at 418nm at the eighth minute for each sample and blank. Absorbance and Hg content in blank experiment 2+ The difference in absorbance of the samples of (1) is one hundred percent.
FIG. 8 is Sub>A circular dichroism plot of three SSAs constructed according to the methods of the present invention (A: A-T11-A; B: A-SULF-A; and C: A-TBA 29-A) before and after addition of hemoglobin and target (A: mercuric ion; B: sulfadimethoxine; C: thrombin). In FIG. 8, from left to right, A-T11-A + heme + mercuric ions are shown; A-SULF-A + heme + sulfadimethoxine; A-TBA29-A + heme + thrombin.
The specific experimental steps of the invention are as follows:
(1) Design of aptamers with structural switching properties: respectively connecting sequences rich in guanine nucleotide (G) to two ends of the aptamer to construct a probe (Apt-G4) with structure switch performance;
(2) Heating Apt-G4 at 95 ℃, and slowly cooling to room temperature;
(3) Adding substances to be detected with different concentrations and Apt-G4 for incubation;
(4) Adding hemin (hemin) and incubating with the above mixture;
(5) With addition of hydrogen peroxide (H) 2 O 2 ) Measuring the ultraviolet absorption at 418nm of the selected time with 2' -hydrazine-bis-3-ethylbenzthiazoline-6-sulfonic Acid (ABTS), and drawing a working curve by taking the ultraviolet absorption value as a vertical coordinate and the concentration of the substance to be measured as a horizontal coordinate;
(6) And (3) selective test: and replacing the substances to be detected with other substances with equal concentrations respectively, and repeating the experiment in 1-5 steps.
Example 1. Working curve of sulfadoxine and selectivity test of the sensor were measured according to the method of the present invention.
A-SULF-A was prepared as Sub>A solution (20 mM Tris-HCl,50mM NaCl,5mM KCl,5mM MgCl) at Sub>A concentration of 0.375 micromol per liter (. Mu.M) 2 pH 8.0), heating at 95 deg.C for 6 min, and slowly cooling to room temperature (about 25 deg.C)). mu.L (μ L) of A-SULF-A solution was taken and 60 μ L of sulfadimethoxine Xin Rongye (20 mM Tris-HCl,50mM NaCl,5mM KCl,5mM MgCl) was added at various concentrations 2 pH 8.0) (0. Mu.M, 0.5. Mu.M, 1. Mu.M, 2. Mu.M, 5. Mu.M, 10. Mu.M, 20. Mu.M, 50. Mu.M, 100. Mu.M, 200. Mu.M, 500. Mu.M, 1000. Mu.M), and incubated for 30 minutes. Heme (10. Mu.L, 3. Mu.M) was then added and incubated for 30 minutes at room temperature in the absence of light. Finally, ABTS (20. Mu.L, 3.75 mM) and H were added simultaneously 2 O 2 (10. Mu.L, 15 mM). At the eighth minute of the reaction, the absorbance value at 418nm was measured by an ultraviolet spectrophotometer. And drawing a working curve.
50 μ L of the heat-treated A-SULF-A solution was added with sulfadoxine, kanamycin A, kanamycin B, ampicillin, and tetracycline (60 μ L,2.5 mM), respectively, and incubated for 30 minutes. Heme (10. Mu.L, 3. Mu.M) was then added and incubated at 37 ℃ for 30 minutes in the absence of light. Finally, ABTS (20. Mu.L, 3.75 mM) and H were added simultaneously 2 O 2 (10. Mu.L, 15 mM). At the eighth minute of the reaction, the absorbance at 418nm was measured using an ultraviolet spectrophotometer. And calculating the relative signal change of each target by taking the difference between the absorbance of the blank experiment and the absorbance of the sample containing sulfadoxine as one hundred percent.
The obtained working curve is shown in FIG. 2, and the detection limit is 0.2. Mu.M, and the detection range is 0.2. Mu.M to 1000. Mu.M. The sulfadoxine sensor has good selectivity (figure 3), and the selectivity for kanamycin A, kanamycin B, ampicillin and tetracycline is respectively 25.8 times, 8.1 times, 10.8 times and 26.3 times.
Example 2 working curves for the detection of thrombin and selectivity tests of the sensor according to the method of the invention.
A-TBA29-A was prepared as a 0.375. Mu.M solution [25mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 20mM KNO 3 ，200mM NaNO 3 0.025% (w/v%) Triton X-100,1% (v/v% (mass/volume%) dimethyl sulfoxide (DMSO), 150mM NH 4 Cl，pH 5.3]Heating at 95 deg.C for 6 min, and slowly cooling to room temperature. Mu.l (. Mu.L) of A-TBA29-A solution was taken and 60. Mu.L of thrombin solution (25mM HEPES,20mM KNO3, 200mM NaNO3,0.025% (w/v%) Triton X-100,1% (v/v%) DMSO was added at various concentrations，150mM NH 4+ pH 5.3) (0. Mu.M, 0.1. Mu.M, 0.2. Mu.M, 0.5. Mu.M, 1. Mu.M, 10. Mu.M, 50. Mu.M), and incubated for 30 minutes. Heme (10. Mu.L, 3. Mu.M) was then added and incubated for 30 minutes at room temperature in the absence of light. Finally, ABTS (20. Mu.L, 3.75 mM) and H were added simultaneously 2 O 2 (10. Mu.L, 15 mM). At the eighth minute of the reaction, the absorbance at 418nm was measured using an ultraviolet spectrophotometer. And drawing a working curve.
50. Mu.L of the heat-treated A-TBA29-A solution was added with thrombin and bovine serum albumin (60. Mu.L, 50. Mu.M), and incubated for 30 minutes. Heme (10. Mu.L, 3. Mu.M) was then added and incubated at 37 ℃ for 30 minutes in the absence of light. Finally, ABTS (20. Mu.L, 3.75 mM) and H were added simultaneously 2 O 2 (10. Mu.L, 15 mM). At the eighth minute of the reaction, the absorbance at 418nm was measured using an ultraviolet spectrophotometer. The relative signal change of each target was calculated using the difference between the absorbance of the blank and the absorbance of the thrombin-containing sample as one hundred percent.
The obtained working curve is shown in FIG. 4, with a detection limit of 0.1. Mu.M and a detection range of 0.1. Mu.M to 50. Mu.M. The thrombin sensor has very good selectivity (fig. 5), with 19.2 times selectivity to bovine serum albumin.
Example 3. Working curve for detection of mercury ions and selectivity test of the sensor according to the method of the invention.
A-T11-A was prepared as a 0.375. Mu.M solution (25mM HEPES,20mM KNO3, 200mM NaNO3,0.025% (w/v%) Triton X-100,1% (v/v%) DMSO,150mM NH 4 Cl, pH 5.3), heating at a constant temperature of 95 ℃ for 6 minutes, and then slowly cooling to room temperature. mu.L of A-T11-A solution was taken and 60. Mu.L of mercuric nitrate solution (25mM HEPES,20mM KNO3, 200mM NaNO3,0.025% (w/v%) Triton X-100,1% (v/v%) DMSO,150mM NH 4+ ，pH 5.3)(0nM、50nM、250nM、
500nM, 1000nM, 2000nM, 5000nM, 10000 nM), incubated for 30 min. Heme (10. Mu.L, 3. Mu.M) was then added and incubated for 30 minutes at room temperature in the absence of light. Finally, ABTS (20. Mu.L, 3.75 mM) and H were added simultaneously 2 O 2 (10. Mu.L, 15 mM). At the eighth minute of the reaction, the absorbance at 418nm was measured by an ultraviolet spectrophotometerAnd (4) measuring values. And drawing a working curve.
Hg was added to 50. Mu.L of the heat-treated A-T11-A solution 2+ 、Pb 2+ 、Fe 2+ 、Fe 3+ 、Mg 2+ 、Ca 2+ 、Cd 2 + 、Co 2+ 、Zn 2+ 、Ni 2+ (60. Mu.L, 5. Mu.M) and incubated for 30 minutes. Heme (10. Mu.L, 3. Mu.M) was then added and incubated at 37 ℃ for 30 minutes in the absence of light. Finally, ABTS (20. Mu.L, 3.75 mM) and H were added simultaneously 2 O 2 (10. Mu.L, 15 mM). At the eighth minute of the reaction, the absorbance value at 418nm was measured by an ultraviolet spectrophotometer. The relative signal change of each target was calculated using the difference between the absorbance of the blank and the absorbance of the thrombin-containing sample as one hundred percent.
The resulting working curve is shown in FIG. 6 with a detection limit of 50nM and a detection range of 50nM to 5. Mu.M. The mercury ion sensor has good selectivity (figure 7), and the selectivity to metal ions is 18.5-37.0 times.
Example 4. Conformational change upon binding of a SSA constructed according to the methods of the invention to a target.
To further confirm that the SSA constructed in accordance with the present invention ((A-SULF-A, AT11A, A-TBA29-A, table 1) has structural switching properties, the conformation of the SSA before and after target addition was determined by binary Chromatography (CD). It was found experimentally that Triton X-100 affected the CD profiling, and thus the binding buffer used removed this component (25mM HEPES,20mM KNO) when studying SSA for thrombin and mercury ions 3 ，200mM NaNO 3 ，1％(v/v％)DMSO，150mM NH 4 Cl, pH = 5.3). The experimental procedure was as follows: heat-treating 2 μ M SSA at 95 deg.C for 6 min, and slowly cooling to room temperature; hemoglobin (1 μ L0.4 mM) was added to SSA (100 μ L,2 μ M) and incubated for 90 minutes; add 100. Mu.L of the target molecule corresponding to SSA (100. Mu.M sulfadimethoxine, 100. Mu.M Hg) 2+ 50 μ M thrombin) for 60 minutes; blank SSA, and hemoglobin incubated SSA were added to target molecule SSA for CD profiling.
As can be seen from FIG. 8, all three SSAs have a parallel G tetrameric structure with a negative peak at 240nm and a positive peak at 260 nm. The parallel G tetramer structure is enhanced after SSA is combined with heme, and is destroyed after target molecules are added, and CD characteristic peaks disappear.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention.
Claims (8)
1. A general method for constructing a structurally switched aptamer comprising the steps of: design of aptamers with structural switching properties: respectively connecting two ends of an aptamer with Sub>A sequence rich in guanine nucleotide G to construct Sub>A probe Apt-G4 with structural switch performance, wherein the probe Apt-G4 with structural switch performance is A-SULF-A; A-TBA29-A, and A-T11-A;
wherein the sequence of the probe A-SULF-A is as follows:
AGGGACGGGACTAGAGAGGGCAACGAGTGTTTATAGAAGGGACGGGA
the sequence of the probe A-TBA29-A is as follows:
AGGGACGGGAAGTCCGTGGTAGGGCAGGTTGGGGTGACTAGGGACGGGA
the sequence of the probe A-T11-A is as follows:
AGGGACGGGATTTTTTTTTTTAGGGACGGGA。
2. a method of constructing a universal sensor, comprising: designing a nucleic acid aptamer with structural switch performance: respectively connecting two ends of an aptamer with Sub>A sequence rich in guanine nucleotide G to construct Sub>A probe Apt-G4 with structural switch performance, wherein the probe Apt-G4 with structural switch performance is A-SULF-A; A-TBA29-A, and A-T11-A; and step (2) heating Apt-G4 at 95 ℃, and slowly cooling to room temperature;
wherein the sequence of the probe A-SULF-A is as follows:
AGGGACGGGACTAGAGAGGGCAACGAGTGTTTATAGAAGGGACGGGA
the sequence of the probe A-TBA29-A is as follows:
AGGGACGGGAAGTCCGTGGTAGGGCAGGTTGGGGTGACTAGGGACGGGA
the sequence of the probe A-T11-A is as follows:
AGGGACGGGATTTTTTTTTTTAGGGACGGGA。
3. the method for constructing the universal sensor according to claim 2, further comprising the steps of (3) adding substances to be tested with different concentrations to incubate with Apt-G4 to form a mixed solution; adding hemin and the mixed solution for incubation in the step (4), simultaneously adding hydrogen peroxide and 2' -hydrazine-bis-3-ethylbenzthiazoline-6-sulfonic acid in the step (5), measuring the ultraviolet absorption at 418nm of a selected time, and drawing a working curve by taking the ultraviolet absorption value as a vertical coordinate and the concentration of the substance to be measured as a horizontal coordinate.
4. The method for constructing a universal sensor according to claim 2, wherein in the step (2), the temperature is kept at 95 ℃ for 6 minutes, and then the temperature is slowly cooled to 25 ℃.
5. The method for constructing a universal sensor according to claim 3, further comprising the step (6) of selectively testing: and (5) replacing the substances to be detected with other substances with equal concentrations respectively, and repeating the experiments in the steps (1) to (5).
6. The method of claim 5, wherein the Apt-G4 probe with structure switching property is configured as a solution with a concentration of 0.375 μ M.
7. A general sensor for constructing Sub>A structure switch type aptamer is characterized by comprising the aptamer with structure switch performance, wherein sequences rich in guanine nucleotide G are respectively connected to two ends of the aptamer to construct Sub>A probe Apt-G4 with structure switch performance, and the probe Apt-G4 with structure switch performance is A-SULF-A; A-TBA29-A, and A-T11-A;
wherein the sequence of the probe A-SULF-A is as follows:
AGGGACGGGACTAGAGAGGGCAACGAGTGTTTATAGAAGGGACGGGA
the sequence of the probe A-TBA29-A is as follows:
AGGGACGGGAAGTCCGTGGTAGGGCAGGTTGGGGTGACTAGGGACGGGA
the sequence of the probe A-T11-A is as follows:
AGGGACGGGATTTTTTTTTTTAGGGACGGGA。
8. the sensor of claim 7, wherein said probe Apt-G4 with structural switching capability is configured as a 0.375 μ M solution.
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| CN107794295A (en) * | 2017-11-09 | 2018-03-13 | 湖南工程学院 | A kind of blood coagulation enzyme assay method and kit that ring mediated isothermal amplification is opened based on double Aptamer interlayer structures |
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| CN107794295A (en) * | 2017-11-09 | 2018-03-13 | 湖南工程学院 | A kind of blood coagulation enzyme assay method and kit that ring mediated isothermal amplification is opened based on double Aptamer interlayer structures |
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| Design of an aptamer – based fluorescence displacement biosensor for selective and sensitive detection of kanamycin in aqueous samples;Long Ma 等;《RSC Adv.》;20171231(第7期);第38512-38518页 * |
| Versatile G-quadruplex-mediated strategies in label-free biosensors and logic systems;Jiangtao Ren 等;《Analyst》;20151231(第140期);第2556-2572页 * |
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