CN108866157B - Biosensor based on strand displacement and dark silver clusters and application method thereof - Google Patents
Biosensor based on strand displacement and dark silver clusters and application method thereof Download PDFInfo
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Abstract
The invention relates to a biosensor based on strand displacement and a dark silver cluster and application thereof, wherein the biosensor comprises three probe strands which are respectively a C-DNA strand, a P-DNA strand and an S-DNA strand, the C-DNA strand consists of two parts, the 5 'end contains a G-rich sequence, the 3' end is partially complementary with the S-DNA of the silver cluster template strand, and is also partially complementary with the P-DNA of the probe strand, and the number of bases complementary with the P-DNA is more than that of bases complementary with the S-DNA; the P-DNA strand contains a sequence that is completely complementary to the Target strand Target; the S-DNA chain consists of two parts, wherein the 5 'end is a C-DNA complementary sequence, and the 3' end is a dark silver cluster synthesis template sequence. In addition, the invention also introduces exonuclease III into the system, and releases Target to participate in the strand displacement of P-DNA/C-DNA again through the cutting of P-DNA in a Target/P-DNA double strand, thereby realizing the cyclic detection of Target and improving the detection sensitivity. The invention utilizes the fluorescence change and strand displacement of the dark silver cluster and the amplification effect of the exonuclease III, can be used for sensitively detecting related gene detection such as gene deletion and the like, and can realize the obvious distinction of three base deletions.
Description
Technical Field
The invention relates to a method for detecting by means of fluorescence, in particular to a biosensor based on strand displacement and a dark silver cluster and a construction method thereof.
Background
A gene deletion is a kind of change in DNA base sequence, and refers to DNA sequence polymorphism caused by deletion of a part of DNA base sequence, and many phenotypic differences in human body, susceptibility to drugs or diseases, and the like may be associated with gene deletion. For example, the amide tRNA synthetase interactive multifunctional protein AIMP2 is a novel tumor suppressor, and the deletion of exon 2 thereof can cause the morphological variation of AIMP2, thereby losing the cancer suppression activity, and further causing the occurrence of cancers such as lung cancer, liver cancer, skin cancer, breast cancer and the like. Therefore, at the molecular level, constructing a high-sensitivity biosensor and realizing gene detection related to diseases have important research significance. The existing biosensor for detecting gene deletion mostly involves the synthesis of fluorescent markers or compounds and even the determination of whole gene sequences, and has complex operation and high cost.
The silver cluster is a silver nanocluster which is composed of several to dozens of silver atoms and has the size of 1-2nm, has excellent photochemical performance and low biological toxicity, and is widely applied to biosensors, biomolecular beacons, molecular logic gates, cell imaging and the like as a potential fluorescent marker substance in recent years. The silver clusters are small in size, and the particles are easy to collide with each other to cause agglomeration, fluorescence quenching and the like. Therefore, the size growth of the silver cluster can be controlled by adding a protective agent such as an organic template into the aqueous solution, and the commonly used organic template comprises an organic ligand, a sulfhydryl-containing compound, a dendrimer and biological macromolecules (DNA, protein and the like), wherein the DNA can greatly widen the light-emitting range and application of the silver cluster due to the structural diversity, so that the silver cluster is widely concerned.
The strand displacement reaction is a reaction of exchange and rehybridization combination between different DNA strands performed depending on the difference in force between DNA molecules or between DNA molecules and other biomolecules, and has the advantages of easy control of operation, relatively simple experimental conditions, short time consumption, high product yield and the like, and has attracted much attention in recent years.
The exonuclease III is exonuclease and acts on the flat end or the 3 ' -sunken end of double-stranded DNA to gradually remove mononucleotide along the 3 ' -5 ' direction, so that the exonuclease III can be used in the cyclic amplification process of a biosensor to realize low-concentration detection of a target.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a biosensor based on strand displacement and dark silver clusters and a construction method thereof, which are efficient, sensitive, simple and quick.
The purpose of the invention can be realized by the following technical scheme:
the invention aims to provide a biosensor based on strand displacement and dark silver clusters.
The biosensor based on strand displacement and dark silver clusters comprises three probe strands which are respectively a C-DNA strand, a P-DNA strand and an S-DNA strand,
the C-DNA chain consists of two parts, wherein the 5 'end contains a G-rich sequence, the 3' end is complementary with the S-DNA part of the silver cluster template chain and is also complementary with the P-DNA part of the probe chain, and the number of the bases complementary with the P-DNA is more than that of the bases complementary with the S-DNA;
the P-DNA strand contains a sequence that is completely complementary to the Target strand (Target);
the S-DNA chain consists of two parts, wherein the 5 'end is a C-DNA complementary sequence, and the 3' end is a dark silver cluster synthesis template sequence.
Due to the acting force difference between DNA chains caused by different complementary base numbers, the Target chain can pull out P-DNA from a P-DNA/C-DNA double chain to form a P-DNA/Target double chain, so that the released C-DNA is combined with S-DNA, a dark silver cluster synthesis template sequence and a G-rich sequence are pulled close, a bright silver cluster is synthesized, and the fluorescence is strong; under the condition that the Target does not exist, due to the strong acting force of a P-DNA/C-DNA double strand, a silver cluster synthesis template in the S-DNA cannot be close to a G-rich sequence, a synthesized silver cluster is in a dark state, the fluorescence is weak, and therefore specific identification of the Target is achieved.
The second purpose of the invention is to provide an application method of the biosensor based on strand displacement and dark silver clusters.
The molar ratio of the C-DNA chain to the P-DNA chain is 1:1, incubation overnight in PB buffer solution to form complementary double-stranded structure (P-DNA/C-DNA). Adding Target and P-DNA/C-DNA according to the molar ratio of 1:1, culturing, and mixing the DNA according to the molar ratio of S-DNA to Target of 1:1 adding S-DNA, adding silver nitrate aqueous solution and ice water to prepare sodium borohydride solution according to the molar ratio of C-DNA to silver nitrate to sodium borohydride being 1:18:18, culturing for a certain time at a certain temperature, and measuring fluorescence.
In one embodiment of the present invention, the PB buffer solution has a pH of 7.4 and a concentration of 20 mmol/L.
In one embodiment of the invention, the concentrations of the silver nitrate aqueous solution and the ice water freshly prepared sodium borohydride solution are prepared according to the concentration of C-DNA, so that the molar ratio of the C-DNA to the silver nitrate to the sodium borohydride in the system is 1:18: 18.
In one embodiment of the invention, the incubation time is generally 1 hour after Target is added, and fluorescence is measured after adding silver nitrate aqueous solution and ice water to prepare sodium borohydride solution, and incubating for 5 hours at 4 ℃.
In one embodiment of the invention, the fluorescence is measured at an excitation wavelength of 475nm and an emission wavelength of 635 nm.
In one embodiment of the invention, fluorescence is detected with parameters set to a slit of 10nm and a voltage of 600 v.
The third purpose of the invention is to provide a biosensor based on strand displacement, a dark silver cluster and exonuclease III.
The biosensor based on strand displacement, dark silver clusters and exonuclease III comprises exonuclease III in addition to C-DNA strand, P-DNA strand and S-DNA strand,
the C-DNA chain consists of two parts, wherein the 5 'end contains a G-rich sequence, the 3' end is complementary with the S-DNA part of the silver cluster template chain and is also complementary with the P-DNA part of the probe chain, and the number of the bases complementary with the P-DNA is more than that of the bases complementary with the S-DNA;
the P-DNA strand contains a sequence that is completely complementary to the Target strand Target;
the S-DNA chain consists of two parts, wherein the 5 'end is a C-DNA complementary sequence, and the 3' end is a dark silver cluster synthesis template sequence.
Due to the acting force difference between the DNA strands caused by different complementary base numbers, the Target strand can pull out P-DNA from the P-DNA/C-DNA double strand to form a P-DNA/Target double strand, but because the 3 'end of the P-DNA is recessed and is the enzyme cutting site of exonuclease III in the P-DNA/Target double strand formed by the Target and the P-DNA, the DE P-DNA in the P-DNA/Target double strand is digested from the 3' end, the Target is released and participates in the strand displacement reaction with the P-DNA/C-DNA double strand again, and the cyclic amplification detection of the Target DNA is realized. The released C-DNA is combined with the S-DNA, and a dark silver cluster synthesis template sequence and a G-rich sequence are drawn close, so that a bright silver cluster is synthesized, and the fluorescence is strong; under the condition that the Target does not exist, due to the strong acting force of a P-DNA/C-DNA double strand, a silver cluster synthesis template in the S-DNA cannot be close to a G-rich sequence, a synthesized silver cluster is in a dark state, the fluorescence is weak, and therefore specific identification of the Target is achieved. And although C-DNA in the P-DNA/C-DNA double strand also has a recessed 3' end, the digestion of a proper amount of C-DNA by a proper amount of exonuclease III can accelerate strand displacement and the digestion of P-DNA in the P-DNA/Target double strand, so that the reaction rate is improved, and no adverse effect is caused on the experimental result.
The fourth purpose of the invention is to provide an application method of the biosensor based on strand displacement, dark silver clusters and exonuclease III.
The molar ratio of the C-DNA chain to the P-DNA chain is 1:1, incubation overnight in PB buffer solution to form complementary double-stranded structure (P-DNA/C-DNA). Adding Target and P-DNA/C-DNA according to the molar ratio of 1:1, culturing, adding 10U of exonuclease III, culturing for a certain time at a certain temperature, and culturing according to the mol ratio of S-DNA to Target of 1:1 adding S-DNA, adding silver nitrate aqueous solution and ice water to prepare sodium borohydride solution according to the molar ratio of C-DNA to silver nitrate to sodium borohydride of 1:90:36, culturing at a certain temperature for a certain time, and measuring fluorescence.
In one embodiment of the present invention, the PB buffer solution has a pH of 7.4 and a concentration of 20 mmol/L.
In one embodiment of the invention, the concentrations of the silver nitrate aqueous solution and the ice water freshly prepared sodium borohydride solution are prepared according to the concentration of C-DNA, so that the molar ratio of the C-DNA to the silver nitrate to the sodium borohydride in the system is 1:18: 18.
In one embodiment of the invention, the culture time is generally 1 hour after Target is added, the culture is generally 2 hours at 37 ℃ after exonuclease III is added, and the fluorescence is measured after the sodium borohydride solution is newly prepared by adding silver nitrate aqueous solution and ice water and is generally cultured for 5 hours at 4 ℃.
As the molar ratio of the C-DNA, the silver nitrate and the sodium borohydride is changed in the silver cluster synthesis process, the luminescent property of the synthesized silver cluster nano material is changed, in one embodiment of the invention, the excitation wavelength is 640nm and the emission wavelength is 700nm during fluorescence measurement.
In one embodiment of the invention, fluorescence is detected with parameters set to a slit of 10nm and a voltage of 600 v.
When Target chain Target lacks a long base, lacks three bases, and even single base changes, the Target chain can not form double chains with P-DNA any more, so that C-DNA and S-DNA form double chains to synthesize a bright silver cluster, and the sensor can generate strong fluorescent signal changes, so the sensor can be used for detecting gene deletion.
The sensor completes the detection of the deletion of the exon 2 of the multifunctional protein 2 interacted with the aminoacyl tRNA synthetase, and can realize good linear and specific detection. Can respectively realize that: linear detection at 0-320nM, limit of detection at 0.703 nmol/L; linear detection at 0-100nM, limit of detection at 0.489 nmol/L. And can be used for detection of fewer base deletions in the future due to the ability to recognize three base differences.
The silver cluster synthesized by the dark-state silver cluster synthesis template under the condition of single strand is dark-state, namely weak fluorescence, and when the silver cluster is close to a G-rich sequence, the silver cluster synthesized is bright-state, namely strong fluorescence. By utilizing the characteristic of the dark silver cluster, the invention designs a probe chain C-DNA, the 5 'end of the probe chain C-DNA contains a G-rich sequence, the 3' end connecting sequence is partially complementary with the silver cluster template chain S-DNA and is partially complementary with the probe chain P-DNA, and the number of bases complementary with the P-DNA is larger than that of bases complementary with the S-DNA. And the P-DNA can be completely complementary with the Target strand Target. Therefore, the S-DNA can not displace the C-DNA from the P-DNA/C-DNA double strand, the silver cluster template strand can not be close to the G-rich sequence, and the synthesized silver cluster has weak fluorescence. When the Target exists, due to stronger acting force between the Target and the P-DNA, the P-DNA is displaced from a P-DNA/C-DNA double strand, and the C-DNA is released from the double strand, so that the C-DNA is complementary and paired with a subsequently added S-DNA base to form a double strand, a G-rich sequence is close to a silver cluster template, and the synthesized silver cluster has stronger fluorescence. In addition, the exonuclease III is introduced into a system, and the Target is released to participate in the strand displacement of the P-DNA/C-DNA again through the cutting of the P-DNA in the Target/P-DNA double strand, so that the cyclic detection of the Target is realized, and the detection sensitivity is improved. The invention utilizes the fluorescence change and strand displacement of the dark silver cluster and the amplification effect of the exonuclease III, can be used for sensitively detecting related gene detection such as gene deletion and the like, and can realize the obvious distinction of three base deletions.
Drawings
FIG. 1 is a schematic diagram of a biosensor application based on strand displacement and dark silver clusters;
FIG. 2 is a schematic diagram of a biosensor application based on strand displacement, dark silver clusters and exonuclease III;
FIG. 3 is a feasible fluorescence plot of a sensor based on strand displacement and dark silver clusters;
FIG. 4 is a graph showing the relationship between the concentration of Target and the fluorescence intensity;
FIG. 5 is a comparison of fluorescence intensity of different target DNAs with P-DNA, S-DNA and C-DNA being kept unchanged;
FIG. 6 is a fluorescence plot of the feasibility of a sensor based on strand displacement, exonuclease III, and dark silver clusters;
FIG. 7 is a graph showing the relationship between the concentration of Target and the fluorescence intensity;
FIG. 8 is a comparison of fluorescence intensity of different target DNAs with P-DNA, S-DNA and C-DNA being kept unchanged.
Detailed Description
The biosensor based on strand displacement and dark silver clusters comprises three probe strands which are respectively a C-DNA strand, a P-DNA strand and an S-DNA strand, wherein the C-DNA strand consists of two parts, the 5 'end of the C-DNA strand is a G-rich sequence containing 20 bases, and the 3' end of the C-DNA strand is a sequence complementary with the part of the P-DNA strand and contains 11 bases; the P-DNA strand is a sequence which contains 18 bases and is completely complementary with a Target strand (Target); the S-DNA chain is composed of two parts, wherein the 5 'end is a C-DNA complementary sequence with 8 basic groups, and the 3' end is a dark silver cluster synthetic template sequence with 14 basic groups.
Referring to fig. 1, methods of application of biosensors based on strand displacement and dark silver clusters:
the molar ratio of the C-DNA chain to the P-DNA chain is 1:1, culturing in PB buffer solution for a certain time to form a complementary double-stranded structure (P-DNA/C-DNA). Adding Target and P-DNA/C-DNA according to the molar ratio of 1:1, culturing for a certain time, and mixing the DNA with the Target according to the molar ratio of 1:1 adding S-DNA, adding silver nitrate aqueous solution and ice water to prepare sodium borohydride solution according to the molar ratio of C-DNA to silver nitrate to sodium borohydride being 1:18:18, culturing for a certain time at a certain temperature, and measuring fluorescence.
Due to the acting force difference between DNA chains caused by different complementary base numbers, the Target chain can pull out P-DNA from a P-DNA/C-DNA double chain to form a P-DNA/Target double chain, so that the released C-DNA is combined with S-DNA, a dark silver cluster synthesis template sequence and a G-rich sequence are pulled close, a bright silver cluster is synthesized, and the fluorescence is strong; under the condition that the Target does not exist, due to the strong acting force of a P-DNA/C-DNA double strand, a silver cluster synthesis template in the S-DNA cannot be close to a G-rich sequence, a synthesized silver cluster is in a dark state, the fluorescence is weak, and therefore specific identification of the Target is achieved.
The biosensor based on strand displacement, dark silver clusters and exonuclease III comprises a C-DNA strand, a P-DNA strand and an S-DNA strand, and also comprises exonuclease III, wherein the C-DNA strand consists of two parts, a G-rich sequence containing 20 bases is arranged at the 5 'end, and a sequence which is complementary with the part of the P-DNA strand and contains 11 bases is arranged at the 3' end; the P-DNA strand is a sequence which contains 18 bases and is completely complementary with a Target strand (Target); the S-DNA chain is composed of two parts, wherein the 5 'end is a C-DNA complementary sequence with 8 basic groups, and the 3' end is a dark silver cluster synthetic template sequence with 14 basic groups.
Referring to fig. 2, the application method of the biosensor based on strand displacement, dark silver clusters and exonuclease III is:
the molar ratio of the C-DNA chain to the P-DNA chain is 1:1, culturing in PB buffer solution for a certain time to form a complementary double-stranded structure (P-DNA/C-DNA). Adding Target and P-DNA/C-DNA according to the molar ratio of 1:1, culturing for a certain time, adding 10U of exonuclease III, culturing for a certain time at a certain temperature, and culturing according to the mol ratio of S-DNA to Target of 1:1 adding S-DNA, adding silver nitrate aqueous solution and ice water to prepare sodium borohydride solution according to the molar ratio of C-DNA to silver nitrate to sodium borohydride of 1:90:36, culturing at a certain temperature for a certain time, and measuring fluorescence.
Due to the acting force difference between the DNA strands caused by different complementary base numbers, the Target strand can pull out P-DNA from the P-DNA/C-DNA double strand to form a P-DNA/Target double strand, but because the 3 'end of the P-DNA is recessed and is the enzyme cutting site of exonuclease III in the P-DNA/Target double strand formed by the Target and the P-DNA, the DE P-DNA in the P-DNA/Target double strand is digested from the 3' end, the Target is released and participates in the strand displacement reaction with the P-DNA/C-DNA double strand again, and the cyclic amplification detection of the Target DNA is realized. The released C-DNA is combined with the S-DNA, and a dark silver cluster synthesis template sequence and a G-rich sequence are drawn close, so that a bright silver cluster is synthesized, and the fluorescence is strong; under the condition that the Target does not exist, due to the strong acting force of a P-DNA/C-DNA double strand, a silver cluster synthesis template in the S-DNA cannot be close to a G-rich sequence, a synthesized silver cluster is in a dark state, the fluorescence is weak, and therefore specific identification of the Target is achieved. And although C-DNA in the P-DNA/C-DNA double strand also has a recessed 3' end, the activity of the exonuclease III is greatly weakened and basically cannot be digested due to the fact that only 2 bases are recessed, and therefore the result cannot be influenced.
The invention is described in detail below with reference to the figures and specific embodiments.
Example 1 (preparation of various stock solutions)
Preparation of PB buffer (PB: 20mM, pH 7.4): accurately weighing Na2HPO4·12H2O7.16 g is put in a 50mL beaker, is completely dissolved by triple distilled water and is transferred to a 100mL volumetric flask, the beaker and a glass rod are washed by ultrapure water for three times, a washing liquid is transferred to the volumetric flask, the volume is constant to 100mL, and 100mL of 0.2M disodium hydrogen phosphate stock solution can be obtained by shaking uniformly for later use; accurately weighing NaH2PO4·2H2Dissolving O3.12 g in a 50mL beaker with triple distilled water completely, transferring the beaker and a glass rod into a 100mL volumetric flask, washing the beaker and the glass rod with ultrapure water for three times, transferring the washing liquid into the volumetric flask, fixing the volume to 100mL, and shaking uniformly to obtain 100mL of 0.2M sodium dihydrogen phosphate stock solution for later use; taking the prepared stock solution: 19mL of 0.2M NaH2PO4And 80mL of 0.2M Na2HPO4The mixture was mixed in a 1000mL volumetric flask and diluted to 1000mL with triple distilled water to obtain 20mM PB buffer solution with pH 7.4 for use.
Preparation of DNA solution: centrifuging P-DNA, C-DNA, S-DNA and Target in a centrifuge at 12000r/min for 1min, dissolving with PB buffer solution of corresponding volume at a concentration of 100 μ M, sealing, culturing in a 90 deg.C oven for 10min, naturally cooling to room temperature, and storing in a 4 deg.C refrigerator.
In the examples, the sequences of P-DNA, C-DNA, S-DNA and Target are shown in Table 1.
TABLE 1
Wherein the C-DNA strand consists of two parts, the 5 'end is a G-rich sequence containing 20 bases, i.e., GGGTGGGGTGGGGTGGGGGT, and the 3' end is a sequence complementary to a part of the P-DNA strand, containing 11 bases, i.e., GCAGGATTACG;
the P-DNA strand is a sequence which contains 18 bases and is completely complementary with a Target strand (Target);
the S-DNA chain is composed of two parts, wherein the 5 'end is a C-DNA complementary sequence of 8 bases, namely TCCTGCAC, and the 3' end is a dark silver cluster synthesis template sequence of 12 bases, namely CCCTTAATCCCC.
In Target, GCTGGCCACGTGCAG, GATTACGGGGCGCTG represent sequences flanking the missing part of the complete sequence.
3. Silver nitrate (AgNO)3) And sodium borohydride (NaBH)4) Preparing a solution: accurately weighing 15.3mg AgNO under dark condition3Dissolving in 50mL centrifuge tube with triple distilled water to constant volume of 50mL to obtain 1.8mM AgNO3Storing the solution at 4 ℃ for later use; accurately weighing 3.4mg NaBH in dark condition4Dissolving in 50mL of centrifuge tube with triple distilled water (ice water mixture) to 50mL to obtain 1.8mM NaBH4The solution is prepared for use in the dark.
Buffer 1 Buffer (50mM Tris-HNO)3，10mM Mg(NO3)21mM DTT, pH 7.0): accurately weigh 15.4Mg DTT, 605.7Mg Tris, 256.4Mg Mg (NO)3)2In a 50mL beaker, mix with triple distilled waterIt is completely dissolved in combination with HNO3Adjusting the pH value to 7.0, transferring the solution into a 100mL volumetric flask, washing a beaker and a glass rod with ultrapure water for three times, transferring the washing solution into the volumetric flask, and performing constant volume to 100mL to obtain Buffer 1 Buffer solution for later use.
5. Preparing an exonuclease III diluent (1000 units/mL): exonuclease III was purchased at 100,000units/mL, 2500units, 25. mu.L, and 2475. mu.L Buffer 1 was quickly removed using a sterilized gun tip to dilute to 2500. mu.L, to prepare a 1000units/mL solution, which was stored at-20 ℃ until use.
Example 2 (sensor construction based on Strand Displacement and dark silver Cluster-fluorescence detection)
1. C-DNA, C-DNA + P-DNA, C-DNA + P-DNA + Target, 10. mu.L of each DNA (100. mu.M) in each sample were added to a 2.0-ml centrifuge tube, and PB buffer solution was added to make the volume of the system 200. mu.L. Culturing at 37 deg.C for 1 h;
2. adding appropriate amount of PB buffer solution to make the volume of the system 970. mu.L, adding 10. mu. L S-DNA (100. mu.M), 10. mu.L AgNO3(1.8mM), shaking by magnetic force for 1min, and culturing at room temperature in a dark state for 30 min;
3. add 10. mu.L of freshly prepared NaBH4(1.8mM), shaking by magnetic force for 1min, and culturing at 4 ℃ in a dark state for 5 h;
4. fluorescence was detected with parameters set to 10nm slit and 600V voltage. (excitation wavelength of 475nm, emission wavelength of 635nm), FIG. 3 was obtained.
Example 3 (detection of deletion of exon 2 Gene of AIMP2 based on strand displacement and sensor of dark silver Cluster)
1. To a 2.0 ml centrifuge tube, 10. mu. L C-DNA + P-DNA (100. mu.M in each case) was added, 10. mu.L of Target of different concentrations and targets of different types (100. mu.M in each case) were added, and a PB buffer solution was added to make the volume of the system 200. mu.L. Culturing at 37 deg.C for 1 h;
2. adding appropriate amount of PB buffer solution to reach volume of 970 μ L, adding 10 μ L S-DNA (100 μ M) and 10 μ L AgNO3(1.8mM), magnetically shaking for 1min, and culturing at room temperature in dark state for 30 min;
3. adding 10 μ L fresh NaBH4(1.8mM), magnetically shaking for 1min, and culturing at 4 deg.C in dark state for 5 hr;
4. fluorescence was detected with parameters set to 10nm slit and 600V voltage. (excitation wavelength is 475nM, emission wavelength is 635nM), and then figure 4 and figure 5 can be obtained respectively, and linear detection of 0-320nM of target chain is realized, the detection limit is 0.703nmol/L, and specific detection can be realized.
TABLE 2
The WT1 and WT2 represent the gene sequences near exon 2 of AIMP2 without exon 2 deletion, and MT1, MT2 and MT3 represent the sequences with 3, 3 and 6 bases deleted from the Target sequence, respectively, and the sequences are used to explore the interference of other sequences on the detection of AIMP2 exon 2 deletion gene.
Example 4 (construction of a sensor based on Strand Displacement, exonuclease III, and dark silver Cluster-fluorescence detection)
1. To a 2.0 mL centrifuge tube were added C-DNA, C-DNA + P-DNA, C-DNA + P-DNA + Target, 10. mu.L of each DNA (100. mu.M) in each sample, Buffer 1 Buffer solution was added to make the volume of the system 198. mu.L, and 2. mu.L of exonuclease solution (1000units/mL) was added. Culturing at 37 deg.C for 2 h;
2. adding appropriate amount of PB buffer solution to make the volume of the system 920. mu.L, adding 10. mu. L S-DNA (100. mu.M), 50. mu.L AgNO3(1.8mM), shaking by magnetic force for 1min, and culturing at room temperature in a dark state for 30 min;
3. add 20. mu.L of freshly prepared NaBH4(1.8mM), shaking by magnetic force for 1min, and culturing at 4 ℃ in a dark state for 5 h;
4. fluorescence was detected with parameters set to 10nm slit and 600V voltage. (excitation wavelength was 640nm, emission wavelength was 700nm), FIG. 6 was obtained.
Example 5 (detection of deletion of exon 2 Gene of AIMP2 based on Strand Displacement, exonuclease III and dark silver Cluster Sensors)
1. To a 2.0 mL centrifuge tube, 10. mu. L C-DNA + P-DNA (100. mu.M each) was added, 10. mu.L of Target and different targets (100. mu.M each) were added at different concentrations, and Buffer 1 Buffer solution was added to make the volume of the system 198. mu.L and 2. mu.L of exonuclease solution (1000units/mL) was added. Culturing at 37 deg.C for 2 h;
2. adding appropriate amount of PB buffer solution to make the volume of the system be 920. mu.L, adding 10. mu. L S-DNA (100. mu.M) and 50. mu.L of AgNO3(1.8mM), magnetically shaking for 1min, and culturing at room temperature in dark state for 30 min;
3. adding 20 μ L fresh NaBH4(1.8mM), magnetically shaking for 1min, and culturing at 4 deg.C in dark state for 5 hr;
4. fluorescence was detected with parameters set to 10nm slit and 600V voltage. (excitation wavelength is 475nM, emission wavelength is 635nM), and then figure 7 and figure 8 can be obtained respectively, which realizes the linear detection of 0-100nM of target chain, the detection limit is 0.489nmol/L, and the specific detection can be realized.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
<110> university of Tongji
<120> biosensor based on strand displacement and dark-state silver cluster and application method thereof
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<213> Artificial Sequence (Artificial Sequence)
<400> 3
tcctgcaccc cttaatcccc 20
<210> 4
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gctggccacg tgcaggatta cggggcgctg 30
<210> 5
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gctggccacg tgcaggaaga gtctaacctg 30
<210> 6
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tcagtgcttg ggaaggatta cggggcgctg 30
<210> 7
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gctggccacg tgcagtacgg ggcgctg 27
<210> 8
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
gctggccacg tggattacgg ggcgctg 27
<210> 9
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gctggccacg tgtacggggc gctg 24
Claims (4)
1. A biosensor based on strand displacement and a dark silver cluster is characterized by comprising three probe strands which are a C-DNA strand, a P-DNA strand and an S-DNA strand respectively,
the C-DNA chain consists of two parts, wherein the 5 'end contains a G-rich sequence, the 3' end is complementary with the S-DNA part of the silver cluster template chain and is also complementary with the P-DNA part of the probe chain, and the number of the bases complementary with the P-DNA is more than that of the bases complementary with the S-DNA;
the P-DNA strand contains a sequence that is completely complementary to the Target strand Target;
the S-DNA chain consists of two parts, wherein the 5 'end is a C-DNA complementary sequence, and the 3' end is a dark silver cluster synthesis template sequence;
wherein the sequence of the C-DNA chain is GGGTGGGGTGGGGTGGGGGT;
the sequence of the P-DNA strand is GCAGGATTACG;
the 5 'end of the S-DNA chain is a C-DNA complementary sequence of 8 bases, namely TCCTGCAC, and the 3' end is a dark silver cluster synthesis template sequence of 12 bases, namely CCCTTAATCCCC.
2. The method of using the strand displacement and dark silver cluster-based biosensor of claim 1 for non-diagnostic purposes, comprising the steps of:
the molar ratio of the C-DNA chain to the P-DNA chain is 1:1, culturing in a PB buffer solution for a certain time to form a complementary double-stranded structure, and adding a Target and P-DNA/C-DNA according to a molar ratio of 1:1, culturing for a certain time, and mixing the DNA with the Target according to the molar ratio of 1:1 adding S-DNA, adding silver nitrate aqueous solution and ice water to prepare sodium borohydride solution according to the molar ratio of C-DNA to silver nitrate to sodium borohydride being 1:18:18, culturing for a certain time at a certain temperature, and measuring fluorescence.
3. A biosensor based on strand displacement, dark silver clusters and exonuclease III, comprising exonuclease III in addition to a C-DNA strand, a P-DNA strand and an S-DNA strand,
the C-DNA chain consists of two parts, wherein the 5 'end contains a G-rich sequence, the 3' end is complementary with the S-DNA part of the silver cluster template chain and is also complementary with the P-DNA part of the probe chain, and the number of the bases complementary with the P-DNA is more than that of the bases complementary with the S-DNA;
the P-DNA strand contains a sequence that is completely complementary to the Target strand Target;
the S-DNA chain consists of two parts, wherein the 5 'end is a C-DNA complementary sequence, and the 3' end is a dark silver cluster synthesis template sequence;
wherein the sequence of the C-DNA chain is GGGTGGGGTGGGGTGGGGGT;
the sequence of the P-DNA strand is GCAGGATTACG;
the 5 'end of the S-DNA chain is a C-DNA complementary sequence of 8 bases, namely TCCTGCAC, and the 3' end is a dark silver cluster synthesis template sequence of 12 bases, namely CCCTTAATCCCC.
4. The method of using the biosensor based on strand displacement, dark silver clusters and exonuclease III for non-diagnostic purposes as claimed in claim 3 comprising the steps of:
the molar ratio of the C-DNA chain to the P-DNA chain is 1:1, culturing in a PB buffer solution for a certain time to form a complementary double-stranded structure, and adding a Target and P-DNA/C-DNA according to a molar ratio of 1:1, culturing for a certain time, adding 10U of exonuclease III, culturing for a certain time at a certain temperature, and culturing according to the mol ratio of S-DNA to Target of 1:1 adding S-DNA, adding silver nitrate aqueous solution and ice water to prepare sodium borohydride solution according to the molar ratio of C-DNA to silver nitrate to sodium borohydride of 1:90:36, culturing at a certain temperature for a certain time, and measuring fluorescence.
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| CN106970229A (en) * | 2017-01-25 | 2017-07-21 | 南京医科大学 | A kind of transcription factor detection method amplified based on DNA silver nanoclusters molecular beacon and exonuclease III cycle signals |
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| CN106970229A (en) * | 2017-01-25 | 2017-07-21 | 南京医科大学 | A kind of transcription factor detection method amplified based on DNA silver nanoclusters molecular beacon and exonuclease III cycle signals |
Non-Patent Citations (3)
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| An approach toward SNP detection by modulating the fluorescence of DNA-templated silver nanoclusters;Juhee Park，et al;《Biosensors and Bioelectronics》;20130104;419-424页 * |
| Ultrasensitive and universal fluorescent aptasensor for the detection of biomolecules (ATP, adenosine and thrombin) based on DNA/Ag nanoclusters fluorescence light-up system;Ying Zhu，et al;《Biosensors and Bioelectronics》;20160515;第79卷;205-212页 * |
| Ying Zhu，et al.Ultrasensitive and universal fluorescent aptasensor for the detection of biomolecules (ATP, adenosine and thrombin) based on DNA/Ag nanoclusters fluorescence light-up system.《Biosensors and Bioelectronics》.2016,第79卷205-212页. * |
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