CN108866157A - Biosensor and its application method based on strand displacement and dark-state silver cluster - Google Patents
Biosensor and its application method based on strand displacement and dark-state silver cluster Download PDFInfo
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- CN108866157A CN108866157A CN201810218591.8A CN201810218591A CN108866157A CN 108866157 A CN108866157 A CN 108866157A CN 201810218591 A CN201810218591 A CN 201810218591A CN 108866157 A CN108866157 A CN 108866157A
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- C12Q1/6825—Nucleic acid detection involving sensors
Abstract
The present invention relates to based on strand displacement and dark-state silver cluster biosensor and its application, biosensor includes three probe chains altogether, it is C-DNA chain, P-DNA chain and S-DNA chain respectively, C-DNA chain consists of two parts, contain rich G sequence in 5 ' ends, 3 ' ends and silver-colored cluster template strand S-DNA partial complementarity, at the same also with probe chain P-DNA partial complementarity, and the base number complementary with P-DNA greater than and S-DNA complementary base number；P-DNA chain contains can be with the sequence of object chain Target complete complementary；S-DNA chain consists of two parts, and 5 ' ends are C-DNA complementary series, and 3 ' ends are dark-state silver cluster synthesis template sequences.In addition, exonucleaseⅢ is also introduced system by the present invention, by the cutting to the P-DNA in Target/P-DNA double-strand, the strand displacement that Target participates in P-DNA/C-DNA again is discharged to realize to the cycle detection of Target and improves the sensitivity of detection.The present invention can be used for the detection of the related genes such as Sensitive Detection gene delection using the change in fluorescence of dark-state silver cluster and the amplification of strand displacement and exonucleaseⅢ, and can be realized the obvious differentiation of three base deletions.
Description
Technical field
Fluorescence detection method is relied on the present invention relates to a kind of, is passed more particularly, to the biology based on strand displacement and dark-state silver cluster
Sensor and its construction method.
Background technique
Gene delection is one kind of the variation of DNA base sequence, is referred to caused by DNA base sequence deletion a part
DNA sequence polymorphism, many phenotypic differences of human body, all may be related with gene delection to neurological susceptibility of drug or disease etc..
If amide tRNA synthesis enzyme interacting multifunctional protein AIMP2 is a kind of novel tumor suppressor, its exon 2 is lacked
Mistake will cause AIMP2 morphological variation, so that tumor-suppression activity is lost, so as to cause cancers such as lung cancer, liver cancer, cutaneum carcinoma, breast cancer
Generation.Therefore, in molecular level, highly sensitive biosensor is constructed, realizes that genetic test relevant to disease has
Important research significance.Current biosensor is related to the conjunction of fluorescent marker or compound for the detection of gene delection more
It is complicated for operation at the measurement of even complete genome sequence, it is at high cost.
Silver-colored cluster refers to and is made of several to dozens of silver atoms that ag nano-cluster of the size between 1-2nm have
Superior photochemical properties, and bio-toxicity is small, in recent years as a kind of potential fluorescence labeling material biosensor,
Biomolecule beacon, Molecular Logic Gates, cell imaging etc. are widely used.Silver-colored cluster is since size is smaller, particle
Between easily collide and cause to reunite and fluorescent quenching etc..Therefore the protective agents such as organic formwork are added in aqueous solution to be controlled
Its dimensional growth, common organic formwork include that organic ligand, compounds containing thiol groups, dendrimer polymer and biology are big
Molecule (DNA, protein etc.), wherein DNA can widen light emitting region and the application of silver-colored cluster due to the diversity of structure significantly, because
This is widely paid close attention to.
Strand replacement reaction be it is a kind of by active force difference between DNA molecular or between DNA molecular and other biological molecule and
Exchange and the reaction of cross combination again between a kind of different DNA chain carried out, have operation opposite vulnerable to control, experiment condition
Simply, the advantages that time-consuming short and products collection efficiency is high, receives significant attention in recent years.
ExonucleaseⅢ is a kind of exonuclease, the flat end of double-stranded DNA or 3 ' recessed ends is acted on, along 3 ' -5 ' sides
It to gradually removing mononucleotide, therefore can be used in biosensor recycling in amplification process, realize to the low dense of object
Degree detection.
Summary of the invention
It is an object of the present invention to overcome the above-mentioned drawbacks of the prior art and provide a kind of efficient and sensibles, letter
List, quickly biosensor and its construction method based on strand displacement and dark-state silver cluster.
The purpose of the present invention can be achieved through the following technical solutions：
The object of the invention one provides a kind of biosensor based on strand displacement and dark-state silver cluster.
Biosensor based on strand displacement and dark-state silver cluster includes three probe chains altogether, is C-DNA chain, P-DNA respectively
Chain and S-DNA chain,
Wherein, C-DNA chain consists of two parts, and containing rich G sequence, 3 ' ends are mutual with the silver part cluster template strand S-DNA at 5 ' ends
It mends, while also with probe chain P-DNA partial complementarity, and the base number complementary with P-DNA is greater than and S-DNA complementary base is a
Number；
P-DNA chain contains can be with the sequence of object chain (Target) complete complementary；
S-DNA chain consists of two parts, and 5 ' ends are C-DNA complementary series, and 3 ' ends are dark-state silver cluster synthesis template sequences.
Active force difference between the DNA chain as caused by different complementary base radixes, object chain can be by P-DNA from P-DNA/
It is pulled out in C-DNA double-strand, forms P-DNA/Target double-strand and further to make the C-DNA released in conjunction with S-DNA
Dark-state silver cluster synthesizes template sequence and rich G sequence, to synthesize illuminated state silver cluster, fluorescence is stronger；And Target the case where being not present
Under, due to the stronger active force of P-DNA/C-DNA double-strand, silver-colored cluster synthesis template cannot be close with rich G sequence in S-DNA, synthesis
Silver-colored cluster is dark-state, and fluorescence is weaker, to realize the specific recognition of Target.
The object of the invention two provides a kind of application method of biosensor based on strand displacement and dark-state silver cluster.
C-DNA chain, P-DNA chain molar ratio are 1：1, the overnight incubation in PB buffer solution forms complementary duplex structure
(P-DNA/C-DNA).Target is added and P-DNA/C-DNA is 1 in molar ratio：1, culture, by S-DNA and Target molar ratio
1：1 is added S-DNA, and then according to C-DNA, silver nitrate, sodium borohydride molar ratio 1:18:18, be added silver nitrate aqueous solution and
Ice water brand-new sodium borohydride solution is cultivated under certain temperature and surveys fluorescence after a certain period of time.
In an embodiment of the invention, the pH=7.4 of the PB buffer solution, concentration 20mmol/L.
In an embodiment of the invention, the concentration of the silver nitrate aqueous solution and ice water brand-new sodium borohydride solution
It is prepared according to C-DNA concentration, makes the molar ratio 1 of C-DNA in system, silver nitrate, sodium borohydride:18:18.
In an embodiment of the invention, incubation time is generally 1 hour after Target being added, and silver nitrate water is added
After solution and ice water brand-new sodium borohydride solution, generally want to survey fluorescence after cultivating 5 hours under 4 degrees Celsius.
In an embodiment of the invention, when surveying fluorescence, excitation wavelength 475nm, launch wavelength 635nm.
In an embodiment of the invention, fluorescence is detected, it is 10nm, voltage 600v that parameter, which is set as slit,.
The object of the invention three provides a kind of biosensor based on strand displacement, dark-state silver cluster and exonucleaseⅢ.
Biosensor based on strand displacement, dark-state silver cluster and exonucleaseⅢ, which removes, includes C-DNA chain, P-DNA chain and S-
It further include exonucleaseⅢ other than DNA chain,
Wherein, C-DNA chain consists of two parts, and containing rich G sequence, 3 ' ends are mutual with the silver part cluster template strand S-DNA at 5 ' ends
It mends, while also with probe chain P-DNA partial complementarity, and the base number complementary with P-DNA is greater than and S-DNA complementary base is a
Number；
P-DNA chain contains can be with the sequence of object chain Target complete complementary；
S-DNA chain consists of two parts, and 5 ' ends are C-DNA complementary series, and 3 ' ends are dark-state silver cluster synthesis template sequences.
Active force difference between the DNA chain as caused by different complementary base radixes, object chain can be by P-DNA from P-DNA/
It is pulled out in C-DNA double-strand, forms P-DNA/Target double-strand, but due to the P-DNA/ of Target and P-DNA formation
In Target double-strand, the 3 ' ends of P-DNA are fallen in, and are the restriction enzyme site of exonucleaseⅢ, to keep P-DNA/Target bis-
DE P-DNA is digested since 3 ' ends in chain, is released Target, is participated in the chain with P-DNA/C-DNA double-strand again
In displacement reaction, to realize the circulation amplification detection of target dna.And make the C-DNA released in conjunction with S-DNA, it furthers
Dark-state silver cluster synthesizes template sequence and rich G sequence, to synthesize illuminated state silver cluster, fluorescence is stronger；And Target the case where being not present
Under, due to the stronger active force of P-DNA/C-DNA double-strand, silver-colored cluster synthesis template cannot be close with rich G sequence in S-DNA, synthesis
Silver-colored cluster is dark-state, and fluorescence is weaker, to realize the specific recognition of Target.Although and C-DNA in P-DNA/C-DNA double-strand
Also there is 3 ' fallen in end, but appropriate exonucleaseⅢ, which digests appropriate C-DNA, can accelerate strand displacement and P-DNA/Target double-strand
The digestion of middle P-DNA improves reaction rate, experimental result is not adversely affected.
The object of the invention four provides a kind of application of biosensor based on strand displacement, dark-state silver cluster and exonucleaseⅢ
Method.
C-DNA chain, P-DNA chain molar ratio are 1：1, the overnight incubation in PB buffer solution forms complementary duplex structure
(P-DNA/C-DNA).Target is added and P-DNA/C-DNA is 1 in molar ratio：1,10U exonucleaseⅢ is added, centainly in culture
At a temperature of cultivate certain time, by S-DNA and Target molar ratio 1：1 be added S-DNA, and then according to C-DNA, silver nitrate,
Sodium borohydride molar ratio 1:90:36, silver nitrate aqueous solution and ice water brand-new sodium borohydride solution is added, cultivates one under certain temperature
Fluorescence is surveyed after fixing time.
In an embodiment of the invention, the pH=7.4 of the PB buffer solution, concentration 20mmol/L.
In an embodiment of the invention, the concentration of the silver nitrate aqueous solution and ice water brand-new sodium borohydride solution
It is prepared according to C-DNA concentration, makes the molar ratio 1 of C-DNA in system, silver nitrate, sodium borohydride:18:18.
In an embodiment of the invention, incubation time is generally 1 hour after Target being added, and excision enzyme is added
It is cultivated 2 hours for general 37 degrees Celsius after III, after silver nitrate aqueous solution and ice water brand-new sodium borohydride solution is added, generally wants 4 to take the photograph
Fluorescence is surveyed after cultivating 5 hours under family name's degree.
Due to the molar ratio variation of C-DNA, silver nitrate, sodium borohydride in silver-colored cluster synthesis process, silver-colored cluster nano material is synthesized
Luminosity change, in an embodiment of the invention, survey fluorescence when, excitation wavelength 640nm, launch wavelength
For 700nm.
In an embodiment of the invention, fluorescence is detected, it is 10nm, voltage 600v that parameter, which is set as slit,.
When three the long section base of object chain Target missing one, missing bases, when even single base changes, due to
Target chain cannot form double-strand with P-DNA again, so that so that C-DNA is formed double-strand with S-DNA synthesizes illuminated state silver cluster, above-mentioned sensing
Device can generate very strong fluorescence signal variation, so the sensor can be used for detecting gene delection.
The sensor completes the detection of the exon 2 missing of aminoacyl tRNA synthetase interaction multifunctional protein 2,
And it can be realized linear and specific detection well.It is able to achieve respectively：The linearity test of 0-320nM, 0.703nmol/L detection
Limit；The linearity test of 0-100nM, 0.489nmol/L detection limit.And due to that can identify three base differences, so future
It can be used for detecting the detection of less base deletion.
The silver-colored cluster that dark-state silver cluster synthesis template synthesizes in single-stranded situation is dark-state, i.e. fluorescence is very weak, when itself and rich G sequence
When column are close, synthesizing silver-colored cluster is illuminated state, i.e. fluorescence is very strong.Using this characteristic of dark-state silver cluster, the present invention devises one
Probe chain C-DNA, its 5 ' end is containing one section of rich G sequence, 3 ' end catenation sequences and silver-colored cluster template strand S-DNA partial complementarity, simultaneously
Also with probe chain P-DNA partial complementarity, and the base number complementary with P-DNA be greater than and S-DNA complementary base number.And P-
DNA can be with object chain Target complete complementary.Therefore S-DNA cannot displace C-DNA from P-DNA/C-DNA double-strand
Come, silver-colored cluster template strand it is very weak cannot to synthesize silver-colored cluster fluorescence close to rich G sequence.In the presence of Target, due to Target and P-
Stronger active force between DNA, P-DNA are replaced out from P-DNA/C-DNA double-strand, and C-DNA is just discharged from double-strand, from
And double-strand is formed with the S-DNA base pair complementarity being then added, keep rich G sequence and silver-colored cluster template close, synthesizes silver-colored cluster fluorescence
It is relatively strong.In addition, exonucleaseⅢ is introduced system by the present invention, by the cutting to the P-DNA in Target/P-DNA double-strand, release
The strand displacement that Target participates in P-DNA/C-DNA again is put, to realize to the cycle detection of Target, improves the sensitive of detection
Property.The present invention can be used for sensitive inspection using the change in fluorescence of dark-state silver cluster and the amplification of strand displacement and exonucleaseⅢ
The related genes detection such as cls gene missing, and can be realized the obvious differentiation of three base deletions.
Detailed description of the invention
Fig. 1 is the biosensor application schematic diagram based on strand displacement and dark-state silver cluster；
Fig. 2 is the biosensor application schematic diagram based on strand displacement, dark-state silver cluster and exonucleaseⅢ；
Fig. 3 is the feasibility fluorogram of the sensor based on strand displacement and dark-state silver cluster；
Fig. 4 is the concentration of Target and the relational graph of fluorescence intensity；
Fig. 5 is to maintain P-DNA, and S-DNA, C-DNA is constant, the fluorescence intensity comparison of different target DNA；
Fig. 6 is the feasibility fluorogram of the sensor based on strand displacement, exonucleaseⅢ and dark-state silver cluster；
Fig. 7 is the concentration of Target and the relational graph of fluorescence intensity；
Fig. 8 is to maintain P-DNA, and S-DNA, C-DNA is constant, the fluorescence intensity comparison of different target DNA.
Specific embodiment
Biosensor based on strand displacement and dark-state silver cluster includes three probe chains altogether, is C-DNA chain, P-DNA respectively
Chain and S-DNA chain, wherein C-DNA chain consists of two parts, and 5 ' ends are one section containing 20 base richness G sequences, and 3 ' ends are one section
It include 11 bases with the sequence of the partial complementarity of P-DNA chain；P-DNA chain is one section and contains 18 bases and and object chain
(Target) sequence of complete complementary；S-DNA chain consists of two parts, and 5 ' ends are the C-DNA complementary series of 8 bases, 3 ' ends
It is the dark-state silver cluster synthesis template sequence of 14 bases.
With reference to Fig. 1, the application method position of the biosensor based on strand displacement and dark-state silver cluster：
C-DNA chain, P-DNA chain molar ratio are 1：1, certain time is cultivated in PB buffer solution, forms complementary double-strand
Structure (P-DNA/C-DNA).Target is added and P-DNA/C-DNA is 1 in molar ratio：1, cultivate certain time, by S-DNA and
Target molar ratio 1：1 is added S-DNA, and then according to C-DNA, silver nitrate, sodium borohydride molar ratio 1:18:18, nitre is added
Sour silver aqueous solution and ice water brand-new sodium borohydride solution are cultivated under certain temperature and survey fluorescence after a certain period of time.
Active force difference between the DNA chain as caused by different complementary base radixes, object chain can be by P-DNA from P-DNA/
It is pulled out in C-DNA double-strand, forms P-DNA/Target double-strand and further to make the C-DNA released in conjunction with S-DNA
Dark-state silver cluster synthesizes template sequence and rich G sequence, to synthesize illuminated state silver cluster, fluorescence is stronger；And Target the case where being not present
Under, due to the stronger active force of P-DNA/C-DNA double-strand, silver-colored cluster synthesis template cannot be close with rich G sequence in S-DNA, synthesis
Silver-colored cluster is dark-state, and fluorescence is weaker, to realize the specific recognition of Target.
Biosensor based on strand displacement, dark-state silver cluster and exonucleaseⅢ, which removes, includes C-DNA chain, P-DNA chain and S-
It further include exonucleaseⅢ other than DNA chain, wherein C-DNA chain consists of two parts, and 5 ' ends are one section and contain 20 base richness G
Sequence, 3 ' ends are the sequences of one section with the partial complementarity of P-DNA chain, include 11 bases；P-DNA chain is one section and contains 18 alkali
Base and sequence with object chain (Target) complete complementary；S-DNA chain consists of two parts, and 5 ' ends are that the C-DNA of 8 bases is mutual
Complementary series, 3 ' ends are the dark-state silver cluster synthesis template sequences of 14 bases.
Application method with reference to Fig. 2, the biosensor based on strand displacement, dark-state silver cluster and exonucleaseⅢ is：
C-DNA chain, P-DNA chain molar ratio are 1：1, certain time is cultivated in PB buffer solution, forms complementary double-strand
Structure (P-DNA/C-DNA).Target is added and P-DNA/C-DNA is 1 in molar ratio：1, certain time is cultivated, is added outside 10U
Enzyme cutting III cultivates certain time under certain temperature, by S-DNA and Target molar ratio 1：1 is added S-DNA, and then according to C-
DNA, silver nitrate, sodium borohydride molar ratio 1:90:36, silver nitrate aqueous solution and ice water brand-new sodium borohydride solution is added, centainly
At a temperature of cultivate and survey fluorescence after a certain period of time.
Active force difference between the DNA chain as caused by different complementary base radixes, object chain can be by P-DNA from P-DNA/
It is pulled out in C-DNA double-strand, forms P-DNA/Target double-strand, but due to the P-DNA/ of Target and P-DNA formation
In Target double-strand, the 3 ' ends of P-DNA are fallen in, and are the restriction enzyme site of exonucleaseⅢ, to keep P-DNA/Target bis-
DE P-DNA is digested since 3 ' ends in chain, is released Target, is participated in the chain with P-DNA/C-DNA double-strand again
In displacement reaction, to realize the circulation amplification detection of target dna.And make the C-DNA released in conjunction with S-DNA, it furthers
Dark-state silver cluster synthesizes template sequence and rich G sequence, to synthesize illuminated state silver cluster, fluorescence is stronger；And Target the case where being not present
Under, due to the stronger active force of P-DNA/C-DNA double-strand, silver-colored cluster synthesis template cannot be close with rich G sequence in S-DNA, synthesis
Silver-colored cluster is dark-state, and fluorescence is weaker, to realize the specific recognition of Target.Although and C-DNA in P-DNA/C-DNA double-strand
Also there is 3 ' fallen in end, but due to only falling in 2 bases, the activity of exonucleaseⅢ weakens significantly, will not be disappeared substantially
Change, so will not be impacted to result.
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
Embodiment 1 (preparations of various stock solutions)
1.PB buffer (PB：20mM, pH=7.4) preparation：Accurately weigh Na2HPO4·12H2O 7.16g is burnt in 50mL
In cup, it is completely dissolved and is transferred in 100mL volumetric flask with triple distillation water, beaker and glass bar milli-Q water three
Secondary, cleaning solution is transferred in volumetric flask, is settled to 100mL, shakes up the disodium hydrogen phosphate stock solution that 100mL 0.2M can be obtained,
It is spare；Accurately weigh NaH2PO4·2H2O 3.12g is completely dissolved and is transferred in 50mL beaker, with triple distillation water
In 100mL volumetric flask, three times with milli-Q water, cleaning solution is transferred in volumetric flask for beaker and glass bar, is settled to 100mL,
The sodium dihydrogen phosphate stock solution that 100mL 0.2M can be obtained is shaken up, it is spare；Take above-mentioned configured stock solution：19mL
The NaH of 0.2M2PO4With the Na of 80mL 0.2M2HPO4, it is mixed in 1000mL volumetric flask, and be settled to triple distillation water
For 1000mL to get 20mM, the PB buffer of pH=7.4 is spare.
The configuration of 2.DNA solution：By P-DNA, C-DNA, S-DNA, Target is in centrifuge with 12000r/min revolving speed
It is centrifuged 1min, is dissolved with the PB buffer solution of corresponding volume, concentration is 100 μM, is put into 90 DEG C of baking ovens and cultivates after sealing
10min is put into 4 DEG C of refrigerators after cooled to room temperature and saves backup.
In embodiment, the sequence of P-DNA, C-DNA, S-DNA, Target are as shown in table 1.
Table 1
Wherein, C-DNA chain consists of two parts, and 5 ' ends are one section containing 20 base richness G sequences, i.e.,
GGGTGGGGTGGGGTGGGGGT, 3 ' ends are the sequences of one section with the partial complementarity of P-DNA chain, include 11 bases, i.e.,
GCAGGATTACG；
P-DNA chain is one section and contains 18 bases and the sequence with object chain (Target) complete complementary；
S-DNA chain consists of two parts, and 5 ' ends are the C-DNA complementary series of 8 bases, i.e. TCCTGCAC, 3 ' ends are 12
The dark-state silver cluster of a base synthesizes template sequence, i.e. CCCTTAATCCCC.
In Target, this two parts of GCTGGCCACGTGCAG, GATTACGGGGCGCTG are not missing portion in complete sequence
Divide the sequence of two sides.
3. silver nitrate (AgNO3) and sodium borohydride (NaBH4) solution preparation：15.3mg is accurately weighed under the conditions of being protected from light
AgNO3In 50mL centrifuge tube, it is settled to 50mL with the dissolution of triple distillation water, the AgNO of 1.8mM can be obtained3Solution, 4 DEG C of preservations
It is spare；3.4mg NaBH is accurately weighed under the conditions of being protected from light4In 50mL centrifuge tube, it is fixed to be dissolved with triple distillation water (mixture of ice and water)
Hold to 50mL, the NaBH of 1.8mM can be obtained4Solution is protected from light ready-to-use.
1 buffer of 4.Buffer (50mM Tris-HNO3, 10mM Mg (NO3)2, 1mM DTT, pH=7.0) preparation：
Accurately weigh 15.4mg DTT, 605.7mg Tris, 256.4mg Mg (NO3)2In 50mL beaker, with triple distillation water by its
It is completely dissolved, and uses HNO3PH to 7.0 is adjusted, is transferred in 100mL volumetric flask, beaker and glass bar milli-Q water three
Secondary, cleaning solution is transferred in volumetric flask, is settled to 100mL, and 1 buffer of Buffer can be obtained, spare.
5. exonucleaseⅢ dilution (1000units/mL) is prepared：Buying exonucleaseⅢ is 100,000units/mL,
2500units, 25 μ L quickly pipette 2475 μ L Buffer, 1 buffer using sterilizing pipette tips and are diluted to 2500 μ L, be configured to
1000units/mL solution, -20 DEG C save backup.
Embodiment 2 (sensor building-fluorescence detection based on strand displacement and dark-state silver cluster)
1. being separately added into C-DNA, C-DNA+P-DNA, C-DNA+P-DNA+Target into 2.0 milliliters of centrifuge tubes, each
The amount of every kind of DNA (100 μM) is 10 μ L in sample, and PB buffer solution is added, makes 200 μ L of system volume.It is cultivated at 37 DEG C
1h；
2. appropriate PB buffer solution is added, make 970 μ L of system volume, 10 μ L S-DNA (100 μM), 10 μ L are added
AgNO3(1.8mM), magnetic force shake 1min, cultivate 30min under room temperature dark-state；
3. 10 μ L brand-new NaBH are added4(1.8mM), magnetic force shake 1min, cultivate 5h under 4 DEG C of dark-state；
4. detecting fluorescence, it is 10nm, voltage 600V that parameter, which is set as slit,.(excitation wavelength 475nm, launch wavelength
For 635nm), Fig. 3 can be obtained.
Embodiment 3 (detects AIMP2 exon 2 gene delection based on the sensor of strand displacement and dark-state silver cluster)
1. 10 μ L C-DNA+P-DNA (being 100 μM) is added into 2.0 milliliters of centrifuge tubes, it is dense to be separately added into 10 μ L differences
Target, variety classes target ((being 100 μM)) are spent, PB buffer solution is added, makes 200 μ L of system volume.At 37 DEG C
Cultivate 1h；
2. appropriate PB buffer solution is added, make 970 μ L of system volume, 10 μ L S-DNA (100 μM), 10 μ L are added
AgNO3 (1.8mM), magnetic force shake 1min, cultivate 30min under room temperature dark-state；
3. 10 μ L brand-new NaBH4 (1.8mM) are added, magnetic force shakes 1min, cultivates 5h under 4 DEG C of dark-state；
4. detecting fluorescence, it is 10nm, voltage 600V that parameter, which is set as slit,.(excitation wavelength 475nm, launch wavelength
For 635nm), Fig. 4 and Fig. 5 can be respectively obtained, realizes the linearity test to object chain 0-320nM, detection is limited to
0.703nmol/L, and it is able to achieve specific detection.
Table 2
The above WT1 and WT2 respectively represents gene order when not lacking exon 2 near AIMP2 exon 2, MT1, MT2
The sequence that opposite 3,3 and 6 bases of Target sequence deletion are respectively represented with MT3, probes into other sequences using the above sequence
For the interference of AIMP2 exon 2 missing gene detection.
Embodiment 4 (sensor building-fluorescence detection based on strand displacement, exonucleaseⅢ and dark-state silver cluster)
1. being separately added into C-DNA, C-DNA+P-DNA, C-DNA+P-DNA+Target into 2.0 milliliters of centrifuge tubes, each
The amount of every kind of DNA (100 μM) is 10 μ L in sample, and 1 buffer solution of Buffer is added, and makes 198 μ L of system volume, and 2 μ L are added
Circumscribed enzyme solutions (1000units/mL).2h is cultivated at 37 DEG C；
2. appropriate PB buffer solution is added, make 920 μ L of system volume, 10 μ L S-DNA (100 μM), 50 μ L are added
AgNO3(1.8mM), magnetic force shake 1min, cultivate 30min under room temperature dark-state；
3. 20 μ L brand-new NaBH are added4(1.8mM), magnetic force shake 1min, cultivate 5h under 4 DEG C of dark-state；
4. detecting fluorescence, it is 10nm, voltage 600V that parameter, which is set as slit,.(excitation wavelength 640nm, launch wavelength
For 700nm), Fig. 6 can be obtained.
(the sensor detection AIMP2 exon 2 gene based on strand displacement, exonucleaseⅢ and dark-state silver cluster lacks embodiment 5
It loses)
1. 10 μ L C-DNA+P-DNA (being 100 μM) is added into 2.0 milliliters of centrifuge tubes, it is dense to be separately added into 10 μ L differences
Target, variety classes target ((being 100 μM)) are spent, 1 buffer solution of Buffer is added, makes 198 μ L of system volume, adds
Enter the 2 circumscribed enzyme solutions of μ L (1000units/mL).2h is cultivated at 37 DEG C；
2. appropriate PB buffer solution is added, make 920 μ L of system volume, 10 μ L S-DNA (100 μM), 50 μ L are added
AgNO3 (1.8mM), magnetic force shake 1min, cultivate 30min under room temperature dark-state；
3. 20 μ L brand-new NaBH4 (1.8mM) are added, magnetic force shakes 1min, cultivates 5h under 4 DEG C of dark-state；
4. detecting fluorescence, it is 10nm, voltage 600V that parameter, which is set as slit,.(excitation wavelength 475nm, launch wavelength
For 635nm), Fig. 7 and Fig. 8 can be respectively obtained, realizes the linearity test to object chain 0-100nM, detection is limited to
0.489nmol/L, and it is able to achieve specific detection.
The above description of the embodiments is intended to facilitate ordinary skill in the art to understand and use the invention.
Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein general
Principle is applied in other embodiments without having to go through creative labor.Therefore, the present invention is not limited to the above embodiments, ability
Field technique personnel announcement according to the present invention, improvement and modification made without departing from the scope of the present invention all should be of the invention
Within protection scope.
<110>Tongji University
<120>Biosensor and its application method based on strand displacement and dark-state silver cluster
<160> 9
<170> SIPOSequenceListing 1.0
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gggtggggtg gggtgggggt gcaggattac g 31
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<213>Artificial sequence (Artificial Sequence)
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cccgtaatcc tgcacgtg 18
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<213>Artificial sequence (Artificial Sequence)
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tcctgcaccc cttaatcccc 20
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<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 (6)
1. a kind of biosensor based on strand displacement and dark-state silver cluster, which is characterized in that include altogether three probe chains, be respectively
C-DNA chain, P-DNA chain and S-DNA chain,
Wherein, C-DNA chain consists of two parts, and 5 ' ends are containing rich G sequence, 3 ' ends and silver-colored cluster template strand S-DNA partial complementarity, together
When also with probe chain P-DNA partial complementarity, and the base number complementary with P-DNA be greater than and S-DNA complementary base number；
P-DNA chain contains can be with the sequence of object chain Target complete complementary；
S-DNA chain consists of two parts, and 5 ' ends are C-DNA complementary series, and 3 ' ends are dark-state silver cluster synthesis template sequences.
2. the application method of the biosensor as described in claim 1 based on strand displacement and dark-state silver cluster, which is characterized in that packet
Include following steps：
C-DNA chain, P-DNA chain molar ratio are 1：1, certain time is cultivated in PB buffer solution, forms complementary duplex structure,
Target is added and P-DNA/C-DNA is 1 in molar ratio：1, certain time is cultivated, by S-DNA and Target molar ratio 1：1 adds
Enter S-DNA, and then according to C-DNA, silver nitrate, sodium borohydride molar ratio 1:18:18, silver nitrate aqueous solution is added and ice water is new
Sodium borohydride solution processed is cultivated under certain temperature and surveys fluorescence after a certain period of time.
3. the application of the biosensor as described in claim 1 based on strand displacement and dark-state silver cluster, which is characterized in that so passing
Sensor is for detecting gene delection.
4. a kind of biosensor based on strand displacement, dark-state silver cluster and exonucleaseⅢ, which is characterized in that remove and include C-DNA
It further include exonucleaseⅢ other than chain, P-DNA chain and S-DNA chain,
Wherein, C-DNA chain consists of two parts, and 5 ' ends are containing rich G sequence, 3 ' ends and silver-colored cluster template strand S-DNA partial complementarity, together
When also with probe chain P-DNA partial complementarity, and the base number complementary with P-DNA be greater than and S-DNA complementary base number；
P-DNA chain contains can be with the sequence of object chain Target complete complementary；
S-DNA chain consists of two parts, and 5 ' ends are C-DNA complementary series, and 3 ' ends are dark-state silver cluster synthesis template sequences.
5. the application method of the biosensor as claimed in claim 4 based on strand displacement, dark-state silver cluster and exonucleaseⅢ,
It is characterized in that, includes the following steps：
C-DNA chain, P-DNA chain molar ratio are 1：1, certain time is cultivated in PB buffer solution, forms complementary duplex structure,
Target is added and P-DNA/C-DNA is 1 in molar ratio：1, it cultivates certain time, addition 10U exonucleaseⅢ, under certain temperature
Certain time is cultivated, by S-DNA and Target molar ratio 1：1 is added S-DNA, and then according to C-DNA, silver nitrate, hydroboration
Sodium molar ratio 1:90:36, silver nitrate aqueous solution and ice water brand-new sodium borohydride solution is added, cultivates certain time under certain temperature
After survey fluorescence.
6. the application of the biosensor as claimed in claim 4 based on strand displacement, dark-state silver cluster and exonucleaseⅢ, feature
It is, so sensor is for detecting gene delection.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109975561A (en) * | 2019-04-25 | 2019-07-05 | 河北医科大学 | A method of the super sensitivity detection dopamine based on aptamer |
| CN114002200A (en) * | 2021-11-01 | 2022-02-01 | 中国科学院苏州纳米技术与纳米仿生研究所 | Near-infrared two-region activated probe and application thereof |
Citations (1)
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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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| Publication number | Priority date | Publication date | Assignee | Title |
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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 (2)
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| JUHEE PARK，ET AL: "An approach toward SNP detection by modulating the fluorescence of DNA-templated silver nanoclusters", 《BIOSENSORS AND BIOELECTRONICS》 * |
| 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》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109975561A (en) * | 2019-04-25 | 2019-07-05 | 河北医科大学 | A method of the super sensitivity detection dopamine based on aptamer |
| CN109975561B (en) * | 2019-04-25 | 2021-11-09 | 河北医科大学 | Ultra-sensitive dopamine detection method based on aptamer |
| CN114002200A (en) * | 2021-11-01 | 2022-02-01 | 中国科学院苏州纳米技术与纳米仿生研究所 | Near-infrared two-region activated probe and application thereof |
| CN114002200B (en) * | 2021-11-01 | 2024-05-07 | 中国科学院苏州纳米技术与纳米仿生研究所 | Near infrared two-region activated probe and application thereof |
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