CN101948927A - Controllable distribution method of gold nanoparticles on DNA origami chip - Google Patents
Controllable distribution method of gold nanoparticles on DNA origami chip Download PDFInfo
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Abstract
The invention relates to a controllable distribution method of gold nanoparticles on the DNA origami chip in the nanometer technical field. The method comprises any one of the following schemes: 1) constructing an asymmetrical two-dimensional graph, preparing a DNA origami chip, preparing a colloidal gold probe, connecting the grain-size colloidal gold probe with the asymmetrical two-dimensional graph DNA origami chip and imaging with an atomic force microscope; 2) constructing an asymmetrical two-dimensional graph, preparing a DNA origami chip, preparing a colloidal gold probe, hybridizing the DNA strands with the colloidal gold probe, connecting the grain-size colloidal gold probe with the asymmetrical two-dimensional graph DNA origami chip and imaging with an atomic force microscope; and 3) constructing an asymmetrical two-dimensional graph, preparing a DNA origami chip, preparing a colloidal gold probe, hybridizing the DNA strands with the colloidal gold probe, connecting the grain-size colloidal gold probe with the asymmetrical two-dimensional graph DNA origami chip and imaging with an atomic force microscope. The controllable distribution method of the invention has wide application ranges in the subject fields such as the development of the slight trace biochemical detecting chip, the development of nano-devices and the research on the fundamental properties of nanoparticles.
Description
Technical field
What the present invention relates to is a kind of location mode of field of nanometer technology, is template based on asymmetric two-dimentional DNA paper folding art chip particularly, makes up the controlled distribution method of 5nm nano particle on DNA paper folding chip.
Background technology
Nanometer gold (nanogold) is meant the molecule of gold, and its diameter is 1～100nm, is generally to be dispersed in the colloidal solution that forms in the water, so claim Radioactive colloidal gold again.Certain chemical substance in the nm gold particles surface adsorption, the monomer nm gold particles is very stable.Golden nanometer particle electron density height has quantum size effect, and small volume effect and surface effects can be passed through multiple action modes such as electrostatic attraction, hydrophobic effect and combine the stable colloidal gold probe of formation with macromole.Over more than 40 year, nm gold particles has been widely used in the biotechnologys such as immunocytochemistry and biomarker in the past.
2006, the Rothemund of California Institute of Technology proposed, designs and realized DNA paper folding art.[Rothemund?PW.Nature?2006，440，297]。Rothemund has folded six kinds of figures in initial article, though differ from one another, be not difficult to find that they all are symmetric figures.The DNA paper folding art nanometer gold of arranging can be divided three classes substantially according to the different of methods: the first kind is an oligonucleotide hybridization assay.On nm gold particles, modify certain oligonucleotide chain (actual gold goal surface can connect a lot of chains), then it is added in the paper folding art figure, owing to can stretch out and this oligonucleotide chain complementary probe on the staple chain, so gold goal will be attached to [Le JD on the specific position of figure by the hybridization of nucleic acid chains, Pinto Y, Seeman NC, et al.Nano Letters 2004,4,2343].Second class is direct construction from part.Since nanometer gold can combine with DNA, so just might allow it participate in paper folding art figure self assembling process directly, when self-assembly is finished, also just formed these micromolecular arranging [Sharma J, Chhabra R, Andersen CS, et al.J Am Chem Soc 2008,130,7820].The 3rd class is an additive method.Other methods that connect nanometer gold also have a lot, such as the molecule photolithography (molecular lithography) [Deng Z, Mao C.Angew Chem Int Ed Engl 2004,43,4068] of Chengde Mao group exploitation, or the like.
Open source literature has been put down in writing Zhang Zhao etc. and has been delivered space by name addressable exempt to disclose DNA paper folding chip staple chain-ordering table [Zhao Z in the asymmetric DNA paper folding of the liquid phase chip article of index on advanced material, Ying W, et al.Adv Mater 2010,22,1], the asymmetric two-dimentional DNA paper folding art of the imitative map of China shape of this article utilization is template, on template the design and draw specific oligonucleotide sequence as connection site, by hybridizing combination with the biotin-avidin system of having modified complementary sequence DNA short chain, nanoparticle is attached on the paper folding chip, simultaneously, also the binding site of nanoparticle is compared.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, the controlled distribution method of a kind of nano particle on DNA paper folding chip is provided.The present invention adopts the identical DNA paper folding chip staple chain-ordering table of prior art, and direction is from left to right to be 5 '-3 '.But, the position of design binding site and stretch out specific oligonucleotide sequence when preparation DNA paper folding chip on the template among the present invention, the staple chain that needs the staple chain that band stretch out oligonucleotide capture probe to be replaced same sequence number in the tabulation gets final product.The method of the present invention by utilizing oligonucleotide sequence to modify and hybridize connects 5nm particle diameter colloid gold particle on the nanometer paper folding art structure of imitative map of China shape, thereby realizes the controlled distribution of nano particle on DNA paper folding chip.
The present invention realizes by following three kinds of technical schemes:
Technical scheme one
Technical scheme one comprises that step is as follows:
The first step is utilized asymmetric X-Y scheme of DNA paper folding art structure, preparation DNA paper folding chip;
Described asymmetric X-Y scheme stretches out oligonucleotide capture probe at the staple chain end of specific position, allows the object chain of it and end modified 5nm particle diameter colloid gold particle hybridize one to one.
Described asymmetric X-Y scheme is the single stranded DNA that relies on the M13mp18 phage, reaches the folding formation of 7249 base scaffolding long-chain grating filling types, and figure is then from 229 that are used to bind short staple chains.
Described DNA paper folding chip production method:
(1) all the 100 μ M staple chains that will use, comprise that forming equal-volume asymmetric X-Y scheme, that stretch out probe mixes, 20 times of redilution are standby;
It is 9 that the staple chain stretches out the oligonucleotide capture probe sequence number.
(2) mixing total amount on 96 orifice plates is the solution of 30 μ l: 0.025 μ M staple chain: 22 μ l; The M13mp18:1 μ l of 1/2 concentration; 1 * TAE-Mg2+buffer:7 μ l.
Second step, the preparation colloidal gold probe;
Colloidal gold probe preparation method described in second step:
1. oligonucleotide adds in the 10mM phosphoric acid buffer with 5nm particle diameter Radioactive colloidal gold molar concentration rate 200 to 1, and room temperature 300rpm shook 24 hours.
2. add final concentration 0.1M NaCl, room temperature 300rpm shook 48 hours.
3. 4 ℃, centrifugal 10 minutes of 15000rpm abandons supernatant.
4. add the 10mM phosphoric acid buffer, final concentration 0.1M NaCl cleans twice.
5. add 1xTE; 10mM Tris-HCl, 1mM EDTA, pH=8.0,4 ℃ of preservations.
6. 5nm particle diameter colloidal gold probe concentration utilizes 520nm wavelength ultraviolet absorption peak to measure.
In the 3rd step, the particle diameter colloidal gold probe is connected with asymmetric X-Y scheme DNA paper folding chip;
Connection described in the 3rd step is meant: 5nm particle diameter colloidal gold probe mixes with asymmetric X-Y scheme DNA paper folding chip molar concentration rate 2 to 1, and 37 ℃ of water-baths were hybridized 1 hour.
The 4th step, the atomic force microscope imaging;
The step of atomic force microscope imaging described in the 4th step: draw 2.5 μ l samples (actual 2-5 μ l all can) and drip to and newly cut mica surface, adsorb and to take imaging under the atomic force microscope after 2 minutes.The unified pattern, liquid phase imaging of rapping of using.
The atomic force microscope imaging results shows, adopts technical scheme one described method, and nm gold particles joint efficiency on DNA paper folding art figure can reach about 10%.
Technical scheme two
Consider that the oligonucleotide probe that is connected on the gold size particle exists about 50 ℃ active temperature threshold value (meltingtemperature), be lower than this temperature oligonucleotide probe and be adsorbed on the gold size particle surface and lost the ability most of and link of capture probe chain, the result causes joint efficiency very low.When hybridization temperature was higher than 50 ℃, imitative map of China paper folding figure the localized heat degraded can occur and be connected with irregular, loses original pattern.So hybridization is fixed near on the oligonucleotide probe of gold grain bottom, influences joint efficiency to avoid oligonucleotide probe to be adsorbed on the gold size particle surface.
Technical scheme two comprises that step is as follows:
The first step is utilized asymmetric X-Y scheme of DNA paper folding art structure, preparation DNA paper folding chip;
Described asymmetric X-Y scheme is the single stranded DNA that relies on the M13mp18 phage, reaches the folding formation of 7249 base scaffolding long-chain grating filling types, and figure is then from 229 that are used to bind short staple chains.
Described DNA paper folding chip production method:
(1) all the 100 μ M staple chains that will use, comprise that forming equal-volume asymmetric X-Y scheme, that stretch out probe mixes, 20 times of redilution are standby;
It is 9 that the staple chain stretches out the oligonucleotide capture probe sequence number.
(2) mixing total amount on 96 orifice plates is the solution of 30 μ l: 0.025 μ M staple chain: 22 μ l; The M13mp18:1 μ l of 1/2 concentration; 1 * TAE-Mg2+buffer:7 μ l.
Second step, the preparation colloidal gold probe;
Described colloidal gold probe, before colloidal gold probe was connected into map, earlier with a 10nt short chain, in the time of 50 ℃, hybridization was fixed near on the oligonucleotide probe of gold grain bottom.
Described colloidal gold probe preparation method may further comprise the steps:
1. oligonucleotide adds in the 10mM phosphoric acid buffer with 5nm particle diameter Radioactive colloidal gold molar concentration rate 200 to 1, and room temperature 300rpm shook 24 hours.
2. add final concentration 0.1M NaCl, room temperature 300rpm shook 48 hours.
3. 4 ℃, centrifugal 10 minutes of 15000rpm abandons supernatant.
4. add the 10mM phosphoric acid buffer, final concentration 0.1M NaCl cleans twice.
5. add 1xTE; 10mM Tris-HCl, 1mM EDTA, pH=8.0,4 ℃ of preservations.
6. 5nm particle diameter colloidal gold probe concentration utilizes 520nm wavelength ultraviolet absorption peak to measure.
The 3rd step, DNA short chain and colloidal gold probe hybridization;
Hybridization described in the 3rd step is meant: 5nm particle diameter colloidal gold probe mixes with 10nt DNA short chain molar concentration rate 1 to 1, and 50 ℃ of water-baths were hybridized 1 hour.
In the 4th step, the particle diameter colloidal gold probe is connected with asymmetric X-Y scheme DNA paper folding chip;
Connection described in the 4th step is meant: 5nm particle diameter colloidal gold probe mixes with asymmetric X-Y scheme DNA paper folding chip molar concentration rate 2 to 1, and 37 ℃ of water-baths were hybridized 1 hour.
The 5th step, the atomic force microscope imaging;
The step of atomic force microscope imaging described in the 5th step: draw 2.5 μ l samples (actual 2-5 μ l all can) and drip to and newly cut mica surface, adsorb and to take imaging under the atomic force microscope after 2 minutes.The unified pattern, liquid phase imaging of rapping of using.
The atomic force microscope imaging results shows, adopts technical scheme two described methods, and nm gold particles joint efficiency on DNA paper folding art figure slightly improves, and can reach about 15%.
Technical scheme three
Technical scheme three comprises that step is as follows:
The first step is utilized asymmetric X-Y scheme of DNA paper folding art structure, preparation DNA paper folding chip;
Described asymmetric X-Y scheme stretches out three staple chain end modified oligonucleotide capture probes near specific position, allow three pairs one hybridization of reporter probe chain of they and same end modified 5nm particle diameter colloid gold particle.
Described asymmetric X-Y scheme is the single stranded DNA that relies on the M13mp18 phage, reaches the folding formation of 7249 base scaffolding long-chain grating filling types, and figure is then from 229 that are used to bind short staple chains.
Described DNA paper folding chip production method:
(1) all the 100 μ M staple chains that will use, comprise that forming equal-volume asymmetric X-Y scheme, that stretch out probe mixes, 20 times of redilution are standby;
It is 9,10,22 that the staple chain stretches out the oligonucleotide capture probe sequence number.
(2) mixing total amount on 96 orifice plates is the solution of 30 μ l: 0.025 μ M staple chain: 22 μ l; The M13mp18:1 μ l of 1/2 concentration; 1 * TAE-Mg2+buffer:7 μ l.
Second step, the preparation colloidal gold probe;
Colloidal gold probe preparation method described in second step:
1. oligonucleotide adds in the 10mM phosphoric acid buffer with 5nm particle diameter Radioactive colloidal gold molar concentration rate 200 to 1, and room temperature 300rpm shook 24 hours.
2. add final concentration 0.1M NaCl, room temperature 300rpm shook 48 hours.
3. 4 ℃, centrifugal 10 minutes of 15000rpm abandons supernatant.
4. add the 10mM phosphoric acid buffer, final concentration 0.1M NaCl cleans twice.
5. add 1xTE; 10mM Tris-HCl, 1mM EDTA, pH=8.0,4 ℃ of preservations.
6. 5nm particle diameter colloidal gold probe concentration utilizes 520nm wavelength ultraviolet absorption peak to measure.
The 3rd step, DNA short chain and colloidal gold probe hybridization;
Hybridization described in the 3rd step is meant: 5nm particle diameter colloidal gold probe mixes with 10nt DNA short chain molar concentration rate 1 to 1, and 50 ℃ of water-baths were hybridized 1 hour.
In the 4th step, the particle diameter colloidal gold probe is connected with asymmetric X-Y scheme DNA paper folding chip;
Connection described in the 4th step is meant: 5nm particle diameter colloidal gold probe mixes with asymmetric X-Y scheme DNA paper folding chip molar concentration rate 2 to 1, and 37 ℃ of water-baths were hybridized 1 hour.
The 5th step, the atomic force microscope imaging;
The step of atomic force microscope imaging described in the 5th step: draw 2.5 μ l samples (actual 2-5 μ l all can) and drip to and newly cut mica surface, adsorb and to take imaging under the atomic force microscope after 2 minutes.The unified pattern, liquid phase imaging of rapping of using.
Near paper folding figure specific position, stretch out three staple chain end modified oligonucleotide capture probes, allow three pairs one hybridization of reporter probe chain of they and same end modified 5nm particle diameter colloid gold particle, association schemes two again, make the gold grain locus be effectively controlled and strengthened connection stability simultaneously.The atomic force microscope imaging results shows, adopts technical scheme three described methods, and nm gold particles joint efficiency on DNA paper folding art figure obviously improves, and can reach more than 95%.
Embodiment
Present embodiment has provided detailed embodiment and process being to implement under the prerequisite with the technical solution of the present invention, but protection scope of the present invention is not limited to following embodiment.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
Embodiment 1
Testing conditions:
Present embodiment adopts DNA paper folding art to construct the shape of map of China, and the atomic force microscope imaging results shows about 150 nanometers of strong point of resulting map of China, and about 120 nanometers of the widest part highly are 2 nanometers.This figure is the folding formation that relies on scaffolding long-chain (single stranded DNA of M13mp18 phage reaches 7249 bases) grating filling type, and the map pattern is then from 229 that are used to bind short staple chains.The characteristics of this figure are that 229 staple chains are exactly more than 200 addressable potential reaction site, and any point on the masterplate can utilize figure self as with reference to accurately locating, and need not index marker.In addition, any one in 229 short dna strands can be stretched out nucleotide sequence and hybridizes as the capture probe and the complementary nucleotide sequence of band nanoparticle label, as the tie point of nano particle on the paper folding figure.When utilizing this asymmetrical graphic to distribute, utilize the template complicacy to reduce the complicacy requirement that nano dot is distributed for template forms nano dot.Asymmetric X-Y scheme described in the present embodiment stretches out oligonucleotide capture probe at the staple chain end of specific position, allows the object chain of it and end modified 5nm particle diameter colloid gold particle hybridize one to one.
Present embodiment is chip base with the map of China, all staple chains, comprise form map, stretch out probe, all order (PAGE purifying), and unified to be diluted to 100 μ M with secondary water standby from Shanghai JaRa company.The scaffolding chain uses the M13mp18 virus strand of NEB company, article No. N4040S.Be total to 7249base, concentration is 0.25 μ g/ μ l, total 5 μ g.Because so 1 μ g ≈ 0.42pmol is the about 0.1pM/ μ of concentration l=0.1 μ M.Actual 1/2 concentration that is diluted to is used.Cut processing without enzyme, retaining ring chain state.5nm particle diameter colloid gold particle uses Sigma company, article No. G1402.Use identical reaction buffer and imaging buffer: at 1 * TAE (Tris, 40mM; Acetate, 20mM; EDTA, 2mM) the middle magnesium acetate powder that adds, the Mg2+ final concentration is 12.5mM, pH8.0.Use multiple-pattern atomic force microscope (Multimode AFM) observation of U.S. Veeco/Digital Instruments company.J-scanner, the NP-S needle point, resonant frequency is between 7-10kHz.
The controlled distribution implementation step of the nano particle of present embodiment is as follows:
First: the imitative map of China shape DNA paper folding chip of preparation
1. all the staple chains that will use (100 μ M) are comprised that forming equal-volume map, that stretch out probe mixes, 20 times of redilution are standby.It is 9 that the staple chain stretches out the oligonucleotide capture probe sequence number.
On 96 orifice plates according to following system mixing solutions: (below be the system of " saving ", can be whole double during actual doing, or double the scaffolding chain separately)
Staple chain (0.025 μ M) 22 μ l
M13mp18 (1/2 concentration) 1 μ l
1×TAE-Mg2+buffer 7μl
Total 30μl
3. be placed on PCR instrument (ABI 9700) and go up annealing, 95 ℃ are cooled to 20 ℃, 0.1 ℃/10s=0.6 ℃/min=1 ℃/100s.
Second: preparation 5nm particle diameter colloidal gold probe
1. oligonucleotide adds in the 10mM phosphoric acid buffer with 5nm particle diameter colloid gold particle molar concentration rate 200 to 1, and room temperature 300rpm shook 24 hours.
2. add final concentration 0.1M NaCl, room temperature 300rpm shook 48 hours.
3.4 ℃, centrifugal 10 minutes of 15000rpm abandons supernatant.
4. add the 10mM phosphoric acid buffer, final concentration 0.1M NaCl cleans twice.
5. add 1xTE (10mM Tris-HCl, 1mM EDTA, pH=8.0), 4 ℃ of preservations.
6.5nm particle diameter colloid gold particle concentration and probe concentration utilizes 0D260 to measure.
The 3rd: 5nm particle diameter colloid gold particle probe is connected with imitative map of China shape DNA paper folding chip
5nm particle diameter colloid gold particle probe mixes with imitative map of China shape DNA paper folding chip molar concentration rate 2 to 1, and 37 ℃ of water-baths were hybridized 1 hour.
The the 4th: the atomic force microscope imaging
Draw 2.5 μ l samples (actual 2-5 μ l all can) and drip to and newly cut mica surface, adsorb and to take imaging under the atomic force microscope after 2 minutes.The unified pattern, liquid phase imaging of rapping of using.
The atomic force microscope imaging results shows that present embodiment nm gold particles joint efficiency on DNA paper folding art figure can reach about 10%.
Embodiment 2
Testing conditions:
Present embodiment adopts DNA paper folding art to construct the shape of map of China, and the atomic force microscope imaging results shows about 150 nanometers of strong point of resulting map of China, and about 120 nanometers of the widest part highly are 2 nanometers.This figure is the folding formation that relies on scaffolding long-chain (single stranded DNA of M13mp18 phage reaches 7249 bases) grating filling type, and the map pattern is then from 229 that are used to bind short staple chains.The characteristics of this figure are that 229 staple chains are exactly more than 200 addressable potential reaction site, and any point on the masterplate can utilize figure self as with reference to accurately locating, and need not index marker.In addition, any one in 229 short dna strands can be stretched out nucleotide sequence and hybridizes as the capture probe and the complementary nucleotide sequence of band nanoparticle label, as the tie point of nano particle on the paper folding figure.When utilizing this asymmetrical graphic to distribute, utilize the template complicacy to reduce the complicacy requirement that nano dot is distributed for template forms nano dot.Consider in the present embodiment that the oligonucleotide probe that is connected on the gold size particle exists about 50 ℃ active temperature threshold value (melting temperature), be lower than this temperature oligonucleotide probe and be adsorbed on the gold size particle surface and lost the ability most of and link of capture probe chain, the result causes joint efficiency very low.When hybridization temperature was higher than 50 ℃, imitative map of China paper folding figure the localized heat degraded can occur and be connected with irregular, loses original pattern.So hybridization is fixed near on the oligonucleotide probe of gold grain bottom, influences joint efficiency to avoid oligonucleotide probe to be adsorbed on the gold size particle surface.
Present embodiment is chip base with the map of China, all staple chains, comprise form map, stretch out probe, all order (PAGE purifying), and unified to be diluted to 100 μ M with secondary water standby from Shanghai JaRa company.The scaffolding chain uses the M13mp18 virus strand of NEB company, article No. N4040S.Be total to 7249base, concentration is 0.25 μ g/ μ l, total 5 μ g.Because so 1 μ g ≈ 0.42pmol is the about 0.1pM/ μ of concentration l=0.1 μ M.Actual 1/2 concentration that is diluted to is used.Cut processing without enzyme, retaining ring chain state.5nm particle diameter colloid gold particle uses Sigma company, article No. G1402.Use identical reaction buffer and imaging buffer: at 1 * TAE (Tris, 40mM; Acetate, 20mM; EDTA, 2mM) the middle magnesium acetate powder that adds, the Mg2+ final concentration is 12.5mM, pH8.0.Use multiple-pattern atomic force microscope (Multimode AFM) observation of U.S. Veeco/Digital Instruments company.J-scanner, the NP-S needle point, resonant frequency is between 7-10kHz.
The controlled distribution implementation step of the nano particle of present embodiment is as follows:
First: the imitative map of China shape DNA paper folding chip of preparation
1. all the staple chains that will use (100 μ M) are comprised that forming equal-volume map, that stretch out probe mixes, 20 times of redilution are standby.It is 9 that the staple chain stretches out the oligonucleotide capture probe sequence number.
On 96 orifice plates according to following system mixing solutions: (below be the system of " saving ", can be whole double during actual doing, or double the scaffolding chain separately)
Staple chain (0.025 μ M) 22 μ l
M13mp18 (1/2 concentration) 1 μ l
1×TAE-Mg2+buffer 7μl
Total 30μl
3. be placed on PCR instrument (ABI 9700) and go up annealing, 95 ℃ are cooled to 20 ℃, 0.1 ℃/10s=0.6 ℃/min=1 ℃/100s.
Second: preparation 5nm particle diameter colloidal gold probe
1. oligonucleotide adds in the 10mM phosphoric acid buffer with 5nm particle diameter colloid gold particle molar concentration rate 200 to 1, and room temperature 300rpm shook 24 hours.
2. add final concentration 0.1M NaCl, room temperature 300rpm shook 48 hours.
3.4 ℃, centrifugal 10 minutes of 15000rpm abandons supernatant.
4. add the 10mM phosphoric acid buffer, final concentration 0.1M NaCl cleans twice.
5. add 1xTE (10mM Tris-HCl, 1mM EDTA, pH=8.0), 4 ℃ of preservations.
6.5nm particle diameter colloid gold particle concentration and probe concentration utilizes OD260 to measure.
The the 3rd: 10nt DNA short chain and 5nm particle diameter colloid gold particle probe hybridization
5nm particle diameter colloid gold particle probe mixes with 10nt DNA short chain molar concentration rate 1 to 1, and 50 ℃ of water-baths were hybridized 1 hour.
The 4th: 5nm particle diameter colloid gold particle probe is connected with imitative map of China shape DNA paper folding chip
5nm particle diameter colloid gold particle probe mixes with imitative map of China shape DNA paper folding chip molar concentration rate 2 to 1, and 37 ℃ of water-baths were hybridized 1 hour.
The the 5th: the atomic force microscope imaging
Draw 2.5 μ l samples (actual 2-5 μ l all can) and drip to and newly cut mica surface, adsorb and to take imaging under the atomic force microscope after 2 minutes.The unified pattern, liquid phase imaging of rapping of using.
The atomic force microscope imaging results shows that present embodiment nm gold particles joint efficiency on DNA paper folding art figure slightly improves, and can reach about 15%.
Embodiment 3
Testing conditions:
Present embodiment adopts DNA paper folding art to construct the shape of map of China, and the atomic force microscope imaging results shows about 150 nanometers of strong point of resulting map of China, and about 120 nanometers of the widest part highly are 2 nanometers.This figure is the folding formation that relies on scaffolding long-chain (single stranded DNA of M13mp18 phage reaches 7249 bases) grating filling type, and the map pattern is then from 229 that are used to bind short staple chains.The characteristics of this figure are that 229 staple chains are exactly more than 200 addressable potential reaction site, and any point on the masterplate can utilize figure self as with reference to accurately locating, and need not index marker.In addition, any one in 229 short dna strands can be stretched out nucleotide sequence and hybridizes as the capture probe and the complementary nucleotide sequence of band nanoparticle label, as the tie point of nano particle on the paper folding figure.When utilizing this asymmetrical graphic to distribute, utilize the template complicacy to reduce the complicacy requirement that nano dot is distributed for template forms nano dot.Near paper folding figure specific position, stretch out three staple chain end modified oligonucleotide capture probes in the present embodiment, allow three pairs one hybridization of reporter probe chain of they and same end modified 5nm particle diameter colloid gold particle, association schemes two again, make the gold grain locus be effectively controlled and strengthened connection stability simultaneously.
Present embodiment is chip base with the map of China, all staple chains, comprise form map, stretch out probe, all order (PAGE purifying), and unified to be diluted to 100 μ M with secondary water standby from Shanghai JaRa company.The scaffolding chain uses the M13mp18 virus strand of NEB company, article No. N4040S.Be total to 7249base, concentration is 0.25 μ g/ μ l, total 5 μ g.Because so 1 μ g ≈ 0.42pmol is the about 0.1pM/ μ of concentration l=0.1 μ M.Actual 1/2 concentration that is diluted to is used.Cut processing without enzyme, retaining ring chain state.5nm particle diameter colloid gold particle uses Sigma company, article No. G1402.Use identical reaction buffer and imaging buffer: at 1 * TAE (Tris, 40mM; Acetate, 20mM; EDTA, 2mM) the middle magnesium acetate powder that adds, the Mg2+ final concentration is 12.5mM, pH8.0.Use multiple-pattern atomic force microscope (Multimode AFM) observation of U.S. Veeco/Digital Instruments company.J-scanner, the NP-S needle point, resonant frequency is between 7-10kHz.
The controlled distribution implementation step of the nano particle of present embodiment is as follows:
First: the imitative map of China shape DNA paper folding chip of preparation
1. all the staple chains that will use (100 μ M) are comprised that forming equal-volume map, that stretch out probe mixes, 20 times of redilution are standby.It is 9,10,22 that the staple chain stretches out the oligonucleotide capture probe sequence number.
On 96 orifice plates according to following system mixing solutions: (below be the system of " saving ", can be whole double during actual doing, or double the scaffolding chain separately)
Staple chain (0.025 μ M) 22 μ l
M13mp18 (1/2 concentration) 1 μ l
1×TAE-Mg2+buffer 7μl
Total 30μl
3. be placed on PCR instrument (ABI 9700) and go up annealing, 95 ℃ are cooled to 20 ℃, 0.1 ℃/10s=0.6 ℃/min=1 ℃/100s.
Second: preparation 5nm particle diameter colloidal gold probe
1. oligonucleotide adds in the 10mM phosphoric acid buffer with 5nm particle diameter colloid gold particle molar concentration rate 200 to 1, and room temperature 300rpm shook 24 hours.
2. add final concentration 0.1M NaCl, room temperature 300rpm shook 48 hours.
3.4 ℃, centrifugal 10 minutes of 15000rpm abandons supernatant.
4. add the 10mM phosphoric acid buffer, final concentration 0.1M NaCl cleans twice.
5. add 1xTE (10mM Tris-HCl, 1mM EDTA, pH=8.0), 4 ℃ of preservations.
6.5nm particle diameter colloid gold particle concentration and probe concentration utilizes 0D260 to measure.
The the 3rd: 10nt DNA short chain and 5nm particle diameter colloid gold particle probe hybridization
5nm particle diameter colloid gold particle probe mixes with 10nt DNA short chain molar concentration rate 1 to 1, and 50 ℃ of water-baths were hybridized 1 hour.
The 4th: 5nm particle diameter colloid gold particle probe is connected with imitative map of China shape DNA paper folding chip
5nm particle diameter colloid gold particle probe mixes with imitative map of China shape DNA paper folding chip molar concentration rate 2 to 1, and 37 ℃ of water-baths were hybridized 1 hour.
The the 5th: the atomic force microscope imaging
Draw 2.5 μ l samples (actual 2-5 μ l all can) and drip to and newly cut mica surface, adsorb and to take imaging under the atomic force microscope after 2 minutes.The unified pattern, liquid phase imaging of rapping of using.
The atomic force microscope imaging results shows that present embodiment nm gold particles joint efficiency on DNA paper folding art figure obviously improves, and can reach more than 95%.
DNA paper folding chip staple chain stretches out the oligonucleotide capture probe sequence, and following (stretched partly is the staple chain, italicized item is to stretch out oligonucleotide capture probe, from left to right be 5 '-3 ', during preparation DNA paper folding chip, replace same sequence number staple chain):
9CGCCTAGTTGGACGGCGACACAGGAGGTAGTGCCGTCGAGAGGG (being used for technical scheme one, two, three)
10CGCCTAGTTGGACGGCGACACAGTACCAGGCGGATATTAGCGGG (being used for technical scheme three)
22CGCCTAGTTGGACGGCGACAGTTTTGCTAAGAGAAGGATTAGGAGAGGCTGA (being used for technical scheme three)
10nt DNA short chain sequence (from left to right being 5 '-3 ')
CGCCTAGTTG
Mercapto groups is modified reporter probe sequence (from left to right being 5 '-3 ')
TGTCGCCGTCCAACTAGGCG-SH
The atomic force microscope imaging results of above embodiment shows respectively: nm gold particles joint efficiency on DNA paper folding art figure can reach 10%-15%, and high energy reaches more than 95%.
Claims (6)
1. the controlled distribution method of a nano particle on DNA paper folding chip is characterized in that described method is any of following three kinds of technical schemes:
Technical scheme one comprises the steps:
The first step is utilized asymmetric X-Y scheme of DNA paper folding art structure, preparation DNA paper folding chip,
Second step, the preparation colloidal gold probe,
In the 3rd step, the particle diameter colloidal gold probe is connected with asymmetric X-Y scheme DNA paper folding chip,
The 4th step, the atomic force microscope imaging,
Described asymmetric X-Y scheme stretches out oligonucleotide capture probe at the staple chain end of specific position, allows the object chain of it and end modified 5nm particle diameter colloid gold particle hybridize one to one;
Technical scheme two comprises the steps:
The first step is utilized asymmetric X-Y scheme of DNA paper folding art structure, preparation DNA paper folding chip,
Second step, the preparation colloidal gold probe,
The 3rd step, DNA short chain and colloidal gold probe hybridization,
In the 4th step, the particle diameter colloidal gold probe is connected with asymmetric X-Y scheme DNA paper folding chip,
The 5th step, the atomic force microscope imaging,
Described colloidal gold probe, before colloidal gold probe was connected into map, earlier with a 10nt short chain, in the time of 50 ℃, hybridization was fixed near on the oligonucleotide probe of gold grain bottom;
Technical scheme three comprises the steps:
The first step is utilized asymmetric X-Y scheme of DNA paper folding art structure, preparation DNA paper folding chip,
Second step, the preparation colloidal gold probe,
The 3rd step, DNA short chain and colloidal gold probe hybridization,
In the 4th step, the particle diameter colloidal gold probe is connected with asymmetric X-Y scheme DNA paper folding chip,
The 5th step, the atomic force microscope imaging,
Described asymmetric X-Y scheme stretches out three staple chain end modified oligonucleotide capture probes near specific position, allow three pairs one hybridization of reporter probe chain of they and same end modified 5nm particle diameter colloid gold particle.
2. the controlled distribution method of nano particle according to claim 1 on DNA paper folding chip is characterized in that, described DNA paper folding chip, and its preparation method is as follows:
(1) all the 100 μ M staple chains that will use, comprise that forming equal-volume asymmetric X-Y scheme, that stretch out probe mixes, 20 times of redilution are standby;
(2) mixing total amount on 96 orifice plates is the solution of 30 μ l: 0.025 μ M staple chain: 22 μ l; The M13mp18:1 μ l of 1/2 concentration; 1 * TAE-Mg2+buffer:7 μ l.
3. the controlled distribution method of nano particle according to claim 1 on DNA paper folding chip is characterized in that, described colloidal gold probe, and its preparation method:
1. oligonucleotide adds in the 10mM phosphoric acid buffer with 5nm particle diameter Radioactive colloidal gold molar concentration rate 200 to 1, and room temperature 300rpm shook 24 hours;
2. add final concentration 0.1M NaCl, room temperature 300rpm shook 48 hours;
3. 4 ℃, centrifugal 10 minutes of 15000rpm abandons supernatant;
4. add the 10mM phosphoric acid buffer, final concentration 0.1M NaCl cleans twice;
5. add 1xTE; 10mM Tris-HCl, 1mM EDTA, pH=8.0,4 ℃ of preservations;
6. 5nm particle diameter colloidal gold probe concentration utilizes 520nm wavelength ultraviolet absorption peak to measure.
4. the controlled distribution method of nano particle according to claim 1 on DNA paper folding chip is characterized in that described hybridization is meant: 5nm particle diameter colloidal gold probe mixes with 10nt DNA short chain molar concentration rate 1 to 1, and 50 ℃ of water-baths were hybridized 1 hour.
5. the controlled distribution method of nano particle according to claim 1 on DNA paper folding chip, it is characterized in that, described connection is meant: 5nm particle diameter colloidal gold probe mixes with asymmetric X-Y scheme DNA paper folding chip molar concentration rate 2 to 1, and 37 ℃ of water-baths were hybridized 1 hour.
6. the controlled distribution method of nano particle according to claim 1 on DNA paper folding chip, it is characterized in that, described atomic force microscope imaging is meant: absorption 2-5 μ l sample drips to newly cuts mica surface, adsorbs and can take imaging under the atomic force microscope after 2 minutes.
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