CN107596436A

CN107596436A - A kind of DNA fluorescence hydrogel and preparation method thereof

Info

Publication number: CN107596436A
Application number: CN201710885237.6A
Authority: CN
Inventors: 仰大勇; 耿金慧; 寇晓虹
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2017-09-26
Filing date: 2017-09-26
Publication date: 2018-01-19

Abstract

The invention discloses a kind of DNA fluorescence hydrogel and preparation method thereof, preparation method is：(1) preparation of DNA hydrogels：(2) DNA hydrogels are put into aqua sterilisa, change water 1 time every 15~30min, change water altogether 3~5 times；(3) step (2) is obtained into DNA hydrogels, adds phosphate buffer, AgNO₃The aqueous solution, reaction；Add NaBH₄The aqueous solution, reaction, obtains DNA fluorescence hydrogel.The DNA fluorescence hydrogel of the present invention, good biocompatibility and material softness, as medical dressing, the secondary damage of the surface of a wound can be effectively avoided, mitigates wound pain；And the hydrogel carries fluorescence, there is certain antibiotic property, no cytotoxicity.Prepare the process of hydrogel simply, conveniently.The fields such as medical dressing, immunization therapy, organizational project, unicellular culture, medicament slow release, acellular albumen synthesis and biology sensor can be applied to.

Description

A kind of DNA fluorescence hydrogel and preparation method thereof

Technical field

The invention belongs to the preparation of DNA (DNA) material functional and application field, it is related to a kind of deoxyribose Nucleic acid fluorescent hydrogel and preparation method thereof.

Background technology

Wound dressing can play protection wound, prevent from infecting, and promote wound to recover the effect of healing, and it widely should Place is treated for various medical treatment and shield.At present, it is generally natural to form the material of aerogel dressing, semi-synthetic or synthesis high score Son, such as：Fiber analog derivative (carboxyl methyl cellulose), gelatin, polypeptide, poly- polysaccharide, alginate, methacrylate Deng.The casting product that these materials are formed is rich in moisture more, can provide moisture to the surface of a wound for drying incrustation, promote autolyzed clear Wound；And can absorbs the unnecessary diffusate of the surface of a wound simultaneously, the surface of a wound is kept appropriate moisture state.But it is used as a kind of Medical coating Material, their biological absorbable and degradability is poor, easily causes unnecessary immune response.DNA has good biology Compatibility, biodegradability, molecular recognition properties and controllability.In recent years, DNA is widely used as a kind of biomaterial In the structure of nanostructured, and DNA hydrogels are then Typical Representatives prepared by DNA materials magnanimity.Because DNA hydrogels had both been kept Biological nature original DNA has the characteristic of Common hydrogels concurrently again, so being led as a kind of functionalization material in biomedicine Domain causes increasing concern, and particularly it can be as a kind of dressing materials of function admirable.

Rolling circle amplification (rolling circle amplification, RCA) is the one of invention application in 1998 Kind nucleic acid amplification technologies [1]." linear RCA " and " index RCA " can be divided into according to the difference of its amplification efficiency.Linear RCA also by Referred to as single primer RCA.Linear RCA is during amplified reaction, and primer is combined with single stranded circle DNA first, in archaeal dna polymerase In the presence of, using single stranded circle DNA as template, synthesize the new DNA being complementary to.After completing a circle, archaeal dna polymerase is again Reach the binding site of primer and template, its strand displacement function primer extension chain that will have been synthesized of having an effect is replaced, weight Building-up process before multiple, the DNA fragmentation repeated so as to synthesizing periodic.The principle of index RCA technologies is identical with linear RCA, On the basis of linear RCA, a primer completely the same with circle DNA sequence, the primer and the first sublinear RCA are further added by Partial sequence complementarity and the enzymatic extension of product, while the primer of hybridized downstream other copy sections to amplified production is displaced, The extension products being replaced afterwards can be expanded as the template of first primer again, so, amplified production amount Exponentially it is incremented by a short period of time.In general, the efficiency of linear amplification is 10⁵, the efficiency of exponential amplification is 10⁹[2]。 Have document report before and DNA hydrogels [3] are formed by rolling circle amplification.

As Fermi's wavelength (the generally De Buluo of gold and silver in Fermi's level being reduced in size to close to electronics of particle Meaning wavelength is 0.5nm) when, unique light, electricity, chemical characteristic can be produced, the particle with this characteristic is referred to as nano-cluster. Noble metal nanocluster has the advantages that sub- nano-scale, has no toxic side effect and photostability, and numerous studies prove silver nanoparticle Cluster has certain antibiotic property.2004, Dickson et al. was first template using DNA, and NaBH4 is prepared for for reducing agent The fluorescence silver nanoclusters [4] having good stability.It is strong that later research finds that silver has to the cytimidine on single stranded DNA and guanine Strong affinity, uses NaBH₄It is generally possible to produce by 2-4 former molecular silver nanoclusters [5] as reducing agent.And with DNA For templated synthesis ag nano-cluster, not only preparation method is simple, and toxicity is low, and bio-compatibility is good, and light is stable, these features It is set to have good application prospect in bio-imaging, chemical detection etc..

With reference to current medical dressing present situation and DNA hydrogels and ag nano-cluster the advantages of, develop a kind of noble metal Nanocluster and DNA composite aquogels are necessary as a kind of new material of biomedical sector.

Bibliography

1.Banér,J.,et al.,Signal amplification of padlock probes by rolling circle replication.Nucleic acids research,1998.26(22):p.5073-5078.

2.Ali,M.M.,et al.,Rolling circle amplification:a versatile tool for chemical biology,materials science and medicine.Chemical Society Reviews, 2014.43(10):p.3324-3341.

3.Lee,J.B.,et al.,A mechanical metamaterial made from a DNA hydrogel.Nature Nanotechnology,2012.7(12):p.816-820.

4.Petty,J.T.,et al.,DNA-templated Ag nanocluster formation.Journal of the American Chemical Society,2004.126(16):p.5207-5212.

5.Gwinn,E.G.,et al.,Sequence-dependent fluorescence of DNA-hosted silver nanoclusters.Advanced Materials,2008.20(2):p.279.

The content of the invention

The purpose of the present invention is overcome the deficiencies in the prior art, there is provided a kind of DNA fluorescence hydrogel.

Second object of the present invention is to provide a kind of preparation method of DNA fluorescence hydrogel.

Technical scheme is summarized as follows：

A kind of preparation method of DNA fluorescence hydrogel, comprises the following steps：

(1) preparation of DNA hydrogels：

A. the final concentration of 50nmol/L cyclic DNA with primary primer, final concentration of 0.05~0.35U/ μ l are added The archaeal dna polymerases of phi 29, the final concentration of 1mmol/L DNA polymerase buffer liquid of dNTPs and 10 × phi 29 and 100 × BSA, final concentration of 16~32mmol/L NaCl solution, surplus are ultra-pure water, stand 100~130min or 200~500rpm 100~130min is shaken, obtains long single stranded DNA；

B. 100 μm of ol/L of the μ l of reaction solution 100 for taking step a to obtain, 2 μ l of the addition ssDNA aqueous solution, standing 100~ 130min or 200~500rpm shakes 100~130min；

C. the ssDNA aqueous solution of 2 μ l of addition, 100 μm of ol/L in repeat step b, 100~130min or 200 is being stood ~500rpm shakes 100~130min and operated 2~5 times, continues standing 18~36h or 200~500rpm and shakes 18~36h, obtains To DNA hydrogels；Inactivate the archaeal dna polymerases of phi 29；

The ssDNA sequences are made up of three parts fragment, and ssDNA total bases are 22~130；

The 3' ends of ssDNA sequences are Part I, and base number is 6~50, the long single stranded DNA part obtained with step a Complementary pairing；

The 5' ends of ssDNA sequences are Part III, and base number is 10~30, has the energy that nanocluster is combined to form with silver Power；

Part II fragment base number among first, Part III fragment is 6~50, is to be used to reduce space bit Any base sequence of resistance；

(2) DNA hydrogels are put into aqua sterilisa, change water 1 time every 15~30min, change water altogether 3~5 times；

(3) step (2) is obtained into DNA hydrogels to be put into container, adds final concentration of 20mmol/L phosphate-buffered Liquid, final concentration of 15~25 μm of ol/L AgNO₃The aqueous solution, at 4~30 DEG C, 15~30min is reacted under 800~1500rpm；Add Enter final concentration of 15~25 μm of ol/L NaBH₄The aqueous solution, at 4~30 DEG C, 0.5~1.5h is reacted under 800~1500rpm, is obtained To DNA fluorescence hydrogel.

Preferably, the cyclic DNA with primary primer is made of following methods：

A. one section of 5 ' end of design synthesis is by the linear ssdna of phosphorylation modification, and base number is 30~200, design one Individual primary primer, base number are 6~30, and the both ends of primary primer are respectively with described 5 ' ends by the linear single-stranded of phosphorylation modification DNA 5 ' ends and 3 ' end complementary pairings；

B. it is 1 by mol ratio:15 ' the end adds by linear ssdna and the primary primer mixing of phosphorylation modification Enter the final concentration of 10~100mmol/L NaCl aqueous solution, enter performing PCR reaction；

C. 2U T4 DNA ligases and 10 × T4 DNA ligases buffering is added in the DNA obtained to every 200ng steps b Liquid, 4 DEG C stand 3h overnight or at 22 DEG C, obtain the cyclic DNA with primary primer.

Base in the nucleotide sequence of Part III in the ssDNA fragments contains guanine continuous arrangement 3 or 3 More than individual；Or contain cytimidine continuous arrangement 3 or more than 3.

Preferably, the nucleotides sequence of the Part III be classified as 5 '-ATCCTCCCACCGGGCCTCCCAC-3 ' or 5 '- CCCCCCCCCC-3’。

Phosphate buffer pH described in step (3) is 6.8.

DNA fluorescence hydrogel prepared by the above method.

Advantages of the present invention：

The DNA fluorescence hydrogel of the present invention, good biocompatibility and material softness, as medical dressing, The secondary damage of the surface of a wound can be effectively avoided, mitigates wound pain；And the hydrogel carries fluorescence, there is certain antibiotic property, nothing Cytotoxicity.Prepare the process of hydrogel simply, conveniently.Medical dressing, immunization therapy, organizational project, slender can be applied to The fields such as born of the same parents' culture, medicament slow release, acellular albumen synthesis and biology sensor.

Brief description of the drawings

Fig. 1 is the electrophoretogram (1 of cyclic DNA of the checking synthesis with primary primer：ladder；2：Phosphorylation is passed through at 5 ' ends The linear ssdna of modification；3：Cyclic DNA with primary primer).

Fig. 2 is the photo of the DNA fluorescence hydrogel of synthesis.

Fig. 3 is the stereoscan photograph of the DNA fluorescence hydrogel of synthesis.

Fig. 4 is the rheology test chart of the DNA fluorescence hydrogel of synthesis.

Fig. 5 is the fluorescence spectra of the DNA fluorescence hydrogel of synthesis.

Fig. 6 is the antibiogram of the DNA fluorescence hydrogel of synthesis.

Fig. 7 is the cytotoxicity figure of the DNA fluorescence hydrogel of synthesis.

Embodiment

Below by specific embodiment, the present invention is further illustrated.The following examples are in order that this area Technical staff better understood when the present invention, but the present invention not imposed any restrictions.

Embodiment 1

The preparation method of cyclic DNA with primary primer, comprises the following steps：

5 ' ends used in cyclic DNA with primary primer by the linear ssdna of phosphorylation modification, primary primer, The particular sequence for the ssDNA that synthesis DNA fluorescence hydrogel is added is shown in Table 1.

The DNA sequence of table 1.

Wherein the nucleotides sequence of Part III is classified as in ssDNA fragments：5’-ATCCTCCCACCGGGCCTCCCAC-3’SEQ ID NO.10

(1) cyclic DNA of the synthesis with primary primer：

A.DNA sample pre-treatments：

5 ' ends are centrifuged respectively by linear ssdna, primary primer and the ssDNA of phosphorylation modification with mini centrifuge 1min, get rid of to centrifuge tube, be then dissolved to 100 μM with ultra-pure water, 4h, condition 30 are vibrated in isothermal vibration blending instrument ℃,1000rpm。

B. sample is mixed by the amount of table 2, i.e., 5 ' ends by phosphorylation modification linear ssdna and primary primer Mol ratio is 1:The final concentration of 10mmol/L of 1, the NaCl aqueous solution.

Table 2

Mixed liquid is placed in PCR instrument and carries out following temperature control program：

95℃ 2min

65℃ 2min

60 DEG C of 5min30s cool 0.5 DEG C per 30s, carry out 80 circulations

20℃ 30s

4℃ 10min

C. connected to obtain the cyclic DNA with primary primer with T4 DNA ligases：

Reaction is attached by the amount addition of table 3,5.68 μ l, the DNA that 1 μm of ol/L steps b is obtained is 200ng.

Table 3

Mixed liquor is placed at 22 DEG C and stands 3h, obtains the cyclic DNA with primary primer.Then place reaction liquid into 70 10min at DEG C, inactivate T4 DNA ligases.

(2) synthesis of cyclic DNA of the checking with primary primer：

The synthesis of the cyclic DNA with primary primer is verified by 12% polyacrylamide gel.Deposition condition is：Electricity Pressure：110V, time：110min.

Applied sample amount such as table 4：

Table 4

See Fig. 1.

Embodiment 2

(1) carry out rolling circle amplification and obtain DNA hydrogels：

A. the final concentration of 50nmol/L cyclic DNA (prepared by embodiment 1) with primary primer is added by the amount of table 5, eventually Concentration be 0.05U/ μ l the archaeal dna polymerases of phi 29, the final concentration of 1mmol/L archaeal dna polymerase of dNTPs and 10 × phi 29 Buffer solution and 100 × BSA, final concentration of 16mmol/L NaCl solution, surplus are ultra-pure water, and reaction condition shakes for 200rpm 100min, carry out rolling circle amplification and react to obtain long single stranded DNA.

Table 5

B. the 100 μ l reaction solutions that step a is obtained are taken, add 2 μ l, the 100 μm of ol/L ssDNA aqueous solution, 200rpm concussions 100min。

SsDNA sequences are made up of three parts fragment,

The 3' ends of ssDNA sequences are Part I, and base number is 22, the long single stranded DNA partial complementarity obtained with step a Pairing；

The 5' ends of ssDNA sequences are Part III, and base number is 22, has the energy that nanocluster is combined to form with silver Power；

Part II fragment base number among first, Part III fragment is 22, is to be used to reduce space bit Any base sequence of resistance；

C. the ssDNA aqueous solution of 2 μ l of addition, 100 μm of ol/L in repeat step b, 200rpm shake 100min operation 3 It is secondary, continue 200rpm concussion 26h, obtain DNA hydrogels.

The container that DNA hydrogels are housed after reaction is then placed in 10min at 65 DEG C, inactivates the archaeal dna polymerases of phi 29.

(2) the DNA hydrogels that step (1) obtains are put into aqua sterilisa, change water 1 time every 15min, change water altogether3It is secondary.Remove Remove the DNA polymerase buffer liquid of phi 29 for having quenching effect to ag nano-cluster fluorescence.

(3) step (2) is obtained into DNA hydrogels to be put into container, adds 147.75 μ l 20mmol/L, pH value thereto For 6.8 phosphate buffer (final concentration of 20mmol/L phosphate buffer), 1.125 μ l 2mmol/L AgNO₃Water Solution (final concentration of 15 μm of ol/L AgNO₃The aqueous solution), react 15min under 4 DEG C, 800rpm.Then 1.125 μ l are added The NaBH that 2mmol/L now matches somebody with somebody₄The aqueous solution (final concentration of 15 μm of ol/L NaBH₄The aqueous solution) continue to react under 4 DEG C, 800rpm 0.5h, obtain DNA fluorescence hydrogel.See Fig. 2, Fig. 3, Fig. 4, Fig. 5.

Embodiment 3

5 ' ends used in cyclic DNA with primary primer by the linear ssdna of phosphorylation modification, primary primer, The particular sequence for the ssDNA that synthesis DNA fluorescence hydrogel is added is shown in Table 6.

The DNA sequence of table 6.

Wherein the nucleotides sequence of Part III is classified as preferably in ssDNA fragments：5’-CCCCCCCCCC-3’SEQ ID NO.11

(1) cyclic DNA of the synthesis with primary primer：

A.DNA sample pre-treatments：

B. sample is mixed by the amount of table 7, i.e., 5 ' ends by phosphorylation modification linear ssdna and primary primer Mol ratio is 1:The final concentration of 40mmol/L of 1, the NaCl aqueous solution.

Table 7

95℃ 2min

65℃ 2min

60 DEG C of 5min30s cool 0.5 DEG C per 30s, carry out 80 circulations

20℃ 30s

4℃ 10min

Reaction is attached by the amount addition of table 8,5.68 μ l, the DNA that 1 μm of ol/L steps b is obtained is 200ng.

Table 8

(2) synthesis of cyclic DNA of the checking with primary primer：

The synthesis of cyclic DNA is verified by 12% polyacrylamide gel.Deposition condition is：Voltage：110V, time： 120min。

Applied sample amount such as table 9：

Table 9

Embodiment 4

(1) carry out rolling circle amplification and obtain DNA hydrogels：

A. the final concentration of 50nmol/L cyclic DNA (prepared by embodiment 3) with primary primer is added by the amount of table 10, eventually Concentration be 0.15U/ μ l the archaeal dna polymerases of phi 29, the final concentration of 1mmol/L archaeal dna polymerase of dNTPs and 10 × phi 29 Buffer solution and 100 × BSA, final concentration of 24mmol/L NaCl solution, surplus are ultra-pure water, and reaction condition shakes for 360rpm 120min, carry out rolling circle amplification and react to obtain long single stranded DNA.

Table 10

B. the 100 μ l reaction solutions that step a is obtained are taken, add 2 μ l, the 100 μm of ol/L ssDNA aqueous solution, 360rpm concussions 120min。

SsDNA sequences are made up of three parts fragment,

The 3' ends of ssDNA sequences are Part I, and base number is 6, the long single stranded DNA partial complementarity obtained with step a Pairing；

The 5' ends of ssDNA sequences are Part III, and base number is 10, has the energy that nanocluster is combined to form with silver Power；

Part II fragment base number among first, Part III fragment is 6, is to be used to reduce steric hindrance Any base sequence；

C. the ssDNA aqueous solution of 2 μ l of addition, 100 μm of ol/L in repeat step b, 360rpm shake 120min operation 2 It is secondary, continue 360rpm concussion 36h, obtain DNA hydrogels.

(2) the DNA hydrogels that step (1) obtains are put into aqua sterilisa, change water 1 time every 20min, change water altogether 4 times.Remove Remove the DNA polymerase buffer liquid of phi 29 for having quenching effect to ag nano-cluster fluorescence.

(3) step (2) is obtained into DNA hydrogels to be put into container, adds 97 μ l 20mmol/L, pH value 6.8 thereto Phosphate buffer (final concentration of 20mmol/L phosphate buffer), 1.5 μ l 2mmol/L AgNO₃The aqueous solution is (eventually Concentration is 20 μm of ol/L AgNO₃The aqueous solution), react 20min under 20 DEG C, 1200rpm.Then 1.5 μ l 2mmol/L are added The NaBH now matched somebody with somebody₄The aqueous solution (final concentration of 20 μm of ol/L NaBH₄The aqueous solution) continue to react 1h under 20 DEG C, 1200rpm, obtain To DNA fluorescence hydrogel.

Embodiment 5

5 ' ends used in cyclic DNA with primary primer by the linear ssdna of phosphorylation modification, primary primer, The particular sequence for the ssDNA that synthesis DNA fluorescence hydrogel is added is shown in Table 11.

The DNA sequence of table 11.

(1) cyclic DNA of the synthesis with primary primer：

A.DNA sample pre-treatments：

B. sample is mixed by the amount of table 12, i.e., the linear ssdna and primary primer of phosphorylation modification are passed through in 5 ' ends Mol ratio be 1:The final concentration of 100mmol/L of 1, the NaCl aqueous solution.

Table 12

95℃ 2min

65℃ 2min

60 DEG C of 5min30s cool 0.5 DEG C per 30s, carry out 80 circulations

20℃ 30s

4℃ 10min

Reaction is attached by the amount addition of table 13,5.68 μ l, the DNA that 1 μm of ol/L steps b is obtained is 200ng.

Table 13

(2) synthesis of cyclic DNA of the checking with primary primer：

The synthesis of the cyclic DNA with primary primer is verified by 12% polyacrylamide gel.Deposition condition is：Electricity Pressure：110V, time：130min.

Applied sample amount such as table 14：

Table 14

Embodiment 6

(1) carry out rolling circle amplification and obtain DNA hydrogels：

A. the final concentration of 50nmol/L cyclic DNA (prepared by embodiment 5) with primary primer is added by the amount of table 15, eventually Concentration be 0.35U/ μ l the archaeal dna polymerases of phi 29, the final concentration of 1mmol/L archaeal dna polymerase of dNTPs and 10 × phi 29 Buffer solution and 100 × BSA, final concentration of 32mmol/L NaCl solution, surplus are ultra-pure water, and reaction condition shakes for 500rpm 130min carries out rolling circle amplification and reacts to obtain long single stranded DNA.

Table 15

B. the 100 μ l reaction solutions that step a is obtained are taken, add 2 μ l, the 100 μm of ol/L ssDNA aqueous solution, 500rpm concussions 130min。

SsDNA sequences are made up of three parts fragment,

The 3' ends of ssDNA sequences are Part I, and base number is 50, the long single stranded DNA partial complementarity obtained with step a Pairing；

The 5' ends of ssDNA sequences are Part III, and base number is 30, has the energy that nanocluster is combined to form with silver Power；

Part II fragment base number among first, Part III fragment is 50, is to be used to reduce space bit Any base sequence of resistance；

C. the ssDNA aqueous solution of 2 μ l of addition, 100 μm of ol/L in repeat step b, 500rpm shake 130min operation 5 It is secondary, continue 500rpm concussion reaction 18h, obtain DNA hydrogels.

(2) the DNA hydrogels that step (1) obtains are put into aqua sterilisa, change water 1 time every 30min, change water altogether 5 times.Remove Remove the DNA polymerase buffer liquid of phi 29 for having quenching effect to ag nano-cluster fluorescence.

(3) step (2) is obtained into DNA hydrogels to be put into container, adds 146.25 μ l 20mmol/L, pH value thereto For 6.8 phosphate buffer (final concentration of 20mmol/L phosphate buffer), 1.875 μ l 2mmol/L AgNO₃Water Solution (final concentration of 25 μm of ol/L AgNO₃The aqueous solution), react 30min under 30 DEG C, 1500rpm.Then 1.875 μ l are added The NaBH that 2mmol/L now matches somebody with somebody₄The aqueous solution (final concentration of 25 μm of ol/L NaBH₄The aqueous solution) continue at 30 DEG C, it is anti-under 1500rpm 1.5h is answered, obtains DNA fluorescence hydrogel.

Embodiment 7

The present invention synthesis with primary primer cyclic DNA used in 5 ' end by phosphorylation modification linear ssdna, The particular sequence for the ssDNA that primary primer, synthesis DNA fluorescence hydrogel are added is shown in Table 1.

(1) cyclic DNA of the synthesis with primary primer：

A.DNA sample pre-treatments：

B. sample is mixed by the amount of table 16, i.e., the linear ssdna and primary primer of phosphorylation modification are passed through in 5 ' ends Mol ratio be 1:The final concentration of 40mmol/L of 1, the NaCl aqueous solution

Table 16

95℃ 2min

65℃ 2min

60 DEG C of 5min30s cool 0.5 DEG C per 30s, carry out 80 circulations

20℃ 30s

4℃ 10min

Reaction is attached by the amount addition of table 17,5.68 μ l, the DNA that 1 μm of ol/L steps b is obtained is 200ng.

Table 17

Mixed liquor is placed in 4 DEG C overnight, obtains the cyclic DNA with primary primer.Then place reaction liquid at 70 DEG C 10min, inactivate T4 DNA ligases.

(3) synthesis of cyclic DNA of the checking with primary primer：

Applied sample amount such as table 18：

Table 18

Embodiment 8

(1) carry out rolling circle amplification and obtain DNA hydrogels：

A. the final concentration of 50nmol/L cyclic DNA (prepared by embodiment 7) with primary primer is added by the amount of table 19, eventually Concentration be 0.15U/ μ l the archaeal dna polymerases of phi 29, the final concentration of 1mmol/L archaeal dna polymerase of dNTPs and 10 × phi 29 Buffer solution and 100 × BSA, final concentration of 24mmol/L NaCl solution, surplus are ultra-pure water, carry out rolling circle amplification and react to obtain Long single stranded DNA, reaction condition are standing 100min.(130min can also be stood)

Table 19

B. the 100 μ l reaction solutions that step a is obtained are taken, add 2 μ l, the 100 μm of ol/L ssDNA aqueous solution, stand 100min (130min can also be stood).

SsDNA sequences are made up of three parts fragment,

C. the ssDNA aqueous solution of 2 μ l of addition, 100 μm of ol/L in repeat step b, standing 100min (can also stand Operation 130min) 2 times, continue to stand 18h (36h can also be stood), obtain DNA hydrogels.

The DNA fluorescence hydrogel obtained with embodiment 2 does following experiments：

1. antibacterial experiment：

DNA fluorescence hydrogel is as follows to the antibacterial experiment step of Escherichia coli：

(1) 10g tryptones, 5g yeast extracts are taken, 10gNaCl solids are added in 1L distilled water, and pH value is adjusted to 7.Then it is 103.4kPa in pressure by the culture medium mixed, the Luria- after sterilizing 20min is sterilized at 121 DEG C Bertani fluid nutrient mediums.

(2) antibacterial test is done in 96 orifice plates.

Three groups of experiments are set：

The orifice plate of 250 μ LLuria-Bertani fluid nutrient mediums and 0.5 μ l Escherichia coli will be added as a control group；

The nitre that 200 μ L Luria-Bertani fluid nutrient mediums, 0.5 μ l Escherichia coli and 50 μ l concentration are 20 μM will be added The silver-colored solution of acid is as one group of experimental group；

The 50 μ l that 200 μ L Luria-Bertani fluid nutrient mediums of addition, 0.5 μ l Escherichia coli and embodiment 2 are obtained DNA fluorescence hydrogel is as another experimental group.

96 orifice plates are placed in ELIASA after the completion of sample-adding, 37 DEG C of temperature setting, determine bacterium solution growth curve.Experimental result See Fig. 6.It was found from the experimental result shown in Fig. 6, DNA fluorescence hydrogel has good antibiotic property.

It is demonstrated experimentally that the deoxidation core that DNA fluorescence hydrogel prepared by embodiment 4,6,8 is prepared with embodiment 2 Ribosomal ribonucleic acid fluorescence hydrogel antibacterial effect is similar.

2. cytotoxicity experiment：

Cytotoxicity experiment operating process is as follows：

9000 smooth muscle cells (MSCs) are added in each hole of 96 orifice plates, adding 50 μ l in each hole is enclosed with not With the DNA fluorescence hydrogel (gel is prepared by embodiment 2) of concentration ag nano-cluster, wherein silver nanoparticle group The concentration of cluster is respectively 0,0.5,1,2,5,10,20 μM.By 96 orifice plates after the completion of sample-adding in CO₂Cultivated in incubator at 37 DEG C 24h.It is careful to suck supernatant, 90ml fresh mediums are added, add 10mlMTT solution, continue to cultivate 4h.Then suck Clearly, 110 μ l Formazan lysates are added per hole, put shaking table low speed concussion 10min.Surveyed at enzyme-linked immunosorbent assay instrument 490nm Measure the light absorption value in each hole.Calculate each sample well cell relative viability.Various kinds sample wells cell relative viability is defined as：Inhale in each hole Light absorption value × 100% of light value/control wells.Experimental result is shown in Fig. 7.It was found from the experimental result shown in Fig. 7, DNA Fluorescence hydrogel does not have cytotoxicity.

It is demonstrated experimentally that DNA fluorescence hydrogel prepared by embodiment 4,6,8 does not have cytotoxicity.

Sequence table

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Claims

1. a kind of preparation method of DNA fluorescence hydrogel, it is characterised in that comprise the following steps：

(1) preparation of DNA hydrogels：

A. the final concentration of 50nmol/L cyclic DNA with primary primer, final concentration of 0.05~0.35U/ μ l phi are added 29DNA polymerases, final concentration of 1mmol/L dNTPs and 10 × phi 29DNA polymerase buffers and 100 × BSA are dense eventually Spend the NaCl solution for 16~32mmol/L, surplus is ultra-pure water, stand 100~130min or 200~500rpm concussion 100~ 130min, obtain long single stranded DNA；

B. the μ l of reaction solution 100 for taking step a to obtain, 2 μ l, the 100 μm of ol/L ssDNA aqueous solution is added, stands 100~130min Or 200~500rpm shakes 100~130min；

C. the ssDNA aqueous solution of 2 μ l of addition, 100 μm of ol/L in repeat step b, stand 100~130min or 200~ 500rpm shakes 100~130min and operated 2~5 times, continues standing 18~36h or 200~500rpm and shakes 18~36h, obtains DNA hydrogels；Inactivate phi 29DNA polymerases；

The 3' ends of ssDNA sequences are Part I, and base number is 6~50, the long single stranded DNA partial complementarity obtained with step a Pairing；

The 5' ends of ssDNA sequences are Part III, and base number is 10~30, has the ability that nanocluster is combined to form with silver；

Part II fragment base number among first, Part III fragment is 6~50, is for reducing steric hindrance Any base sequence；

(3) step (2) is obtained into DNA hydrogels to be put into container, adds final concentration of 20mmol/L phosphate buffer, eventually Concentration is 15~25 μm of ol/L AgNO₃The aqueous solution, at 4~30 DEG C, 15~30min is reacted under 800~1500rpm；Add eventually Concentration is 15~25 μm of ol/L NaBH₄The aqueous solution, at 4~30 DEG C, 0.5~1.5h is reacted under 800~1500rpm, is taken off Oxygen ribonucleic acid fluorescence hydrogel.

2. according to the method for claim 1, it is characterized in that the following method systems of the cyclic DNA with primary primer Into：

A. one section of 5 ' end of design synthesis is by the linear ssdna of phosphorylation modification, and base number is 30~200, at the beginning of design one Level primer, base number are 6~30, and the linear ssdna of phosphorylation modification is passed through at the both ends of primary primer with described 5 ' ends respectively 5 ' end and 3 ' end complementary pairings；

B. it is 1 by mol ratio:15 ' the end adds eventually by linear ssdna and the primary primer mixing of phosphorylation modification Concentration is the 10~100mmol/L NaCl aqueous solution, enters performing PCR reaction；

C. addition 2U T4DNA ligases and 10 × T4DNA ligase buffer solutions in the DNA obtained to every 200ng steps b, 4 DEG C 3h is stood overnight or at 22 DEG C, obtains the cyclic DNA with primary primer.

3. according to the method for claim 1, it is characterized in that in the nucleotide sequence of Part III in the ssDNA fragments Base contain guanine continuous arrangement 3 or more than 3；Or contain cytimidine continuous arrangement 3 or more than 3.

4. according to the method for claim 3, it is characterized in that the nucleotides sequence of the Part III be classified as 5 '- ATCCTCCCACCGGGCCTCCCAC-3 ' or 5 '-CCCCCCCCCC-3 '.

5. according to the method for claim 1, it is characterised in that the phosphate buffer pH described in step (3) is 6.8.

6. DNA fluorescence hydrogel prepared by one of claim 1-5 method.