CN104391119A - Preparation method of pH sensitive element based on DNA molecule configuration change - Google Patents
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Abstract
The invention relates to a preparation method of a pH sensitive element based on DNA molecule configuration change. The preparation method comprises the following steps: preparing a DNA self-assembling structure; and adsorbing a DNA molecule and a gold base to obtain the pH sensitive element. The DNA molecule is subjected to the action of H<+1> under an acidic condition and a chain segment has the configuration change to a certain extent; and meanwhile, some DNA chain segments which are sensitive to pH, such as i-motig chain segments, are folded under the action the H<+1>, so that the change of the pH can be directly reflected through the change of the configuration by the DNA molecule. According to the preparation method, the DNA molecule is modified on a gold surface, a material preparation process is developed and the maneuverability is strong; the sensitivity to the change of the pH is high and a rapid reaction can be realized; and a proton-driven environment-friendly and clean reversible biological change-over switch device is provided and has a wide application prospect in the field of biosensors and the field of clean energy sources.
Description
Technical field
The present invention proposes a kind of preparation method of the pH sensitive element based on DNA molecular change of configuration, and being specifically related to a kind of is substrate with gold, modifies the DNA molecular of different configuration on its surface respectively.
Background technology
PH value has very important effect for the normal operation of biosome, relate to much physiology and pathologic process, such as, the proliferation and apoptosis of cell, endocytosis, ion transportation and mobile equilibrium, multidrug resistance etc., once pH value is abnormal, cell functional disorders will be caused.The pH distribution of human body is very complicated, and blood pH scope is between 7.35 ~ 7.45, and pancreas is between 8.0 ~ 8.3, and gastric juice is then 0.8 ~ 1.5; PH in cell also can be divided into two scopes, the pH scope 4.5 ~ 6.0 of acidic organelles (endosome, lysosome), tenuigenin 6.8 ~ 7.4; In addition, the secretion of some tumour cell can cause the change of surrounding environment pH.Therefore, pH sensitive material research will to biology and medical science significant.
PH sensitive materials are widely used in pH probe in detecting, demonstrate higher sensitivity, and part probe is also applied in living body detection.But the three-dimensional paper folding structure of DNA is as a kind of good biocompatibility, Stability Analysis of Structures, the biological controlled-released material that response is responsive, have no by its to the responsive response application of pH value in detection or other any fields.
Summary of the invention
For overcoming the deficiencies in the prior art, the invention provides a kind of preparation method of the pH sensitive element based on DNA molecular change of configuration.
Based on a preparation method for the pH sensitive element of DNA molecular change of configuration, it is characterized in that, comprise the following steps:
(1) preparation of DNA self-assembled structures:
Respectively getting the different DNA single chain of 2 μ L joins in 42 μ L Tris-MgCl solution, 50 μMs, DNA single chain, Tris-MgCl solution Tris 10mM, MgCl
250mM, pH8; Mixed solution is placed in PCR instrument, and temperature of reaction is 95 DEG C, is cooled to 4 DEG C after 10 minutes, continues reaction 30 minutes; Namely DNA single chain is self-assembled into the Small molecular of different configuration by base pair complementarity;
(2) absorption of DNA molecular and gold substrate:
Get sulfydryl coupling agent (SH-(PEG)
7-OCH
3, 1mg/mL) and be modified at gold surface, leave standstill 30 minutes; Then take from the DNA molecular solution assembled, be added drop-wise to above-mentioned gold surface, fully reaction 2 hours, can obtain this pH sensitive material;
(3) change of configuration of DNA molecular:
DNA molecular in acid condition, is subject to H
+effect, will change of configuration to a certain degree be there is in segment; Meanwhile, some are for pH than more sensitive DNA segment, and as i-motif segment, meeting is due to H
+effect and produce folding; Thus, the change of pH can be reacted in the change of configuration by DNA molecular intuitively.
Described gold substrate is nano-Au solution, or is coated with the quartz wafer of gold thin film.
The mode that described DNA molecular is connected with surface is that the key between sulfydryl-Jin connects.
Described DNA single chain is the sequence in sequence table.
These DNA moleculars there occurs reversible change of configuration along with the change of pH, and the biological controlled-released switch of the clean energy resource driven as a kind of proton has broad application prospects.Meanwhile, gold and DNA molecular all have good biocompatibility, and thus the present invention can be applied to nano biological medical material field, and be specifically expected to has good application in pH detection and biological intelligence controlled release etc.
And achievement in research of the present invention will provide new approaches to structure new bio pH sensor.Can be there is reversible change in DNA molecular configuration under different pH environment, and the DNA molecular of multiple configuration still can keep the sensitivity suitable to pH after the environment of experience biosome complexity.Therefore the present invention has higher Research Significance and using value.
The object of the invention is to build a kind of Novel pH Sensitive element, is expected to be applied in the fields such as pH detection and biological intelligence controlled release.The method, by choosing different DNA chains, is self-assembled into the DNA molecular of different configuration, as DNA tetrahedron (TDN), and i-motif, DNA double chain (dS) etc.It is even folding in acid condition to be there is change of configuration in these DNA moleculars, demonstrates and distinct molecular configuration under neutral or meta-alkalescence condition, and after pH recovers, its configuration also can recover thereupon.The present invention has very high sensitivity for pH, can tackle more complicated biological internal environment simultaneously.
The invention has the advantages that:
The present invention is at gold surface modified dna molecule, and material preparation process is ripe, workable;
Material prepared by the present invention for pH change highly sensitive, can realize rapid reaction, be the reversible biological transfer switch unit that cleans of green that a kind of proton drives, in field of biosensors, clean energy resource field is with a wide range of applications;
Multiple dna molecular configuration in the present invention is stablized, and has certain rigidity, still can keep the sensitivity suitable to pH, meet the detection in vitro and body after the environment of experience biosome complexity.
Accompanying drawing explanation
The mechanism of action schematic diagram of the gold-plated quartz crystal-DNA pH sensitive element of Fig. 1 prepared by embodiment 1.
The mechanism of action schematic diagram of the gold-plated quartz crystal-DNA pH sensitive element of Fig. 2 prepared by embodiment 2.
The mechanism of action schematic diagram of the gold-plated quartz crystal-DNA pH sensitive element of Fig. 3 prepared by embodiment 3.
The mechanism of action schematic diagram of the nm of gold-DNA pH sensitive element of Fig. 4 prepared by embodiment 4.
The mechanism of action schematic diagram of the nm of gold-DNA pH sensitive element of Fig. 5 prepared by embodiment 5.
The mechanism of action schematic diagram of the nm of gold-DNA pH sensitive element of Fig. 6 prepared by embodiment 6.
Embodiment
Below by way of specific embodiment, technical scheme of the present invention is further described.Following embodiment further illustrates of the present invention, and do not limit the scope of the invention.
embodiment 1:
Respectively get 2 μ L single stranded DNAs (50 μMs) 1,2,3,4 and join 42 μ L Tris-MgCl(Tris 10mM, MgCl
250mM, pH8) in solution.Mixed solution is placed in PCR instrument, and temperature of reaction is 95 DEG C, is cooled to 4 DEG C after 10 minutes, continues reaction 30 minutes.Namely DNA single chain is self-assembled into the DNA tetrahedron containing i-motif by base pair complementarity.
Get 100 μ L sulfydryl coupling agent (SH-(PEG)
7-OCH
3, 0.1mg/mL) drip on the surface of gold-plated quartz crystal, leave standstill 30 minutes.Get the DNA molecular solution that 100 μ L self assemblies complete, be added drop-wise to above-mentioned gold-plated strand DNA on Surface of Quartz crystal, fully modify 5 hours (spending the night).
This TDN i-motif molecule in acid condition, is subject to H
+effect, will deformation to a certain degree be there is in DNA tetrahedron.I-motif segment simultaneously, can due to H
+effect and produce folding.Thus, the change of pH can be reacted in the change of configuration by TDN i-motif molecule intuitively, becomes a kind of biological sensing material of pH sensitivity.
embodiment 2:
Respectively get 2 μ L single stranded DNAs (50 μMs) 2,3,4,5 and join 42 μ L Tris-MgCl(Tris 10mM, MgCl
250mM, pH8) in solution.Mixed solution is placed in PCR instrument, and temperature of reaction is 95 DEG C, is cooled to 4 DEG C after 10 minutes, continues reaction 30 minutes.Namely DNA single chain is self-assembled into DNA tetrahedron by base pair complementarity.
Get 100 μ L sulfydryl coupling agent (SH-(PEG)
7-OCH
3, 0.1mg/mL) drip on the surface of gold-plated quartz crystal, leave standstill 30 minutes.Get the DNA molecular solution that 100 μ L self assemblies complete, be added drop-wise to above-mentioned gold-plated strand DNA on Surface of Quartz crystal, fully modify 5 hours (spending the night).
This gold-plated quartz crystal TDN molecule in acid condition, is subject to H
+effect, will deformation to a certain degree be there is in DNA tetrahedron.Thus, the change of pH can be reacted in the change of configuration by TDN molecule intuitively.
embodiment 3:
Respectively get 2 μ L single stranded DNAs (50 μMs) 6,7 and join 46 μ L Tris-MgCl(Tris 10mM, MgCl
250mM, pH8) in solution.Namely DNA single chain is self-assembled into double chain DNA molecule by base pair complementarity.
Get 100 μ L sulfydryl coupling agent (SH-(PEG)
7-OCH
3, 0.1mg/mL) drip on the surface of gold-plated quartz crystal, leave standstill 30 minutes.Get the DNA molecular solution that 100 μ L self assemblies complete, be added drop-wise to above-mentioned gold-plated strand DNA on Surface of Quartz crystal, fully modify 5 hours (spending the night).
This gold-plated quartz crystal-double chain DNA molecule in acid condition, is subject to H
+effect, will to a certain degree curling be there is in double chain DNA molecule segment.Thus, the change of pH can be reacted in the change of configuration by double chain DNA molecule intuitively.
embodiment 4:
Respectively get 2 μ L single stranded DNAs (50 μMs) 1,2,3,4 and join 42 μ L Tris-MgCl(Tris 10mM, MgCl
250mM, pH8) in solution.Mixed solution is placed in PCR instrument, and temperature of reaction is 95 DEG C, is cooled to 4 DEG C after 10 minutes, continues reaction 30 minutes.Namely DNA single chain is self-assembled into the DNA tetrahedron containing i-motif by base pair complementarity.
Get 1 μ L sulfydryl coupling agent (SH-(PEG)
7-OCH
3, 1mg/mL) join in the solution of 1mL nm of gold (13nm, 3nM), leave standstill 30 minutes.Get the DNA molecular solution that 20 μ L self assemblies complete, be added drop-wise in the solution of above-mentioned nm of gold, fully reaction 2 hours, can obtain this pH sensitive material.
This nm of gold-TDN i-motif molecule in acid condition, is subject to H
+effect, will deformation to a certain degree be there is in DNA tetrahedron.I-motif segment simultaneously, can due to H
+effect and produce folding.Thus, the change of pH can be reacted in the change of configuration by TDN i-motif molecule intuitively.
embodiment 5:
Respectively get 2 μ L single stranded DNAs (50 μMs) 2,3,4,5 and join 42 μ L Tris-MgCl(Tris 10mM, MgCl
250mM, pH8) in solution.Mixed solution is placed in PCR instrument, and temperature of reaction is 95 DEG C, is cooled to 4 DEG C after 10 minutes, continues reaction 30 minutes.Namely DNA single chain is self-assembled into DNA tetrahedron by base pair complementarity.
Get 1 μ L sulfydryl coupling agent (SH-(PEG)
7-OCH
3, 1mg/mL) join in the solution of 1mL nm of gold (13nm, 3nM), leave standstill 30 minutes.Get the DNA molecular solution that 20 μ L self assemblies complete, be added drop-wise in the solution of above-mentioned nm of gold, fully reaction 2 hours, can obtain this pH sensitive material.
This nm of gold-TDN molecule in acid condition, is subject to H
+effect, will deformation to a certain degree be there is in DNA tetrahedron.Thus, the change of pH can be reacted in the change of configuration by TDN molecule intuitively.
embodiment 6:
Respectively get 2 μ L single stranded DNA (50 μMs) Double Strains-A, Double Strains-B and join 46 μ L Tris-MgCl(Tris 10mM, MgCl
250mM, pH8) in solution.Mixed solution is placed in PCR instrument, and temperature of reaction is 95 DEG C, is cooled to 4 DEG C after 10 minutes, continues reaction 30 minutes.Namely DNA single chain is self-assembled into double chain DNA molecule by base pair complementarity.
Get 1 μ L sulfydryl coupling agent (SH-(PEG)
7-OCH
3, 1mg/mL) join in the solution of 1mL nm of gold (13nm, 3nM), leave standstill 30 minutes.Get the DNA molecular solution that 20 μ L self assemblies complete, be added drop-wise in the solution of above-mentioned nm of gold, fully reaction 2 hours, can obtain this pH sensitive material.
This nm of gold-double chain DNA molecule in acid condition, is subject to H
+effect, will to a certain degree curling be there is in double chain DNA molecule segment.Thus, the change of pH can be reacted in the change of configuration by double chain DNA molecule intuitively.
Claims (4)
1., based on a preparation method for the pH sensitive element of DNA molecular change of configuration, it is characterized in that, comprise the following steps:
(1) preparation of DNA self-assembled structures:
Respectively getting the different DNA single chain of 2 μ L joins in 42 μ L Tris-MgCl solution, 50 μMs, DNA single chain, Tris-MgCl solution Tris 10mM, MgCl
250mM, pH8; Mixed solution is placed in PCR instrument, and temperature of reaction is 95 DEG C, is cooled to 4 DEG C after 10 minutes, continues reaction 30 minutes; Namely DNA single chain is self-assembled into the Small molecular of different configuration by base pair complementarity;
(2) absorption of DNA molecular and gold substrate:
Get sulfydryl coupling agent (SH-(PEG)
7-OCH
3, 1mg/mL) and be modified at gold surface, leave standstill 30 minutes; Then take from the DNA molecular solution assembled, be added drop-wise to above-mentioned gold surface, fully reaction 2 hours, can obtain this pH sensitive material;
(3) change of configuration of DNA molecular:
DNA molecular in acid condition, is subject to H
+effect, will change of configuration to a certain degree be there is in segment; Meanwhile, some are for pH than more sensitive DNA segment, and as i-motif segment, meeting is due to H
+effect and produce folding; Thus, the change of pH can be reacted in the change of configuration by DNA molecular intuitively.
2., according to claim 1 based on the preparation method of the pH sensitive element of DNA molecular change of configuration, it is characterized in that, described gold substrate is nano-Au solution, or is coated with the quartz wafer of gold thin film.
3. according to claim 1 based on the preparation method of the pH sensitive element of DNA molecular change of configuration, it is characterized in that, the mode that described DNA molecular is connected with surface is that the key between sulfydryl-Jin connects.
4. according to claim 1 based on the preparation method of the pH sensitive element of DNA molecular change of configuration, it is characterized in that, described DNA single chain is the sequence in sequence table.
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| CN110331188A (en) * | 2019-07-16 | 2019-10-15 | 上海纳米技术及应用国家工程研究中心有限公司 | A kind of detection method of internal pH |
| CN111763673A (en) * | 2020-07-09 | 2020-10-13 | 南方科技大学 | C-quadruplex deoxyribozyme and preparation method and application thereof |
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| CN106729710A (en) * | 2016-12-20 | 2017-05-31 | 上海纳米技术及应用国家工程研究中心有限公司 | A kind of thermochemotherapy Integral rice grain and preparation and application |
| CN106729710B (en) * | 2016-12-20 | 2019-12-03 | 上海纳米技术及应用国家工程研究中心有限公司 | A kind of thermochemotherapy Integral rice grain and preparation and application |
| CN110331188A (en) * | 2019-07-16 | 2019-10-15 | 上海纳米技术及应用国家工程研究中心有限公司 | A kind of detection method of internal pH |
| CN111763673A (en) * | 2020-07-09 | 2020-10-13 | 南方科技大学 | C-quadruplex deoxyribozyme and preparation method and application thereof |
| CN111763673B (en) * | 2020-07-09 | 2023-11-24 | 南方科技大学 | C-quadruplex deoxyribozyme and preparation method and application thereof |
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