CN104391119B - The preparation method of pH sensing element based on DNA molecular change of configuration - Google Patents
The preparation method of pH sensing element based on DNA molecular change of configuration Download PDFInfo
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- CN104391119B CN104391119B CN201410661290.4A CN201410661290A CN104391119B CN 104391119 B CN104391119 B CN 104391119B CN 201410661290 A CN201410661290 A CN 201410661290A CN 104391119 B CN104391119 B CN 104391119B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Abstract
The present invention relates to the preparation method of a kind of pH sensing element based on DNA molecular change of configuration, including the preparation of DNA self-assembled structures；DNA molecular and the absorption of gold substrate；This pH sensitive material can be obtained；DNA molecular in acid condition, by H+Effect, segment will occur a certain degree of change of configuration；Meanwhile, some are for pH than more sensitive DNA section, and such 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.The present invention is at gold surface modified dna molecule, and material preparation process is ripe, workable；Highly sensitive for pH change, can realize fast reaction, be the reversible biotransformation switching device of the green cleaning that a kind of proton drives, and in field of biosensors, clean energy resource field is with a wide range of applications.
Description
Technical field
The present invention proposes the preparation method of a kind of pH sensing element based on DNA molecular change of configuration, is specifically related to a kind of with gold as substrate, modifies the DNA molecular of various configuration on its surface respectively.
Background technology
PH value has very important effect for the normal operation of organism, relate to much physiology and pathological process, such as, the propagation of cell and apoptosis, endocytosis, ion transport and dynamic equilibrium, multidrug resistance etc., once pH value is abnormal, it will cause cell functional disorders.The pH distribution of human body is extremely complex, 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), Cytoplasm 6.8 ~ 7.4；Additionally, the secretions of some tumor cell can cause the change of surrounding pH.Therefore, the research of pH sensitive material will be significant to biology and medical science.
PH sensitive materials are widely used in pH probe in detecting, demonstrate higher sensitivity, and part probe is also employed in living body detection.But DNA three-dimensional paper folding structure is as a kind of good biocompatibility, Stability Analysis of Structures, the biological controlled-released material that response is sensitive, have no by its to the sensitive response application of pH value in detection or other any fields.
Summary of the invention
For overcoming the deficiencies in the prior art, the present invention provides the preparation method of a kind of pH sensing element based on DNA molecular change of configuration.
The preparation method of a kind of pH sensing element based on DNA molecular change of configuration, it is characterised in that comprise the following steps:
(1) preparation of DNA self-assembled structures:
Respectively take 2 DNA strands different for μ L and join in 42 μ L Tris-MgCl solution, DNA strand 50 μMs, Tris-MgCl solution Tris 10mM, MgCl2
50mM, pH8；Being placed in PCR instrument by mixed solution, reaction temperature is 95 DEG C, is cooled to 4 DEG C after 10 minutes, continues reaction 30 minutes；DNA strand can be self-assembled into the little molecule of various configuration by base pair complementarity；
(2) DNA molecular and the absorption of gold substrate:
Take sulfydryl coupling agent (SH-(PEG)7-OCH3, 1mg/mL) modify in gold surface, stand 30 minutes；Then taking from the DNA molecular solution assembled, be added drop-wise to above-mentioned gold surface, fully reaction 2 hours, i.e. can obtain this pH sensitive material；
(3) change of configuration of DNA molecular:
DNA molecular in acid condition, by H+Effect, segment will occur a certain degree of change of configuration；Meanwhile, some are for pH than more sensitive DNA section, and such 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 bonded between sulfydryl-gold.
Described DNA strand 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 as the clean energy resource of a kind of proton driving has broad application prospects.Meanwhile, gold and DNA molecular are respectively provided with good biocompatibility, thus present invention could apply to nano biological medical material field, are specifically expected to there is good application at aspects such as pH detection and biological intelligence controlled releases.
And the achievement in research of the present invention will provide new approaches to building new bio pH sensor.DNA molecular configuration under different pH environment can occur reversible change, and the DNA molecular of many Reconstructeds remains to keep the sensitivity suitable to pH after the interior environment that experience organism is complicated.Therefore the present invention has higher Research Significance and using value.
Present invention aim at building a kind of Novel pH Sensitive element, be expected to apply in fields such as pH detection and biological intelligence controlled releases.The method, by choosing different DNA, is self-assembled into the DNA molecular of various configuration, such as DNA tetrahedron (TDN), i-motif, DNA double chain (dS) etc..These DNA moleculars can occur change of configuration even to fold in acid condition, demonstrate with neutral or meta-alkalescence under the conditions of distinct molecular configuration, and after pH recovers, its configuration also can recover therewith.The present invention has the highest sensitivity for pH, can tackle increasingly complex biological internal environment simultaneously.
It is an advantage of the current invention 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, fast reaction can be realized, be the reversible biotransformation switching device of the green cleaning 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 stable, has certain rigidity, remains to keep the sensitivity suitable to pH, meet in vitro and internal detection after the interior environment that experience organism is complicated.
Accompanying drawing explanation
Fig. 1 is the mechanism of action schematic diagram of the gold-plated quartz crystal-DNA pH sensing element prepared by embodiment 1.
Fig. 2 is the mechanism of action schematic diagram of the gold-plated quartz crystal-DNA pH sensing element prepared by embodiment 2.
Fig. 3 is the mechanism of action schematic diagram of the gold-plated quartz crystal-DNA pH sensing element prepared by embodiment 3.
Fig. 4 is the mechanism of action schematic diagram of the nanometer gold-DNA pH sensing element prepared by embodiment 4.
Fig. 5 is the mechanism of action schematic diagram of the nanometer gold-DNA pH sensing element prepared by embodiment 5.
Fig. 6 is the mechanism of action schematic diagram of the nanometer gold-DNA pH sensing element prepared by embodiment 6.
Detailed description of the invention
Below by way of specific embodiment, technical scheme is further described.Below example is to further illustrate the present invention, and does not limits the scope of the invention.
Embodiment
1
:
Respectively take 2 μ L single stranded DNAs (50 μMs) 1,2,3,4 and join 42 μ L Tris-MgCl(Tris 10mM, MgCl2
50mM, pH8) in solution.Being placed in PCR instrument by mixed solution, reaction temperature is 95 DEG C, is cooled to 4 DEG C after 10 minutes, continues reaction 30 minutes.DNA strand can be self-assembled into the DNA tetrahedron containing i-motif by base pair complementarity.
Take 100 μ L sulfydryl coupling agent (SH-(PEG)7-OCH3, 0.1mg/mL) drip on the surface of gold-plated quartz crystal, stand 30 minutes.Take 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 (overnight).
This TDN i-motif molecule in acid condition, by H+Effect, DNA tetrahedron will occur a certain degree of deformation.I-motif segment simultaneously, can be 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 the sensitive biological sensing material of a kind of pH.
Embodiment
2
:
Respectively take 2 μ L single stranded DNAs (50 μMs) 2,3,4,5 and join 42 μ L Tris-MgCl(Tris 10mM, MgCl2
50mM, pH8) in solution.Being placed in PCR instrument by mixed solution, reaction temperature is 95 DEG C, is cooled to 4 DEG C after 10 minutes, continues reaction 30 minutes.DNA strand can be self-assembled into DNA tetrahedron by base pair complementarity.
Take 100 μ L sulfydryl coupling agent (SH-(PEG)7-OCH3, 0.1mg/mL) drip on the surface of gold-plated quartz crystal, stand 30 minutes.Take 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 (overnight).
This gold-plated quartz crystal TDN molecule in acid condition, by H+Effect, DNA tetrahedron will occur a certain degree of deformation.Thus, the change of pH can be reacted in the change of configuration by TDN molecule intuitively.
Embodiment
3
:
Respectively take 2 μ L single stranded DNAs (50 μMs) 6,7 and join 46 μ L Tris-MgCl(Tris 10mM, MgCl2
50mM, pH8) in solution.DNA strand can be self-assembled into double chain DNA molecule by base pair complementarity.
Take 100 μ L sulfydryl coupling agent (SH-(PEG)7-OCH3, 0.1mg/mL) drip on the surface of gold-plated quartz crystal, stand 30 minutes.Take 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 (overnight).
This gold-plated quartz crystal-double chain DNA molecule in acid condition, by H+Effect, double chain DNA molecule segment will occur a certain degree of curling.Thus, the change of pH can be reacted in the change of configuration by double chain DNA molecule intuitively.
Embodiment
4
:
Respectively take 2 μ L single stranded DNAs (50 μMs) 1,2,3,4 and join 42 μ L Tris-MgCl(Tris 10mM, MgCl2
50mM, pH8) in solution.Being placed in PCR instrument by mixed solution, reaction temperature is 95 DEG C, is cooled to 4 DEG C after 10 minutes, continues reaction 30 minutes.DNA strand can be self-assembled into the DNA tetrahedron containing i-motif by base pair complementarity.
Take 1 μ L sulfydryl coupling agent (SH-(PEG)7-OCH3, 1mg/mL) join in the solution of 1mL nanometer gold (13nm, 3nM), stand 30 minutes.Taking the DNA molecular solution that 20 μ L self assemblies complete, be added drop-wise in the solution of above-mentioned nanometer gold, fully reaction 2 hours, i.e. can obtain this pH sensitive material.
This nanometer gold-TDN i-motif molecule in acid condition, by H+Effect, DNA tetrahedron will occur a certain degree of deformation.I-motif segment simultaneously, can be 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 take 2 μ L single stranded DNAs (50 μMs) 2,3,4,5 and join 42 μ L Tris-MgCl(Tris 10mM, MgCl2
50mM, pH8) in solution.Being placed in PCR instrument by mixed solution, reaction temperature is 95 DEG C, is cooled to 4 DEG C after 10 minutes, continues reaction 30 minutes.DNA strand can be self-assembled into DNA tetrahedron by base pair complementarity.
Take 1 μ L sulfydryl coupling agent (SH-(PEG)7-OCH3, 1mg/mL) join in the solution of 1mL nanometer gold (13nm, 3nM), stand 30 minutes.Taking the DNA molecular solution that 20 μ L self assemblies complete, be added drop-wise in the solution of above-mentioned nanometer gold, fully reaction 2 hours, i.e. can obtain this pH sensitive material.
This nanometer gold-TDN molecule in acid condition, by H+Effect, DNA tetrahedron will occur a certain degree of deformation.Thus, the change of pH can be reacted in the change of configuration by TDN molecule intuitively.
Embodiment
6
:
Respectively take 2 μ L single stranded DNA (50 μMs) Double Strains-A, Double Strains-B and join 46 μ L Tris-MgCl(Tris 10mM, MgCl2
50mM, pH8) in solution.Being placed in PCR instrument by mixed solution, reaction temperature is 95 DEG C, is cooled to 4 DEG C after 10 minutes, continues reaction 30 minutes.DNA strand can be self-assembled into double chain DNA molecule by base pair complementarity.
Take 1 μ L sulfydryl coupling agent (SH-(PEG)7-OCH3, 1mg/mL) join in the solution of 1mL nanometer gold (13nm, 3nM), stand 30 minutes.Taking the DNA molecular solution that 20 μ L self assemblies complete, be added drop-wise in the solution of above-mentioned nanometer gold, fully reaction 2 hours, i.e. can obtain this pH sensitive material.
This nanometer gold-double chain DNA molecule in acid condition, by H+Effect, double chain DNA molecule segment will occur a certain degree of curling.Thus, the change of pH can be reacted in the change of configuration by double chain DNA molecule intuitively.
Claims (2)
1. the preparation method of a pH sensing element based on DNA molecular change of configuration, it is characterised in that comprise the following steps:
(1) preparation of DNA self-assembled structures:
Respectively take 2 DNA strands different for μ L and join 42 μ L Tris-MgCl2In solution, DNA strand 50 μMs, Tris-MgCl2Solution Tris 10mM, MgCl250mM, pH8；Being placed in PCR instrument by mixed solution, reaction temperature is 95 DEG C, is cooled to 4 DEG C after 10 minutes, continues reaction 30 minutes；DNA strand can be self-assembled into the DNA tetrahedron molecule containing i-motif, DNA tetrahedron molecule or double chain DNA molecule by base pair complementarity；
(2) DNA molecular and the absorption of gold substrate:
Take sulfydryl coupling agent SH-(PEG) of 1mg/mL7-OCH3Modify in gold surface, stand 30 minutes；Then taking from the DNA molecular assembled, be added drop-wise to above-mentioned gold surface, fully reaction 2 hours, i.e. can obtain this pH sensing element；
(3) change of configuration of DNA molecular:
The described DNA tetrahedron molecule containing i-motif in acid condition, by H+Effect, DNA tetrahedron will occur a certain degree of deformation, and i-motif segment can be due to H simultaneously+Effect and produce folding；Or described DNA tetrahedron molecule is in acid condition, by H+Effect, DNA tetrahedron will occur a certain degree of deformation；Or described double chain DNA molecule is in acid condition, by H+Effect, double chain DNA molecule segment will occur a certain degree of curling；
The mode that described DNA molecular is connected with surface is bonded between sulfydryl-gold.
The preparation method of pH sensing element based on DNA molecular change of configuration the most according to claim 1, it is characterised in that described gold substrate is nano-Au solution, or is coated with the quartz wafer of gold thin film.
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| US9881786B2 (en) * | 2015-04-02 | 2018-01-30 | Micron Technology, Inc. | Methods of forming nanostructures using self-assembled nucleic acids, and nanostructures thereof |
| 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 |
| 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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| CN101561398A (en) * | 2008-04-18 | 2009-10-21 | 中国科学院上海应用物理研究所 | Target molecule detection method based on nano-Au and nucleic acid structure |
| WO2011082419A2 (en) * | 2010-01-04 | 2011-07-07 | Life Technologies Corporation | Dna sequencing methods and detectors and systems for carrying out the same |
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