CN105136875A - Method utilizing nano-channel sensor modified by nucleic acid probe having super sandwich structure to high-sensitively and high-specifically detect Zn2+ - Google Patents
Method utilizing nano-channel sensor modified by nucleic acid probe having super sandwich structure to high-sensitively and high-specifically detect Zn2+ Download PDFInfo
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- CN105136875A CN105136875A CN201510464904.4A CN201510464904A CN105136875A CN 105136875 A CN105136875 A CN 105136875A CN 201510464904 A CN201510464904 A CN 201510464904A CN 105136875 A CN105136875 A CN 105136875A
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Abstract
The invention disclose a method utilizing a nano-channel sensor modified by a nucleic acid probe having a super sandwich structure to high-sensitively and high-specifically detect Zn2+. The method comprises the following steps: (1) preparing a polymer nano channel; (2) designing a nucleic acid probe with a super sandwich structure, wherein the probe has a specific recognition function on Zn2+; (3) carrying out functional modification on the nano channel; (4) carrying out Zn2+ quantitative detection. The invention provides a novel method for detecting specific ions. The detection sensitive of Zn2+ can reach 1 nM, the specificity is high, other ions can be well identified, moreover, the sensor has a stable property, and the repeatability is high.
Description
Technical field
The invention belongs to field of biological detection, be specifically related to one and utilize the highly sensitive high specific of super sandwich nucleic acid probe nano pore sensor to detect Zn
2+method.
Background technology
Zinc is one of trace element of needed by human, is to maintain the growth of organism normal growth, metabolic important substance.Scientific research shows, zinc ion is one of important meals ion of composition more than 300 kind of bioenzyme activity catalytic center; It can as the structure factor of metalloproteinases or transcription factor; Meanwhile, zinc ion, at modulation of ion channels, participates in all play very important effect in nerve conduction process and central nervous system.But, when zinc ion in human body excessive exist time, then can make the immunity degradation of people, can bring out iron content in body inner blood, kidney and liver and reduce, copper ion lacks, thus causes the generation of various diseases.Therefore, the zinc ion of real-time follow-up and monitoring bio body weight has important scientific value.
At present to Zn
2+detection method have a lot, wherein the most commonly fluorescence probe method.Although fluorescence probe method detects have the advantages such as simple to operate, quick, in testing process, institute uses synthesis and the modification of fluorescent dye, and be but a process for individual complexity, preparation cost is higher.And this method still will depend on instrument carries out, thus limit the usable range of the method.Be necessary to utilize biology sensor, develop a kind of quick, accurate, real-time zinc ion detection method, make it have the features such as testing cost is low, highly sensitive, high specificity.
Summary of the invention
Goal of the invention: the object of the invention is to for the deficiencies in the prior art, provides one to utilize the highly sensitive high specific of super sandwich nucleic acid probe nano pore sensor to detect Zn
2+method.
Technical scheme: in order to achieve the above object, the present invention is specifically achieved like this: utilize the highly sensitive high specific of super sandwich nucleic acid probe nano pore sensor to detect Zn
2+method, it is characterized in that, comprise the following steps:
(1) preparation in high molecular nanometer duct
To have polyphosphazene polymer ethylene glycol terephthalate (PET) film of heavy ion bombardment track, first each irradiation of pros and cons one hour under uviol lamp, makes it in chemical etching, have better stability; During etching, irradiated PET film is immersed in the NaOH solution of 6M/L, and holding temperature is at 50 ± 5 DEG C, the time taking-up PET film that interval is different, and by the NaOH solution on deionized water rinsing surface, then soak 4h in deionized water and, to remove the NaOH with remained on surface in duct, just obtain the PET film containing nanohole array etched; When etching time is 5min, characterized by scanning electron microscope to nano pore, obtain the nano pore of cylindricality, through the statistics to aperture, the same terms lower 50 ducts, obtaining constructed nano pore aperture is 34 ± 3nm;
(2) have Zn
2+the design of the super sandwich nucleic acid probe of specific recognition effect:
On the basis of traditional sandwich, by design increase by two assist probes, make it be self-assembled into super sandwich structure, simultaneously substrate probe can with DNA enzymatic formed special, to ion Zn
2+there is the structure of specific recognition effect, when there being Zn
2+when existing in system, this special construction will be sheared, and super sandwich structure is untied thereupon;
(3) functional modification of nano pore:
Through the PET film of over etching, carboxyl is rich in duct, can through carbodiimide hydrochloride and the activation of N-hydroxy thiosuccinimide containing amino capture probe, form the ester of transition state, be modified at the inside surface of nano pore, then by base pair complementarity principle, add other nucleic acid probe, self assembly forms super sandwich structure, has constructed nano pore senser element;
(4) to Zn
2+quantitatively detect:
Organic glass electrolytic tank is adopted to be used for the test of ion responsitivity experiment, electrolytic tank is made up of two chambers, the film with functional nano duct is fixed in the middle of two chambers, hole on chamber cross section contacts with film, ion can detect through duct, membrance current proving installation is bipolar electrode system, what adopt in ion responsitivity test is Ag/AgCl electrode, the operating system of instrument is KeithleyInstrumentsExceLINXsoftwarefortheModel6487 software, according to special requirement of experiment, with the field sweep voltage of-2V-+2V, detection transmembrane current changes.The electrolytic solution used of test is Tris buffer solution (pH=7.5,100mMNaCl10mMMgCl
2).Super sandwich structure nucleic acid probe is assembled 10h at nano pore, then adds the Zn of variable concentrations
2+(from 1nM to 1mM) reacts 1h, detects curent change.By the Zn of 1mM
2+, Pb
2+, Cu
2+, Hg
2+detect with the nano pore sensor of super sandwich structure respectively.Experimental result confirms, this sensor achieves Zn
2+real-time highly sensitive high specific detects.This detection method have easy and simple to handle, consumption costs is low, good stability, and goes for the detection of other different ions by the design of probe, the advantages such as applied range.
Beneficial effect: the present invention is compared with conventional art, and tool has the following advantages:
1. the present invention utilizes the nano pore sensor that the nucleic acid probe of super sandwich structure is modified, for Zn
2+detection, improve detect Zn
2+sensitivity and specificity, minimumly can detect 1nM;
2. sensor used in this detection method, not only have signal amplification mechanism, and the mechanism that signal is opened also makes it have good stability;
3. by the design to sandwich structure amplifying nucleic acid probe super in this sensor, go for detecting other different target molecules, there is universality, more utilize and promote the use of.
Accompanying drawing explanation
Fig. 1 is principle schematic of the present invention;
Fig. 2 is to Zn
2+the sensitivity results figure detected;
Fig. 3 is to Zn
2+the specific outcome figure detected.
Embodiment
Embodiment:
(1) preparation in high molecular nanometer duct
To have polyphosphazene polymer ethylene glycol terephthalate (PET) film of heavy ion bombardment track, first each irradiation of pros and cons one hour under uviol lamp, makes it in chemical etching, have better stability.During etching, irradiated PET film is immersed in the NaOH solution of 2M/L, and holding temperature is at 50 ± 5 DEG C, the time taking-up PET film that interval is different, and by the NaOH solution on deionized water rinsing surface, then soak 4h in deionized water and, to remove the NaOH with remained on surface in duct, just obtain the PET film containing nanohole array etched.When etching time is 6min, by scanning electron microscope, nano pore is characterized, obtain the nano pore of cylindricality.Through under the same terms to the statistics in aperture, duct, obtain constructed nano pore aperture and be about 80nm.
(2) have Zn
2+the design of the super sandwich nucleic acid probe of specific recognition effect:
On the basis of traditional sandwich, by design increase by two assist probes, it is made to be self-assembled into super sandwich structure.Simultaneously substrate probe can with DNA enzymatic formed special, to ion Zn
2+there is the structure of specific recognition effect, when there being Zn
2+when existing in system, this special construction will be sheared, and super sandwich structure is untied thereupon.In system, the nucleic acid probe of self assembly, the concentration of DNA enzymatic are 1uM.Designed nucleic acid probe sequence is bought in precious bioengineering (Dalian) company limited, is respectively:
Capture probe: 5 '-CAGTGTGGAAAATCTCTAGC-(CH
2)
6-NH
2-3 '
Substrate probe: 5 '-GCTAGAGATTTTCCACACTGATGCAGACGTTGAAGGATTATCTACTAAAAGGGTCT GAGGG-3 '
Assist probes 1:5 '-TACTCCCCCAGGTGCCCCTCAGACCCTTTTAGT-3 '
Assist probes 2:5 '-GCACCTGGGGGAGTAACTAAAAGGGTCTGAGGG-3 '
DNA enzymatic: 5 '-AGATAATCTAGTTGAGCTGTCTGCAT-3 '
(3) functional modification of nano pore:
Through the PET film of over etching, in duct, be rich in carboxyl-COOH.Therefore, through carbodiimide hydrochloride and the activation of N-hydroxy thiosuccinimide, the ester of transition state can be formed, is modified at the inside surface of nano pore finally by two step chemical reactions containing amino capture probe.Then by base pair complementarity principle, add other nucleic acid probe, self assembly forms super sandwich structure, has constructed nano pore senser element.
(4) to Zn
2+quantitatively detect:
Self-control transmembrane current proving installation is bipolar electrode system, and what adopt in ion responsitivity test is Ag/AgCl electrode.The operating system of instrument is KeithleyInstrumentsExceLINXsoftwarefortheModel6487 software.According to special requirement of experiment, with the field sweep voltage of-2V-+2V, detect transmembrane current change.The electrolytic solution used of test is Tris buffer solution (pH=7.5,100mMNaCl10mMMgCl
2).Super sandwich structure nucleic acid probe is assembled 10h at nano pore, and the transmembrane current in detection system obviously reduces.Current value is now set to starting point, then adds the Zn of variable concentrations
2+(from 1nM to 1mM) reacts 1h, detects transmembrane current change, presents the trend that signal progressively increases.By the Zn of 1mM
2+, Pb
2+, Cu
2+, Hg
2+detect with the nano pore sensor of super sandwich structure respectively.
Claims (1)
1. utilize the highly sensitive high specific of super sandwich nucleic acid probe nano pore sensor to detect Zn
2+method, it is characterized in that, comprise the following steps:
(1) preparation in high molecular nanometer duct
To have polyphosphazene polymer ethylene glycol terephthalate (PET) film of heavy ion bombardment track, first each irradiation of pros and cons one hour under uviol lamp, makes it in chemical etching, have better stability; During etching, irradiated PET film is immersed in the NaOH solution of 6M/L, and holding temperature is at 50 ± 5 DEG C, the time taking-up PET film that interval is different, and by the NaOH solution on deionized water rinsing surface, then soak 4h in deionized water and, to remove the NaOH with remained on surface in duct, just obtain the PET film containing nanohole array etched; When etching time is 5min, characterized by scanning electron microscope to nano pore, obtain the nano pore of cylindricality, through the statistics to aperture, the same terms lower 50 ducts, obtaining constructed nano pore aperture is 34 ± 3nm;
(2) have Zn
2+the design of the super sandwich nucleic acid probe of specific recognition effect:
On the basis of traditional sandwich, by design increase by two assist probes, make it be self-assembled into super sandwich structure, simultaneously substrate probe can with DNA enzymatic formed special, to ion Zn
2+there is the structure of specific recognition effect, when there being Zn
2+when existing in system, this special construction will be sheared, and super sandwich structure is untied thereupon;
(3) functional modification of nano pore:
Through the PET film of over etching, carboxyl is rich in duct, can through carbodiimide hydrochloride and the activation of N-hydroxy thiosuccinimide containing amino capture probe, form the ester of transition state, be modified at the inside surface of nano pore, then by base pair complementarity principle, add other nucleic acid probe, self assembly forms super sandwich structure, has constructed nano pore senser element;
(4) to Zn
2+quantitatively detect:
Organic glass electrolytic tank is adopted to be used for the test of ion responsitivity experiment, electrolytic tank is made up of two chambers, the film with functional nano duct is fixed in the middle of two chambers, hole on chamber cross section contacts with film, ion can detect through duct, membrance current proving installation is bipolar electrode system, what adopt in ion responsitivity test is Ag/AgCl electrode, the operating system of instrument is KeithleyInstrumentsExceLINXsoftwarefortheModel6487 software, according to special requirement of experiment, with the field sweep voltage of-2V-+2V, detection transmembrane current changes.The electrolytic solution used of test is Tris buffer solution (pH=7.5,100mMNaCl10mMMgCl
2).
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| CN107966423A (en) * | 2017-10-27 | 2018-04-27 | 中国农业大学 | A kind of colorimetric sensor of resistance to high salt of functional nucleic acid based on zinc and its application |
| CN107993917A (en) * | 2017-12-11 | 2018-05-04 | 中国建筑材料科学研究总院有限公司 | Organic material microchannel plate and preparation method thereof |
| CN109082084A (en) * | 2018-07-04 | 2018-12-25 | 温州大学 | A kind of polymeric membrane and preparation method thereof with nano pore |
| CN109986089A (en) * | 2019-03-14 | 2019-07-09 | 厦门大学 | A kind of preparation method and applications of bismuth selenide nanogold composite material |
| CN111218497A (en) * | 2020-01-16 | 2020-06-02 | 台州学院 | Construction of polymer nano-pore and detection of miRNA (micro ribonucleic acid) by using same |
| CN112179954A (en) * | 2020-09-27 | 2021-01-05 | 西北工业大学 | Method for preparing nano-fluid diode responding to pH and temperature based on gelatin modified solid-state nano-pores |
| CN112394089A (en) * | 2020-11-20 | 2021-02-23 | 台州学院 | Construction of label-free nanopore sensor and application of label-free nanopore sensor in tetracycline detection |
| CN112858434A (en) * | 2021-01-11 | 2021-05-28 | 北京大学口腔医学院 | Cysteine protease inhibitor B detection device and preparation method and application thereof |
| CN114606295A (en) * | 2022-01-26 | 2022-06-10 | 湖南大学 | Deoxyriboribozyme-based demethylase activity detection method and application |
| CN117110401A (en) * | 2023-05-16 | 2023-11-24 | 中国环境科学研究院 | Used for detecting Cr by electrode method 3+ Nuclear pore membrane, and preparation method and application thereof |
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Cited By (15)
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| CN107966423B (en) * | 2017-10-27 | 2020-10-02 | 中国农业大学 | Zinc-based high-salt-resistance colorimetric sensor of functional nucleic acid and application thereof |
| CN107966423A (en) * | 2017-10-27 | 2018-04-27 | 中国农业大学 | A kind of colorimetric sensor of resistance to high salt of functional nucleic acid based on zinc and its application |
| CN107993917A (en) * | 2017-12-11 | 2018-05-04 | 中国建筑材料科学研究总院有限公司 | Organic material microchannel plate and preparation method thereof |
| CN109082084A (en) * | 2018-07-04 | 2018-12-25 | 温州大学 | A kind of polymeric membrane and preparation method thereof with nano pore |
| CN109082084B (en) * | 2018-07-04 | 2021-06-29 | 温州大学 | Polymer film with nano-pore and preparation method thereof |
| CN109986089A (en) * | 2019-03-14 | 2019-07-09 | 厦门大学 | A kind of preparation method and applications of bismuth selenide nanogold composite material |
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| CN112179954A (en) * | 2020-09-27 | 2021-01-05 | 西北工业大学 | Method for preparing nano-fluid diode responding to pH and temperature based on gelatin modified solid-state nano-pores |
| CN112179954B (en) * | 2020-09-27 | 2024-06-04 | 西北工业大学 | Method for preparing nano fluid diode responsive to pH and temperature based on gelatin modified solid state nano hole |
| CN112394089A (en) * | 2020-11-20 | 2021-02-23 | 台州学院 | Construction of label-free nanopore sensor and application of label-free nanopore sensor in tetracycline detection |
| CN112858434A (en) * | 2021-01-11 | 2021-05-28 | 北京大学口腔医学院 | Cysteine protease inhibitor B detection device and preparation method and application thereof |
| CN112858434B (en) * | 2021-01-11 | 2022-08-16 | 北京大学口腔医学院 | Cysteine protease inhibitor B detection device and preparation method and application thereof |
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| CN114606295B (en) * | 2022-01-26 | 2024-04-12 | 湖南大学 | Demethylation enzyme activity detection method based on deoxyriboribozyme and application thereof |
| CN117110401A (en) * | 2023-05-16 | 2023-11-24 | 中国环境科学研究院 | Used for detecting Cr by electrode method 3+ Nuclear pore membrane, and preparation method and application thereof |
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