CN110824161B - Nanocomposite for detecting silver ions and preparation method thereof - Google Patents
Nanocomposite for detecting silver ions and preparation method thereof Download PDFInfo
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- CN110824161B CN110824161B CN201810915712.4A CN201810915712A CN110824161B CN 110824161 B CN110824161 B CN 110824161B CN 201810915712 A CN201810915712 A CN 201810915712A CN 110824161 B CN110824161 B CN 110824161B
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- 229910052709 silver Inorganic materials 0.000 title claims abstract description 17
- -1 silver ions Chemical class 0.000 title claims abstract description 14
- 239000004332 silver Substances 0.000 title claims abstract description 13
- 238000002360 preparation method Methods 0.000 title claims description 10
- 239000002114 nanocomposite Substances 0.000 title abstract description 6
- 238000005516 engineering process Methods 0.000 claims abstract description 46
- 229910052737 gold Inorganic materials 0.000 claims abstract description 34
- 239000010931 gold Substances 0.000 claims abstract description 34
- 108060002716 Exonuclease Proteins 0.000 claims abstract description 29
- 102000013165 exonuclease Human genes 0.000 claims abstract description 29
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 28
- 230000003321 amplification Effects 0.000 claims abstract description 23
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 23
- 239000002131 composite material Substances 0.000 claims abstract description 18
- 239000000243 solution Substances 0.000 claims description 21
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 claims description 12
- 239000007993 MOPS buffer Substances 0.000 claims description 8
- 230000015572 biosynthetic process Effects 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 8
- 108020004707 nucleic acids Proteins 0.000 claims description 8
- 102000039446 nucleic acids Human genes 0.000 claims description 8
- 150000007523 nucleic acids Chemical class 0.000 claims description 8
- 238000004140 cleaning Methods 0.000 claims description 7
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical group [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 claims description 7
- 229940043267 rhodamine b Drugs 0.000 claims description 7
- 229940104302 cytosine Drugs 0.000 claims description 6
- 238000013461 design Methods 0.000 claims description 6
- 238000003786 synthesis reaction Methods 0.000 claims description 6
- 229920000371 poly(diallyldimethylammonium chloride) polymer Polymers 0.000 claims description 5
- 239000011324 bead Substances 0.000 claims description 4
- 238000007885 magnetic separation Methods 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 3
- 108020004414 DNA Proteins 0.000 claims 1
- 102000053602 DNA Human genes 0.000 claims 1
- 239000013066 combination product Substances 0.000 claims 1
- 229940127555 combination product Drugs 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 22
- 239000002539 nanocarrier Substances 0.000 abstract description 18
- 238000000034 method Methods 0.000 abstract description 14
- 230000035945 sensitivity Effects 0.000 abstract description 14
- 125000004122 cyclic group Chemical group 0.000 abstract description 13
- 238000010008 shearing Methods 0.000 abstract description 10
- 239000003814 drug Substances 0.000 abstract description 6
- 235000013305 food Nutrition 0.000 abstract description 5
- 239000011148 porous material Substances 0.000 abstract description 4
- 230000005284 excitation Effects 0.000 abstract description 3
- 239000006228 supernatant Substances 0.000 abstract description 3
- 239000012488 sample solution Substances 0.000 abstract description 2
- 238000000926 separation method Methods 0.000 abstract 1
- 239000007850 fluorescent dye Substances 0.000 description 6
- 229910021645 metal ion Inorganic materials 0.000 description 6
- 239000000975 dye Substances 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 108010052305 exodeoxyribonuclease III Proteins 0.000 description 4
- 230000008876 conformational transition Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- PUAQLLVFLMYYJJ-UHFFFAOYSA-N 2-aminopropiophenone Chemical compound CC(N)C(=O)C1=CC=CC=C1 PUAQLLVFLMYYJJ-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000013399 early diagnosis Methods 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- 239000003607 modifier Substances 0.000 description 2
- 239000002088 nanocapsule Substances 0.000 description 2
- 125000003396 thiol group Chemical group [H]S* 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- FOIXSVOLVBLSDH-UHFFFAOYSA-N Silver ion Chemical compound [Ag+] FOIXSVOLVBLSDH-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000001479 atomic absorption spectroscopy Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000004993 emission spectroscopy Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 238000002354 inductively-coupled plasma atomic emission spectroscopy Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 238000000918 plasma mass spectrometry Methods 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 238000003969 polarography Methods 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000000844 transformation Methods 0.000 description 1
Classifications
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
- G01N33/553—Metal or metal coated
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
Abstract
The invention provides a nano gold composite material based on an exonuclease cycle amplification technology and a base mismatch recognition technology, which can be used in the fields of food, environment, medicine and health and the like. The invention utilizes nano gold with hollow and porous structure and identifiable Ag + To construct a nanocomposite with "pore caps". When the sample solution containing silver ions is added, the silver ions are separated from the surface of the nano carrier due to the action of the biomolecules on the surface of the nano carrier, dye molecules in the nano carrier are released, after separation, the supernatant generates fluorescence emission under the irradiation of excitation light with a certain wavelength, and the detection of the silver ions is realized according to the intensity of fluorescence emission signals. Meanwhile, the invention realizes the cyclic amplification of fluorescent signals by utilizing the shearing action of exonuclease. The method is simple, efficient, high in sensitivity, good in selectivity, convenient and quick, low in cost and wide in application range.
Description
Technical Field
The invention relates to a nano-gold composite material based on an exonuclease cycle amplification technology and a base mismatch recognition technology and a preparation method thereof, in particular to a nano-composite material for detecting silver ions and a preparation method thereof.
Background
Silver ion (Ag) as heavy metal ion + ) With mercury ions (Hg) 2+ ) Similarly, it is a highly toxic metal ion and a widely distributed environmental pollutant. Even at low concentrations, serious and permanent poisoning of the environment and humans can occur. More seriously, byContaminated water source, ag + Can be accumulated in agricultural products and aquatic products continuously and then enter the human food chain. If exposed to Ag for a long time + In the existing environment, it may lead to slow onset of degenerative diseases in humans in the body and nervous system. Thus, efficient, sensitive and economical Ag is established + The detection method of the method has very important significance in the fields of environmental monitoring, food safety, clinical diagnosis and the like.
Currently, used for Ag + Conventional methods of detection mainly include plasma mass spectrometry (ICP-AES), atomic absorption/emission spectrometry, polarography, and the like. However, these methods often require complicated operations, time-consuming analysis, expensive, complicated equipment, etc., rendering them unusable in environments where resources are limited, and in addition, these methods have yet to be improved in terms of detection sensitivity and selectivity. To overcome these disadvantages, there is an urgent need to develop a simple, sensitive, economical and efficient assay method to meet the requirements of the fields of biology, medicine, environment, etc. on Ag + Is a detection requirement of (1).
Because metal ions can form coordination bonds with base pairs and can replace hydrogen bonds in conventional Watson-Crick base pairs to form metal-base pairs, the effect has great significance for rapid and efficient detection of metal ions, and therefore, the metal ions are paid attention to widely. Wherein cytosine (C) can be combined with Ag + Formation of C-Ag + The binding is stable and the generation of this particular structure can be used to achieve the binding to Ag + Is detected. Moreover, only Ag is recognized due to the C-C base mismatch + Thereby forming Ag + Base pairs of the backbone such that Ag + Has very high specificity and selectivity. The invention is based on Ag + Combines the reaction characteristic with nucleic acid biomolecules rich in cytosine, is skillfully applied to the field of nano materials with hollow and porous structures, combines the shearing action of biological enzyme on the basis of a base mismatch recognition technology to improve the detection sensitivity, and is Ag + Provides a novel, specific and efficient detection technology. Nano gold with hollow and porous structure is used as nanoRice carrier, ag paired by C-C base mispairing + Construction of nanocomposite for detection of Ag by specific biological recognition + The technology of (2) is not reported in the literature.
Disclosure of Invention
In order to overcome the defects existing in the prior art, the method aims at detecting Ag by the nano gold composite material based on the exonuclease cycle amplification technology and the base mismatch recognition technology + The technology of (2) is not reported, and therefore, the first object of the present invention is: provides and constructs a novel nano gold composite material based on an exonuclease cycle amplification technology and a base mismatch recognition technology, specifically designs and synthesizes Ag-capable nano gold by using hollow and porous nano gold as a nano carrier + The identified biological molecules are assembled on the surface of the nano-carrier, and on one hand, the biological molecules are used as a 'hole cap' for blocking the hole opening of the nano-carrier to prevent the leakage of substances in the hole; on the other hand as Ag + Can be combined with Ag + Specific base mismatch recognition reaction occurs to form C-Ag + The simultaneous conformational transition of the C base pairs is separated from the surface of the nanocapsule, so that the plugged 'hole caps' are opened, dye molecules in the nanocapsule are released, supernatant liquid is separated, fluorescent emission is generated under the irradiation of excitation light with a certain wavelength, and Ag is realized according to the intensity of fluorescent emission signals + Is detected. Meanwhile, in order to further increase the sensitivity, the invention realizes the cyclic amplification of fluorescent signals by utilizing the shearing action of the exonuclease on the basis of biological recognition.
Since exonuclease can be used for preparing the double-chain structure of the biological molecule-Ag + Shearing the combination, and after shearing, ag + Is released, these Ag + Can be subjected to recognition reaction with other recognition probes again and then sheared … … again, resulting in Ag + And the porous cap can be recycled, more porous caps are opened, and more porous substances are released. Ag is due to the action of the shearing enzyme + Is recycled, so that the detection sensitivity is obviously enhanced. The detection system based on the exonuclease cyclic amplification technology and the base mismatch recognition technology can detect trace Ag + The sample realizes high sensitivity and highAnd (5) selectively detecting. Even if the sample contains very small amount of Ag + Satisfactory detection results can also be obtained. The second object of the present invention: provides a method for detecting trace Ag + Is based on exonuclease cyclic amplification technology and base mismatch recognition technology.
The invention achieves the aim through the following technical proposal. The invention provides the method for detecting Ag + The nano gold composite material of (2) takes nano gold with a hollow and porous structure as a nano carrier, and utilizes the hollow and porous structural characteristics of the nano gold to load guest molecules such as fluorescent dye in the nano gold composite material, wherein the fluorescent dye is rhodamine B. In order to prevent the leakage of fluorescent dye, the invention designs and synthesizes the fluorescent dye which can be coated with Ag + The identified biological molecules are assembled on the surface of the nano carrier to form a 'hole cap' for blocking the hole opening, so as to play a role in preventing substances in the hole from leaking out; wherein the catalyst can be Ag + The identified biomolecule is a specially designed and synthesized nucleic acid biomolecule rich in cytosine with a certain base length, the base sequence of which is 5'-TCC TCC CTC CTTAAG GAA CCACCC ACC A-3', and the biomolecule is used as Ag + The recognition probe of (2) is assembled on the nano-gold surface with a hollow and porous structure to form a 'hole cap', and the assembly of the 'hole cap' is realized by a method of pre-modifying a positive charge modifier on the nano-carrier surface, wherein the positive charge modifier is preferably polydiallyl dimethyl ammonium chloride. In order to open more 'hole caps' and release more fluorescent dyes, the invention is designed and synthesized to be capable of being used by Ag + Recognized biomolecules and Ag + The combined product can be acted by exonuclease, i.e. the invention uses exonuclease to make double-chain structure of biological molecule-Ag + Shearing the combination to obtain Ag + From mismatched base pairs C-Ag + Released in C, recycled back into solution and combined with other Ag + The recognition probe is combined, so that more rhodamine B is released, amplification of fluorescent signals is realized, and the exonuclease is preferably exonuclease ExoIII.
The detection mark provided by the invention is preparedQuantity of Ag + The preparation method of the nano gold composite material based on the exonuclease cyclic amplification technology and the base mismatch recognition technology comprises the following steps:
(1) Mixing magnetic beads with a nano gold carrier solution with a hollow and porous structure, adding a polydiallyl dimethyl ammonium chloride solution, magnetically separating after 10-12 hours, and cleaning;
(2) Adding dye molecule solution, adding identifiable Ag after 10-12 hr + After 10-12h, magnetically separating and cleaning;
wherein the identifiable Ag + The base sequence of the biological molecule is 5'-TCC TCC CTC CTTAAG GAACCACCCACCA-3', and the biological molecule and Ag + Can be cleaved by exonuclease to release Ag + 。
The invention has the beneficial effects that: the nano gold composite material based on the exonuclease cyclic amplification technology and the base mismatch recognition technology provided by the invention can recognize Ag + The biomolecules of the (2) are combined with nano gold material with hollow and porous structure, and can be coated with Ag by design and synthesis + The identified biological molecule is assembled on the surface of the nano carrier to form a 'hole cap', and the identifiable Ag is utilized + Biomolecules of (2) and Ag + The base mismatch recognition reaction occurs to form C-Ag + The simultaneous conformational transition of the-C base pairs breaks away from the surface of the nano-carrier, so that the blocked 'pore cap' is opened, dye molecules in the nano-carrier are released, and in order to further increase the sensitivity, the invention utilizes exonuclease to carry out the preparation of the double-chain structure of the biological molecule-Ag + The shearing action of the conjugate realizes the cyclic amplification and detection of fluorescent signals.
The method makes Ag + The detection sensitivity of (2) is obviously improved, and the Ag can be realized + High sensitivity and high selectivity detection. The nano gold composite material based on the exonuclease cyclic amplification technology and the base mismatch recognition technology has the advantages of simple structure, easy synthesis, excellent performance, stability, economy, high efficiency, sensitivity and the like, and can not be subjected to other common interfering substances such as Cd 2+ ，Hg 2+ ，Pb 2+ ，Cu 2+ ，Fe 3+ ，Zn 2+ The influence of the plasma metal ions has high specificity and selectivity. Experimental results show that compared with other common technical methods, the nano-gold composite material based on the exonuclease cycle amplification technology and the base mismatch recognition technology provided by the invention has high sensitivity and excellent selectivity, and is 1.0x10 -13 ～8.0×10 - 11 Detection of Ag in the concentration range of mol/L + The logarithm of the concentration and the fluorescence signal intensity show good linear relation, and the detection limit is as low as 1.0 multiplied by 10 -13 mol/L. Compared with literature values, the invention has the advantages of no pollution to Ag + The detection sensitivity of (2) is improved by nearly 100 times. It is expected that the nano gold composite material based on the exonuclease cycle amplification technology and the base mismatch recognition technology, the preparation method and the detection technology thereof have great medical application potential and wide application prospect, and play an important role in the fields of early diagnosis and treatment of serious diseases, foods, biological medicines, medicine, environment and the like.
Drawings
FIG. 1 Ag + A plot of the logarithm of the concentration versus the fluorescence signal intensity.
Detailed Description
The following are specific examples related to the present invention, and the technical solutions of the present invention will be further described, but the scope of the present invention is not limited to these examples. All changes and equivalents that do not depart from the gist of the invention are intended to be within the scope of the invention.
The present invention will be specifically described with reference to the following examples, but the present invention is not limited to the examples.
Experimental instrument: THZ-82A air bath constant temperature oscillator (medical instruments works in gold altar); f-4600 fluorescence spectrophotometer (Hitachi, japan); magnetic separation rack (Tianjin Beisi le chromatography technology development center).
Experimental reagent: exonuclease Exo iii (Thermo Scientific, usa); polydiene-based propylene-dimethyl-ammonium chloride (Shanghai Ala Biochemical technologies Co., ltd.); 3-4 mu m sulfhydryl magnetic bead (Tianjin Sile color)Spectral technology development center); rhodamine B (Shanghai Ala Biochemical technologies Co., ltd.); can be coated with Ag + The identified biomolecules are specifically designed and synthesized cytosine-rich nucleic acid biomolecules having a base sequence of 5'-TCC TCC CTC CTTAAG GAACCACCCACCA-3' (Shanghai Biotechnology Co., ltd.) and MOPS buffer solution of 0.01M (pH 7.0, shanghai Ala Biotechnology Co., ltd.).
Example 1:
the invention provides a method for preparing a nano gold composite material based on an exonuclease cyclic amplification technology and a base mismatch recognition technology, which comprises the following steps:
(1) Design and synthesis of Ag and Ag + Nucleic acid biomolecules with a specific base length, which are recognized by base mismatches and are rich in cytosine, which nucleic acid biomolecules are capable of interacting with Ag + Specific base mismatch recognition reactions occur;
(2) Cleaning sulfhydryl magnetic beads (20 μl) with MOPS buffer solution, mixing with nano gold carrier solution (400 μl) with hollow and porous structure, adding polydiallyl dimethyl ammonium chloride solution (200 μl,11.664 mg/mL), magnetically separating after 10h at 37deg.C, and cleaning with MOPS buffer solution;
(3) Rhodamine B solution (2. Mu.L, final concentration 1.0X10) was added -4 mol/L), diluted to 100. Mu.L with MOPS buffer solution having pH=7.0, and recognizable Ag was added after 10 hours at 37 ℃ + Is used (10. Mu.L, final concentration 1.0X10) -6 mol/L), magnetic separation is carried out after 10 hours at 37 ℃, and MOPS buffer solution is used for cleaning;
wherein the identifiable Ag + Is a nucleic acid biomolecule of which the base sequence is 5'-TCC TCC CTC CTTAAGGAACCACCCACC A-3', and Ag + Can be sheared by exonuclease ExoIII to release Ag + . The nano gold material with a hollow and porous structure is obtained according to a literature method (W.Wang, C.Chen, X.X.Li, S.Y.Wang and X.L.Luo.chem.Commun.,2015,51,9109-9112.).
Example 2:
the invention is adopted to put forwardIs based on exonuclease cyclic amplification technology and base mismatch recognition technology + The method of (2) is as follows:
(1) 10 mu L of the mixture containing Ag + Adding the sample solution to be detected into the nano-gold composite material based on the exonuclease cycle amplification technology and the base mismatch recognition technology, diluting to 100 mu L by using a MOPS buffer solution (pH=7.0), adding exonuclease ExoIII (20U), oscillating at a constant temperature of 37 ℃ for 2h, and positioning identifiable Ag on the surface of a nano-gold carrier with a hollow and porous structure + Biomolecules of (2) and Ag + Specific recognition reaction occurs to form C-Ag + Concurrent conformational transformations of the C base pairs off the nanocarrier surface, such that the blocked "pore cap" is opened and the dye molecule rhodamine B within the nanocarrier is released; meanwhile, the resultant biomolecule-Ag having a double-stranded structure due to the pair of exonuclease ExoIII + Shearing the combination to obtain Ag + From mismatched base pairs C-Ag + Released in C, recycled back into solution and combined with other identifiable Ag + The biological molecules of the fluorescent dye are combined, so that more rhodamine B is released, and the fluorescent signal is obviously enhanced;
(2) Magnetic separation, taking supernatant, diluting to 2.0mL with secondary water for fluorescence detection, and detecting conditions: the excitation wavelength and emission wavelength were 530 and 575nm, respectively.
FIG. 1 is Ag + A linear relation graph of the logarithm of the concentration and the fluorescence signal intensity shows that Ag + The concentration is 1.0X10 -13 ～8.0×10 -11 At mol/L, fluorescence signal intensity and Ag + The logarithm of the concentration shows a good linear relationship, and the linear equation is: fl=354.1304+121.7803 lgc Ag + (10 -13 ) The linear correlation coefficient was 0.9935.
The invention combines the exonuclease cyclic amplification technology with the base mismatch recognition technology and the nano-carrier technology, and can be used for Ag by design and synthesis + The identified biological molecule is assembled on the surface of the nano carrier to form a 'hole cap', and the C-C base mismatching is utilized to match with Ag + Specific recognition reaction, shapeC-Ag formation + The simultaneous conformational transition of the-C base pairs breaks away from the surface of the nano-carrier, so that the blocked 'pore cap' is opened, dye molecules in the nano-carrier are released, and in order to further increase the sensitivity, the invention utilizes exonuclease to carry out the preparation of the double-chain structure of the biological molecule-Ag + The shearing action of the conjugate realizes the cyclic amplification and detection of fluorescent signals.
The nano gold composite material based on the exonuclease cyclic amplification technology and the base mismatch recognition technology has the advantages of simple structure, easy synthesis, excellent performance, stability, economy, sensitivity, high efficiency and the like, and can not be subjected to other common interfering substances such as Cd 2+ ，Hg 2+ ，Pb 2+ ，Cu 2+ ，Fe 3+ ，Zn 2+ The influence of plasma metal ions has high specificity and selectivity, and can be used for micro samples with low content to extremely low content of Ag + The nano gold composite material based on the exonuclease cycle amplification technology and the base mismatch recognition technology, the preparation method and the detection technology thereof have great medical application potential and wide application prospect, and play an important role in the fields of early diagnosis and treatment of serious diseases, foods, biological medicines, medicine, environment and the like.
Sequence listing
<110> Qingdao university of science and technology
<120> a nanocomposite for detecting silver ions and a method for preparing the same
<141> 2018-08-13
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
tcctccctcc ttaaggaacc acccacca 28
Claims (1)
1.A nano gold composite material based on an exonuclease cycle amplification technology and a base mismatch recognition technology is characterized in that: the nano gold composite material comprises nano gold with a hollow and porous structure, dye molecules and biomolecules capable of recognizing silver ions through base mismatch;
the biological molecule capable of recognizing silver ions is a DNA molecule with a base sequence of 5'-TCC TCC CTC CTT AAG GAA CCA CCC ACC A-3';
the biological molecule capable of identifying silver ions is assembled on the surface of the nano-gold with a hollow and porous structure by adopting polydiallyl dimethyl ammonium chloride;
the dye molecule is rhodamine B;
the preparation method of the nano gold composite material is characterized by comprising the following steps:
(1) Design and synthesis of Ag and Ag + Nucleic acid biomolecules with a specific base length, which are recognized by base mismatches and are rich in cytosine, which nucleic acid biomolecules are capable of interacting with Ag + Specific base mismatch recognition reactions occur;
(2) Mixing magnetic beads with a nano gold carrier solution with a hollow and porous structure, adding a polydiallyl dimethyl ammonium chloride solution, performing magnetic separation after 10-12h, and cleaning with a MOPS buffer solution;
(3) Adding dye molecule solution, adding biological molecule solution capable of recognizing silver ions after 10-12 and h, magnetically separating after 10-12 and h, and cleaning with MOPS buffer solution;
wherein, the base sequence of the biological molecule capable of recognizing silver ions is 5'-TCC TCC CTC CTT AAG GAA CCA CCC ACC A-3', and the combination product of the biological molecule and silver ions can be sheared by exonuclease.
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