CN106596484B - A kind of detection Hg2+Method - Google Patents

A kind of detection Hg2+Method Download PDF

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Publication number
CN106596484B
CN106596484B CN201611127910.1A CN201611127910A CN106596484B CN 106596484 B CN106596484 B CN 106596484B CN 201611127910 A CN201611127910 A CN 201611127910A CN 106596484 B CN106596484 B CN 106596484B
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detection
base
biosensor
stranded dna
single stranded
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CN106596484A (en
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王卫
万均
罗细亮
申欣
王凤英
李会平
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Guangdong Youzhi Testing Technology Co.,Ltd.
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Qingdao University of Science and Technology
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells

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  • Life Sciences & Earth Sciences (AREA)
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Abstract

The present invention provides a kind of detection Hg2+Method, this method can be used for the Hg in the fields such as environmental monitoring, health care, food safety, life science2+Highly sensitive, highly selective detection.Method proposed by the present invention is to carry out Hg using biosensor2+Detection, used biosensor is built-up by nano-carrier, fluorescent material and single stranded DNA rich in T base.Single stranded DNA rich in T base is as Hg2+Recognition unit, be the gold nanometer cage surface being assembled by electrostatic interaction as nano-carrier, thus construct the biosensor with controlled release function.Pass through Hg2+Specific molecular recognition is reacted between recognition unit, is caused the single stranded DNA rich in T base that configuration transformation occurs, so that molecule door be made to be opened, is released fluorescent molecule, realize Hg2+Fluorescence detection.Therefore, it using this method, can be realized to Hg2+Highly sensitive, highly selective detection.

Description

A kind of detection Hg2+Method
Technical field
The present invention relates to a kind of detection Hg2+Method, and in particular to one kind can in vivo, food, medicine and ring That applies in border is used to detect Hg2+Method.
Background technique
It is well known that heavy metal pollution causes the influence got worse to human health and living environment.Mercury is A kind of heavy metal with serious physiological-toxicity has high migration and bioconcentration, is distributed widely in all kinds of surrounding mediums In food chain, by migration and transformation, it is circulated in water body, soil, atmosphere and biosphere etc..Hg2+Wastewater Pollutant is The important component of water pollution is constituted, the electrodeless mercury contaminants being discharged into water cannot be biodegradable, but participate in food Chain circulation can be the stronger methyl mercury of toxicity by microorganism conversion, after being largely enriched in vivo (especially by food chain Fish) enter human body, the health of the mankind is seriously threatened, the damage of central nervous system, endocrine system and brain can be caused, In addition, it can may also destroy human immune system, death is even resulted in.For this purpose, Environmental Protection Agency (EPA) and Europe are total The maximum permissible value that body proposes Mercury in Drinking Water respectively is 10nM and 5nM.
Therefore, quick, sensitive, accurate Hg is established2+Detection method not only facilitates environmental monitoring, cures simultaneously for clinic The numerous areas such as, life science, food hygiene, medicine and cosmetics also have practical significance.
Currently, Hg2+Detection method specifically include that spectrophotometry, Atomic absorption/emission spectrometry, inductive coupling etc. Ionomer emission spectrum method (ICPMS), cold steam atomic fluorescence spectrometry, chromatography, electrochemical process etc., however, compared to high Expensive instrument and reagent, many and diverse sample pretreatment, time-consuming testing process etc., fluorescence method is more suitable for Hg2+Detection, not only Better meet convenience, economy, quickly testing requirements, meanwhile, also have the advantages that it is highly sensitive, highly selective, in water ring Border improvement, clinical medicine create conservation culture environment and are of great immediate significance in terms of ensureing people's health.
In recent years, the development of modern molecular biology technique and nanometer new material is Hg2+Highly sensitive, specific biological pass Sense detection technique provides new thinking and advantage, emerges many new technologies, new method.As gold nanoparticle family In the Cenozoic, gold nanometer cage once come out just caused extensive concern and research.Studies have shown that including Hg2+Inside more Metal ion species can be allowed to form stable metal ion-base-pair specifically with nucleotide " bridging ".Wherein, Hg2+ It can interact with the DNA base of mispairing to T-T, form stable T-Hg2+- T base-pair, this characteristic is in bio-sensing Design and application aspect show huge potentiality, be Hg2+Detection provide novel, special, efficient detection platform.It adopts The gold nanometer cage for using positive surface charge to modify utilizes the single stranded DNA and gold nanometer cage surface for being rich in T base as nano container Electrostatic interaction building biosensor detection Hg2+Technology yet there are no document report.
Summary of the invention
In order to overcome the shortcomings of the prior art, for based on the single stranded DNA-gold nanometer cage biology for being rich in T base Sensor has not been reported, and therefore, the first object of the present invention: proposing and constructs and a kind of novel can be used for detecting Hg2+Biology Sensor, i.e., by gold nanometer cage and Hg2+Identification probe and positive surface charge modification technique combine, and have the biosensor Can biological response molecule door, once target molecule to be measured such as Hg is added2+, it will Hg occurs2+With rich in T base single stranded DNA it Between specific molecular identify reaction, i.e., in T-Hg2+Under the coordination of-T, the single stranded DNA rich in T base is transformed into similar hair The double-spiral structure of card-type causes the single stranded DNA rich in T base to be detached from from gold nanometer cage surface, makes the molecule door in plugging cage hole It is opened, and is released the guest molecule such as fluorescent molecule in hole, the fluorescence signal of system is enhanced；Therefore, may be used Hg is indicated by the increase of the fluorescence intensity of system2+Presence and its concentration；Using the biosensor, can be realized pair Hg2+Highly sensitive, highly selective detection；The second object of the present invention: a kind of detection Hg is provided2+Biosensor preparation Method；The third object of the present invention: a kind of application biosensor detection Hg is provided2+Method.By inspection proposed by the present invention Survey method is used for Hg2+Fluorescence detection, Hg can be improved significantly2+The sensitivity and accuracy of detection are environmental monitoring, food The application and research of the numerous areas such as product health, medicine and cosmetics provide new method.
The present invention is achieved through the following technical solutions goal of the invention.Detection Hg proposed by the present invention2+Bio-sensing Device is, using its hollow porous architectural characteristic, it is for example glimmering to load guest molecule inside it using gold nanometer cage as nano-carrier Optical molecule, fluorescent molecule leaks in order to prevent, by being rich in the single strand dna door of T base in its surface-assembled, by Jenner The hole on rice cage surface blocks；Wherein, the single stranded DNA rich in T base is specially designed, is enabled and Hg2+ The identification reaction of specificity occurs, its base sequence is 3 '-TTT GTT TGT TGG CCC CCC TTC TTT CTT A- 5 ', it can be assembled into gold nanometer cage surface by electrostatic interaction, specifically can be by gold nanometer cage surface modification positive electricity The method of lotus and be assembled into gold nanometer cage surface, preferably use positive charge dressing agent diallyl dimethyl ammoniumchloride In gold nanometer cage surface modification one strata cation.
Preferably, above-mentioned detection Hg2+Biosensor, the fluorescent molecule is rhodamine B.
It is a kind of to prepare detection Hg proposed by the present invention2+Biosensor preparation method, include the following steps:
(1) magnetic bead is mixed with gold nanometer cage solution, shaken at room temperature 8-12h, Magneto separate, remove supernatant, be added sun from Sub- coating material diallyl dimethyl ammoniumchloride solution, is shaken at room temperature overnight；
(2) Magneto separate removes supernatant, and fluorescent material rhodamine B solution is added, is shaken at room temperature overnight；
(3) the single stranded DNA solution for being rich in T base is added, is shaken at room temperature overnight；
(4) Magneto separate, cleaning obtain detection Hg2+Biosensor.
It is a kind of to detect Hg using biosensor of the invention2+Method, steps are as follows:
(1) by Hg2+Sample solution is added in biosensor of the invention, and 37 DEG C of constant temperature oscillations react 1-3h, in T- Hg2+Under the coordination of-T, Hg occurs2+It reacts, makes rich in T alkali with the identification of the specific molecular of the single stranded DNA rich in T base The single stranded DNA of base is detached from from gold nanometer cage surface, and molecule door is opened, and the rhodamine B molecule in gold nanometer cage is released；
(2) the above-mentioned solution of Magneto separate collects supernatant, detects its fluorescence signal.
Beneficial effects of the present invention: detection Hg proposed by the present invention2+Biosensor be by gold nanometer cage with rich in T alkali The single stranded DNA of base combines, and forms molecular biosciences door by electrostatic interaction, utilizes target molecule Hg2+It is special between molecule door Property molecular recognition reaction, so that the single strand dna door rich in T base is opened, release fluorescent molecule, realize fluorescence letter Number detection, this method makes Hg2+Detection sensitivity be significantly improved, it can be achieved that target molecule Hg2+Highly sensitive, high selection The detection of property.Detection Hg proposed by the present invention2+Biosensor have structure it is simple, stability is good, and controllability is strong, fluorescence The advantages that signal is sensitive, meanwhile, not by other common interference substance such as Pb2+, Zn2+, Cd2+, Ni2+, Fe3+, Ca2+, Cu2+Deng from The influence of son has high selectivity, can be applied to the fields Hg such as environmental monitoring, health care2+Fluorescence detection.Experimental result Show the detection Hg for proposing and preparing using the present invention2+Biosensor can be 5.0 × 10-12~1.0 × 10-9M range Interior realization is to Hg2+Highly sensitive, highly selective detection.Detection Hg2+Biosensor and preparation method thereof and detection technique Before the fields such as environmental monitoring, health care, food safety, life science have biggish application potential and wide application Scape.
Detailed description of the invention
Fig. 1: different Hg2+The fluorescence signal intensity of concentration.
Fig. 2: Hg2+The linear relationship of concentration and fluorescence signal intensity.
Specific embodiment
It is specific embodiment of the present invention below, technical solution of the present invention is described further, but this hair Bright protection scope is not limited to these examples.It is all to be included in this without departing substantially from the change of present inventive concept or equivalent substitute Within the protection scope of invention.
The present invention is specifically described below by embodiment, but the present invention is not limited by following embodiments.
Laboratory apparatus: THZ-82A gas bath constant temperature oscillator (medical apparatus and instruments factory, Jintan City)；F-4600 sepectrophotofluorometer (Hitachi, Japan)；Magnetic separation rack (thinks happy chromatographic technique development centre in Tianjin) again.
Experiment reagent: 3-4 μm of sulfydryl modification magnetic bead (thinking happy chromatographic technique development centre again in Tianjin)；Rhodamine B (I Fourth)；Single stranded DNA rich in T base: (the raw work in Shanghai is raw by 3 '-TTT GTT TGT TGG CCC CCC TTC TTT CTT A-5 ' Object Engineering stock Co., Ltd), PBS buffer solution is 0.01M (pH 7.4, Na2HPO4-NaH2PO4)。
Embodiment 1:
It is a kind of to prepare detection Hg proposed by the present invention2+Biosensor method, include the following steps:
(1) 20 μ L sulfydryl magnetic beads are taken, are uniformly mixed after being cleaned with PBS buffer solution with 400 μ L gold nanometer cages, shaken at room temperature 10h, Magneto separate are cleaned with PBS buffer solution, are removed supernatant, are added into the compound of acquired magnetic bead-gold nanometer cage 200 μ L concentration are the diallyl dimethyl ammoniumchloride solution of 11.664mg/mL, shaken at room temperature 10h；
(2) 100 L1.0 × 10 μ are added in the above-mentioned solution of Magneto separate after being cleaned with PBS buffer solution-5Mol/L rhodamine B PBS solution, shaken at room temperature 10h；
(3) it is 1.0 × 10 that 10 μ L concentration, which are added, in Xiang Shangshu solution-5The single stranded DNA solution rich in T base of M, 37 DEG C of oscillations 12h, Magneto separate remove supernatant, detect Hg2+Biosensor prepare complete；
Wherein, the sulfydryl magnetic bead is the commodity (thinking happy chromatographic technique development centre again in Tianjin) of purchase；Described Gold nanometer cage obtains (G.D.Moon, S.W.Choi, X.Cai, W.Y.Li, E.C.Cho, U.Jeong, L.V. by literature method Wang and Y.N.Xia.J.Am.Chem.Soc.2011,133,4762–4765)。
Embodiment 2:
It is a kind of to utilize biosensor proposed by the present invention for Hg2+Detection, the method is as follows:
(1) by Hg2+Sample solution is added to detection Hg proposed by the present invention2+Biosensor in, with PBS buffer it is molten Hg occurs for liquid (PH=7.4) dilution, 37 DEG C of constant temperature oscillation 2h2+Specific molecular between the single stranded DNA rich in T base is known It does not react, i.e., in T-Hg2+Under the coordination of-T, the single stranded DNA rich in T base is transformed into the double helix knot of similar hair fastener type Structure causes the single stranded DNA rich in T base to be detached from from gold nanometer cage surface, so that the molecule door in plugging cage hole is opened, and make in hole Guest molecule rhodamine B be released；
(2) the above-mentioned solution of Magneto separate collects supernatant, fluorescence detection, testing conditions: excitation wavelength and launch wavelength difference For 530,573nm.
Fig. 1 is the Hg of various concentration2+Corresponding fluorescence signal intensity, Hg2+Concentration be respectively (0；1.0×10-13；5.0 ×10-13； 1.0×10-12；5.0×10-12；1.0×10-11；4.0×10-11；8.0×10-11；1.0×10-10；5.0×10-10；1.0×10-9；1.0×10-8； 1.0×10-7M)；Fig. 2 is Hg2+The linear relationship of concentration and fluorescence signal intensity.As a result table It is bright, Hg2+Concentration is 5.0 × 10-12~1.0 × 10-9When M, fluorescence signal intensity and Hg2+The logarithm of concentration is in good linear Relationship, linear equation are as follows: FL=332.6791+64.6606lgCHg 2+(10-13M), linearly dependent coefficient 0.9963.
The present invention combines nanotechnology with molecular biotechnology, by specificity molecular recognition reaction, it can be achieved that To Hg2+Highly sensitive, highly selective detection.Specifically gold nanometer cage is repaired with the single stranded DNA and positive surface charge for being rich in T base Decorations technology combines, make the biosensor have can biological response molecule door, once have Hg2+In the presence of Hg then occurs2+With Specific molecular between single stranded DNA rich in T base identifies reaction, i.e., in T-Hg2+Under the coordination of-T, it is rich in T base Single stranded DNA be transformed into the double-spiral structure of similar hair fastener type, cause the single stranded DNA rich in T base de- from gold nanometer cage surface From making the molecule door in plugging cage hole be opened, and be released the guest molecule rhodamine B in hole, therefore, utilize the life Object sensor, can be realized to Hg2+Highly sensitive, highly selective detection.
Proposed by the present invention to have structure simple by the biosensor of carrier of gold nanometer cage, stability is good, controllably The advantages that property is strong, and fluorescence signal is sensitive, meanwhile, not by other common interference substance such as Pb2+, Zn2+, Cd2+, Ni2+, Fe3+, Ca2+, Cu2+The influence of plasma has high selectivity, can be used for Hg2+Highly sensitive, specific detection, be not only able to for environment prison The detection of the fields such as survey, health care, food safety, life science heavy metal provides new approaches and methods, meanwhile, based on this The controlled release technologies of guest molecule are also cancer target in the nano-carrier of invention, drug conveys, the clinic of major disease is examined It is disconnected to carry out new thinking with treatment zone.
1
Sequence table
SEQUENCE LISTING
<110>Qingdao University of Science and Technology
<120>a kind of method for detecting Hg2+
<160> 1
<210> 1
<211> 28
<212> DNA
<213>artificial sequence
<400> 1
attctttctt ccccccggtt gtttgttt 28

Claims (2)

1. a kind of detection Hg2+Method, it is characterised in that steps are as follows:
(1) by Hg2+Sample solution is added to by magnetic bead, nano-carrier, fluorescent material and Hg2+Identify the built-up biology of probe In sensor, 37 DEG C of constant temperature oscillations react 1-3h；
(2) the above-mentioned solution of Magneto separate collects supernatant, detects its fluorescence signal；
The biosensor is by magnetic bead, gold nanometer cage, fluorescent material and Hg2+Identify that probe is built-up；
The Hg2+Identification probe is that base sequence is 3 '-TTT GTT TGT TGG CCC CCC TTC TTT CTTA-5 ' Single stranded DNA rich in T base.
2. a kind of detection Hg as described in claim 12+Method, it is characterised in that: the Hg2+Identify that probe is by quiet Electro ultrafiltration is assembled into gold nanometer cage surface.
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CN106990081A (en) * 2017-04-10 2017-07-28 江苏大学 One kind is based on graphene oxide sensor and its to Hg2+The method of detection
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CN113252620A (en) * 2020-02-12 2021-08-13 青岛科技大学 Hg can be detected simultaneously2+And Ag+Method (2)
CN113252622A (en) * 2020-02-12 2021-08-13 青岛科技大学 Hg simultaneous detection based on single excitation2+And Ag+Method (2)
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CN111778244B (en) * 2020-05-29 2023-10-27 南京邮电大学 DNA probe combined with gold nanospheres and detection of Hg based on solid-state nanopores 2+ Application in (a)

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