CN109444397A - A kind of detection method of mercury ion - Google Patents

A kind of detection method of mercury ion Download PDF

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CN109444397A
CN109444397A CN201811285305.6A CN201811285305A CN109444397A CN 109444397 A CN109444397 A CN 109444397A CN 201811285305 A CN201811285305 A CN 201811285305A CN 109444397 A CN109444397 A CN 109444397A
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dna enzymatic
gns
dna
nucleic acid
ferroheme
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CN109444397B (en
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云雯
杨丽珠
杨哲涵
尤琳烽
刘学成
吴虹
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Chongqing Technology and Business University
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    • 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
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • 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
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry

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Abstract

The present invention provides a kind of mercury ion detecting methods associated with graphene oxide complex catalysts chromogenic reaction without enzyme amplifying technique and ferroheme-that fragment DNA is formed to DNA enzymatic.Wherein, the ferroheme-graphene oxide composite surface adsorbs a large amount of DNA fragmentations, prevents from assembling, and the DNA fragmentation recycles cutting nucleic acid molecules hair fastener ring by DNA enzymatic and formed, and the DNA enzymatic is that the DNA enzymatic sequence of two divisions passes through T-Hg2+T-phase interaction is formed.A large amount of ferroheme-graphene oxide complex catalysts chromogenic reactions make supernatant show navy blue.It is measured by ultraviolet-visible absorption spectroscopy, Hg can be calculated2+Concentration.This method shows the range of linearity of 50pM to 1200pM.Limit detection can be down to 33pM.This method can detect Hg rapidly and sensitively2+。

Description

A kind of detection method of mercury ion
Technical field
The present invention relates to the detection fields of mercury ion, especially by fragment DNA formation DNA enzymatic without enzyme amplifying technique and blood Red pigment-mercury ion detecting method field associated with graphene oxide complex catalysts chromogenic reaction.
Background technique
Mercury is caused as one of maximum heavy metal of toxicity, environmentally safe and human health problems adverse effects The extensive concern of the public.Various natures and the generally existing mercury pollution of mankind's activity.The greatest contamination object of Mercury in Drinking Water is horizontal (MCL) 30nM is set as by the World Health Organization (WHO).The prior art is by Hg2+It is embedded in thymidine-thymidine (T-T) T-Hg is formd in mispairing2+- T base-pair establishes various Hg2+Determination method, including colorimetric method, fluorescence, surface enhanced Raman scattering and electrochemical detection method.However, detection limit is both greater than 30nM, detection sensitivity is not high.In order to improve sensitivity, No enzyme amplifying technique is amplified for signal, such as cross chain reaction and the catalysis dress self assembly of nucleic acid molecules hair clip.Due to isothermy, It is low in cost and without enzyme participate in etc. conditions cause many concerns.However, most of no enzyme amplifying techniques need to design complexity Helper nucleic acid molecule hair clip.
Since the solubility of ferroheme in aqueous solution is low and molecular aggregation is high, it is difficult to directly using it as catalysis Agent.Pass through π-π interaction synthesis ferroheme-graphene oxide compound between ferroheme and GO in the prior art.GO is used Make the carrier of ferroheme, provides electric conductivity for ferroheme.Therefore, it shows peroxidase activity, is catalyzed peroxide The chromogenic reaction of zymolyte.But due to the easily aggregation sedimentation in salting liquid of ferroheme-graphene oxide compound, so that from Supernatant catalytic effect after the heart is not obvious.In addition, ferroheme-graphene oxide compound is shown in the presence of sodium chloride To the different dispersing characteristics of single-stranded or double-stranded DNA sequence dna.Therefore it be used to detect hydrogen peroxide, glucose, enzymatic activity, bis-phenol A and DNA damage.
It there is no and fragment DNA is formed into developing the color without enzyme amplifying technique and ferroheme-graphene oxide complex catalysts for DNA enzymatic Two kinds of technology combinations are reacted to detect the report of mercury ion.
Summary of the invention
To solve the above problems, the present invention provide it is a kind of by fragment DNA formed DNA enzymatic without enzyme amplifying technique and blood red Element-mercury ion detecting method associated with graphene oxide complex catalysts chromogenic reaction.It is above-mentioned that " ferroheme-graphene oxide is multiple Close object " it is hereinafter referred to as H-GNs.
Testing principle of the invention are as follows: in Hg2+In the presence of, two DNA enzymatic sequence fragments can pass through T-Hg2+T-phase is mutual Intermediate formation duplex portions are acted on, become two complete DNA enzymatic chains at both ends.DNA enzymatic chain and nucleic acid molecules hair fastener ring The substrate chain-ordering in portion, which combines, generates DNA enzymatic structure.DNA enzymatic can be generated a large amount of with the ring portion of circulation shear nucleic acid molecules hair fastener DNA fragmentation.Obtained DNA fragmentation can prevent H-GNs from assembling in salting liquid by being adsorbed on the surface H-GNs.Cause This contains the H-GNs of more polydispersion in centrifuged supernatant, after chromogenic reaction, supernatant shows navy blue.However, There is no Hg2+In the case where, H-GNs assembles in the case where not having DNA fragmentation protection, and there are also a small amount of in centrifuged supernatant H-GNs, generate it is light blue.The cyclic annular cutting of nucleic acid molecules hair fastener leads to Hg2+The significant signal of detection amplifies.This method provides A kind of colour developing and without enzyme amplification mode, can detect Hg rapidly and sensitively2+, designed without complicated auxiliary hair clip.
The present invention includes the following steps:
(1) synthesis of H-GNs synthesizes H-GNs using the prior art；
(2) 5 '-CACCACAAATTCTCTCTrAGGACAAAAAAAGT GGTG-3 ' of nucleic acid molecules hair fastener sequence is heated It is kept for 5 minutes to 90 DEG C, then cools down 2 hours to room temperature formation nucleic acid molecules hairpin structure；
(3) formation of DNA enzymatic, the 50nM fragment DNA enzymatic sequence that will be bought in 10mM Tris-HCl (pH 7.5) buffering 1 and fragment DNA enzymatic sequence 2 and Hg2+Prepare liquid and 200nM step (2) obtained by nucleic acid molecules hair fastener hybrid reaction, formed DNA Enzymatic structure,
Above-mentioned fragment DNA enzymatic sequence 1 is 5 '-TTTTGTCAGCGATCCGGAATTGTGGTTGGTGCGGCACCCATGTG AG AGAA-3 ',
Above-mentioned fragment DNA enzymatic sequence 2 is 5 '-TTTTGTCAGCGATCCGGAACTCCTTCCTCTTCGGCACCCATGTG AG AGAA-3'；
(4) shearing of nucleic acid molecules hair fastener ring, the shearing of nucleic acid molecules hair fastener ring, by step (3) acquired solution and 10mM Mg2+Solution hybrid reaction 15 minutes；
(5) formation of H-GNs catalyst, the H-GNs obtained by Tris-HCl solution dilution mixing step (1) and step (4), Incubate and be added appropriate NaCl, centrifuging and taking supernatant, the supernatant, that is, H-GNs catalyst；
(6) it develops the color and measures, by H-GNs catalyst obtained by step (5) for being catalyzed (3,3', 5, the 5'- tetramethyls containing TMB Benzidine) and H2O2Tris-HCl buffer solution chromogenic reaction, measure its UV-vis absorption spectrum, and use calibration curve method Calculate Hg2+Concentration.
Preferably, step (1) specifically: specifically: 20mL is contained to the water of 10mg graphene oxide (hereinafter referred to as GO) Ultrasonic treatment 1 hour, then, by the 0.5mg mL of 20mL-1Haemachrome solution is mixed and is shaken a few minutes with above-mentioned GO dispersion, Then 200 μ L ammonia spirits and 30 μ L hydrazine hydrates are sequentially added, are stirred 3.5 hours at 60 DEG C, are centrifuged 30 minutes, precipitating is used Ultrapure water is for several times.0.3mg mL is diluted to ultrapure water-1It is spare.
Preferably, Tris-HCl solution ph used in step (5) is 7.5, Tris-HCl solution pH value used in step (6) It is 5.
Preferably, the UV-vis absorption spectrum of step (6) measurement is within the scope of 500 to 800nm.
Fragment DNA is creatively formed being combined without enzyme amplifying technique and H-GNs Catalytic color reaction for DNA enzymatic by the present invention Mercury ion is detected, as indivisible entirety, not only specific recognition mercury ion and amplification detection signal, more eliminate Complicated helper nucleic acid molecule hair clip design, it is bad to solve the problems, such as that H-GNs easily assembles catalytic effect.
Detailed description of the invention
Fig. 1 is detection process schematic diagram of the present invention.
Fig. 2 (A) GO, the uv-visible absorption spectra of ferroheme and H-GNs.The AFM figure of the H-GNs of Fig. 2 (B) synthesis Picture.Fig. 2 (C) is the afm image of GO.
Fig. 3 is that the absorbance of different detection methods compares.
Specific embodiment
Below with reference to embodiment, the present invention is described in further detail.
Embodiment 1
There is provided a kind of fragment DNA formed DNA enzymatic without mercury ion associated with enzyme amplifying technique and H-GNs Catalytic color reaction Detection method, detection schematic diagram referring to Fig. 1, the specific steps are that:
(1) the 20mL water for containing 10mg GO is ultrasonically treated 1 hour, then, by the 0.5mg of 20mL by the synthesis of H-GNs mL-1Haemachrome solution is mixed and is shaken a few minutes with above-mentioned GO dispersion, then sequentially adds 200 μ L ammonia spirits and 30 μ L water Close hydrazine, stir 3.5 hours at 60 DEG C, centrifugation 30 minutes, precipitating with ultrapure water for several times.
The graphene complex of synthesis is characterized by UV-vis absorption spectrum.There are strong absworption peak (figures near 230nm 2A), corresponding to the π-π * transition of aromatics C=C and the shoulder at 290-300nm, correspond to n- π * transition.C=O key.This Outside, ferroheme shows two characteristic absorptions, the Q-band peak value near B wave band peak value and 480-670nm at 386nm.Synthesis Graphene complex shows the peak the GO red shift of 35nm to 265nm, and the absorption peak 418nm at corresponds to 32nm red shift Ferroheme B absorption band.Red shift indicates the π-π interaction between GO and ferroheme.These results and previous report one It causes, i.e. the interaction of cationic porphyrin derivative and the graphene of chemical conversion leads to the red shift of porphyrin Soret band.
AFM is used to characterize the surface (Fig. 2 B) of graphene complex nanometer sheet.The average thickness of H-GN is about 1.2nm.With Naked GO is compared, thickness increase about 0.25nm (Fig. 2 C).This may be to be caused by the 0.2nm single layer ferroheme absorbed on the surface GO 's.Ultraviolet-visible spectrum and AFM show that haemachrome molecule is successfully attached to the surface GO.
(2) 5 '-CACCACAAATTCTCTCTrAGGACAAAAAAAGT GGTG-3 ' of nucleic acid molecules hair fastener sequence is heated It is kept for 5 minutes to 90 DEG C, then cools down 2 hours to room temperature formation nucleic acid molecules hairpin structure；
(3) formation of DNA enzymatic, the 50nM fragment DNA enzymatic sequence that will be bought in 10mM Tris-HCl (pH 7.5) buffering Nucleic acid molecules hair fastener hybrid reaction obtained by the prepare liquid and 200nM step (2) of 1 and fragment DNA enzymatic sequence 2 and Hg2+, forms DNA Enzymatic structure,
Above-mentioned fragment DNA enzymatic sequence 1 is 5 '-TTTTGTCAGCGATCCGGAATTGTGGTTGGTGCGGCACCCATGTG AG AGAA-3 ',
Above-mentioned fragment DNA enzymatic sequence 2 is 5 '-TTTTGTCAGCGATCCGGAACTCCTTCCTCTTCGGCACCCATGTG AG AGAA-3'；
(4) nucleic acid molecules obtained by step (3) acquired solution and 200nM step (2) are sent out in the shearing of nucleic acid molecules hair fastener ring Card and Mg2+Solution hybrid reaction 15 minutes；
(5) formation of H-GNs catalyst mixes H- obtained by appropriate step (1) with Tris-HCl solution (pH7.5) dilution GNs and 40 μ L step (4) acquired solutions incubate and are added appropriate NaCl, centrifuging and taking supernatant, the supernatant, that is, H-GNs catalysis Agent；
(6) develop the color and measure, by H-GNs catalyst obtained by 30 μ L steps (5) for be catalyzed 800 μ Μ TMB (3,3', 5, 5'- tetramethyl benzidine) and 10mM H2O2With the chromogenic reaction of 760 μ L Tris-HCl solution (pH 5), spectrophotometric is used The UV-vis absorption spectrum of meter record 500 to 800nm, measures Hg2+Concentration quantitative.
Embodiment 2
This method is used to detect the Hg in Chinese herbal medicine Rhizoma Atractylodis Macrocephalae2+.In brief, 1g Rhizoma Atractylodis Macrocephalae is crushed and is led to and sieved with 100 mesh sieve.It will Powder is 10 hours dry at 120 DEG C.Then, 100mL HNO is added3And it heats.Then, H is added dropwise2O2Until solution becomes It is transparent.Solution is diluted to 200mL with ultrapure water, and is filtered with 0.45 micron filter.Before test by the pH of solution 7.5 are adjusted to as prepare liquid.Remaining detection process is the same as embodiment 1.The results show that Hg in two different Rhizoma Atractylodis Macrocephalae samples2+It is dense Degree is respectively 68.7 μ gkg-1With 72.1 μ gkg-1.The μ of mark-on 10 and 20 gkg-1The rate of recovery is 91% and 97% afterwards.
Comparative example
For the technical effect for embodying above-described embodiment, provide following comparative example: comparative example 1: casting out H-GNs, remaining survey Examination process and embodiment 1 are identical；Comparative example 2: H-GNs is replaced with GO, remaining test process and embodiment 1 are identical；Comparative example 3: and embodiment 1 is identical；Comparative example 4: blank sample is free of mercury ion, 1 phase of remaining test process and embodiment that is, in prepare liquid Together；Comparative example 5: fragment DNA enzymatic sequence and the molar ratio of nucleic acid molecules hair fastener are down to 1:2,1 phase of remaining test process and embodiment Together；Comparative example 6: the cleavage reaction time was reduced to 5 minutes, remaining test process and embodiment 1 are identical.
As the result is shown in Fig. 3, No. 1 be comparative example 1 absorbance signal, since there is no H-GN in the case where without aobvious Colour response.No. 2 absorbance signals for comparative example 2 show absorbance similar with comparative example 1, it means that ferroheme pair It is had a significant impact in chromogenic reaction, in the presence of Hg2+, it can induce formation and the nucleic acid molecules hair fastener of entire DNA enzymatic Catalysis shearing, generating a large amount of DNA fragmentation and chromogenic reaction is navy blue, such as comparative example 3, i.e. the absorbance letter of embodiment 1 Number -- shown in No. 3.No. 4 absorbance signals for comparative example 4, i.e. blank signal, no Hg2+ are unable to inducing catalysis cleavage reaction, Cause a small amount of DNA fragmentation and chromogenic reaction be it is light blue, show that method of the invention is specific to the identification of Hg2+.No. 5 For the absorbance signal of comparative example 5, when the molar ratio of fragment DNA enzymatic sequence and nucleic acid molecules hair fastener is down to 1:2；This means that DNA enzymatic and the molar ratio of nucleic acid molecules hair fastener are 1:1, and cannot cause circulation shear iodine.No. 6 are comparative example 6 Absorbance signal, a small amount of DNA fragmentation due to caused by cleavage reaction, Δ absorbance intensity are relatively low.When the cleavage reaction time When reducing to 5 minutes, Δ absorbance strength reduction.This incubation time for being attributable to one third causes circulation shear amplification anti- It should not complete.
The foregoing is merely preferred embodiment of the present application, are not intended to limit this application, for the skill of this field For art personnel, various changes and changes are possible in this application.Within the spirit and principles of this application, made any to repair Change, equivalent replacement, improvement etc., should be included within the scope of protection of this application.

Claims (4)

1. a kind of mercury ion detecting method, includes the following steps:
(1) ferroheme-graphene oxide compound synthesis synthesizes H-GNs, above-mentioned ferroheme-oxidation stone using the prior art Black alkene compound abbreviation H-GNs；
(2) 5 '-CACCACAAATTCTCTCTrAGGACAAAAAAAGT GGTG-3 ' of nucleic acid molecules hair fastener sequence is heated to 90 DEG C keep 5 minutes, then cool down 2 hours to room temperature formed nucleic acid molecules hairpin structure；
(3) formation of DNA enzymatic, 1 He of 50nM fragment DNA enzymatic sequence that will be bought in 10mM Tris-HCl (pH 7.5) buffering Fragment DNA enzymatic sequence 2 and Hg2+Prepare liquid and 200nM step (2) obtained by nucleic acid molecules hair fastener hybrid reaction, formed DNA enzymatic knot Structure,
Above-mentioned fragment DNA enzymatic sequence 1 is 5 '-TTTTGTCAGCGATCCGGAATTGTGGTTGGTGCGGCACCCATGTGAG AGAA-3 ',
Above-mentioned fragment DNA enzymatic sequence 2 is 5 '-TTTTGTCAGCGATCCGGAACTCCTTCCTCTTCGGCACCCATGTGAG AGAA-3'；
(4) shearing of nucleic acid molecules hair fastener ring, by step (3) acquired solution and 10mM Mg2+Solution hybrid reaction 15 minutes；
(5) formation of H-GNs catalyst, the H-GNs obtained by Tris-HCl solution dilution mixing step (1) and step (4), incubate And appropriate NaCl is added, and centrifuging and taking supernatant, the supernatant, that is, H-GNs catalyst；
(6) develop the color and measure, by H-GNs catalyst obtained by step (5) for being catalyzed containing 3,3', 5,5'- tetramethyl benzidines and H2O2Tris-HCl buffer solution chromogenic reaction, measure its UV-vis absorption spectrum, and calculate Hg with calibration curve method2+It is dense Degree, above-mentioned 3,3', 5,5'- tetramethyl benzidine abbreviation TMB.
2. the method as described in claim 1, it is characterised in that step (1) specifically: 20mL is contained 10mg graphene oxide (GO) water is ultrasonically treated 1 hour, then, by the 0.5mg mL of 20mL-1Haemachrome solution and above-mentioned graphene oxide dispersion It mixes and shakes a few minutes, then sequentially add 200 μ L ammonia spirits and 30 μ L hydrazine hydrates, stirred 3.5 hours at 60 DEG C, from The heart 30 minutes, precipitating with ultrapure water for several times.0.3mg mL is diluted to ultrapure water-1It is spare.
3. the method as described in one of preceding claims, it is characterised in that Tris-HCl solution ph used in step (5) is 7.5, Tris-HCl solution ph used in step (6) is 5.
4. the method as described in one of preceding claims, it is characterised in that the UV-vis absorption spectrum of step (6) measurement is 500 To 800nm.
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CN106191042A (en) * 2016-07-16 2016-12-07 湖南工程学院 Two-way Cycle series signals based on exonuclease III auxiliary amplifies DNA combination probe compositions and preparation method and application
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