CN101936905A

CN101936905A - Mercury ion detection reagent and detection method

Info

Publication number: CN101936905A
Application number: CN2009100167475A
Authority: CN
Inventors: 陈令新; 王国庆; 李金花; 张卫卫
Original assignee: YANTAI HAIANDAI SUSTAINABLE DEVELOPMENT INSTITUTE
Current assignee: Yantai Institute of Coastal Zone Research of CAS
Priority date: 2009-07-03
Filing date: 2009-07-03
Publication date: 2011-01-05
Anticipated expiration: 2029-07-03
Also published as: CN101936905B

Abstract

The invention discloses a mercury ion detection reagent and a detection method. The reagent comprises nano sol, nucleic acid adaptors marked by Raman active molecules and a charge modifier. The detection method comprises the following steps of: firstly, synthesizing the nano sol, then adding a proper quantity of adaptors to a proper quantity of fresh nano sol and uniformly mixing; continuing to add a proper quantity of charge modifier and uniformly mixing; then adding a sample to be tested and incubating for 1-5 minutes; and finally, taking the nano sol to test a Roman scattering spectral signal and carrying out data processing. In the invention, based on the addition of Hg<2+>, the structures of the nano particle surface nucleic acid adaptors are changed, i.e. nano particle aggregation in the silver sol and surface enhanced Roman scattering signals of signal molecules are enhanced. In the invention, the SERS (Surface Enhanced Raman Scattering) technology is adopted, the detection limit to the Hg<2+> is at the nmol/L level, thus the invention has the advantages of high detection speed of the linear response range of Hg<2+> concentration and high selectivity and is suitable for the quick detection of trace Hg<2+> in environmental water.

Description

A kind of mercury ion detecting reagent and detection method

Technical field

The present invention relates to a kind of detectable and detection method, is a kind of mercury ion detecting reagent and detection method specifically.

Background technology

Common heavy metal generally has bigger toxicity.The excess accumulation of heavy metal can influence the metabolism and the biosome normal operation of cellular level in the human body, causes the irreversible damage of histoorgan.Mercury is discharged in the environment by approach such as coal-fired, metallurgy, volcanic eruption and refuse abandon in a large number as one of the most general heavy metal contaminants.The same with other heavy metals, mercury not only can not be degraded by microorganisms, the opposite but bigger metal organic compound (as methyl mercury) of generation toxicity under the aquatic microorganisms effect, and under the biological magnification of food chain, through thousands of hundred times of ground enrichments, thereby the ecosystem and human health are caused significant damage.Therefore, to Hg in the water ²⁺Migration, conversion and detection research seem particularly important.From the fifties in last century, Hg ²⁺The pollution problem of water environment has promptly been caused people's attention and analytical work person's attention.Present domestic Hg ²⁺The examination criteria method mainly adopts cold atomic absorption spectrometry and dithizone colourimetry.The former adopts stannous chloride with the Hg in the water sample ²⁺Be reduced into simple substance mercury, form mercuryvapour, and make carrier, it is blown into the mercury analyzer, carry out the cold atom absorption measurement with nitrogen or dry air.The latter then utilizes dithizone and mercury ion to generate orange red complex compound in acid solution, is dissolved in methenyl choloride through extraction, surveys 490nm wavelength place absorbance.These methods or relative effort, time-consuming need the instrument of relative complex and skilled person skilled can obtain reliable result; Perhaps belong to the microanalysis method, be difficult to satisfy current growing environmental monitoring requirement.

Species of metal ion is various and valence state is changeable in the aqueous solution, and conventional Raman analysis method can't directly detect, and selectivity detects the challenging especially difficult problem of a certain metallic ion.These limiting factors have hindered conventional Raman analysis method becomes the pervasive analytical technology that detects metallic ion.

Summary of the invention

Technical matters to be solved by this invention provides a kind of mercury ion detecting reagent and detection method, and it can be simple and efficient, high sensitivity, alternatively detect Hg in the water ²⁺, overcome traditional Hg ²⁺The shortcoming of detection method and detectable.

In order to address the above problem, the present invention adopts following technical scheme:

A kind of mercury ion detecting reagent, this reagent comprise the aptamer and the electric charge dressing agent of Nano sol, raman active molecule mark, and three's mol ratio is 1～6: 100～500: 5000～20000.

The aptamer of Nano sol, raman active molecule mark and electric charge dressing agent three's mol ratio is 3: 200: 10000.

This Nano sol is gold or silver-colored Nano sol;

Raman active molecule be carboxyl tetramethyl rhodamine (6-carboxytetramethylrhodamine, TAMRA), rhodamine B (RhodamineB), rhodamine 6G rhodamine classes such as (Rhodamine 6G) (Rhodamine), texas Red (Texas Red), anthocyan (Cyanine), fluoresceins (Fluorescein) etc.;

Aptamer is for containing thymine (Thymine, nucleic acid strand T);

The electric charge dressing agent is spermine (spermine), acids or salt, as sodium chloride etc.;

Use this detectable to detect the method for mercury ion, comprise the steps:

A. synthesis of nano colloidal sol, its granularity is between 10-100 nanometer (nm); These colloidal sols are the nano particles with surface enhanced Raman scattering effect;

B. in fresh Nano sol, add the aptamer of raman active molecule mark, and mix, wherein determine the relation of Nano sol and aptamer amount in the ratio in the above-mentioned detectable;

C. continue in Nano sol, to add the electric charge dressing agent, and mix;

D. add testing sample to above-mentioned system, hatched 1-5 minute, hatch and be mixed reactant and wait for that certain hour reacts reactant;

E. get above-mentioned Nano sol, test Raman diffused light spectrum signal, the line data of going forward side by side is handled.This detection is based on Raman scattering intensity or the colloidal sol change color that nano particle concentration class difference causes.

Compared with prior art, the present invention has following advantage:

1, the present invention adopt Raman scattering (Surface-enhanced Raman Scattering, SERS) analytical technology,, to Hg ²⁺Response is fast, and detection limit has high selectivity simultaneously in the nmol/L level, and the whole operation process only needs 10 minutes, and is simple and efficient, is suitable for environment underwater trace Hg ²⁺Fast detecting.

2, Nano sol synthetic method of the present invention (N.Leopold and B.Lendl.J.Phys.Chem.B., 2003, the Nano sol after 107:5723-5727) synthetic then has surface-enhanced Raman effects.

3, the present invention adopts fresh Nano sol, has then avoided leaving with of long durationly because of Nano sol, and nano particle is assembled, and influences the enhancing of Raman scattering effect.

4, the present invention adopts spermine as the electric charge dressing agent, both can make electronegative aptamer be adsorbed onto electronegative nanoparticle surface, has the function of assembling nano particle again, plays the effect of aggregating agent prepared therefrom.

The present invention adopts following principle:

The present invention is based on Hg ²⁺Existence cause that (Surface-enhanced Raman Scattering, SERS) signal changes this principle, realizes Hg for nano particle concentration class and Raman scattering ²⁺Detect.Heavy metals such as mercury exist with the ionic state form in Environmental Water mostly, and common Raman analysis means can't directly detect.Rely on the electromagnetism on nanoparticle submanifold surface " focus " (hot spots) and the SERS of chemical enhancement effect to detect, can obtain highly sensitive considerable detection signal, do not assemble or the low Nano sol of concentration class can't produce effective enhancing and have.Usually metal-sol such as gold, silver is because of the repulsive interaction of surface negative charge (difference according to the preparation method also can be positively charged) stable existence dispersedly.When an amount of aggregating agent prepared therefrom spermine existed, the electronegative property aptamers that is marked with raman active molecule (for example TAMRA) can effectively cover nanoparticle surface by electric charge modification and " bridge " effect of spermine, and plays the effect of stable sol.At this moment because of the colloidal sol concentration class is low, can detect more weak SERS signal.Hg ²⁺When existing, based on T-Hg ²⁺-T coordination, aptamers and Hg ²⁺In conjunction with and follow that conformation is folding to be changed, (the Silver nanoparticles for example so nano particle, AgNPs) surface portion exposes and makes repulsive interaction and steric hindrance reduce, nano particle is in the leading gathering down of Van der Waals force, increasing of " focus " quantity sharply strengthens the TAMRA Raman signal, and with Hg under aptamers and the finite concentration scope ²⁺Strengthen in conjunction with the increase of quantity, thereby can set up SERS signal and Hg ²⁺The relation of concentration.

Description of drawings

Fig. 1: Hg ²⁺Detect principle schematic.

Fig. 2: to blank and finite concentration Hg ²⁺The SERS response spectrogram of solution, interior illustration is a SERS response intensity situation (study on the stability) over time.

Fig. 3: to 5nmol/L-5 μ mol/L concentration range Hg ²⁺The SERS response collection of illustrative plates of solution, the SERS relative intensity increases successively from bottom to top.

Fig. 4: 1651cm ^-1The SERS intensity and the Hg at place ²⁺Concentration relationship and response curve (3 measurements), wherein interior illustration shows Hg ²⁺In the 5-100nmol/L concentration range with SERS response intensity linear (linearly dependent coefficient is 0.982).

Fig. 5: to Hg ²⁺Selectivity experiment and the response of other common metal ion.

Embodiment

Embodiment 1:

A. synthesize silver-colored Nano sol (N.Leopold and B.Lendl.J.Phys.Chem.B. under the room temperature, 2003,107:5723-5727): at first in the conical flask that contains the 84mL deionized water, add 5mL oxammonium hydrochloride (0.03mol/L) and 1mL NaOH (0.6mol/L), and stir with brute force.And then to wherein dripping/at the uniform velocity drip second AgNO with 2-3 ₃(0.01mol/L) be total to 10mL.Continue stirring and obtain glassy yellow AgNPs after 1 hour, granularity is 35 ± 8nm, and is as the storing solution nanoparticle sol, standby.

B. in containing the fresh AgNPs of 200 μ L, add 1 μ L aptamers solution (100 μ mol/L), and mix.The aptamers sequence is: 5 '-TAMRA-TTCTT TCTTCCCTTG TTTGTT-3 '.

C. in above-mentioned solution, add 5 μ L spermine (100 μ mol/L), and mix (D.Graham, W.E.Smith, A.M.T.Linacre, C.H.Munro, N.D.Watson and P.C.White.Anal.Chem., 1997,69:4703-4707).

D. in 90 μ L colloidal sols, add certain density Hg respectively ²⁺ Solution 10 μ L are hatched 5min.

E. adopt glass capillary to draw a little colloidal sol, collect the Raman scattering signal.The instrument parameter that testing process is collected Raman scattering is respectively: optical maser wavelength-632.8nm, laser power-12 microwatt, integral time-10 second (the detection principle under this condition is referring to Fig. 1); To blank and finite concentration Hg ²⁺The SERS response spectrogram difference of solution, and SERS response intensity stable fine (referring to Fig. 2) in time.

F. quantitative experiment: repeated experiments steps d-e obtains variable concentrations Hg ²⁺SERS response signal (referring to Fig. 3), with 1651cm ^-1Place's Raman peaks is a base peak, carries out data processing, can obtain Hg ²⁺Concentration within the specific limits with SERS intensity linear (referring to Fig. 4).

G. selectivity experiment: implementation process is identical with the a-e step of experimental procedure, with solution to be measured by Hg ²⁺Become isocyatic other metallic ion respectively.These metallic ions are respectively: K ⁺, Mg ²⁺, Ca ⁺, Zn ²⁺, Fe ³⁺, Cu ²⁺, Co ²⁺, Ni ²⁺, Ba ²⁺, Cd ²⁺, Pb ²⁺The present invention is used for Hg ²⁺Detection has high selectivity (referring to Fig. 5).

This embodiment is to Hg ²⁺Detection limit is lower than 5nmol/L, to Hg ²⁺The linear response range of concentration is 5-100nmol/L, while tool high selectivity, and whole testing process only needs 5-10 minute.

Embodiment 2:

After adopting embodiment 1 silver sol synthetic method to obtain fresh silver sol in the implementation process, it is centrifugal, add low amounts of water and obtain the AgNPs that particle concentration is 2.5 times of initial concentrations.In containing the fresh AgNPs of 200 μ L, add 2.5 μ L aptamers solution (100 μ mol/L), and mix.Identical among aptamers sequence and the embodiment 1.In above-mentioned solution, add 10 μ L spermine (100 μ mol/L), and mix.In above-mentioned colloidal sol, add high concentration Hg respectively ²⁺Solution, 1: 1 (volume ratio) mixes, and hatches 5min.Adopt glass capillary to draw a little colloidal sol, collect the Raman scattering signal.Experiment condition is identical with embodiment 1.With 1651cm ^-1Place's Raman peaks intensity is base peak, carries out data processing.This embodiment is to Hg ²⁺Detection limit is lower than 5nmol/L, to Hg ²⁺The linear response range of concentration is 5-150nmol/L, while tool high selectivity, and whole testing process only needs 10-15 minute.

Embodiment 3:

A. with embodiment 1; B. in containing the fresh AgNPs of 200 μ L, add 1 μ L aptamers solution (100 μ mol/L), and mix.The aptamers sequence is: 5 '-TAMRA-TTTTTTTTTTTTTTTTTTTTT-3 '; C-g is with embodiment 1.This embodiment is to Hg ²⁺Detection limit is lower than 2nmol/L, to Hg ²⁺The linear response range of concentration is 5-80nmol/L, while tool high selectivity, and whole testing process only needs 5-10 minute.

Claims

1. mercury ion detecting reagent, it is characterized in that: this reagent comprises the aptamer and the electric charge dressing agent of Nano sol, raman active molecule mark, and three's mol ratio is 1～6: 100～500: 5000～20000.

2. mercury ion detecting reagent according to claim 1 is characterized in that: the aptamer of Nano sol, raman active molecule mark and electric charge dressing agent three's mol ratio are 3: 200: 10000.

3. mercury ion detecting reagent according to claim 1 is characterized in that: this Nano sol is gold or silver-colored Nano sol.

4. mercury ion detecting reagent according to claim 1 is characterized in that: raman active molecule is carboxyl tetramethyl rhodamine, rhodamine B, rhodamine 6G, texas Red, anthocyan or fluoresceins.

5. mercury ion detecting reagent according to claim 1 is characterized in that: aptamer is the nucleic acid strand that contains thymine.

6. mercury ion detecting reagent according to claim 1 is characterized in that: the electric charge dressing agent is spermine, acids or salt.

7. use the described detectable of claim 1 to detect the method for mercury ion, comprise the steps:

A. synthesis of nano colloidal sol;

B. in fresh Nano sol, add the aptamer of raman active molecule mark, and mix;

C. continue in Nano sol, to add the electric charge dressing agent, and mix;

D. add testing sample to above-mentioned system, hatched 1-5 minute;

E. get above-mentioned Nano sol, test Raman diffused light spectrum signal, the line data of going forward side by side is handled.