CN107589085B - Method for measuring Hg by using aptamer to regulate and control nano-silica activity-absorption spectrum2+Method (2) - Google Patents
Method for measuring Hg by using aptamer to regulate and control nano-silica activity-absorption spectrum2+Method (2) Download PDFInfo
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Abstract
The invention discloses a method for measuring Hg by using aptamer to regulate and control nano-silica activity-absorption spectrum2+The method is characterized by comprising the following steps: (1) preparation of known concentrations of Hg2+Measuring the absorbance value of a standard solution system to be A; (2) preparing blank control solution system, and measuring its absorbance value to be A0(ii) a (3) Calculate Δ A = A-A0(ii) a (4) As Δ A to Hg2+Making a working curve according to the concentration relation of the active carbon; (5) preparing a sample solution to be detected, and determining the absorbance value of the sample solution to be ASample (I)Calculating Δ ASample (I)=ASample (I)‑A0(ii) a (6) Calculating the Hg of the sample solution according to the working curve of the step (4)2+The content of (a). Compared with the existing method, the method does not need a complex process for constructing the aptamer nanoprobe, and is simpler, more convenient and faster; the non-metal nano-enzyme is catalyzed, the sensitivity is high, and the system is more stable.
Description
Technical Field
The invention relates to the field of analytical chemistry, in particular to a method for measuring Hg by using aptamer to regulate and control nano-silica activity-absorption spectrum2+The method of (1).
Background
The aptamer is a small section of oligonucleotide sequence obtained through in vitro screening, can be combined with a plurality of target substances with high specificity, high affinity and high selectivity, when the aptamer is specifically combined with the target substances, the configuration of the aptamer can be changed, and the detection signal of the aptamer is changed accordingly, so that the detection of the target substances is realized. Mercury is a heavy metal with strong toxicity, and can easily generate an enrichment effect in human bodies and animals, particularly fishes through a food chain, and even under the condition of low concentration, mercury can also cause great and long-term toxic effects on human bodies and the environment. Therefore, the research on the measuring method of the trace mercury is of great significance.
At present, the method of measuring mercuryThe method mainly comprises a combination method of atomic absorption spectrometry and gas chromatography-mass spectrometry and a method combined with immunoreaction, such as colorimetry, fluorescence spectrometry, resonance Rayleigh scattering spectrometry and surface enhanced Raman scattering spectrometry. Among these methods, atomic absorption spectroscopy has a poor precision although it is popular and has good stability; the gas-liquid chromatography-mass spectrometry combined technology is more sensitive than the atomic absorption spectrometry, has the advantages of wide linear range and the like, but has complex process and higher cost, and the detection sensitivity depends on post-column detection technology; the immune colorimetric method is simple, convenient and easy to implement and economical, but the sensitivity is not good enough; the fluorescence spectroscopy process is complicated and the cost is high; the resonance Rayleigh scattering spectrometry is sensitive, rapid and simple and has application in the aspects of biochemistry, analytical chemistry, nano material research and the like. However, the above immunoassay method is basically based on Hg2+T-Hg formation with thymine in aptamers2+-T structure, construction of the immunoprobe molecule, followed by the corresponding experimental manipulations, which are all complex.
The nano enzyme is a mimic enzyme which not only has the unique performance of nano materials, but also has a catalytic function. The nano enzyme has the characteristics of high catalytic efficiency, stability, economy and large-scale preparation. Since the report of HRP nanoenzyme in 2007, the research of the nanoenzyme is rapidly rising, and the research related surface is gradually wide, and the research comprises different fields of material science, physics, chemistry, biology, medicine, environment and the like. Compared with natural enzyme, the nano material has the advantages of high stability, high catalytic activity, low price, easy obtaining and the like, particularly avoids the characteristics of instability and easy denaturation of the natural enzyme, and increases the application prospect of the natural enzyme in the fields of process catalysis and enzymatic kinetics, so that the nano material with the simulated enzyme activity has important significance in analytical chemistry. The nano enzyme is applied to analytical chemistry, mainly relates to heavy metal ion detection, biomolecule detection and immunoassay, but is more in research application based on the metal nano enzyme, and has little research on the nonmetal nano enzyme. Surface Plasmon Resonance (SPR) is generated by the resonance of free electrons of metal generated by irradiating incident light onto the Surface of medium with different refractive indexes from critical angleThe interaction-specific signals between the molecules of the molecule allow qualitative and quantitative analysis of the molecular components. The nano silicon dioxide is increasingly widely applied as a new nano material, and the catalytic action of the non-metal nano enzyme of the nano silicon dioxide is regulated and controlled by an aptamer and the surface plasma resonance absorption spectroscopy technology is applied to quantitative determination of Hg2+The analysis method (2) has not been reported.
Disclosure of Invention
The invention aims at measuring Hg2+The prior art has the defects that the method for measuring Hg by using aptamer to regulate and control the activity-absorption spectrum of nano-silica2+The method of (1). The method does not need a complex process of constructing the aptamer nanoprobe, and is simpler, more convenient and faster; the non-metal nano-enzyme is catalyzed, the sensitivity is high, and the system is more stable.
The technical scheme for realizing the purpose of the invention is as follows:
method for measuring Hg by using aptamer to regulate and control silicon dioxide activity-absorption spectrum2+The method comprises the following steps:
(1) preparation of known concentrations of Hg2+Standard solution system: sequentially adding 10-200 μ L100 nmol/L Hg into a graduated test tube2+Mixing the standard solution, 50-120 μ L of 66ng/mL of mercury aptamer with sequence 5'-TTTCTTCTTTCTTCCCCCCTTGTTTGTTGTTT-3' and 10-20 μ L of 100 μ g/mL nano silicon dioxide, and standing for 10 min; then, 100 to 200 mul of 0.5 moL/L glucose, 100 to 180 mul of 0.01 moL/L LHCl and 100 to 120 mul of 84 mul/L HAuCl are added into each test tube in turn4Mixing, fixing the volume to 1.5mL by using secondary distilled water, reacting in 75 ℃ water bath for 20 minutes, taking out the test tube, cooling by using ice water to stop the reaction, and fixing the volume to 2.0 mL by using the secondary distilled water;
(2) preparation of blank control solution system: hg is not added by the method of the step (1)2+Preparing a blank control solution system by using the standard solution;
(3) taking Hg prepared according to the steps (1) and (2) respectively2+Pouring the standard solution system and the blank control solution system into a cuvette, setting the slit of the instrument parameter to be 5nm on a spectrophotometer, and scanning to obtainObtaining the absorption spectrum of the system, measuring the absorbance value at 580nm as A, and measuring the absorbance value of the blank control solution system as A0Calculate Δ A = A-A0；
(4) As Δ A to Hg2+Making a working curve according to the concentration relation of the active carbon;
(5) preparing a sample solution to which Hg is added according to the method of step (1)2+Replacing the standard solution with the sample solution, and determining the absorbance value of the sample solution as A according to the method in the step (3)Sample (I)Calculating Δ ASample (I)= ASample (I)- A0;
(6) Calculating the Hg of the sample solution according to the working curve of the step (4)2+The content of (a).
The principle for realizing the technical scheme is as follows:
under the condition of the technical scheme, the nano silicon dioxide is used for treating glucose-HAuCl4The reaction for generating the gold nanoparticles has stronger catalytic action; when the aptamer is adsorbed on the surface of the silicon dioxide nanoenzyme, the glucose-HAuCl is inhibited4A reaction of generating gold nanoparticles; when Hg is added into the system2+When combined with an aptamer, form a stable aptamer-Hg2+The conjugate is separated from the surface of the silica nanoenzyme, and the catalytic activity of the silica is recovered. The Hg is accompanied in the system2+The concentration is increased, the catalytic activity of the silicon dioxide is enhanced, the generated gold nanoparticles are subjected to surface plasmon resonance absorption, and the absorbance value is increased. Hg is a mercury vapor2+The concentration and the absorbance enhancement value of the system are in a linear relationship, and the Hg determination is established according to the linear relationship2+The absorption spectrum method for regulating and controlling the activity of the nano silicon dioxide by the aptamer.
The method has the advantages that: compared with the existing method, the determination method does not need the complex process of constructing the aptamer nanoprobe, and is simpler, more convenient and faster; the non-metal nano-enzyme is catalyzed, the sensitivity is high, and the system is more stable.
Drawings
FIG. 1 is a graph showing an absorption spectrum in examples.
In the figure, a.3.3 ng/mL mercury aptamer + 0.6. mu.g/mL silica +0.75 mmoL/L HCl +37.5mmoL/L glucose + 4.2. mu. moL/L HAuCl4b.a+0.5 nmoL/L Hg2+c.a+2.5 nmoL/L Hg2+d.a+5nmoL/L Hg2+e.a+7.5 nmoL/L Hg2+f.a+10 nmoL/L Hg2+。
Detailed Description
The invention will be further elucidated with reference to the embodiments and the drawings, without however being limited thereto.
Example (b):
method for measuring Hg by using aptamer to regulate and control nano-silica activity-absorption spectrum2+The method comprises the following steps:
(1) preparation of known concentrations of Hg2+Standard solution system: 10. mu.L, 50. mu.L, 100. mu.L, 150. mu.L, 200. mu.L of 100nmol/L Hg were added to 7 graduated tubes, respectively2+Sequentially adding 100 mu L of 66ng/mL of mercury aptamer with the sequence of 5'-TTTCTTCTTTCTTCCCCCCTTGTTTGTTGTTT-3' and 12 mu L of 100 mu g/mL of nano silicon dioxide into each graduated test tube, uniformly mixing, and standing for 10 minutes; then 150. mu.L of 0.5 moL/L glucose, 150. mu.L of 0.01 moL/L HCl and 100. mu.L of 84. mu.mol/L HAuCl were added to each tube in this order4Mixing, fixing the volume to 1.5mL by using secondary distilled water, reacting in 75 ℃ water bath for 20 minutes, taking out the test tube, cooling by using ice water to stop the reaction, and fixing the volume to 2.0 mL by using the secondary distilled water;
(2) preparation of blank control solution system: hg is not added by the method of the step (1)2+Preparing a blank control solution system by using the standard solution;
(3) taking Hg prepared according to the steps (1) and (2) respectively2+Pouring the standard solution system and the blank control solution system into a cuvette, setting instrument parameters on a spectrophotometer, scanning to obtain the absorption spectrum of the system, measuring the absorbance value at 580nm as A, and measuring the absorbance value of the blank control solution system as A0Calculate Δ A = A-A0；
(4) In Delta I to Hg2+Making a working curve according to the concentration relation of the active carbon; obtaining a linear regression equation of Δ A580nm=0.026C +0.035, Hg among them2+Concentration ofCHas the unit ofnmoL/L, the linear range of measurement is 0.5-10 nmoL/L, and the detection limit is 0.09 nmoL/L;
(5) preparing a sample solution to which Hg is added according to the method of step (1)2+Replacing the standard solution with the sample solution, and determining the absorbance value of the sample solution as A according to the method in the step (3)Sample (I)Calculating Δ ASample (I)= ASample (I)- A0;
(6) Calculating the Hg of the sample solution according to the working curve of the step (4)2+The content of (a).
(5) And (3) sample determination: taking pond water and river water from suburb of Guilin, filtering with filter paper, measuring appropriate amount of filtrate, and preparing the sample to be detected according to the method in step (1), wherein Hg is added2+And (4) replacing the standard solution with the sample to be detected, and operating according to the steps (2) to (4). Calculating the Delta of the measured sampleA Sample (I)=A Sample (I)-A 0;
(6) Calculating the Hg of the sample to be detected according to the working curve of the step (4)2+Content of Hg in pond water2+The content of Hg in river water is 3.9 nmol/L2+The content was 5.3 nmol/L.
The detection method of the technical scheme comprises the following steps:
three parts of each of the two water samples obtained in the step (5) of the above example were taken, and Hg at concentrations of 3 nmol/L and 5 nmol/L was added thereto2+The standard solutions were subjected to a standard recovery test to determine recovery rates of 97.7%, 98.4%, 101.2%, and 99.3%, 98.5%, and 97.6%, respectively, and relative standard deviations of 4.7% and 4.9%.
The technical scheme is accurate and reliable.
Claims (1)
1. Method for measuring Hg by using aptamer to regulate and control nano-silica activity-absorption spectrum2+The method is characterized by comprising the following steps:
(1) preparation of known concentrations of Hg2+Standard solution system: sequentially adding 10-200 μ L100 nmol/L Hg into a graduated test tube2+The standard solution, 50-120. mu.L 66ng/mL sequence is 5' -TTTCTTCTTTCTTCCCCCCTTGTTTGMixing the mercury aptamer of TTGTTT-3' and 10-20 muL 100 mug/mL nano silicon dioxide, and standing for 10 minutes; then, 100 mu L-200 mu L of 0.5 moL/L glucose, 100 mu L-180 mu L of 0.01 moL/L HCl and 100 mu L-120 mu L of 84 mu moL/L HAuCl are added into each test tube in turn4Mixing, fixing the volume to 1.5mL by using secondary distilled water, reacting in 75 ℃ water bath for 20 minutes, taking out the test tube, cooling by using ice water to stop the reaction, and fixing the volume to 2.0 mL by using the secondary distilled water;
(2) preparation of blank control solution system: hg is not added by the method of the step (1)2+Preparing a blank control solution system by using the standard solution;
(3) taking Hg prepared according to the steps (1) and (2) respectively2+Pouring the standard solution system and the blank control solution system into a cuvette, setting an instrument parameter slit to be 5nm on a spectrophotometer, scanning to obtain an absorption spectrum of the system, measuring the absorbance value at 580nm to be A, and measuring the absorbance value of the blank control solution system to be A0Calculate Δ A = A-A0；
(4) As Δ A to Hg2+Making a working curve according to the concentration relation of the active carbon;
(5) preparing a sample solution to which Hg is added according to the method of step (1)2+Replacing the standard solution with the sample solution, and determining the absorbance value of the sample solution as A according to the method in the step (3)Sample (I)Calculating Δ ASample (I)= ASample (I)- A0;
(6) Calculating the Hg of the sample solution according to the working curve of the step (4)2+The content of (a).
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