Resonance Rayleigh scattering spectrum method for determining Hg < 2 + >

Abstract

The invention belongs to the technical field of determination of Hg < 2 + >, and particularly discloses a resonance Rayleigh scattering spectrum method for determining Hg < 2 + >, which comprises the following steps: preparing an Hg < 2 + > standard solution reaction system and a blank control system; calculating an intensity difference value delta I; drawing a working curve according to the relationship between the intensity difference value delta I and the concentration of the corresponding Hg < 2 + > standard solution; preparing an actual sample solution; calculating an intensity difference value delta I sample; and calculating the working curve and the delta I sample to obtain the content of Hg < 2 + > in the actual sample solution. According to the resonance Rayleigh scattering spectrum method for determining the Hg < 2 + >, the resonance Rayleigh scattering method for detecting the Hg < 2 + > is constructed by taking the nano-silver generated by a light wave method as a resonance Rayleigh scattering indicating probe and by virtue of an unmarked aptamer reaction (AptHg-Hg < 2 + >) to regulate an aggregation phenomenon of nano-silver (AgNPs) sol in a salt solution; the method is high in sensitivity, good in stability, simple and fast.

Description

Technical Field

[0001] The present invention belongs to the technical field of Hg 2+ detection, and particularly relates to a resonance Rayleigh scattering spectroscopy method for determining Hg 2+ . Background Art

[0002] Resonance Rayleigh scattering (RRS) is a special elastic scattering that occurs when the wavelength of Rayleigh scattering lies at or near the wavelength absorbed by the molecule. RRS is a simple and highly sensitive analytical technique that has been widely applied in the analysis of metal ions, anions, surfactants, proteins, biomolecules, and other fields. In the prior art, the RRS detection of phenol has been achieved by utilizing the resonance energy transfer mechanism between Cr(III) metal-organic frameworks and red quinone imine (QI) products; an RRS sensor for detecting organic small molecules has been constructed by using the catalytic effect of covalently organic framework doped with palladium nanoclusters on the reaction of gold-core nickel-phosphorus shell (Au@NiP) alloy and the regulation effect of aptamer reaction on this catalytic amplification reaction.

[0003] Mercury is a common and highly toxic environmental pollutant generated in human daily life activities. Due to its accumulation in the environment, it has posed a major risk to wildlife and human health. Moreover, mercury ions cannot be metabolized and can accumulate in organisms, leading to various diseases such as kidney and liver diseases. In addition, studies have also proven its association with neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease and its acute toxicity to fetuses and infants. Therefore, it is particularly important to construct a sensitive, simple, and intuitive detection method. Currently, many detection methods have been developed, among which the most common ones are chromatography, electrochemistry, atomic absorption, surface-enhanced Raman scattering (SERS), ultraviolet-visible spectrophotometry (Abs), and fluorescence. Chromatography, atomic absorption, electrochemistry, and surface-enhanced Raman scattering (SERS) have high sensitivity but expensive analytical instruments and complex operations. Fluorescence and ultraviolet-visible spectrophotometry are simple to operate but have low sensitivity. These methods cannot achieve rapid, simple, and sensitive detection of mercury ions. Currently, an RRS spectroscopy method for detecting Hg 2+ based on the regulation effect of label-free aptamer reaction on the aggregation phenomenon of silver nanoparticles (AgNPs) sol in salt solution has not been reported yet.

[0004] Therefore, there is a need in the art to develop a resonance Rayleigh scattering spectroscopy method for determining Hg 2+ that can effectively solve the above problems. Summary of the Invention

[0005] The object of the present invention is to provide a method for determining Hg 2+Resonance Rayleigh scattering spectral method, which uses the silver nanoparticles generated by the light wave method as the resonance Rayleigh scattering indicator probe and the label-free aptamer reaction (Apt Hg -Hg 2+ ) to construct a resonance Rayleigh scattering method for detecting Hg 2+ by regulating the aggregation phenomenon of silver nanoparticle (AgNPs) sol in salt solution; this method has high sensitivity, good stability, and is simple and fast.

[0006] To achieve the above object, the present invention provides a resonance Rayleigh scattering spectral method for determining Hg based on the regulation of the aggregation phenomenon of silver nanoparticle (AgNPs) sol in salt solution by label-free aptamer reaction 2+ , comprising the following steps:

[0007] Step S1, configure a reaction system of Hg 2+ standard solution with known concentration and a blank control system;

[0008] Step S2, add the reaction system of Hg 2+ standard solution with known concentration and the blank control system configured in Step S1 into a four-way quartz cuvette respectively, measure the intensity value of the reaction system of Hg 2+ standard solution with known concentration as I, the intensity value of the blank control system as I0, and calculate the intensity difference ΔI between the two;

[0009] Step S3, make a working curve of the relationship between the intensity difference ΔI and the concentration of the corresponding Hg 2+ standard solution;

[0010] Step S4, repeat Step S1 to prepare an actual sample solution; wherein, replace the Hg 2+ standard solution added in Step S1 with the actual sample solution;

[0011] Repeat Step S2 to obtain the resonance Rayleigh scattering intensity value I 样品 of the actual sample solution; calculate the intensity difference ΔI 样品 between the intensity value I0 of the blank control system and the intensity value I 样品 of the actual sample solution; wherein, ΔI 样品 = I0 - I 样品 ;

[0012] Step S5, perform calculation on the working curve obtained in Step S3 and the intensity difference ΔI 样品 obtained in Step S4, and then the content of Hg 2+ in the actual sample solution can be obtained.

[0013] Preferably, Step S1 is specifically:

[0014] Step S11: Accurately pipette 400 - 600 μL of the prepared silver nanoparticles into each of the 7 colorimetric test tubes. Then add 30 - 100 μL of a 100 nmol / L mercury ion aptamer (Apt Hg ) solution. After shaking well, let it stand for 4 - 8 minutes;

[0015] Among them, the silver nanoparticles (AgNPs) sol is prepared by the existing technology. The specific preparation method is as follows: Add 44 mL of secondary distilled water into a triangular flask. While stirring, sequentially add 2 mL of a 10 mmol / L AgNO3 solution, 2 mL of a 100 mmol / L trisodium citrate solution, 600 μL of a 30% H2O2 solution, and 600 μL of a 0.1 mol / L NaBH4 solution. Stir rapidly until the color turns blue. Immediately transfer the prepared blue silver nanoparticle colloid into a microwave oven and irradiate it with light waves at 250 °C for 10 minutes to obtain a red transparent silver nanoparticle sol. After natural cooling, make up the volume to 50 mL for standby, that is, a silver nanoparticle (AgNPs) sol with a concentration of 4.0×10 -4 mol / L is obtained.

[0016] Step S12: Subsequently, add Hg 2+ standard solution, tris(hydroxymethyl)aminomethane hydrochloride (Tris - HCl), and sodium chloride, and make up the volume to 2 mL. Let it stand for 2 - 4 minutes, that is, a reaction system of Hg 2+ standard solution with a known concentration is obtained;

[0017] Step S13: Repeat Step S11 - Step S12; among them, without adding Hg 2+ standard solution, and keep other operations the same, that is, a blank control reaction system is obtained.

[0018] Preferably, in Step S12, the concentrations of the Hg 2+ standard solution are 0, 2.5×10 -3 , 0.60, 1.25, 2.60, 5.00, 7.50 μmol / L respectively; the volume of tris(hydroxymethyl)aminomethane hydrochloride is 110 - 150 μL, and its pH value is 6.79; the volume of sodium chloride is 130 - 160 μL, and its concentration is 0.1 mol / L.

[0019] Preferably, Step S2 is specifically as follows:

[0020] Step S21: Set the instrument parameters on the fluorescence spectrophotometer;

[0021] Step S22: Synchronously scan to obtain the resonance Rayleigh scattering spectra of the reaction system of the Hg 2+ standard solution with a known concentration and the blank control system;

[0022] Step S23: Measure the resonance Rayleigh scattering intensity value I at 455 nm, and simultaneously measure the resonance Rayleigh scattering intensity value I0 of the blank control reaction system at 455 nm;

[0023] Step S24: Calculate the intensity difference ΔI between the standard solution reaction system and the blank control system; where, ΔI = I0 - I. 2+ Preferably, in step S21, the instrument parameters are volt = 350 V, excited slit = emission slit = 5 nm, emission filter = 1% T attenuator, λex - λem = Δλ = 0.

[0024] The present invention adopts the above-mentioned resonance Rayleigh scattering spectral method for measuring Hg

[0025] and has the following beneficial effects: 2+

[0026] (1) In the present invention, under the action of the electrostatic shielding effect, AgNPs will aggregate in a salt solution with a given concentration, resulting in a change in the RRS signal of the silver nano-sol, and the color changes from orange-red to gray; when the aptamer (Apt(1) In the present invention, under the action of the electrostatic shielding effect, AgNPs will aggregate in a salt solution with a given concentration, resulting in a change in the RRS signal of the silver nano-sol, and the color changes from orange-red to gray; when the aptamer (Apt Hg ) is added, the N atoms of the bases on the surface of the aptamer coordinate with AgNPs and adsorb on the surface of the silver nanoparticles, so that AgNPs are kept stable; when the target (Hg 2+ ) is added, the aptamer will bind to the target molecule with high affinity and form a stable complex structure (Apt Hg -Hg 2+ ), at this time the aptamer cannot protect AgNPs, resulting in a change in the RRS signal of the system again. Based on this principle, the present invention establishes a simple and rapid RRS method for detecting Hg 2+ ;

[0027] (2) Compared with the existing methods, the method in the present invention realizes the RRS detection of Hg 2+ by using the regulatory effect of the specific reaction of the label-free aptamer on the aggregation phenomenon of AgNPs in the salt solution. This method is simple, rapid, highly sensitive, and has a wide linear range.

[0028] The technical solution of the present invention will be further described in detail below through the drawings and embodiments. Brief Description of the Drawings

[0029] Figure 1 It is the RRS spectral schematic diagram in the embodiment of the resonance Rayleigh scattering spectral method for measuring Hg 2+ of the present invention; where, a is 100.00 μmol / L AgNPs + 2.50 nmol / L AptHg +7.50 mmol / L NaCl + 3.25 mmol / L Tris-HCl, where b is a + 2.5×10 -3 μmol / L Hg 2+ , c is a + 0.60 μmol / L Hg 2+ , d is a + 1.25 μmol / L Hg 2+ , e is a + 2.60 μmol / L Hg 2+ , f is a + 5.00 μmol / L Hg 2+ , g is a + 7.50 μmol / L Hg 2+ . Specific implementation mode

[0030] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0031] Unless otherwise defined, the technical terms or scientific terms used in the present invention shall have the ordinary meaning understood by those of ordinary skill in the field to which the present invention belongs.

[0032] Example 1

[0033] A resonance Rayleigh scattering spectroscopic method for measuring Hg by the regulatory effect of aptamer reaction on the aggregation of AgNPs in salt solution 2+ comprises the following steps:

[0034] Step S1, configure a reaction system of Hg 2+ standard solution and a blank control system.

[0035] In 7 graduated colorimetric test tubes, accurately pipette 500 μL of the prepared 4.0×10 -4 mol / L silver nanoparticles into the test tubes, add 50 μL of 100 nmol / L mercury ion aptamer (Apt Hg ) solution, shake well and let stand for 6 min. Then add Hg 2+ standard solution (the concentrations of Hg 2+ standard solution are 0, 2.5×10 -3 , 0.60, 1.25, 2.6, 5.00, 7.50 μmol / L respectively), 130 μL of tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) with a pH value of 6.79, and 150 μL of 0.1 mol / L sodium chloride. Make up the volume to 2 mL and let stand for 3 min to obtain a reaction system of Hg 2+ standard solution with known concentration. At the same time, prepare a blank control reaction system without adding Hg 2+ standard solution, and other operations are the same as those for configuring the reaction system of Hg 2+The reaction systems of the standard solutions are the same.

[0036] Among them, prepare silver nanoparticle (AgNPs) sol by using the existing technology. The specific preparation method is as follows: Add 44 mL of secondary distilled water into a triangular flask. While stirring, sequentially add 2 mL of AgNO3 with a concentration of 10 mmol / L, 2 mL of trisodium citrate with a concentration of 100 mmol / L, 600 μL of H2O2 with a concentration of 30%, and 600 μL of NaBH4 with a concentration of 0.1 mol / L. Stir rapidly until the color turns blue. Immediately transfer the prepared blue silver nanoparticle colloid into a microwave oven and irradiate it with 250 °C for 10 min to obtain a red transparent silver nanoparticle sol. After natural cooling and volume fixation to 50 mL, it is reserved for use, that is, a silver nanoparticle (AgNPs) sol with a concentration of 4.0×10 -4 mol / L is obtained.

[0037] Step S2: Add the reaction systems of the known-concentration Hg 2+ standard solution and the blank control reaction system into a four-way quartz cuvette respectively. On a fluorescence spectrophotometer, set the instrument parameters: volt = 350 V, excitedslit = emissionslit = 5 nm, emission filter = 1% Tattenuator, λex - λem = Δλ = 0, and perform synchronous scanning to obtain the resonance Rayleigh scattering spectrum of the reaction system of the known-concentration Hg 2+ standard solution. Measure the RRS intensity value at 455 nm as I. At the same time, measure the RRS intensity value of the blank control reaction system as I0, and calculate ΔI = I0 - I, as Figure 1 shown.

[0038] Step S3: Based on the relationship between ΔI obtained in Step S2 and the concentration of the corresponding reaction system of the known-concentration Hg 2+ standard solution, make a working curve.

[0039] In this example, the obtained linear regression equation is:

[0040] ΔI = 0.08232C + 63.69672;

[0041] Among them, ΔI is the intensity difference between the reaction systems of the Hg 2+ standard solution and the blank control system; C is the concentration of Hg 2+ with the unit of μmol / L.

[0042] The measured linear range is 2.5×10 -3 -7.5 μmol / L, and the detection limit is 1.22 nmol / L.

[0043] Step S4, Sample Determination: Three river water samples were used as the actual test samples for this method. Three water samples (10 mL) were accurately transferred into three 10 mL centrifuge tubes and filtered twice with filter paper to remove suspended solids. Subsequently, the water samples were filtered through a 0.45 μm microporous membrane and centrifuged at 12,000 r / min for 10 minutes. Then the supernatant was extracted and used as the sample test solution. At the same time, the same operations were performed on the reagent blank solution without Hg 2+ Then, the actual samples to be measured were prepared according to the method of Step S1, where the Hg 2+ standard solution was replaced with the actual samples to be measured, and the resonance Rayleigh scattering intensity value I 样品 of the actual sample solution was obtained.

[0044] Subsequently, I0 obtained in Step S2 was subtracted from I obtained in Step S4 样品 to calculate the intensity difference ΔI 样品 of the sample to be measured, that is, ΔI 样品 = I0 - I 样品 .

[0045] Step S5. Substitute ΔI 样品 obtained in Step S4 into the working curve obtained in Step S3 to calculate the Hg 2+ content of the actual sample to be measured. In this example, the Hg 2+ contents of the actual samples to be measured were calculated to be 0.308 μmol / L, 2.623 μmol / L, and 2.383 μmol / L respectively.

[0046] Verify the detection method of this example:

[0047] Take the supernatant sample in Step S4 of this example, add the Hg 2+ standard solution with a concentration of 2.50 μmol / L, conduct a standard addition recovery experiment, and calculate that the standard addition recovery rates are 96.9%, 102.6%, and 97.3% respectively, and the relative standard deviations are 3.91%, 5.29%, and 4.46% respectively.

[0048] According to the above verification results, it shows that the determination method of this example is accurate and reliable.

[0049] Therefore, the present invention adopts the above-mentioned resonance Rayleigh scattering spectral method for measuring Hg 2+ , which constructs a resonance Rayleigh scattering method for detecting Hg Hg -Hg 2+ by using the silver nanoparticles generated by the optical wave method as the resonance Rayleigh scattering indicator probe and the regulation of the aggregation phenomenon of silver nanoparticle (AgNPs) sol in the salt solution by the label-free aptamer reaction (Apt 2+ ); this method has high sensitivity, good stability, and is simple and fast.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions of the present invention or make equivalent replacements, and these modifications or equivalent replacements do not cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for determining Hg 2+ by resonance Rayleigh scattering spectroscopy, characterized in that Including the following steps: Step S1: Configure a reaction system of Hg standard solution with a known concentration and a blank control system; 2+ ​ Step S2: Add the reaction systems of the known-concentration Hg standard solution configured in Step S1 and the blank control system into a four-way quartz cuvette respectively, and measure the intensity value of the known-concentration Hg standard solution reaction system as I and the intensity value of the blank control system as I0, and calculate the intensity difference ΔI between the two. 2+ standard solution reaction system and the blank control system into a four-way quartz cuvette respectively, and measure the known-concentration Hg 2+ standard solution reaction system as I, the intensity value of the blank control system as I0, and calculate the intensity difference ΔI between the two; Step S3: Plot a working curve based on the concentration relationship between the intensity difference ΔI and the Hg standard solution reaction system with a corresponding known concentration. 2+ ​ Step S4. Repeat Step S1 to prepare an actual sample solution; wherein, replace the Hg 2+ standard solution added in Step S1 with the actual sample solution; Repeat step S2 to obtain the resonance Rayleigh scattering intensity value I of the actual sample solution 样品 ; Calculate the intensity difference ΔI between the intensity value I0 of the blank control system and the intensity value I of the actual sample solution 样品 ; Wherein, ΔI 样品 ; 样品 = I0 - I 样品 ; Step S5: Calculate the working curve obtained in step S3 and the intensity difference ΔI obtained in step S4, and then the content of Hg in the actual sample solution can be obtained. 样品 That is, the content of Hg in the actual sample solution can be obtained. 2+ in the actual sample solution can be obtained.

2. A resonance Rayleigh scattering spectroscopy method for measuring Hg according to claim 1 2+ , characterized in that Specifically, step S1 is as follows: In step S11, accurately pipette 400 - 600 μL of the prepared silver nanoparticles into 7 cuvettes for colorimetric analysis, add 30 - 100 μL of a 100 nmol / L mercury ion aptamer solution, shake well, and let stand for 4 - 8 min; Among them, the concentration of silver nanoparticles is 4.0×10 -4 mol / L; Step S12. Subsequently, add Hg 2+ standard solution, tris(hydroxymethyl)aminomethane hydrochloride, and sodium chloride, and make up the volume to 2 mL, and let it stand for 2 - 4 min to obtain a reaction system of Hg 2+ standard solution; Step S13. Repeat Step S11 - Step S12; wherein, Hg is not added. 2+ For the standard solution, with other operations being the same, a blank control reaction system is obtained.

3. A resonance Rayleigh scattering spectroscopy method for determining Hg according to claim 2 2+ , characterized in that: In step S12, Hg 2+ The concentrations of the standard solutions are 0, 2.5×10 -3 , 0.60, 1.25, 2.60, 5.00, 7.50 μmol / L respectively; the volume of tris(hydroxymethyl)aminomethane hydrochloride is 110 - 150 μL and its pH value is 6.79; the volume of sodium chloride is 130 - 160 μL and its concentration is 0.1 mol / L.

4. A resonance Rayleigh scattering spectroscopy method for measuring Hg according to claim 1, characterized in that 2+ Specifically, step S2 is as follows: ​ In step S21, set the instrument parameters on a fluorescence spectrophotometer; Step S22. Synchronously scan to obtain Hg with a known concentration 2+ Resonance Rayleigh scattering spectra of the standard solution reaction system and the blank control system; In step S23, measure the resonance Rayleigh scattering intensity value I at 455 nm, and simultaneously measure the resonance Rayleigh scattering intensity value I0 of the blank control reaction system at 455 nm; Step S24, calculate Hg 2+ The intensity difference ΔI between the standard solution reaction system and the blank control system; where ΔI = I0 - I.

5. A resonance Rayleigh scattering spectroscopy method for determining Hg according to claim 1 2+ , characterized in that: In step S21, the instrument parameters are volt = 350 V, excitedslit = emission slit = 5 nm, emission filter = 1% T attenuator, λex - λem = Δλ = 0.

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