Method for detecting 17bata-estradiol by using resonance scattering spectrometry based on functional nucleic acids

A resonance scattering, functional nucleic acid technology, applied in fluorescence/phosphorescence, material excitation analysis, etc., can solve the problems of prone to false positives, long detection time, high cost, and achieve the effect of significant technological progress, low cost, and good stability.

Inactive Publication Date: 2015-06-03

SHANGHAI APPLIED TECHNOLOGIES COLLEGE

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AI-Extracted Technical Summary

Problems solved by technology

[0005] Aiming at the above-mentioned technical problems in the prior art, the present invention provides a method for detecting 17β-estradiol based on functional nucleic acid using resonance sca...

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Method used

(7) verification: measure 3 parts with the inventive method and contain 17β-estradiol concentration and be respectively 100nM, 300nM and 500nM each part of milk after pr...

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Abstract

The invention discloses a method for detecting 17bata-estradiol by using resonance scattering spectrometry based on functional nucleic acids. The method comprises the following steps: preparing an aptamer-nanogold probe sequence, wherein the aptamer is a section of functional nucleotide sequence, and the nucleotide sequence is 5'-GCT-TCC-AGC-TTA-TTG-AAT-TAC-ACG-CAG-AGG-GTA-GCG-GCT-CTG-CGC-ATT-CAA-TTG-CTG-CGC-GCT-GAA-GCG-CGG-AAG-C-3'; combining nanogold with the aptamer to form a stable aptamer-nanogold probe; and adding a to-be-detected aqueous solution which is suspected to contain 17bata-estradiol to a probe solution, performing incubation, adding a sodium chloride solution, and judging the content of 17bata-estradiol in the to-be-detected solution by measuring the strength of a resonance scattering signal. According to the method disclosed by the invention, the detection of 17bata-estradiol can be achieved by comparing the changes of resonance scattering values.

Application Domain

Fluorescence/phosphorescence

Technology Topic

Estradiols17 beta estradiol +10

Image

Examples

Experimental program(4)

Example Embodiment

[0028] Example 1 Sodium chloride concentration in the optimized system

[0029] In a 1.5mL centrifuge tube, add 100μL of nano-gold solution, then add propanesulfonic acid buffer (concentration is 10mmol/L, pH is 7.0), mix well, and sequentially add sodium chloride solution with a concentration range of 0-160mmol/L, After five minutes, the resonance scattering value was measured, the relative resonance scattering value was obtained, and a curve was drawn. The sodium chloride concentration corresponding to the highest peak was 120 mmol/L, such as figure 2 shown.

Example Embodiment

[0030] Example 2 Optimizing the concentration of 17β-estradiol aptamer in the system

[0031] The 17β-estradiol aptamer was purchased from Shanghai Shenggong. The received sample was prepared into a 1 μmol/L mother solution, and then the aptamer with a concentration range of 0-40 nmol/L was added to 100 μL of nano-gold solution. For aptamer-gold nanoparticles, after incubation for 15 minutes, add a certain concentration of estradiol solution, incubate for 15 minutes, add the optimized sodium chloride solution, adjust the volume to 500 μL with propanesulfonic acid buffer, and measure the resonance scattering value after five minutes , draw a curve, the aptamer concentration corresponding to the highest peak is 20nmol/L, such as image 3 shown.

Example Embodiment

[0032] Embodiment 3 The process of using the method of the present invention to detect whether the milk sample contains 17β-estradiol specifically includes the following steps:

[0033](1) Prepare a detection system with known 17β-estradiol concentration: take 11 centrifuge tubes to which 10 μL and 1 μmol/L 17β-estradiol aptamer were added respectively, add 100 μL nano-gold solution, mix well and place the centrifuge tubes into the centrifuge tubes. Incubate at 25°C for 15min, and then add 10μL of 17β-estradiol solutions of different concentrations respectively, so that the content of 17β-estradiol in the entire detection system is maintained at 0.1-3.0μmol/L, and then fully mixed before Incubate the centrifuge tube at 25°C for 15 min, add 30 μL, 2 mol/L sodium chloride solution to the above mixture, mix well, and adjust the buffer volume to 500 μL, which is reserved for the following determinations;

[0034] (2) Take another centrifuge tube and replace the 17β-estradiol solution with 10 μL of ultrapure water, and use it as a blank control system solution after processing according to the method of the above step (1);

[0035] (3) Take 500 μL of the standard solution and blank control solution prepared in steps (1) and (2) respectively, and place them in a quartz fluorescence cuvette (inner diameter is 0.5cm×0.5cm, outer diameter is 1.0cm×1.0cm) , the signal was measured by scanning with a fluorophotometer. The specific scanning conditions are as follows: the excitation light slit is 2.5 nm, the emission light slit is 2.5 nm, and the excitation light wavelength is 550 nm for scanning to obtain the resonance scattering signal spectrum. The resonance scattering spectral signals of 17β-estradiol and blank control solution are I respectively 550nm and (I 550nm ) b , calculate the relative resonance scattering intensity value ΔI=I 550nm -(I 550nm ) b.

[0036] (4) 17β-estradiol (C E2 ) and the corresponding relative resonance scattering intensity value ΔI to draw a standard curve, the regression equation is: ΔI=1893C E2 +0.61. (like Figure 4 shown)

[0037] (5) Prepare a sample detection system: add a certain brand of pure milk to trichloroacetic acid to remove the protein in the milk, take 10 μL of the supernatant after centrifugation, and add it to the nano-gold-adaptation incubated in the specific embodiment step (1) method In the centrifuge tube of the body probe detection system, mix well and then incubate the centrifuge tube at 25 °C for 15 minutes, add 30 μL, 2 mol/L sodium chloride solution to the mixed solution, mix well, and adjust the buffer solution. The volume was adjusted to 500 μL, and the resonance scattering signal was measured according to the method in step (3) and ΔI was calculated.

[0038] (6) According to the calculated ΔI value, check the standard curve ΔI=1779C E2 -5.54, the content of 17β-estradiol in milk samples can be obtained.

[0039] (7) Verification: The method of the present invention is used to measure three parts of processed milk containing 17β-estradiol with concentrations of 100 nM, 300 nM, and 500 nM, respectively, and the average recoveries obtained are 108.3%, 93.9%, and 107.6%, respectively. , the RSD values are 5.8%, 6.1%, and 4.6%, respectively, which proves the reliability of the method.

[0040] The concentration range of 17β-estradiol in milk determined by the method of the invention is 0.1-3.0 μM, and the minimum detection limit is 9.0 nM.

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PUM

Property	Measurement	Unit
Concentration	10.0	mmol/l
tensile		MPa
Particle size		Pa
strength	10

Description & Claims & Application Information

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Method for detecting 17bata-estradiol by using resonance scattering spectrometry based on functional nucleic acids

AI-Extracted Technical Summary

Problems solved by technology

Method used

Abstract

Image

Examples

Example Embodiment

Example Embodiment

Example Embodiment

PUM

Description & Claims & Application Information

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