Methods for detecting endocrine disruptors using dual modes of colorimetric and fluorometric analysis

Patent metadata

Field	Value
Publication number	US-10801960-B2
Application number	US-201815884868-A
Country	US
Kind code	B2
Filing date	Jan 31, 2018
Priority date	Jan 31, 2017
Publication date	Oct 13, 2020
Grant date	Oct 13, 2020

Abstract

Provided herein is a method of effectively quantifying a target material by performing both colorimetry and fluorescence analysis on the same sample, based on metal nanoparticles and an aptamer.

First claim

What is claimed is:

1. A method of detecting a target material, the method comprising:
reacting a sample comprising a target material with an aptamer-metal nanoparticle complex; wherein the aptamer-metal nanoparticle complex reacts with the target material when present in the sample so as for the aptamer to be separated from the complex, and the separated aptamer forms an aptamer-target material complex by binding to the target material;
adding a fluorescent dye to a reaction product of the reacting and measuring fluorescence; wherein either the aptamer-target material complex formed when the sample contains the target material does not react with the fluorescent dye so that no fluorescence is produced, or the aptamer-metal nanoparticle complex reacts with the fluorescent dye so as for the aptamer to be separated from the complex when no target material is present in the sample, and the separated aptamer exhibits fluorescence by binding to the fluorescent dye; and
adding a salt to a reaction product of the adding and measuring absorbance, wherein metal nanoparticles that do not have an aptamer bound exhibit a color change by being agglomerated with each other and the color change is measured by absorbance.

2. The method of claim 1 , wherein the target material is one selected from bisphenol A (BPA), bisphenol S (BPS), bisphenol F (BPF), β-estradiol, phthalate, and thrombin.

3. The method of claim 1 , wherein the target material has a concentration ranging from 0.001 ng/ml to 10,000 ng/ml.

4. The method of claim 1 , wherein in metal nanoparticles, a metal is gold or silver.

5. The method of claim 1 , wherein the fluorescent dye is one selected from SYBR Green, Gel Red, and Gel green.

6. The method of claim 1 , wherein the salt is sodium chloride.

7. A method of quantifying a target material to be detected, the method comprising:
measuring a fluorescence value and absorbance according to a concentration of a target material to be detected, using the method according to claim 1 ;
calculating fluorescence⋅absorbance of Equation 1 below by using the fluorescence value and the absorbance; and
using changes in fluorescence⋅absorbance according to concentration as a standard curve for quantification of the target material,
Fluorescence⋅absorbance={(OD x /OD y ) n −(OD x /OD y ) o }/( Fn/Fo )  <Equation 1>

wherein, in Equation 1, OD x denotes absorbance at x nm, OD y denotes absorbance at y nm, (OD x /OD y )o and Fo denote an absorbance ratio and a fluorescence value, respectively, of a sample not comprising the target material, and (OD x /OD y )n and Fn denote an absorbance ratio and a fluorescence value, respectively, of a sample comprising the target material.

8. The method of claim 7 , wherein, when the target material is bisphenol A, the fluorescence⋅absorbance is calculated by Equation 2 below:
Fluorescence⋅absorbance={(OD 650 /OD 530 ) n −(OD 650 /OD 530 ) o }/( Fn/Fo )  <Equation 2>

wherein, in Equation 2, OD 650 denotes absorbance at 650 nm, OD 530 denotes absorbance at 530 nm, (OD 650 /OD 530 )o and Fo denote an absorbance ratio and a fluorescence value, respectively, of a sample not comprising bisphenol A, and (OD 650 /OD 530 )n and Fn denote an absorbance ratio and a fluorescence value, respectively, of a sample comprising bisphenol A.

9. The method of claim 7 , wherein a concentration of a target material in the sample comprising the target material is obtained by finding the calculated fluorescence-absorbance of the sample on the standard curve and then matching with a corresponding concentration of the target material that results in said fluorescence-absorbance.

Assignees

• Univ Hanyang Ind Univ Coop Found

Inventors

• Kim Young Pil
• Gye Myung Chan
• Lee Eunsong
• Kim Gae Baik

Classifications

• G01N21/6428

  Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53) · CPC title

• G01N21/31 Primary

  Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry {(G01N21/72 takes precedence)} · CPC title

• C12N2310/16

  Aptamers · CPC title

• G01N31/00 Primary

  Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods · CPC title

• G01N21/78

  producing a change of colour · CPC title