DNA aptamer-cyanine complexes as mephedrone and cannabinoid colorimetric sensors

Patent metadata

Field	Value
Publication number	US-11656235-B2
Application number	US-202117396663-A
Country	US
Kind code	B2
Filing date	Aug 7, 2021
Priority date	Aug 7, 2021
Publication date	May 23, 2023
Grant date	May 23, 2023

Abstract

The subject invention provides materials and methods for single-step detection of target molecules in a sample. The methods and assays of the subject invention employ a dye-displacement strategy, in which aptamers complexed with a cyanine dye for sensitive and rapid detection of targets of interest. In the presence of a target, aptamer-target binding liberates the non-covalently bound aptamer-binding dye, resulting in optical changes that can be observed spectrophotometrically or with the naked eye. The methods and assays of the subject invention enable the colorimetric detection of targets of interest regardless of their structure, sequence, target-binding affinity, and physicochemical properties of their targets.

First claim

We claim:

1. A method for detecting mephedrone in a sample, the method comprising
a) mixing an aptamer with a dye to form an aptamer-dye complex, the aptamer comprising a nucleic acid sequence selected from SEQ ID NO: 7 and sequences sharing at least 95% sequence identity with SEQ ID NO: 7, and the dye being MTC;
b) mixing/contacting the aptamer-dye complex with the sample; and
c) detecting mephedrone in the sample by detecting an optical change generated upon the assembly of an aptamer-mephedrone complex and the disassembly of the aptamer-dye complex.

2. The method of claim 1 , wherein the optical change can be observed with the naked eye or detected by a spectrometer.

3. The method of claim 1 , wherein the dye non-covalently binds to the aptamer as a monomer or dimer.

4. The method of claim 1 , the sample being a biological sample or an environmental sample.

5. The method of claim 4 , the biological sample being selected from blood, plasma, urine, tears, and saliva.

6. The method of claim 1 , the aptamer being SEQ ID NO: 7.

7. The method of claim 1 , comprising in step c) detecting a decrease of absorbance at 770±10 nm and an increase of absorbance at 600 nm, or a decrease of absorbance at 585-590 nm and an increase of absorbance at 650-655 nm.

8. The method of claim 1 , wherein the optical change can be detected spectrophotometrically.

9. A method for detecting mephedrone in a sample, the method comprising
mixing/contacting non-covalent assemblies of aptamer-dye complex with the sample, the dye being MTC, and the aptamer comprising a nucleic acid sequence selected from SEQ ID NO: 7 and sequences sharing at least 95% sequence identity with SEQ ID NO: 7;
measuring absorbance at 585-590 nm and 650-655 nm; and
detecting methedrone in the sample by detecting a change in absorbance measured at 585-590 nm and 650-655 nm.

10. The method of claim 9 , the sample being a biological sample or an environmental sample.

11. The method of claim 10 , the biological sample being selected from blood, plasma, urine, tears, and saliva.

12. The method of claim 9 , wherein the change in absorbance further leads to a color change that can be observed with the naked eye.

13. The method of claim 9 , further comprising, prior to mixing/contacting non-covalent assemblies of aptamer-dye complex with the sample, mixing the aptamer with MTC, and detecting an increase of absorbance at 585-590 nm and a decrease of absorbance at 650-655 nm.

14. The method of claim 9 , the change in absorbance being a decrease of absorbance at 585-590 nm and an increase of absorbance at 650-655 nm.

15. A method for detecting a cannabinoid in a sample, the method comprising
a) mixing an aptamer with a dye to form an aptamer-dye complex, the aptamer comprising a nucleic acid sequence selected from SEQ ID NO: 4 and sequences sharing at least 95% sequence identity with SEQ ID NO: 4, and the dye being MTC;
b) mixing/contacting the aptamer-dye complex with the sample; and
c) detecting the cannabinoid in the sample by detecting an optical change generated upon the assembly of an aptamer-cannabinoid complex and the disassembly of the aptamer-dye complex.

16. The method of claim 15 , wherein the optical change can be observed with the naked eye or detected by a spectrometer.

17. The method of claim 15 , the sample being selected from blood, plasma, urine, tears, and saliva.

18. The method of claim 15 , the biological sample being the aptamer being SEQ ID NO: 4.

19. The method of claim 15 , the aptamer being used at a concentration of up to 2.5 μm.

20. The method of claim 15 , the cannabinoid being selected from tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THCA), cannabinol (CBN), and tetrahydrocannabivarin (THCV).

Assignees

• Xiao Yi
• Alkhamis Obtin
• Yu Haixiang
• The Florida International Univ Board Of Trustees

Inventors

• Xiao Yi
• Alkhamis Obtin
• Yu Haixiang

Classifications

• G01N33/583

  with non-fluorescent dye label · CPC title

• G01N33/946

  CNS-stimulants, e.g. cocaine, amphetamines · CPC title

• G01N33/9413

  Dopamine · CPC title

• G01N33/948

  Sedatives, e.g. cannabinoids, barbiturates (opiates G01N33/9486) · CPC title

• C12Q1/6816

  characterised by the detection means (C12Q1/6804 takes precedence) · CPC title