METHOD FOR ISOLATING CROSS-REACTIVE APTAMER AND USE THEREOF

• US 20200131514A1
• Filed: 10/30/2018
• Published: 04/30/2020
• Est. Priority Date: 10/30/2018
• Status: Active Grant

• Current Assignee: The Florida International University Board of Trustees (State University System of Florida)
• Original Assignee: The Florida International University Board of Trustees (State University System of Florida)
• Inventors: Yang, Weijuan, Yu, Haixiang, Liu, Yingzhu, Xiao, Yi
• Granted Patent: US 10,655,132 B1
• Time in Patent Office: Days
• CPC Class Codes: A61K 31/137 Arylalkylamines, e.g. amphetamine, epinephrine, salbutamol, ephedrine or methadone
• CPC Class Codes: A61K 31/167 having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
• CPC Class Codes: C12N 15/115 Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
• CPC Class Codes: C12N 2310/16 Aptamers
• CPC Class Codes: C12N 2320/11 for the determination of target sites, i.e. of active nucleic acids
• CPC Class Codes: C12N 2330/31 Libraries, arrays
• CPC Class Codes: G01N 27/26 by investigating electrochemical variables; by using electrolysis or electrophoresis
• CPC Class Codes: G01N 27/3276 being a hybridisation with immobilised receptors using a FET type sensor G01N27/4145; concerning the hybridisation C12Q1/68

Abstract

The subject invention provides a novel SELEX strategy for isolating cross-reactive aptamers that recognize a core structure of a small-molecule family and bind to each structurally-similar molecule in said family. The subject invention also provides methods, assays, and products for detecting small-molecule targets of the family in a sample in both clinical and field settings. Such method is based on an aptamer sensor that reports the presence of small-molecule targets via a sensitive colorimetric signal for naked-eye detection. The subject invention further provides exonuclease-based methods for generating structure-switching aptamers from fully folded aptamers and developing electrochemical aptamer-based (E-AB) sensors for rapid and sensitive detection of synthetic cathinones.

Claims

1. A method for isolating a cross-reactive aptamer for synthetic cathinones, the method comprising:

providing a DNA library;

selecting at least three synthetic cathinone targets;

performing at least one round of parallel selection against each selected synthetic cathinone target to isolate a pool of aptamers for each selected synthetic cathinone target;

performing at least one cycle of serial selection against each selected synthetic cathinone target in each round sequentially; and

collecting the cross-reactive aptamer.

3. The method according to claim 1, the DNA library being SEQ ID No:

1.

2. (canceled)

4-7. -7. (canceled)

8. A method for detecting one or more synthetic cathinones in a sample comprising contacting the sample with a cross-reactive aptamer that binds Cy7, and detecting one or more synthetic cathinones in the sample by naked-eye if a change in color occurs, a change in color being indicative of the presence of synthetic cathinones in the sample.

9. (canceled)

10. An electrochemical aptamer-based (E-AB) sensor comprising a structure-switching cross-reactive aptamer and an electrode, the structure-switching cross-reactive aptamer being conjugated to the surface of the electrode, the structure-switching cross-reactive aptamer comprising SEQ ID No:

19.

11. The E-AB sensor according to claim 10, the electrode being a gold electrode.

12. The E-AB sensor according to claim 10, the structure-switching cross-reactive aptamer being labeled with a thiol group at 5′

end, and methylene blue redox tag at 3′

end.

13. The E-AB sensor according to claim 10, further comprising a backfiller selected from 6-mercapto-1-hexanol (MCH), dithiothreitol (DTT), and combination thereof.

14. The E-AB sensor according to claim 10, further comprising a sensor buffer comprising Mg²⁺ and/or Na⁺.

15. The E-AB sensor according to claim 10, the structure-switching cross-reactive aptamer having SEQ ID NO:

18 or 19.

16. A method for detecting one or more synthetic cathinones in a sample comprising contacting the sample with the E-AB sensor according to claim 10, and detecting one or more synthetic cathinones in the sample by measuring a current generated upon binding of synthetic cathinones to the E-AB sensor.

17. The method according to claim 16, the sample being a biological sample or an environmental sample.

18. The method according to claim 16, the sample being a seized sample.

19. The method according to claim 16, the biological sample being selected from blood, plasma, urine, tears, sweat, and saliva.

20. The method according to claim 16, the one or more synthetic cathinones being selected from 3,4-methylenedioxypyrovalerone (MDPV), α

-PVP, pyrovalerone, methylone, pentylone, 3,4-methylenedioxy-α

-pyrrolidinobutiophenone (MDPBP), mephedrone, 4-methyl-α

-pyrrolidinobutiophenone (MPBP), 4′

-methyl-α

-pyrrolidinohexanophenone (MPHP), naphyrone, methedrone, ethylone, butylone, 4-methylmethcathinone (4-MMC), 4-fluoromethcathinone (4-FMC), 3-FMC, methcathinone, and 4-methyl-α

-pyrrolidinobutiophenone (MEPBP).

21. The E-AB sensor according to claim 10, the structure-switching cross-reactive aptamer being modified with a linker.

22. The E-AB sensor according to claim 21, the linker having from 1 to 10 carbons.

23. The E-AB sensor according to claim 22, the linker having 6 or 7 carbons.

24. The E-AB sensor according to claim 10, the structure-switching cross-reactive aptamer being conjugated to the electrode via a functional group.

25. The E-AB sensor according to claim 24, the functional group being selected from thiol, amide, ester, alkenyl, alkynyl, carbonyl, aldehyde, carboxylate, carboxyl, and carbonate ester groups.

26. The E-AB sensor according to claim 10, the electrode being made of gold, silver, or platinum.