Method for isolating cross-reactive aptamer and use thereof
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.
1. 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.
2. The E-AB sensor according to claim 1 , the electrode being a gold electrode.
3. The E-AB sensor according to claim 1 , the structure-switching cross-reactive aptamer being labeled with a thiol group at 5′ end, and methylene blue redox tag at 3′end.
4. The E-AB sensor according to claim 1 , further comprising a backfiller selected from 6-mercapto-1-hexanol (MCH), dithiothreitol (DTT), and combination thereof.
5. The E-AB sensor according to claim 1 , further comprising a sensor buffer comprising Mg 2+ and/or Na + .
6. The E-AB sensor according to claim 1 , the structure-switching cross-reactive aptamer having SEQ ID NO: 18 or 19.
7. A method for detecting one or more synthetic cathinones in a sample comprising contacting the sample with the E-AB sensor according to claim 1 , 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.
8. The method according to claim 7 , the sample being a biological sample or an environmental sample.
9. The method according to claim 7 , the sample being a seized sample.
10. The method according to claim 1 , the biological sample being selected from blood, plasma, urine, tears, sweat, and saliva.
11. The method according to claim 7 , 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).
12. The E-AB sensor according to claim 1 , the structure-switching cross-reactive aptamer being modified with a linker.
13. The E-AB sensor according to claim 12 , the linker having from 1 to 10 carbons.
14. The E-AB sensor according to claim 13 , the linker having 6 or 7 carbons.
15. The E-AB sensor according to claim 1 , the structure-switching cross-reactive aptamer being conjugated to the electrode via a functional group.
16. The E-AB sensor according to claim 15 , the functional group being selected from thiol, amide, ester, alkenyl, alkynyl, carbonyl, aldehyde, carboxylate, carboxyl, and carbonate ester groups.
17. The E-AB sensor according to claim 1 , the electrode being made of gold, silver, or platinum.