Material and method for colorimetric detection of small-molecule targets
View Patent ↗The subject invention provides methods, assays, and products for visual detection of small-molecule targets in a sample in both clinical and field settings within minutes. The subject invention is based on an aptamer sensor that reports the presence of small-molecule target via a sensitive colorimetric signal for naked-eye detection. The aptamer sensor is a CBSAzyme-based sensor having both target-mediated cooperative behavior of the CBSA and peroxidase-mimicking catalytic activity of DNAzyme. The subject invention also provides methods of using the CBSAzyme-based sensor.
1. A cooperative binding split aptamer (CBSA)-DNAzyme conjugate (CBSAzyme) comprising a first fragment and a second fragment, the first fragment comprising a first segment of a split DNAzyme and a long fragment of a CBSA, and the second fragment comprising a second segment of the split DNAzyme and a short fragment of the CBSA, the first and second segments of the split DNAzyme being split from a DNAzyme, the short and long fragments of the CBSA assembling upon binding of a small-molecule target. the CBSA being MDPV- 6335 having the long fragment of SEQ ID NO: 25 and the short fragment of SEQ ID NO: 26 .
2. The CBSAzyme according to claim 1 , the long fragment of the CBSA being linked to the first segment of the DNAzyme via a first linker, and the short fragment of the CBSA being grafted to the second segment of the DNAzyme via a second linker.
3. The CBSAzyme according to claim 2 , the first and second linkers are independently selected from A, C, T, AA, AC, AT, CC, CA, CT, TA, TC, and TT.
4. The CBSAzyme according to claim 1 , the DNAzyme having a sequence of SEQ ID NO: 43 or a sequence sharing at least 90% identity thereof.
5. The CBSAzyme according to claim 1 , the DNAzyme being split in a 1:3 or 2:2 mode.
6. The CBSAzyme according to claim 1 , which is
MDPV-CBSAzyme having a first fragment of SEQ ID NO: 28 and a second fragment of SEQ ID NO: 29 .
7. A method for visually detecting one or more synthetic cathinones in a sample, the method comprising:
contacting the sample with a CBSAzyme-based sensor comprising a signal reporter and a CBSAzyme according to claim 1 , the CBSAzyme being specific for one or more synthetic cathinones, and
detecting one or more synthetic cathinones in the sample by determining whether a signal occurs upon one or more synthetic cathinones binding to the CBSAzyme, the signal being a color change, which is indicative of the presence of one or more synthetic cathinones in the sample, the synthetic cathinone having a core structure of:
R 1 and R 2 , are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, and hydroxylalkyl; or R 1 and R 2 , taken together with the carbon atoms to which they are attached, form a substituted or unsubstituted 5- or 6-membered homocyclic or heterocyclic ring;
R 3 is hydrogen, or alkyl;
R 4 , and R 5 are each independently selected from the group consisting of hydrogen, and alkyl; or R 4 and R 5 , taken together with the nitrogen atom to which they are attached, form a substituted or unsubstituted 5- or 6-membered heterocyclic ring; and
R 6 is hydrogen, or alkyl.
8. The method according to claim 7 , the sample being a seized sample or a biological sample selected from urine and saliva.
9. The method according to claim 7 , the signal reporter being a mixture of a peroxidase substrate and H 2 O 2 .
10. The method according to claim 9 , the peroxidase substrate being 2, 2′-azinobis(3-ethylbenzthiazo-line)-6-sulfonic acid (ABTS).
11. The method according to claim 7 , the one or more synthetic cathinones being selected from 3,4- methylenedioxypyrovalerone (MDPV), penthylone, mephedrone, naphyrone, MDPBP, methylone, methedrone, ethylone, butylone, 4′-methyl-a-pyrrolidinohexanophenone (MPHP), and 4-methyl-α-pyrrolidinobutiophenone (MEPBP).
12. The method according to claim 7 , the CBSAzyme comprising a first fragment having a sequence of SEQ ID NO: 28 , and a second fragment having a sequence of SEQ ID NO: 29 .
13. A cooperative binding split aptamer (CBSA) comprising a long fragment of SEQ ID NO: 25 and a short fragment of SEQ ID NO: 26 , the short and long fragments of the CBSA assembling upon binding of a small-molecule target.
14. A method for detecting one or more synthetic cathinones in a sample, the method comprising contacting the sample with the CBSA of claim 13 , the CBSA further comprising a signal reporter; and detecting one or more synthetic cathinones in the sample by detecting a signal generated from the signal reporter upon binding of one or more synthetic cathinones to the CBSA, the signal reporter being a fluorophore and quencher pair.