Material and method for colorimetric detection of small-molecule targets

Patent metadata

Field	Value
Publication number	US-10725058-B2
Application number	US-201816179336-A
Country	US
Kind code	B2
Filing date	Nov 2, 2018
Priority date	Nov 2, 2018
Publication date	Jul 28, 2020
Grant date	Jul 28, 2020

Abstract

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.

First claim

What is claimed is:

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.

Assignees

• Xiao Yi
• Luo Yingping
• Yu Haixiang
• The Florida International Univ Board Of Trustees

Inventors

• Xiao Yi
• Luo Yingping
• Yu Haixiang

Classifications

• C12N2310/3519

  Fusion with another nucleic acid · CPC title

• C12N2310/16

  Aptamers · CPC title

• C12N2310/127

  DNAzymes · CPC title

• C12N15/115

  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 title

• C12N15/113

  Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} · CPC title