System and method for iterative detection of biological molecules

Patent metadata

Field	Value
Publication number	US-9933431-B2
Application number	US-201514820875-A
Country	US
Kind code	B2
Filing date	Aug 7, 2015
Priority date	Aug 22, 2014
Publication date	Apr 3, 2018
Grant date	Apr 3, 2018

Abstract

A system for the iterative detection of biological molecules includes a probe, and a fluorophore tethered to the probe with an azide-based linker. The linker is configured to be cleaved in the presence of tris(2-carboxyethyl)phosphine (TCEP), and the on/off ratio between a signal measured before treatment with TCEP and a signal measured after treatment with TCEP is at least about 20:1.

First claim

What is claimed is:

1. A system for iterative in situ fluorescence detection of biological molecules, comprising:
a multi-nucleotide or antibody probe; and
a fluorophore tethered to the probe by an azide-based linker;
wherein the azide-based linker is configured to be cleaved by tris(2-carboxyethyl)phosphine (TCEP) at a plurality of sequential TCEP-specific cleavage sites within said linker thereby cleaving the fluorophore from the multi-nucleotide or antibody probe upon exposure to TCEP, and
wherein the on/off ratio between a fluorescent signal produced by said fluorophore measured before treatment with TCEP and a fluorescent signal produced by said fluorophore measured after treatment with TCEP is at least about 20:1.

2. The system of claim 1 , wherein the linker is:

wherein R 1 is linkage between one of the probe and the fluorophore and wherein R 2 is a linkage between the other of the probe and the fluorophore.

3. The system of claim 1 , wherein the multi-nucleotide or antibody probe is selected for the detection of at least one of a DNA target, an RNA target, a protein target and a small molecule target.

4. A method for iterative in situ fluorescence detection a biological molecule, comprising:
incubating a sample including a target molecule with a detection system, the detection system including a multi-nucleotide or antibody probe and a fluorophore tethered to the probe by an azide-based linker comprising a plurality of TCEP-specific cleavage sites;
exciting the incubated sample at a first wavelength;
detecting the emission of a second wavelength from the excited, incubated sample; and
contacting tris(2-carboxyethyl)phosphine (TCEP) to the excited, incubated sample, whereby the TCEP cleaves at one or more of the plurality of sequential TCEP-specific cleavage sites, thereby cleaving the fluorophore from the probe, wherein the on/off ratio between the emission of the second wavelength detected before the contacting step and an emission of the second wavelength after the contacting step is at least about 20:1.

5. The method of claim 4 , further including, repeating the steps of incubating, exciting and detecting.

6. The method of claim 4 , wherein the linker is:

wherein R 1 is linkage between one of the probe and the fluorophore and wherein R 2 is a linkage between the other of the probe and the fluorophore.

7. The method of claim 4 , wherein the probe is selected for the in situ detection of at least one of a DNA target, an RNA target, a protein target and a small molecule target.

8. The method of claim 4 , further including binding the target molecule with the probe.

9. The method of claim 4 , wherein the on/off ratio between a signal measured before treatment with TCEP and a signal measured after treatment with TCEP is at least about 50:1.

Assignees

• Guo Jia
• Univ Arizona State

Inventors

• Guo Jia

Classifications

• G01N33/582 Primary

  with fluorescent label · CPC title

• C12Q1/6823

  Release of bound markers · CPC title

• C12Q2563/107

  fluorescence · CPC title

• C12Q2527/125

  Specific component of sample, medium or buffer · CPC title