System and method for iterative detection of biological molecules

• US 9,933,431 B2
• Filed: 08/07/2015
• Issued: 04/03/2018
• Est. Priority Date: 08/22/2014
• Status: Active Grant

• Current Assignee: Arizona Board of Regents (University of Arizona)
• Original Assignee: Arizona Board of Regents (University of Arizona)
• Inventors: Guo, Jia
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US14/820,875
• Publication Number:
  US 20160054308A1
• Time in Patent Office: 970 Days
• Field of Search: 435 61, 536 266
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: G01N 33/582 with fluorescent label

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.

Claims

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:

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:

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.