Methods and Devices for Detection and Measurement of Analytes

Patent metadata

Field	Value
Publication number	US-2016299135-A1
Application number	US-201615189263-A
Country	US
Kind code	A1
Filing date	Jun 22, 2016
Priority date	Jan 31, 2012
Publication date	Oct 13, 2016
Grant date	—

Abstract

Sensors for target entities having functionalized thereon, at least one aptamer specific to the target entity, and methods of making and using the same are described for use in glycated protein monitoring and/or biomarkers.

First claim

What is claimed is:

1 . A method for optimizing sensitivity and/or selectivity of a sensor for one or more analytes, comprising:
linking one or more types of aptamers to a substrate with a self-assembled monolayer (SAM) linkage, the SAM linkage having a desired linking spacing and/or length to form a functionalized surface on the substrate, the desired linkage spacing and/or length being chosen in order to optimize at least one of surface plasmon resonance (SPR), Raman spectroscopy, electrochemical spectroscopy, or fluorescence spectroscopy sensitivity and selectivity based on analyte and/or aptamer characteristics.

2 . The method of claim 1 , wherein at least one packing density and/or length of the SAM linkage affects a surface plasmon resonance (SPR) signal.

3 . The method of claim 1 , wherein linkage is through a binary SAM and reductive desorption process.

4 . The method of claim 3 , wherein the desorption process comprises: exposing the functionalized surface of the substrate to a material resistant to protein adsorption to inhibit non-specific adsorption of protein on the functionalized surface.

5 . The method of claim 1 , wherein the SAM linkage is formed using dithiobis-N-succinimidyl propionate (DTSP) and (1-mercapto-11-undecyl) tri(ethylene glycol) (PEG3),
wherein PEG3 prevents non-specific adsorption of proteins, and wherein a carboxylic moiety on DTSP forms a stable bonding with the aptamer.

6 . A kit for the detection of one or more analytes, comprising: a sensor comprising one or more aptamers linked to a substrate with a self-assembled monolayer (SAM) linkage; and at least one container including the sensor, where a sample may be added to the container.

7 . The kit of claim 6 , further comprising one or more solid supports, one or more separating agents for separating the sensor from an elute, and one or more reagents for separating an aptamer from the sensor.

8 . A method of identifying a single target-site binding aptamer from a pool of nucleic acids having single-target-site-binding-aptamers and non-target-protein-binding-aptamers therein, comprising:
adding to a pool of nucleic acids a single-site-target-protein-complex, wherein both the single-target-site-binding-aptamers and the non-target-protein-binding-aptamers present in the pool bind to the single-site-target-protein-complex and form a single-target-site-binding-aptamer+non-target-protein-binding-aptamer+single-site-target-protein-complex; separating the single-target-site-binding-aptamer+non-target-protein-binding-aptamer+single-site-target-protein-complex from the pool; eluting the single-target-site-binding-aptamers and the non-target-protein-binding-aptamers from the single-site-target-protein-complex; adding to the elute of the previous step a non-target-protein-complex, wherein the non-target-protein-binding-aptamers present in the elute bind to the non-target-protein-complex and form a non-target-protein-binding-aptamer+non-target-protein-complex; separating the non-target-protein-binding-aptamer+non-target-protein-complex from the elute of the previous step, leaving the single-target-site-binding-aptamer in the elute; and separating the single-target-site-binding-aptamers from the elution; optionally, further amplifying the single-target-site-binding-aptamers.

9 . The method of claim 8 , wherein single-target-site-binding-aptamers are used to select for one of: hemoglobin, immunoglobulin G (IgG), immunoglobulin M (IgM), and albumin.

10 . The method of claim 8 , wherein single-target-site-binding-aptamers are selected from: SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15.

11 . The method of claim 8 , wherein the single-site-target-protein is immobilized on a solid support.

12 . The kit of claim 6 , wherein the aptamer binds to glycated hemoglobin, wherein the aptamer comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4 and 5.

13 . The kit of claim 12 , wherein the aptamer comprises at least one chemical modification selected from the group consisting of: a chemical substitution at a sugar position, a chemical substitution at an internucleotide linkage, and a chemical substitution at a base position.

14 . The kit of claim 6 , wherein the aptamer has at least 70% identity to the entire sequence of any one of SEQ ID NOs: 4 and 5, and binds to human glycated hemoglobin.

15 . The kit of claim 6 , wherein the aptamer binds to non-glycated hemoglobin, wherein the aptamer comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6 and 7.

16 . The kit of claim 6 , wherein the aptamer has at least 70% identity to the entire sequence of any one of SEQ ID NOs: 6 and 7, and binds to human non-glycated hemoglobin.

17 . The kit of claim 6 , wherein the aptamer binds to glycated serum albumin, wherein the aptamer comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3, 8, and 9.

18 . The kit of claim 6 , wherein the aptamer has at least 70% identity to the entire sequence of any one of SEQ ID NOS: 3, 8, and 9, and binds to human glycated serum albumin.

19 . The kit of claim 6 , wherein the aptamer binds to non-glycated serum albumin, wherein the aptamer comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12, 13, 14, and 15.

20 . The kit of claim 6 , wherein the aptamer has at least 70% identity to the entire sequence of any one of SEQ ID NOS: 10, 11, 12, 13, 14, and 15, and binds to human non-glycated serum albumin.

Assignees

• Univ Toledo

Inventors

• Cameron Brent D
• Kim Dong-Shik

Classifications

• G01N2333/805

  Haemoglobins; Myoglobins · 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

• G01N2333/765

  Serum albumin, e.g. HSA · CPC title

• C12N2320/10

  in screening processes · CPC title

• C12N2310/16

  Aptamers · CPC title