Multiplex microarray of serially deposited biomolecules on a microarray

• US 9,927,434 B2
• Filed: 01/20/2011
• Issued: 03/27/2018
• Est. Priority Date: 01/20/2010
• Status: Active Grant

• Current Assignee: CustomArray, Inc. (Genscript Biotech Corp.)
• Original Assignee: CustomArray, Inc. (Genscript Biotech Corp.)
• Inventors: Cooper, John
• Primary Examiner(s): Crow, Robert T.
• Application Number:
  US12/930,977
• Publication Number:
  US 20110281766A1
• Time in Patent Office: 2,623 Days
• Field of Search: None
• CPC Class Codes: G01N 33/5438 Electrodes

Abstract

Disclosed herein is a multiplex microarray having serially attached non-functionalized biomolecules attached to a polymer coating covering each electrode of an array of electrodes for assays and a method of making the multiplex microarray. The method comprises serially blocking the electrodes of the microarray with a blocking protein, electropolymerizing pyrrole or a functionalized pyrrole on the electrodes where the biomolecule is not present during polymerization, exposing the microarray to a biomolecular solution containing a non-functionalized biomolecule for attachment to the polymer coating, and then repeating the steps to form the multiplex microarray.

Claims

1. A method of making a multiplex microarray of serially attached different biomolecules on a microarray, comprising:

(a) blocking a microarray with a blocking protein, wherein the microarray is a device having an array of addressable microelectrodes on a surface of the device;

(b) electropolymerizing a monomer on one or more microelectrodes of the microarray using an electropolymerizing solution with one or more electropolymerizing parameters to form a first thickness on at least a first microelectrode and electropolymerizing the monomer with one or more different electropolymerizing parameters to form at least a second thickness on at least a second microelectrode, where the first microelectrode and the second microelectrode are selected from the one or more microelectrodes, where the first thickness is different than the second thickness, where the one or more electropolymerizing parameters are selected from the group consisting of potential, current and time of electropolymerizing, where the electropolymerizing solution containing the monomer does not contain any biomolecules;

(c) exposing the microarray to a biomolecular solution containing a biomolecule for attachment to the first thickness on the one or more electrodes;

(d) blocking the microarray with a blocking protein; and

(e) exposing the microarray to a different biomolecular solution containing a different biomolecule for attachment to the second thickness on the one or more electrodes, where one or both the first thickness and the second thickness facilitates detection of one or both the biomolecule and the different biomolecule.

2. The process of claim 1, wherein steps (a) and (d) further comprise:

washing the microarray to remove the blocking protein.

3. The process of claim 1, wherein step (b) further comprises:

washing the microarray to remove the electropolymerizing solution.

4. The process of claim 1, wherein step (c) further comprises:

washing the microarray to remove the biomolecular solution.

5. The process of claim 1, wherein the monomer is selected from the group consisting of pyrrole and functionalized pyrrole and combinations thereof.

6. The process of claim 1, wherein the monomer is selected from the group consisting of thiophenol, aniline, phenylene sulfide, monomers electropolymerizing to a conducting polymer, phenylenediamine, diaminohaphthalene, phenol, monomers electropolymerizing to a nonconducting polymer and phenolic derivatives, and combinations thereof.

7. The process of claim 1, wherein the biomolecules are selected from the group consisting of antibodies, antigens, proteins, Ricin, anti-SEB MAb, enzymes, and HRP, and combinations thereof.

8. The process of claim 1, wherein the biomolecules are selected from the group consisting of carbohydrates, sugars, dextrane, PEG, and poly-L lysine and combinations thereof.

9. The process of claim 1, wherein the device is a CMOS device.

18. The process of claim 1, where the one or more electropolymerizing parameters include a constant current between 10 and 260 nA and electropolymerizing time periods selected from the group consisting of 0.1, 0.5, 1.0, and 2.0 seconds of electropolymerizing.

19. The process of claim 1, where the one or more electropolymerizing parameters include a constant current between 0 and 980 nA and an electropolymerizing time period of 1.0 second.

20. The process of claim 1, where the one or more electropolymerizing parameters include increasing constant currents between 10 and 130 nA and an electropolymerizing time period of 1.0 second.

10. A method of making a multiplex microarray of serially attached different oligonucleotides on a microarray, comprising:

(a) blocking a microarray with a blocking protein, wherein the microarray is a device having an array of addressable microelectrodes on a surface of the device;

(b) electropolymerizing a monomer on one or more microelectrodes of the microarray using an electropolymerizing solution with one or more electropolymerizing parameters to form a first thickness on a first microelectrode and electropolymerizing the monomer with one or more different electropolymerizing parameters to form at least a second thickness on a second microelectrode, where the first microelectrode and the second microelectrode are selected from the one or more microelectrodes, where the first thickness is different than the second thickness, where the one or more electropolymerizing parameters are selected from the group consisting of potential, current and time of electropolymerizing, where the electropolymerizing solution containing the monomer does not contain any oligonucleotides;

(c) exposing the microarray with an oligonucleotide solution containing an oligonucleotide for attachment to the first thickness on the one or more electrodes;

(d) blocking the microarray with a blocking protein; and

(e) exposing the microarray to a different oligonucleotide solution containing a different oligonucleotide for attachment to the second thickness on the one or more electrodes, where one or both the first thickness and the second thickness facilitates detection of one or both the oligonucleotide and the different oligonucleotide.

11. The process of claim 10, wherein steps (a) and (d) further comprise:

washing the microarray to remove the blocking protein.

12. The process of claim 10, wherein step (b) further comprises:

washing the microarray to remove the electropolymerizing solution.

13. The process of claim 10, wherein step (c) further comprises:

washing the microarray to remove the oligonucleotide solution.

14. The process of claim 10, wherein the monomer is selected from the group consisting of pyrrole and functionalized pyrrole and combinations thereof.

15. The process of claim 10, wherein the monomer is selected from the group consisting of thiophenol, aniline, phenylene sulfide, monomers electropolymerizing to a conducting polymer, phenylenediamine, diaminohaphthalene, phenol, monomers electropolymerizing to a nonconducting polymer and phenolic derivatives, and combinations thereof.

16. A microarray for multiplex oligonucleotide hybridization assay, comprising:

(a) a microarray having on a surface an array of addressable microelectrodes having an exposed surface;

(b) an electropolymerized polymer attached to the exposed surface of the array of addressable microelectrodes, where the electropolymerized polymer attached to a first microelectrode has a first thickness and the electropolymerized polymer attached to a second microelectrode has a second thickness, where the first thickness is thicker than the second thickness; and

(c) a first preformed oligonucleotide serially-attached to the first thickness and a second preformed oligonucleotide serially attached to the second thickness, wherein the different preformed oligonucleotides are not synthesized in situ on the microarray.

17. The microarray of claim 16, where one or more electropolymerizing parameters are different for the first thickness than the second thickness, where the electropolymerizing parameters are selected from the group consisting of potential, current and time of electropolymerizing.