Phosphoprotein detection using a chip-based pillar array

• US 10,386,276 B2
• Filed: 09/20/2016
• Issued: 08/20/2019
• Est. Priority Date: 09/20/2016
• Status: Active Grant

• Current Assignee: International Business Machines Corporation
• Original Assignee: International Business Machines Corporation
• Inventors: Gifford, Stacey M., Stolovitzky, Gustavo A., Wunsch, Benjamin H.
• Primary Examiner(s): Crawford, Erik B
• Application Number:
  US15/270,478
• Publication Number:
  US 20180080857A1
• Time in Patent Office: 1,064 Days
• Field of Search: None
• CPC Class Codes: B01D 15/34 Size selective separation, e.g. size exclusion chromatography, gel filtration, permeation
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/0652 Sorting or classification of particles or molecules
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 3/50273 characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• CPC Class Codes: B01L 3/502753 characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation separation in general B01D; microapparatus for analysis using electrophoresis G01N27/44791; sample preparation G01N1/28
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: G01N 1/34 Purifying; Cleaning processes or apparatus for extracting or separating nucleic acids from biological samples C12N15/1003
• CPC Class Codes: G01N 1/40 Concentrating samples
• CPC Class Codes: G01N 30/0005 Field flow fractionation
• CPC Class Codes: G01N 33/537 with separation of immune complex from unbound antigen or antibody

Abstract

Techniques for phosphoprotein detection, quantification, and purification using a chip-based pillar array are provided. In one aspect, a method for purifying a protein sample includes: introducing a mixture including the protein sample and an antibody to a nanoDLD array having a plurality of pillars separated by a gap g, wherein the antibody and proteins in the protein sample form antibody-protein complexes having a size that is greater than a size threshold of the nanoDLD array created by the gap g which permits size-based separation of the antibody-protein complexes as the mixture flows through the nanoDLD array; and collecting a purified protein sample containing the antibody-protein complexes from the nanoDLD array. A lab-on-a-chip (LOC) device including the nanoDLD array is also provided.

Claims

1. A method for purifying a protein sample, the method comprising:

introducing a mixture comprising the protein sample and an antibody conjugated to a microbead to a deterministic lateral displacement (DLD) array comprising a plurality of pillars separated by a gap, wherein the antibody conjugated to the microbead and proteins in the protein sample bind to form protein-antibody-microbead complexes having a size that is greater than a size threshold of 500 nm created by the gap which permits size-based separation of the protein-antibody-microbead complexes as the mixture flows through the DLD array, with the protein-antibody-microbead complexes passing through the DLD array in a bump mode and unbound proteins, which are smaller than the size threshold created by the gap, passing through the DLD array in a zig-zag mode;

applying a negative to positive charge gradient to the DLD array for charge separation of the protein-antibody-microbead complexes, wherein the negative to positive charge gradient is applied to the DLD array perpendicular to a direction of flow of the mixture through the DLD array; and

collecting a purified protein sample containing the protein-antibody-microbead complexes from the DLD array into a collection reservoir located downstream from the DLD array at a positive end of the negative to positive charge gradient,

wherein the microbeads are selected such that the protein-antibody-microbead complexes are greater in size than the size threshold and smaller in size than the gap.

2. The method of claim 1, wherein the gap is from about 1 micrometer to about 5 micrometers.

3. The method of claim 1, wherein the proteins in the protein sample comprise both phosphorylated proteins and non-phosphorylated proteins.

4. The method of claim 3, wherein the antibody comprises a phospho-specific antibody.

5. The method of claim 3, wherein antibody comprises a non-phospho-specific antibody.

6. The method of claim 1, wherein the microbeads are polystyrene beads.

7. The method of claim 1, wherein the antibody is labeled with a fluorescent tag.

8. The method of claim 1, further comprising:

analyzing the purified protein sample collected from the DLD array.

9. The method of claim 1, further comprising:

mixing the protein sample with a solution containing the antibody conjugated to the microbead to form the mixture prior to introducing the mixture to the DLD array.

10. The method of claim 1, further comprising:

preloading the antibody conjugated to the microbead upstream from the DLD array.