Phosphoprotein Detection Using a Chip-Based Pillar Array

• US 20190331561A1
• Filed: 07/09/2019
• Published: 10/31/2019
• 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.
• Granted Patent: US 11,161,112 B2
• Time in Patent Office: Days
• 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 lab-on-a-chip (LOC) device for purifying a protein sample, comprising:

a nanoscale deterministic lateral displacement (nanoDLD) array comprising a plurality of pillars separated by a gap g; and

an antibody preloaded onto the LOC device upstream from the nanoDLD array, 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.

2. The LOC device of claim 1, further comprising:

a loading reservoir located upstream from the nanoDLD array in which the antibody is preloaded.

3. The LOC device of claim 1, further comprising:

at least one collection reservoir located downstream from the nanoDLD array configured to collect a purified protein sample containing the antibody-protein complexes from the nanoDLD array.

4. The LOC device of claim 1, wherein the gap g is from about 1 micrometer to about 5 micrometers, and ranges therebetween.

5. The LOC device of claim 1, wherein the antibody comprises a phospho-specific antibody.

6. The LOC device of claim 1, wherein the antibody comprises a non-phospho-specific antibody.

7. The LOC device of claim 1, further comprising:

polystyrene beads preloaded onto the device upstream from the nanoDLD array on which the antibody is present.

8. The LOC device of claim 7, wherein the polystyrene beads comprise micro-beads having a diameter of from about 1 micrometer to 1,000 micrometers, and ranges therebetween.

9. The LOC device of claim 7, wherein the polystyrene beads comprise nano-beads having a diameter of from about 1 nanometer to about 100 nanometers, and ranges therebetween.

10. The LOC device of claim 1, wherein the antibody is labeled with a fluorescent tag.

11. A LOC device for purifying a protein sample, comprising:

a nanoDLD array comprising a plurality of pillars separated by a gap g;

an antibody preloaded onto the LOC device upstream from the nanoDLD array, 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;

a loading reservoir located upstream from the nanoDLD array in which the antibody is preloaded; and

at least one collection reservoir located downstream from the nanoDLD array configured to collect a purified protein sample containing the antibody-protein complexes from the nanoDLD array.

12. The LOC device of claim 11, wherein the gap g is from about 1 micrometer to about 5 micrometers, and ranges therebetween.

13. The LOC device of claim 11, wherein the antibody comprises a phospho-specific antibody.

14. The LOC device of claim 11, wherein the antibody comprises a non-phospho-specific antibody.

15. The LOC device of claim 11, further comprising:

polystyrene beads preloaded onto the device upstream from the nanoDLD array on which the antibody is present.

16. The LOC device of claim 15, wherein the polystyrene beads comprise micro-beads having a diameter of from about 1 micrometer to 1,000 micrometers, and ranges therebetween.

17. The LOC device of claim 15, wherein the polystyrene beads comprise nano-beads having a diameter of from about 1 nanometer to about 100 nanometers, and ranges therebetween.

18. The LOC device of claim 11, wherein the antibody is labeled with a fluorescent tag.

19. A LOC device for purifying a protein sample, comprising:

a nanoDLD array comprising a plurality of pillars separated by a gap g;

an antibody preloaded onto the LOC device upstream from the nanoDLD array, wherein the antibody is labeled with a fluorescent tag, and 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;

a loading reservoir located upstream from the nanoDLD array;

polystyrene beads on which the antibody is present preloaded in the loading reservoir; and

at least one collection reservoir located downstream from the nanoDLD array configured to collect a purified protein sample containing the antibody-protein complexes from the nanoDLD array.

20. The LOC device of claim 19, wherein the gap g is from about 1 micrometer to about 5 micrometers, and ranges therebetween.