Nanoscale process to generate reagents selective for individual protein variants

Patent metadata

Field	Value
Publication number	US-9938330-B2
Application number	US-201314388209-A
Country	US
Kind code	B2
Filing date	Mar 12, 2013
Priority date	Mar 29, 2012
Publication date	Apr 10, 2018
Grant date	Apr 10, 2018

Abstract

The present invention provides devices and methods to separate and concentrate target protein species at a microliter scale and to generate reagents to those variants with exquisite selectivity for specific protein isoforms using only picograms of target material.

First claim

What is claimed is:

1. A direct current insulating gradient dielectrophoresis (DC-iGDEP) device for separating a target protein species in a biological sample based on a chemical or physical parameter, the device comprising: a sawtooth microfluidic channel configured to separate a target protein species from a biological sample, the channel having an inlet port and an outlet port, the sawtooth microfluidic channel including a plurality of pairs of opposing teeth lining both sides of the microfluidic channel, wherein the teeth have a triangular, asymmetric, crescent, starred or rounded shape, each of the plurality of pairs of opposing teeth including a gap between each of the plurality of pairs of opposing teeth, each of the gaps associated with each of the pairs of opposing teeth decreasing from about 1 mm to about 0.5 mm along the sawtooth microfluidic channel.

2. The device of claim 1 , wherein the teeth have a triangular shape.

3. The device of claim 2 , wherein the teeth have an equilateral triangular shape having a base and a height.

4. The device of claim 3 , wherein the smallest teeth have a base length of 2-10 μm and a height of 2-10 μm, and wherein after every 1-10 repeats successive teeth are 25-50 μm larger in their side length and width.

5. The device of claim 1 , wherein the spacing of the initial gaps has a distance of about 945 pm and the spacing of the final gaps has a distance of about 27 pm.

6. The device of claim 1 , wherein the spacing of the initial gaps has a distance of about 50 pm and the spacing of the final gaps has a distance of about 1 nm.

7. The device of claim 1 , wherein the spacing of the initial gaps has a distance of about 50 pm and the spacing of the final gaps has a distance of about 1 pm.

8. The device of claim 1 , wherein the microfluidic channel has a depth of about 10 to 20 μm.

9. A method of separating from a biological sample a target species based on a chemical or physical parameter comprising,
(a) providing the device of claim 1 ,
(b) loading a loading volume of the sample into the inlet port,
(c) applying a field to the device to separate particles or molecules in the sample based on the chemical or physical parameter, and
(d) recovering the target species.

10. The method of claim 9 , wherein the chemical or physical parameter is charge, size, permittivity, deformation, or shape.

11. The method of claim 9 , wherein the target species is an Aβ aggregate.

12. The method of claim 11 , further comprising contacting the recovered Aβ aggregates with an antibody to confirm the size of the Aβ aggregate.

13. The method of claim 12 , wherein the antibody is specific for oligomeric Aβ aggregates.

14. The method of claim 12 , wherein the antibody is a nanobody, and the nanobody is a C6, A4, E1, D5, 10H, 6E, D10 or BSEC1 nanobody.

15. The method of claim 9 , wherein the biological sample has a volume of less than 100 microliters.

16. The method of claim 9 , wherein the biological sample is brain tissue, serum, cerebrospinal fluid (CSF), urine or saliva.

17. The method of claim 9 , wherein the field is applied for a period of time that is less than 20 minutes.

18. The method of claim 9 , wherein the target species is concentrated by several orders of magnitude as compared to the loading volume.

19. The method of claim 9 , wherein the target species is concentrated by 10 6 as compared to the loading volume.

20. The method of claim 9 , wherein the target species is a protein.

21. The method of claim 20 , wherein the protein is p53, islet amyloid polypeptide, beta-amyloid, tau, alpha-synuclein, huntingtin, or superoxide dismutase.

Assignees

• Univ Arizona State
• Arizona Board Of Regents A Body Corporate Of The State Of Arizona Acting For And On Behalf Of Arizon

Inventors

• Sierks Michael
• Hayes Mark

Classifications

• B03C5/005 Primary

  Dielectrophoresis, i.e. dielectric particles migrating towards the region of highest field strength · CPC title

• G01N27/447

  using electrophoresis · CPC title

• B01L3/5027

  by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip · CPC title

• C07K14/4711 Primary

  Alzheimer's disease; Amyloid plaque core protein · CPC title

• B03C5/026

  using open-gradient differential dielectric separation, i.e. using electrodes of special shapes for non-uniform field creation, e.g. Fluid Integrated Circuit [FIC] · CPC title