Enzymatic sample purification

Patent metadata

Field	Value
Publication number	US-10845276-B2
Application number	US-201615770456-A
Country	US
Kind code	B2
Filing date	Feb 29, 2016
Priority date	Feb 29, 2016
Publication date	Nov 24, 2020
Grant date	Nov 24, 2020

Abstract

An enzymatic purification method involves the introduction of a sample comprising a target analyte and amino acids into a porous matrix of a reaction chamber. The reaction chamber includes first pores and second pores. The first pores contain polypeptide synthesis enzymes that react with the amino acids to form polypeptides. First pores having a first size to be accessible by amino acids but inaccessible by the subsequently formed polypeptides. The second pores have a second size greater than the first size, are in contact with the first pores and form a series extending from within the reaction chamber to a waste chamber. The formed polypeptides are migrated through the series of second pores to the waste chamber. The target analyte of the sample is extracted from the reaction chamber.

First claim

What is claimed is:

1. An enzymatic sample purifier comprising:
a waste chamber;
a reaction chamber having a port to receive a sample comprising a protein degradation product (PDP), and the reaction chamber containing a polypeptide synthesis enzyme, an enzyme cofactor, and a cosolvent, wherein polypeptides are formed in the reaction chamber when the sample is received in the reaction chamber and reacts with the polypeptide synthesis enzyme; and
a porous matrix within the reaction chamber, wherein the porous matrix comprises first pores having a first size and second pores having a second size larger than the first size, the first size being smaller than a molecular size of the polypeptides, the second pores being larger than the molecular size of the polypeptides, in contact with the first pores and forming a polypeptide passage comprising a series or chain of the second pores, wherein the polypeptide passage extends from within the reaction chamber to the waste chamber.

2. The enzymatic sample purifier of claim 1 , wherein the first pores comprise at least 10% and no greater than 95% of the porous matrix.

3. The enzymatic sample purifier of claim 1 , wherein the first pores comprise at least 80% and no greater than 95% of the porous matrix.

4. An enzymatic sample purifier comprising:
a waste chamber;
a reaction chamber containing a polypeptide synthesis enzyme, an enzyme cofactor and a cosolvent, the reaction chamber having a port to receive a sample comprising a protein degradation product (PDP), and polypeptides are formed in the reaction chamber when the sample is received in the reaction chamber and reacts with the polypeptide synthesis enzyme;
a porous matrix within the reaction chamber, wherein the porous matrix comprises first pores having a first size and second pores having a second size larger than the first size, the first size being smaller than a molecular size of the polypeptides, the second pores being larger than the molecular size of the polypeptides, in contact with the first pores, and forming a polypeptide passage comprising a series or chain of the second pores, wherein the polypeptide passage extends from within the reaction chamber to the waste chamber; and
a field generator to apply a field to accelerate transport of the formed polypeptides through the second pores to the waste chamber.

5. The enzymatic sample purifier of claim 4 , wherein the field generator is selected from a group of field generators consisting of: an electric potential gradient field generator, a thermal gradient field generator, an osmotic gradient field generator and a pressure gradient field generator.

6. An enzymatic sample purifier comprising:
a waste chamber;
a reaction chamber containing a polypeptide synthesis enzyme, an enzyme cofactor and a cosolvent, the reaction chamber having a port to receive a sample comprising a protein degradation product (PDP), and polypeptides are formed in the reaction chamber when the sample is received in the reaction chamber and reacts with the polypeptide synthesis enzyme;
a porous matrix within the reaction chamber, wherein the porous matrix comprises first pores having a first size and second pores having a second size larger than the first size, the first size being smaller than a molecular size of the polypeptides, the second pores being larger than the molecular size of the polypeptides, in contact with the first pores, and forming a polypeptide passage comprising a series or chain of the second pores, wherein the polypeptide passage extends from within the reaction chamber to the waste chamber; and
a buffer within the waste chamber to inhibit polypeptide breakdown.

7. The enzymatic sample purifier of claim 6 further comprising a partition between the reaction chamber and the waste chamber, the partition being permeable to molecules greater than the first size and impermeable to molecules less than the first size, wherein the partition omits the polypeptide synthesis enzyme.

8. The enzymatic sample purifier of claim 6 , wherein the polypeptide passage carries the polypeptides to the waster chamber, and does not carry a target analyte retained in the first size pores.

9. The enzymatic sample purifier of claim 8 , comprising:
a persistent non-equilibrium state in the reaction chamber between the polypeptides and the polypeptide synthesis enzyme, wherein the persistent non-equilibrium state causes additional polypeptides to be formed in the reaction chamber and expelled through the polypeptide passage to the waste chamber.

10. The enzymatic sample purifier of claim 9 , wherein the additional polypeptides are formed in the reaction chamber and expelled through the polypeptide passage to the waste chamber over a plurality of cycles.

11. The enzymatic sample purifier of claim 1 , wherein the reaction chamber comprises serpentine paths formed from the first pores, and the serpentine paths are longer than the polypeptide passage formed from the second pores.

12. The enzymatic sample purifier of claim 11 , wherein an amount of first pores is greater than an amount of second pores in the reaction chamber.

13. The enzymatic sample purifier of claim 11 , comprising:
a field generator to apply a field to accelerate transport of the formed polypeptides through the second pores to the waste chamber.

14. The enzymatic sample purifier of claim 13 , wherein the field generator is selected from a group of field generators consisting of: an electric potential gradient field generator, a thermal gradient field generator, an osmotic gradient field generator and a pressure gradient field generator.

15. The enzymatic sample purifier of claim 11 , comprising:
a buffer within the waste chamber to inhibit polypeptide breakdown.

16. The enzymatic sample purifier of claim 1 , comprising:
a partition between the reaction chamber and the waste chamber, the partition being permeable to molecules greater than the first size and impermeable to molecules less than the first size, wherein the partition omits the polypeptide synthesis enzyme.

Assignees

• Hewlett Packard Development Co
• Hewlett Packard Development Lp

Inventors

• Shkolnikov Viktor
• Rogacs Anita

Classifications

• C07K1/34

  by filtration, ultrafiltration or reverse osmosis · CPC title

• G01N1/4044 Primary

  by chemical techniques; Digestion; Chemical decomposition · CPC title

• G01N2001/4038

  electric methods, e.g. electromigration, electrophoresis, ionisation · CPC title

• C12Q1/37

  involving peptidase or proteinase · CPC title