# US 9,518,983 B2 · Layering for separating particles

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- Number: US 9,518,983 B2
- Document ID: US9518983B2
- Status: Granted
- Kind: B2
- Application number: 13/257,188
- Filed: 2010-03-25
- Published: 2012-03-29
- Granted: 2016-12-13
- Earliest claimed: 2009-03-25
- Assignees: Qiagen GmbH
- Applicants: Janina Cramer
- Inventors: Janina Cramer
- Web page: https://patentgrep.com/patent/US9518983B2

## CPC

- G01N 33/54326: {Magnetic particles}
- B03C 1/00: Magnetic separation
- C12N 15/1013: {by using magnetic beads}
- B03C 2201/18: Magnetic separation whereby the particles are suspended in a liquid
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- B03C: MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS (separating isotopes B01D59/00; combinations of magnetic or electrostatic separation with separation of solids by other means B03B, B07B)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention relates to a method for purifying biomolecules or for analyzing whether an aqueous phase contains biomolecules by means of magnetic separation. The invention further relates to uses, to devices, and to kits that relate to the method according to the invention.

## Claims

### Claim 1

A method for purifying target biomolecules from an aqueous phase, or for analyzing whether an aqueous phase contains target biomolecules, the method comprising:
(a) providing a vessel comprising an aqueous phase containing magnetic particles and possibly target biomolecules, wherein the aqueous phase is overlaid with an organic phase and wherein target biomolecules present in the aqueous phase bind to the magnetic particles;
(b) guiding a magnetic rod through the organic phase;
(c) binding the magnetic particles to the magnetic rod, wherein the magnetic articles are not fixed to a side of the vessel by a magnet prior to the binding;
(d) removing the bound magnetic particles from the aqueous phase by passing the magnetic rod and the bound magnetic particles through the organic phase, thereby displacing any aqueous phase from the magnetic particles so as to prevent carry-over of the aqueous phase; and
(e) when the method is for analyzing, further comprising analyzing whether the target biomolecules are present.

### Claim 2 (depends on 1)

The method as claimed in claim 1, wherein the target biomolecules are or comprise nucleic acids, proteins, or sugars.

### Claim 3 (depends on 1)

The method as claimed in claim 1, wherein the aqueous phase comprises a cell lysate, a bacteria lysate or a tissue lysate, a body fluid, or an in vitro reaction mixture.

### Claim 4 (depends on 1)

The method as claimed in claim 1, wherein the organic phase contains hydrocarbons and/or silicone oil.

### Claim 5 (depends on 1)

The method as claimed in claim 1, wherein the magnetic particles are silica particles.

### Claim 6 (depends on 1)

The method as claimed in claim 1, wherein the aqueous phase is completely overlaid with the organic phase.

### Claim 7 (depends on 1)

The method as claimed in claim 1, wherein after the providing of the vessel in (a) and before the removal of the bound magnetic particles in (d), there is no carrying out of a chemical reaction or an enzymatic reaction in the aqueous phase.

### Claim 8 (depends on 1)

The method as claimed in claim 1, wherein the method is automated.

### Claim 9 (depends on 1)

The method as claimed in claim 1, wherein the biomolecule to be isolated is not a PCR product.

### Claim 10 (depends on 9)

The method as claimed in claim 9, wherein no PCR reaction is carried out in the aqueous phase after adding the magnetic particles to the aqueous phase.

### Claim 11

A method for purifying target biomolecules from an aqueous phase, or for analyzing whether an aqueous phase contains target biomolecules, the method comprising:
(a) providing a first vessel comprising a first aqueous phase, which possibly contains target biomolecules,
(b) adding magnetic particles to the first aqueous phase, wherein target biomolecules present in the first aqueous phase bind to the magnetic particles,
(c) binding the magnetic particles to a magnetic rod, wherein the magnetic particles are not fixed to a side of the vessel by a magnet prior to the binding,
(d) removing the bound magnetic particles with target biomolecules bound thereto from the first aqueous phase,
(e) transferring the magnetic particles with the target biomolecules bound thereto to a second reaction vessel comprising a second aqueous phase,
(f) eluting the target biomolecules from the magnetic particles into the second aqueous phase,
(g) binding the magnetic particles to a magnetic rod, wherein the magnetic particles are not fixed to a side of the vessel by a magnet prior to the binding,
(h) removing the bound magnetic particles from the second reaction vessel using the magnetic rod, with the target biomolecules remaining in the second aqueous phase, and
(i) when the method is for analyzing, further comprising analyzing whether the target biomolecules are present,
wherein prior to the binding in (c) the first aqueous phase is overlaid with an organic phase, and the magnetic rod is guided through the organic phase prior to the removal of the bound magnetic particles from the first aqueous phase, and/or prior to the binding in (g) the second aqueous phase is overlaid with an organic phase, and the magnetic rod is guided through the organic phase prior to the removal of the bound particles from the second aqueous phase, and wherein the removing the bound magnetic particles in (d) and/or in (h) comprises passing the magnetic rod and the bound magnetic particles through the organic phase thereby displacing any aqueous phase from the magnetic particles so as to prevent carry-over of the aqueous phase, wherein the organic phase contains hydrocarbons and/or silicone oil.

### Claim 12 (depends on 11)

The method as claimed in claim 11, wherein the target biomolecules are or comprise nucleic acids, proteins, or sugars.

### Claim 13 (depends on 11)

The method as claimed in claim 11, wherein the aqueous phase comprises a cell lysate, a bacterial lysate, or a tissue lysate, a body fluid, or an in vitro reaction. mixture.

### Claim 14 (depends on 11)

The method as claimed in claim 11, wherein the magnetic particles are silica particles.

### Claim 15 (depends on 11)

The method as claimed in claim 11, wherein the aqueous phase is completely overlaid with the organic phase.

### Claim 16 (depends on 11)

The method as claimed in claim 11, wherein, after the providing of the first vessel in (a) and before the removal of the bound magnetic particles in (d), there is no carrying out of a chemical reaction or an enzymatic reaction in the first aqueous phase.

### Claim 17 (depends on 11)

The method as claimed in claim 11, wherein the method is automated.

### Claim 18 (depends on 11)

The method as claimed in claim 11, wherein the biomolecule to be isolated is not a PCR product.

### Claim 19 (depends on 18)

The method as claimed in claim 18, wherein no PCR reaction is carried out in the aqueous phase after adding the magnetic particles to the first or second aqueous phase.