# US 10,308,929 B2 · Method of preparing biological material

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- Number: US 10,308,929 B2
- Document ID: US10308929B2
- Status: Granted
- Kind: B2
- Application number: 14/343,019
- Filed: 2012-09-06
- Published: 2014-12-18
- Granted: 2019-06-04
- Earliest claimed: 2011-09-06
- Assignees: North-West University
- Applicants: Anne Frederica Grobler; Oksana Levanets
- Inventors: Anne Frederica Grobler; Oksana Levanets
- Web page: https://patentgrep.com/patent/US10308929B2

## CPC

- C12N 15/1003: {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor}
- C12N 15/1006: {by means of a solid support carrier, e.g. particles, polymers}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- G01N 1/34: Purifying; Cleaning {(processes or apparatus for extracting or separating nucleic acids from biological samples C12N15/1003)}
- G01N 33/54386: {Analytical elements}
- Y10T 436/255: including use of a solid sorbent, semipermeable membrane, or liquid extraction
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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)
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

This invention pertains to a method of preparing biological material from a biological sample selected from the group consisting of blood and sputum samples. The method includes the step of altering at least one constitutive characteristic of the biological sample in the presence of a capturing scaffold by adding a lysis buffer containing a solubilising agent and a detergent to the biological sample, for simultaneously inhibiting coagulation of the biological sample; lysing the biological sample to release the biological material from the biological sample, thus making the biological material available; and capturing at least one fraction of the biological material on the capturing scaffold.

## Claims

### Claim 1

A method of preparing biological material from a biological sample selected from the group consisting of blood and sputum samples, the method including the step of altering at least one constitutive characteristic of the biological sample in the presence of a capturing scaffold by adding a lysis buffer containing a solubilising agent in the form of a chaotropic salt selected from the group consisting of urea, thiourea, guanidine hydrochloride, lithium perchlorate, sodium iodine, sodium perchlorate, guanidine isothiocyanate, guanidine carbonate, guanidine thiocyanate, derivatives of said chaotropic salts and combinations thereof and a detergent to the biological sample, and concomitantly adding purified starch to the biological material together with the lysis buffer for neutralising any PCR inhibitors present in the biological material, and concomitantly physically treating the biological sample through agitation and elevating the temperature of the biological sample above 40 degrees Celsius and up to a 100 degrees Celsius; for simultaneously:
inhibiting coagulation of the biological sample;
lysing the biological sample to release biological material from the biological sample thus making the biological material available; and
capturing at least one fraction of the biological material on the capturing scaffold in a single chamber or well such that the biological material captured on the capturing scaffold is usable directly for analysis without any purification required and wherein the biological material captured on the capturing scaffold can be subsequently air dried and stored at ambient temperature.

### Claim 2 (depends on 1)

A method according to claim 1 including the subsequent step of removing the capturing scaffold together with the captured fraction of the biological sample from the remainder of the biological sample.

### Claim 3 (depends on 1)

A method according to claim 1 wherein the concentration of the chaotropic salt in the lysis buffer is in the range of from 2 M to 8 M.

### Claim 4 (depends on 1)

A method according to claim 1 wherein the lysis buffer is selected from the group consisting of phosphate buffers, 4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid (HEPES), N-Cyclohexyl-2-aminoethanesulfonic acid (CHES), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS) and piperazine-N/, N-bis(2-ethanesulfonic acid) (PIPES), Tris-HCI, as well as other tris(hydroxymethyl)aminomethane (Tris) buffers containing ethylene diamine tetra-acetic acid (EDTA), ethylene glycol tetra-acetic acid (EGTA), deoxycholate, sodium chloride (MCI), sodium phosphate, octylphenoxypolyethoxyethanol, and non-ionic surfactants provided with a hydrophilic polyethylene oxide group and a hydrocarbon lipophilic or hydrophobic group, and combinations thereof.

### Claim 5 (depends on 4)

A method according to claim 4 wherein the lysis buffer has a pH of between 4 and 12.

### Claim 6 (depends on 1)

A method according to claim 1 wherein the detergent is selected from the group consisting of 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), a nonionic surfactant and emulsifier derived from polyethoxylated sorbitan and oleic acid, a nonionic surfactant which has a hydrophilic polyethylene oxide group and a hydrocarbon lipophilic or hydrophobic group, saponin, sodium deoxycholate, SDS, octyl glucoside, octyl thioglucoside, laurly maltose, octylphenoxypolyethoxyethanol, and combinations thereof.

### Claim 7 (depends on 6)

A method according to claim 6 wherein the detergent has a concentration of between 0.3% and 6%.

### Claim 8 (depends on 1)

A method according to claim 1 wherein the biological material is captured in the form of nucleic acids, protein, serum, cells, tissue, plasma, antigens, antibodies, or reaction products.

### Claim 9 (depends on 1)

A method according to claim 1 including the step of concomitantly adding a reducing agent selected from the group consisting of 2-mecarptoethanol, dithiothreitol (DTT), 2- mercaptoethylamine, tris(2-carboxyl)phosphine (TCEP), cysteine HCI N-ethylmaleimide, Nacystelyn, dornase alfa, thymosin β4, guaifenesin TCEP HCl, and combinations thereof, to the biological sample together with the lysis buffer containing the solubilising agent and the detergent.

### Claim 10 (depends on 1)

A method according to claim 1 wherein the capturing scaffold is treated chemically and/or physically prior to the capturing of the biological material such that a batch of capturing scaffolds is pre-prepared and stored for later use.

### Claim 11 (depends on 10)

A method according to claim 10 wherein the step of pre-treating the capturing scaffold physically includes the further step of increasing the outer surface area of the capturing scaffold.

### Claim 12 (depends on 10)

A method according to claim 10 wherein the capturing scaffold is chemically pre-treated with a cross-linking agent selected from the group consisting of ethyldimethylaminopropyl carbodiimide (EDC), N,N′-dicyclohexylcarbodiimide (DCC), N,N′-diisopropylcarbodiimide (DIC), 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiitnide metho-p- toluenesulfonate (CMC), aldehyde and combinations thereof.

### Claim 13 (depends on 10)

A method according to claim 10 wherein the step of pre-preparing the capturing scaffold chemically and/or physically includes the further steps of washing the treated capturing scaffold with Tris buffer (containing NaCI, a non-ionic surfactant and emulsifier derived from polyethoxylated sorbitan and oleic acid), de-ionised water, phosphate buffer containing a non-ionic surfactant emulsifier and combinations thereof.

### Claim 14 (depends on 1)

A method according to claim 1 wherein the capturing scaffold is selected from the group consisting of nano- or micro-particles and a body of a polymeric material.

### Claim 15 (depends on 14)

A method according to claim 14 wherein the nano-particles are prepared from polylactic acid or chitosan derivatives.

### Claim 16 (depends on 14)

A method according to claim 14 wherein the polymeric material is selected from the group consisting of polyethylene, polystyrene, polypropylene, polyvinyl chloride, nylon, teflon (poly tetra polyethylene), polychloroprene and polyacrylonitrile.