Centrifugal method and apparatus for isolating a substance from a mixture of substances in a sample liquid

• US 5,610,074 A
• Filed: 02/24/1993
• Issued: 03/11/1997
• Est. Priority Date: 02/24/1993
• Status: Expired due to Fees

• Current Assignee: David R. Beritashvili, Gennady M. Ershov, George I. Kapanadze, George P. Georgiev, Maxim V. Myakishev
• Original Assignee: David R. Beritashvili, Gennady M. Ershov, George I. Kapanadze, George P. Georgiev, Maxim V. Myakishev
• Inventors: Myakishev, Maxim V., Kapanadze, George I., Ershov, Gennady M., Beritashvili, David R., Georgiev, George P.
• Primary Examiner(s): BEISNER, WILLIAM H
• Application Number:
  US08/022,047
• Time in Patent Office: 1,476 Days
• Field of Search: 435/4, 435/5, 435/6, 435/29, 435/30, 435/287, 435/288, 435/289, 435/291, 435/311, 435/310, 435/312, 435/803, 435/240.1, 435/259, 435/261, 435/287.2, 435/288.2, 435/288.5 , 435/308.1, 435/306.1, 422/68.1, 422/70, 422/72, 422/99, 422/101, 494/23, 494/27, 494/29, 494/36, 494/37, 494/42, 494/56, 494/67, 494/68, 494/69, 494/70, 494/17, 210/512.1, 210/656, 210/657, 210/198.2, 210/198.3, 210/781, 210/787, 73/863.21, 436/45, 436/94, 436/177, 436/178, 436/180, 436/175, 536/25.4, 935/19, 935/85
• US Class Current: 436/177
• CPC Class Codes: B01D 21/262 by using a centrifuge
• CPC Class Codes: B01D 2221/10 Separation devices for use in medical, pharmaceutical or laboratory applications, e.g. separating amalgam from dental treatment residues apparatus for dental treatment A61C17/065
• CPC Class Codes: B01L 2200/026 Fluid interfacing between devices or objects, e.g. connectors, inlet details
• CPC Class Codes: B01L 2200/0621 Control of the sequence of chambers filled or emptied
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2300/0681 Filter
• CPC Class Codes: B01L 2300/0803 Disc shape
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/087 Multiple sequential chambers
• CPC Class Codes: B01L 2400/0412 using additionally coriolis forces
• CPC Class Codes: B01L 3/502 with fluid transport, e.g. in multi-compartment structures centrifugal-type cuvettes G01N21/07; analysis by separation into components G01N30/00; automatic analysers G01N35/00
• CPC Class Codes: B04B 5/0407 for liquids contained in receptacles B04B5/0442 takes precedence
• CPC Class Codes: C12M 47/04 Cell isolation or sorting purging biological preparations of unwanted cells C12N5/0081, determining the presence or kind of microorganism C12Q1/04
• CPC Class Codes: C12Q 1/24 Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
• CPC Class Codes: G01N 1/40 Concentrating samples
• CPC Class Codes: Y10T 436/25375 Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
• CPC Class Codes: Y10T 436/255 including use of a solid sorbent, semipermeable membrane, or liquid extraction

Abstract

A centrifugal rotor effects the isolation, in a sequence of steps, of a substance from a mixture of substances dissolved, suspended or dispersed in a sample liquid. Multiple samples are processed simultaneously by means of a plurality of fractionation cells, each of which contains a series of interconnected, chambered and vented compartments in which individual steps of the fractionation and isolation procedure take place. Specific steps in the preferred embodiments include lysis, sedimentation, aggregation, sorption, rinsing, and desorption. The specific compartment occupied by the sample liquid or one of its fractions at any stage of the process is governed by the speed and direction of rotation of the rotor and by gravitational force. The interconnections, chambers and passages of each compartment are sized and angled to prevent predetermined amounts of sample and reagent liquids from overflowing the compartment. Predetermined amounts of the reagent liquids required to contact the sample in individual steps of the isolation process are simultaneously delivered to specific compartments of every cell by means of a dynamic flow distributor which accepts partially aerosolized injections of said reagents pipetted from a dosator while the centrifugal rotor is rotating. A method of using the rotor to isolate deoxyribonucleic acid is described. The operation of the centrifuge and the dosator can be controlled and coordinated automatically.

Claims

1. A method of isolating a substance sought to be isolated from a mixture of substances in a sample liquid comprising the following steps:

(a) loading a first compartment of a fractionation cell with a predetermined volume of the sample liquid by way of an entry port opening into the first compartment;

(b) inserting the cell into a centrifugal rotor equipped with a dynamic flow distributor;

(c) rotating the rotor in a first direction at a first predetermined speed to cause the sample liquid to flow into a second compartment of the cell;

(d) while continuing to rotate the rotor, injecting a predetermined volume of a first liquid reagent into a first flow distributor, said first flow distributor being aligned to permit a flow of the first reagent into the first compartment of the cell and therethrough to the second compartment where the first reagent contacts and mixes with the sample liquid to form a conditioned sample liquid;

(e) stopping rotation of the rotor to permit the conditioned sample liquid to flow away from a material sedimented in a chamber in the second compartment during step (d) and into a third compartment of the cell;

(f) rotating the rotor again in the first direction at the first predetermined speed to cause the conditioned sample liquid to flow into a fourth compartment wherein the conditioned sample liquid contacts a retaining means fixed within the fourth compartment for releasably retaining, selectively, the substance sought to be isolated;

(g) while continuing to rotate the rotor to cause the conditioned sample liquid to pass through the fourth compartment into a waste collection chamber, injecting a predetermined volume of a second reagent into a second flow distributor, said second flow distributor being aligned to permit a flow of the second reagent into the third compartment and, therethrough, into the fourth compartment, to elute substances from the retaining means, which retaining means, when contacted by the second reagent, selectively retains the substance sought to be isolated;

(h) while continuing to rotate the rotor to permit the second reagent to flow out of the fourth compartment into the waste collection chamber, injecting a third reagent into the second flow distributor to rinse traces of the second reagent into the waste collection chamber;

(i) rotating the rotor in a second direction at a second predetermined speed less than the first predetermined speed and injecting a fourth reagent into the second flow distributor to cause the retaining means to release the substance sought to be isolated into the fourth reagent;

(j) rotating the rotor in the second direction at about the first predetermined speed to cause the fourth reagent with the isolated substance contained therein to flow out of the fourth compartment into a sample collection chamber.

2. The method according to claim 1 wherein the substance sought to be isolated is selected from the group comprising ribonucleic acid and deoxyribonucleic acid.

3. The method according to claim 2 wherein the sample liquid comprises a suspension of viral particles.

4. The method according to claim 2 wherein the sample liquid comprises a suspension of bacteria.

5. The method according to claim 2 wherein the sample liquid comprises a suspension of plant cells.

6. The method according to claim 2 wherein the sample liquid comprises a suspension of animal cells.

7. The method according to claim 2 wherein the sample liquid is a physiological fluid containing cells suspended therein.

8. The method of claim 2 wherein the first reagent is an aqueous acidic solution.

9. The method of claim 2 wherein the first reagent is a lysing reagent.

10. The method of claim 2 wherein the second reagent is an aqueous solution of sodium perchlorate.

11. The method of claim 2 wherein the third reagent is an aqueous solution of ethanol wherein the concentration of ethanol is about more than 50%.

12. The method of claim 2 wherein the fourth reagent is an aqueous ionic solution of ionic strength about less than 200 milliequivalents per liter.

13. The method claim 2 wherein the retaining means is glass fibers.

14. The method of claim 2 wherein the retaining means is glass beads.

15. The method of claim 1 wherein the temperature of the rotor is controlled between a temperature of about 0°

C. and about 100°

C.

16. The method of claim 1 wherein steps (c) through (j) are automatically controlled.

17. The method according to claim 1 wherein the substance sought to be isolated is a biopolymer.

18. An apparatus for isolating a substance sought to be isolated from a mixture of substances in a sample liquid, said apparatus comprising, in combination:

a) a centrifugal rotor having a superior region, an inferior region, an axis of rotation disposed in controllable relation to a gravitational force, and a reversible impelling means for applying a predetermined centrifugal force thereto;

b) a fractionation cell contained in said rotor, said cell comprising walls defining an entry port and a plurality of interconnected chambers and passages, said chambers and passages being sized, angled and disposed relative to one another to provide, in combination with said impelling means, a means for controlling a flow therebetween of a predetermined liquid volume under a centrifugal force and said gravitational force, wherein the centrifugal force is a means for constraining and for advancing the flow and wherein the gravitational force is a means for constraining and for advancing the flow, and wherein said interconnected chambers and passages in the cell are distributed among a plurality of contiguous compartments comprising;

1) a first compartment comprising;

a first chamber for containing said sample liquid and, axially, a first passage connecting the first chamber with a first entry port, the first passage and the first chamber being sized and angled to prevent said liquid volume from overflowing the first compartment when said rotor is static;

a first centrifugal surface, said centrifugal surface being angled such that centrifugal force urges said liquid volume out of the first compartment into

2) a second compartment, disposed generally in a radially outward direction relative to the first compartment, comprising;

a second chamber and, superiorly, a first vent passage connecting the second cheer to a first vent port, the second chamber being sized and the first vent passage being sized and angled to prevent said liquid volume from flowing out of the cell under the centrifugal force;

a second centrifugal surface disposed in parallel to and radially outward from the first centrifugal surface such that gravitational force urges said liquid volume out of the second compartment into

3) a third compartment disposed inferiorly to the first and second compartments, the third compartment comprising;

a third chamber and, axially, a second passage connecting the third chamber with a second entry port, the third chamber and second passage being sized and angled to prevent said liquid volume from overflowing said third compartment when said rotor is static;

a third centrifugal surface angled such that centrifugal force urges substantially all of said liquid volume in the third chamber into

4) a fourth compartment disposed radially outward and superiorly in relation to the third compartment, the fourth compartment comprising a fourth chamber and a means for controllably permitting an outflow of said liquid volume from the fourth chamber, whereby, upon sequential exposure of said rotor to said centrifugal and gravitational forces, said sample liquid is urged through said interconnected chambers and passages thereby isolating a substance present in said sample liquid into said fourth chamber.

19. The apparatus of claim 18 wherein the second chamber is sized and angled to retain substantially all of and substantially only a sedimentable material dispersed in said liquid volume, which material is sedimentable under said predetermined centrifugal force.

20. The apparatus of claim 18 wherein said outflow-permitting means is sized to provide a predetermined resistance to flow of said liquid volume.

21. The apparatus according to claim 18 wherein the outflow-permitting means is a capillary.

22. The apparatus of claim 18 wherein the fourth chamber contains a means of selectively retaining, releasably, the substance sought to be isolated.

23. The apparatus of claim 22 wherein the fourth compartment further comprises:

an antechamber disposed radially outward from the outflow-permitting means;

a sample collection chamber disposed radially outward from, and in open communication with, the antechamber;

a waste collection chamber disposed radially outward from, and in open communication with, the antechamber, the waste collection chamber being disposed in closely spaced relationship to the sample collection chamber and lying in substantially the same plane as the sample collection chamber;

a second vent passage connecting the waste collection chamber to a second vent port.

24. The apparatus of claim 18 wherein the fractionation cell is removably contained in the rotor.

25. The apparatus of claim 18 wherein the fractionation cell contains a vent port.