DIAGNOSTIC AND SAMPLE PREPARATION DEVICES AND METHODS

• US 20170253912A1
• Filed: 01/17/2017
• Published: 09/07/2017
• Est. Priority Date: 03/10/2011
• Status: Active Grant

• Current Assignee: General Atomics, Inc.
• Original Assignee: Xagenic Inc
• Inventors: KELLEY, Shana O., BORTOLIN, Susan, ORTON, Reginald James McKenzie, Wiechula, Stefan Christopher
• Granted Patent: US 10,301,666 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 1/066 by physical methods
• CPC Class Codes: C12N 13/00 Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2523/307 Denaturation or renaturation by electric current/voltage
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

Contemplated methods and devices are drawn to preparation and analysis of analytes from biological samples. In a preferred embodiment the analytes are nucleic acids that are both released from biological compartments present in the sample and fragmented through the use of a voltage potential applied to a pair of electrodes. The nucleic acids thus prepared are subsequently characterized.

Claims

1. A method of preparing nucleic acids from a biological sample comprising:

collecting a biological sample;

transferring at least a portion of the biological sample to an extraction zone, wherein the extraction zone comprises a pair or set of multiple electrodes;

applying a modulated potential difference to a pair of electrodes, such that a plurality of nucleic acids are released from the portion of the biological sample and fragmented within the extraction zone to produce a plurality of fragmented nucleic acids;

wherein the plurality of fragmented nucleic acids have an average length that is equal or less than 75% of a length of the respective plurality of fragmented nucleic acids prior to fragmentation.

2. The method of claim 1, wherein the average length is equal or less than 50% of the length of the respective plurality of fragmented nucleic acids prior to fragmentation.

3. The method of claim 1, wherein the plurality of nucleic acids are fragmented to produce a plurality of respective fragments having an average length of equal or less than 200 bases.

4. The method of claim 1 further comprising a step of adjusting the modulated potential difference to thereby adjust the average length.

5. The method of claim 4 wherein the step of adjusting the modulated potential difference comprises adjusting at least one of a magnitude of a voltage applied to the pair of electrodes, a duration of a voltage pulse applied to the pair of electrodes, a frequency at which a voltage pulse is applied to the pair of electrodes and the overall treatment time.

6. The method of claim 4 wherein the step of adjusting the modulated potential difference is effective to reduce hybridization time of at least one of the plurality of fragmented nucleic acids to a solid-phase bound capture probe at least 50% relative to a hybridization time of at least one of the plurality of nucleic acids prior to fragmentation.

7. The method of claim 1, further comprising a step of contacting the biological sample in the extraction zone with an enhancing reagent.

8. The method of claim 7, wherein the enhancing reagent is selected from the group consisting of a metal ion, a free radical promoting compound, a chaotropic salt, an ionic detergent, and a nonionic detergent.

9. A device for preparation of nucleic acids from a biological sample, comprising:

a fluid reservoir comprising a pliant wall, the pliant wall having an external surface;

an extraction zone comprising a pair of electrodes;

a sample receiving zone configured to receive and retain at least a portion of a biological sample; and

wherein the sample receiving zone is in fluid communication with the fluid reservoir and the extraction zone and is fluidly interposed between the fluid reservoir and the extraction zone.

10. The device of claim 9, wherein the fluid reservoir further comprises an extraction buffer.

11. The device of claim 10, wherein the fluid reservoir is configured such that pressure applied to the external surface of the pliant wall results in deformation of the pliant wall into an interior volume of the fluid reservoir, thereby inducing a flow of the extraction buffer through the sample receiving zone.

12. The device of claim 9, wherein the sample receiving zone is configured to receive and retain the biological sample and at least a portion of a sample collecting device.

13. The device of claim 9 wherein the pair of electrodes in the extraction zone are arranged such as to effect release of a plurality of nucleic acids from the biological sample and to effect fragmentation of the released nucleic acids.

14. A method for analysis of a biological sample comprising:

providing a device having an extraction zone that is fluidly coupled to an analysis zone, wherein the extraction zone comprises a pair of electrodes, and wherein the analysis zone comprises a sensing electrode and a reference electrode;

introducing at least a portion of a biological sample into the extraction zone;

applying a first charge or current to the pair of electrodes under a protocol effective to release a plurality of nucleic acids from the portion of the biological sample and to fragment the plurality of nucleic acids within the extraction zone to thereby produce a plurality of fragmented nucleic acids;

moving the plurality of fragmented nucleic acids to the analysis zone and applying a second charge or current flow to the sensing electrode and the reference electrode; and

measuring a third charge or current flow between the sensing electrode and the reference electrode to thereby quantify or ascertain presence of a nucleic acid in the biological sample.

15. The method of claim 14, wherein the plurality of respective fragments have an average length of equal or less than 200 bases.

16. The method of claim 14, wherein the sensing electrode is a nanostructured microelectrode, and wherein the nanostructure is spiky, rough or fractal.

17. The method of claim 14, further comprising a step of using a reporter system with the sensing electrode that is responsive to nucleic acid hybridization.

18. The method of claim 17, wherein the sensing electrode further comprises a probe molecule that is at least partially complementary to the fragmented nucleic acid.

19. The method of claim 14, further comprising a step of adjusting the first charge or current flow to thereby adjust an average length of the plurality of fragmented nucleic acids.

20. The method of claim 19, wherein the step of adjusting the first charge or current flow comprises adjusting at least one of a magnitude of a voltage applied to the pair of electrodes, a duration of a voltage pulse applied to the pair of electrodes, a frequency at which a voltage pulse is applied to the pair of electrodes and the total time of applied voltage.