DEVICES AND METHODS FOR DETERMINING THE LENGTH OF BIOPOLYMERS AND DISTANCES BETWEEN PROBES BOUND THERETO

• US 20100078325A1
• Filed: 09/03/2009
• Published: 04/01/2010
• Est. Priority Date: 09/03/2008
• Status: Active Grant

• Current Assignee: NABsys, Inc.
• Original Assignee: NABsys, Inc.
• Inventors: Oliver, John S.
• Granted Patent: US 8,262,879 B2
• Time in Patent Office: Days
• US Class Current: 204/452
• CPC Class Codes: G01N 27/3278 involving nanosized elements, e.g. nanogaps or nanoparticles nanopores G01N33/48721; magnetic beads G01N27/745
• CPC Class Codes: G01N 27/4473 by electric means
• CPC Class Codes: G01N 33/48721 Investigating individual macromolecules, e.g. by translocation through nanopores Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68
• CPC Class Codes: Y10S 977/957 Of chemical property or presence

Abstract

Devices and methods for detecting the length of analytes and/or sequencing analytes are provided in which two or more electrical signals are obtained as an analyte traverses a fluidic channel. Detection of the relative position of probes hybridized to a biopolymer and/or the length of the analyte (e.g., a biopolymer) does not rely on the absolute time between detection events of a given electrical signal to determine a distance associated with the biopolymer. Instead, multiple signals are obtained (e.g., as functions of time) corresponding to a plurality of detector volumes at known locations along a fluidic channel through which the biopolymer passes, and the distances are determined from the multiple signals.

Claims

1. A device for determining a length of an analyte by detecting electrical signals, the device comprising:

a fluidic channel defined in a substrate; and

a plurality of sensing electrodes disposed along a length of the fluidic channel for detection of two or more electrical signals corresponding to two or more detector volumes disposed along the fluidic channel,

wherein (i) said sensing electrodes are configured for connection to a measurement tool for capturing said electrical signals corresponding to said detector volumes, (ii) the fluidic channel is a nanochannel or a microchannel, (iii) relative positions of the sensing electrodes are known, and (iv) the captured electrical signals in conjunction with the relative positions of the sensing electrodes indicate the length of the analyte.

2. The device of claim 1, further comprising:

a data collection device configured for recording electrical signals captured by the measurement tool as a function of time; and

a computer in electrical communication with the data collection device, the computer programmed to determine which detector volumes record a change in electrical signal at the same time.

3. The device of claim 1, further comprising an electronic circuit configured to output a signal only when two electrical signals corresponding to two detector volumes change at the same time.

4. The device of claim 1, further comprising a pair of electromotive electrodes disposed at a first end and a second end of the fluidic channel.

5. The device of claim 4, wherein the pair of electromotive electrodes disposed at the first and second ends of the channel comprises macroscopic electrodes arranged to generate a constant, changing, or oscillating electrophoretic force in the fluidic channel for translocation of the analyte disposed therein.

6. The device of claim 1, wherein the device is configured such that positive pressure drives the analyte through the fluidic channel.

7. The device of claim 1, wherein the device is configured such that a chemical gradient drives the analyte through the fluidic channel.

8. The device of claim 1, wherein the substrate comprises a material selected from the group consisting of silicon, silicon dioxide, fused silica, and gallium arsenide.

9. The device of claim 1, wherein at least one of the sensing electrodes comprises a material selected from the group consisting of platinum, gold, chrome, titanium, silver chloride, silver, and graphene.

10. The device of claim 1, wherein a sensing element corresponding to a given detector volume comprises two sensing electrodes disposed on opposing sides of the fluidic channel.

11. The device of claim 1, wherein a sensing element corresponding to a given detector volume comprises two sensing electrodes disposed on a first side of the fluidic channel.

12. The device of claim 1, wherein a sensing element corresponding to a given detector volume comprises two sensing electrodes transversing the fluidic channel.

13. The device of claim 1, wherein a sensing element corresponding to a given detector volume comprises a first sensing electrode transversing the fluidic channel, and a second sensing electrode on a side of the fluidic channel.

14. The device of claim 1 wherein the measurement tool comprises at least one of a voltmeter, an ammeter, or a field-effect transistor.

15. The device of claim 1, further comprising a plurality of fluidic channels defined in the substrate.

16. The device of claim 1, further comprising a voltage amplifier configured to amplify the two or more electrical signals.

17. The device of claim 1, wherein the fluidic channel has a width selected from a range of 1 nm to 5 μ

m.

18. The device of claim 1, wherein the fluidic channel has a depth selected from a range of 1 nm to 5 μ

m.

19. The device of claim 1, wherein the fluidic channel has a length selected from a range of 1 μ

m to 10 cm.

20. The device of claim 1, wherein (i) the device comprises at least three pairs of sensing electrodes, and (ii) a distance between detector volumes corresponding to a first and second pair of sensing electrodes is unequal to a distance between detector volumes corresponding to the second and third pair of electrodes.

21. The device of claim 1, wherein (i) the device comprises at least three pairs of sensing electrodes, (ii) a detector volume corresponding to a first pair of sensing electrodes is unequal to a detector volume corresponding to a second pair of sensing electrodes, and (iii) a detector volume corresponding to the second pair of sensing electrodes is unequal to a detector volume corresponding to a third pair of sensing electrodes.

52. A system for determining a length of an analyte, the system comprising an analyzing module that determines the length of the analyte based at least in part on a plurality of electrical signals captured by the device of claim 1.

53. An apparatus for determining a length of an analyte, the apparatus comprising:

(a) a memory that stores code defining a set of instructions; and

(b) a processor that executes the instructions thereby to determine the length of the analyte from two or more detected electrical signals captured by the device of claim 1.

22. (canceled)

23. A method for determining a length of an analyte, the method comprising the steps of:

disposing the analyte in a fluidic channel;

applying a potential along the fluidic channel;

translocating the analyte from a first end of the fluidic channel to a second end of the fluidic channel;

detecting two or more electrical signals as the analyte moves through the fluidic channel, said two or more electrical signals corresponding to two or more detector volumes of the fluidic channel, said two or more electrical signals being detected using a plurality of sensing electrodes disposed along the length of the fluidic channel; and

determining the length of the analyte by analyzing the two or more detected electrical signals,

wherein the fluidic channel is a nanochannel or a microchannel.

24. The method of claim 23, wherein applying the potential along the fluidic channel generates an electrophoretic force therein.

25. The method of claim 23, wherein translocating the analyte comprises using a chemical gradient.

26. The method of claim 23, wherein translocating the analyte comprises using a pressure differential.

27. The method of claim 23, wherein determining the length of the analyte comprises identifying at least two detector volumes in which the analyte is sensed at a given time, and determining a distance between sensing electrodes corresponding to said at least two detector volumes.

28. The method of claim 23, wherein an amount that the analyte partially fills the detector volume is determined by comparing the electrical signal caused by the analyte to a maximum signal caused by a sample biopolymer long enough to fill the detector volume entirely.

29. The method of claim 23, further comprising applying a correction factor to a measured length to determine an actual length of the analyte.

30. The method of claim 23, wherein the analyte comprises a biopolymer selected from the group consisting of deoxyribonucleic acids, ribonucleic acids, and polypeptides.

31. The method of claim 30, wherein the biopolymer is a single-stranded molecule.

32. The method of claim 23, wherein the analyte is at least partially hybridized, and the detected electrical signals indicate the presence of a probe bound to the analyte.

33. A method for sequencing a biopolymer, the method comprising:

preparing an analyte by hybridizing a first plurality of probes with the biopolymer such that said first plurality of probes attaches to portions of the biomolecule to produce a partially hybridized biomolecule;

disposing the analyte in a fluidic channel;

applying a potential along the fluidic channel;

translocating the analyte from a first end of the fluidic channel to a second end of the fluidic channel;

detecting two or more electrical signals as the analyte moves through the fluidic channel, said two or more electrical signals corresponding to two or more detector volumes of the fluidic channel, said two or more electrical signals being detected by using a plurality of sensing electrodes disposed along the length of the fluidic channel, the detected electrical signals indicating locations of the hybridized probes along the biopolymer;

analyzing the electrical signals to determine in which detector volumes probes bound to the biomolecule are located;

determining at least a portion of the sequence of the biopolymer using a distance between sensing electrodes corresponding to said detector volumes in which the probes are located, wherein the fluidic channel is a nanochannel or a microchannel.

34. The method of claim 33 wherein applying the potential along the fluidic channel generates an electrophoretic force therein.

35. The method of claim 33, wherein translocating the analyte comprises using a chemical gradient.

36. The method of claim 33, wherein translocating the analyte comprises using a pressure differential.

37. The method of claim 33, further comprising determining a distance between probes using a coincident response of electrical signals corresponding to two or more detector volumes.

38. The method of claim 37, wherein a spacing between sensing electrodes is used to determine a maximum distance between probes.

39. The method of claim 37, wherein a spacing between sensing electrodes is used to determine a minimum distance between probes.

40. The method of claim 33, wherein the electrical signal initially changes when the biopolymer moves through a detector volume associated with two sensing electrodes and further changes when a portion of the biopolymer comprising a hybridized probe moves through the detector volume.

42. The method of claim 33, further comprising hybridizing a second plurality of probes with the biopolymer and repeating the detecting, analyzing, and determining steps with said second plurality of probes.

43. The method of claim 42, further comprising using the two or more electrical signals to detect and record complexed and uncomplexed regions of the biopolymer to create a first probe map of the first plurality of probes and a second probe map of the second plurality of probes, the first probe map and the second probe map respectively comprising information about the relative positions of the hybridized first and second plurality of probes.

44. The method of claim 43, further comprising determining a candidate sequence by ordering at least two probe sequences using at least one of positional information or a combination of overlapping probe binding sequences and positional information.

41. (canceled)

45. (canceled)

46. The method of claim 45, further comprising determining a candidate sequence by ordering at least two probe sequences using at least one of (i) positional information and parameters relating to the error in positional information or (ii) a combination of overlapping sequences of the probe molecules and positional information and the error in positional information.

47. (canceled)

48. The method of claim 47, wherein preparing the analyte comprises contacting the biopolymer with a first probe having a first probe specificity for recognition sites of the biopolymer to form a first plurality of local ternary complexes, the first probe having a first known recognition site sequence.

49. The method of claim 48, further comprising using the electrical signal to determine positional information of the first plurality of local ternary complexes.

50. The method of claim 48, wherein preparing the analyte further comprises contacting the biopolymer with a second probe having a second probe specificity for recognition sites of the biopolymer to form a second plurality of local ternary complexes, the second probe having a second known recognition site sequence.

51. The method of claim 50, further comprising aligning positional information of at least the first and second plurality of local ternary complexes to determine a sequence of the biopolymer.

54. A system for sequencing a biopolymer, the system comprising:

(a) a fluidic channel defined in a substrate;

(b) a plurality of sensing electrodes disposed along a length of the fluidic channel for detection of two or more electrical signals corresponding to two or more detector volumes disposed along the fluidic channel,

wherein the fluidic channel is configured such that a biopolymer with at least a first plurality of probes attached thereto may pass therethrough, wherein said sensing electrodes are configured for connection to a measurement tool for capturing said electrical signals corresponding to said detector volumes as said biopolymer passes through the fluidic channel, wherein the fluidic channel is a nanochannel or a microchannel, and wherein relative positions of the sensing electrodes are known; and

(c) an analyzing module that determines at least a portion of the sequence of the biopolymer based at least in part on a plurality of the captured electrical signals.

55. (canceled)

56. An apparatus for sequencing a biopolymer, the apparatus comprising:

(a) a fluidic channel defined in a substrate; and

(b) a plurality of sensing electrodes disposed along a length of the fluidic channel for detection of two or more electrical signals corresponding to two or more detector volumes disposed along the fluidic channel,

wherein the fluidic channel is configured such that a biopolymer with at least a first plurality of probes attached thereto may pass therethrough, wherein said sensing electrodes are configured for connection to a measurement tool for capturing said electrical signals corresponding to said detector volumes as said biopolymer passes through the fluidic channel, wherein the fluidic channel is a nanochannel or a microchannel, wherein relative positions of the sensing electrodes are known, and wherein the captured electrical signals in conjunction with the relative positions of the sensing electrodes indicate at least a portion of the sequence of the biopolymer.

57. A device for voltage sensing of analytes, the device comprising:

a fluidic channel defined in a substrate;

a first and a second pair of sensing electrodes disposed in the channel for sensing voltage therein, the second pair of sensing electrodes being disposed distal to the first pair of sensing electrodes, and each pair of sensing electrodes comprising a first and a second electrode disposed at two discrete locations along a length of the channel; and

a pair of electromotive electrodes disposed at a first end and a second end of the channel for applying a potential along the channel,

wherein the channel comprises a nanochannel or a microchannel.

58. The device of claim 57, wherein the substrate comprises a material selected from the group consisting of silicon, silicon dioxide, and fused silica.