Devices and methods for determining the length of biopolymers and distances between probes bound thereto

• US 8,926,813 B2
• Filed: 08/06/2012
• Issued: 01/06/2015
• Est. Priority Date: 09/03/2008
• Status: Active Grant

• Current Assignee: NABsys, Inc.
• Original Assignee: NABsys, Inc.
• Inventors: Oliver, John S.
• Primary Examiner(s): Van, Luan
• Assistant Examiner(s): ROSENWALD, STEVEN ERIC
• Application Number:
  US13/567,595
• Publication Number:
  US 20130026038A1
• Time in Patent Office: 883 Days
• Field of Search: 204/549, 204/645, 204/452, 204/602, 204/450
• 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 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 by a trench in a substrate;

a pair of electromotive electrodes disposed at a first end and a second end of the fluidic channel; and

a plurality of sensing electrodes disposed at discrete laterally offset locations 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, each detector volume being defined by two laterally offset sensing electrodes,

wherein (i) said sensing electrodes are connected to a voltmeter for capturing said electrical signals corresponding to said detector volumes, with the sensing electrodes not being connected to a voltage source, (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, 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.

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

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

7. 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.

8. 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.

9. 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.

10. 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.

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

12. 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.

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

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

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

m.

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

m.

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

m to 10 cm.

18. 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.

19. 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.

29. A system for determining a length of an analyte, the system comprising the device of claim 1 and 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.

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

(a) the device of claim 1;

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

(c) 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.

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

disposing the analyte in a fluidic channel defined by a trench in a substrate;

applying a potential along the fluidic channel between a pair of electromotive electrodes disposed at a first end and a second end of 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 at discrete laterally offset locations along the length of the fluidic channel, each detector volume being defined by two sensing electrodes; and

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

wherein (i) said sensing electrodes are connected to a voltmeter for capturing said electrical signals corresponding to said detector volumes, with the sensing electrodes not being connected to a voltage source, (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.

21. The method of claim 20, wherein applying the potential along the fluidic channel generates an electrophoretic force therein.

22. The method of claim 20, wherein translocating the analyte comprises using a chemical gradient.

23. The method of claim 20, wherein translocating the analyte comprises using a pressure differential.

24. The method of claim 20, 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.

25. The method of claim 20, 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.

26. The method of claim 20, further comprising applying a correction factor to a measured length to determine an actual length of the analyte.

27. The method of claim 20, wherein the analyte comprises a biopolymer selected from the group consisting of deoxyribonucleic acids, ribonucleic acids, and polypeptides.

28. The method of claim 20, wherein the analyte is at least partially hybridized, and the detected electrical signals indicate the presence of a probe bound to the analyte.

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

(a) a fluidic channel defined by a trench in a substrate;

(b) a pair of electromotive electrodes disposed at a first end and a second end of the fluidic channel; and

(c) a plurality of sensing electrodes disposed at discrete laterally offset locations 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, each detector volume being defined by two laterally offset sensing electrodes,

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 connected to a voltmeter for capturing said electrical signals corresponding to said detector volumes as said biopolymer passes through the fluidic channel, with the sensing electrodes not being connected to a voltage source, wherein the fluidic channel is a nanochannel or a microchannel, and wherein relative positions of the sensing electrodes are known; and

(d) 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.

32. The system of claim 31, wherein the analyzing module is configured to perform one or more of the following steps

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 the plurality of sensing electrodes disposed at discrete laterally offset locations along the length of the fluidic channel;

and

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

wherein (i) said sensing electrodes are connected to a voltmeter for capturing said electrical signals corresponding to said detector volumes, with the sensing electrodes not being connected to a voltage source, (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.

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

(a) a fluidic channel defined by a trench in a substrate;

(b) a pair of electromotive electrodes disposed at a first end and a second end of the fluidic channel; and

(c) a plurality of sensing electrodes disposed at discrete laterally offset locations 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, each detector volume being defined by two laterally offset sensing electrodes,

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 are connected to a voltmeter for capturing said electrical signals corresponding to said detector volumes as said biopolymer passes through the fluidic channel, with the sensing electrodes not being connected to a voltage source, 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.

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

a fluidic channel defined by a trench 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 offset along a length of the channel and defining a detector volume therebetween; 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 and said sensing electrodes are connected to a voltmeter for capturing said sensed voltage corresponding to said detector volumes as said analyte passes through the fluidic channel, with the sensing electrodes not being connected to a voltage source.

35. The device of claim 34, wherein the substrate comprises a material selected from the group consisting of silicon, silicon dioxide, and fused silica.