Method and System for Characterizing or Identifying Molecules and Molecular Mixtures

• US 20130071837A1
• Filed: 08/12/2010
• Published: 03/21/2013
• Est. Priority Date: 10/06/2004
• Status: Abandoned Application

• Current Assignee: Robert L. Adelman, Stephen N. Winters-Hilt
• Original Assignee: Robert L. Adelman, Stephen N. Winters-Hilt
• Inventors: Winters-Hilt, Stephen N., Adelman, Robert L.
• Application Number:
  US12/855,635
• Publication Number:
  US 20130071837A1
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2521/345 DNAzyme
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 27/26 by investigating electrochemical variables; by using electrolysis or electrophoresis
• 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

Abstract

A system and method for identifying a material passing through a nanopore filter wherein an electrical signal is detected as a result of the passage and that signal is processed in real-time using mathematical and statistical tools to identify the molecule. A carrier molecule is preferably attached to one or more molecule(s) under consideration using a non-covalent bond and the pore in the nanopore filter is sized so that the molecule rattles around in the pore before being discharged without passing through the filter pore. The present invention includes not only a method and system for identifying the molecule(s) under consideration but also a kit for setting up the filter as well as mathematical tools for analyzing the signals from the sensing circuitry for the molecule(s) under consideration.

Claims

1. A device for identifying at least one molecule, the device comprising two chambers of buffer separated by a membrane over an aperture having at least one nanometer-scale nanopore channel in the membrane, with an applied potential applied between the two chambers, a single blockade molecule that enters the nanopore channel but does not pass immediately therethrough, remaining in the nanopore channel for a period of time and modulating the nanopore channel, a sensor generating electrical signals associated with the blockading molecule and at least one processor using an algorithm for analyzing the electrical signal to characterize the blockade molecule.

2. The device according to claim 1, wherein the membrane includes a plurality of nanopore-scale nanopore channels.

3. The device according to claim 1 further including a system to externally excite the nanopore-scale nanopore channel.

4. The device according to claim 1 further including a sensor for identifying a binding event in the blockade molecule.

5. The device according to claim 2 further including a selector to read one nanopore channel at a selected time.

6. The device, according to claim 1 further including signal processing calibration protocols, data structures, and data schemas for reference molecules.

7. A method for analysis of at least one molecule comprising the steps of:

Positioning a membrane with at least one nanopore channel opening adjacent a solution containing a molecule to be analyzed, with size of transducer molecule and channel chosen such that channel inner-diameter and blockading-molecular width are comparable, such that the molecule to be analyzed has some portion interacting within the channel for an extended period;

Establishing an ionic current flow through that nanopore channel;

Capturing from the solution, within the nanopore channel, at least one molecular portion to be identified;

Introducing at least one bifunctional transduction molecule into the solution, said transduction molecule having one end which can be captured in the channel and modulate the channel current while rattling around in the channel for an extended period of time, while the other, extra-channel-exposed end has information for event detection.

Using electrophoresis to draw at least one bifunctional transducer molecule into the nanopore channel to modulate the ionic current flow through the nanopore channel;

Generating an electrical signal of the ionic current flow based on the state of the transducer molecule captured by the nanopore channel;

Analyzing the electrical signal using computational methods and pattern recognition to characterize the molecule; and

Releasing the captured molecule and resetting the nanopore channel for capture of another molecule.

8. The method according to claim 7 wherein the method is repeated to identify different types of molecules in the solution to determine a relationship between the different types of molecules.

9. The method according to claim 7 further including introducing a biosensing sensitivity gain into the system using a molecular-capture matrix comprising at least one of an antibody-capture matrix, an aptamer-capture matrix, and a molecularly-imprinted polymer capture matrix.

10. The method according to claim 7 further including introducing a biosensing sensitivity gain into the system using an enzyme acting on a substrate.

11. The method according to claim 7 further including introducing a biosensing sensitivity gain using an enzyme turn-over rate and real-time signal tracking.

12. The method, according to claim 7, where the membrane includes multiple channels and the method includes processing signals from the multiple channels.

13. The method according to claim 7 further including producing standard biochemistry sample-analysis gel-analogs from observations with buffer-shift population measurements.

14. The method according to claim 7 further including using orientation selection for direct antibody utilization as transducer and binding moiety.

15. The method according to claim 7 further including establishing a chemical computation device with parallelized, ‘

chemical’

computation loaded with choice of buffer and changes in that buffer, and sampling the output for CCC analyte recognition and SSA program/data processing.

16. The method according to claim 7 further including the step of introducing Y-shaped nucleic acid molecules into the solution for direct, annealed to modulator, reporting on SNPs and single-point mutations.

17. The method, according to claim 7 further including the step of transducing a DNA enzyme signal by channel current observation involving at least one of direct observation of enzyme-channel interactions and indirect transduction of enzyme state when linked to a channel modulator to establish a DNA sequencing capability.

18. The method according to claim 7, further including using nanopore transduction detection for direct channel-interaction nanopore detector-to-target assays and in combination with indirect channel-interaction NTD-to-target assays via transducer molecule intervening between channel and target.

19. The method, according to claim 7 further including performing active multichannel signal processing with HMMD heavy-tail encoding modulation.

20. A method of identifying a molecule by analyzing electrical signals from a nanopore transducer blockade molecule that is producing stochastic sequential data by using training data, the method comprising the steps of:

Identifying signal regions in the stochastic sequential data using at least one of HMM-based methods and FSA-based methods;

Extracting feature vectors from the identified signal regions using at least one of a generalized clique HMM analysis, gap-interpolated and hash-interpolated Markov models, and HMM-with-binned-duration models;

Classifying the extracted feature vectors using training data and at least one of SVM-based methods and HMM-based methods to identify the molecule; and

Clustering the extracted features in instances where there is no training data to reference, using at least one of SVM-based-methods, and clustering methods including kernel k-means.

21. The method according to claim 20 further including using a holistic signal-acquisition approach for extracting features.

22. The method according to claim 20 further including the steps of coding an adaptive self-tuning explicit hidden Markov model with Duration process is coded on a data processing apparatus and accomplishing HMMD computations like the standard HMM computations.

23. The method according to claim 20 further including the step of using at least one of an HMM with pMM/SVM sensors, an HMM with Martingale/SVM sensors, an HMMBD with pMM/SVM sensors, and an HMMBD with Martingale/SVM sensors.

24. The method according to claim 20 further including the step of using at least one of an HMM with EVA, an HMM with Emission Inversion, an HMMBD with EVA, and an HMMBD with Emission Inversion.

25. The method according to claim 20 further including the step of using a meta-HMM with a footprint sufficient to strengthen contrast resolution at the start of self-transition regions and heavy-tail resolution at the end of self-transition regions.

26. The method according to claim 20 further including the step of using HMMD extensions to capture side-information.

27. The method according to claim 20 further including the step of using multi-track HMM emissions.

28. The method according to claim 20 further including the step of performing distributed HMM processing in single-pass table-processing, via segment-join tests.

29. The method according to claim 20 further including the step of using HMMD modeling on data exhibiting non-geometric length profiles.

30. The method according to claim 20 further including the step of performing HMMD-based stochastic carrier wave communications.

31. The method according to claim 20 further including the step of choosing SVM kernels complimentary to feature vector attributes, including feature vectors comprising probability vectors and including Martingale vectors.

32. The method according to claim 20 further including the step of using SVM clustering with at least two convergence results prior to re-label/re-train operations using the convergence results.

33. The method according to claim 20 further including the step of using SVM clustering with multiclass SVM using at least one of label flipping, tuning, and multiple convergences.

34. The method according to claim 20 further including the step of using at least one of data structures, related data schemas, and databases to implement at least some of the tasks including data acquisition, feature extraction, selection, calibration, classification and classification methods using the SSA methods and protocols.

35. The method according to claim 20 further including the step of using the SSA Protocol on a data processing apparatus for improving real-time signal processing.

36. The method according to claim 20 further including the step of using an SSA Protocol and Algorithms'"'"' signal processing process.