# US 10,871,467 B2 · Cannabinoid profiling using nanopore transduction

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- Number: US 10,871,467 B2
- Document ID: US10871467B2
- Status: Granted
- Kind: B2
- Application number: 16/219,182
- Filed: 2018-12-13
- Published: 2019-12-12
- Granted: 2020-12-22
- Earliest claimed: 2017-12-13
- Assignees: Cannaptic Biosciences, LLC
- Applicants: Cannaptic Biosciences, LLC
- Inventors: Robert Adelman; Stephen Winters-Hilt
- Web page: https://patentgrep.com/patent/US10871467B2

## CPC

- G01N 27/4473: {by electric means}
- G01N 27/44791: {Microapparatus (sample containers with integrated microfluidic structures B01L3/5027)}
- 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)}
- G01N 33/948: {Sedatives, e.g. cannabinoids, barbiturates (opiates G01N33/9486)}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Embodiments profile cannabinoids using a nanopore detector. Embodiments use an applied potential to electrophoretically draw a nanopore blockade reporter molecule into a nanopore channel to establish an electrophoretic molecular channel-capture for the reporter molecule. The reporter molecule includes one of an aptamer or a monoclonal antibody and has a specific binding to at least one of: a specific cannabinoid, a particular cannabinoid isoform; or a specific cannabinoid family. Embodiments receive a blockade channel current signal in response to a nanoscale membrane channel of the nanopore detector being partially blockaded by a presence of the reporter molecule non-covalently bonded to the membrane channel. Embodiments receive a blockade signal for the reporter molecule that is toggling between more than one level.

## Claims

### Claim 1

A method for profiling cannabinoids using a nanopore detector, the method comprising:
using an applied potential to electrophoretically draw a nanopore blockade reporter molecule into a nanopore channel to establish an electrophoretic molecular channel-capture for the reporter molecule;
wherein the reporter molecule comprises one of an aptamer or a monoclonal antibody and has a specific binding to at least one of: a specific cannabinoid, a particular cannabinoid isoform; or a specific cannabinoid family;
receiving a blockade channel current signal in response to a nanoscale membrane channel of the nanopore detector being partially blockaded by a presence of the reporter molecule non-covalently bonded to the membrane channel;
receiving a blockade signal for the reporter molecule that is toggling between more than one level;
using applied potential polarity-reversals to eject reporter blockades with channel blockade analysis to generate at least one of an open-channel reset process or a solution sampling process;
using the applied potential to establish a reporter capture state with an electrophoretic restoring force that is tuned to obtain blockade states having stationary statistics over observation periods;
using the nanopore blockade reporter molecule to transduce molecular state and track molecular state;
transducing binding and conformational states of the reporter molecule into electrical signals;
classifying the electrically signals automatically by use of a stochastic carrier wave signal method for identifying information in sequential data that has phases of stationary signal;
exposing the channel to a test solution during ionic current flow while extracting from the blockaded channel current signal a set of at least one pattern feature to establish over a period of time at least one of a blockaded channel current signal pattern and a change in the blockaded channel current signal pattern;
having a test condition comprising an introduction of test molecules to a conductive medium and having at least one test molecule specifically bound by the reporter molecule, and upon specific binding of at least one test molecule to the reporter molecule a toggling signal is altered from one pattern to another pattern, having at least one of different rates of toggle between blockade levels and different blockade levels; and
using the blockade signals to analyze at least one of the reporter molecule, the test molecule, or the molecular complex comprising the test molecule bound to a reporter molecule;
wherein the reporter molecule comprises a modulatory function separated from a sensing-moiety function by use of at least one of the following: a covalent linker between molecular functionalities; a nucleic-acid annealing based linker between these molecular functionalities; or a nucleic-acid annealing based complex that provides these molecular functionalities upon annealing; and the reporter molecule is a DNA hairpin modulator that is covalently linked to an aptamer-based sensing moiety.

### Claim 2 (depends on 1)

The method of claim 1, further comprising:
comparing the blockade channel current signal pattern to the blockade channel current signal pattern associated with at least one known condition and correlate the blockade channel current signal pattern with at least one known test condition.

### Claim 3 (depends on 2)

The method of claim 2 wherein the known condition comprises the presence of a known molecule.

### Claim 4 (depends on 3)

The method of claim 3 further comprising correlating the blockade channel current signal pattern with a characteristic of interaction between the test molecule and the reporter molecule.

### Claim 5 (depends on 1)

The method of claim 1, further comprising using at least one of chaotropes, viscosity agents, or injected laser modulations from a low-power laser beam directed at the channel with a beam chopper at a tunable frequency.

### Claim 6 (depends on 5)

The method of claim 5 wherein the viscosity agents comprise polyethylene glycol (PEG).

### Claim 7 (depends on 5)

The method of claim 5 wherein the laser modulations comprise a laser-tweezer tugging on a magnetic bead attached to the reporter molecule.

### Claim 8 (depends on 7)

The method of claim 7 wherein the magnetic bead is attached to the reporter molecule using a streptavidin-coated magnetic bead and a biotinylated variant of the reporter molecule that together provides a streptavidin-biotin linkage between modulator and binding functions.

### Claim 9 (depends on 1)

The method of claim 1 further comprising using at LNA/DNA chimeras to generate at least one of: longer transducer lifetime; choice of twist mode event transmission with the most discernible modulatory states; or choice of modulator states with the most discernibly different modulations with stationary signal statistics over long time periods.

### Claim 10 (depends on 1)

The method of claim 1, wherein a collection of reporter molecules with specific binding to different molecules is used to perform multiplex analysis of a molecular profile by differentiating the reporter molecules according to their different channel modulation signals.

### Claim 11 (depends on 1)

The method of claim 1, wherein a collection of reporter molecules with specific binding to different cannabinoids is used to perform multiplex analysis of the cannabinoid profile by differentiating the reporter molecules according to their different channel modulation signals.

### Claim 12 (depends on 1)

The method of claim 1, the classifying the electrically signals comprising:
identifying signal regions in stochastic sequential data using at least one of Hidden Markov model (HMM)-based methods or Finite State Automata (FSA)-based methods, and using the signal region identification in later stages of signal processing to focus more intense computational efforts on only the regions indicated;
identifying sub-states and features in the signal regions using at least one of an FSA; an HMM; an HMM with Emission Variance Amplification (EVA) filtering; an HMM with Duration (HMMD);
using identified signal sub-states and features to define a feature vector with a fixed number of parameters regardless of the duration and type of signal region examined so as to provide a standard feature vector extraction for use by Support Vector Machine (SVM) processing;
extracting feature vectors from the identified signal regions using at least one of an FSA, an HMM, a generalized clique HMM (meta-HMM), a gap-interpolated Markov model, a hash-interpolated Markov model, and an HMM-with-binned-duration model (HMMBD);
using generalized SVM-based methods for signal learning in at least one of three contexts: (1) classifying the extracted feature vectors using labeled training data; (2) clustering the extracted features in instances where there is no labeled training data to reference, using external SVM-based clustering methods; and (3) classifying some of the extracted features in instances where there is incomplete label information on the signal classes, using SVM-based projection-classification methods;
using the trained (from signal learning) generalized SVM-based method to accomplish at least one of two learning tasks: signal region classification and signal region clustering, to thereby establish known channel current signal patterns.

### Claim 13

A method for profiling cannabinoids using a nanopore detector, the method comprising:
using an applied potential to electrophoretically draw a nanopore blockade reporter molecule into a nanopore channel to establish an electrophoretic molecular channel-capture for the reporter molecule;
wherein the reporter molecule comprises one of an aptamer or a monoclonal antibody and has a specific binding to at least one of: a specific cannabinoid, a particular cannabinoid isoform; or a specific cannabinoid family;
receiving a blockade channel current signal in response to a nanoscale membrane channel of the nanopore detector being partially blockaded by a presence of the reporter molecule non-covalently bonded to the membrane channel;
receiving a blockade signal for the reporter molecule that is toggling between more than one level;
using applied potential polarity-reversals to eject reporter blockades with channel blockade analysis to generate at least one of an open-channel reset process or a solution sampling process;
using the applied potential to establish a reporter capture state with an electrophoretic restoring force that is tuned to obtain blockade states having stationary statistics over observation periods;
using the nanopore blockade reporter molecule to transduce molecular state and track molecular state;
transducing binding and conformational states of the reporter molecule into electrical signals;
classifying the electrically signals automatically by use of a stochastic carrier wave signal method for identifying information in sequential data that has phases of stationary signal;
exposing the channel to a test solution during ionic current flow while extracting from the blockaded channel current signal a set of at least one pattern feature to establish over a period of time at least one of a blockaded channel current signal pattern and a change in the blockaded channel current signal pattern;
having a test condition comprising an introduction of test molecules to a conductive medium and having at least one test molecule specifically bound by the reporter molecule, and upon specific binding of at least one test molecule to the reporter molecule a toggling signal is altered from one pattern to another pattern, having at least one of different rates of toggle between blockade levels and different blockade levels;
using the blockade signals to analyze at least one of the reporter molecule, the test molecule, or the molecular complex comprising the test molecule bound to a reporter molecule; and
using LNA/DNA chimeras to generate at least one of: longer transducer lifetime; choice of twist mode event transmission with the most discernible modulatory states; or choice of modulator states with the most discernibly different modulations with stationary signal statistics over long time periods.

### Claim 14 (depends on 13)

The method of claim 13, further comprising:
comparing the blockade channel current signal pattern to the blockade channel current signal pattern associated with at least one known condition and correlate the blockade channel current signal pattern with at least one known test condition.

### Claim 15 (depends on 14)

The method of claim 14 wherein the known condition comprises the presence of a known molecule, further comprising correlating the blockade channel current signal pattern with a characteristic of interaction between the test molecule and the reporter molecule.

### Claim 16 (depends on 13)

The method of claim 13, further comprising using at least one of chaotropes, viscosity agents, or injected laser modulations from a low-power laser beam directed at the channel with a beam chopper at a tunable frequency.

### Claim 17 (depends on 13)

The method of claim 13, wherein a collection of reporter molecules with specific binding to different cannabinoids is used to perform multiplex analysis of the cannabinoid profile by differentiating the reporter molecules according to their different channel modulation signals.

### Claim 18 (depends on 13)

The method of claim 13, the classifying the electrically signals comprising:
identifying signal regions in stochastic sequential data using at least one of Hidden Markov model (HMM)-based methods or Finite State Automata (FSA)-based methods, and using the signal region identification in later stages of signal processing to focus more intense computational efforts on only the regions indicated;
identifying sub-states and features in the signal regions using at least one of an FSA; an HMM; an HMM with Emission Variance Amplification (EVA) filtering; an HMM with Duration (HMMD);
using identified signal sub-states and features to define a feature vector with a fixed number of parameters regardless of the duration and type of signal region examined so as to provide a standard feature vector extraction for use by Support Vector Machine (SVM) processing;
extracting feature vectors from the identified signal regions using at least one of an FSA, an HMM, a generalized clique HMM (meta-HMM), a gap-interpolated Markov model, a hash-interpolated Markov model, and an HMM-with-binned-duration model (HMMBD);
using generalized SVM-based methods for signal learning in at least one of three contexts: (1) classifying the extracted feature vectors using labeled training data; (2) clustering the extracted features in instances where there is no labeled training data to reference, using external SVM-based clustering methods; and (3) classifying some of the extracted features in instances where there is incomplete label information on the signal classes, using SVM-based projection-classification methods;
using the trained (from signal learning) generalized SVM-based method to accomplish at least one of two learning tasks: signal region classification and signal region clustering, to thereby establish known channel current signal patterns.