# US 8,771,491 B2 · Ultrafast sequencing of biological polymers using a labeled nanopore

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- Number: US 8,771,491 B2
- Document ID: US8771491B2
- Status: Granted
- Kind: B2
- Application number: 13/426,515
- Filed: 2012-03-21
- Published: 2012-10-18
- Granted: 2014-07-08
- Earliest claimed: 2009-09-30
- Assignees: Quantapore, Inc.
- Applicants: Martin Huber
- Inventors: Martin Huber
- Web page: https://patentgrep.com/patent/US8771491B2

## CPC

- C12Q 1/6869: Methods for sequencing
- G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)}
- 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 2021/6432: {Quenching}
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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

Methods and systems for sequencing a biological molecule or polymer, e.g., a nucleic acid, are provided. One or more donor labels, which are attached to a pore or nanopore, may be illuminated or otherwise excited. A polymer having a monomer labeled with one or more acceptor labels, may be translocated through the pore. Either before, after or while the labeled monomer of the polymer passes through, exits or enters the pore, energy may be transferred from the excited donor label to the acceptor label of the monomer. As a result of the energy transfer, the acceptor label emits energy, and the emitted energy is detected in order to identify the labeled monomer of the translocated polymer and to thereby sequence the polymer.

## Claims

### Claim 1

A method for sequencing a polymer comprising:
exciting a donor label attached to a nanopore wherein the donor label emits energy at a first wavelength;
translocating a polymer through the nanopore, wherein the polymer comprises a monomer labeled with an acceptor label;
transferring energy from the excited donor label to the acceptor label of the monomer after the labeled monomer passes through and exits the nanopore, wherein the energy transfer causes the acceptor label to emit energy at a second wavelength which is different from the first wavelength;
detecting the energy emitted by the acceptor label as a result of the energy transfer, wherein the energy emitted by the acceptor label is associated with a single monomer; and
deducing the polymer sequence based on the detection of the emitted energy from the acceptor label and identification of the monomer.

### Claim 2 (depends on 1)

The method of claim 1, wherein the labeled polymer is threaded through the nanopore by applying an electric field through the nanopore.

### Claim 3 (depends on 1)

The method of claim 1, wherein the donor label is a Quantum dot.

### Claim 4 (depends on 1)

The method of claim 1, wherein the acceptor label is a fluorophore.

### Claim 5 (depends on 1)

The method of claim 1, wherein a plurality of donor labels exhibiting different spectral emission maxima are attached to the nanopore and a plurality of acceptor labels having distinct absorption/emission spectra each associated with a specific monomer are attached to the polymer, and wherein each donor label and corresponding acceptor label undergo a FRET (Förster Resonance Energy Transfer) interaction such that each acceptor labeled monomer of the polymer emits a specific wavelength that is detected and used to sequence the polymer.

### Claim 6 (depends on 1)

The method of claim 1, wherein detecting the energy emitted by the acceptor label comprises optical detection of a specific wavelength emitted by the acceptor label and associated with a specific nucleotide.

### Claim 7 (depends on 1)

The method of claim 1, wherein the polymer is a nucleic acid and the monomer is a nucleotide.

### Claim 8 (depends on 7)

The method of claim 7, wherein one of four nucleotide bases is labeled and the time dependent signal arising from a nucleotide label emission is converted into a nucleic acid sequence corresponding to positions of the labeled nucleotide in the nucleic acid sequence.

### Claim 9 (depends on 8)

The method of claim 8, wherein the method is repeated for each of the four nucleotides in separate samples and four partial sequences are deduced and then aligned to assemble an entire nucleic acid sequence.

### Claim 10

A system for sequencing a polymer comprising:
a nanopore membrane assembly having a donor labeled nanopore which spans a lipid bilayer membrane, the nanopore membrane assembly having a negatively charged side and a positively charged side;
an aqueous buffer solution surrounding the nanopore membrane assembly;
a negative electrode positioned on the negatively charged (cis) side of the nanopore membrane assembly;
a positive electrode positioned on the positively charged (trans) side of the nanopore membrane assembly;
a polymer comprising a monomer having an acceptor label, wherein the polymer is loaded into a port positioned on the negatively charged side of the nanopore membrane assembly;
an energy source for exciting the nanopore donor label, wherein the nanopore donor label is configured to emit energy at a first wavelength;
an electric field applied by the electrodes to force the labeled polymer to translocate through the nanopore from the negatively charged side to the positively charged side in a single file, wherein energy is transferred from the nanopore donor label to the monomer acceptor label after the labeled monomer passes through and exits the nanopore and comes in proximity to the nanopore donor label, wherein the energy transfer causes the monomer acceptor label to emit energy at a second wavelength which is different from the first wavelength;
an optical detector for detecting the energy emitted from the monomer acceptor label as a result of the transfer of energy from the nanopore donor label to the monomer acceptor label, wherein the emitted energy is associated with a single monomer and facilitates the sequencing of the polymer.

### Claim 11 (depends on 10)

The system of claim 10, wherein the donor label comprises one or more quantum dots and the acceptor label comprises one or more fluorophores.

### Claim 12 (depends on 10)

The system of claim 10, wherein a plurality of nanopores are incorporated into the membrane in parallel, such that a plurality of polymers may be sequenced simultaneously.

### Claim 13 (depends on 10)

The system of claim 10, wherein a plurality of nanopores are incorporated into a rotatable disc, such that the nanopores may be rotatably interchanged.

### Claim 14 (depends on 10)

The method of claim 10, wherein the polymer is a nucleic acid and the monomer is a nucleotide.