# US 11,441,182 B2 · Multiplexed and recyclable single-molecule sensors for quantitative analysis of nucleic-acid biomarkers

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- Number: US 11,441,182 B2
- Document ID: US11441182B2
- Status: Granted
- Kind: B2
- Application number: 16/535,722
- Filed: 2019-08-08
- Published: 2020-02-13
- Granted: 2022-09-13
- Earliest claimed: 2018-08-08
- Assignees: Virginia Commonwealth University
- Applicants: Virginia Commonwealth University
- Inventors: Soma Dhakal; Anisa Kaur
- Web page: https://patentgrep.com/patent/US11441182B2

## CPC

- C12Q 1/6816: characterised by the detection means (C12Q1/6804 takes precedence)
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- 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)}
- 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

Disclosed herein is a simple, sensitive, and fully-recyclable fluorescence resonance energy transfer (FRET)-based multiplex detection platform that overcomes current requirements of complex labeling schemes and complicated data analysis algorithms for employing single-molecule FRET (smFRET) microscopy in multiplexing. While conventional smFRET detection techniques allow for the analysis of one target at a time, the disclosed approach utilizes the gaps between high- and low-FRET signals to provide simultaneous detection and quantification of multiple nucleic acid targets.

## Claims

### Claim 1

A hairpin-based sensor, comprising
a first single-stranded DNA oligonucleotide (“first oligo”) having a 5′ end and a 3′ end and comprising a DNA hairpin region flanked by a 5′ flanking region and a 3′ flanking region;
a second single-stranded DNA oligonucleotide (“second oligo”) having a 5′ end and a 3′ end and comprising a nucleic acid sequence complementary to at least a portion of the 3′ flanking region;
a third single-stranded DNA oligonucleotide (“third oligo”) having a 5′ end and a 3′ end and comprising a nucleic acid sequence complementary to at least a portion of the 5′ flanking region;
a fourth single-stranded DNA oligonucleotide (“probe”) having a 5′ end and a 3′ end, comprising a nucleic acid sequence complementary to at least a portion of the DNA hairpin region, and comprising a nucleic acid sequence complementary to at least a portion of a target single stranded DNA or RNA molecule;
a first fluorescent molecule conjugated to the second oligo; and a second fluorescent molecule conjugated to the third oligo;
wherein the first fluorescent molecule and the second fluorescent molecule together form a fluorescence resonance energy transfer (FRET) pair,
wherein the FRET pair emit at a first-FRET efficiency when the probe is binding the DNA hairpin region, and
wherein the FRET pair emit at a higher second-FRET efficiency when the probe binds the target DNA or RNA molecule, displacing the probe from the DNA hairpin region, allowing the DNA hairpin region to form a DNA hairpin, and shortening the distance between the first fluorescent molecule and the second fluorescent molecule.

### Claim 2 (depends on 1)

The hairpin-based sensor of claim 1, wherein the first fluorescent molecule is conjugated to the 3′ end of the second oligo.

### Claim 3 (depends on 1)

The hairpin-based sensor of claim 1, wherein the second fluorescent molecule is conjugated to the 5′ end of the third oligo.

### Claim 4 (depends on 1)

The hairpin-based sensor of claim 1, wherein the FRET pair comprise Cy3 and Cy5.

### Claim 5 (depends on 1)

The hairpin-based sensor of claim 1, further comprising a fifth single-stranded DNA oligonucleotide (“fifth oligo”) having a 5′ end and a 3′ end and comprising a nucleic acid sequence complementary to at least a portion of the second oligo, wherein the 3′ flanking region of the first oligo and the fifth oligo together form a complementary strand for the second oligo.

### Claim 6 (depends on 1)

The hairpin-based sensor of claim 1, further comprising a sixth single-stranded DNA oligonucleotide (“sixth oligo”) having a 5′ end and a 3′ end and comprising a nucleic acid sequence complementary to at least a portion of the third oligo, wherein the 5′ flanking region of the first oligo and the sixth oligo together form a complementary strand for the third oligo.

### Claim 7 (depends on 1)

The hairpin-based sensor of claim 1, further comprising a seventh single-stranded DNA oligonucleotide (“seventh oligo”) having a 5′ end and a 3′ end and an eighth single-stranded DNA oligonucleotide (“eighth oligo”) having a 5′ end and a 3′ end, wherein the seventh oligo comprises a nucleic acid sequence complementary to at least a portion of either the fifth or the sixth oligo and a nucleic acid sequence complementary to at least a portion of the eight oligo.

### Claim 8 (depends on 1)

The hairpin-based sensor of claim 1, wherein the eight oligo is conjugated to a biotin molecule.

### Claim 9 (depends on 1)

A multiplexed detection system, comprising a plurality of unique hairpin-based sensors according to claim 1, wherein each of the unique hairpin-based sensors has a unique probe designed to recognize a different DNA or RNA target molecule, wherein each of the unique hairpin-based sensors has a unique spacing between the first fluorescent molecule and the second fluorescent molecule when the DNA hairpin is formed, and wherein each of the unique hairpin-based sensors have the same FRET pair but emit a unique second-FRET efficiency.

### Claim 10 (depends on 9)

The system of claim 9, wherein the hairpin-based sensors are attached to a microscope slide.

### Claim 11 (depends on 9)

A method for detecting a DNA or RNA target molecule in a sample, comprising contacting the system of claim 9 with the sample under conditions suitable for DNA binding, exciting the FRET pair with a light source, and measuring FRET efficiency.