# US 10,768,111 B2 · Fluorescent supramolecular biosensor and preparation method thereof

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- Number: US 10,768,111 B2
- Document ID: US10768111B2
- Status: Granted
- Kind: B2
- Application number: 15/988,616
- Filed: 2018-05-24
- Published: 2019-02-28
- Granted: 2020-09-08
- Earliest claimed: 2017-08-23
- Assignees: Industry-Academic Cooperation Foundation, Yonsei University
- Applicants: Industry-Academic Cooperation Foundation, Yonsei University
- Inventors: Yong-beom Lim; Woo-jin Jeong
- Web page: https://patentgrep.com/patent/US10768111B2

## CPC

- 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)}
- C07K 14/003: {Peptide-nucleic acids (PNAs)}
- C07K 19/00: Hybrid peptides {, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes}
- C12Q 1/04: Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor {(C12Q1/6897 takes precedence)}
- G01N 33/542: with steric inhibition or signal modification, e.g. fluorescent quenching
- G01N 33/56911: {Bacteria}
- C07K 1/13: Labelling of peptides
- C12Q 1/6834: Enzymatic or biochemical coupling of nucleic acids to a solid phase
- G01N 2021/6439: {with indicators, stains, dyes, tags, labels, marks}
- 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)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- 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

## Abstract

The present disclosure relates to a fluorescent supramolecular biosensor, more particularly to a fluorescent supramolecular biosensor which shows change in fluorescence signal through binding to a specific bacterium and a method for preparing the same. The fluorescent supramolecular biosensor is advantageous in that it exhibits remarkably superior affinity and selectivity for a target bacterium. In addition, because a secondary coiled coil stereostructure is highly stabilized as the supramolecular building block is self-assembled to form the fluorescent supramolecular biosensor, the fluorescent supramolecular biosensor can be stored for a long period of time without structural change even at high temperatures.

## Claims

### Claim 1

A fluorescent supramolecular biosensor for detecting a bacterium, comprising a plurality of supramolecular building blocks composed of:
(1) a first peptide having a coiled coil structure;
(2) an α-helical second peptide binding specifically to a specific bacterium; and
(3) a third peptide having a fluorophore bound, wherein the supramolecular building block is a lariat-type polypeptide wherein the first and second peptides are bound centered around a lysine residue to form a linear peptide, the linear peptide forms a cyclic structure as both ends of are bound through a cyclization process and the third peptide is bound to a nitrogen atom of a main chain amine group (α-amine group) of the lysine residue of the linear peptide.

### Claim 2 (depends on 1)

The fluorescent supramolecular biosensor according to claim 1, wherein the fluorescent supramolecular biosensor is a tetramer formed from the four supramolecular building blocks.

### Claim 3 (depends on 1)

The fluorescent supramolecular biosensor according to claim 1, wherein the supramolecular building block further comprises a linker connecting the first and second peptides.

### Claim 4 (depends on 1)

The fluorescent supramolecular biosensor according to claim 1, wherein the first peptide is represented by SEQ ID NO 1 or 2.

### Claim 5 (depends on 1)

The fluorescent supramolecular biosensor according to claim 1, wherein the second peptide is represented by SEQ ID NO 3 or 4.

### Claim 6 (depends on 1)

The fluorescent supramolecular biosensor according to claim 1, wherein the third peptide is represented by SEQ ID NO 5 or 6.

### Claim 7 (depends on 1)

The fluorescent supramolecular biosensor according to claim 1, wherein the supramolecular building block forms a tetramer through self-assembly when pH is 6.0-7.0 or ionic strength is 0.01-0.3 M.

### Claim 8 (depends on 1)

The fluorescent supramolecular biosensor according to claim 1, wherein the fluorescent supramolecular biosensor shows change in the fluorescence signal of the fluorophore as a folded structure formed in the supramolecular building block is changed through binding to a target bacterium.

### Claim 9 (depends on 1)

The fluorescent supramolecular biosensor according to claim 1, wherein the fluorescent supramolecular biosensor is a spherical nanoparticle with an average particle diameter of 5-20 nm.

### Claim 10 (depends on 1)

The fluorescent supramolecular biosensor according to claim 1, wherein the supramolecular building block is represented by Chemical Formula 1:
wherein R1 is DEABA (p-(N,N-diethylamino)benzoic acid) or Dapoxyl.

### Claim 11 (depends on 1)

The fluorescent supramolecular biosensor according to claim 1, wherein the fluorescent supramolecular biosensor maintains a molar ellipticity ([θ]222/[θ]208) of 1-0.8 at 20-90° C.

### Claim 12 (depends on 1)

The fluorescent supramolecular biosensor according to claim 1, wherein the fluorophore is one selected from a group consisting of DEABA (p-(N,N-diethylamino)benzoic acid), SNAFL, SNARF, SNAFL, Calcium Green, Amplex Red, Texas Red, BIODIPY, Oregon Green, Alexa Fluor, Cascade Blue, Dapoxyl, coumarin, rhodamine, N-methyl-4-hydrazino-7-nitrobenzofurazan, dansylethylenediamine, dansylcadaverine and dansylhydrazine.

### Claim 13 (depends on 1)

A method for detecting a bacterium using the fluorescent supramolecular biosensor according to claim 1, comprising:
irradiating light for fluorescence excitation; and
detecting fluorescence emission.