# US 2014/0193830 A1 · Optical biosensors

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- Number: US 2014/0193830 A1
- Document ID: US20140193830A1
- Status: Published application
- Kind: A1
- Application number: 14/146,575
- Filed: 2014-01-02
- Published: 2014-07-10
- Earliest claimed: 2007-01-24
- Assignees: Carnegie Mellon University
- Inventors: Brigitte F. Schmidt; Christopher S. Szent-Gyorgyi; Alan S. Waggoner; Peter B. Berget; Marcel P. Bruchez; Jonathan W. Jarvik
- Web page: https://patentgrep.com/patent/US20140193830A1

## CPC

- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- A61K 49/0021: {the fluorescent group being a small organic molecule}
- A61K 49/0023: {Di-or triarylmethane dye (xanthene dyes A61K49/0041)}
- A61K 49/0032: {Methine dyes, e.g. cyanine dyes}
- A61K 49/0056: {Peptides, proteins, polyamino acids}
- A61K 49/0058: {Antibodies}
- C07K 16/44: against material not provided for elsewhere {, e.g. haptens, metals, DNA, RNA, amino acids}
- C09B 69/00: Dyes not provided for by a single group of this subclass
- G01N 33/52: Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper {and including single- and multilayer analytical elements (immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72)}
- G01N 33/5308: {for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites}
- C07K 2317/21: from primates, e.g. man
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2319/00: Fusion polypeptide
- 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)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C09B: ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES {, e.g. PIGMENTS}; MORDANTS; LAKES (fermentation or enzyme using processes to synthesise a desired chemical compound C12P)
- 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

Provided are biosensors, compositions comprising biosensors, and methods of using biosensors in living cells and organisms. The biosensors are able to be selectively targeted to certain regions or structures within a cell. The biosensors may provide a signal when the biosensor is targeted and/or in response to a property of the cell or organism such as membrane potential, ion concentration or enzyme activity.

## Claims

### Claim at position 1

1-36. (canceled)

### Claim 37

A ligand-dye complex, comprising a cognate ligand of a dye non covalently bound to the dye, wherein the cognate ligand comprises an antibody fragment comprising a polypeptide sequence having at least 90% sequence identity to the polypeptide of one of SEQ ID NO: 13, wherein the dye that binds the antibody fragment comprises one of a malachite green or an analog of malachite green, and wherein the bound dye and ligand exhibit an increase in detectable fluorescence signal at least ten times greater than the detectable fluorescence signal of the dye when not bound to the ligand.

### Claim 38 (depends on 37)

The ligand-dye complex of claim 37, wherein the ligand-dye complex further comprises the antibody fragment bound to a protein.

### Claim 39 (depends on 37)

The ligand-dye complex of claim 37, wherein the bound dye and ligand exhibit an extinction coefficient of greater than 30,000 and a maximum absorption at a wave length of greater than 350 nm.

### Claim 40 (depends on 37)

The ligand-dye complex of claim 37, wherein the bound dye emits photons having a changed polarization upon binding the ligand.

### Claim 41 (depends on 40)

The ligand-dye complex of claim 40 wherein the polarization change is greater than or equal to 20%.

### Claim 42 (depends on 37)

The ligand-dye complex of claim 37, wherein the dye of the bound dye and ligand exhibits an increased emission wave length of at least 10 nm.

### Claim 43 (depends on 37)

The ligand-dye complex of claim 37, wherein the ligand is fixed to a substrate.

### Claim 44 (depends on 43)

The ligand-dye complex of claim 43, wherein the substrate comprises molecules of formula X(a)-R(b)-Y(c), wherein R is a spacer, X is a functional group that binds R to a surface, Y is a functional group for binding to the ligand, (a) is an integer from 0 to about 4, (b) is an integer from 0 or 1, and (c) is an integer not equal to 0.

### Claim 45 (depends on 37)

The ligand-dye complex of claim 37, wherein the ligand-dye complex occupies a microenvironment and wherein the dye provides the increase in detectable fluorescence signal in response to pH, polarity, restriction, or mobility properties within the microenvironment.

### Claim 46

A method of forming a ligand-dye complex for use in detecting expression of a gene in a cell, the method comprising:
adding a vector to a cell, wherein the vector comprises a nucleic acid gene encoding an antibody fragment comprising a polypeptide sequence having at least 90% sequence identity to the polypeptide of SEQ ID NO: 13;
causing expression of the gene to produce a gene product comprising the antibody fragment;
adding dye to the cell to bind to the gene product, wherein the dye comprises one of a malachite green or an analog thereof.

### Claim 47 (depends on 46)

The method of claim 46, wherein the gene further encodes a second protein, and wherein the gene product comprises a fusion protein comprising the antibody fragment and the second protein.

### Claim 48 (depends on 47)

The method of claim 47, wherein the dye is substantially excluded from an interior of the cell, and wherein fluorescence is detected only when a portion of the second protein that is fused to the antibody fragment is exposed at a surface of the cell.

### Claim 49

A method of forming and using a ligand-dye complex for detecting interaction of two proteins in a cell comprising:
adding a vector to a cell, wherein the vector comprises a nucleic acid gene encoding a first protein fused to a first portion of a polypeptide sequence having at least 90% sequence identity to the polypeptide of SEQ ID NO: 13;
adding a vector to a cell, wherein the vector comprises a nucleic acid gene encoding a second protein fused to a second portion of the polypeptide sequence;
causing expression of the genes to produce fusion proteins comprised of the first and second proteins fused to first and second portions, respectively, of the polypeptide; and
adding dye to the cell, wherein the dye comprises one of malachite green or an analog thereof; and
detecting a fluorescence signal indicative of the dye binding to the fusion proteins, wherein the fluorescence signal is at least ten times greater than the detectable fluorescence signal of the dye when not bound to the fusion proteins.

### Claim 50 (depends on 49)

The method of claim 49, wherein the first polypeptide portion and the second polypeptide portion interact to form a molecule comprising a polypeptide sequence having at least 90%-sequence identity to the polypeptide of SEQ ID NO: 13, and wherein fluorescence is detectable when interaction between the first protein and the second protein brings the first polypeptide portion into proximity of the second polypeptide portion.