# US 8,465,989 B2 · Method and system for detecting a target within a population of molecules

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- Number: US 8,465,989 B2
- Document ID: US8465989B2
- Status: Granted
- Kind: B2
- Application number: 12/312,071
- Filed: 2007-10-25
- Published: 2010-02-18
- Granted: 2013-06-18
- Earliest claimed: 2006-10-27
- Assignees: Ramot at Tel-Aviv University Ltd.
- Applicants: Ady Arie; Amos Danielli
- Inventors: Ady Arie; Amos Danielli
- Web page: https://patentgrep.com/patent/US8465989B2

## CPC

- G01N 33/54333: {Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction}
- C12Q 1/6818: involving interaction of two or more labels, e.g. resonant energy transfer
- G01N 21/1717: {with a modulation of one or more physical properties of the sample during the optical investigation, e.g. electro-reflectance}
- G01N 21/64: Fluorescence; Phosphorescence
- 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 33/54326: {Magnetic particles}
- G01N 33/54353: {with ligand attached to the carrier via a chemical coupling agent (coatings G01N33/54393)}
- G01N 33/582: {with fluorescent label}
- 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)
- 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

A method (800) of detecting a target within a population of molecules comprising: contacting a plurality of labeled probe molecules with the population of molecules potentially containing a target of the probe molecules (810); acquiring a probe specific signal emitted by said labeled probe molecules that bound to said target together with a background signal (820); modulating said probe specific signal by at least one of modulating said acquisition and modulating an emission of said probe specific signal (830); and detecting said probe specific signal over said background signal using said preferential modulation (840). The target comprises one or more molecule type selected from the group consisting of a nucleic acid sequence, an amino acid sequence, a carbohydrate sequence, an ion and a feature of a protein determined by non-primary structure. The probe specific signal may be a fluorescent signal.

## Claims

### Claim 1

A system for analysis of a population of molecules, comprising:
a vessel adapted to contain a plurality of labeled probe molecules, each selectively binds to at least one target molecule, in contact with a population of molecules potentially containing a target comprising said at least one target molecule so that at least one of said plurality of labeled probe molecules being bound to at least a portion of said target;
a fluorescent excitation source adapted to direct an excitation beam through the vessel, the beam configured to cause at least some of said at least one labeled probe molecule to emit a probe specific signal;
a detector adapted to detect said probe specific signal and produce a detection output accordingly;
a signal modulator configured to perform a preferential modulation to at least one of the probe specific signal and said detection output, wherein the preferential modulation is configured to apply power at a level suitable for frequency modulation sufficient for moving said at least one bound probe molecule out of the excitation beam; and
an analysis module adapted to analyze the detection output in consideration of said preferential modulation.

### Claim 2 (depends on 1)

The system of claim 1, wherein said analysis module is configured to determine the presence or absence of said at least one target molecule within the population of molecules.

### Claim 3 (depends on 1)

The system of claim 1, comprising a modulation indication source adapted to provide a modulation indication to the signal modulator and to the detector.

### Claim 4 (depends on 1)

The system of claim 1, wherein the signal modulator is adapted to:
monitor an output of the signal modulator; and
provide a modulation indication to said detector responsive to the output of the signal modulator.

### Claim 5 (depends on 1)

The system of claim 1, wherein said signal modulator comprises an alternating field generator selected from a group consisting of an electric field generator and a magnetic field generator.

### Claim 6 (depends on 1)

The system of claim 1, wherein said preferential modulation comprises moving said at least one labeled probe molecule in and out of an excitation beam.

### Claim 7 (depends on 1)

The system of claim 1, wherein said preferential modulation comprises temporal modulation.

### Claim 8 (depends on 1)

The system of claim 1, wherein said preferential modulation comprises spatial modulation.

### Claim 9 (depends on 1)

The system of claim 1, wherein said preferential modulation comprises modulation of a pH of a solution containing the labeled probe molecules.

### Claim 10 (depends on 1)

The system of claim 1, wherein said preferential modulation comprises multiple modulation cycles.

### Claim 11 (depends on 1)

The system of claim 1, wherein said preferential modulation comprises a modification of a response of said at least one labeled probe molecule to excitation energy applied thereon.

### Claim 12 (depends on 1)

The system of claim 1, wherein said plurality of probe molecules comprise a molecule elected from a group consisting of a polynucleotide, a polypeptide, a carbohydrate and an ion chelator.

### Claim 13 (depends on 12)

The system of claim 12, wherein the polypeptide comprises an antibody.

### Claim 14 (depends on 12)

The system of claim 12, wherein the polynucleotide is selected from a group consisting of DNA, RNA and synthetic oligonucleotide.

### Claim 15 (depends on 1)

The system of claim 1, wherein said at least one target molecule comprises a molecule selected from a group consisting of a nucleic acid sequence, an amino acid sequence, a carbohydrate and a feature of a protein determined by non-primary structure.

### Claim 16 (depends on 1)

The system of claim 1, wherein said modulator is configured to apply an alternating field for establishing a periodic motion of said at least one bound probe molecule.

### Claim 17 (depends on 16)

The system of claim 16, wherein said alternating field is selected from a group consisting of a magnetic field and an electric field to said probe.

### Claim 18 (depends on 1)

The system of claim 1, wherein said plurality of probe molecules is attached to a magnetic particle.

### Claim 19 (depends on 1)

The system of claim 1, wherein the probe specific signal is a fluorescent signal.

### Claim 20 (depends on 1)

The system of claim 1, wherein said plurality of probe molecules comprises at least one fluorescence-modifying moiety.

### Claim 21 (depends on 1)

The system of claim 1, wherein the probe specific signal employs an energy transfer mechanism selected from a group consisting of fluorescent energy transfer (FET) and fluorescence resonance energy transfer (FRET).

### Claim 22 (depends on 1)

The system of claim 1, wherein said detector is configured to detect said probe specific signal over a background signal within one minute of the contacting a labeled probe with the population of molecules.

### Claim 23 (depends on 1)

The system of claim 1, wherein said detector is configured to detect at least one detection type selected from a group consisting of binary detection, amplitude detection and synchronous detection.

### Claim 24 (depends on 1)

The system of claim 1, wherein said probe specific signal is acquired together with a background signal comprising Raman scattering.

### Claim 25 (depends on 1)

The system of claim 1, wherein said detector is configured to detect said probe specific signal if said preferential modulation produces a probe specific signal being ten times greater in amplitude than a background signal.

### Claim 26 (depends on 1)

The system of claim 1, wherein the preferential modulation comprises increasing a local concentration of said plurality of labeled probe molecules.