# US 9,518,982 B2 · Sequential analysis of biological samples

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- Number: US 9,518,982 B2
- Document ID: US9518982B2
- Status: Granted
- Kind: B2
- Application number: 12/611,299
- Filed: 2009-11-03
- Published: 2010-02-25
- Granted: 2016-12-13
- Earliest claimed: 2006-11-16
- Assignees: General Electric Company
- Applicants: Anup Sood; Michael Christopher Montalto; Michael Gerdes; Maximilian Lewis Seel; Robert John Filkins
- Inventors: Anup Sood; Michael Christopher Montalto; Michael Gerdes; Maximilian Lewis Seel; Robert John Filkins
- Web page: https://patentgrep.com/patent/US9518982B2

## CPC

- G01N 33/53: Immunoassay; Biospecific binding assay; Materials therefor
- G01N 33/542: with steric inhibition or signal modification, e.g. fluorescent quenching
- G01N 33/582: {with fluorescent label}
- C12Q 1/68: involving nucleic acids
- Y10T 436/143333: Saccharide [e.g., DNA, etc.]
- 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
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

Methods for detecting multiple targets in a biological sample are provided. The methods includes contacting the sample with a first probe; physically binding the first probe to a first target; observing a first signal from the first probe; applying a chemical agent to modify the first signal; contacting the sample with a second probe; physically binding the second probe to a second target; and observing a second signal from the second probe. The methods disclosed herein also provide for multiple iterations of binding, observing, signal modification for deriving information about multiple targets in a single sample. An associated kit and device are also provided.

## Claims

### Claim 1

A kit for detection of multiple targets in a biological sample, comprising:
multiple probes, wherein each probe comprises a binder and a fluorophore, wherein each probe is configured to specifically bind to a target via the binder, wherein the binder comprises a primary antibody and wherein the fluorophore is coupled to a secondary antibody that binds to the target via the primary antibody; and
at least one chemical agent in a concentration that, when applied to each probe, is configured to modify the fluorescence signal provided by the fluorophore of at least one probe without affecting the integrity of the binder in the at least one probe such that the primary antibody, when bound to the target, remains bound to the target after modification of the fluorescent signal by the at least one chemical agent under conditions in which the fluorophore is destroyed.

### Claim 2 (depends on 1)

The kit of claim 1, wherein the at least one chemical agent is configured to modify the fluorescence signal provided by the fluorophores of all of the multiple probes.

### Claim 3 (depends on 1)

The kit of claim 1, wherein the multiple probes comprise a first probe comprising a first binder and a first fluorophore, wherein the first probe is configured to specifically bind to a first target, and wherein the first signal generator is configured to provide a first fluorescence signal; and a second probe comprising a second binder and a second fluorophore, wherein the second probe is configured to specifically bind to a second target, and wherein the second signal generator is configured to provide a second fluorescence signal.

### Claim 4 (depends on 3)

The kit of claim 3, wherein the first target is a protein and the second target is a nucleic acid.

### Claim 5 (depends on 1)

The kit of claim 1, wherein the target is a protein antigen.

### Claim 6 (depends on 1)

The kit of claim 1, further comprising a morphological stain configured to provide a distinguishable signal that does not overlap with the fluorescence signal produced by the fluorophores of multiple probes.

### Claim 7 (depends on 6)

The kit of claim 6, wherein the morphological stain is a compartmental marker.

### Claim 8 (depends on 6)

The kit of claim 6, wherein the at least one chemical agent is incapable of modifying the signal provided by the morphological stain.

### Claim 9 (depends on 1)

The kit of claim 1, wherein the at least one chemical agent is chosen from hydrogen peroxide, sodium hydroxide, or a periodate salt.

### Claim 10 (depends on 1)

The kit of claim 1, wherein the multiple probes comprises at least one probe, the fluorophore of which is configured to provide a fluorescence signal that is not modified by the at least one chemical agent.

### Claim 11 (depends on 1)

The kit of claim 1, wherein the binder is an antibody and the fluorophore is a cyanine dye.

### Claim 12

A kit for detection of multiple targets in a biological sample, comprising:
a first probe, comprising a first binder and a first fluorophore, wherein the first probe is configured to specifically bind to a first target via the first binder, and wherein the first fluorophore is configured to provide a first fluorescence signal;
a second probe, comprising a second binder and a second fluorophore, wherein the second probe is configured to specifically bind to a second target via the second binder, and wherein the second fluorophore is configured to provide a second fluorescence signal;
a first chemical agent in a concentration that, when applied to each probe, is configured to modify the first fluorescence signal provided by the first fluorophore without affecting the integrity of the first binder such that the first binder, when bound to the first target, remains bound to the first target after modification of the first fluorescent signal by the first chemical agent under conditions in which the first fluorophore is destroyed; and
a second chemical agent configured to modify the second fluorescence signal provided by the second fluorophore without affecting the integrity of the second binder such that the second binder, when bound to the second target, remains bound to the second target after modification of the second fluorescent signal by the second chemical agent.

### Claim 13 (depends on 12)

The kit of claim 12, wherein the first fluorophore is a Cy5 dye or a Cy7 dye, and the second fluorophore is a Cy3 dye.

### Claim 14 (depends on 12)

The kit of claim 12, wherein the first target is a protein and the second target is a nucleic acid.

### Claim 15 (depends on 12)

The kit of claim 12, wherein either the first chemical agent or the second chemical agent is an oxidizing agent.

### Claim 16

A kit for detection of multiple proteins in a single tissue section, comprising:
a plurality of cyanine dye-labeled antibodies; and
at least one oxidizing agent in a concentration that, when applied to the cyanine dye-labeled antibodies, is configured to modify the cyanine fluorescence of at least one of the plurality of cyanine dye-labeled antibodies without affecting the integrity of the antibodies.

### Claim 17 (depends on 1)

The kit of claim 1, wherein the at least one chemical agent configured to modify the fluorescence signal is capable of the modifying at room temperature.

### Claim 18 (depends on 1)

The kit of claim 1, wherein the at least one chemical agent configured to modify the fluorescence signal is a buffered sodium hydroxide solution having a pH of about 12.

### Claim 19 (depends on 1)

The kit of claim 1, wherein the at least one chemical agent configured to modify the fluorescence signal is a hydrogen peroxide solution having a volume percentage of about 3%.

### Claim 20 (depends on 1)

The kit of claim 1, wherein the at least one chemical agent configured to modify the fluorescence signal comprises a first solution comprising the at least one chemical agent configured to modify the fluorescence signal and a second solution comprising the at least one chemical agent configured to modify the fluorescence signal.

### Claim 21 (depends on 1)

The kit of claim 1, wherein destruction of the fluorophore does not affect binding of the primary antibody with the secondary antibody such that the secondary antibody remains bound to the primary antibody after the destruction of the fluorophore.

### Claim 22 (depends on 1)

The kit of claim 1, wherein the chemical agent does not destroy a fluorophore associated with a morphological stain.

### Claim 23 (depends on 1)

The kit of claim 1, wherein the morphological stain is DAPI.

### Claim 24 (depends on 1)

The kit of claim 1, wherein the chemical agent is configured to destroy the fluorophore but not the morphological stain after contact times of at least 30 minutes.

### Claim 25 (depends on 1)

The kit of claim 1, wherein the chemical agent is configured to destroy the fluorophore such that an average pixel intensity of an imaged sample including the target after the destruction is less than 7× a background average pixel intensity observed from the sample before application of the fluorophore.