# US 10,770,170 B2 · Signal encoding and decoding in multiplexed biochemical assays

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- Number: US 10,770,170 B2
- Document ID: US10770170B2
- Status: Granted
- Kind: B2
- Application number: 15/914,356
- Filed: 2018-03-07
- Published: 2019-01-31
- Granted: 2020-09-08
- Earliest claimed: 2012-02-03
- Assignees: California Institute of Technology
- Applicants: California Institute of Technology
- Inventors: Emil P. Kartalov; Aditya Rajagopal; Axel Scherer
- Web page: https://patentgrep.com/patent/US10770170B2

## CPC

- C12Q 1/6825: Nucleic acid detection involving sensors
- C12Q 1/68: involving nucleic acids
- C12Q 1/6851: Quantitative amplification
- G01N 21/6486: {Measuring fluorescence of biological material, e.g. DNA, RNA, cells (G01N21/6428 takes precedence)}
- G16B 5/00: ICT specially adapted for modelling or simulations in systems biology, e.g. gene-regulatory networks, protein interaction networks or metabolic networks
- G16B 25/00: ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
- G16B 25/20: Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
- G16B 40/10: Signal processing, e.g. from mass spectrometry [MS] or from PCR
- G06F 17/10: Complex mathematical operations {(function generation by table look-up G06F1/03; evaluation of elementary functions by calculation G06F7/544)}
- G06F 17/11: for solving equations {, e.g. nonlinear equations, general mathematical optimization problems (optimization specially adapted for a specific administrative, business or logistic context G06Q10/04)}
- G16B 40/00: ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- 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)
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G06F: ELECTRIC DIGITAL DATA PROCESSING (computer systems based on specific computational models G06N)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

This disclosure provides methods, systems, compositions, and kits for the multiplexed detection of a plurality of analytes in a sample. In some examples, this disclosure provides methods, systems, compositions, and kits wherein multiple analytes may be detected in a single sample volume by acquiring a cumulative measurement or measurements of at least one quantifiable component of a signal. In some cases, additional components of a signal, or additional signals (or components thereof) are also quantified. Each signal or component of a signal may be used to construct a coding scheme which can then be used to determine the presence or absence of any analyte.

## Claims

### Claim 1

A method of unambiguously detecting any unique combination of presence or absence of at least five polynucleotide analytes in a plurality of droplets, the method comprising:
(a) providing a sample comprising, or potentially comprising, at least one of said at least five polynucleotide analytes;
(b) forming a mixture of said sample and at least five hybridization probes, wherein each of said at least five hybridization probes further comprises at least one fluorophore and at most four fluorophores;
(c) partitioning said mixture into said plurality of droplets;
(d) exciting said at least one fluorophore to generate one or more signals if one or more of said at least five polynucleotide analytes is present in said plurality of droplets, wherein said one or more signals comprise at least one signal generated by excitement of said at least one fluorophore;
(e) measuring said one or more signals to generate a cumulative intensity measurement, wherein said cumulative intensity measurement corresponds to the presence of a unique combination of presence or absence of said at least five polynucleotide analytes in said sample; and
(f) determining whether each of said least five polynucleotide analytes is present, in any unique combination of presence or absence, based on said cumulative intensity measurement,
wherein the method does not require any step of immobilization of said at least five polynucleotide analytes or mass spectrometry.

### Claim 2 (depends on 1)

The method of claim 1, wherein each of said at least five hybridization probes is complementary to one of said at least five polynucleotide analytes.

### Claim 3 (depends on 1)

The method of claim 1, wherein said at least five hybridization probes comprise one or more fluorophores selected from one, two, three, or four fluorophores.

### Claim 4 (depends on 1)

The method of claim 1, wherein, in (b), a droplet of said plurality of droplets comprises said at least five hybridization probes.

### Claim 5 (depends on 1)

The method of claim 1, wherein, in (b), a droplet of said plurality of droplets comprises one or more of said at least five polynucleotide analytes.

### Claim 6 (depends on 1)

The method of claim 1, wherein, in (b), a droplet of said plurality of droplets comprises at most one of said at least five polynucleotide analytes.

### Claim 7 (depends on 1)

The method of claim 1, wherein said one or more signals is generated during a polymerase chain reaction.

### Claim 8 (depends on 1)

The method of claim 1, wherein said at least five polynucleotide analytes are at least seven polynucleotide analytes.

### Claim 9 (depends on 1)

The method of claim 1, wherein said at least five hybridization probes are at least seven hybridization probes.

### Claim 10 (depends on 1)

The method of claim 1, wherein said one or more of said at least five polynucleotide analytes comprise cell-free nucleic acid, tumor nucleic acid, fetal nucleic acid, viral nucleic acid, or any combination thereof.

### Claim 11 (depends on 1)

The method of claim 1, wherein said sample is a biological sample derived from a subject.

### Claim 12 (depends on 11)

The method of claim 11, wherein detecting the presence or absence of said at least five polynucleotide analytes indicates the presence of cancer or an infection with a pathogen in said subject.

### Claim 13 (depends on 1)

The method of claim 1, further comprising transmitting information concerning the presence or absence of said at least five polynucleotide analytes through a computer network.

### Claim 14 (depends on 1)

The method of claim 1, further comprising providing information concerning the presence or absence of said at least five polynucleotide analytes to a health care professional, wherein said health care professional makes a clinical decision based on said information.

### Claim 15 (depends on 14)

The method of claim 14, wherein said clinical decision is a treatment decision.

### Claim 16 (depends on 15)

The method of claim 15, wherein said treatment decision comprises treatment with an antibiotic or an anticancer therapeutic.

### Claim 17 (depends on 1)

The method of claim 1, wherein at least part of said method is performed using a computer.

### Claim 18 (depends on 17)

The method of claim 17, wherein said computer is located on a remote server.

### Claim 19 (depends on 17)

The method of claim 17, wherein said computer is located on a droplet reader.

### Claim 20 (depends on 1)

The method of claim 1, wherein each of said at least five hybridization probes are provided at a given concentration such that said cumulative intensity measurement corresponds to said presence of said unique combination.