# US 2023/0272003 A1 · Methods and apparatus for simultaneously detecting a large range of protein concentrations

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2023/0272003 A1
- Document ID: US20230272003A1
- Status: Published application
- Kind: A1
- Application number: 18/301,858
- Filed: 2023-04-17
- Published: 2023-08-31
- Earliest claimed: 2016-11-29
- Assignees: ProteinSimple
- Applicants: ProteinSimple
- Inventors: Hui XU; Kenneth SWARTZ
- Web page: https://patentgrep.com/patent/US20230272003A1

## CPC

- C07K 1/26: Electrophoresis
- C07K 1/28: Isoelectric focusing
- G01N 1/20: for flowing or falling materials ({G01N1/2035} G01N1/12, G01N1/14 take precedence)
- G01N 1/40: Concentrating samples
- G01N 21/76: Chemiluminescence; Bioluminescence
- G01N 33/54366: {Apparatus specially adapted for solid-phase testing}
- G01N 33/6803: {General methods of protein analysis not limited to specific proteins or families of proteins}
- G06V 10/141: Control of illumination
- G06V 10/507: {Summing image-intensity values; Histogram projection analysis}
- G06V 20/693: {Acquisition}
- G06V 20/695: {Preprocessing, e.g. image segmentation}
- G01N 27/44721: {by optical means}
- G01N 27/44726: {using specific dyes, markers or binding molecules}
- G01N 2001/4038: {electric methods, e.g. electromigration, electrophoresis, ionisation}
- G01N 2021/1765: {Method using an image detector and processing of image signal}
- G06V 10/16: {using multiple overlapping images; Image stitching}
- 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)
- 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)
- G06V: IMAGE OR VIDEO RECOGNITION OR UNDERSTANDING

## Abstract

Some embodiments described herein relate to a method that includes separating an analyte-containing sample via electrophoresis in a capillary. The capillary is loaded with a chemiluminescence agent, such as luminol, that is configured to react with the analyte (e.g., HRP-conjugated proteins) to produce a signal indicative of a concentration and/or quantity of analyte at each location along the length of the capillary. A first image of the capillary containing the analytes and the chemiluminescence agent is captured over a first period of time. A second image of the capillary containing the analytes and the chemiluminescence agent is captured over a second, longer, period of time. A concentration and/or quantity of a first population of analytes at a first location is determined using the first image, and a concentration and/or quantity of a second population of analytes at a second location is determined using the second image.

## Claims

### Claim 1

A method, comprising:
separating a sample containing a first population of proteins and a second population of proteins via capillary electrophoresis such that the first population of proteins is located at a first position along a length of a capillary and the second population of proteins is located at a second position along the length of the capillary;
capturing a plurality of images of the capillary, each image from the plurality of images having a different exposure period;
selecting a first image from the plurality of images in which a strength of a first optical signal indicative of a quantity of the first population of proteins exceeds a predetermined threshold, the first optical signal changing with time;
determining an initial intensity of the first optical signal by dividing the strength of the first optical signal by an exposure period for the first image;
calculating a quantity of the first population of proteins based on the initial intensity of the first optical signal;
selecting a second image from the plurality of images in which a strength of a second optical signal indicative of a quantity of the second population of proteins exceeds the predetermined threshold, the second optical signal changing with time; and
calculating a quantity of the second population of proteins based on the second optical signal and an exposure period for the second image.

### Claim 2 (depends on 1)

The method of claim 1, wherein a quantity of the first population of proteins is at least three times greater than a quantity of the second population of proteins.

### Claim 3 (depends on 1)

The method of claim 1, wherein a quantity of the first population of proteins is at least ten times greater than a quantity of the second population of proteins.

### Claim 4 (depends on 1)

The method of claim 1, further comprising:
determining an initial intensity of the second optical signal by dividing the strength of the second optical signal by the exposure period for the second image, the quantity of the second population calculated based on the initial intensity of the second optical signal.

### Claim 5 (depends on 1)

The method of claim 1, wherein the plurality of images is divided into subsets each having a nominal exposure time, an exposure time of each image from each subset varying by 50% or less from the nominal exposure time for that subset.

### Claim 6 (depends on 1)

The method of claim 1, wherein each subset of images from the plurality of images has a nominal exposure time of twice a nominal exposure time of an immediately prior subset of images from the plurality of images.

### Claim 7 (depends on 1)

The method of claim 1, wherein each image from the plurality of images has an exposure time of twice an exposure time of an immediately prior image from the plurality of images.

### Claim 8 (depends on 1)

The method of claim 1, further comprising:
adding one of a chemiluminescence agent or a detection agent to the capillary prior to capturing an image from the plurality of images.

### Claim 9 (depends on 1)

The method of claim 1, further comprising:
adding one of a chemiluminescence agent or a chromogenic agent to the capillary prior to capturing each image from the plurality of images.

### Claim 10 (depends on 1)

The method of claim 1, wherein the first optical signal and the second optical signal are each associated with a detection agent, the detection agent at the second position depleted by during the exposure period for the first image.

### Claim 11 (depends on 1)

The method of claim 1, wherein the second optical signal is below a detection threshold in the first image.

### Claim 12 (depends on 1)

The method of claim 1, wherein the first position of the capillary and the second position of the capillary both appear in each of the first image and the second image.

### Claim 13 (depends on 1)

The method of claim 1, wherein:
the second image is captured after the first image; and
the exposure period for the second image is longer than the exposure period for the first image.

### Claim 14 (depends on 1)

The method of claim 1, wherein the plurality of images is divided into subsets of images, each subset of images including a plurality of images sharing an exposure time that is different from each other subset of images.

### Claim 15 (depends on 1)

The method of claim 1, wherein:
the plurality of images includes a first subset of images and a second subset of images, each image from the first subset of images having a first exposure time and each image from the second subset of images having a second exposure time, the first subset of images captured before the second subset of images.

### Claim 16

A non-transitory processor-readable medium storing code, the code comprising instructions to cause a processor to:
send a signal to a voltage source to cause the voltage source to apply an electric potential across a capillary to cause a sample containing a first population of proteins and a second population of proteins to be separated via capillary electrophoresis such that the first population of proteins is located at a first position along a length of a capillary and the second population of proteins is located at a second position along the length of the capillary;
receive, from a detector, a plurality of images of the capillary, each image from the plurality of images having a different exposure period;
select a first image from the plurality of images in which a strength of a first optical signal indicative of a quantity of the first population of proteins exceeds a predetermined threshold, the first optical signal changing with time;
determine an initial intensity of the first optical signal by dividing the strength of the first optical signal by an exposure period for the first image;
calculate a quantity of the first population of proteins based on the initial intensity of the first optical signal;
select a second image from the plurality of images in which a strength of a second optical signal indicative of a quantity of the second population of proteins exceeds the predetermined threshold, the second optical signal changing with time; and
calculate a quantity of the second population of proteins based on the second optical signal and an exposure period for the second image.

### Claim 17 (depends on 16)

The non-transitory processor-readable medium of claim 16, the code further comprising instructions to cause the processor to:
determine an initial intensity of the second optical signal by dividing the strength of the second optical signal by the second duration, the quantity of the second population of proteins calculated based on the initial intensity of the second optical signal.

### Claim 18 (depends on 16)

The non-transitory processor-readable medium of claim 16, the code further comprising instructions to cause the processor to:
send a signal to cause one of a chemiluminescence agent or a detection agent to be added to the capillary prior to capturing an image from the plurality of images.

### Claim 19 (depends on 16)

The non-transitory processor-readable medium of claim 16, the code further comprising instructions to cause the processor to:
send a signal to cause one of a chemiluminescence agent or a detection agent to be added to the capillary prior to capturing each image from the plurality of images.

### Claim 20 (depends on 16)

The non-transitory processor-readable medium of claim 16, wherein:
the second image is captured after the first image; and
the exposure period for the second image is longer than the exposure period for the first image.