# US 11,906,526 B2 · Systems and methods for sample preparation, data generation, and protein corona analysis

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- Number: US 11,906,526 B2
- Document ID: US11906526B2
- Status: Granted
- Kind: B2
- Application number: 18/365,674
- Filed: 2023-08-04
- Published: 2023-11-30
- Granted: 2024-02-20
- Earliest claimed: 2019-08-05
- Assignees: Seer, Inc.
- Applicants: Seer, Inc.
- Inventors: William Manning; Young Kim; Brandon Kwan-Leong; Hope Liou; Xiaoyan Zhao; Daniel Hornburg; Martin Goldberg
- Web page: https://patentgrep.com/patent/US11906526B2

## CPC

- G01N 33/6848: {Methods of protein analysis involving mass spectrometry}
- B01L 3/5085: {for multiple samples, e.g. microtitration plates}
- B01L 7/00: Heating or cooling apparatus (autoclaves B01J3/04); Heat insulating devices
- G01N 1/34: Purifying; Cleaning {(processes or apparatus for extracting or separating nucleic acids from biological samples C12N15/1003)}
- G01N 1/44: Sample treatment involving radiation, e.g. heat
- G01N 33/5432: {Liposomes or microcapsules}
- G01N 33/54326: {Magnetic particles}
- G01N 33/54346: {Nanoparticles}
- G01N 33/68: involving proteins, peptides or amino acids {(involving lipoproteins G01N33/92)}
- G01N 33/6842: {Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins}
- B01L 2400/043: magnetic forces
- B82Y 5/00: Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
- B82Y 25/00: Nanomagnetism, e.g. magnetoimpedance, anisotropic magnetoresistance, giant magnetoresistance or tunneling magnetoresistance
- B82Y 30/00: Nanotechnology for materials or surface science, e.g. nanocomposites
- B82Y 35/00: Methods or apparatus for measurement or analysis of nanostructures
- B82Y 40/00: Manufacture or treatment of nanostructures
- 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)
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B82Y: SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES

## Abstract

Systems and methods for automated sample preparation and processing of protein corona are described herein, as well as its application in the discovery of advanced diagnostic tools as well as therapeutic agents.

## Claims

### Claim 1

An automated system for sample preparation, the system comprising:
(a) a fluid transfer unit comprising a multichannel fluid transfer instrument for transferring fluids between units within the system;
(b) a sensor array comprising a magnetic support and a magnetic sensor array, wherein the magnetic sensor array comprises a plurality of partitions, wherein at least one of the plurality of partitions comprises (i) a biological sample, (ii) a diluent comprising a buffer, and (iii) a plurality of magnetic particles, wherein the plurality of magnetic particles comprise silica and a carboxyl functionalized surface, wherein at least one of the plurality of magnetic particles is configured to bind to at least 300 different proteins from the biological sample, and wherein the magnetic support is configured to immobilize the plurality of magnetic particles when the magnetic sensor array is transferred to the magnetic support;
(c) a reagent storage unit comprising supports for storing a plurality of reagents;
(d) a waste unit comprising supports for storing a reagent to be disposed of;
(e) a consumable storage unit comprising supports for storing a plurality of consumables for use by the multichannel fluid transfer instrument; and
(f) a control unit comprising one or more processors programmed to perform steps comprising:
(i) incubating the biological sample and the diluent with the plurality of magnetic particles in the at least one of the plurality of partitions of the magnetic sensor array such that proteins from the biological sample interact with at least one of the plurality of magnetic particles, wherein the incubating is performed using agitation and/or mixing at a temperature of 4° C. to 40° C. for at least 20 minutes;
(ii) removing a supernatant from the at least one of the plurality of partitions of the magnetic sensor array to isolate the proteins interacting with at least one of the plurality of magnetic particles;
(iii) washing the proteins interacting with at least one of the plurality of magnetic particles to obtain washed proteins interacting with the plurality of magnetic particles; and
(iv) preparing a population of peptides for mass spectrometry, wherein the preparing comprises incubating a digestion solution with the washed proteins and at least one of the plurality of magnetic particles.

### Claim 2 (depends on 1)

The automated system of claim 1, wherein the biological sample is diluted with the diluent by at least 2-fold.

### Claim 3 (depends on 2)

The automated system of claim of claim 2, wherein the biological sample is diluted with the diluent no more than 8-fold.

### Claim 4 (depends on 1)

The automated system of claim 1, wherein the biological sample comprises plasma or serum.

### Claim 5 (depends on 4)

The automated system of claim 4, wherein the biological sample comprises plasma.

### Claim 6 (depends on 1)

The automated system of claim 1, wherein the plurality of magnetic particles comprises a plurality of magnetic particles having a size of at least 900 nm.

### Claim 7 (depends on 1)

The automated system of claim 1, wherein the plurality of magnetic particles comprises a negative surface charge.

### Claim 8 (depends on 1)

The automated system of claim 1, wherein the plurality of magnetic particles comprises a carboxyl functionalized polymer.

### Claim 9 (depends on 1)

The automated system of claim 1, wherein the automated instrument is configured to perform steps (i) to (iv) in less than 8 hours.

### Claim 10 (depends on 1)

The automated system of claim 1, wherein the plurality of reagents comprises TE 150 mM KCL 0.05% CHAPS buffer.

### Claim 11 (depends on 1)

The automated system of claim 1, wherein the plurality of magnetic particles comprises a polydispersity index of 0.5 to 1.

### Claim 12 (depends on 1)

The automated system of claim 1, wherein the digestion solution comprises trypsin.

### Claim 13 (depends on 1)

The automated system of claim 1, wherein the incubating in step (i) is performed above room temperature.

### Claim 14 (depends on 1)

The automated system of claim 1, wherein the magnetic sensor array comprises a multi-well plate.

### Claim 15 (depends on 1)

The automated system of claim 1, wherein the plurality of magnetic particles are configured to bind to at least 500 different proteins from the biological sample.

### Claim 16 (depends on 1)

The automated system of claim 1, wherein the plurality of magnetic particles are configured to bind to from 300 to 2200 different proteins from the biological sample.

### Claim 17 (depends on 1)

The automated system of claim 1, wherein the plurality of magnetic particles comprises a homogeneous size distribution.

### Claim 18 (depends on 1)

The automated system of claim 1, wherein the plurality of magnetic particles comprises a heterogeneous size distribution.

### Claim 19 (depends on 1)

The automated system of claim 1, wherein the at least one of the plurality of partitions comprises about 10 microliters to about 100 microliters of the biological sample.

### Claim 20 (depends on 1)

The automated system of claim 1, wherein the plurality of reagents comprises at least two buffers having a different pH.

### Claim 21 (depends on 1)

The automated system of claim 1, wherein the at least one of the plurality of partitions comprises 1 mg/mL to 3 mg/mL of the plurality of magnetic particles.

### Claim 22 (depends on 1)

The automated system of claim 1, wherein a portion of the plurality of partitions comprises the biological sample and a plurality of poly(N-(3-(dimethylamino)propyl)methacrylamide) (PDMAPMA)-coated particles.

### Claim 23 (depends on 1)

The automated system of claim 1, wherein the plurality of magnetic particles comprises a non-polymeric outer shell.

### Claim 24 (depends on 1)

The automated system of claim 1, wherein the preparing in step (iv) further comprises incubating the washed proteins and the plurality of magnetic particles with chemical reagents at about 95° C. for about 10 minutes to lyse, reduce, and alkylate the washed proteins.

### Claim 25 (depends on 12)

The automated system of claim 12, wherein the digestion solution further comprises LysC.

### Claim 26 (depends on 1)

The automated system of claim 1, wherein the at least one of the plurality of partitions comprises: about 10 to 100 microliters of plasma or serum, wherein the diluent is an aqueous buffer, wherein the plasma or serum is diluted about 3-fold, about 4-fold or about 5-fold with the diluent, wherein the plurality of magnetic particles comprises a plurality of magnetic particles having a size of about 1000 nm to about 10000 nm, and wherein the at least one of the plurality of partitions comprises 1 mg/mL to 5 mg/mL of the plurality of magnetic particles.