# US 12,768,835 B2 · Method and apparatus for measuring physiological properties of biological samples

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- Number: US 12,768,835 B2
- Document ID: US12768835B2
- Status: Granted
- Kind: B2
- Application number: 18/126,809
- Filed: 2023-03-27
- Published: 2023-07-20
- Granted: 2026-10-20
- Earliest claimed: 2017-04-06
- Assignees: Agilent Technologies, Inc.
- Applicants: Agilent Technologies, Inc.
- Inventors: Andrew C Neilson; Paul D McGarr
- Web page: https://patentgrep.com/patent/US12768835B2

## CPC

- G01N 33/5302: {Apparatus specially adapted for immunological test procedures}
- B01L 3/50853: {with covers or lids (closures for test tubes B01L3/50825)}
- C12M 41/46: {of cellular or enzymatic activity or functionality, e.g. cell viability}
- G01N 21/03: Cuvette constructions
- G01N 21/6452: {Individual samples arranged in a regular 2D-array, e.g. multiwell plates}
- G01N 33/5304: {Reaction vessels, e.g. agglutination plates (for solid-phase systems G01N33/543)}
- B01L 2300/0609: Holders integrated in container to position an object
- B01L 2300/0627: Sensor or part of a sensor is integrated
- B01L 2300/0829: Multi-well plates; Microtitration plates
- G01N 2021/0325: {Cells for testing reactions, e.g. containing reagents}
- 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
- C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS}

## Abstract

The present disclosure relates to methods and apparatus for measuring of multiple physiological properties of biological samples, such as measuring biological flux.

## Claims

### Claim 1

A system for measuring one or more physiological properties of a biological sample, the system comprising:
a well plate having a plurality of wells, the wells having an open end and a closed end opposite the open end;
an apparatus comprising:
a lid having a first surface and a second surface;
a plurality of spines connected to and extending from the first surface of the lid, each of the plurality of spines comprising a body having a distal end wherein at least one spine of the plurality of spines comprises one or more sensors, and wherein the lid forms a continuous surface once connected to the plurality of spines;
wherein the plurality of spines extend into the wells through the open end to a pre-determined depth within the well when the lid is positioned on the well plate;
wherein the distal end of the spines are spaced-apart from a respective well bottom of the well plate to form a sub-chamber within the well;
wherein the one or more sensors are disposed on at least one spine of the plurality of spines, wherein the one or more sensors engage and provide a signal responsive to a gradient of one or more analytes that are formed from the biological sample consuming or producing the one or more analytes;
wherein the lid has a coupling for allowing movement of the lid from a first position to one or more second positions with respect to the well; and
a processor in communication with the coupling to provide automated motion of the lid so as to move the lid from the first position to a second position, thereby modifying the depth of the distal end of the spine into its respective well;
the one or more sensors coupled to a signal processor, the signal processor configured to:
interrogate the one or more sensors to obtain readings at the first position and second position; and
compute a change in concentration in the gradient from the readings.

### Claim 2 (depends on 1)

The system of claim 1, wherein at least one of the at least sensors is a fluorescent indicator.

### Claim 3 (depends on 1)

The system of claim 1, wherein at least one of the at least twe sensors is an indicator embedded in a permeable medium.

### Claim 4 (depends on 1)

The system of claim 1, wherein at least one of the at least twe sensors is a capture reagent.

### Claim 5 (depends on 1)

The system of claim 1, wherein each of the plurality of spines has a length, and the length is between 1 mm and 20 mm.

### Claim 6 (depends on 5)

The system of claim 5, wherein the plurality of spines collectively has a mean length, and the length of each of the plurality of spines is within 20% of the mean length.

### Claim 7 (depends on 1)

The system of claim 1, wherein each of the plurality of spines comprises an impeller on the body.

### Claim 8 (depends on 1)

The system of claim 1, wherein the plurality of spines extend into the wells, wherein the distal end of each of the plurality of spines is between 50 micrometers and 800 micrometers from the closed end of the well.

### Claim 9 (depends on 1)

The system of claim 1, further comprising a signal detector in a position to detect a signal from the one or more sensors.

### Claim 10 (depends on 9)

The system of claim 9, wherein at least one of the sensors is positioned on an opposite side of the closed end of the well plate from the signal detector.

### Claim 11 (depends on 1)

The system of claim 1, wherein the coupling comprises a handle attached to the lid.

### Claim 12 (depends on 1)

The system of claim 1, wherein each of the plurality of spines is solid.

### Claim 13

A method for measuring one or more physiological properties of a biological sample, the method comprising:
providing a lid having a first surface and a second surface and a plurality of spines connected to and extending from the first surface of the lid, each of the plurality of spines comprising a body having a distal end, and wherein the lid forms a continuous surface once connected to the plurality of spines; and
applying one or more sensors to the distal end of at least one of the plurality of spines, wherein the one or more sensors engage and provide a signal responsive to one or more analytes that are formed from the biological sample consuming or producing the one or more analytes;
positioning the lid on a well plate having a plurality of wells, the wells having an open end and a closed end opposite the open end;
wherein the plurality of spines extend into the wells through the open end to a pre-determined depth within the well when the lid is positioned on the well plate;
wherein the distal end of the spines are spaced-apart from a respective well bottom for the well plate to form a sub-chamber within the well;
wherein the lid has a coupling for allowing controlled movement of the lid via a processor in communication with the coupling;
controlling motion of the lid from a first position to one or more second positions with respect to the well, thereby modifying the depth of the distal end of the spine into its respective well;
interrogating the one or more sensors to obtain readings at the first position and second position; and
computing a change in concentration in the gradient from the readings.

### Claim 14 (depends on 13)

The method of claim 13, wherein the one or more sensors are applied as a mixture of a fluorescent indicator in a medium, and the one or more sensors are attached to the plurality of spines by solidifying or removing the medium.

### Claim 15 (depends on 13)

The method of claim 13, wherein the one or more sensors are applied by dipping or spotting the distal end of at least one of the plurality of spines in a mixture of a fluorescent indicator in a medium.

### Claim 16 (depends on 13)

The method of claim 13, wherein the lid is provided by molding a polymer material to form the lid and the plurality of spines.

### Claim 17 (depends on 13)

The method of claim 13, wherein the coupling comprises a handle attached to the lid.

### Claim 18 (depends on 13)

The method of claim 13, wherein each of the plurality of spines is solid.

### Claim 19

A system for measuring one or more physiological properties of a biological sample, the system comprising:
a well plate having a plurality of wells, the wells having an open end and a closed end opposite the open end;
an apparatus comprising:
a lid having a first surface and a second surface;
a plurality of spines connected to and extending from the first surface of the lid, each of the plurality of spines comprising a body having a distal end wherein at least one spine of the plurality of spines comprises one or more sensors, each of the spines being solid such that the spines do not include cavities or hollow regions;
wherein the plurality of spines extend into the wells through the open end to a pre-determined depth within the well when the lid is positioned on the well plate;
wherein the distal end of the spines are spaced-apart from a respective well bottom of the well plate to form a sub-chamber within the well;
wherein the one or more sensors are disposed on at least one spine of the plurality of spines, wherein the one or more sensors engage and provide a signal responsive to a gradient of one or more analytes that are formed from a biological sample consuming or producing the one or more analytes;
wherein the lid has a coupling for allowing movement of the lid from a first position to one or more second positions with respect to the well; and
a processor in communication with the coupling to provide automated motion of the lid so as to move the lid from the first position to a second position, thereby modifying the depth of the distal end of the spine into its respective well;
the one or more sensors coupled to a signal processor, the signal processor configured to:
interrogate the one or more sensors to obtain readings at the first position and second position; and
compute a change in concentration in the gradient from the readings.