Determining extracellular analyte concentration with nanoplasmonic sensors
Inventors
Raphael, Marc P. • Christodoulides, Joseph A. • Byers, Jeff M. • Delehanty, James B.
Assignees
Government Of United States Represented By Department Of Navy AS • US Department of Navy
Publication Number
US-12007328-B2
Publication Date
2024-06-11
Expiration Date
2036-06-20
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Abstract
Methods and systems for determining extracellular concentration data of an analyte are disclosed. A method for determining extracellular concentration data of an analyte includes receiving sensor data from one or more arrays of functionalized plasmonic nanostructures on a localized surface plasmon resonance imaging chip in contact with a fluid containing at least one living cell for a plurality of times, determining intensity data for the one or more arrays, determining fractional occupancy based on the intensity data, and determining extracellular concentration data based on the fractional occupancy data. A system for determining extracellular concentration data of an analyte includes a LSPRi chip, a sensor component, an intensity component, a fractional occupancy component, a concentration component, and a processor to implement the components.
Core Innovation
The invention provides methods and systems for determining extracellular concentration data of an analyte using arrays of functionalized plasmonic nanostructures on a localized surface plasmon resonance imaging (LSPRi) chip in contact with a fluid containing at least one living cell. The method receives sensor data from one or more arrays of nanostructures for multiple time points, determines intensity data based on the sensor data, calculates fractional occupancy from the intensity data, and then derives extracellular concentration data from the fractional occupancy over time. A system implementing this method comprises an LSPRi chip, sensor, intensity, fractional occupancy, concentration components, and a processor to execute the method steps.
The problem being solved addresses the difficulty in real-time, non-invasive measurement of extracellular protein concentrations and gradients produced by living cells without disrupting signaling pathways. Existing techniques using fluorescent labeling are limited by size, diffusion, and interference with downstream signaling, while spectrometry-based methods are restrictive and require significant light exposure harmful to live cells. Solid-state nanosensors, such as nanoplasmonic sensors, offer potential solutions but had challenges in quantifying spatio-temporal extracellular protein concentrations precisely.
This invention overcomes these limitations by enabling quantification of extracellular analyte concentrations in space and time without the need for fluorescent tags or spectrometers. It takes advantage of the linear relationship between normalized image intensity data from LSPRi and fractional occupancy, allowing concentration determination from CCD camera data alone. The approach utilizes temporal filtering and kinetic rate constants to estimate concentration distributions dynamically, enabling imaging of single-cell secretions with high spatial and temporal resolution.
Claims Coverage
The claims present two independent inventions: a computer-implemented system for determining extracellular concentration data of an analytes, and a method of determining extracellular concentrations of an analyte in fluid based on sensor data without using a spectrometer.
A system for extracellular analyte concentration determination without spectrometer
A localized surface plasmon resonance imaging (LSPRi) chip with functionalized plasmonic nanostructures on a substrate in contact with fluid containing living cells, a sensor component that receives sensor data (including brightfield or fluorescent images) without using a spectrometer, an intensity component that derives intensity data from sensor data over multiple times, a fractional occupancy component that calculates fractional occupancy data from the intensity, and a concentration component that determines extracellular concentration data based on fractional occupancy, all implemented by a processor.
Determining analyte movement by mapping concentration data
In addition to the system above, a movement component determines the movement of the analyte in the fluid by mapping extracellular concentration data over time for each array of nanostructures.
Using charge-coupled device to capture emissions from nanostructures
The system includes a charge-coupled device positioned to receive emissions from arrays of functionalized plasmonic nanostructures to obtain the sensor data.
A method for determining extracellular concentrations using nanoplasmonic sensors without spectrometer
Providing arrays of functionalized plasmonic nanostructures on an LSPRi chip contacting fluid with living cells, receiving sensor data for multiple times (including secretions in contact with nanostructures and brightfield or fluorescent cell images) without a spectrometer, determining fractional occupancy data for nanostructures from sensor data, and spatially and temporally mapping extracellular analyte concentration based on fractional occupancy.
The claims collectively cover a computer-implemented system and method leveraging localized surface plasmon resonance imaging chips with functionalized nanostructures to measure extracellular analyte concentrations and movements without using spectrometers, employing sensor data processing components to derive intensity, fractional occupancy, and concentration data from multiple time points.
Stated Advantages
Enables real-time, non-invasive measurement of extracellular analyte concentrations without disrupting cellular signaling pathways.
Avoids the need for fluorescent tagging, eliminating issues related to labeling size, diffusion, and photobleaching.
Does not require the use of a spectrometer, allowing lower light exposure that protects live cells.
Provides high spatial and temporal resolution, enabling imaging of single-cell secretions and burst secretions over a wide dynamic range.
Integrates with standard microscopy techniques including brightfield and fluorescence imaging for combined label and label-free studies.
Documented Applications
Real-time imaging and quantification of secreted protein concentrations from single living cells in culture.
Mapping the extracellular concentration gradients and dynamics of proteins, lipids, and DNA in fluids containing biological cells.
Correlation of secretions from one cell type in a co-culture with responses of another cell type to study causal relationships in cell signaling.
Applications in developmental biology and cell migration by identifying polarized secretions at the single-cell level.
Compatible integration with fluorescence microscopy for multiplexed and combined label and label-free cell investigations.
Potential use in printing applications such as inkjet and dip-pen lithography for multiplexed protein quantification assays.
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