Light microscopy chips and data analysis methodology for quantitative localzied surface plasmon resonance (LSPR) biosensing and imaging
Inventors
Raphael, Marc P. • Christodoulides, Joseph A • Byers, Jeff M
Assignees
Publication Number
US-11067573-B2
Publication Date
2021-07-20
Expiration Date
2033-09-27
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Abstract
A chip for localized surface plasmon resonance (LSPR) biosensing and imaging having a glass coverslip compatible for use in a standard microscope and at least one array of functionalized plasmonic nanostructures patterned onto the glass coverslip with electron beam nanolithography. The nanostructures can be regenerated allowing the chip to be used multiple times. Also disclosed is a method for determining the fractional occupancy values for surface-bound receptors as a function of time for LSPR biosensing from the spectroscopic response of the array and modeling the photon count in each spectrometer channel, allowing for a functional relationship to be determined between the acquired spectrum and the fractional occupancy of binding sites on the array. Additionally disclosed is a method for the spatiotemporal mapping of receptor-ligand binding kinetics in LSPR imaging using the chip and projecting a magnified image of the array to a CCD camera and monitoring the binding kinetics of the array.
Core Innovation
The present invention provides a chip for localized surface plasmon resonance (LSPR) biosensing and imaging comprising a glass coverslip compatible for use in a standard microscope, onto which at least one array of functionalized plasmonic nanostructures has been patterned using electron beam nanolithography. These nanostructures can be regenerated, allowing repeated use of the chip, and are calibrated for quantitative determination of concentration as a function of time and space.
Additionally, the invention discloses a method for determining the fractional occupancy values for surface-bound receptors over time for LSPR biosensing and imaging by spectroscopically characterizing the array. It involves modeling the photon count in each spectrometer channel as the sum of a baseline scattering rate and a scattering term responsive to small dielectric perturbations, thereby relating the acquired spectrum functionally to the fractional occupancy of binding sites using a non-linear least squares approach.
The invention aims to devise a light microscopy-based instrumental and quantitative analytical methodology for LSPR biosensing that determines surface-receptor fractional occupancy, alongside an LSPR imaging technique for spatiotemporal mapping of binding events. Notably, it accomplishes this on a commercially-available light microscope platform compatible with other microscopy techniques such as fluorescence and differential interference contrast (DIC). This innovation addresses the lack of methods for measuring fractional occupancy of surface-bound receptors at the space and time resolution provided herein.
Claims Coverage
The patent discloses one independent claim covering a method for determining fractional occupancy in LSPR biosensing using a specialized chip.
Chip comprising patterned functionalized plasmonic nanostructure arrays on a glass coverslip
The use of a chip with a glass coverslip compatible with standard microscopes, featuring at least one array of functionalized plasmonic nanostructures patterned by electron beam nanolithography, calibrated for quantitative concentration measurements over time and space.
Spectroscopic characterization via focused image projection
Projecting a focused image of the nanostructure array onto a spectrometer to obtain spectroscopic characterization of the array.
Modeling photon counts as sum of baseline and dielectric perturbation scattering terms
Using a model in which the photon count in each spectrometer channel is expressed as a sum of a baseline scattering rate and a scattering term sensitive to small dielectric perturbations, with the summation performed over hundreds of spectrometer channels at each time point.
Relating acquired spectra to fractional occupancy using non-linear least squares
Determining the fractional occupancy of binding sites by functionally relating the acquired spectrum to occupancy values via a non-linear least squares calculation.
The claims cover a method using a calibrated chip with functionalized plasmonic nanostructure arrays, spectroscopic characterization, detailed photon count modeling, and a non-linear least squares approach to accurately determine fractional occupancy in LSPR biosensing and imaging.
Stated Advantages
Measurements are made in real-time, enabling dynamic monitoring of binding events.
Nanostructures are lithographically patterned onto standard glass coverslips, facilitating integration with traditional imaging techniques like fluorescence and bright field microscopy.
Nanostructures are calibrated to allow quantitative determination of analyte concentration as a function of time and space.
Documented Applications
Quantitative localized surface plasmon resonance (LSPR) biosensing to determine fractional occupancy of surface-bound receptors over time.
Spatiotemporal mapping of receptor-ligand binding kinetics using LSPR imaging on an inverted light microscope.
Real-time monitoring of biomolecular interactions such as biotin-neutravidin binding with varying analyte concentrations.
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