Light microscopy chips and data analysis methodology for quantitative localized surface plasmon resonance (LSPR) biosensing and imaging
Inventors
Raphael, Marc P. • Christodoulides, Joseph A. • Byers, Jeff M.
Assignees
Publication Number
US-9915654-B2
Publication Date
2018-03-13
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 invention provides a chip for localized surface plasmon resonance (LSPR) biosensing and imaging comprising a glass coverslip compatible with a standard microscope and at least one array of functionalized plasmonic nanostructures patterned onto the glass coverslip using electron beam nanolithography. This chip enables quantitative determination of concentration as a function of time and space through calibrated nanostructures.
A method is disclosed for determining fractional occupancy values for surface-bound receptors over time by spectroscopically characterizing the array and modeling photon counts in each spectrometer channel. This modeling allows for a functional relationship to be established between the acquired spectrum and the fractional occupancy of binding sites on the array. An additional method enables spatiotemporal mapping of receptor-ligand binding kinetics by projecting a magnified image of the array onto a CCD camera, allowing monitoring of binding kinetics.
The problem addressed is the lack of a method for measuring fractional occupancy of surface-bound receptors with simultaneous spatial and temporal resolution on a commercially-available light microscopy platform compatible with other microscopy techniques such as fluorescence and differential interference contrast (DIC). Current biosensing technologies like SPR have limitations including lower spatial resolution and complexity of integration with standard microscopes. The invention overcomes these issues by providing reproducible, regenerable nanostructure arrays patterned on glass coverslips suitable for real-time, quantitative LSPR biosensing and imaging.
Claims Coverage
The patent claims center around a chip for LSPR biosensing and imaging and its key features. Two primary inventive features are identified based on the independent claims.
Chip comprising a glass coverslip with arrays of functionalized plasmonic nanostructures patterned by electron beam nanolithography
The chip includes a glass coverslip compatible with standard microscopes and at least one array of functionalized plasmonic nanostructures calibrated for quantitative determination of concentration in space and time. The arrays are patterned onto the coverslip using electron beam nanolithography, enabling precise control and compatibility with microscopy systems.
Nanostructure arrays capable of regeneration and reuse through plasma ashing
The functionalized plasmonic nanostructure arrays on the chip can be regenerated and reused by plasma ashing to remove organic layers and biofunctionalization, preserving the physical structure and calibration of the nanostructures for repeated use.
The claims cover a microscopy-compatible chip with calibrated functionalized plasmonic nanostructure arrays produced by electron beam nanolithography that enable quantitative LSPR biosensing and imaging, including a regeneration process for repeated use of the same nanostructure arrays to ensure measurement reproducibility and calibration consistency.
Stated Advantages
Real-time measurements of analyte binding kinetics.
Nanostructures lithographically patterned onto standard glass coverslips allowing integration with traditional imaging techniques such as fluorescence and bright field microscopy.
Calibrated nanostructures enable quantitative determination of concentration as a function of time and space.
Documented Applications
Quantitative localized surface plasmon resonance biosensing to determine fractional occupancy values of surface-bound receptors over time.
Spatiotemporal mapping of receptor-ligand binding kinetics through LSPR imaging by projecting magnified nanostructure array images onto a CCD camera and monitoring binding kinetics.
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