Calibrating single plasmonic nanostructures for quantitative biosening
Inventors
Raphael, Marc P. • Christodoulides, Joseph A. • Byers, Jeff M.
Assignees
Publication Number
US-10345287-B2
Publication Date
2019-07-09
Expiration Date
2033-09-27
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Abstract
A method for calibrating multiple nanostructures in parallel for quantitative biosensing using a chip for localized surface plasmon resonance (LSPR) biosensing and imaging. The chip is a glass coverslip compatible for use in a standard microscope with at least one array of functionalized plasmonic nanostructures patterned onto it using electron beam nanolithography. The chip is used to collect CCD-based LSPR imagery data of each individual nanostructure and LSPR spectral data of the array. The spectral data is used to determine the fractional occupancy of the array. The imagery data is modeled as a function of fractional occupancy to determine the fractional occupancy of each individual nanostructure.
Core Innovation
The invention provides a method for calibrating multiple nanostructures in parallel for quantitative biosensing using a chip configured for localized surface plasmon resonance (LSPR) biosensing and imaging. The chip comprises a glass coverslip compatible with standard microscopes, patterned with at least one array of functionalized plasmonic nanostructures fabricated by electron beam nanolithography. This setup enables the simultaneous acquisition of CCD-based LSPR imagery data for each nanostructure and spectral data of the entire array.
The spectral data from the array is used to determine the fractional occupancy of the array. The imagery data, which encodes similar biochemical information, is modeled as a function of this fractional occupancy to ascertain the fractional occupancy of individual nanostructures. Thus, the method allows quantitative calibration of single nanostructures with nanomolar sensitivity and a temporal resolution of 225 milliseconds, facilitating the spatial mapping of surface-bound receptor occupancy.
The background articulates that while LSPR biosensing offers label-free detection with high sensitivity, a significant challenge is calibrating each individual nanostructure for quantitative analysis. Sequential calibration of numerous nanostructures is time-consuming and impractical. The invention solves this by providing a batch-mode calibration allowing simultaneous calibration of hundreds of nanostructures. This enables their use in quantitative measurements of biochemical parameters such as kinetic rates and analyte concentrations, thus advancing LSPR applications in complex biological environments.
Claims Coverage
The patent contains one independent claim detailing a method for parallel calibration of nanostructures for quantitative biosensing. The main inventive features focus on fabrication, simultaneous measurement, analysis, and selective calibration steps.
Fabrication of nanostructured arrays on glass coverslips by electron beam nanolithography
Fabricating at least one array of functionalized plasmonic nanostructures for LSPR biosensing and imaging on a glass coverslip compatible for use in a standard microscope using electron beam nanolithography.
Simultaneous measurement of LSPR imagery and spectral data
Simultaneously measuring CCD-based LSPR imagery data of each individual nanostructure and LSPR spectral data of the array to gather comprehensive sensor information.
Determination of fractional occupancy of entire array based on spectral data
Analyzing the LSPR spectral data of the array to determine the fractional occupancy of the entire array.
Determination of fractional occupancy of individual nanostructures from imagery
Analyzing the imagery data to determine the fractional occupancy of individual nanostructures by correlating imagery with spectral measurements.
Creation of image map highlighting deviations between individual and array fractional occupancy
Creating an image map to show deviations between fractional occupancy of the entire array and the fractional occupancy of individual nanostructures for quality assessment.
Selective calibration of nanostructures based on deviation threshold
Calibrating for quantitative biosensing only individual nanostructures within a defined deviation threshold while ignoring nanostructures outside this defined deviation to ensure accuracy and reliability.
The inventive features encompass fabrication of functionalized plasmonic nanostructure arrays, dual simultaneous data acquisition, analytical determination of fractional occupancy both at the array and individual nanostructure levels, and a methodology for selective calibration using deviation mapping to enable efficient and precise quantitative biosensing.
Stated Advantages
Fabrication by electron-beam lithography results in highly uniform nanostructures, confirmed by size and spectral characterization.
Hundreds of individual nanostructures can be calibrated simultaneously in parallel.
The method integrates into standard wide-field microscopy setups enabling complementary imaging techniques such as fluorescence and differential interference contrast imaging.
The LSPR imaging mode offers spatial resolutions limited only by nanostructure size and temporal resolutions in the hundreds of milliseconds, allowing measurement of fast biomolecular kinetics.
The technique functions effectively in complex environments such as live-cell microscopy without sensor biofouling, maintaining sensor performance in serum-free medium.
Documented Applications
Quantitative biosensing of anti-c-myc antibodies, including secreted antibodies directly from hybridoma cell supernatants, demonstrating applicability to molecular and cell biology.
High-density proteomic arrays enabling multiplexed biosensing with nanomolar sensitivity and minimal sample volumes.
Imaging and mapping of analyte concentration gradients in complex live-cell environments.
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