Label-Free Biomolecule Detection via Plasmonic Scattering Microscopy
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Solution Overview
Problem
Current techniques for detecting intracellular proteins and protein complexes, such as single-cell western blotting, require large numbers of cells and denature protein complexes, limiting the detection of rare or low-abundant proteins and altering their native composition and function.
Innovation Solution
A label-free imaging approach using plasmonic scattering microscopy to detect unlabeled biomolecules by measuring scattered light from single molecules, which allows for non-destructive analysis of intact protein complexes and distinguishes specific from nonspecific binding, enabling the detection of unlabeled biomolecules with minimal sample disruption.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional PAGE and western blot are used to detect proteins, then the detection method is simple and established, but a substantial number of cells (at least thousands) are required to reach sufficient signal-to-noise ratio, limiting the detection of rare cells or low-abundant proteins
Solution Approach 1:
The patent replaces conventional mechanical/chemical separation methods (PAGE, western blot) with optical detection methods. Specifically, it uses light scattering microscopy to directly image individual protein complexes in situ within cells, eliminating the need for bulk sample preparation and cell lysis. This substitution enables detection with far fewer cells (as few as 1-10 cells) while maintaining or improving measurement precision.
Solution Approach 2:
The patent changes the detection parameter from bulk signal averaging (requiring thousands of cells) to single-molecule light scattering signal detection. By detecting the scattering intensity of individual protein complexes, the method achieves sufficient signal-to-noise ratio with minimal cells. The scattering intensity serves as a direct measure of molecular weight, providing both sensitivity and compositional information.
2Measurement precision
If single-cell western-blot (scWB) is used to improve detection sensitivity, then single cell resolution is achieved, but protein complexes are denatured, making it difficult to interpret their native composition and function in cells
Solution Approach 1:
The patent replaces the denaturing chemical processes of scWB (SDS-PAGE, Western blotting) with non-destructive optical imaging. Light scattering microscopy allows direct visualization of intact protein complexes within living or fixed cells without chemical denaturation. The scattered light intensity provides information about molecular weight and composition while the complexes remain in their native conformation.
Solution Approach 2:
The patent enables protein complexes to serve their own detection function through their intrinsic light scattering properties. No external labels, fluorophores, or tags are required - the complexes themselves scatter light proportionally to their molecular weight, allowing self-detection and self-characterization while maintaining native structure and function.
3Stability of the object's composition
If single-molecule fluorescence techniques (SiMPull, single-molecule FRET) are used for sensitive and non-destructive detection, then protein complexes can be measured with intact structure, but fluorescent tags may interact with off-target proteins or alter interaction affinity, and fluorescence is not applicable for long-term imaging due to photobleaching
Solution Approach 1:
The patent eliminates the need for fluorescent tags by utilizing the intrinsic light scattering properties of protein complexes themselves. The complexes scatter light based on their molecular weight without requiring any external labeling. This self-service detection method avoids all tag-related artifacts including off-target interactions, altered binding affinity, and photobleaching, enabling reliable long-term imaging and binding kinetics analysis.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables the detection of unlabeled biomolecules with high sensitivity and specificity, retaining the native structure of protein complexes, and allows for real-time binding kinetics analysis using as few as several cells, improving the detection of rare or low-abundant proteins.
Implementation Method 1
The methods and related aspects of the present disclosure typically measure scattered light from single molecules that are not fluorescently labelling
Implementation Method 2
the chamber comprises a fluidic material and at least first and second inner surfaces, wherein a cell population is disposed on the first inner surface, and wherein the second inner surface is coated with a metallic layer that is configured to create surface plasmon resonance
Data Source
AI summary
Provided herein are methods of detecting unlabeled biomolecules. In some embodiments, the methods include disrupting a cell population sufficient to release unlabeled biomolecules from the cell population to produce released biomolecules in which the cell population is disposed on a first inner surface of a chamber that is disposed substantially within a fluidic device and in which the chamber comprises a fluidic material. In some embodiments, the methods also include binding the released biomolecules to a second inner surface of the chamber to produce surface-bound biomolecules, introducing an incident light toward the second inner surface of the chamber concurrent with, and/or after, producing the surface-bound biomolecules, and detecting light scattered by the surface-bound biomolecules over a duration to produce a set of biomolecule imaging data. Related fluidic devices, systems, and computer readable media are also provided.