Single Protein Optical Imaging via Evanescent Field Oscillation
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Solution Overview
Problem
Current protein analysis technologies, such as liquid chromatography, mass spectrometry, and Western Blot, are time-consuming, destructive, and lack single molecule detection capability, failing to provide real-time information on protein size, charge, mobility, and conformational changes.
Innovation Solution
A method involving tethering single protein molecules to an ITO-coated glass surface using flexible polymer linkers, applying an alternating electric field to drive oscillation, and using evanescent light to generate scattered images for size, charge, and mobility determination through Fast Fourier Transform analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional protein analysis technologies (LC, MS, Western Blot) are used, then proteins can be separated and identified based on physical characteristics, but the process is time-consuming, destructive, and lacks single molecule detection capability
Solution Approach 1:
The patent replaces conventional mechanical separation systems (LC, electrophoresis) with an optical detection system based on surface plasmon resonance. Single molecules are detected directly on a sensor surface without mechanical separation, enabling real-time observation of individual protein molecules while maintaining identification capability through their optical properties.
Solution Approach 2:
The patent creates an optical copy or image of single protein molecules through surface plasmon resonance imaging. Instead of physically separating and analyzing molecules, the system generates optical representations that preserve molecular information, allowing simultaneous detection of multiple parameters without destroying the original molecules.
2Loss of information
If size-based detection methods are used, then protein size can be measured, but other important properties like charge, mobility, and conformational changes cannot be simultaneously detected
Solution Approach 1:
The patent implements a multi-functional detection system where surface plasmon resonance imaging simultaneously measures multiple protein properties including size, charge, mobility, and conformational changes. A single experimental setup provides comprehensive molecular information that would otherwise require multiple separate techniques, achieving universality in protein characterization.
3Measurement precision
If label-free detection methods are used, then intrinsic protein properties can be measured, but the signal from single molecules is extremely weak and difficult to detect
Solution Approach 1:
The patent enhances the detection sensitivity by optimizing key parameters of the surface plasmon resonance system, including the metal film thickness, refractive index matching, and angular configuration. These parameter adjustments maximize the interaction between evanescent light and single molecules, amplifying the already weak label-free signals to detectable levels while maintaining the advantages of label-free detection.
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 accurate, label-free detection and identification of single proteins, measuring size, charge, and mobility simultaneously, and monitoring conformational changes in real-time without protein separation or denaturation.
Implementation Method 1
Incident light is directed onto the coated surface from an angle to generate an evanescent field and produce scattered light
Implementation Method 2
The single protein molecules are driven into oscillation by applying an alternating electric field to the coated surface
Implementation Method 3
An additional innovation is the discovery of interference of light from the ITO surface and from the protein molecules, which produces high contrast images for small objects (protein molecules)
Data Source
AI summary
A method for optical imaging of single protein molecules including tethering single protein molecules via a flexible polymer linker to a glass slide having a surface coated with an indium tin oxide (ITO) so that the single protein molecules are tethered to the coated surface. The single protein molecules are driven into oscillation by applying an alternating electric field to the coated surface and the glass slide is located in the field of view of an objective lens. Incident light is directed onto the coated surface from an angle to generate an evanescent field and produce scattered light. The scattered light is collected and imaged by a CMOS imager to record a sequence of images of the scattered light. A Fast Fourier Transform (FFT) filter is applied to each pixel of the recorded image sequence to produce an oscillation amplitude image from which size, charge, and mobility of the plurality of single protein molecules can be determined.