Label-Free Single-Molecule Immunoassay for Direct Protein Detection
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Solution Overview
Problem
Current single-molecule immunoassays rely on end-point detection and require signal amplification through labeling, which leads to matrix effects, autofluorescence interference, and inability to measure biomarkers directly in undiluted complex sample matrices like whole blood.
Innovation Solution
A real-time mass imaging-based label-free single-molecule immunoassay (LFSMiA) using plasmonic scattering microscopy for background reduction and dynamic tracking of single binding events, combined with a Gaussian Bayes algorithm for improved measurement precision.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If signal amplification through labeling is used, then detection sensitivity is improved, but matrix effects and autofluorescence interference increase
Solution Approach 1:
The invention extracts and removes the labeling step from the immunoassay process, using label-free detection methods that directly measure binding events without fluorescent or enzymatic labels, thereby eliminating autofluorescence interference and matrix effects while maintaining single-molecule detection sensitivity
Solution Approach 2:
The invention replaces optical detection methods (fluorescence) with mass-based detection (surface plasmon resonance), substituting the mechanical/optical system with a different physical principle that is not susceptible to autofluorescence and matrix effects, enabling direct detection in complex sample matrices
2Measurement precision
If fluorescence-based signal amplification is used, then detection limit is improved, but photobleaching and autofluorescence occur
Solution Approach 1:
The invention replaces fluorescence-based optical detection with surface plasmon resonance mass detection, eliminating photobleaching issues entirely by using a non-optical excitation mechanism that measures mass changes directly, providing stable long-term signal detection without signal decay
3Measurement precision
If enzyme amplification and nanoparticle labeling are used, then signal detection is improved, but nonspecific binding to substrate increases
Solution Approach 1:
The invention extracts and eliminates the enzyme amplification and nanoparticle labeling steps, using direct label-free detection that measures binding events without additional reagents, thereby removing the sources of nonspecific binding to substrate while maintaining adequate signal detection through single-molecule sensitivity
Solution Approach 2:
The invention enables the binding complexes to serve their own detection function without requiring external labels or amplification reagents, where the mass of the binding event itself provides the detection signal through surface plasmon resonance, eliminating nonspecific interactions introduced by additional reagents
4Device complexity
If end-point detection is used, then assay simplicity is maintained, but real-time kinetic information is lost
Solution Approach 1:
The invention transitions from discrete end-point detection to continuous real-time monitoring of binding events, where the surface plasmon resonance signal continuously reports on binding kinetics as events occur, providing uninterrupted kinetic information without complicating the assay workflow
Solution Approach 2:
The invention implements real-time feedback by continuously monitoring binding events and using this information to track association and dissociation kinetics dynamically, allowing the system to provide immediate information about binding processes rather than waiting for end-point measurement
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 ultra-sensitive and direct protein detection at the single-molecule level in neat blood sample matrices with a sub-femtomolar limit of detection and an eight-log dynamic range, while minimizing matrix effects and eliminating the need for signal amplification.
Implementation Method 1
Featuring plasmonic scattering microscopy-based mass imaging
Data Source
AI summary
Provided herein are methods of detecting target molecules. The methods include contacting a sample comprising the target molecule with a substrate that comprises a plurality of capture antibodies, or antigen binding portions thereof, that specifically bind to the target molecule to form captured target molecules, and contacting the captured target molecules with a plurality of detection antibodies, or antigen binding portions thereof, that bind to the captured target molecules to form target molecule complexes. The methods also include taking images of the target molecule complexes to produce imaged target molecule complexes, and quantifying an amount of target molecules in the sample using the imaged target molecule complexes. Additional methods as well as related devices and systems are also provided.