Addressable Protein Arrays for Single-Molecule Affinity Kinetics
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Solution Overview
Problem
Existing methods for selecting and characterizing affinity reagents often focus on high affinity and specificity for a single epitope, limiting their versatility and applicability in applications requiring promiscuous binding or high avidity, which is crucial for identifying a variety of analytes and reducing dissociation rates.
Innovation Solution
A method and system for characterizing affinity reagents by contacting them with binding targets, detecting their binding at single-analyte resolution at multiple timepoints, and determining association and dissociation rates based on quantity differences, using a solid support with resolvable addresses, fluidics, optical detection, and a processor to analyze optical signals.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If selection methods focus on high affinity and specificity for a single epitope, then binding strength is improved, but versatility and promiscuity are reduced
Solution Approach 1:
The system dynamically characterizes affinity reagents by measuring association and dissociation rates at multiple timepoints, allowing the same reagent to be evaluated for both high affinity (slow dissociation) and promiscuity (binding to multiple epitopes), thus resolving the contradiction between binding strength and versatility
Solution Approach 2:
The invention changes the evaluation parameters from static affinity measurements to dynamic kinetic measurements (association rate kon and dissociation rate koff), enabling comprehensive characterization of reagents that can exhibit both high affinity and promiscuous binding behavior
2Measurement precision
If single-analyte resolution detection is used, then measurement precision is improved, but device complexity increases
Solution Approach 1:
The system segments the detection process into discrete addressable locations on a solid support, with each location containing a single binding target. This spatial segmentation enables single-analyte resolution by allowing independent detection at each address while using standardized detection components
Solution Approach 2:
The invention introduces an intermediary solid support with addressable locations as a mediator between the affinity reagents and the detection system. This intermediary enables precise localization and single-analyte resolution while simplifying the overall system architecture by providing a structured platform for controlled interactions
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 efficient selection and characterization of affinity reagents with desired binding properties, allowing for high avidity and promiscuity, and identifying subpopulations with varying binding behaviors, enhancing research and clinical applications.
Implementation Method 1
each affinity reagent comprises a detectable label that is configured to produce optical signals
Data Source
AI summary
The present disclosure provides methods of determining association rates or dissociation rates between affinity reagents and proteins. The methods can be configured to monitor a large number of proteins in parallel, for example, using arrays of proteins that are contacted with solutions containing affinity reagents. The methods can be further configured to detect the arrayed proteins at single-molecule resolution. Accordingly the methods allow a large population of proteins to be monitored on an individual basis. As such binding kinetics and thermodynamics can be determined on a population level while allowing individual interactions to be evaluated.