Blocking probes for analyte binding specificity
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Solution Overview
Problem
Current methods for analyzing spatial heterogeneity in biological samples face challenges in enhancing the specificity of analyte binding, particularly when a second capture step is involved, as they fail to effectively address background noise and premature hybridization of capture binding domains with capture probes, leading to reduced sensitivity and specificity.
Innovation Solution
The method involves using a substrate with capture probes featuring spatial barcodes and capture domains, where the capture binding domain is initially blocked using blocking probes or artificial nucleic acids like caged nucleotides, allowing for controlled hybridization and enhanced specificity of analyte binding by preventing premature interactions and releasing the block at the appropriate time for specific binding.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the capture binding domain is left unblocked to increase binding efficiency, then the capture efficiency improves, but background noise increases due to premature hybridization with capture probes
Solution Approach 1:
The capture binding domain is pre-blocked with a blocking probe before the analyte binding assay is performed. This preliminary blocking action prevents premature hybridization between the capture binding domain and capture probes on the substrate, thereby reducing background noise while maintaining high capture efficiency when the blocking probe is subsequently removed
Solution Approach 2:
A blocking probe is introduced as an intermediary molecule that temporarily occupies the capture binding domain. This intermediary prevents direct interaction between the capture binding domain and capture probes during storage and preparation, reducing background noise. The blocking probe is then removed to allow specific analyte binding
2Measurement precision
If the capture binding domain is blocked to reduce background noise, then the specificity improves, but the binding efficiency decreases
Solution Approach 1:
The blocking probe is periodically applied and removed in a controlled manner. The blocking probe is applied before the assay to establish high specificity, then removed at the appropriate time to enable efficient analyte binding. This periodic action allows the system to switch between high specificity mode and high efficiency mode as needed
3Measurement precision
If a second capture step is added to enhance specificity, then the measurement precision improves, but the device complexity increases
Solution Approach 1:
The blocking probe application step is merged with the sample preparation phase, and the removal of the blocking probe is combined with the initiation of the analyte binding assay. This merging of steps reduces the overall workflow complexity while maintaining the enhanced specificity provided by the second capture step
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
This approach significantly increases the specificity of analyte binding, reducing background noise and improving the resolution of spatial analysis by ensuring that analytes bind specifically to their target locations, thereby enhancing the accuracy of analyte localization and abundance determination in biological samples.
Implementation Method 1
a capture binding domain that hybridizes to the capture domain of the capture probe
Implementation Method 2
wherein a portion of the capture binding domain is blocked
Data Source
AI summary
Methods for enhancing specificity of an analyte binding moiety or probe oligonucleotide to an analyte are provided herein. For example, methods provided herein include blocking a capture binding domain, thereby preventing hybridization to the capture domain of the capture probe affixed to a substrate. Further methods include releasing the block from the capture binding domain, thereby allowing the capture binding domain to specifically bind to the capture domain of the capture probe on the substrate.