Spatial Array Capture Efficiency via Padlock Probe Rolling Circle Amplification
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Solution Overview
Problem
Current spatial analysis methods using capture probes are limited in capturing multiple analytes simultaneously due to low probe density and selection bias, which restricts the discovery of spatial heterogeneity in biological samples and is costly and laborious.
Innovation Solution
The method involves preparing a spatial array by contacting a substrate with capture probes and padlock probes, where the padlock probes are ligated and amplified using rolling circle amplification to increase capture efficiency, allowing for the detection of multiple analytes at high spatial resolution.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If traditional capture probes are used on spatial arrays, then the array can be manufactured with current technology, but the capture efficiency and sensitivity are limited due to low probe density
Solution Approach 1:
The probe construction is segmented into modular components: a capture sequence specific to the target analyte, a docking sequence for array attachment, and a padlock probe region. This modular design allows independent optimization of each component and simplifies the manufacturing process by enabling separate synthesis and assembly of probe elements.
Solution Approach 2:
Padlock probes are pre-synthesized and prepared before array assembly. The padlock probes contain predetermined sequences that will complementary hybridize to the capture probes, allowing for pre-optimization of probe design and reducing manufacturing complexity during the array assembly process.
2Adaptability or versatility
If traditional spatial analysis methods are used, then the methodology is simpler, but the ability to detect multiple analytes simultaneously is limited
Solution Approach 1:
The spatial array platform is designed with universal docking sequences that can bind multiple different padlock probes targeting various analytes. This universal docking mechanism allows a single array to simultaneously detect multiple analytes through different padlock probes, achieving multiplexing capability without requiring separate arrays for each analyte.
Solution Approach 2:
The padlock probe serves as an intermediary molecule that bridges the capture probe (attached to the array) and the target analyte. The padlock probe contains a capture sequence that hybridizes to the analyte and a docking sequence that binds to the capture probe, enabling flexible and specific detection of multiple analytes on a single array.
3Ease of operation
If pre-defined markers are used for spatial analysis, then the analysis protocol is simpler, but selection bias is introduced that limits discovery
Solution Approach 1:
The system enables dynamic changes in the captured parameters by allowing users to select different padlock probes with different capture sequences specific to various analytes of interest. This flexibility allows the same array platform to be reconfigured for different analytical goals without being limited to pre-defined markers, thereby maintaining operational simplicity while enhancing discovery capability.
4Productivity
If rolling circle amplification is used to amplify padlock probes, then capture efficiency increases, but the amplification process becomes more complex
Solution Approach 1:
The rolling circle amplification process is designed to be self-sustaining once initiated. The padlock probe structure with its complementary docking sequences enables automatic circularization and continuous amplification without requiring external intervention or complex control mechanisms, thereby achieving high capture efficiency with relatively simple process implementation.
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 enhances the capture efficiency and sensitivity of spatial arrays, enabling the simultaneous detection of multiple analytes at high spatial resolution, thereby overcoming the limitations of existing methods.
Implementation Method 1
hybridizing the first docking sequence to the first docking padlock sequence and hybridizing the second docking sequence to the second docking padlock sequence
Implementation Method 2
amplifying the ligated padlock probe, thereby generating a padlock probe sequence with multiple copies of the capture domain
Data Source
AI summary
This disclosure relates to methods for increasing capture efficiency of a spatial array using rolling circle amplification of a padlock probe that hybridizes to a capture probe. Also provided are methods for using such spatial arrays to detect a biological analyte in a biological sample.