Crowding Agents for Molecule Loading into Sample Wells
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Solution Overview
Problem
Current methods for preparing biological and chemical samples for analysis, particularly in next-generation sequencing, are laborious and time-consuming due to the complexity of biological samples and the restrictive size of sample wells, limiting the analysis of larger molecules.
Innovation Solution
The use of crowding agents, such as polysaccharides and polyethers, to selectively exclude molecules of interest from bulk solvent volumes, facilitating their loading into sample wells by reducing bulk volume and increasing local concentration, thereby improving the efficiency of sequencing template preparation and loading.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional single molecule technologies are used, then sample preparation can be performed, but the process becomes laborious and time-consuming
Solution Approach 1:
The invention changes the physical-chemical parameters of the sample environment by introducing crowding agents (polymers, proteins, or other macromolecules) that alter the volumetric availability and concentration dynamics, enabling faster and more efficient sample preparation without manual intervention
Solution Approach 2:
The system enables self-loading of molecules into sample wells through the autonomous action of crowding agents that naturally drive molecules into available volume spaces, eliminating the need for manual or mechanically assisted loading procedures
2Productivity
If sample well size is reduced for single molecule analysis, then parallel analysis capability increases, but the size of molecules that can be analyzed becomes restricted
Solution Approach 1:
The invention creates different local environments within the sample well: regions with high crowding agent concentration that exclude large molecules from bulk solution, and regions at the well bottom with available volume that accommodate large molecules for analysis, thus enabling both small well size and large molecule analysis
Solution Approach 2:
The invention utilizes the vertical dimension within the sample well by creating a gradient of molecular exclusion zones, where crowding agents occupy upper regions and exclude large molecules from bulk solution, while leaving the bottom region accessible for molecule capture and analysis
3Productivity
If crowding agent concentration is increased to improve loading efficiency, then molecule exclusion from bulk volume improves, but selectivity between target molecules and other components may be compromised
Solution Approach 1:
The invention optimizes the parameter of crowding agent concentration to achieve maximum loading efficiency while preserving selectivity, using sufficient but not excessive concentrations that maintain the ability to distinguish target sequencing templates from other sample components
Solution Approach 2:
The crowding agent acts as an intermediary that indirectly facilitates molecule loading by modifying the bulk solution properties and creating volumetric exclusion zones, rather than directly interacting with or binding to target molecules, thus preserving selectivity
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 loading efficiency of sequencing templates into sample wells, allowing for the analysis of larger molecules and reducing the time required for sample preparation, while maintaining the integrity of the sequencing reaction.
Implementation Method 1
contacting the sample with a crowding agent that excludes the molecule of interest from bulk volumes of the sample
Implementation Method 2
a layer of crowding agent is applied at the upper surface of the sample... the crowding agent excludes the molecule of interest from bulk volumes of the sample
Data Source
Figure 1
Figure 2
Figure 3A~3B
AI summary
Methods of loading a molecule of interest into a sample well are provided. In some aspects, methods of loading a molecule of interest into a sample well involve loading a molecule of interest into a sample well in the presence of a crowding agent and/or a condensing agent. In some aspects, methods of loading a sequencing template into a sample well are provided.