Cleavable Biological Marker for Multiplexed Cell Identification
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Solution Overview
Problem
Current fluorescence microscopy techniques are limited by the low number of available fluorescent dyes, leading to poor identification of cell types and increased time and cost when trying to resolve ambiguities among a high number of markers in biological samples.
Innovation Solution
A marker system with a marker base and attachment structure that includes cleavage sites, allowing for sequential removal and replacement of reporters with unique dye combinations, enabling reliable identification of multiple structures without the need for reintroducing affinity reagents.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple fluorescent dyes are distributed to all markers in the same experiment, then the number of identifiable cell types increases, but the number of available fluorescent dyes is limited to only 1-5
Solution Approach 1:
The marker system is segmented into a marker base with affinity reagent and separate reporter molecules with fluorescent dyes. The attachment structure contains multiple attachment sites that can sequentially bind different reporters, allowing one marker base to represent multiple cell types through sequential marking cycles rather than requiring multiple dyes simultaneously
Solution Approach 2:
The system employs periodic marking cycles where reporters are sequentially introduced, imaged, then removed via cleavage sites. This periodic action allows the same marker base to be reused for identifying different cell types in successive cycles, effectively multiplying the identification capacity beyond the limited number of available fluorescent dyes
2Adaptability or versatility
If a large number of markers are used in close proximity, then more cell types can be identified, but ambiguities arise that require removing markers and repeating image acquisition
Solution Approach 1:
The system uses combinatorial dye codes where each reporter contains a unique combination of fluorescent dyes. This feedback mechanism allows unambiguous identification of markers even in close proximity through spectral unmixing and code decoding, eliminating the need to remove and reintroduce markers to resolve ambiguities
Solution Approach 2:
The attachment structure includes cleavage sites that allow selective removal of reporters after imaging. This enables the marker base to be recovered and reused in subsequent marking cycles with different reporters, reducing the need to reintroduce affinity reagents and minimizing time loss
3Measurement precision
If markers are removed and reintroduced to resolve ambiguities, then identification accuracy improves, but cost and time expenditure increase
Solution Approach 1:
The system performs preliminary action by introducing a combination of fluorescent dyes in unique codes during the initial marking cycle. This preliminary coding allows unambiguous identification of all markers in subsequent cycles without needing to remove and reintroduce them, thereby maintaining identification accuracy while eliminating repetitive experiments and associated costs
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 allows for the reliable identification of a high number of predetermined structures at reduced cost and time expenditure by resolving ambiguities through sequential marking and imaging cycles.
Implementation Method 1
a marker base having an affinity reagent configured to attach to the predetermined structure of the sample
Implementation Method 2
The attachment structure is capable of being cut at the at least one cleavage site by a cleving agent in order to remove at least one attachment site from the marker base
Implementation Method 3
Each reporter includes a linker structure having a complementary attachment site configured to attach to one of the two attachment sites of the attachment structure
Implementation Method 4
one or more fluorescent dyes that are either directly conjugated to the affinity reagent or attached to the affinity reagent by other means
Data Source
AI summary
A marker for marking a predetermined structure within a biological sample includes a marker base having an affinity reagent, and an attachment structure connected to the affinity reagent having two attachment sites. The attachment structure includes a cleavage site arranged between the attachment sites. The attachment structure is capable of being cut at the cleavage site by a cleaving agent in order to remove an attachment site from the marker base. The marker further includes at least two reporters. Each reporter includes a linker structure having a complementary attachment site configured to attach to one of the attachment sites, and a combination of at least two different fluorescent dyes. The combination of the at least two different fluorescent dyes is unique for each reporter. The complementary attachment site is unique for each reporter and configured such that each reporter attaches to a different attachment site of the marker base.