Enzymatically Erasable Detection Conjugates With Cell Barcodes
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Solution Overview
Problem
Existing methods fail to analyze the genetic information of a single cell within a tissue or cell culture context, and repeated staining with conjugates interferes with subsequent detection steps.
Innovation Solution
A conjugate with an enzymatically degradable spacer linking a detection moiety and an antigen recognizing moiety with an oligonucleotide barcode, allowing erasable labeling and sequencing of single cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If a conjugate with detection moiety and antigen recognizing moiety is used for cell detection, then detection capability is improved, but the detection moiety cannot be removed to allow repeated detection steps
Solution Approach 1:
The conjugate is divided into separate functional components: an antigen recognizing moiety (e.g., antibody) and a detection moiety (e.g., fluorophore), connected by a degradable spacer. This segmentation allows the detection moiety to be removed after use while preserving the antigen recognizing moiety on the cell, enabling repeated detection steps with different detection moieties.
Solution Approach 2:
A degradable spacer acts as an intermediary component between the antigen recognizing moiety and the detection moiety. This spacer contains cleavable bonds (e.g., peptide bonds, ester bonds) that can be hydrolyzed by cellular enzymes or external agents, allowing controlled separation of the detection moiety from the antigen recognizing moiety after detection.
2Adaptability or versatility
If multiple conjugates are used for sequential detection, then multiple phenotypes can be detected, but previous labels interfere with subsequent detection
Solution Approach 1:
After detection, the degradable spacer is cleaved to release and remove the detection moiety from the cell surface, while the antigen recognizing moiety remains bound to the cell. This discarding of the detection moiety eliminates interference with subsequent detection steps, allowing multiple phenotypes to be detected sequentially using different conjugates.
3Measurement precision
If barcode moiety is added for genetic information identification, then single cell identification capability is improved, but conjugate complexity increases
Solution Approach 1:
The conjugate merges multiple functional moieties into a single molecular assembly: the antigen recognizing moiety for cell targeting, the degradable spacer for controlled release, and the oligonucleotide barcode for genetic identification. This merging allows simultaneous achievement of cell-specific targeting and genetic information encoding without requiring separate conjugates.
Solution Approach 2:
The degradable spacer provides multi-functionality by serving as both a structural connector during detection and a controlled-release mechanism after detection. This universal component enables the conjugate to perform both detection and barcode preservation functions without requiring additional specialized components.
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 multiple identification steps on single cells within tissues by erasing the detection moiety, preserving barcode moieties for genetic information linkage, facilitating sequential analysis without interference.
Implementation Method 1
the conjugate is provided with an enzymatically degradable spacer
Data Source
AI summary
The invention is directed to a conjugate having the general formula (I)Xn—P—YmBo (I)with X is an detection moiety,P is a spacer unit,Y an antigen recognizing moiety,B an oligonucleotide comprising 2 to 100 nucleotide residuesand n, m, o are independent integers between 1 and 100wherein P and B are covalently bound to Y and X is covalently bound to P and wherein X is erasable.Further, the invention is directed to a library of such conjugates and a method of detecting target cells utilizing the conjugates or the library of conjugates.