Sortase-Mediated Ligation for Multiplex Biological Detection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for detecting the co-expression of multiple disease markers in biological samples are complex, expensive, and require specialized equipment, making them difficult to perform and costly.
Innovation Solution
A method utilizing sortase-mediated ligation technology, where binding agents linked to sortase molecules and recognition sequences specifically bind to targets in a biological sample, allowing for the detection of concurrent presence of multiple targets through a simple and cost-effective process.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional methods (FRET, BiFC, PLA) are used to detect co-expression of multiple markers, then detection specificity and sensitivity are improved, but device complexity and cost increase
Solution Approach 1:
The detection system is segmented into separate functional components: binding agents with first sortase molecules for detecting first targets, and binding agents with second sortase molecules for detecting second targets. This segmentation allows independent optimization of each detection pathway while maintaining overall system simplicity and reducing the need for complex specialized equipment.
Solution Approach 2:
Sortase molecules serve as intermediary enzymes that mediate the detection process. The binding agents are operably linked to sortase molecules, which facilitate the detection of targets through a standardized mechanism. This intermediary approach simplifies the detection system by using a common enzymatic platform rather than requiring multiple specialized detection mechanisms.
2Measurement precision
If conventional methods are used to detect multiple targets, then measurement precision is improved, but reagent cost increases
Solution Approach 1:
The sortase molecule serves multiple functions: it acts as a detection mediator, enables target identification, and facilitates the binding agent operation. This multi-functionality reduces the need for multiple specialized reagents, thereby lowering overall reagent costs while maintaining detection sensitivity through the standardized sortase-based mechanism.
Solution Approach 2:
The detection system utilizes parameter changes in the sortase enzyme activity and binding agent interaction to achieve sensitive detection. By monitoring changes in sortase-mediated binding and signal generation, the system achieves high detection sensitivity without requiring expensive specialized reagents, as the sortase-based platform can be optimized through parameter adjustment rather than reagent expenditure.
3Measurement precision
If specialized equipment is required for conventional detection methods, then measurement precision is improved, but ease of operation deteriorates
Solution Approach 1:
The binding agents are designed to self-assemble with sortase molecules and automatically detect targets through the sortase-mediated mechanism. The system performs detection through inherent biochemical interactions without requiring complex external equipment or specialized operational procedures. This self-service approach maintains detection accuracy while significantly improving ease of operation, as the detection process occurs through natural biochemical binding and signal generation.
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 the specific and efficient detection of multiple targets in biological samples, reducing the need for expensive reagents and specialized equipment, while maintaining high specificity and sensitivity.
Implementation Method 1
a first binding agent operably linked to a first sortase molecule... a first sortase-mediated ligation of the sortase substrate to the first sortase recognition sequence
Data Source
AI summary
The disclosure provides methods for detecting the concurrent presence of at least two targets within a biological sample. The method includes contacting said biological sample with a first binding agent, said first binding agent operably linked to a first sortase molecule, wherein said first binding agent specifically binds to a first target; contacting said biological sample with a second binding agent, said second binding agent operably linked to a first sortase recognition sequence peptide, wherein said second binding agent specifically binds to a second target; adding a sortase substrate under conditions where a first sortase-mediated ligation of the sortase substrate to the first sortase recognition sequence will produce a ligation product, and detecting the ligation product wherein detection of said ligation product indicates the concurrent presence of the first target and the second target in the biological sample. Also disclosed are kits comprising reagents for performing the methods as claimed.