Microwell Array for Multiplexed Single-Cell Protein Detection
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Solution Overview
Problem
Current technologies lack the capability for high-throughput, highly multiplexed measurement of secreted proteins from single cells, which is essential for accurate monitoring of cellular immunity and effective pharmacological therapies.
Innovation Solution
A device comprising a microwell array and a capture agent array, where the microwell array has a plurality of individual microwells for containing single cells, and the capture agent array has immobilized capture agents that specifically bind to secreted proteins, allowing for spatially encoded multiplexed detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If intracellular cytokine staining (ICS) is used to detect secreted proteins, then single cell analysis capability is achieved, but cells must be fixed and are no longer alive for further studies
Solution Approach 1:
The system segments the detection process by using microwell arrays to physically isolate individual cells while maintaining their viability. Each microwell acts as an independent compartment that allows single-cell analysis without requiring cell fixation, thus resolving the contradiction between single-cell detection capability and cell recovery for further studies.
2Adaptability or versatility
If ELISpot is used for single cell secretion analysis, then cell viability is maintained, but multiplexing capability is limited
Solution Approach 1:
The system transitions from traditional planar ELISpot formats to a three-dimensional microwell array configuration. This dimensional change enables simultaneous single-cell isolation and multiplexed protein detection by stacking multiple capture agent layers around each microwell, thereby increasing multiplexing capability while maintaining cell viability.
Solution Approach 2:
The system implements a nested structure where capture agents are organized in concentric layers or nested compartments within and around each micrawell. This nesting allows multiple capture agents targeting different proteins to be accommodated in a compact space, enabling high-level multiplexing without compromising the single-cell isolation and viability.
3Quantity of substance
If highly multiplexed measurement is implemented, then detection of multiple proteins is achieved, but throughput and scalability are reduced
Solution Approach 1:
The system uses identical, replicated microwell units arranged in large arrays, where each micrawell functions as an independent detection unit. This copying approach allows parallel processing of thousands of single cells simultaneously, maintaining high throughput while enabling multiplexed detection of multiple proteins through the standardized capture agent configurations in each replicated unit.
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 simultaneous detection of multiple secreted proteins from a large number of single cells, providing insights into cellular heterogeneity and phenotypic variability, which is crucial for disease diagnosis and therapeutic monitoring.
Implementation Method 1
a microwell array having a plurality of individual microwells in uniform arrangement, at least some of the plurality of individual microwells having a length of greater than 50 μm and configured to contain an isolated single cell in a sub-nanoliter volume of contents
Implementation Method 2
The capture agent array comprises a plurality of immobilized capture agents, each immobilized capture agent capable of specifically binding to one of the plurality of compounds
Data Source
AI summary
The present invention relates to a system, device, and method for the high throughput multiplexed detection of a wide number of compounds. The invention comprises of a microwell array coupled to a capture agent array to form a plurality of interfaces between a microwell and a set of immobilized capture agents. The set of capture agents comprises a plurality of distinguishable features, with each feature corresponding to the detection of a particular compound of interest. In certain embodiments, each microwell is configured to contain a single cell. The invention is therefore capable of performing a high throughput analysis of single cell profiles, including profiles of secreted compounds.