DESI-MS Enzyme Assays for High-Throughput Label-Free Screening
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Solution Overview
Problem
Current enzyme assays are limited in their ability to efficiently monitor enzymatic reactions and screen for inhibitors or reactivators in a high-throughput and label-free manner, particularly for drug discovery and biochemical studies.
Innovation Solution
A high-throughput platform combining desorption electrospray ionization-mass spectrometry (DESI-MS) to create droplets and thin films for enzymatic reactions, allowing for the analysis of product distribution and monitoring of enzymatic reactions by generating ions from discrete spots on a substrate, which can be used to determine the progress of enzymatic reactions and assess the effect of test compounds as inhibitors or reactivators.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional enzyme assays are used to monitor enzymatic reactions, then the measurement can be performed with simple equipment, but the throughput is low and labeling compounds are required
Solution Approach 1:
The assay plate is divided into multiple discrete spots, each containing a separate enzymatic reaction. The DESI-MS system sequentially analyzes each spot independently, enabling high-throughput screening of 384 reactions simultaneously while maintaining simple reaction setups in each spot
Solution Approach 2:
The mass spectrometry platform serves multiple functions: it creates droplets for reactions, monitors reaction progress through product detection, and provides quantitative analysis without requiring different equipment for each function. This multi-functionality increases throughput while managing system complexity
2Measurement precision
If labeled compounds are used in enzyme assays, then the detection sensitivity can be improved, but the assay complexity and cost increase
Solution Approach 1:
The mass spectrometry detector inherently provides high sensitivity detection without requiring external labels on the compounds. The system uses the intrinsic mass-to-charge ratio of molecules for detection, eliminating the need for fluorescent, radioactive, or enzymatic labels while maintaining excellent measurement precision
Solution Approach 2:
The labeling step is completely removed from the assay procedure. The DESI-MS system directly detects and quantifies substrates and products based on their mass characteristics, extracting the detection function from label-dependent methods to label-free analysis
3Productivity
If high-throughput enzyme assays are performed, then the screening efficiency increases, but the analysis time per sample must be reduced
Solution Approach 1:
The DESI-MS system continuously scans across the plate, analyzing spots in rapid succession without interruption. This continuous scanning approach minimizes idle time between samples and maintains high throughput while achieving sufficient analysis depth for each spot
Solution Approach 2:
The system uses periodic scanning cycles where the DESI probe rapidly moves between spots in a systematic pattern. This periodic action allows efficient time management across 384 spots, balancing throughput requirements with adequate analysis time for each reaction
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 rapid and sensitive monitoring of enzymatic reactions, allowing for the completion of 384 inhibition reactions in less than 15 minutes with excellent sensitivity, and facilitates the determination of kinetic parameters and inhibition constants without the need for labeled compounds, thereby supporting drug development for conditions like Alzheimer's disease and counteracting chemical warfare agents.
Implementation Method 1
directing sequentially a discharge from an ionization source onto each of the plurality of discrete spots to sequentially desorb the substrate and/or product(s) from each of the discrete spots and sequentially generate ions of the substrate and/or product(s)
Data Source
AI summary
The invention generally relates to high-throughput label-free enzymatic bioassays using desorption electrospray ionization-mass spectrometry (DESI-MS).