Enzyme-Activated G-Quadruplex Synthesis for in Situ Label-Free Detection and Bioimaging of Cell Apoptosis

Mass spectrometry (MS) is known for its label-free detection of substrates and products from a variety of enzyme reactions. Recent hardware improvements have increased interest in the use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS for high-throughput drug discovery. Despite interest in this technology, several challenges remain and must be overcome before MALDI-MS can be integrated as an automated “in-line reader” for high-throughput drug discovery. Two such hurdles include in situ sample processing and deposition, as well as integration of MALDI-MS for enzymatic screening assays that usually contain high levels of MS-incompatible components. Here we adapt our c-MET kinase assay to optimize for MALDI-MS compatibility and test its feasibility for compound screening. The pros and cons of the Echo (Labcyte) as a transfer system for in situ MALDI-MS sample preparation are discussed. We demonstrate that this method generates robust data in a 1536-grid format. We use the MALDI-MS to directly measure the ratio of c-MET substrate and phosphorylated product to acquire IC50 curves and demonstrate that the pharmacology is unaffected. The resulting IC50 values correlate well between the common label-based capillary electrophoresis and the label-free MALDI-MS detection method. We predict that label-free MALDI-MS-based high-throughput screening will become increasingly important and more widely used for drug discovery.

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Comparison of DNAzyme activity for the development of an immobilized heme sensor

MRS Advances ◽

10.1557/adv.2017.637 ◽

2018 ◽

Vol 3 (26) ◽

pp. 1491-1496

Author(s):

Natalie Hughes ◽

Nancy Nguyen ◽

Deanna-Kaye Daley ◽

Justin Grennell ◽

Amira Gee ◽

...

Keyword(s):

Self Assembly ◽

Point Of Care ◽

Label Free ◽

Detection Range ◽

Chemical Complexity ◽

Detection Strategy ◽

Detection Approach ◽

Label Free Detection ◽

G Quadruplex ◽

Care Systems

ABSTRACTPoint-of-care systems require highly sensitive, quantitative and selective detection platforms for the real-time multiplexed monitoring of target analytes. To ensure facile development of a sensor, it is preferable for the detection assay to have minimal chemical complexity, contain no wash steps and provide a wide and easily adaptable detection range for multiple targets. Current studies involve label-free detection strategy for relevant clinical molecules such as heme using G-quadruplex based self-assembly. We have explored the measurement of binding and kinetic parameters of various G-quadruplex/heme complexes which are able to self-associate to form a DNAzyme with peroxidase mimicking capabilities and are critical to nucleic acid research. The detection strategy includes immobilizing the G-quadruplex sequences within a polymer matrix to provide a self-assembly based detection approach for heme that could be translated towards other clinically relevant targets.

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