A sol-gel-derived acetylcholinesterase microarray for nanovolume small-molecule screening

Area of Science:

• Biochemistry
• Analytical Chemistry
• Materials Science

Background:

• Acetylcholinesterase (AChE) is a key enzyme in neurotransmission, making it a target for drug development.
• Existing AChE assays can be limited in throughput and applicability for screening large compound libraries.

Purpose of the Study:

• To develop a novel, sensitive, and high-throughput fluorimetric assay for acetylcholinesterase (AChE).
• To create functional AChE microarrays using sol-gel-derived silica for enzyme activity monitoring and inhibitor screening.

Main Methods:

• Developed a fluorimetric assay based on a disulfide-thiol interchange reaction using BODIPY FL l-cystine and thiocholine.
• Fabricated AChE microarrays by entrapping the enzyme in sol-gel-derived silica using contact pin-printing.
• Screened numerous sol-gel formulations and surfaces to optimize microarray fabrication and enzyme activity.
• Validated the assay by comparing it with the Ellman assay and determining the Z' factor for on-array performance.

Main Results:

• The fluorimetric assay demonstrated high sensitivity and was validated against the established Ellman assay.
• Optimized sol-gel/surface combinations yielded robust and highly active AChE microarrays.
• The on-array assay achieved a Z' factor of 0.60, indicating suitability for screening applications.
• Successfully screened two libraries of small molecules, including alkaloids and known bioactive compounds, for AChE inhibitory activity.

Conclusions:

• The developed sol-gel-based AChE microarrays provide a powerful platform for quantitative screening of enzyme inhibitors.
• This assay facilitates the identification of potential therapeutic agents targeting AChE activity.
• The methodology is adaptable for screening diverse compound libraries in a high-throughput format.