Experimental and Analytical Framework for "Mix-and-Read" Assays Based on Split Luciferase.

Area of Science:

• Biochemistry
• Molecular Biology
• Assay Development

Background:

• Traditional immunodetection assays like ELISA require protein immobilization and multiple steps, limiting point-of-care applications.
• There is a need for simpler, more sensitive detection methods for analytes in solution.

Purpose of the Study:

• To develop a "mix-and-read" enzymatic complementation assay using split luciferase for sensitive analyte detection and quantification.
• To establish an analytical framework for optimizing and understanding the assay's performance.

Main Methods:

• Engineered protein binders targeting specific analytes were fused to nonactive luciferase fragments, creating biosensor probes.
• Yeast surface display was used to isolate high-affinity binding proteins against model targets (lysozyme and Sso6904).
• Analyte detection was achieved through luciferase complementation and subsequent chemiluminescence measurement.

Main Results:

• The split luciferase assay enabled sensitive detection of analytes in solution at low picomolar concentrations.
• An equilibrium binding model was developed to correlate binding affinities, assay parameters, and detection limits.
• The assay demonstrated modularity and simplicity, reducing the need for extensive washing steps.

Conclusions:

• The developed experimental and analytical framework provides a foundation for creating versatile split luciferase-based biosensors.
• This approach offers a simplified and sensitive alternative to traditional immunoassays for various detection applications.