A novel fluorescence sensing system using a photochromism-based assay (P-CHROBA) technique for the detection of target proteins

Abstract

In the post-genomic era a number of biological technologies, including protein-detecting microarrays which can detect molecular interactions based on changes in fluorescence intensity, have been developed to investigate complicated protein functions and networks. However, the ability of such techniques to obtain reproducible and quantitative results can be compromised due to the need for the immobilization of capture agents and the labelling analytes with chromophores. In the present study, first we report the design and synthesis of photochromic spiropyran-containing peptides and then demonstrate a unique fluorescence sensing system comprising a photochromism-based assay (P-CHROBA) technique to distinguish between target proteins. The spiropyran moiety in the peptides exhibited characteristic physicochemical properties in the SP-to-MC isomerization (thermocoloration) and the MC-to-SP photoisomerization (photobleaching) depending upon changes in micro-environments such as the dielectric constants of solvents and steric hindrances generated by molecular interactions. We attempted to detect protein–peptide interactions using reproducible MC-to-SP photoisomerization properties by monitoring the fluorescence decay of the MC form in the peptide. This can reduce background fluorescence signals caused by emission from excess reagents and avoid the laborious introduction of probing molecules to analytes and the immobilization of capture agents onto solid surfaces. The protein fingerprints (PFPs) based on the photoisomerization properties could successfully distinguish between six different model proteins, and the combination of the P-CHROBA and PFP technique would be a powerful tool for profiling target proteins with reproducible and reliable results.