A Bioassay Based on the Ultrafast Response of a Reporter Molecule

Abstract

The capability of using ultrafast detection technologies for a fast analysis of biomolecular reactions has been explored. As an example, the ultrafast response of tetramethylrhodamine (TMR)-labeled bovine serum albumin (BSA) as a function of different extents in proteolytic cleavage was investigated. The authors compared 4 samples of masses differing over several orders of magnitude: untreated, TMR-labeled BSA (66 kDa), TMR-labeled BSA treated with elastase (6-33 kDa) and with subtilisin (< 3 kDa), and the pure label TMR (0.4 kDa). A direct comparison with gel electrophoresis revealed that various ultrafast parameters give robust information about the progress of the proteolytic cleavage. The authors found the ratio of the transient absorption signal observed at 0 psec and 50 psec after excitation (lambda(Pump) = 540 nm, lambda(Probe) = 570 nm) to be the most precise parameter for determining the cleavage. This parameter allowed determining the mass accurately within 1 sec (Z' factor of 0.83) or 600 msec (Z' factor of 0.64), measuring time per sample. This indicates that many of the known ultrafast detection technologies might be used for monitoring biochemical reactions, probably even without any labeling procedure. The authors also discuss briefly which ultrafast processes contribute to the signals and how they are affected by changes in the biomolecular environment.

Two‐Color Two‐Photon Excitation of Intrinsic Protein Fluorescence: Label‐Free Observation of Proteolytic Digestion of Bovine Serum Albumin

“…As an example we monitored the progress of the proteolytic cleavage of bovine serum albumin (BSA) by subtilisin. [27] The strong fluorescence signal allows short acquisition times even with a rather simple experimental setup. A more sophisticated setup using high numerical aperture objectives will enhance the efficiency of excitation as well as the fluorescence detection, and thus allow bioassays fast enough for highthroughput screening.…”