An integrated laser-tweezers instrument for microanalysis of individual protein aggregates

Herein we introduce a new laser-tweezers instrument capable of providing both physical and chemical information of individually trapped protein aggregates. These capabilities were demonstrated by investigating the aggregation process of α-elastin droplets of nanometers to micrometers in diameter. Individual droplets of aggregated elastin molecules were trapped at the focus of a 1064 nm near infrared (NIR) laser in the instrument. The size of these individual elastin aggregates was inferred by recording their thermal motion at a frequency of 100 kHz using a position sensitive photodetector (PSPD). The chemical information of the trapped particles was revealed by confocal fluorescence and confocal Raman spectroscopies using 532-nm laser excitation. Background signals outside trapped particles were eliminated by a pinhole in back reflected geometry. Both physical and confocal fluorescence measurements revealed a transition temperature of 38 °C for a fast process in individual elastin aggregates, a value consistent with the clouding temperature in bulk. Confocal Raman spectroscopy yielded a transition temperature of 35 °C for a slow process characteristic of the formation of elastin aqueous two phase systems (ATPS).