Laser Transmission Spectroscopy Based on Tunable-Gain Dual-Channel Dual-Phase LIA for Biological Nanoparticles Characterization.

Size and absolute concentration of suspensions of nanoparticles are important information for the study and development of new materials and products in different industrial applications spanning from biotechnology and pharmaceutics to food preparation and conservation. Laser Transmission Spectroscopy (LTS) is the only methodology able to measure nanoparticle size and concentration by performing a single measurement. In this paper we report on a new variable gain calibration procedure for LTS-based instruments allowing to decrease of an order of magnitude the experimental indetermination of the particle size respect to the conventional LTS based on the double ratio technique. The variable gain calibration procedure makes use of a specifically designed tunable-gain, dual-channel, dual-phase Lock-In Amplifier (LIA) whose input voltage signals are those ones generated by two Si photodiodes that measure the laser beam intensities passing through the sample containing the nanoparticles and a reference optical path. The LTS variable gain calibration procedure has been validated by firstly using a suspension of NIST standard polystyrene nanoparticles even 36 hours after the calibration procedure was accomplished. The paper reports in detail the LIA implementation describing the design methodologies and the electronic circuits. As a case example of the characterization of biological nanostructures, we demonstrate that a single LTS measurement allowed to determine size density distribution of a population of extracellular vesicles extracted from orange juice (25 nm in size) with the presence of their aggregates having a size of 340 nm and a concentration smaller than 3 orders of magnitude.