# LLPS droplet size estimation via UV–Vis spectroscopy using a microplate reader

## TL;DR

A new high-throughput method uses UV–Vis spectroscopy to estimate the size of LLPS droplets, offering a simpler alternative to traditional techniques.

## Contribution

A label-free, high-throughput method for LLPS droplet size estimation using UV–Vis spectroscopy and Mie scattering theory.

## Key findings

- The method was validated using peptide droplets of known sizes.
- It successfully monitored the growth of protein condensates formed by VAPB.
- The approach is minimally invasive and scalable for various biomolecular systems.

## Abstract

Liquid-liquid phase separation (LLPS) is critical to the formation of membraneless organelles within cells. Monitoring the size and dynamics of LLPS droplets is essential to understanding their function. However, conventional methods, such as microscopy and dynamic light scattering (DLS), have limitations, including low throughput and complex setups. Here, we present a simple, label-free, high-throughput method for estimating droplet size using ultraviolet-visible (UV–Vis) spectra obtained using a standard microplate reader. This method is theoretically supported by Mie scattering theory. By analyzing the spectral shape and comparing it to that of glass beads of known sizes, our method enables the quantitative estimation of LLPS droplet size in a minimally invasive manner. We validated our approach using peptide droplets of known sizes, then applied it to monitor the growth of protein condensates formed by vesicle-associated membrane protein-associated protein B (VAPB). This technique offers a convenient, scalable alternative for screening LLPS dynamics in various biomolecular systems.

The online version contains supplementary material available at 10.1038/s41598-025-33638-8.