Characterization of Biocondensate Microfluidic Flow using FCS with Sub-resolution Spatial Correlation, Paving the Way for Single-Molecule Exploration

Session

Session 4 - New Technologies: Recent advances from Acquisition to Analysis

Authors

Stijn Dilissen (2, 1), Pedro L. Silva (2), Anastastia Smolentseva (2), Tom Kache (2), Ronald Thoelen (1, 3), Jelle Hendrix (2)

Affiliations

1. UHasselt, Biomedical Device Engineering group, Institute for Materials Research (IMO-IMOMEC)
2. UHasselt, Dynamic Bioimaging Group, Biomedical Research Institute
3. IMOMEC Division, IMEC vzw

Keywords

Liquid-liquid phase separation; Microfluidics; Fluorescence Correlation Spectroscopy; Tau; Single-molecule research 

Abstract text

Biocondensation via liquid-liquid phase separation (LLPS) is a colloidal process by which dynamic liquid-like droplets of concentrated proteins are spontaneously and reversibly formed within solutions and cells. It has become clear that biocondensation is crucial for cellular homeostasis and is functionally involved in various biological processes. Since biocondensation has been associated with pathologies such as dementia and cancer, a better understanding of the mechanisms underlying biocondensate formation will help to elucidate disease aetiology. Although the rheological heterogeneity of biocondensates and the structural dynamics of their constituents carry critical functional information, methods to quantitatively study biocondensates are lacking. Single-molecule fluorescence research on biomolecules in condensates represents a way to gain insight into LLPS mechanisms. Unfortunately, as dense condensates tend to sink inside their dilute aqueous surroundings, studying their properties via methods relying on Brownian diffusion are prone to fail. To address this issue, we took a first step towards single-molecule research on biocondensates under flow in a microfluidic channel. 

For this study, the microtubule-associated protein Tau, which tends to condensate in cellular and in vitro models in the presence of negatively charged molecules or crowding agents, was used as a model system. Fluorescence correlation spectroscopy (FCS), a well-known technique to collect molecular characteristics within a sample, was utilized with a newly commercialized technology. This method performs FCS on an array detector (AD-FCS), providing detailed information on diffusion and flow. By integrating this technique with a straight-channel microfluidic chip, optimal parameters were established for investigating dense molecular organizations in a single-molecule study under pressure-controlled microfluidic flow.

First, the AD-FCS technology effectively characterized a microfluidic chip, revealing 3D flow velocity profiles consistent with predicted microfluidic flow simulations. Subsequently, a Tau condensate sample was introduced into the microfluidic chip to replicate conditions used in a single-molecule experimental setup. AD-FCS mapped the condensate flow while measuring the burst durations of these biocondensates through the stationary laser, verifying the ability to distinguish between the dense and dilute phases. The microfluidic chip and pump pressures used in this study provided a sufficient rate of condensate detections per minute for meaningful conformational analysis, with burst durations suitable for molecular dynamic analysis. Finally, using the AD-FCS data on flow velocity and burst durations, we demonstrated the feasibility of estimating the size distribution of the condensates within the LLPS sample via FCS.

In conclusion, studying Tau condensates under microfluidic flow through FCS with sub-resolution spatial correlation demonstrates the promising application in single-molecule experiments. Furthermore, the commercialized AD-FCS technology enables the assessment of size distribution in condensate samples, prompting a new way of investigating LLPS solutions and their corresponding phase diagrams.

Graphical Abstract