Bouzigues, C. I. | Search bioRxiv

bioRxiv2025

Ultrasensitive quantitative protein detection using Eu-ion doped vanadate nanoparticles

Disease prevention, diagnosis, and treatment monitoring often require ultrasensitive (sub-)femtomolar biomarker detection and quantification. While standard ELISA assays yield picomolar sensitivity, existing ultrasensitive approaches reach fM, aM or even zM sensitivity. This, however, is obtained at the expense of increased complexity and cost which hampers their biomedical applications. We propose a novel approach, NLISA, combining ultrasensitive, fM/sub-fM, quantitative detection with simplicity and ease of use based on 38-nm YVO4:Eu (20%) crystalline nanoparticles used as detection probes. These particles possess an extremely strong absorption in the UV leading to bright Eu3+-ion emission. We developed a transportable, multi-well plate reader providing LED excitation and detection with a photomultiplier enabling detection down to 16,000 nanoparticle probes/well. We obtained sensitivity gain factors with respect to ELISA ranging from 65 to 35,000 for insulin, IFN-{gamma}, and HIV-GAG-p24 while maintaining the same antibodies. We demonstrated femtomolar LOD and a dynamic range of 4-5 orders of magnitude and NLISA efficiency for HIV-positive patient diagnosis. This approach for straightforward, ultrasensitive polypeptide/protein detection is easily generalizable paving the way for a new generation of diagnostic tests.

bioengineering↗

bioRxiv2023

Quantitative classification of energy landscapes inferred from single nanoparticle tracking of membrane receptors inside nanodomains reveals confinement functional and molecular features

The cell membrane organization has been hypothesized for a long time to have an essential functional role, through the control of membrane receptor confinement in micro- or nanodomains. Several mechanisms have been proposed to account for these properties, though some features of the resulting organization have remained controversial, notably the nature, size, and stability of cholesterol- and sphingolipid-rich domains called rafts. Here, we quantitatively probed the energy landscape experienced by single nanoparticle-labeled membrane receptors - epidermal growth factor receptors (EGFR), transferrin receptors (TfR), and receptors of {varepsilon}-toxin produced by C. perfringens and -toxin of C.Septicum (CP{varepsilon}TR and CSTR, respectively) - through the development of new computational methods. By establishing a new analysis pipeline combining Bayesian inference, decision trees and clustering approaches, we indeed systematically classified single protein trajectories according to the type of confining energy landscape. This revealed the existence of only two distinct organization modalities: (A) confinement in a quadratic energy landscape for EGF, CP{varepsilon}T and CST receptors and (B) free diffusion in confinement domains resulting from the steric hindrance due to F-actin barriers for transferrin receptors. The characterization of confinement energy landscapes by Bayesian inference furthermore revealed the role of interactions with the domain environment in cholesterol- and sphingolipid-rich domains with (in the case of EGFR) or without (for CP{varepsilon}T and CST receptors) parallel interactions with F-actin, to regulate the confinement energy depth. Strikingly, these two distinct mechanisms result in the same organization type (A). We furthermore revealed that the apparent domain sizes for these receptor trajectories resulted from Brownian exploration of the energy landscape in a steady-state like regime at a common effective temperature, independently of the underlying molecular mechanisms. These results highlight that the membrane organization in confinement domains may be more adequately described as interaction hotspots rather than rafts with abrupt domain boundaries. Altogether, these results establish a new computational approach, which paves the way to the constitution of an atlas of energy landscape of membrane proteins and of their control mechanisms, and support a new general model for functional receptor confinement in membrane nanodomains.

biophysics↗