Resolving molecule-specific information in dynamic lipid membrane processes with multi-resonant infrared metasurfaces

A multitude of biological processes are enabled by complex interactions between lipid membranes and proteins. To understand such dynamic processes, it is crucial to differentiate the constituent biomolecular species and track their individual time evolution without invasive labels. Here, we present a label-free mid-infrared biosensor capable of distinguishing multiple analytes in heterogeneous biological samples with high sensitivity. Our technology leverages a multi-resonant metasurface to simultaneously enhance the different vibrational fingerprints of multiple biomolecules. By providing up to 1000-fold near-field intensity enhancement over both amide and methylene bands, our sensor resolves the interactions of lipid membranes with different polypeptides in real time. Significantly, we demonstrate that our label-free chemically specific sensor can analyze peptide-induced neurotransmitter cargo release from synaptic vesicle mimics. Our sensor opens up exciting possibilities for gaining new insights into biological processes such as signaling or transport in basic research as well as provides a valuable toolkit for bioanalytical and pharmaceutical applications.

Infrared nanoplasmonic metasurfaces augmented by artificial intelligence for universal biosensing

Aurélian Michel John-Herpin

Nanoplasmonic metasurfaces have shown outstanding light-matter interaction enhancement capabilities, leading to their emergence as powerful platforms for highly sensitive biospectroscopy. Metasurface-enhanced biospectroscopy offers unprecedented opportunities for biological studies, and its full potential remains to be unleashed. Mid-IR metasurfaces, in particular, are very promising because they can act as amplifiers of fingerprint-like molecule vibrations, which are plentiful in this rich spectral range. In this thesis, we develop novel nanoplasmonic designs coupled with custom microfluidics and artificial intelligence-based data analysis models to demonstrate real-time, label-free, chemically-specific, and non-destructive monitoring of biomolecules and their interactions in aqueous media. Our first nanoplasmonic design combines optimized grating order-coupled nanoantenna arrays with protein-accessible nanogaps to enable the high sensitivity monitoring of proteins and their three-dimensional structures in aqueous media. The engineered nanoantennas reach electric field intensity enhancements of up to five orders of magnitude and provide chemically specific detection of proteins and their secondary structures down to picograms and nanograms per milliliter, respectively.In the next part of the thesis, we develop multiresonant metasurfaces to monitor interactions between biomolecules with vibrational fingerprints in different parts of the mid-IR range. Our first effort focuses on developing a nanoplasmonic design for simultaneous monitoring of both proteins and lipid molecules. Lipids are another important class of biomolecules as they are the building blocks of biological membranes, and lipid-protein interactions are at the core of many cellular processes. New analytical tools for their study in water and at the monolayer level are of fundamental importance. Therefore, we introduce a dual-resonant nanoplasmonic design coupled to machine learning-based data analysis to overcome current sensor challenges. We apply our technology to a dynamic system involving synaptic vesicle mimics and demonstrate that we can resolve complex mass-preserving biological interactions in real-time. This is a remarkable feat that traditional non-chemically specific analytical measurement tools such as surface plasmon resonance or quartz crystal microbalance spectroscopy could not achieve. In the final part of the thesis, we develop yet another multiresonant design for broadband coverage of the whole mid-IR range. We couple our sensor to a deep learning model to resolve a dynamic biological system including all major classes of biomolecules simultaneously. Specifically, we resolve the toxic peptide-induced release of carbohydrates and nucleotides from exosome-like bionanoparticles.

EPFL2022

Two-Dimensional Label-Free Affinity Analysis of Tumor-Specific CD8 T Cells with a Biomimetic Plasmonic Sensor

Hatice Altug, George Coukos, Aurélian Michel John-Herpin, Xiaokang Li, Maria Soler Aznar

The screening and analysis of T cells functional avidity for specific tumor-associated antigens is crucial for the development of personalized immunotherapies against cancer. The affinity and kinetics of a T cell receptor (TCR) binding to the peptide-major histocompatibility complex (pMHC), expressed on tumor or antigen-presenting cells, have shown major implications in T cell activation and effector functions. We introduce an innovative methodology for the two-dimensional affinity analysis of TCR-pMHC in a label-free configuration by employing a multiparametric Surface Plasmon Resonance biosensor (MP-SPR) functionalized with artificial cell membranes. The biomimetic scaffold created with planar lipid bilayers is able to efficiently capture the specific and intact tumor-specific T cells and monitor the formation of the immunological synapse in situ. We have achieved excellent limits of detection for in-flow cell capturing, up to 2 magnitude below the current state-of-the-art for plasmonic sensing. We demonstrate the accuracy and selectivity of our sensor for the analysis of CD8(+) T cells bioengineered with TCR of incremental affinities specific for the HLA-A0201/NY-ESO-I157-165 pMHC complex. The study confirmed the significance of providing a biomimetic microenvironment, compared to the traditional molecular analysis, and showed fine agreement with previous results employing flow cytometry. Our methodology is reliable and versatile; thus, it can be applied to more sophisticated photonic and nanoplasmonic technologies for the screening of multiple cell types and boost the development of novel treatments for cancer.

AMER CHEMICAL SOC2018

Dynamic Imaging of Lipid Membranes by Means of Water

Orly Bagunu Tarun

Lipid membranes are self-assembled structures whose composition determines the properties of membranes of cells and organelles. The molecular level understanding of lipid membranes is based on spectroscopy and MD simulations of lipid monolayer systems. As spectroscopy rely on spatial and temporal averaging and are necessarily linked to mean field models, information about the molecular interactions and their spatiotemporal evolution in real membranes is currently unavailable. In this thesis, we use high throughput wide-field second harmonic (SH) microscopy to image water-membrane interactions at sub-second time scale to follow the spatiotemporal evolution of membranes in freestanding membranes. We improve the throughput of nonlinear SH microscope by 2-3 orders and integrate the apparatus of freestanding membranes for simultaneous electrical and optical characterization of membranes. We show that the non-resonant response of water can be SH imaged on sub-second time scales and use this response as a contrast mechanism to image membrane dynamics. We investigate how oil redistributes within bilayers after formation. Using SH imaging, there is less oil present within bilayers prepared with hexadecane versus squalene. Diffusion of excess hexadecane droplets within bilayer follow directed diffusion whereas squalene show no directed motion. Hexadecane can diffuse within a single leaflet whereas squalene span both leaflets and more branched, couples to both leaflets and moves slower. We probe the diffusion of charged lipid domains at sub-second time scale and construct electrostatic potential maps of asymmetric membranes. The average membrane potential is quadratic to an applied external bias, modeled by nonlinear optical theory. We observe fluctuations in the membrane potential of ~100 mV implying that membranes are dynamic in structure and in their potential landscape. We probe the interactions of divalent cations with water and negatively charged membranes and show that Ca2+, Ba2+ and Mg2+ induce short-lived ( Mg2+ > Ba2+, for all quantities. We quantify KD and observe domain values that deviate up to 4 orders of magnitude from average KD. The transient domains exhibit potential fluctuations of up to -386 mV (dG = 28.6 kT), induce strain in the membrane resulting to fluctuations in membrane curvature. We SH image the opening and closing of voltage-gated alamethicin ion channels in freestanding membranes. The SH intensity is due to changes in the orientational distribution of water molecules induced by electric field gradients. Only 0.01 % of the transported ions arrives at the membrane interface, leading to interfacial electrostatic changes on the time scale of a second. We quantify the distribution of ion channels and observe that regions with high ion channel density exhibit a lower rate of interfacial charge build-up, likely caused by crowding. Ion transport along the membrane, thought to be involved in the propagation of action potential, is taking place over seconds. The observation suggests a more complex mechanism for the propagation of action potentials. On a fundamental level, structural and temporal heterogeneity needs to be included in biochemical, physical and molecular models of membranes.