Disc antenna enhanced infrared spectroscopy: From felf-assembled monolayers to membrane proteins

Plasmonic surfaces have emerged as a powerful platform for biomolecular sensing applications and can be designed to optimize the plasmonic resonance for probing molecular vibrations at utmost sensitivity. Here, we present a facile procedure to generate metallic microdisc antenna arrays that are employed in surface-enhanced infrared absorption (SEIRA) spectroscopy of biomolecules. Transmission electron microscopy (TEM) grids are used as shadow mask deployed during physical vapor deposition of gold. The resulting disc-shaped antennas exhibit enhancement factors of the vibrational bands of 4 × 104 giving rise to a detection limit <1 femtomol (10–15 mol) of molecules. Surface-bound monolayers of 4-mercaptobenzoic acid show polyelectrolyte behavior when titrated with cations in the aqueous medium. Conformational rigidity of the self-assembled monolayer is validated by density functional theory calculations. The membrane protein sensory rhodopsin II is tethered to the disc antenna arrays and is fully functional as inferred from the light-induced SEIRA difference spectra. As an advance to previous studies, the accessible frequency range is improved and extended into the fingerprint region

Approaching the Single-Molecule Limit

Infrared (IR) spectroscopy is one of the most powerful tools in life science. It delivers molecular information about structure and functionality in a non-invasive manner. However, its sensitivity and spatial resolution remain insufficient for interrogating single biomolecules. Surface and tip-enhanced spectroscopy exploit nanoscopic localization of both the probing light and the sample to address these shortcomings. Diffraction limited surface-enhanced methods offer high enhancement factors for ensembles of molecules even in aqueous environments but typically come along with high costs of production. Tip-enhanced methods provide even higher sensitivities down to hundreds of molecules and nanometric spatial resolution but are so far slow and require dry samples. I present in this work several routes towards single-molecule IR spectroscopy using both approaches. A cost effective and reproducible method for preparation of surface-enhanced infrared absorption spectroscopy substrates was developed. These resonant disc antenna arrays allowed the microspectroscopic characterization of sub-fmol (10^-15 mol or 10^9 molecules) of active membrane proteins. Their applicability to a variety of biologically important environments highlights their relevance for spectroscopy in life science. However, surface-enhanced techniques lack spatial resolution necessary for single-molecule detection and localization. Therefore, I have designed a scattering-type scanning near-field optical microscope (sSNOM) for IR nanoimaging and spectroscopy. A lateral resolution of 30 nm was achieved on protein loaded membrane patches pushing the sensitivity beyond zmol (10^-21 mol or 600 molecules). The imaging speed was improved by a factor of 20 compared to conventional setups enabling µs time-resolved studies on biomolecules. The obvious combination of resonant substrates and sSNOM yielded no appreciable enhancement of sensitivity and calls for alternative strategies. As an application to life science, whole cell nanoimaging and spectroscopy of the archeon Halobacterium salinarum was accomplished from which a homogeneous protein density within the cell wall could be inferred. Adapting a total internal reflection illumination scheme provided first experimental evidence towards sSNOM in aqueous environments. Those experiments lay the foundation for the analysis of complex membrane systems in living cells. The sSNOM setup was modified to record the locally deposited heat via the anomalous Nernst effect to expand the scope of tip-enhanced methods. The domain wall within a ferromagnetic micro device was localized as a proof of principle. This method cannot only be applied to antiferromagnetic systems but bears great potential for near-field IR spectroscopy. In conclusion, I believe that these results pave the way towards single-molecule IR spectroscopy by combining surface-, tip-enhancement and novel spectroscopic readouts

Thermoelectric nanospectroscopy for the imaging of molecular fingerprints

We present a nanospectroscopic device platform allowing simple and spatially resolved thermoelectric detection of molecular fingerprints of soft materials. Our technique makes use of a locally generated thermal gradient converted into a thermoelectric photocurrent that is read out in the underlying device. The thermal gradient is generated by an illuminated atomic force microscope tip that localizes power absorption onto the sample surface. The detection principle is illustrated using a concept device that contains a nanostructured strip of polymethyl methacrylate (PMMA) defined by electron beam lithography. The platform's capabilities are demonstrated through a comparison between the spectrum obtained by on-chip thermoelectric nanospectroscopy with a nano-FTIR spectrum recorded by scattering-type scanning near-field optical microscopy at the same position. The subwavelength spatial resolution is demonstrated by a spectral line scan across the edge of the PMMA layer

Magneto-Seebeck microscopy of domain switching in collinear antiferromagnet CuMnAs

Antiferromagnets offer spintronic device characteristics unparalleled in ferromagnets owing to their lack of stray fields, THz spin dynamics, and rich materials landscape. Microscopic imaging of antiferromagnetic domains is one of the key prerequisites for understanding physical principles of the device operation. However, adapting common magnetometry techniques to the dipolar-field-free antiferromagnets has been a major challenge. Here we demonstrate in a collinear antiferromagnet a thermoelectric detection method by combining the magneto-Seebeck effect with local heat gradients generated by scanning far-field or near-field techniques. In a 20-nm epilayer of uniaxial CuMnAs we observe reversible 180∘ switching of the Néel vector via domain wall displacement, controlled by the polarity of the current pulses. We also image polarity-dependent 90∘ switching of the Néel vector in a thicker biaxial film, and domain shattering induced at higher pulse amplitudes. The antiferromagnetic domain maps obtained by our laboratory technique are compared to measurements by the established synchrotron-based technique of x-ray photoemission electron microscopy using x-ray magnetic linear dichroism

Infrared Scattering-Type Scanning Near-Field Optical Microscopy in Water

Infrared (IR) absorption spectroscopy detects state and chemical composition of biomolecules solely by their inherent vibrational fingerprints. Major disadvantages like the lack of spatial resolution and sensitivity were compensated lately by the use of pointed probes as local sensors enabling the detection of quantities as few as hundreds of proteins with nanometer precision. This makes infrared scattering-type scanning near-field optical microscopy a very powerful tool in life science. The strong absorption of infrared radiation of liquid water, however, still prevents to simply access the measured quantity – light scattered at the probing atomic force microscope tip. Here we report on the local IR response of biological membranes immersed in aqueous bulk solution. We make use of a silicon solid immersion lens as substrate and focusing optics to achieve detection efficiencies sufficient to yield IR near-field maps of purple membranes. We scrutinized our experimental findings by applying theoretical models. Finally, we suggest a means to improve the imaging quality by laser scanning assisted scattering-type scanning near-field optical microscopy. We believe that IR scattering-type scanning near-field optical microscopy will resolve biological structures in their native environments at nm resolution without the need for labeling.</div

Characterization of membrane protein interactions by peptidisc-mediated mass photometry

Summary: Membrane proteins perform numerous critical functions in the cell, making many of them primary drug targets. However, their preference for a lipid environment makes them challenging to study using established solution-based methods. Here, we show that peptidiscs, a recently developed membrane mimetic, provide an ideal platform to study membrane proteins and their interactions with mass photometry (MP) in detergent-free conditions. The mass resolution for membrane protein complexes is similar to that achievable with soluble proteins owing to the low carrier heterogeneity. Using the ABC transporter BtuCD, we show that MP can quantify interactions between peptidisc-reconstituted membrane protein receptors and their soluble protein binding partners. Using the BAM complex, we further show that MP reveals interactions between a membrane protein receptor and a bactericidal antibody. Our results highlight the utility of peptidiscs for membrane protein characterization in detergent-free solution and provide a rapid and powerful platform for quantifying membrane protein interactions

Disc Antenna Enhanced Infrared Spectroscopy: From Self-Assembled Monolayers to Membrane Proteins

Plasmonic surfaces have emerged as a powerful platform for biomolecular sensing applications and can be designed to optimize the plasmonic resonance for probing molecular vibrations at utmost sensitivity. Here, we present a facile procedure to generate metallic microdisc antenna arrays that are employed in surface-enhanced infrared absorption (SEIRA) spectroscopy of biomolecules. Transmission electron microscopy (TEM) grids are used as shadow mask deployed during physical vapor deposition of gold. The resulting disc-shaped antennas exhibit enhancement factors of the vibrational bands of 4 × 10⁴ giving rise to a detection limit <1 femtomol (10^–15 mol) of molecules. Surface-bound monolayers of 4-mercaptobenzoic acid show polyelectrolyte behavior when titrated with cations in the aqueous medium. Conformational rigidity of the self-assembled monolayer is validated by density functional theory calculations. The membrane protein sensory rhodopsin II is tethered to the disc antenna arrays and is fully functional as inferred from the light-induced SEIRA difference spectra. As an advance to previous studies, the accessible frequency range is improved and extended into the fingerprint region