Christian Santschi

Fast three-dimensional imaging of gold nanoparticles in living cells with photothermal optical lock-in Optical Coherence Microscopy

We introduce photothermal optical lock-in Optical Coherence Microscopy (poli-OCM), a volumetric imaging technique, which combines the depth sectioning of OCM with the high sensitivity of photothermal microscopy while maintaining the fast acquisition speed inherent to OCM. We report on the detection of single 40 nm gold particles with a 0.5 μm lateral and 2 μm axial resolution over a 50 μm depth of field and the three-dimensional localization of gold colloids within living cells. In combination with intrinsic sample contrast measured with dark-field OCM, poli-OCM offers a versatile platform for functional cell imaging.

Biosensor based on chemically-designed anchorable cytochrome c for the detection of H2O2 released by aquaticcells

A novel third generation biosensor was developed based on one-shot adsorption of chemically-modified cytochrome c (cyt c) onto bare gold electrodes. In contrast to the classic approach which consists of attaching cyt c onto an active self-assembled monolayer (SAM) priory chemisorbed on gold, here short-chain thiol derivatives (mercaptopropionic acid, MPA) were chemically introduced on cyt c protein shell via its lysine residues enabling the very fast formation (< 5 min) of an electroactive biological self-assembled monolayer (SAM) exhibiting a quasi-reversible electrochemical behavior and a fast direct electron transfer (ET). The heterogeneous ET rate constant was estimated to be k(s)=1600 s⁻¹, confirming that short anchors facilitate the ET via an efficient orientation of the heme pocket. In comparison, no ET was observed in the case of native and long-anchor (mercaptoundecanoic acid, MUA) modified cyt c directly adsorbed on gold. However, in both cases the ET was efficiently restored upon in-bulk generation of gold nanoparticles which acted as electron shuttles. This observation emphasizes that the lack of electroactivity might be caused by either an inappropriate orientation of the protein (native cyt c) or a critical distance (MUA-cyt c). Finally, the sensitivity of the bare gold electrode directly modified with MPA-cyt c to hydrogen peroxide (H₂O₂) was evaluated by amperometry and the so-made amperometric biosensor was able to perform real-time and non-invasive detection of endogeneous H₂O₂ released by unicellular aquatic microorganisms, Chlamydomonas reinhardtii, upon cadmium exposure.

Coupling Strength Can Control the Polarization Twist of a Plasmonic Antenna

The far-field polarization of the optical response of a plasmonic antenna can be tuned by subtly engineering of its geometry. In this paper, we develop design rules for nano antennas which enable the generation of circular polarized light via the excitation of circular plasmonic modes in the structure. Two initially orthogonal plasmonic modes are coupled in such a way that a rotational current is excited in the structure. Modifying this coupling strength from a weak to a strong regime controls the helicity of the scattered field. Finally, we introduce an original sensing approach that relies on the rotation of the incident polarization and demonstrates a sensitivity of 0.23 deg·nm(-1) or 33 deg·RIU(-1), related to changes of mechanical dimensions and the refractive index, respectively.

Sensing the dynamics of oxidative stress using enhanced absorption in protein-loaded random media

Reactive oxygen species play a key role in cell signalling and oxidative stress mechanisms, therefore, sensing their production by living organisms is of fundamental interest. Here we describe a novel biosensing method for extracellular detection of endogenous hydrogen peroxide (H2O2). The method is based on the enhancement of the optical absorption spectrum of the hemoprotein cytochrome c when loaded into a highly scattering random medium. Such a configuration enables, in contrast to existing techniques, non-invasive and dynamic detection of the oxidation of cyt c in the presence of H2O2 with unprecedented sensitivity. Dynamic information on the modification of the cell oxidative status of Chlamydomonas reinhardtii, an aquatic green algae, was obtained under oxidative stress conditions induced by the presence of trace concentrations of Cd(II). Furthermore, the dynamics of H2O2 production was investigated under different lighting conditions confirming the impact of Cd(II) on the photosynthetic activity of those phytoplanktonic cells.

A Zeptoliter Volume Meter for Analysis of Single Protein Molecules

A central goal in bioanalytics is to determine the concentration of and interactions between biomolecules. Nanotechnology allows performing such analyses in a highly parallel, low-cost, and miniaturized fashion. Here we report on label-free volume, concentration, and mobility analysis of single protein molecules and nanoparticles during their diffusion through a subattoliter detection volume, confined by a 100 nm aperture in a thin gold film. A high concentration of small fluorescent molecules renders the aqueous solution in the aperture brightly fluorescent. Nonfluorescent analytes diffusing into the aperture displace the fluorescent molecules in the solution, leading to a decrease of the detected fluorescence signal, while analytes diffusing out of the aperture return the fluorescence level. The resulting fluorescence fluctuations provide direct information on the volume, concentration, and mobility of the nonfluorescent analytes through fluctuation analysis in both time and amplitude.

Full Color Generation Using Silver Tandem Nanodisks

Plasmonic effects associated with metallic nanostructures have been widely studied for color generation. It became apparent that highly saturated and bright colors are hard to obtain, and very small nanostructures need to be fabricated. To address this issue, in this study, we employ metal-insulator-metal sandwich nanodisks that support enhanced in-phase electric dipole modes, which are blue-shifted with respect to a single metal disk. The blue shift enables the generation of short wavelength colors with larger nanostructures. The radiation modes hybridize with the Woods anomaly in periodic structures, creating narrow and high-resonance peaks in the reflection and deep valleys in the transmission spectra, thus producing vivid complementary colors in both cases. Full colors can be achieved by tuning the radius of the nanodisks and the periodicity of the arrays. Good agreement between simulations and experiments is demonstrated and analyzed in CIE1931, sRGB, and HSV color spaces. The presented method has potential for applications in imaging, data storage, ultrafine displays, and plasmon-based biosensors.

Portable oxidative stress sensor: Dynamic and non-invasive measurements of extracellular H2O2 released by algae

Reactive oxygen species (ROS) generated by aerobic organisms are essential for physiological processes such as cell signaling, apoptosis, immune defense and oxidative stress mechanisms. Unbalanced oxidant/antioxidant budgets are involved in many diseases and, therefore, the sensitive measurement of ROS is of great interest. Here, we present a new device for the real-time monitoring of oxidative stress by measuring one of the most stable ROS, namely hydrogen peroxide (H2O2). This portable oxidative stress sensor contains the heme protein cytochrome c (cyt c) as sensing element whose spectral response enables the detection of H2O2 down to a detection limit of 40 nM. This low detection limit is achieved by introducing cyt c in a random medium, enabling multiscattering that enhances the optical trajectory through the cyt c spot. A contact microspotting technique is used to produce reproducible and reusable cyt c spots which are stable for several days. Experiments in static and microfluidic regimes, as well as numerical simulations demonstrate the suitability of the cyt c/H2O2 reaction system for the real-time sensing of the kinetics of biological processes without H2O2 depletion in the measurement chamber. As an example, we detect the release of H2O2 from the green alga Chlamydomonas reinhardtii exposed to either 180 nM functionalized CdSe/ZnS core shell quantum dots, or to 10 mg/l TiO2 nanoparticles. The continuous measurement of extracellular H2O2 by this optical sensor with high sensitivity is a promising new means for real-time cytotoxicity tests, the investigation of oxidative stress and other physiological cell processes.

Mass sensor for in situ monitoring of focused ion and electron beam induced processes

A cantilever-based mass sensor for in situ monitoring of deposition and milling using focused ion and electron beams is presented. Carefully designed experiments allowed for mass measurements with a noise level of ±10fg by tracking the resonance frequency of a temperature stabilized piezoresistive cantilever using phase locking. The authors report on measurements of precursor surface coverage, residence time, mass deposition rates, yields, and deposit density using the (CH3)3PtCpCH3 precursor.

Comparison of fabrication methods of sub-100nm nano-optical structures and devices

Nano-optical devices are raising more and more interest for a variety of applications. From single molecule detection at high molecular concentration by Fluorescence Correlation Spectroscopy (FCS) through optical multiplexing with photonic crystal structures into the exciting field of negative index of refraction materials, the hardware functional dimensions and surely the tolerances are reaching the lower tens of nanometer range. The fabrication of such devices, i.e. the machining of optically interesting materials and material combinations (dielectric, semiconducting, or metallic) at this scale needs adaptation of classical nanostructuring technologies like Electron Beam Lithography (EBL), or the application of serial direct machining technologies like Focused Charged Particle Beam Etching or Deposition with electrons or Ga ions. For low excitation volume FCS measurements, EBL is used for production of high quality nanoscale sub-wavelength apertures in optically opaque (150 nm thick) metal films. The process consists in high aspect ratio patterning of a thick negative e-beam resist film followed by metal lift off. The optically transparent substrate allows the production of any 2D aperture geometry. Difficulties of the production process and their limits are presented. Direct serial machining with charged particle beams shows excellent flexibility and is an interesting 3D alternative method. Deposition by decomposing volatile chemicals under an ion/electron probe, which can be as small as 7nm/1nm, this technique allows for rapid, local prototyping of 2D and 3D nano-structures with highest lateral and axial resolution. The deposited material can be tuned to homogeneous, nanocomposite or crystalline, metallic or transparent, opening the way to applications in photonic crystals and plasmonics. An original in-situ micro-reflectometry method permits the real time control of the growth of the deposits.

Absorbance enhancement in microplate wells for improved-sensitivity biosensors

A generic optical biosensing strategy was developed that relies on the absorbance enhancement phenomenon occurring in a multiple scattering matrix. Experimentally, inserts made of glass fiber membrane were placed into microplate wells in order to significantly lengthen the trajectory of the incident light through the sample and therefore increase the corresponding absorbance. Enhancement factor was calculated by comparing the absorbance values measured for a given amount of dye with and without the absorbance-enhancing inserts in the wells. Moreover, the dilution of dye in solutions with different refractive indices (RI) clearly revealed that the enhancement factor increased with the ΔRI between the membrane and the surrounding medium, reaching a maximum value (EF>25) when the membranes were dried. On this basis, two H2O2-biosensing systems were developed based on the biofunctionalization of the glass fiber inserts either with cytochrome c or horseradish peroxidase (HRP) and the analytical performances were systematically compared with the corresponding bioassay in solution. The efficiency of the absorbance-enhancement approach was particularly clear in the case of the cytochrome c-based biosensor with a sensitivity gain of 40 folds and wider dynamic range. Therefore, the developed strategy represents a promising way to convert standard colorimetric bioassays into optical biosensors with improved sensitivity.