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In bio-imaging by electron microscopy, damage of the sample and limited contrast are the two main hurdles for reaching high image quality. We extend a new preparation method based on nanofabrication and super-hydrophobicity to the imaging and structural studies of nucleic acids, nucleic acid–protein complexes (DNA/Rad51 repair protein complex) and neuronal ion channels (gap-junction, K+ and GABAA channels) as paradigms of biological significance and increasing complexity. The preparation method is based on the liquid phase and is compatible with physiological conditions. Only in the very last stage, samples are dried for TEM analysis. Conventional TEM and high-resolution TEM (HRTEM) were used to achieve a resolution of 3.3 and 1.5 Å, respectively. The EM dataset quality allows the determination of relevant structural and metrological information on the DNA structure, DNA–protein interactions and ion channels, allowing the identification of specific macromolecules and their structure.

The detection of a few molecules in a highly diluted solution is of paramount interest in fields including biomedicine, safety and eco-pollution in relation to rare and dangerous chemicals. Nanosensors based on plasmonics are promising devices in this regard, in that they combine the features of high sensitivity, label-free detection and miniaturization. However, plasmonic-based nanosensors, in common with general sensors with sensitive areas on the scale of nanometres, cannot be used directly to detect molecules dissolved in femto- or attomolar solutions. In other words, they are diffusion-limited and their detection times become impractical at such concentrations. In this Article, we demonstrate, by combining super-hydrophobic artificial surfaces and nanoplasmonic structures, that few molecules can be localized and detected even at attomolar (10−18 mol l−1) concentration. Moreover, the detection can be combined with fluorescence and Raman spectroscopy, such that the chemical signature of the molecules can be clearly determined.

Early detection of diseases has great importance in terms of success of the disease treatment. In fact, it has a profound positive influence on the response provided by the patient, leading to shorter and less invasive treatment regimes. We consider here the Raman detection of low (atto-molar) concentrates of molecules by applying nanofabrication techniques in the fabrication of plasmonic devices fulfilling the requirement of superhydrophobicity. Plasmonic resonances will have the effect of substantially increasing the local electric field around the fabricated nano-device which, in turn, will positively affect the Raman signal. Similarly, the superhydrophobicity will play the crucial role in localizing the few molecules of the analyte around the plasmonic device, therefore allowing their detection in a manner otherwise impossible in diffusion-based devices. We will theoretically explain the concept of superhydrophobicity by providing also a roadmap for defining the optimal superhydrophobic device, then we will introduce the fabrication process to realize such a device and, finally, we will provide the Raman counting of a series of analytes together with electromagnetic simulations illustrating the role of the electric field in the formation of the Raman signal.

Exosomes are very small vesicles that are shed from a variety of cell types. They are present in body fluids comprising blood, urine and ascites, and they hold the potential to serve as indicators in the diagnosis, prognosis and surveillance of a variety of health conditions. In consideration of this, there is broad interest worldwide in deriving and understanding their biochemical composition. Here, small drops of solutions containing exosomes, isolated from either healthy (CCD841-CoN) or tumor (HCT116) colon cells, were manipulated using super-hydrophobic surfaces (SHSs) decorated with nano-geometry based photonics structures. SHSs are surfaces with superior properties, including a reduced friction coefficient, on account of which they can be used to control diluted solutions of biological or medical interest. The combination of this technology with optical resonance phenomena, open up terrific opportunities in the detection, analysis or characterization of biological compounds. Therefore, exosomes were conveniently concentrated and conveyed onto the Surface Enhanced Raman Spectroscopy (SERS) active areas of the substrate, whereby they were analyzed and characterized with extreme precision. Results indicate a difference in the concentration of some biological compounds, and namely: exosomes derived from healthy cells exhibit higher intensities of the peaks corresponding to lipids vibrations, while exosomes extracted from tumor cell lines exhibit a richer RNA content.

Micro and nano-patterned devices were fabricated which disclose the possibility of concentrating, localizing, detecting and thus analyzing with unprecedented accuracy few molecules (at the limit a single molecule) of biological and medical interest. Important applications are envisioned where proteins poorly concentrated in blood are detected thus (early) revealing the appearance of diseases. The major advance of the paper is that it combines micro/nano-fabrication and spectroscopical nano-optics based techniques to study and individualize the biomolecules at study. Super hydrophobic surface would concentrate the diluted solution into a small area whereas spectroscopical nano-optics techniques would gather the molecular/chemical information of the residual solute. Fluorescence and SERS techniques were used to analyze Rhodamine molecules initially highly diluted (10^-^1^8M).