Highly efficient human serum filtration with water-soluble nanoporous nanoparticles

Antonella Pujia1, Francesco De Angelis1,2, Domenica Scumaci3, Marco Gaspari3, Carlo Liberale1,2, Patrizio Candeloro1, Giovanni Cuda3, Enzo Di Fabrizio1,21BIONEM Laboratory, Department of Experimental and Clinical Medicine, University of Catanzaro “Magna Graecia”, Germaneto (CZ), Italy; 2IIT, Italian Institute of Technology, Genova, Italy; 3Proteomics and Mass Spectrometry Laboratory, Department of Experimental and Clinical Medicine, University of Catanzaro “Magna Graecia”, Germaneto (CZ), ItalyBackground: Human serum has the potential to become the most informative source of novel biomarkers, but its study is very difficult due to the incredible complexity of its molecular composition. We describe a novel tool based on biodegradable nanoporous nanoparticles (NPNPs) that allows the harvesting of low-molecular-weight fractions of crude human serum or other biofluids. NPNPs with a diameter of 200 nm and pore size of a few nm were obtained by ultrasonication of nanoporous silicon. When incubated with a solution, the NPNPs harvest only the molecules small enough to be absorbed into the nanopores. Then they can be recovered by centrifugation and dissolved in water, making the harvested molecules available for further analyses.Results: Fluorescence microscopy, gel electrophoresis, and mass spectrometry were used to show the enrichment of low-molecular-weight fraction of serum under physiological conditions, with a cut-off of 13 kDa and an enrichment factor >50.Conclusion: From these findings, we conclude that ability to tune pore size, combined with the availability of hundreds of biomolecule cross-linkers, opens up new perspectives on complex biofluid analysis, discovery of biomarkers, and in situ drug delivery.Keywords: nanoporous silicon, nanoparticle, biomarker discovery, human serum proteomics, harvestin

Detection of single amino acid mutation in human breast cancer by disordered plasmonic self-similar chain

Control of the architecture and electromagnetic behavior of nanostructures offers the possibility of designing and fabricating sensors that, owing to their intrinsic behavior, provide solutions to new problems in various fields. We show detection of peptides in multicomponent mixtures derived from human samples for early diagnosis of breast cancer. The architecture of sensors is based on a matrix array where pixels constitute a plasmonic device showing a strong electric field enhancement localized in an area of a few square nanometers. The method allows detection of single point mutations in peptides composing the BRCA1 protein. The sensitivity demonstrated falls in the picomolar (10−12 M) range. The success of this approach is a result of accurate design and fabrication control. The residual roughness introduced by fabrication was taken into account in optical modeling and was a further contributing factor in plasmon localization, increasing the sensitivity and selectivity of the sensors. This methodology developed for breast cancer detection can be considered a general strategy that is applicable to various pathologies and other chemical analytical cases where complex mixtures have to be resolved in their constitutive components

Science communication and concept of risk in bio-tech-sciences: Is it a part of neo-liberalism, or foucaultian bio-politics?

In this work a Raman flow cytometer is presented. It consists of a microfluidic device that takes advantages of the basic principles of Raman spectroscopy and flow cytometry. The microfluidic device integrates calibrated microfluidic channels- where the cells can flow one-by-one -, allowing single cell Raman analysis. The microfluidic channel integrates plasmonic nanodimers in a fluidic trapping region. In this way it is possible to perform Enhanced Raman Spectroscopy on single cell. These allow a label-free analysis, providing information about the biochemical content of membrane and cytoplasm of the each cell. Experiments are performed on red blood cells (RBCs), peripheral blood lymphocytes (PBLs) and myelogenous leukemia tumor cells (K562)

Mechanical Stress Downregulates MHC Class I Expression on Human Cancer Cell Membrane

In our body, cells are continuously exposed to physical forces that can regulate different cell functions such as cell proliferation, differentiation and death. In this work, we employed two different strategies to mechanically stress cancer cells. The cancer and healthy cell populations were treated either with mechanical stress delivered by a micropump (fabricated by deep X-ray nanolithography) or by ultrasound wave stimuli. A specific down-regulation of Major Histocompatibility Complex (MHC) class I molecules expression on cancer cell membrane compared to different kinds of healthy cells (fibroblasts, macrophages, dendritic and lymphocyte cells) was observed, stimulating the cells with forces in the range of nano-newton, and pressures between 1 and 10 bar (1 bar = 100.000 Pascal), depending on the devices used. Moreover, Raman spectroscopy analysis, after mechanical treatment, in the range between 700-1800 cm(-1), indicated a relative concentration variation of MHC class I. PCA analysis was also performed to distinguish control and stressed cells within different cell lines. These mechanical induced phenotypic changes increase the tumor immunogenicity, as revealed by the related increased susceptibility to Natural Killer (NK) cells cytotoxic recognition

The Five Ws (and one H) of Super-Hydrophobic Surfaces in Medicine

Super-hydrophobic surfaces (SHSs) are bio-inspired, artificial microfabricated interfaces, in which a pattern of cylindrical micropillars is modified to incorporate details at the nanoscale. For those systems, the integration of different scales translates into superior properties, including the ability of manipulating biological solutions. The five Ws, five Ws and one H or the six Ws (6W), are questions, whose answers are considered basic in information-gathering. They constitute a formula for getting the complete story on a subject. According to the principle of the six Ws, a report can only be considered complete if it answers these questions starting with an interrogative word: who, why, what, where, when, how. Each question should have a factual answer. In what follows, SHSs and some of the most promising applications thereof are reviewed following the scheme of the 6W. We will show how these surfaces can be integrated into bio-photonic devices for the identification and detection of a single molecule. We will describe how SHSs and nanoporous silicon matrices can be combined to yield devices with the capability of harvesting small molecules, where the cut-off size can be adequately controlled. We will describe how this concept is utilized for obtaining a direct TEM image of a DNA molecule

Micro-Optics Fabrication on Top of Optical Fibers Using Two-Photon Lithography

We describe the fabrication of different micro-optical structures on top of optical fibers using two-photon polymerization. We show the convenience of this approach to quickly create generic three-dimensional shapes using a single setup by comparison to previous shape-dependent methods. A set of different structures, designed for different optical functions, are fabricated and characterized to demonstrate the versatility of this approach and their high optical quality

Electroless deposition of metal nanoparticle clusters: Effect of pattern distance

Electroless plating is a deposition technique in which metal ions are reduced as atoms on specific patterned sites of a silicon surface to form metal nanoparticles (NPs) aggregates with the desired characteristics. Those NPs, in turn, can be used as constituents of surface enhanced Raman spectroscopy substrates, which are devices where the electromagnetic field and effects thereof are giantly amplified. Here, the electroless formation of nanostructures was studied as a function of the geometry of the substrate. High resolution, electron beam lithography techniques were used to obtain nonperiodic arrays of circular patterns, in which the spacing of patterns was varied over a significant range. In depositing silver atoms in those circuits, the authors found that the characteristics of the aggregates vary with the pattern distance. When the patterns are in close proximity, the interference of different groups of adjacent aggregates cannot be disregarded and the overall growth is reduced. Differently from this, when the patterns are sufficiently distant, the formation of metal clusters of NPs is independent on the spacing of the patterns. For the particular subset of parameters used here, this critical correlation distance is about three times the pattern diameter. These findings were explained within the framework of a diffusion limited aggregation model, which is a simulation method that can decipher the formation of nanoaggregates at an atomic level. In the discussion, the authors showed how this concept can be used to fabricate ordered arrays of silver nanospheres, where the size of those spheres may be regulated on varying the pattern distance, for applications in biosensing and single molecule detection