Maria Laura Coluccio

Superhydrophobic Surfaces as Smart Platforms for the Analysis of Diluted Biological Solutions

The aim of this paper is to expound on the rational design, fabrication and development of superhydrophobic surfaces (SHSs) for the manipulation and analysis of diluted biological solutions. SHSs typically feature a periodic array or pattern of micropillars; here, those pillars were modified to incorporate on the head, at the smallest scales, silver nanoparticles aggregates. These metal nanoclusters guarantee superior optical properties and especially SERS (surface enhanced Raman scattering) effects, whereby a molecule, adsorbed on the surface, would reveal an increased spectroscopy signal. On account of their two scale-hybrid nature, these systems are capable of multiple functions which are (i) to concentrate a solution, (ii) to vehicle the analytes of interest to the active areas of the substrate and, therefore, (iii) to measure the analytes with exceptional sensitivity and very low detection limits. Forasmuch, combining different technologies, these devices would augment the performance of conventional SERS substrates and would offer the possibility of revealing a single molecule. In this work, similar SHSs were used to detect Rhodamine molecules in the fairly low atto molar range. The major application of this novel family of devices would be the early detection of tumors or other important pathologies, with incredible advances in medicine.

FT-IR, Raman, RRS measurements and DFT calculation for doxorubicin.

Doxorubicin (DOXO) is a powerful anthracycline antibiotic used to treat many human neoplasms, including acute leukemias, lymphomas, stomach, breast and ovarian cancer, and bone tumors, yet causing cardiotoxicity at the same time. For this reason, there is a great interest in medical field to gain deep insight and knowledge of this molecule. Raman, Fourier Transform Infrared (FT‐IR) absorption spectroscopy, and Resonance Raman scattering were performed for the vibrational characterization of DOXO molecule. Density function theorem (DFT) modeling of Raman and FT‐IR spectra were used for the assignment of the vibrational frequencies. The optimized molecular structured was obtained, first, on the basis of potential energy distribution. The simulation for vibrational bands is based on the calculations for internal force constants and potential energy distribution matrices. The calculated DOXO vibrational bands show qualitative agreement with the experimental observations (FT‐IR absorption and Raman scattering). Microsc. Res. Tech. 73:991–995, 2010.

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

Novel nanoarray for single molecule detection from peptide mixture. 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.

Electroless Deposition and Nanolithography Can Control the Formation of Materials at the Nano-Scale for Plasmonic Applications

The new revolution in materials science is being driven by our ability to manipulate matter at the molecular level to create structures with novel functions and properties. The aim of this paper is to explore new strategies to obtain plasmonic metal nanostructures through the combination of a top down method, that is electron beam lithography, and a bottom up technique, that is the chemical electroless deposition. This technique allows a tight control over the shape and size of bi- and three-dimensional metal patterns at the nano scale. The resulting nanostructures can be used as constituents of Surface Enhanced Raman Spectroscopy (SERS) substrates, where the electromagnetic field is strongly amplified. Our results indicate that, in electroless growth, high quality metal nanostructures with sizes below 50 nm may be easily obtained. These findings were explained within the framework of a diffusion limited aggregation (DLA) model, that is a simulation model that makes it possible to decipher, at an atomic level, the rules governing the evolution of the growth front; moreover, we give a description of the physical mechanisms of growth at a basic level. In the discussion, we show how these findings can be utilized to fabricate dimers of silver nanospheres where the size and shape of those spheres is controlled with extreme precision and can be used for very large area SERS substrates and nano-optics, for single molecule detection.

Microfluidic devices modulate tumor cell line susceptibility to NK cell recognition.

This study aims to adoptively reduce the major histocompatibility complex class I (MHC-I) molecule surface expression of cancer cells by exposure to microfluid shear stress and a monoclonal antibody. A microfluidic system is developed and tumor cells are injected at different flow rates. The bottom surface of the microfluidic system is biofunctionalized with antibodies (W6/32) specific for the MHC-I molecules with a simple method based on microfluidic protocols. The antibodies promote binding between the bottom surface and the MHC-I molecules on the tumor cell membrane. The cells are injected at an optimized flow rate, then roll on the bottom surface and are subjected to shear stress. The stress is localized and enhanced on the part of the membrane where MHC-I proteins are expressed, since they stick to the antibodies of the system. The localized stress allows a stripping effect and consequent reduction of the MHC-I expression. It is shown that it is possible to specifically treat and recover eukaryotic cells without damaging the biological samples. MHC-I molecule expression on treated and control cell surfaces is measured on tumor and healthy cells. After the cell rolling treatment a clear reduction of MHC-I levels on the tumor cell membrane is observed, whereas no changes are observed on healthy cells (monocytes). The MHC-I reduction is investigated and the possibility that the developed system could induce a loss of these molecules from the tumor cell surface is addressed. The percentage of living tumor cells (viability) that remain after the treatment is measured. The changes induced by the microfluidic system are analyzed by fluorescence-activated cell sorting and confocal microscopy. Cytotoxicity tests show a relevant increased susceptibility of natural killer (NK) cells on microchip-treated tumor cells.

Poly(ethylene‐co‐vinyl alcohol) Membranes with Specific Adsorption Properties for Potential Clinical Application

Abstract The preparation of novel polymeric systems through Molecular Imprinting Technology (MIT) for potential application in extracorporeal blood purification is described. Membranes based on poly(ethylene‐co‐vinyl alcohol) material, produced through a phase inversion method, were modified introducing in their structure specific binding sites for lipid and/or protein molecules. Membranes prepared are intended to selectively remove low density lipoproteins and cholesterol (LDL) from the plasma, by using interactions at a molecular level, between the molecularly imprinted membrane and specific target molecules, created during the preparation procedure. The binding performances of membranes and their potentiality as adsorbents for two different model target compounds, a phospholipid (phosphotidylcholine, PC) and a protein (α‐amylase enzyme) were investigated, showing improved adsorption capacity with respect to unmodified control membranes. In addition, molecularly imprinted poly(ethylene‐co‐vinyl alcohol) materials in the shape of microparticles, using the same templates, were prepared and studied for their potential use as adsorbers into a column.

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 thi...

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.

Silver self aggregation in a nanodevice for enhanced Raman spectroscopy: experiments vs. simplified modeling via molecular dynamics

We present a study, via experiments and exploratory molecular dynamics simulations, of self aggregation in cylindrical nanostructures obtained experimentally by combining high resolution electron beam lithography with electroless silver deposition. This process is key to the fabrication of a nanolens device, where a strong surface enhancement can be exploited for Raman spectroscopy. In order to investigate the process, we introduce a simple theoretical model and compare the results of simulations with the fabricated silver nanostructures during the growth phase. Our simulations qualitatively agree with the experiments and allow a general characterization of the process at length scales smaller than those easily accessible by microscopy. We identify a geometrical parameter, the aspect ratio of the cylinder, that relates two different types of growth with different characteristics and, possibly, different Raman enhancements.

Folic Acid Functionalized Surface Highlights 5‐Methylcytosine‐Genomic Content within Circulating Tumor Cells

Although the detection of methylated cell free DNA represents one of the most promising approaches for relapse risk assessment in cancer patients, the low concentration of cell-free circulating DNA constitutes the biggest obstacle in the development of DNA methylation-based biomarkers from blood. This paper describes a method for the measurement of genomic methylation content directly on circulating tumor cells (CTC), which could be used to deceive the aforementioned problem. Since CTC are disease related blood-based biomarkers, they result essential to monitor tumors stadiation, therapy, and early relapsing lesions. Within surfaces bio-functionalization and cells isolation procedure standardization, the presented approach reveals a singular ability to detect high 5-methylcytosine CTC-subset content in the whole CTC compound, by choosing folic acid (FA) as transducer molecule. Sensitivity and specificity, calculated for FA functionalized surface (FA-surface), result respectively on about 83% and 60%. FA-surface, allowing the detection and characterization of early metastatic dissemination, provides a unique advance in the comprehension of tumors progression and dissemination confirming the presence of CTC and its association with high risk of relapse. This functionalized surface identifying and quantifying high 5-methylcytosine CTC-subset content into the patients blood lead significant progress in cancer risk assessment, also providing a novel therapeutic strategy.