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The homeostatic control of their environment is an essential task of living cells. It has been hypothesized that when microenvironmental pH inhomogeneities are induced by high cellular metabolic activity, diffusing protons act as signaling molecules, driving the establishment of cross-feeding networks sustained by the cell-to-cell shuttling of overflow products such as lactate. Despite their fundamental role, the extent and dynamics of such networks is largely unknown due to the lack of methods in single cell flux analysis. In this study we provide direct experimental characterization of such exchange networks. We devise a method to quantify single cell fermentation fluxes over time by integrating high-resolution pH microenvironment sensing via ratiometric nanofibers with constraint-based inverse modeling. We apply our method to cell cultures with mixed populations of cancer cells and fibroblasts. We find that the proton trafficking underlying bulk acidification is strongly heterogeneous, with maximal single cell fluxes exceeding typical values by up to 3 orders of magnitude. In addition, a crossover in time from a networked phase sustained by densely connected “hubs” (corresponding to cells with high activity) to a sparse phase dominated by isolated dipolar motifs (i.e. by pair-wise cell-to-cell exchanges) is uncovered, which parallels the time course of bulk acidification. Our method promises to shed light on issues ranging from the homeostatic function of proton exchange to the metabolic coupling of cells with different energetic demands, and paves the way for real-time non-invasive single cell metabolic flux analysis.

The tumour microenvironment (TME) strongly influences tumorigenesis and metastasis. Two of the most characterized properties of the TME are acidosis and hypoxia, both of which are considered hallmarks of tumours as well as critical factors in response to anticancer treatments. Currently, various imaging approaches exist to measure acidosis and hypoxia in the TME, including magnetic resonance imaging (MRI), positron emission tomography and optical imaging. In this review, we will focus on the latest fluorescent-based methods for optical sensing of cell metabolism and MRI as diagnostic imaging tools applied both in vitro and in vivo. The primary emphasis will be on describing the current and future uses of systems that can measure intra- and extra-cellular pH and oxygen changes at high spatial and temporal resolution. In addition, the suitability of these approaches for mapping tumour heterogeneity, and assessing response or failure to therapeutics will also be covered., Comment: 36 pages, 13 figures

Fine manipulation of fluid flows at the microscale has a tremendous impact on mass transport phenomena of chemical and biological processes inside microfluidic platforms. Fluid mixing in the laminar flow regime at low Reynolds is poorly effective due to the inherently slow diffusive mechanism. As a strategy to enhance mixing and prompt mass transport, here, we focus on polyelectrolyte multilayer capsules (PMCs) embodying a catalytic polyoxometalate as microobjects to create elastic turbulence and as micromotors to generate chaotic flows by fuel-fed propulsions. The effects of the elastic turbolence and of the artificial propulsion on some basic flow parameters, such as pressure and volumetric flow rate are studied by a microfluidic set-up including pressure and flow sensors. Numerical-handling and physical models of the experimental data are presented and discussed to explain the measured dependence of the pressure drop on the flow rate in presence of the PMCs. As a practical outcome of the study, a strong decrease of the mixing time in a serpentine microreactor is demonstrated. Unlike our previous reports dealing with capillarity flow studies, the present paper relies on hydrodynamic pumping experiments, that allows us to both develop a theoretical model for the understanding of the involved phenomena and demonstrate a successfully microfluidic mixing application. All of this is relevant in the perspective of developing microobject based methods to overcome microscale processes purely dominated by diffusion with potential improvements of mass trasport in microfluidic platforms., In Press, Corrected Proof

The realization of microfluidic platforms with liquid pumping and fluid transport independent on external power sources is the goal of a major part of research in the lab-on-chip (LOC) field. Autonomous pumping, indeed, has a strong impact on the cost, usability and portability of LOCs. In this context, power-free pumping is exploited herein by the use of chemically-responsive flexible thin membranes (TMs) as tool to push liquids inside the microchannels of a LOC platform. The assembled device consists of a closed poly(dimethylsiloxane) (PDMS) micro-chamber in which H2O2 dismutation occurs by an artificial catalase (ACat) system, evolving oxygen and generating a pressure gradient. This pressure is then used to push a liquid contained within an upper chamber and inject it into a tailored microfluidic channel. The two chambers are overlapped and separated by a PDMS TM, whose flexibility allows the conversion of the chemical energy into mechanical forces. Thanks to the fine-tuning of the reaction conditions by modulating the ACat catalyst and/or reagents concentrations, a precise control over the injection time and forces of the liquid can be achieved.

We report on the folding properties of the protein azurin, deposited onto SiO2 surfaces and subsequently dehydrated. The molecular films have been maintained at ambient conditions through several days, and the ageing effects have been investigated by fluorescence spectroscopy. The experimental results show a modest initial conformational rearrangement, followed by long-term stability. Interestingly, upon rehydration of the biomolecular films at the end of the investigated period (approximately one month), azurin returns to exhibit a native-like conformation. This study indicates a rather surprising resilience of proteins to ambient conditions and sheds a somewhat unexpected positive light on reliability in biomolecular electronics. (C) 2005 Elsevier B.V. All rights reserved.

Liquid flow in microchannels is completely laminar and uniaxial, with a very low Reynolds number regime and long mixing lengths. To increase fluid mixing and solubility of reactants, as well as to reduce reaction time, complex three-dimensional networks inducing chaotic advection have to be designed. Alternatively, turbulence in the liquid can be generated by active mixing methods (magnetic, acoustic waves, etc.) or adding small quantities of elastic materials to the working liquid. Here, polyelectrolyte multilayer capsules embodying a catalytic polyoxometalate complex have been suspended in an aqueous solution and used to create elastic turbulence and to propel fluids inside microchannels as an alternative to viscoelastic polymers. The overall effect is enhanced and controlled by feeding the polyoxometalate-modified capsules with hydrogen peroxide, H2O2, thus triggering an on-demand propulsion due to oxygen evolution resulting from H2O2 decomposition. The quantification of the process is done by analysing some structural parameters of motion such as speed, pressure, viscosity, and Reynolds and Weissenberg numbers, directly obtained from the capillary dynamics of the aqueous mixtures with different concentrations of H2O2. The increases in fluid speed as well as the capsule-induced turbulence effects are proportional to the H2O2 added and therefore dependent on the kinetics of H2O2 dismutation.

Molecular flexibility and rigidity are required to determine the function and specificity of protein molecules. Some psychrophilic enzymes demonstrate a higher catalytic efficiency at low temperatures, compared to the efficiency demonstrated by their meso/thermophilic homologous. The emerging picture suggests that such enzymes have an improved flexibility of the structural catalytic components, whereas other protein regions far from functional sites may be even more rigid than those of their mesophilic counterparts. To gain a deeper insight in the analysis of the activity-flexibility/rigidity relationship in protein structure, psychrophilic carbonic anhydrase of the Antarctic teleost Chionodraco hamatus has been compared with carbonic anhydrase II of Bos taurus through fluorescence studies, three-dimensional modeling, and activity analyses. Data demonstrated that the cold-adapted enzyme exhibits an increased catalytic efficiency at low and moderate temperatures and, more interestingly, a local flexibility in the region that controls the correct folding of the catalytic architecture, as well as a rigidity in the hydrophobic core. The opposite result was observed in the mesophilic counterpart. These results suggest a clear relationship between the activity and the presence of flexible and rigid protein substructures that may be useful in rational molecular and drug design of a class of enzymes playing a key role in pathologic processes.

We report a widely applicable and highly controlled approach, based on electron beam lithography (EBL), to interconnect single nano-objects, previously immobilized onto solid surfaces, and to investigate the transport properties at the level of single nanostructures. In particular, a three-step EBL-procedure was used for this purpose by patterning two planar contacts on the sides of an individual nano-object. To demonstrate this approach, we use two different kinds of active elements: a semiconductor nanocrystal (tetrapod) and a thin triangular gold nanoprism (NT). (c) 2007 Elsevier B.V. All rights reserved.

In this work we demonstrate the possibility of obtaining a significant increase of the photoluminescence of colloidal semiconductor nanocrystals (NCs) by means of metallic nanopatterns. Highly ordered triangular-shaped gold nanopatterns (typical dimensions 200 nm) were fabricated on planar substrates by electron beam lithography (EBL). Colloidal semiconductor nanocrystals (core/shell CdSe/ZnS quantum dots or CdSe nanorods) dispersed in a polymer matrix (PMMA) were subsequently deposited on the substrates by spin-coating. The coupling between the surface plasmons (SPs) resonance band of the metallic nanostructures and the excitation/emission bands of the nanocrystals resulted in a strong enhancement of the fluorescence from the quantum emitters, as probed by confocal microscopy analyses. Importantly, the proposed approach allows a precise control of the shape and dimensions of the single metallic nanostructure (and consequently of the SPs resonances), thanks to the nanometer resolution of the EBL. Moreover, the concentration of the NCs dispersed in the blend, as well as the thickness of the active layer, can be finely tuned. These results may open interesting perspectives for a wide range of applications, such as photonic devices, LEDs, sensor technology, microarrays, single/few molecules experiments, and biochemical/biophysical investigations. (C) 2006 Elsevier B.V. All rights reserved.

Electrical conduction in solid state disordered multilayers of non-redox proteins is demonstrated by two-terminal transport experiments at the nanoscale and by scanning tunneling microscopy (STM/STS experiments). We also show that the conduction of the biomolecular films can be modulated by means of a gate field. These results may lead to the implementation of protein-based three-terminal nanodevices and open important new perspectives for a wide range of bioelectronic/biosensing applications. (c) 2006 American Institute of Physics.

We show the possibility of realizing a hybrid system composed of a semiconductor nanoparticle (NP) and a metalloprotein, in which the photophysical properties of the two species can be exploited to elicit fluorescence resonance energy transfer mechanisms from the biomolecule to the NP. A specific conjugation process between CdSe/ZnS core/shell water soluble NPs, functionalized with surface exposed thiol groups (-SH), and the apo form of the metalloprotein azurin (Az) has been achieved, resulting in a fixed distance in the donor-acceptor pairs. The increase in the NP fluorescence intensity was found to be dependent on the Az to NP molar ratio. (c) 2005 Elsevier B.V. All rights reserved.

The authors report a highly controlled approach, based on electron-beam lithography, to interconnect individual nano-objects for transport experiments. The process is based on a three-step procedure, consisting of fabrication of four alignment markers, localization of the nano-object after its immobilization onto functionalized surfaces, and interconnection of the single nanostructure by patterning two nanoelectrodes on its sides. The approach is highly reproducible and widely applicable and allows an alignment accuracy of 15-20 nm. Here they demonstrate the reliability of such technique by using a thin triangular gold nanoprism as the active element and show the I-V characteristics of the single nanostructure. © 2006 American Vacuum Society.