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The DNA‐based parallelogram is a self‐assembling nanostructure that was first designed and prepared almost 10 years ago. Even though a variety of different and better‐bahaved DNA tiles have been proposed and realized, the DNA parallelogram still holds interest. It can be used as template for the assembly of nucleic‐acids or non‐nucleic acid functional elements and it can be polymerized into supramolecular assemblies of complex hierarchy.

This paper is concerned with an investigation of electron transfer between cytochrome P450scc (CYP11A1) immobilized on nanostructured rhodium-graphite electrodes. Multi-walled carbon nanotubes (MWCNT) were deposited onto the rhodium-graphite electrodes by drop casting. Cytochrome P450scc was deposited onto MWCNT-modified rhodium-graphite electrodes. Cytochrome P450scc was also deposited onto both gold nanoparticle-modified and bare rhodium-graphite electrodes, in order to have a comparison with our previous works in this field. Cyclic voltammetry indicated largest enhanced activity of the enzyme at the MWCNT-modified surface. The role of the nanotubes in mediating electron transfer to the cytochrome P450scc was verified as further improved with respect to the case of rhodium-graphite electrodes modified by the use of gold nanoparticles. The sensitivity of our system in cholesterol sensing is higher by orders of magnitude with respect to other similar systems very recently published that are based on cholesterol oxidase and esterase. The electron transfer improvement attained by the use of MWCNT in P450-based cholesterol biosensors was demonstrated to be larger than 2.4 times with respect to the use of gold nanoparticles and 17.8 times larger with respect to the case of simple bare electrodes. The sensitivity was equal to 1.12 mu A/(mM mm(2)) and the linearity of the biosensor response was improved with respect to the use of gold nanoparticles. (C) 2008 Elsevier B.V. All rights reserved.

A brief survey of thein vitro methods of preparing DNA mesomorphic phases is presented. The significance of thesein vitro preparations for thein vivo condensed states of DNA is also discussed. The procedure of preparing a dispersion of mesomorphic condensates of unsonicated calf thymus DNA by using polyethylene glycol was investigated by the circular dichroism (CD) technique. The operational conditions able to provide at the same time good packing of the DNA chains and reproducible CD signals were searched. The morphology of the condensates thus obtained was studied by transmission electron microscopy.

Human α-Synuclein (αSyn) is a natively unfolded protein whose aggregation into amyloid fibrils is involved in the pathology of Parkinson disease. A full comprehension of the structure and dynamics of early intermediates leading to the aggregated states is an unsolved problem of essential importance to researchers attempting to decipher the molecular mechanisms of αSyn aggregation and formation of fibrils. Traditional bulk techniques used so far to solve this problem point to a direct correlation between αSyn's unique conformational properties and its propensity to aggregate, but these techniques can only provide ensemble-averaged information for monomers and oligomers alike. They therefore cannot characterize the full complexity of the conformational equilibria that trigger the aggregation process. We applied atomic force microscopy–based single-molecule mechanical unfolding methodology to study the conformational equilibrium of human wild-type and mutant αSyn. The conformational heterogeneity of monomeric αSyn was characterized at the single-molecule level. Three main classes of conformations, including disordered and “β-like” structures, were directly observed and quantified without any interference from oligomeric soluble forms. The relative abundance of the “β-like” structures significantly increased in different conditions promoting the aggregation of αSyn: the presence of Cu2+, the pathogenic A30P mutation, and high ionic strength. This methodology can explore the full conformational space of a protein at the single-molecule level, detecting even poorly populated conformers and measuring their distribution in a variety of biologically important conditions. To the best of our knowledge, we present for the first time evidence of a conformational equilibrium that controls the population of a specific class of monomeric αSyn conformers, positively correlated with conditions known to promote the formation of aggregates. A new tool is thus made available to test directly the influence of mutations and pharmacological strategies on the conformational equilibrium of monomeric αSyn., Author Summary Natively unstructured proteins defy the classical “one sequence–one structure” paradigm of protein science. In pathological conditions, monomers of these proteins can aggregate in the cell, a process that underlies neurodegenerative diseases such as Alzheimer and Parkinson. A key step in the aggregation process—the formation of misfolded intermediates—remains obscure. To shed light on this process, we characterized the folding and conformational diversity of αSyn, a natively unstructured protein involved in Parkinson disease, by mechanically stretching single molecules of this protein and recording their mechanical properties. These experiments permitted us to observe directly and quantify three main classes of conformations that, under in vitro physiological conditions, exist simultaneously in the αSyn sample. We found that one class of conformations, “β-like” structures, is directly related to αSyn aggregation. In fact, their relative abundance increases drastically in three different conditions known to promote the formation of αSyn fibrils. We expect that a critical concentration of αSyn with a “β-like” structure must be reached to trigger fibril formation. This critical concentration is therefore controlled by a chemical equilibrium. Novel pharmacological strategies can now be tailored to act upstream, before the aggregation process ensues, by targeting this equilibrium. To this end, single-molecule force spectroscopy can be an effective tool to tailor and test new pharmacological agents., A single-molecule study detects structured and unstructured conformers in equilibrium in monomeric α-synuclein. The β-like conformers increase with pathological mutations and under other conditions known to promote aggregation.

Several authors demonstrated that an oligonucleotide based pH-sensitive construct can act as a switch between an open and a closed state by changing the pH. To validate this process, specially designed fluorescence dye-quencher substituted oligonucleotide constructs were developed to probe the switching between these two states. This paper reports on bulk and single molecule fluorescence investigations of a duplex-triplex pH sensitive oligonucleotide switch. On the bulk level, only a partial quenching of the fluorescence is observed, similarly to what is observed for other published switches and is supposed to be due to intermolecular interactions between oligonucleotide strands. On the single molecule level, each DNA-based nanometric construct shows a complete switching. These observations suggest the tendency of the DNA construct to associate at high concentration.

There are many processes in biology in which mechanical forces are generated. Force-bearing networks can transduce locally developed mechanical signals very extensively over different parts of the cell or tissues. In this article we conduct an overview of this kind of mechanical transduction, focusing in particular on the multiple layers of complexity displayed by the mechanisms that control and trigger the conversion of a mechanical signal into a biochemical function. Single molecule methodologies, through their capability to introduce the force in studies of biological processes in which mechanical stresses are developed, are unveiling subtle intertwining mechanisms between chemistry and mechanics and in particular are revealing how chemistry can control mechanics. The possibility that chemistry interplays with mechanics should be always considered in biochemical studies., 50 pages, 18 figures

We wish to propose a novel mechanism by which the triggering of a biochemical signal can be controlled by the hierarchical coupling between a protein redox equilibrium and an external mechanical force. We have characterized this mechanochemical mechanism in angiostatin, and we have evidence that it can switch the access to partially unfolded structures of this protein. We have identified a metastable intermediate that is specifically accessible under thioredoxin-rich reducing conditions, like those met by angiostatin on the surface of a tumor cell. The structure of the same intermediate accounts for the unexplained antiangiogenic activity of angiostatin. These findings demonstrate a new link between redox biology and mechanically regulated processes.

Silicon technology is rapidly progressing toward device sizes in the range of 10 nm. The evolution rate of silicon technology in next decades will require the use of new materials and processes to develop revolutionary CMOS devices. Organic nanostructures onto semiconductors or metals may provide interesting solutions to address the technological issues of nanoscale devices. In particular, technologies and devices based on organic nanostructures such as carbon nanotubes, semiconductor or metallic nanoparticles, DNA-templated nanowires may supply solutions for on-chip interconnections. The aim of the this work is to present recent results on hybrid nanostructures based on gold nanoparticles stabilized by organic coating. Particular emphasis is devoted to the problem of the interconnections at the nanoscale. The resulting hybrid devices are suitable to develop Single Electron Transistors and memories.

Atomic force microscopy (AFM) has proved to be an essential tool of structural biology, being able not only to image but also to manipulate single biological molecules. These techniques make it possible to investigate the nanometer scale structure of single biological macromolecules and to study how an external force drives single biological molecules towards nonequilibrium conformations, by stretching and breaking bonds and interactions. This chapter focuses on the capabilities of the AFM-based single molecule methodologies to bring us into the nanometer-scale world of the single DNA molecules and into the pico-Newton force-scales of the interactions that sustain the protein folding.

The self-assembly of oligodeoxynucleotides is a versatile and powerful tool for the construction of objects in the nanoscale. The strictly information-driven pairing of DNA fragments can be used to rationally design and build nanostructures with planned topologies and geometries. Taking advantage of the steadily expanding library of well-characterized DNA motifs, several examples of structures with different dimensionalities have appeared in the literature in the past few years, laying the foundations for a promising DNA-mediated, bottom-up approach to nanotechnology. This article focuses on recent developments in this area of research and proposes a classification of DNA nanostructures based on topological considerations in addition to describing strategies for tackling the inherent complexities of such an endeavor.

We present a set of DNA supramolecular architectures based on the polymerization of discrete DNA tiles having the shape of parallelograms and designed to have a one-dimensional inter-tile connectivity. Tiles bind to each other with two connections, which have different thermal stabilities. We discuss how this difference in stability implies that the same monomeric tile can yield supramolecular polymers of different shapes just by changing the polymerization conditions. We show how this system reacts to external stimuli by interconverting between some of its possible states. Concurrently, we show how performing the polymerization on a surface can influence its outcome.

A multitude of DNA-based nanostructures of different size and dimensionality have appeared in the literature in the last few years. The programmed self-assembly of oligodeoxynucleotides can be employed to build DNA nanostructures by design in a very successful approach towards bottom-up nanofabrication. This review paper will focus on the developments in this area of research and on some of the properties of DNA that elicited the birth of this new area of research.

By studying with the single molecule force spectroscopy (SMFS) methodology the mechanical behaviour of single biomolecules, we are learning how mechanical forces like those present in the extracellular space modify the conformation of proteins, possibly leading to functional switches. We also understand that the functional efficiency of those mechanical switches can rely on their coupling to some independent biochemical control of the protein conformational changes. The disulfide bonds have been recently proposed to act as potential redox switches, even if their structural bases are unclear. Here we discuss, also on the basis of experimental evidences based on SMFS, the possibility that disulfide bond switching and mechanical deformation of extracellular proteins can be coupled, thus leading to an efficient and highly tuned switch for protein function. We propose this as one of the biological mechanisms that regulate extracellular protein functionality. (c) 2006 Elsevier Ltd. All rights reserved.

This paper presents a fully electronic label-free DNA chip in 0.5-mum CMOS technology, with 5-V supply voltage, suitable for low-cost highly integrated applications. The chip features an array of 128 sensor sites with gold electrodes and integrated measurement, conditioning, multiplexing and analog-to-digital conversion circuitry. The circuits measure capacitance variations due to DNA hybridization on the gold electrodes which are bio-modified by covalently attaching probes of known sequence. Specificity, repeatability and parallel detection capability of the fabricated chip are successfully demonstrated

Hierarchical self-assembly is nature’s solution to the build up of complex structures starting from components that are orders of magnitude smaller [4]. The components are brought together in a precisely defined way by recognition processes based on non-covalent interactions. This offers the possibility for error correction through a continuous sequence of trial and error steps to optimize functionality. Under a set of hierarchical assembly nstructions, the functional aggregates produced at each level are the building blocks for the self-assembly at the next higher level of complexity. The information needed for the formation of the resulting structures is encoded within the covalent structure of the subunits. This integration of components over disparate length scales might include inorganic matter. The inorganic matter can be a surface upon which the assembled organic structure is transferred from a 3D to a quasi-2D space. This could lead to functional nanodevices with properties that do not exist, not only in the individual components but also in the organic assembled structure alone.

Critical information on several biological processes such as DNA-protein interactions and DNA transcription can be derived from analysis of DNA curvature. Under thermal perturbation, the curvature is composed of static and dynamic contributions, thus, can be described as the sum of intrinsic curvature and a fluctuation contribution. Without considering thermal agitations, the DNA curvature is reducible to the intrinsic component, which is a function of the DNA nucleotide sequence only. In this paper, we present an automated algorithm to determine the DNA intrinsic curvature profiles and the molecular spatial orientations in Atomic Force Microscope images. The algorithm allows to reconstruct the intrinsic curvature profile by filtering the thermal contribution. It detects fragment orientation on atomic force microscope images without labels with a percentage of correct molecular-orientation detection of 96.79% in computer-generated benchmarks, for molecules with a high curvature peak. The automated algorithm reconstructs the intrinsic curvature profile of DNA molecules with a mean square error of 3.8122/spl middot/10/sup -4/ rads over a profile with a central peak value of 0.196 rads, and 6.1/spl middot/10/sup -3/ rads over a curvature profile with two symmetric peaks of about 0.08rads. Moreover, it correctly detects the location of the peaks in the molecules with a deviation of about 1% of molecule length.

We here report that the formation and breakdown of an intramolecular cytosine-thymine (CT) motif DNA triple-helix can be performed repeatedly, quickly and independently of its local concentration without performance reduction over successive cycles; as a consequence, we propose that this set of characteristics makes the DNA duplex-triplex transition an ideal candidate to power simple nanometer-scale devices capable of maintaining effective performance regardless of their local concentration.

The nanometer scale is a special place where all sciences meet and develop a particularly strong interdisciplinarity. While biology is a source of inspiration for nanoscientists, chemistry has a central role in turning inspirations and methods from biological systems to nanotechnological use. DNA is the biological molecule by which nanoscience and nanotechnology is mostly fascinated. Nature uses DNA not only as a repository of the genetic information, but also as a controller of the expression of the genes it contains. Thus, there are codes embedded in the DNA sequence that serve to control recognition processes on the atomic scale, such as the base pairing, and others that control processes taking place on the nanoscale. From the chemical point of view, DNA is the supramolecular building block with the highest informational content. Nanoscience has therefore the opportunity of using DNA molecules to increase the level of complexity and efficiency in self-assembling and self-directing processes.

This paper presents a new electronic methodology to detect DNA hybridization for rapid identification of diseases, as well as food and environmental monitoring on a genetic base. The proposed solution exploits a new (electrical) capacitive measurement circuit, not requiring any prior labeling of the DNA (as it is often the case with the commonly employed optical detection). The sensitivity, the reliability, and the reproducibility of this device have been evaluated by experiments performed with a (non-integrated) prototype implementation, easily integrable in IC and/or micro-fabricated lab-on-a-chip. (C) 2003 Elsevier B.V. All rights reserved.

The supercoiling of topologically constrained DNA molecules is altered by modifications of their original B structure. Contact mode, scanning force microscope (SFM) imaging requires a fairly tight binding of the DNA molecules to a flat surface, generally mica, and is often accomplished using divalent or trivalent cations and dehydrating the sample thoroughly immediately upon deposition, following procedures known to cause at least a partial B‐DNA to A‐DNA transition. We report a modification of the DNA deposition method presented by Koller et al. [Biopolymers 13, 995 (1974)] that is suitable to SFM imaging of DNA and allows complete control of the conditions of deposition (ionic strength, temperature). We have measured the contour length of linear DNA molecules in SFM images of depositions prepared with the reported method. Our data show that the B structure is preserved.

Fibronectin (Fn) is an important mediator of bacterial invasions and of persistent infections like that of Staphylococcus epidermis . Similar to many other types of cell-protein adhesion, the binding between Fn and S. epidermidis takes place under physiological shear rates. We investigated the dynamics of the interaction between individual living S. epidermidis cells and single Fn molecules under mechanical force by using the scanning force microscope. The mechanical strength of this interaction and the binding site in the Fn molecule were determined. The energy landscape of the binding/unbinding process was mapped, and the force spectrum and the association and dissociation rate constants of the binding pair were measured. The interaction between S. epidermidis cells and Fn molecules is compared with those of two other protein/ligand pairs known to mediate different dynamic states of adhesion of cells under a hydrodynamic flow: the firm adhesion mediated by biotin/avidin interactions, and the rolling adhesion, mediated by L-selectin/P-selectin glycoprotein ligand-1 interactions. The inner barrier in the energy landscape of the Fn case characterizes a high-energy binding mode that can sustain larger deformations and for significantly longer times than the correspondent high-strength L-selectin/P-selectin glycoprotein ligand-1 binding mode. The association kinetics of the former interaction is much slower to settle than the latter. On this basis, the observations made at the macroscopic scale by other authors of a strong lability of the bacterial adhesions mediated by Fn under high turbulent flow are rationalized at the molecular level.

Staphylococcus epidermidis is able to adhere onto biomaterials and to cause implant infections. Recently, host matrix proteins, which in vivo cover the implants, have been indicated as substrates for adhesion by specific bacterial adhesins. Here, the binding of S. epidermidis to fibronectin, a main protein of the extracellular matrix, and the effect of heparin on this interaction were studied by dynamic force spectroscopy (DFS). Novelties are that S. epidermidis strains analysed by DFS were clinical isolates from prosthesis-associated infections, genotyped and phenotyped for their adhesion properties to fibronectin and examined as living cells. Thus, fibronectin-binding staphylococci adhered to the fibronectin-coated substratum and formed a continuous layer assuring their contact with the fibronectin-coated cantilever tip during the approach-retraction cycles of the DFS measurements. Results show that only a single molecular binding site of fibronectin is involved in the interaction with S. epidermidis, that it takes place at the domain near the C-terminus and that it is specifically inhibited by heparin.

Both the processes of adsorption of double‐stranded DNA and its desorption from the surface of freshly cleaved mica can be studied thanks to the Scanning Force Microscope (SFM). The adsorption can be investigated under different conditions with methods ranging from the simple count of adsorbed DNA molecules to the more elaborate study of the local curvature of the adsorbed DNA chains: this high‐resolution information is shedding light on the process of structure‐dependent DNA adsorption on crystal surfaces. SFM‐based Single‐Molecule Force Spectroscopy is very useful for studying the desorption process of DNA from the same surfaces, with the same level of environmental control.

The genome of parvovirus B19 is a 5600-base-long single-stranded DNA molecule with peculiar sequence symmetries. Both complementary forms of this single-stranded DNA are contained in distinct virions and they hybridize intermolecularly to double-stranded DNA if extracted from the capsids with traditional methods, thus losing some of their native structural features. A scanning force microscopy analysis of these double-stranded DNA molecules after thermal denaturation and renaturation gave us the chance to study the possible states that this DNA can assume in both its single-stranded and double-stranded forms. A novel but still poorly reproducible in situ lysis experiment that we have conducted on single virions with the scanning force microscope made it possible to image the totally unpaired state that the single-stranded DNA molecule most likely assumes inside the viral particle. Structural considerations on single molecules offer the opportunity for the formulation of plausible hypotheses on the interaction between the DNA and the viral structural proteins that could prove important for the DNA packaging in the capsid and, possibly, the viral infection mechanisms.

The availability of a number of methods to controllably adsorb DNA on solid surfaces is useful to researchers working in different fields, such as structural biology, biophysical chemistry, diagnostics, sensorics, and nanotechnology. In this paper, we review some of the methods that have been devised in the last years to solve this problem. Thanks to the Scanning Force Microscope, we have recently been able to study the dynamics of DNA molecules adsorbed on mica. From the statistical analysis of the shapes of properly designed DNA molecules, we have also discovered an unexpected base‐sequence specificity in the adsorption of intrinsically curved DNA molecules on mica.

The large demand for DNA analysis calls for the development of portable, low-cost, easy-to-perform assays. We developed and performed preliminary assessments of an integrable biosensor for the direct detection of DNA sequences through capacitance measurements. The device behaves consistently with a proposed electrical model and it reliably detects DNA hybridization with high specificity. We have also verified the reproducibility of the experimental results and the reusability of the DNA biosensor.

Scanning force microscopy was used to study in fluid the conformational fluctuations of two double-stranded DNA molecules resulting from differently cut pBR322 circular DNAs. A new approach was conceived to monitor the thermodynamic equilibrium of the chain dynamics on different scale lengths. This method made it possible to demonstrate that both the observed DNA molecules were allowed to equilibrate only on their local small-scale dynamics during the time of the experiment. This capability of monitoring the length scale and the time scale of the equilibration processes in the dynamics of a DNA chain is relevant to give an insight in the thermodynamics of the DNA binding with proteins and synthetic ligands. It was also shown that the small-scale equilibration of the DNA chain during surface-restricted dynamics is enough to allow a valid measurement of the local sequence-dependent curvature.

The energy of DNA deformation plays a crucial and active role in its packaging and its function in the cell. Considerable effort has gone into developing methodologies capable of evaluating the local sequence-directed curvature and flexibility of a DNA chain. These studies thus far have focused on DNA constructs expressly tailored either with anomalous flexibility or curvature tracts. Here we demonstrate that these two structural properties can be mapped also along the chain of a “natural” DNA with any sequence on the basis of its scanning force microscope (SFM) images. To know the orientation of the sequence of the investigated DNA molecules in their SFM images, we prepared a palindromic dimer of the long DNA molecule under study. The palindromic symmetry also acted as an internal gauge of the statistical significance of the analysis carried out on the SFM images of the dimer molecules. It was found that although the curvature modulus is not efficient in separating static and dynamic contributions to the curvature of the population of molecules, the curvature taken with its direction (its sign in two dimensions) permits the direct separation of the intrinsic curvature from the flexibility contributions. The sequence-dependent flexibility seems to vary monotonically with the chain's intrinsic curvature; the chain rigidity was found to modulate as its local thermodynamic stability and does not correlate with the dinucleotide chain rigidities evaluation made from x-ray data by other authors.

Individual molecules can be stretched with a scanning force microscope and the forces required to rupture bonds and to mechanically drive their structures towards new conformations and states can be measured. The tailoring of the experiments, the possibility of carrying them out in quasi-equilibrium conditions, the relationships between single molecule force measurements, and macroscopic kinetics or thermodynamic data are discussed. Mechanochemical experiments are expanding chemistry into new realms between biology and material science.

Reproducible images of pBR322 plasmid molecules have been recorded by Atomic Force Microscopy (AFM) under n-propanol. By applying a stratigraphic analysis which takes advantage of the height information contained in the AFM images, it is possible to assign the chirality of the local supercoiling of the individual molecules.

Reproducible images of pBR322 plasmid molecules have been recorded by scanning force microscopy under 1-propanol. Most of the plasmids were found in a coiled state. The supercoiled molecules of our samples look like branched or unbranched interwound superhelixes. This is consistent with available electron microscopy data on circular DNA molecules. By applying a stratigraphic analysis which takes advantage of the height information contained in the scanning force microscopy images, it is possible to assign the chirality of the local supercoiling of the individual molecules.

A general approach is developed to interpret linear dichroism (LD) spectra of ubiquinones (Qn) in host bilayers. Information is reported in terms of guest-host mutual orientation and localization. The overall orientational anisotropy of guest ubiquinone molecules is described by a basic set of limiting orientation/localization modes. Assignments of the UV transitions of the ubiquinone chromophore were obtained by the liquid crystal-linear dichroism technique and molecular orbital (CNDO/S) calculations. The LD spectra of Qn in the bilayers provided by the lyotropic nematic mesophase exhibited by water solutions of potassium laurate and decanol were interpreted on the basis of the above assignments. The resulting experimental evidence showed a multisite distribution in the host bilayer for the aromatic heads of all the investigated Qn derivatives except Q0. The orientational distribution suggested by the LD spectra fits the solubilization model recently proposed by G. Lenaz [J. Membrane Biol. (1988) 104:193-209] for ubiquinone in lipid membranes. Within this model Qn molecules are located in the midplane and their headgroups oscillate transversally across the membrane. Q0 instead has a single site location, close to the polar bilayer interface. Experimental evidence that the headgroup carbonyls tend to grasp the polar interface of the host bilayer was also obtained. Orientation and location distributions of Qn guest molecules are therefore likely to result from the tendency of their aromatic heads to grasp the polar heads of the host bilayer and from the concurrent tendency of their chains to settle into the hydrocarbon host interior.

Summary Point-of-care biosensing for multiple marker detection requires multiple-site detection, ease of use, portability, and low cost. State-of-the-art measuring systems are based on optical detection, but such techniques demand bulky, expensive, and often delicate instrumentation. Systems based on electronic detection are good candidates for the next generation technology for biosensors. Integration of electrodes and electronics onto the same silicon die for biosensing and molecular diagnostic applications has advantages in terms of high parallelism, small probe volumes and low noise. On the other hand, VLSI solutions have high non-recurring-engineering cost and are not suited for small volume production. Hybrid electronic circuits connected to a passive disposable support featuring a limited number of electrodes represent a more cost-effective solution for moderate-parallelism (less than 100 sites) applications, such as protein biomarker monitoring. The main challenge in this case is lower accuracy and higher noise levels due to connectors and limited device matching. In this work we demonstrate a flexible system based on hybrid electronics for capacitive biosensing that achieves signal-to-noise ratios compatible with micro-fabricated arrays of passive electrodes. Biosensor operating principle Our biosensing approach relies on the principle of chronoamperometry, i.e., the measurement of the time constant associated with the electrical transient which follows the application of a voltage step to a pair of bio-functionalized electrodes immersed in a solution. The system presented in this work is based on a printed circuit board and allows addressing and excitation of multiple electrode couples (in this prototype implementation up to 16), as well as the parallel measurement of the transient characteristics. The key design specification is detection of capacitive impedance variations between 4% and 20%. Results The measurement circuit provides a square wave through a follower stage and switches to excite several sensing sites of the biosensor independently. The response RC decay signal is amplified and sampled by an NI-DAQ acquisition device (sampling rate: 400 kHz). The load resistor and the amplification factor can be selected to tune the decay constant in a certain range (around 100 μsec). The addressing devices on the PCB allow an independent measurement of each site. The switches and the follower stage can be considered transparent to the excitation signal, and the noise is independent of the excitation voltage between −100 mV and 100 mV. Signal distortion caused by the limited bandwidth of the amplifier is negligible for transients longer than 1 μs decay time. Parasitic elements influence the response transient when the measured capacitance is lower than 100 pF. This lower capacitance limit corresponds to an electrode size of 100 μm × 100 μm, which is fully adequate to moderate density arrays. As an example of the use of the system, 1 mm2 gold electrodes with a self-assembled monolayer of ethylene glycols were measured. The response transient was well represented by an exponential, and the capacitance value was 11.6 nF and was determined with a relative standard deviation of 1.5%, which fully meets the design specification.

Introduction The progress in multi-marker biosensors is expected to induce changes in medicine beyond the mere innovation of diagnostic screening tests. In the near future, decentralized point-of-care testing and therapy is expected to foster the birth of personalized medicine, revolutionizing the classical hospital-centered analysis laboratory. Low-cost, automatic, fool-proof diagnostic systems and parallelized analyses will represent key technologies for the implementation of such epochal change. Most multi-marker assays are nowadays performed with surface-bound strategies. These derive directly from the parallelization of classical techniques that involve complex multi-step procedures. In a more modern, but still largely experimental approach, most of the assay specificity relies on the specific binding of the analyte to its probing molecule that has been covalently immobilized on a designed surface. The detection of such binding can then be measured with label-free techniques that are sensitive to a change of a physical property related to the binding of the analyte material. This can be a change in dielectric constant, mass, or ion conductivity. In order for such a strategy to be successful, it is at least required that i) the probe molecules are highly specific, exposed efficiently to the surface and stably bound, ii) the solid-liquid interface is stable over time and is not subject to non-specific interactions with molecules in the analyte, iii) the measuring technique is sensitive enough. As an additional set of desired features, the entire process should be amenable to parallelization, easy to run, inexpensive, and possibly provide real-time data. Results The instrumentation we developed responds to variations of electrical properties as a consequence of the affinity binding of biological material on an derivatized electrode. The biosensing principle relies on a custom application of 2-electrode chronoamperometry. Such instrumentation can work in parallel on a large number of measuring microelec-trodes embedded in a microfluidic system. Time-drift is a common instability problem of electrochemical measurements for biosensing, as the measurement can modify the interface. Our instrumentation applies such a low excitation signal that it does not perturb the sensitive interface, hence minimizing time-drift to a value negligible in the timeframe of the analysis (about 0.1 %/min). A simple variation of the standard electronics used for chronoamperometry makes the measurement independent of the specific composition of the measuring buffer, and makes it possible to perform tests in moderately complex buffers. Along with the low time-drift, this is an essential feature for real-time analysis, which can be performed with this setup. The biosensor was employed to study the dynamic assembly and substitution of self-assembled monolayers on a clean gold surface, and it proved sensitive enough to follow such processes in real time. Likewise, the dynamics of the non-specific adsorption of proteins on gold electrodes has been observed. Such experiments can be performed with many techniques, but our simple, parallel and compact electronic approach is a valuable practical improvement. For the purpose of biological assays, the microelectrodes have been functionalized with a monolayer based on mixed oli-goethyleneglycol alkanethiols, used to immobilize and expose biological probe molecules to the analyte. Such monolayer reduces unwanted non-specific adsorption. The advances presented herein, in particular the high stability, parallelism and the possibility to measure in moderately complex buffers, could open new perspectives in the development of electrochemical techniques for diagnostic multi-marker applications.

The effect of daunomycin upon DNA condensed states induced by poly(ethylene glycol) (PEG) was studied by circular dichroism (CD) and circular intensity differential scattering (CIDS). The CD spectra of these aggregates showed psi-type anomalies and intensities 10-100 times greater than those obtained with the dispersed DNA solutions in the absence of PEG. Increasing concentrations of daunomycin, added to the DNA solution prior to its aggregation, led, in the presence of PEG, to CD and CIDS signals which gradually decreased in magnitude and eventually inverted sign. The coincidence of the transition point of both signals and a careful characterization of the CD spectrum at the transition point clearly indicated that the inversion observed corresponds to an inversion of the handedness of the aggregates. The latter result suggests that the structure of the aggregates at the inversion point should resemble that of a nematic liquid-crystalline structure. The characteristic B-DNA spectrum obtained in this case further suggests that the packing process does not affect the secondary structure of the DNA molecules and that small changes in their local structure can induce dramatic changes in their long-range tertiary packing. The results obtained in this study represent a confirmation of a recent theory of psi-type CD in which the anomalous signals are interpreted as a manifestation of the long-range chirality of the aggregates.

The binding between Distamycin 3 and the palindromic duplexes d(CGTTTAAACG)2 and d(CGTACGTACG)2 was investigated by two independent techniques: UV-Vis absorption in the Job's plot approach and Induced Circular Dichroism. Both decamers bind two molecules of peptide per duplex, with close overall affinities. This result indicates that a row of six A:T base pairs can accommodate two molecules of drug and that the minimal binding site of Distamycin 3 can consist of just two A:T base pairs.

Scanning force microscopy can routinely image supercoiled DNA molecules under liquids or in air and the complete three‐dimensional path of the chain can be identified. This makes it possible to evaluate the writhing number (Wr) of the coiled curve described by the chain axis in the imaged conformation of the molecule. Wr is a much better descriptor of supercoiling than the linking deficit (ΔLk) which has been commonly used so far. Wr of the DNA axis curve can be roughly estimated by the signed‐intersection approach or calculated as the Gauss integral of the curve itself. We applied both methods to a model closed simple curve and to coiled DNA molecules exhibiting one or two turns of the chain about their superhelical axis.