Moria Kwiat

Biorecognition Layer Engineering: Overcoming Screening Limitations of Nanowire-Based FET Devices

Detection of biological species is of great importance to numerous areas of medical and life sciences from the diagnosis of diseases to the discovery of new drugs. Essential to the detection mechanism is the transduction of a signal associated with the specific recognition of biomolecules of interest. Nanowire-based electrical devices have been demonstrated as a powerful sensing platform for the highly sensitive detection of a wide-range of biological and chemical species. Yet, detecting biomolecules in complex biosamples of high ionic strength (>100 mM) is severely hampered by ionic screening effects. As a consequence, most of existing nanowire sensors operate under low ionic strength conditions, requiring ex situ biosample manipulation steps, that is, desalting processes. Here, we demonstrate an effective approach for the direct detection of biomolecules in untreated serum, based on the fragmentation of antibody-capturing units. Size-reduced antibody fragments permit the biorecognition event to occur in closer proximity to the nanowire surface, falling within the charge-sensitive Debye screening length. Furthermore, we explored the effect of antibody surface coverage on the resulting detection sensitivity limit under the high ionic strength conditions tested and found that lower antibody surface densities, in contrary to high antibody surface coverage, leads to devices of greater sensitivities. Thus, the direct and sensitive detection of proteins in untreated serum and blood samples was effectively performed down to the sub-pM concentration range without the requirement of biosamples manipulation.

Non-covalent Monolayer-Piercing Anchoring of Lipophilic Nucleic Acids: Preparation, Characterization, and Sensing Applications

Functional interfaces of biomolecules and inorganic substrates like semiconductor materials are of utmost importance for the development of highly sensitive biosensors and microarray technology. However, there is still a lot of room for improving the techniques for immobilization of biomolecules, in particular nucleic acids and proteins. Conventional anchoring strategies rely on attaching biomacromolecules via complementary functional groups, appropriate bifunctional linker molecules, or non-covalent immobilization via electrostatic interactions. In this work, we demonstrate a facile, new, and general method for the reversible non-covalent attachment of amphiphilic DNA probes containing hydrophobic units attached to the nucleobases (lipid-DNA) onto SAM-modified gold electrodes, silicon semiconductor surfaces, and glass substrates. We show the anchoring of well-defined amounts of lipid-DNA onto the surface by insertion of their lipid tails into the hydrophobic monolayer structure. The surface coverage of DNA molecules can be conveniently controlled by modulating the initial concentration and incubation time. Further control over the DNA layer is afforded by the additional external stimulus of temperature. Heating the DNA-modified surfaces at temperatures >80 °C leads to the release of the lipid-DNA structures from the surface without harming the integrity of the hydrophobic SAMs. These supramolecular DNA layers can be further tuned by anchoring onto a mixed SAM containing hydrophobic molecules of different lengths, rather than a homogeneous SAM. Immobilization of lipid-DNA on such SAMs has revealed that the surface density of DNA probes is highly dependent on the composition of the surface layer and the structure of the lipid-DNA. The formation of the lipid-DNA sensing layers was monitored and characterized by numerous techniques including X-ray photoelectron spectroscopy, quartz crystal microbalance, ellipsometry, contact angle measurements, atomic force microscopy, and confocal fluorescence imaging. Finally, this new DNA modification strategy was applied for the sensing of target DNAs using silicon-nanowire field-effect transistor device arrays, showing a high degree of specificity toward the complementary DNA target, as well as single-base mismatch selectivity.

Highly Ordered Large-Scale Neuronal Networks of Individual Cells – Toward Single Cell to 3D Nanowire Intracellular Interfaces

The use of artificial, prepatterned neuronal networks in vitro is a promising approach for studying the development and dynamics of small neural systems in order to understand the basic functionality of neurons and later on of the brain. The present work presents a high fidelity and robust procedure for controlling neuronal growth on substrates such as silicon wafers and glass, enabling us to obtain mature and durable neural networks of individual cells at designed geometries. It offers several advantages compared to other related techniques that have been reported in recent years mainly because of its high yield and reproducibility. The procedure is based on surface chemistry that allows the formation of functional, tailormade neural architectures with a micrometer high-resolution partition, that has the ability to promote or repel cells attachment. The main achievements of this work are deemed to be the creation of a large scale neuronal network at low density down to individual cells, that develop intact typical neurites and synapses without any glia-supportive cells straight from the plating stage and with a relatively long term survival rate, up to 4 weeks. An important application of this method is its use on 3D nanopillars and 3D nanowire-device arrays, enabling not only the cell bodies, but also their neurites to be positioned directly on electrical devices and grow with registration to the recording elements underneath.

Monolithic Integration of a Silicon Nanowire Field-Effect Transistors Array on a Complementary Metal-Oxide Semiconductor Chip for Biochemical Sensor Applications

We present a monolithic complementary metal-oxide semiconductor (CMOS)-based sensor system comprising an array of silicon nanowire field-effect transistors (FETs) and the signal-conditioning circuitry on the same chip. The silicon nanowires were fabricated by chemical vapor deposition methods and then transferred to the CMOS chip, where Ti/Pd/Ti contacts had been patterned via e-beam lithography. The on-chip circuitry measures the current flowing through each nanowire FET upon applying a constant source-drain voltage. The analog signal is digitized on chip and then transmitted to a receiving unit. The system has been successfully fabricated and tested by acquiring I-V curves of the bare nanowire-based FETs. Furthermore, the sensing capabilities of the complete system have been demonstrated by recording current changes upon nanowire exposure to solutions of different pHs, as well as by detecting different concentrations of Troponin T biomarkers (cTnT) through antibody-functionalized nanowire FETs.

Nanotechnology meets electrophysiology.

Recording of electrical signals from electrogenic cells is an essential aspect to many areas, ranging from fundamental biophysical studies of the function of the brain and heart, through medical monitoring and intervention. Over the past decades, these studies have been primarily carried out by various well-established techniques that have greatly advanced the field, yet pose handicapping technical limitations. Nanotechnology allows the fabrication of devices small enough to enable recording of single cells, and even single neurites. The rise in knowledge in controlling nanostructures allows their tailoring to match cellular components, thus offering high level of interfacing to single cells. We will cover the latest developments in electrophysiology, applying new nanotechnology-based approaches for cellular electrical recordings, both extracellularly and intracellularly.

2 Interfacing Biomolecules, Cells and Tissues with Nanowire-based Electrical Devices

Detection and qualification of biological and chemical species are critical to many areas of health care and the life sciences, from diagnostic disease to the discovery and screening of new drug molecules. Central to detection is the transduction of a signal associated with the selective recognition of a species of interest. Several approaches have been reported for the detection of biological molecules, including ELISA, surface plasmon resonance, nanoparticles, chemically sensitive field-effect transistors and microcantilevers. Although all have shown feasibility and promising progress applicability, none has yet demonstrated the combination of features required for rapid, highly sensitive multiplexed detection of biomolecules.

Noncellulosomal cohesin from the hyperthermophilic archaeon Archaeoglobus fulgidus

The increasing numbers of published genomes has enabled extensive survey of protein sequences in nature. During the course of our studies on cellulolytic bacteria that produce multienzyme cellulosome complexes designed for efficient degradation of cellulosic substrates, we have investigated the intermodular cohesin–dockerin interaction, which provides the molecular basis for cellulosome assembly. An early search of the genome databases yielded the surprising existence of a dockerin‐like sequence and two cohesin‐like sequences in the hyperthermophilic noncellulolytic archaeon, Archaeoglobus fulgidus, which clearly contradicts the cellulosome paradigm. Here, we report a biochemical and biophysical analysis, which revealed particularly strong‐ and specific‐binding interactions between these two cohesins and the single dockerin. The crystal structure of one of the recombinant cohesin modules was determined and found to resemble closely the type‐I cohesin structure from the cellulosome of Clostridium thermocellum, with certain distinctive features: two of the loops in the archaeal cohesin structure are shorter than those of the C. thermocellum structure, and a large insertion of 27‐amino acid residues, unique to the archaeal cohesin, appears to be largely disordered. Interestingly, the cohesin module undergoes reversible dimer and tetramer formation in solution, a property, which has not been observed previously for other cohesins. This is the first description of cohesin and dockerin interactions in a noncellulolytic archaeon and the first structure of an archaeal cohesin. This finding supports the notion that interactions based on the cohesin–dockerin paradigm are of more general occurrence and are not unique to the cellulosome system. Proteins 2010.