Raphaël Lévy

Measuring the spring constant of atomic force microscope cantilevers: thermal fluctuations and other methods

Knowledge of the interaction forces between surfaces gained using an atomic force microscope?(AFM) is crucial in a variety of industrial and scientific applications and necessitates a precise knowledge of the cantilever spring constant. Many methods have been devised to experimentally determine the spring constants of AFM cantilevers. The thermal fluctuation method is elegant but requires a theoretical model of the bending modes. For a rectangular cantilever, this model is available (Butt and Jaschke). Detailed thermal fluctuation measurements of a series of AFM cantilever beams have been performed in order to test the validity and accuracy of the recent theoretical models. The spring constant of rectangular cantilevers can also be determined easily with the method of Sader and White. We found very good agreement between the two methods. In the case of the V-shaped cantilever, we have shown that the thermal fluctuation method is a valid and accurate approach to the evaluation of the spring constant. A comparison between this method and those of Sader-Neumeister and of Ducker has been established. In some cases, we found disagreement between these two methods; the effect of non-conservation of material properties over all cantilevers from a single chip is qualitatively invoked.

Peptide-capped Gold Nanoparticles: Towards Artificial Proteins…

Peptides can be designed to form self‐assembled monolayers on gold nanoparticles to give nanomaterials with some chemical properties analogous to those of proteins. A variety of molecular‐recognition properties are readily integrated within the peptide monolayer. Monofunctionalized nanoparticles are obtained by using separation methods that have been optimized for proteins. Recent applications as artificial enzymes and artificial enzyme substrates are presented. The limitations and long‐term potential of peptide‐capped nanoparticles as artificial proteins are discussed.

Gold nanoparticles delivery in mammalian live cells: a critical review

Functional nanomaterials have recently attracted strong interest from the biology community, not only as potential drug delivery vehicles or diagnostic tools, but also as optical nanomaterials. This is illustrated by the explosion of publications in the field with more than 2,000 publications in the last 2 years (4,000 papers since 2000; from ISI Web of Knowledge, ‘nanoparticle and cell’ hit). Such a publication boom in this novel interdisciplinary field has resulted in papers of unequal standard, partly because it is challenging to assemble the required expertise in chemistry, physics, and biology in a single team. As an extreme example, several papers published in physical chemistry journals claim intracellular delivery of nanoparticles, but show pictures of cells that are, to the expert biologist, evidently dead (and therefore permeable). To attain proper cellular applications using nanomaterials, it is critical not only to achieve efficient delivery in healthy cells, but also to control the intracellular availability and the fate of the nanomaterial. This is still an open challenge that will only be met by innovative delivery methods combined with rigorous and quantitative characterization of the uptake and the fate of the nanoparticles. This review mainly focuses on gold nanoparticles and discusses the various approaches to nanoparticle delivery, including surface chemical modifications and several methods used to facilitate cellular uptake and endosomal escape. We will also review the main detection methods and how their optimum use can inform about intracellular localization, efficiency of delivery, and integrity of the surface capping. Raphaël Lévy is a BBSRC David Phillips Research Fellow at the University of Liverpool. He graduated in Physics at the University Louis Pasteur in Strasbourg (France). In 2002, after a Master in Soft Condensed Matter Physics, he obtained a PhD in Physics at the University Louis Pasteur. He then moved to the University of Liverpool as a Post-doctoral Marie Curie Research Fellow. In 2006, he obtained a prestigious David Phillips Fellowship, to develop single particle-based imaging in living cells (photothermal microscopy). His research interests include the design and characterization of nanomaterials and their interactions with living cells. Umbreen Shaheen completed her Master in Zoology and then lectured at the University of Balochistan. She studied biotechnology at the National Institute of Biotechnology and Genetic Engineering (NIBGE, Pakistan) and is currently doing her PhD at the University of Liverpool, on intracellular delivery of peptide-capped gold nanoparticles. Yann Cesbron is a PhD student at the University of Liverpool, developing photothermal microscopy for biological imaging. He graduated at the University Louis Pasteur (Strasbourg, France) with a Master of Science in Condensed Matter Physics and a second Master of Science in Polymer Materials. He moved to Liverpool in 2006 to start his PhD. Violaine Sée is a BBSRC David Phillips Research Fellow at the University of Liverpool. She graduated in Chemistry and Molecular and Cellular Biology at the University Louis Pasteur in Strasbourg (France). After a Master in Pharmacology, in 2001 she obtained her PhD in Pharmacology and Neurobiology at the University Louis Pasteur. She was then assistant lecturer and subsequently moved to the University of Liverpool as a Post-doctoral Research Fellow. In 2005, she obtained a prestigious David Phillips Fellowship, to develop her work on intracellular signaling dynamics. She is focusing on the imaging of single living cells in order to understand regulation of gene transcription and cell fate. She has recently been interested in using new techniques for single molecule imaging in live cells based on the use of gold nanoparticles.

TAT and HA2 Facilitate Cellular Uptake of Gold Nanoparticles but Do Not Lead to Cytosolic Localisation.

The methods currently available to deliver functional labels and drugs to the cell cytosol are inefficient and this constitutes a major obstacle to cell biology (delivery of sensors and imaging probes) and therapy (drug access to the cell internal machinery). As cell membranes are impermeable to most molecular cargos, viral peptides have been used to bolster their internalisation through endocytosis and help their release to the cytosol by bursting the endosomal vesicles. However, conflicting results have been reported on the extent of the cytosolic delivery achieved. To evaluate their potential, we used gold nanoparticles as model cargos and systematically assessed how the functionalisation of their surface by either or both of the viral peptides TAT and HA2 influenced their intracellular delivery. We evaluated the number of gold nanoparticles present in cells after internalisation using photothermal microscopy and their subcellular localisation by electron microscopy. While their uptake increased when the TAT and/or HA2 viral peptides were present on their surface, we did not observe a significant cytosolic delivery of the gold nanoparticles.

Nanoparticles for Imaging, Sensing, and Therapeutic Intervention

Nanoparticles have the potential to contribute to new modalities in molecular imaging and sensing as well as in therapeutic interventions. In this Nano Focus article, we identify some of the current challenges and knowledge gaps that need to be confronted to accelerate the developments of various applications. Using specific examples, we journey from the characterization of these complex hybrid nanomaterials; continue with surface design and (bio)physicochemical properties, their fate in biological media and cells, and their potential for cancer treatment; and finally reflect on the role of animal models to predict their behavior in humans.

Fluorescent or not? Size-dependent fluorescence switching for polymer-stabilized gold clusters in the 1.1-1.7 nm size range

The synthesis of fluorescent water-soluble gold nanoparticles by the reduction of a gold salt in the presence of a designed polymer ligand is described, the size and fluorescence of the particles being controlled by the polymer to gold ratio; the most fluorescent nanomaterial has a 3% quantum yield, a 1.1 nm gold core and a 6.9 nm hydrodynamic radius.

Fmoc-diphenylalanine hydrogels: understanding the variability in reported mechanical properties

Fmoc-diphenylalanine (FmocFF or FmocPhePhe) is an important low molecular weight hydrogelator. Gelation can be induced by either lowering the pH of an aqueous solution of FmocFF or by the addition of water to a solution of FmocFF in a solvent such as DMSO. Despite the volume of literature on FmocFF, the mechanical properties reported for the gels vary significantly over four orders of magnitude and the origins of this variability is unclear. Here, we study systematically the mechanical properties of FmocFF gels prepared with different protocols. We demonstrate that the final pH of the gels is the principal determinant of the mechanical properties independently of the method of gel formation. We also show that additional variability arises from experimental factors such as the fraction of DMSO or the nature of the buffers used in selected systems.

A generic approach to monofunctionalized protein-like gold nanoparticles based on immobilized metal ion affinity chromatography

Due to their chemical, optical and electronic properties, metal nanoparticles (NPs) are essential components of new biosensors and self-assembled nanodevices. The ability to vary and control the surface composition of NPs with molecular accuracy is crucial for many envisioned applications and has been a major focus of research. The average composition can be varied through synthesis or ligand exchange by using different capping-ligand mixtures. Pioneering work by Alivisatos’ group led to the separation of NPs with a defined number of DNA strands by gel electrophoresis. The drawback of gel electrophoresis is that it is not easily scalable and is limited to DNA. Monofunctionalization based on solid-phase coupling and solid-phase ligand-exchange reactions has recently been reported for small amphiphilic NPs. We have recently developed a route to protein-like metal NPs based on self-assembled monolayers of peptides. The protein-like behaviour of NPs enables a wide range of biochemistry techniques to be applied. Separation of proteins has been a major challenge in the past decades, and powerful tools have been created to achieve this task. In this communication, we report the power of immobilized metal ion affinity chromatography (IMAC), a method developed and optimized for the purification of recombinant proteins, to separate peptide-capped NPs with a given number of molecular labels. This simple, scalable and analytical strategy is applicable to the preparation of NPs bearing a predefined number of virtually any water-soluble chemical moieties. Peptide-capped NPs were prepared by mixing 6 nm gold NPs with peptide solutions. The excess peptide was removed by size-exclusion chromatography. The peptide solutions comprised different proportions of the peptide CALNN (“matrix” peptide) and of an extended peptide, namely CALNNGHHHHHHGKbiotinG (“functional” peptide). The extension of the functional peptide is composed of two independent entities: the His-tag (a sequence of 6 histidines), which is used for its ability to bind to immobilized chelated transition metal ions such as nickel, followed by a label (Figure 1). Because

High-Resolution Sizing of Monolayer-Protected Gold Clusters by Differential Centrifugal Sedimentation

Differential centrifugal sedimentation (DCS) has been applied to accurately size ligand-protected gold hydrosols in the 10 to 50 nm range. A simple protocol is presented to correct for particle density variations due to the presence of the ligand shell, which is formed here by either polyethylene glycol-substituted alkane thiols (PEG-alkane thiols) of different chain length or oligopeptides. The method gives reliable data for all particle sizes investigated and lends itself to rapid routine sizing of nanoparticles. Unlike TEM, DCS is highly sensitive to small changes in the thickness of the organic ligand shell and can be applied to monitor shell thickness variations of as little as 0.1 nm on particles of a given core size.

Amyloid-derived peptide forms self-assembled monolayers on gold nanoparticle with a curvature-dependent β-sheet structure.

Using a combination of Fourier transform infrared (FTIR) spectroscopy and solid-state nuclear magnetic resonance (SSNMR) techniques, the secondary structure of peptides anchored on gold nanoparticles of different sizes is investigated. The structure of the well-studied CALNN-capped nanoparticles is compared to the structure of nanoparticles capped with a new cysteine-terminated peptide, CFGAILSS. The design of that peptide is derived from the minimal amyloidogenic sequence FGAIL of the human islet polypeptide amylin. We demonstrate that CFGAILSS forms extended fibrils in solution. When constrained at a nanoparticle surface, CFGAILSS adopts a secondary structure markedly different from CALNN. Taking into account the surface selection rules, the FTIR spectra of CFGAILSS-capped gold nanoparticles indicate the formation of β-sheets which are more prominent for 25 nm diameter nanoparticles than for 5 nm nanoparticles. No intermolecular (13)C-(13)C dipolar coupling is detected with rotational resonance SSNMR for CALNN-capped nanoparticles, while CALNN is in a random coil configuration. Coupling is detected for CFGAILSS-capped gold nanoparticles, however, consistent with an intermolecular (13)C-(13)C distance of 5.0 ± 0.3 Å, in agreement with intermolecular hydrogen bonding in a parallel β-sheet structure.