Direct Detection of Toxic Contaminants in Minimally Processed Food Products Using Dendritic Surface-Enhanced Raman Scattering Substrates.

We present a method for the surface-enhanced Raman scattering (SERS)-based detection of toxic contaminants in minimally processed liquid food products, through the use of a dendritic silver nanostructure, produced through electrokinetic assembly of nanoparticles from solution. The dendritic nanostructure is produced on the surface of a microelectrode chip, connected to an AC field with an imposed DC bias. We apply this chip for the detection of thiram, a toxic fruit pesticide, in apple juice, to a limit of detection of 115 ppb, with no sample preprocessing. We also apply the chip for the detection of melamine, a toxic contaminant/food additive, to a limit of detection of 1.5 ppm in milk and 105 ppb in infant formula. All the reported limits of detection are below the recommended safe limits in food products, rendering this technique useful as a screening method to identify liquid food with hazardous amounts of toxic contaminants.

The organization of melatonin in lipid membranes.

Melatonin is a hormone that has been shown to have protective effects in several diseases that are associated with cholesterol dysregulation, including cardiovascular disease, Alzheimer's disease, and certain types of cancers. We studied the interaction of melatonin with model membranes made of dimyristoylphosphatidylcholine (DMPC) at melatonin concentrations ranging from 0.5mol% to 30mol%. From 2-dimensional X-ray diffraction measurements, we find that melatonin induces a re-ordering of the lipid membrane that is strongly dependent on the melatonin concentration. At low melatonin concentrations, we observe the presence of melatonin-enriched patches in the membrane, which are significantly thinner than the lipid bilayer. The melatonin molecules were found to align parallel to the lipid tails in these patches. At high melatonin concentrations of 30mol%, we observe a highly ordered melatonin structure that is uniform throughout the membrane, where the melatonin molecules align parallel to the bilayers and one melatonin molecule associates with 2 lipid molecules. Understanding the organization and interactions of melatonin in membranes, and how these are dependent on the concentration, may shed light into its anti-amyloidogenic, antioxidative and photoprotective properties and help develop a structural basis for these properties.

Amyloid-ß(25-35) peptides aggregate into cross-ß sheets in unsaturated anionic lipid membranes at high peptide concentrations.

One of the hallmarks of Alzheimer's disease is the formation of protein plaques in the brain, which mainly consist of amyloid-ß peptides of different lengths. While the role of these plaques in the pathology of the disease is not clear, the mechanism behind peptide aggregation is a topic of intense research and discussion. Because of their simplicity, synthetic membranes are promising model systems to identify the elementary processes involved. We prepared unsaturated zwitterionic/anionic lipid membranes made of 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC) and 1,2-dimyristoyl-sn-glycero-3-phospho-l-serine (DMPS) at concentrations of POPC/3 mol% DMPS containing 0 mol%, 3 mol%, 10 mol%, and 20 mol% amyloid-ß25-35 peptides. Membrane-embedded peptide clusters were observed at peptide concentrations of 10 and 20 mol% with a typical cluster size of ∼11 µm. Cluster density increased with peptide concentration from 59 (±3) clusters per mm(2) to 920 (±64) clusters per mm(2), respectively. While monomeric peptides take an α-helical state when embedded in lipid bilayers at low peptide concentrations, the peptides in peptide clusters were found to form cross-ß sheets and showed the characteristic pattern in X-ray experiments. The presence of the peptides was accompanied by an elastic distortion of the bilayers, which can induce a long range interaction between the peptides. The experimentally observed cluster patterns agree well with Monte Carlo simulations of long-range interacting peptides. This interaction may be the fundamental process behind cross-ß sheet formation in membranes and these sheets may serve as seeds for further growth into amyloid fibrils.

Cholesterol expels ibuprofen from the hydrophobic membrane core and stabilizes lamellar phases in lipid membranes containing ibuprofen.

There is increasing evidence that common drugs, such as aspirin and ibuprofen, interact with lipid membranes. Ibuprofen is one of the most common over the counter drugs in the world, and is used for relief of pain and fever. It interacts with the cyclooxygenase pathway leading to inhibition of prostaglandin synthesis. From X-ray diffraction of highly oriented model membranes containing between 0 and 20 mol% ibuprofen, 20 mol% cholesterol, and dimyristoylphosphatidylcholine (DMPC), we present evidence for a non-specific interaction between ibuprofen and cholesterol in lipid bilayers. At a low ibuprofen concentrations of 2 mol%, three different populations of ibuprofen molecules were found: two in the lipid head group region and one in the hydrophobic membrane core. At higher ibuprofen concentrations of 10 and 20 mol%, the lamellar bilayer structure is disrupted and a lamellar to cubic phase transition was observed. In the presence of 20 mol% cholesterol, ibuprofen (at 5 mol%) was found to be expelled from the membrane core and reside solely in the head group region of the bilayers. 20 mol% cholesterol was found to stabilize lamellar membrane structure and the formation of a cubic phase at 10 and 20 mol% ibuprofen was suppressed. The results demonstrate that ibuprofen interacts with lipid membranes and that the interaction is strongly dependent on the presence of cholesterol.

Hierarchical, self-similar structure in native squid pen.

The structure of native squid pen (gladius) was investigated in two different species on different length scales. By combining microscopy, atomic force microscopy (AFM), and X-ray diffraction, the experiments probed length scales from millimetres down to nanometres. The gladii showed a hierarchical, self-similar structure in the optical experiments with fibres of different size oriented along the long axis of the gladius. The fibre-like structure was reproduced at the nanoscale in AFM measurements and fibres with diameters of 500 µm, 100 µm, 10 µm, 2 µm and 0.2 µm were observed. Their molecular structure was determined using X-ray diffraction. In the squid gladius, the chitin molecules are known to form nano-crystallites of monoclinic lattice symmetry wrapped in a protein layer, resulting in ß-chitin nano-fibrils. Signals corresponding to the α-coil protein phase and ß-chitin crystallites were observed in the X-ray experiments and their orientation with respect to the fibre-axis was determined. The size of a nano-fibril was estimated from the X-ray experiments to be about 150 × 300 Å. About 100 of these nano-fibrils are needed to form a 0.2 µm thick micro-fibre. We found that the molecular structure is highly anisotropic with ∼90% of the α-coils and ß-chitin crystallites oriented along the fibre-axis, indicating a strong correlation between the macroscale structure and molecular orientation.

Acetylsalicylic acid (ASA) increases the solubility of cholesterol when incorporated in lipid membranes.

Cholesterol has been well established as a mediator of cell membrane fluidity. By interacting with lipid tails, cholesterol causes the membrane tails to be constrained thereby reducing membrane fluidity, well known as the condensation effect. Acetylsalicylic acid (ASA), the main ingredient in aspirin, has recently been shown to increase fluidity in lipid bilayers by primarily interacting with lipid head groups. We used high-resolution X-ray diffraction to study both ASA and cholesterol coexisting in model membranes of dimyristoylphosphatidylcholine (DMPC). While a high cholesterol concentration of 40 mol% cholesterol leads to the formation of immiscible cholesterol bilayers, as was reported previously, increasing the amount of ASA in the membranes between 0 to 12.5 mol% was found to significantly increase the fluidity of the bilayers and dissolve the cholesterol plaques. We, therefore, present experimental evidence for an interaction between cholesterol and ASA on the level of the cell membrane at elevated levels of cholesterol and ASA.

Electrokinetically-Driven Assembly of Gold Colloids into Nanostructures for Surface-Enhanced Raman Scattering.

Surface-enhanced Raman scattering (SERS) enables the highly sensitive detection of (bio)chemical analytes in fluid samples; however, its application requires nanostructured gold/silver substrates, which presents a significant technical challenge. Here, we develop and apply a novel method for producing gold nanostructures for SERS application via the alternating current (AC) electrokinetic assembly of gold nanoparticles into two intricate and frequency-dependent structures: (1) nanowires, and (2) branched "nanotrees", that create extended sensing surfaces. We find that the growth of these nanostructures depends strongly on the parameters of the applied AC electric field (frequency and voltage) and ionic composition, specifically the electrical conductivity of the fluid. We demonstrate the sensing capabilities of these gold nanostructures via the chemical detection of rhodamine 6G, a Raman dye, and thiram, a toxic pesticide. Finally, we demonstrate how these SERS-active nanostructures can also be used as a concentration amplification device that can electrokinetically attract and specifically capture an analyte (here, streptavidin) onto the detection site.

In situ assembly of active surface-enhanced Raman scattering substrates via electric field-guided growth of dendritic nanoparticle structures.

Surface-enhanced Raman scattering (SERS) can provide ultrasensitive detection of chemical and biological analytes down to the level of a single molecule. The need for costly, nanostructured, noble-metal substrates, however, poses a major obstacle in the widespread application of the method. Here we present for the first time a novel type of metallic nanostructured substrates that, not only exhibit a remarkable SERS activity, but are also produced in a facile, cost-effective and nanofabrication-free manner. The substrates are formed through an electric field-guided assembly process of silver nanocolloids, which results in extended and interconnected dendritic nanoparticle structures with a high density of "hot spots". A unique and significant performance attribute of these nanostructures is their ability to also function as concentration amplification devices, thereby further enhancing their analyte detection efficiency. This major advantage against conventional SERS substrates is illustrated experimentally here with the concentration and detection of proteins from solution. Low limits of detection for illicit drugs, food contaminants and pesticides in relevant matrices are also demonstrated. The SERS-active dendrites are reusable and can be removed and replaced in a few minutes. The SERS substrates presented herein constitute a significant advance towards more effective and less expensive diagnostic tools.

Effect of Cholesterol on the Structure of a Five-Component Mitochondria-Like Phospholipid Membrane.

Cellular membranes have a complex phospholipid composition that varies greatly depending on the organism, cell type and function. In spite of this complexity, most structural data available for phospholipid bilayers concern model systems containing only one or two different phospholipids. Here, we examine the effect of cholesterol on the structure of a complex membrane reflecting the lipid composition of mitochondrial membranes, with five different types of headgroups (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS) and cardiolipin (CL)) and a variety of hydrocarbon tails. This particular system was chosen because elevated cholesterol contents in mitochondrial membranes have been linked to a breaking down of Bax-mediated membrane permeabilization and resistance to cancer treatments. High resolution electron density profiles were determined by X-ray reflectivity, while the area per phospholipid chain, Apc, and the chain order parameter, SX-ray, were determined by wide-angle X-ray scattering (WAXS). We show that chain order increases upon the addition of cholesterol, resulting in both a thickening of the lipid bilayer and a reduction in the average surface area per phospholipid chain. This effect, well known as cholesterol's condensation effect, is similar, but not as pronounced as for single-component phospholipid membranes. We conclude by discussing the relevance of these findings for the insertion of the pro-apoptotic protein Bax in mitochondrial membranes with elevated cholesterol content.

The interaction between amyloid-ß peptides and anionic lipid membranes containing cholesterol and melatonin.

One of the hallmarks of Alzheimer's disease is the formation of senile plaques, primarily consisting of amyloid-ß (Aß) peptides. Peptide-membrane and peptide-lipid interactions are thought to be crucial in this process. We studied the interaction of Aß1₋42 and Aß25₋35 peptides with anionic lipid membranes made of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphoserine (DMPS) using X-ray diffraction. We compare the experimentally determined electron densities in the gel state of the membranes with density calculations from peptide structures reported in the Protein Data Bank in order to determine the position of the peptide in the bilayers. The full length peptide Aß1₋42 was found to embed in the hydrocarbon core of the anionic lipid bilayers. Two populations were found for the Aß25₋35 peptide: (1) membrane-bound states in the hydrophilic head group region of the bilayers, where the peptides align parallel to the membranes, and (2) an embedded state in the bilayer center. Aging plays an important role in the development of Alzheimer's, in particular with respect to changes in cholesterol and melatonin levels in the brain tissue. Immiscible cholesterol plaques were created by addition of 30 mol% cholesterol to the anionic membranes. The Aß25₋35 peptides were found to strongly interact with the lipid bilayers, displacing further cholesterol molecules into the plaques, effectively lowering the cholesterol concentration in the membranes and increasing the total fraction of cholesterol plaques. Addition of 30 mol% melatonin molecules to the anionic membranes drastically reduced the population of the membrane-embedded Aß state. These results present experimental evidence for an interaction between Aß peptides, melatonin and cholesterol in lipid membranes.

Adenosine monophosphate forms ordered arrays in multilamellar lipid matrices: insights into assembly of nucleic acid for primitive life.

A fundamental question of biology is how nucleic acids first assembled and then were incorporated into the earliest forms of cellular life 4 billion years ago. The polymerization of nucleotides is a condensation reaction in which phosphodiester bonds are formed. This reaction cannot occur in aqueous solutions, but guided polymerization in an anhydrous lipid environment could promote a non-enzymatic condensation reaction in which oligomers of single stranded nucleic acids are synthesized. We used X-ray scattering to investigate 5'-adenosine monophosphate (AMP) molecules captured in a multilamellar phospholipid matrix composed of dimyristoylphosphatidylcholine. Bragg peaks corresponding to the lateral organization of the confined AMP molecules were observed. Instead of forming a random array, the AMP molecules are highly entangled, with the phosphate and ribose groups in close proximity. This structure may facilitate polymerization of the nucleotides into RNA-like polymers.

The Observation of Highly Ordered Domains in Membranes with Cholesterol.

Rafts, or functional domains, are transient nano- or mesoscopic structures in the exoplasmic leaflet of the plasma membrane, and are thought to be essential for many cellular processes. Using neutron diffraction and computer modelling, we present evidence for the existence of highly ordered lipid domains in the cholesterol-rich (32.5 mol%) liquid-ordered ([Formula: see text]) phase of dipalmitoylphosphatidylcholine membranes. The liquid ordered phase in one-component lipid membranes has previously been thought to be a homogeneous phase. The presence of highly ordered lipid domains embedded in a disordered lipid matrix implies non-uniform distribution of cholesterol between the two phases. The experimental results are in excellent agreement with recent computer simulations of DPPC/cholesterol complexes [Meinhardt, Vink and Schmid (2013). Proc Natl Acad Sci USA 110(12): 4476-4481], which reported the existence of nanometer size [Formula: see text] domains in a liquid disordered lipid environment.