Polyamorphism Mirrors Polymorphism in the Liquid-Liquid Transition of a Molecular Liquid.

Liquid-liquid transitions between two amorphous phases in a single-component liquid have courted controversy. All known examples of liquid-liquid transitions in molecular liquids have been observed in the supercooled state, suggesting an intimate connection with vitrification and locally favored structures inhibiting crystallization. However, there is precious little information about the local molecular packing in supercooled liquids, meaning that the order parameter of the transition is still unknown. Here, we investigate the liquid-liquid transition in triphenyl phosphite and show that it is caused by the competition between liquid structures that mirror two crystal polymorphs. The liquid-liquid transition is found to be between a geometrically frustrated liquid and a dynamically frustrated glass. These results indicate a general link between polymorphism and polyamorphism and will lead to a much greater understanding of the physical basis of liquid-liquid transitions and allow the systematic discovery of other examples.

Surface-enhanced coherent anti-Stokes Raman imaging of lipids.

This work describes in detail a wide-field surface-enhanced coherent anti-Stokes Raman scattering (CARS) microscope, which enables enhanced detection of sample structures in close proximity (∼100 nm) of the substrate interface. Unlike conventional CARS microscopy, where the sample is illuminated with freely propagating light, the current implementation uses evanescent fields to drive Raman coherences across the entire object plane. By coupling the pump and Stokes excitation beams to the surface plasmon-polariton mode at the interface of a 30 nm thick gold film, we obtained strong CARS signals from cholesteryl oleate droplets adhered to the surface. The surface-enhanced CARS imaging system visualizes lipid structures with vibrational selectivity using illumination doses per unit area that are more than four orders of magnitude lower than in point-scanning CARS microscopy.

Quantitative characterization of individual microdroplets using surface-enhanced resonance Raman scattering spectroscopy.

Surface-enhanced resonance Raman scattering (SERRS) spectroscopy is a highly sensitive optical technique capable of detecting multiple analytes rapidly and simultaneously. There is significant interest in SERRS detection in micro- and nanotechnologies, as it can be used to detect extremely low analyte concentrations in small volumes of fluids, particularly in microfluidic systems. There is also rapidly growing interest in the field of microdroplets, which promises to offer the analyst many potential advantages over existing technologies for both design and control of microfluidic assays. While there have been rapid advances in both fields in recent years, the literature on SERRS-based detection of individual microdroplets remains lacking. In this paper, we demonstrate the ability to quantitatively detect multiple variable analyte concentrations from within individual microdroplets in real time using SERRS spectroscopy. We also demonstrate the use of a programmable pump control algorithm to generate concentration gradients across a chain of droplets.

Chiral Quantum Metamaterial for Hypersensitive Biomolecule Detection.

Chiral biological and pharmaceutical molecules are analyzed with phenomena that monitor their very weak differential interaction with circularly polarized light. This inherent weakness results in detection levels for chiral molecules that are inferior, by at least six orders of magnitude, to the single molecule level achieved by state-of-the-art chirally insensitive spectroscopic measurements. Here, we show a phenomenon based on chiral quantum metamaterials (CQMs) that overcomes these intrinsic limits. Specifically, the emission from a quantum emitter, a semiconductor quantum dot (QD), selectively placed in a chiral nanocavity is strongly perturbed when individual biomolecules (here, antibodies) are introduced into the cavity. The effect is extremely sensitive, with six molecules per nanocavity being easily detected. The phenomenon is attributed to the CQM being responsive to significant local changes in the optical density of states caused by the introduction of the biomolecule into the cavity. These local changes in the metamaterial electromagnetic environment, and hence the biomolecules, are invisible to "classical" light-scattering-based measurements. Given the extremely large effects reported, our work presages next generation technologies for rapid hypersensitive measurements with applications in nanometrology and biodetection.

Monitoring the uptake and redistribution of metal nanoparticles during cell culture using surface-enhanced Raman scattering spectroscopy.

We describe the uptake of silver nanoparticles by CHO (Chinese hamster ovary) cells and their subsequent fate as a result of cell division during culture, as monitored by surface-enhanced Raman scattering (SERS) spectroscopy. Mapping of populations of cells containing both labeled and native nanoparticles by SERS spectroscopy imaging provided a quantitative method by which the number of intracellular nanoparticles could be monitored. Initially, for a given amount of nanoparticles, the relationship between the number taken up into the cell and the time of incubation was explored. Subsequently, the redistribution of intracellular nanoparticles upon multiple rounds of cell division was investigated. Intracellular SERS signatures remained detectable in the cells for up to four generations, although the abundance and intensity of the signals declined rapidly as nanoparticles were shared with daughter cells. The intensity of the SERS signal was dependent both on stability of the label and their abundance (nanoparticle aggregation increases the extent of the SERS enhancement). The data show that while the labeled nanoparticles remain stable for prolonged periods, during cell division, the changes in signal could be attributed both to a decrease in abundance and distribution (and hence aggregation).

Copper(II) binding to amyloid-beta fibrils of Alzheimer's disease reveals a picomolar affinity: stoichiometry and coordination geometry are independent of Abeta oligomeric form.

Cu(2+) ions are found concentrated within senile plaques of Alzheimer's disease patients directly bound to amyloid-beta peptide (Abeta) and are linked to the neurotoxicity and self-association of Abeta. The affinity of Cu(2+) for monomeric Abeta is highly disputed, and there have been no reports of affinity of Cu(2+) for fibrillar Abeta. We therefore measured the affinity of Cu(2+) for both monomeric and fibrillar Abeta(1-42) using two independent methods: fluorescence quenching and circular dichroism. The binding curves were almost identical for both fibrillar and monomeric forms. Competition studies with free glycine, l-histidine, and nitrilotriacetic acid (NTA) indicate an apparent (conditional) dissociation constant of 10(-11) M, at pH 7.4. Previous studies of Cu-Abeta have typically found the affinity 2 or more orders of magnitude weaker, largely because the affinity of competing ligands or buffers has been underestimated. Abeta fibers are able to bind a full stoichiometric complement of Cu(2+) ions with little change in their secondary structure and have coordination geometry identical to that of monomeric Abeta. Electron paramagnetic resonance studies (EPR) with Abeta His/Ala analogues suggest a dynamic view of the tetragonal Cu(2+) complex, with axial as well as equatorial coordination of imidazole nitrogens creating an ensemble of coordination geometries in exchange between each other. Furthermore, the N-terminal amino group is essential for the formation of high-pH complex II. The Abeta(1-28) fragment binds an additional Cu(2+) ion compared to full-length Abeta, with appreciable affinity. This second binding site is revealed in Abeta(1-42) upon addition of methanol, indicating hydrophobic interactions block the formation of this weaker carboxylate-rich complex. A Cu(2+) affinity for Abeta of 10(11) M(-1) supports a modified amyloid cascade hypothesis in which Cu(2+) is central to Abeta neurotoxicity.

Chlorophyll triplet states associated with Photosystem I and Photosystem II in thylakoids of the green alga Chlamydomonas reinhardtii.

The analysis of FDMR spectra, recorded at multiple emission wavelengths, by a global decomposition technique, has allowed us to characterise the triplet populations associated with Photosystem I and Photosystem II of thylakoids in the green alga Chlamydomonas reinhardtii. Three triplet populations are observed at fluorescence emissions characteristic of Photosystem II, and their zero field splitting parameters have been determined. These are similar to the zero field parameters for the three Photosystem II triplets previously reported for spinach thylakoids, suggesting that they have a widespread occurrence in nature. None of these triplets have the zero field splitting parameters characteristic of the Photosystem II recombination triplet observed only under reducing conditions. Because these triplets are generated under non-reducing redox conditions, when the recombination triplet is undetectable, it is suggested that they may be involved in the photoinhibition of Photosystem II. At emission wavelengths characteristic of Photosystem I, three triplet populations are observed, two of which are attributed to the P(700) recombination triplet frozen in two different conformations, based on the microwave-induced fluorescence emission spectra and the triplet minus singlet difference spectra. The third triplet population detected at Photosystem I emission wavelengths, which was previously unresolved, is proposed to originate from the antenna chlorophyll of the core or the unusually blue-shifted outer antenna complexes of this organism.

Solution 1H NMR investigation of Zn2+ and Cd2+ binding to amyloid-beta peptide (Abeta) of Alzheimer's disease.

Elevated levels of zinc2+ and copper2+ are found chelated to the amyloid-beta-peptide (Abeta) in isolated senile plaque cores of Alzheimer's disease (AD) patients. However, the precise residues involved in Zn2+ ligation are yet to be established. We have used 1H NMR and CD to probe the binding of Zn2+ to Abeta(1-28). Zinc binding to Abeta causes a number of 1H NMR resonances to exhibit intermediate exchange broadening upon Zn2+ addition, signals in slow and fast exchange are also observed. In addition, there is a general loss of signal for all resonances with Zn2+ addition, suggestive of the formation of high molecular weight polymeric species. Perturbations in specific 1H NMR resonances between residues 6 and 14, and analysis of various Abeta analogues in which each of the three His residues have been replaced by alanine, indicates that His6, His13 and His14 residues are implicated in Zn-Abeta binding. Complementary studies with Cd2+ ions cause perturbations to 1H NMR spectra that are strikingly similar to that observed for Zn2+. Binding monitored at Val12 indicates a 1:1 stoichiometry with Abeta for both Zn2+ and Cd2+ ions. Circular Dichroism (CD) studies in the far-UV indicate quite minimal ordering of the main-chain with Zn2+ or Cd2+ addition. Changes in the far-UV are quite different from that obtained with Cu2+ additions indicating that Zn2+ coordination is distinct from that of Cu2+ ions. Taken together, these observations seem to suggest that Zn2+ coordination is dominated by inter-molecular coordination and the formation of polymeric species.

Frustration of crystallisation by a liquid-crystal phase.

Frustration of crystallisation by locally favoured structures is critically important in linking the phenomena of supercooling, glass formation, and liquid-liquid transitions. Here we show that the putative liquid-liquid transition in n-butanol is in fact caused by geometric frustration associated with an isotropic to rippled lamellar liquid-crystal transition. Liquid-crystal phases are generally regarded as being "in between" the liquid and the crystalline state. In contrast, the liquid-crystal phase in supercooled n-butanol is found to inhibit transformation to the crystal. The observed frustrated phase is a template for similar ordering in other liquids and likely to play an important role in supercooling and liquid-liquid transitions in many other molecular liquids.

Order Parameter of the Liquid-Liquid Transition in a Molecular Liquid.

Liquid-liquid transitions (LLTs) between amorphous phases of a single (chemically unchanged) liquid were predicted to occur in most molecular liquids but have only been observed in triphenyl phosphite (TPP) and n-butanol, and even these examples have been dismissed as "aborted crystallization". One of the foremost reasons that LLTs remain so controversial is the lack of an obvious order parameter, that is, a physical parameter characterizing the phase transition. Here, using the technique of fluorescence lifetime imaging, we show for the first time that the LLT in TPP is characterized by a change in polarity linked to changes in molecular ordering associated with crystal polymorphs. We conclude that the LLT in TPP is a phase transition associated with frustrated molecular clusters, explaining the paucity of examples of LLTs seen in nature.

Acoustically controlled enhancement of molecular sensing to assess oxidative stress in cells.

We demonstrate a microfluidic platform for the controlled aggregation of colloidal silver nanoparticles using surface acoustic waves (SAWs), enabling surface enhanced Raman scattering (SERS) analysis of oxidative damage in cells. We show that by varying the frequency and the power of the acoustic energy, it is possible to modulate the aggregation of the colloid within the sample and hence to optimise the SERS analysis.

The origin of off-resonance non-linear optical activity of a gold chiral nanomaterial.

We demonstrate that engineered artificial gold chiral nanostructures display significant levels of non-linear optical activity even without plasmonic enhancement. Our work suggests that although plasmonic excitation enhances the intensity of second harmonic emission it is not a prerequisite for significant non-linear (second harmonic) optical activity. It is also shown that the non-linear optical activities of both the chiral nanostructures and simple chiral molecules on surfaces have a common origin, namely pure electric dipole excitation. This is a surprising observation given the significant difference in length scales, three orders of magnitude, between the nanostructures and simple chiral molecules. Intuitively, given that the dimensions of the nanostructures are comparable to the wavelength of visible light, one would expect non-localised higher multipole excitation (e.g. electric quadrupole and magnetic dipole) to make the dominant contribution to non-linear optical activity. This study provides experimental evidence that the electric dipole origin of non-linear optical activity is a generic phenomenon which is not limited to sub-wavelength molecules and assemblies. Our work suggests that viewing non-plasmonic nanostructures as "meta-molecules" could be useful for rationally designing substrates for optimal non-linear optical activity.

Investigation of the stability of labelled nanoparticles for SE(R)RS measurements in cells.

We explore the long-term stability of two different classes of labelled nanoparticles as intracellular SE(R)RS probes. Whilst chemisorbed labels gave stable responses inside cells for extended periods of time, signals from physisorbed labels could only be measured for short periods of time. These results help inform strategies for cellular imaging using vibrational spectroscopies.

SERS mapping of nanoparticle labels in single cells using a microfluidic chip.

We report for the first time the time-resolved mapping of intracellular nanoparticle labels from within living cells retained in a microstructured trap using Raman spectroscopy. The methods employed here also demonstrate the ability to rapidly discriminate between cell populations containing different SERS labels.

Copper binding to the amyloid-beta (Abeta) peptide associated with Alzheimer's disease: folding, coordination geometry, pH dependence, stoichiometry, and affinity of Abeta-(1-28): insights from a range of complementary spectroscopic techniques.

There is now direct evidence that copper is bound to amyloid-beta peptide (Abeta) in senile plaque of Alzheimer's disease. Copper is also linked with the neurotoxicity of Abeta and free radical damage, and Cu(2+) chelators represent a possible therapy for Alzheimer's disease. We have therefore used a range of complementary spectroscopies to characterize the coordination of Cu(2+) to Abeta in solution. The mode of copper binding is highly pH-dependent. EPR spectroscopy indicates that both coppers have axial, Type II coordination geometry, square-planar or square-pyramidal, with nitrogen and oxygen ligands. Circular dichroism studies indicate that copper chelation causes a structural transition of Abeta. Competition studies with glycine and l-histidine indicate that copper binds to Abeta-(1-28) at pH 7.4 with an affinity of K(a) approximately 10(7) m(-1). (1)H NMR indicates that histidine residues are involved in Cu(2+) coordination but that Tyr(10) is not. Studies using analogues of Abeta-(1-28) in which each of the histidine residues have been replaced by alanine or in which the N terminus is acetylated suggest that the N terminus and His(13) are crucial for Cu(2+) binding and that His(6) and His(14) are also implicated. Evidence for the link between Alzheimer's disease and Cu(2+) is growing, and our studies have made a significant contribution to understanding the mode of Cu(2+) binding to Abeta in solution.

Molecular structures of viruses from Raman optical activity.

A vibrational Raman optical activity (ROA) study of a range of different structural types of virus exemplified by filamentous bacteriophage fd, tobacco mosaic virus, satellite tobacco mosaic virus, bacteriophage MS2 and cowpea mosaic virus has revealed that, on account of its sensitivity to chirality, ROA is an incisive probe of their aqueous solution structures at the molecular level. Protein ROA bands are especially prominent from which, as we have shown by comparison with the ROA spectra of proteins with known structures and by using a pattern recognition program, the folds of the major coat protein subunits may be deduced. Information about amino acid side-chain conformations, exemplified here by the determination of the sign and magnitude of the torsion angle chi(2,1) for tryptophan in fd, may also sometimes be obtained. By subtracting the ROA spectrum of the empty protein capsid (top component) of cowpea mosaic virus from those of the intact middle and bottom-upper components separated by means of a caesium chloride density gradient, the ROA spectrum of the viral RNA was obtained, which revealed that the RNA takes up an A-type single-stranded helical conformation and that the RNA conformations in the middle and bottom-upper components are very similar. This information is not available from the X-ray crystal structure of cowpea mosaic virus since no nucleic acid is visible.

A Raman optical activity study of rheomorphism in caseins, synucleins and tau. New insight into the structure and behaviour of natively unfolded proteins.

The casein milk proteins and the brain proteins alpha-synuclein and tau have been described as natively unfolded with random coil structures, which, in the case of alpha-synuclein and tau, have a propensity to form the fibrils found in a number of neurodegenerative diseases. New insight into the structures of these proteins has been provided by a Raman optical activity study, supplemented with differential scanning calorimetry, of bovine beta- and kappa-casein, recombinant human alpha-, beta- and gamma-synuclein, together with the A30P and A53T mutants of alpha-synuclein associated with familial cases of Parkinson's disease, and recombinant human tau 46 together with the tau 46 P301L mutant associated with inherited frontotemporal dementia. The Raman optical activity spectra of all these proteins are very similar, being dominated by a strong positive band centred at approximately 1318 cm(-1) that may be due to the poly(l-proline) II (PPII) helical conformation. There are no Raman optical activity bands characteristic of extended secondary structure, although some unassociated beta strand may be present. Differential scanning calorimetry revealed no thermal transitions for these proteins in the range 15-110 degrees C, suggesting that the structures are loose and noncooperative. As it is extended, flexible, lacks intrachain hydrogen bonds and is hydrated in aqueous solution, PPII helix may impart a rheomorphic (flowing shape) character to the structure of these proteins that could be essential for their native function but which may, in the case of alpha-synuclein and tau, result in a propensity for pathological fibril formation due to particular residue properties.

Solution structures of potato virus X and narcissus mosaic virus from Raman optical activity.

Potato virus X (PVX) and narcissus mosaic virus (NMV) were studied using vibrational Raman optical activity (ROA) in order to obtain new information on the structures of their coat protein subunits. The ROA spectra of the two intact virions are very similar to each other and similar to that of tobacco mosaic virus (TMV) studied previously, being dominated by signals characteristic of proteins with helix bundle folds. In particular, PVX and NMV show strong positive ROA bands at approximately 1340 cm(-1) assigned to hydrated alpha-helix and perhaps originating in surface exposed helical residues, together with less strong positive ROA intensity in the range approximately 1297-1312 cm(-1) assigned to alpha-helix in a more hydrophobic environment and perhaps originating in residues at helix-helix interfaces. The positive approximately 1340 cm(-1) ROA band of TMV is less intense than those of PVX and NMV, suggesting that TMV contains less hydrated alpha-helix. Small differences in other spectral regions reflect differences in some loop, turn and side-chain compositions and conformations among the three viruses. A pattern recognition program based on principal component analysis of ROA spectra indicates that the coat protein subunit folds of PVX and NMV may be very similar to each other and similar to that of TMV. These results suggest that PVX and NMV may have coat protein subunit structures based on folds similar to the TMV helix bundle and hence that the helical architecture of the PVX and NMV particles may be similar to that of TMV but with different structural parameters.