Showing papers on "Circular dichroism published in 2014"

Application of electronic circular dichroism in the study of supramolecular systems

Abstract: Chiral supramolecular architectures constitute crucial structural and functional elements in living systems and have been long mimicked by chemists to synthesize new artificial systems endowed with desired properties and functions. Among several techniques to study noncovalent chiral assemblies or aggregates, electronic circular dichroism (ECD) plays a key role because many mechanisms responsible for the appearance of ECD bands occur through space, and therefore are intrinsically sensitive to intermolecular interactions, from short to long-range. The aim of this tutorial review is to emphasize the different kinds of information which can be obtained specifically when chiral supramolecular species are characterized by means of ECD spectroscopy. We will survey several typical applications of ECD in the context of supramolecular chemistry, ranging from the simple detection of chiral aggregates or complexes, to the definition of stoichiometric ratios between the partners, the derivation of thermodynamic and kinetic parameters such as binding and rate constants, and ultimately to the refinement of the most plausible structure of the supramolecular species.

Circularly Polarized Luminescence in Chiral Aggregates: Dependence of Morphology on Luminescence Dissymmetry

Abstract: The self-assembly of a chiral perylene bisimide bichromophoric derivative possessing a 1,1′-binaphthalene bridge was investigated by adopting two different methodologies, leading to the formation of aggregates with dissimilar morphologies. The chiral nature of the aggregated structures was optically probed with the help of circular dichroism (CD), vibrational circular dichroism (VCD), and circularly polarized luminescence (CPL). The one-dimensional aggregates formed in methylcyclohexane (MCH) exhibited twice the value of luminescence dissymmetry factor (glum) when compared with the spherical aggregates formed in chloroform at higher concentration. The summation of excitonic couplings between the individual chromophoric units in an aggregated system is responsible for the remarkably high luminescence dissymmetry exhibited by the chiral aggregates. The nanostructures could be successfully embedded into polymer films, leading to the fabrication of solid-state materials with high CPL dissymmetry that can find...

Secondary structure of corona proteins determines the cell surface receptors used by nanoparticles.

Abstract: Nanoparticles used for biological and biomedical applications encounter a host of extracellular proteins. These proteins rapidly adsorb onto the nanoparticle surface, creating a protein corona. Poly(ethylene glycol) can reduce, but not eliminate, the nonspecific adsorption of proteins. As a result, the adsorbed proteins, rather than the nanoparticle itself, determine the cellular receptors used for binding, the internalization mechanism, the intracellular transport pathway, and the subsequent immune response. Using fluorescence microscopy and flow cytometry, we first characterize a set of polystyrene nanoparticles in which the same adsorbed protein, bovine serum albumin, leads to binding to two different cell surface receptors: native albumin receptors and scavenger receptors. Using a combination of circular dichroism spectroscopy, isothermal titration calorimetry, and fluorescence spectroscopy, we demonstrate that the secondary structure of the adsorbed bovine serum albumin protein controls the cellular ...

New Insights into the Mechanism of the DNA/Doxorubicin Interaction

Abstract: Doxorubicin (DOX) is an important anthracycline antibiotic whose intricate features of binding to DNAs, not yet fully understood, have been the object of intense debate. The dimerization equilibrium has been studied at pH = 7.0, I = 2.5 mM, and T = 25 °C. A thermodynamic and kinetic study of the binding of doxorubicin to DNA, carried out by circular dichroism, viscometry, differential scanning calorimetry, fluorescence, isothermal titration calorimetry, and T-jump relaxation measurements, has enabled us to characterize for the first time two different types of calf thymus DNA (ctDNA)/DOX complexes: PD1 for C(DOX)/C(DNA) < 0.3, and PD2 for higher drug content. The nature of the PD1 complex is described better in light of the affinity of DOX with the synthetic copolymers [poly(dA-dT)]2 and [poly(dG-dC)]2. The formation of PD1 has been categorized kinetically as a two-step mechanism in which the fast step is the groove binding in the AT region, and the slow step is the intercalation into the GC region. This bifunctional nature provides a plausible explanation for the high PD1 constant obtained (K1 = 2.3 × 10(8) M(-1)). Moreover, the formation of an external aggregate complex ctDNA/DOX (PD2) at the expense of PD1, with K2 = 9.3 × 10(5) M(-1), has been evinced.

A G4·K+ Hydrogel Stabilized by an Anion

Abstract: Supramolecular hydrogels derived from natural products have promising applications in diagnostics, drug delivery, and tissue engineering. We studied the formation of a long-lived hydrogel made by mixing guanosine (G, 1) with 0.5 equiv of KB(OH)4. This ratio of borate anion to ligand is crucial for gelation as it links two molecules of 1, which facilitates cation-templated assembly of G4·K+ quartets. The guanosine–borate (GB) hydrogel, which was characterized by cryogenic transmission electron microscopy and circular dichroism and 11B magic-angle-spinning NMR spectroscopy, is stable in water that contains physiologically relevant concentrations of K+. Furthermore, non-covalent interactions, such as electrostatics, π-stacking, and hydrogen bonding, enable the incorporation of a cationic dye and nucleosides into the GB hydrogel.

Comparative α‐Helicity of Cyclic Pentapeptides in Water

Abstract: Helix-constrained polypeptides have attracted great interest for modulating protein-protein interactions (PPI). It is not known which are the most effective helix-inducing strategies for designing PPI agonists/antagonists. Cyclization linkers (X1-X5) were compared here, using circular dichroism and 2D NMR spectroscopy, for α-helix induction in simple model pentapeptides, Ac-cyclo(1,5)-[X1-Ala-Ala-Ala-X5]-NH2, in water. In this very stringent test of helix induction, a Lys1→Asp5 lactam linker conferred greatest α-helicity, hydrocarbon and triazole linkers induced a mix of α- and 3₁₀-helicity, while thio- and dithioether linkers produced less helicity. The lactam-linked cyclic pentapeptide was also the most effective α-helix nucleator attached to a 13-residue model peptide.

N-Alpha-Acetylation of α-Synuclein Increases Its Helical Folding Propensity, GM1 Binding Specificity and Resistance to Aggregation

Abstract: A switch in the conformational properties of α-synuclein (αS) is hypothesized to be a key step in the pathogenic mechanism of Parkinson’s disease (PD). Whereas the beta-sheet-rich state of αS has long been associated with its pathological aggregation in PD, a partially alpha-helical state was found to be related to physiological lipid binding; this suggests a potential role of the alpha-helical state in controlling synaptic vesicle cycling and resistance to β-sheet rich aggregation. N-terminal acetylation is the predominant post-translational modification of mammalian αS. Using circular dichroism, isothermal titration calorimetry, and fluorescence spectroscopy, we have analyzed the effects of N-terminal acetylation on the propensity of recombinant human αS to form the two conformational states in interaction with lipid membranes. Small unilamellar vesicles of negatively charged lipids served as model membranes. Consistent with previous NMR studies using phosphatidylserine, we found that membrane-induced α-helical folding was enhanced by N-terminal acetylation and that greater exothermic heat could be measured upon vesicle binding of the modified protein. Interestingly, the folding and lipid binding enhancements with phosphatidylserine in vitro were weak when compared to that of αS with GM1, a lipid enriched in presynaptic membranes. The resultant increase in helical folding propensity of N-acetylated αS enhanced its resistance to aggregation. Our findings demonstrate the significance of the extreme N-terminus for folding nucleation, for relative GM1 specificity of αS-membrane interaction, and for a protective function of N-terminal-acetylation against αS aggregation mediated by GM1.

Distinct circular dichroism spectroscopic signatures of polyproline II and unordered secondary structures: Applications in secondary structure analyses

Abstract: Circular dichroism (CD) spectroscopy is a valuable method for defining canonical secondary structure contents of proteins based on empirically-defined spectroscopic signatures derived from proteins with known three-dimensional structures. Many proteins identified as being “Intrinsically Disordered Proteins” have a significant amount of their structure that is neither sheet, helix, nor turn; this type of structure is often classified by CD as “other”, “random coil”, “unordered”, or “disordered”. However the “other” category can also include polyproline II (PPII)-type structures, whose spectral properties have not been well-distinguished from those of unordered structures. In this study, synchrotron radiation circular dichroism spectroscopy was used to investigate the spectral properties of collagen and polyproline, which both contain PPII-type structures. Their native spectra were compared as representatives of PPII structures. In addition, their spectra before and after treatment with various conditions to produce unfolded or denatured structures were also compared, with the aim of defining the differences between CD spectra of PPII and disordered structures. We conclude that the spectral features of collagen are more appropriate than those of polyproline for use as the representative spectrum for PPII structures present in typical amino acid-containing proteins, and that the single most characteristic spectroscopic feature distinguishing a PPII structure from a disordered structure is the presence of a positive peak around 220nm in the former but not in the latter. These spectra are now available for inclusion in new reference data sets used for CD analyses of the secondary structures of soluble proteins.

Circular dichroism calculation for natural products

Abstract: Determination of the absolute configuration (AC) is often a challenging aspect in the structure elucidation of natural products. When chiral compounds possess appropriate chromophore(s), electronic circular dichroism (ECD) may provide a powerful approach to the determination of their absolute configuration. Recently, ECD calculations by time-dependent density functional theory (TDDFT) have come to be used more commonly. In the present review, we give several examples of recent studies using TDDFT-calculated ECD spectra for the AC determination of natural products.

Effects of surface compositional and structural heterogeneity on nanoparticle-protein interactions: different protein configurations.

Abstract: As nanoparticles (NPs) enter into biological systems, they are immediately exposed to a variety and concentration of proteins. The physicochemical interactions between proteins and NPs are influenced by the surface properties of the NPs. To identify the effects of NP surface heterogeneity, the interactions between bovine serum albumin (BSA) and gold NPs (AuNPs) with similar chemical composition but different surface structures were investigated. Different interaction modes and BSA conformations were studied by dynamic light scattering, circular dichroism spectroscopy, fluorescence quenching and isothermal titration calorimetry (ITC). Depending on the surface structure of AuNPs, BSA seems to adopt either a "side-on" or an "end-on" conformation on AuNPs. ITC demonstrated that the adsorption of BSA onto AuNPs with randomly distributed polar and nonpolar groups was primarily driven by electrostatic interaction, and all BSA were adsorbed in the same process. The adsorption of BSA onto AuNPs covered with alternating domains of polar and nonpolar groups was a combination of different interactions. Overall, the results of this study point to the potential for utilizing nanoscale manipulation of NP surfaces to control the resulting NP-protein interactions.

Helical Sense-Responsive and Substituent-Sensitive Features in Vibrational and Electronic Circular Dichroism, in Circularly Polarized Luminescence, and in Raman Spectra of Some Simple Optically Active Hexahelicenes

Abstract: Four different hexahelicenes, 5-aza-hexahelicene (1), hexahelicene (2), 2-methyl-hexahelicene (3), and 2-bromo-hexahelicene (4), were prepared and their enantiomers, which are stable at r.t., were separated. Vibrational circular dichroism (VCD) spectra were measured for compound 1; for all the compounds, electronic circular dichroism (ECD) and circularly polarized luminescence (CPL) spectra were recorded. Each type of experimental spectrum was compared with the corresponding theoretical spectrum, determined via Density Functional Theory (DFT). Following the recent papers by Nakai et al., this comparison allowed to identify some features related to the helicity and some other features typical of the substituent groups on the helical backbone. The Raman spectrum of compound 1 is also examined from this point of view.

Straightforward access to mono- and bis-cycloplatinated helicenes displaying circularly polarized phosphorescence by using crystallization resolution methods

Abstract: Enantiopure mono-cycloplatinated-[8]helicene and bis-cycloplatinated-[6]helicene derivatives were prepared through column chromatography combined with crystallization of diastereomeric complexes using a chiral ancillary sulfoxide ligand. The UV-visible spectra, circular dichroism, molar rotations, and (circularly polarized) luminescence activity of these new helical complexes have been examined in detail and analysed with the help of first-principles quantum-chemical calculations.

Chiral Phase Transfer and Enantioenrichment of Thiolate-Protected Au102 Clusters

Abstract: The Au102(p-MBA)44 cluster (p-MBA: para-mercaptobenzoic acid) is observed as a chiral compound comprised of achiral components in its single-crystal structure. So far the enantiomers observed in the crystal structure are not isolated, nor is the circular dichroism spectrum known. A chiral phase transfer method is presented which allows partial resolution of the enantiomers by the use of a chiral ammonium bromide, (−)-1R,2S-N-dodecyl-N-methylephedrinium bromide ((−)-DMEBr). At sufficiently low concentration of (−)-DMEBr, the phase transfer from water to chloroform is incomplete. Both the aqueous and organic phases show optical activity of near mirror image relationship. Differences in the spectra are ascribed to the formation of diastereomeric salts. At high concentrations of (−)-DMEBr, full phase transfer is observed. The organic phase, however, still displays optical activity. We assume that one of the diastereomers has very strong optical activity, which overrules the cancelation of the spectra with opp...

Chirality effects at each amino acid position on tripeptide self-assembly into hydrogel biomaterials

Abstract: Hydrogels formed by ultrashort peptides are emerging as cost-effective materials for cell culture. However, L-peptides are labile to proteases, while their D-isomers are thought to not support cell growth as well. In contrast, the self-assembly behaviour and biological performance of heterochiral peptides (i.e., made of both D and L amino acids) are largely unknown. In this study, we evaluate the effects of amino acid chirality on tripeptide self-assembly and hydrogelation at physiological pH, and cytocompatibility in fibroblast cell culture. A series of uncapped hydrophobic tripeptides with all combinations of D, L amino acids was prepared, tested for self-assembly under physiological conditions, and analysed by circular dichroism, FT-IR, cryo-TEM, AFM, and Thioflavin T fluorescence imaging. Amino acid chirality has a profound effect on the peptides' supramolecular behaviour. Only selected isomers form hydrogels, and of amyloid structure, as confirmed by rheology and XRD. Importantly, they are able to maintain the viability and proliferation of fibroblasts in vitro. This study identifies two heterochiral gels that perform well in cell culture and will assist in the design of innovative and cost-effective peptide gel biomaterials.

Stitched α-helical peptides via bis ring-closing metathesis.

Abstract: Conformationally stabilized α-helical peptides are capable of inhibiting disease-relevant intracellular or extracellular protein–protein interactions in vivo. We have previously reported that the employment of ring-closing metathesis to introduce a single all-hydrocarbon staple along one face of an α-helical peptide greatly increases α-helical content, binding affinity to a target protein, cell penetration through active transport, and resistance to proteolytic degradation. In an effort to improve upon this technology for stabilizing a peptide in a bioactive α-helical conformation, we report the discovery of an efficient and selective bis ring-closing metathesis reaction leading to peptides bearing multiple contiguous staples connected by a central spiro ring junction. Circular dichroism spectroscopy, NMR, and computational analyses have been used to investigate the conformation of these “stitched” peptides, which are shown to exhibit remarkable thermal stabilities. Likewise, trypsin proteolysis assays co...

L-Valine methyl ester-containing tetraphenylethene: aggregation-induced emission, aggregation-induced circular dichroism, circularly polarized luminescence, and helical self-assembly

Abstract: L-Valine methyl ester-containing tetraphenylethene (Val-TPE) has been designed and synthesized. This novel molecule exhibits aggregation-induced emission (AIE), aggregation-induced circular dichroism (AICD), circularly polarized luminescence (CPL) and the capacity to self-assemble into helical nanofibers.

Structural characteristics of thermostable immunogenic outer membrane protein from Salmonella enterica serovar Typhi

Abstract: In this work, we explored the acid-induced unfolding pathway of non-porin outer membrane protein (OMP), an immunogenic protein from Salmonella Typhi, by monitoring the conformational changes over a pH range of 1.0–7.0 by circular dichroism, intrinsic fluorescence, ANS binding, acrylamide quenching, and dynamic light scattering. The spectroscopic measurements showed that OMP in its native state at pH 7.0 exists in more stable and compact conformation. In contrast, at pH 2.0, OMP retains substantial amount of secondary structure, disrupted side chain interactions, increased hydrodynamic radii, and nearly four-fold increase in ANS fluorescence with respect to the native state, indicating that MG state exists at pH 2.0. Quenching of tryptophan fluorescence by acrylamide further confirmed the accumulation of a partially unfolded state between native and unfolded state. The effect of pH on the conformation and thermostability of OMP points towards its heat resistance at neutral pH (T m ~ 69 °C at pH 7.0, monitored by change in MRE222 nm). Acid unfolded state was also characterized by the lack of a cooperative thermal transition. All these results suggested that acid-induced unfolded state of OMP at pH 2.0 represented the molten globule state. The chemical denaturation studies with GuHCl and urea as denaturants showed dissimilar results. The chemical unfolding experiments showed that in both far-UV CD and fluorescence measurements, GuHCl is more efficient than urea. GuHCl is characterized by low C m (~1 M), while urea is characterized by high C m (~3 M). The fully unfolded states were reached at 2 M GuHCl and 4 M urea concentration, respectively. This study adds to several key considerations of importance in the development of therapeutic agents against typhoid fever for clinical purposes.

Triple helix formation in amphiphilic discotics: demystifying solvent effects in supramolecular self-assembly.

Abstract: A set of chiral, amphiphilic, self-assembling discotic molecules based on the 3,3′-bis(acylamino)-2,2′-bipyridine-substituted benzene-1,3,5-tricarboxamide motif (BiPy-BTA) was prepared. Amphiphilicity was induced into the discotic molecules by an asymmetrical distribution of alkyl and oligo(ethylene oxide) groups in the periphery of the molecules. Small-angle X-ray scattering, cryogenic transmission electron microscopy, and circular dichroism spectroscopy measurements were performed on the discotic amphiphiles in mixtures of water and alcohol at temperatures between 0 °C an 90 °C. The combined results show that these amphiphilic discotic molecules self-assemble into supramolecular fibers consisting of either one or three discotic molecules in the fiber cross-section and that the presence of water induces the bundling of the supramolecular fibers. The rich phase behavior observed for these molecules proves to be intimately connected to the mixing thermodynamics of the water–alcohol mixtures.

Circular dichroism spectroscopy as a tool for monitoring aggregation in monoclonal antibody therapeutics.

Abstract: Aggregation continues to be a critical quality attribute for a monoclonal antibody therapeutic product due to its perceived significant impact on immunogenicity. This paper aims to establish the versatility of circular dichorism (CD) spectroscopy toward understanding aggregation of monoclonal antibody (mAb) therapeutics. The first application involves the use of far-UV CD as a complementary analytical technique to size exclusion chromatography (SEC) for understanding protein aggregation. The second application uses thermal scanning CD as a high throughput screening tool for examining stability of a mAb therapeutic in various formulation and downstream buffers. For establishing far-UV CD as an orthogonal technique, a mAb was incubated in different downstream processing buffers and another mAb in formulation buffers, and they were analyzed by SEC and far-UV CD for aggregate content and conformational stability, respectively. To examine thermal scanning as a high throughput screening tool, ellipticity as a function of the temperature was measured at 218 nm from 20 to 90 °C. Far-UV CD was found to display high sensitivity toward early detection of conformational changes in mAb. CD measurements were also able to elucidate the different aggregation mechanisms. Furthermore, thermal stability scan allowed us to estimate T(onset) which has been found to correlate with aggregation induced by salt, low pH, and buffer species. T(onset) temperature from thermal scanning at 218 nm using CD was correlated successfully to aggregate content measured by SEC. Results from both the studies demonstrate the usefulness of CD for assessing stability of therapeutic proteins during process development, formulation development, and product characterization.

Binding of alkaloids berberine, palmatine and coralyne to lysozyme: a combined structural and thermodynamic study

Abstract: The interaction between three isoquinoline alkaloids, berberine, palmatine and coralyne, and the protein lysozyme was studied using fluorescence, absorption, circular dichroism and isothermal titration calorimetry under physiological conditions. The three alkaloids caused strong quenching of the fluorescence of lysozyme by a static quenching mechanism, but with differing quenching efficiencies. The binding constants (K) at 25 °C are 5.37 × 104, 4.22 × 104, and 1.15 × 105 M−1, respectively, for berberine, palmatine and coralyne with binding sites (n) of approximately 1. We have demonstrated strong conformational changes in the secondary structure of the lysozyme molecule on alkaloid binding using synchronous fluorescence spectra, 3D fluorescence results and circular dichroism spectroscopic measurements. Interestingly, binding of the positively charged lysozyme to the positively charged alkaloid was endothermic and entropy driven. The negative standard molar Gibbs energy change (ΔGo) in all the cases revealed that the binding process was spontaneous. The corresponding ΔHo and TΔSo values were 0.58 ± 0.03, 7.09 and 2.37 ± 0.03, 8.67 and 4.31 ± 0.03, 11.23 kcal mol−1, respectively, for berberine, palmatine and coralyne. The thermodynamic parameters (ΔHo and TΔSo) of the reaction further indicated that both van der Waals forces and hydrogen bonds play a key role in the interaction. Spectroscopic evidence suggests that Trp62 and Trp63 in the β-domain of the protein are closer to the binding site of the alkaloids. Based on the Forster's theory of non-radiation energy transfer, the binding distances (r) between donor (protein) and acceptor (berberine, palmatine and coralyne) are 3.30, 3.09 and 3.06 nm, respectively. The results provide some valuable information on the interaction of these ligands with the protein.

Electric field induced changes in protein conformation

Abstract: The effect of a low strength oscillating electric field on the conformation of Bovine Serum Albumin (BSA) and Lysozyme in solution has been measured. A purpose built cell has been used to measure the real time autofluorescence and Circular Dichroism of the protein solutions exposed to electric fields of differing strength and frequency. Exposure to the electric fields results in protein unfolding for both Lysozyme and BSA. The applied field strengths are extremely small compared to the protein inter-chain intra-molecular forces. We propose a model whereby the electrophoretic motion of the proteins leads to a frictional force that results in protein unfolding. For BSA and Lysozyme in the electric fields used in this study, the shear rates at the protein surface under electrophoretic motion are of order 10(3) and 10(4) s(-1) respectively. Prolonged electric field exposure results in significant frictional energy dissipation in the proteins. The energy dissipated in the proteins results in protein unfolding, which is a critical initial step for protein aggregation and potentially amyloid fibril formation.

Angular momentum-induced circular dichroism in non-chiral nanostructures

Abstract: Circular dichroism, that is, the differential absorption of a system to left and right circularly polarized light, is one of the only techniques capable of providing morphological information of certain samples. In biology, for instance, circular dichroism spectroscopy is widely used to study the structure of proteins. More recently, it has also been used to characterize metamaterials and plasmonic structures. Typically, circular dichorism can only be observed in chiral objects. Here we present experimental results showing that a non-chiral sample such as a subwavelength circular nanoaperture can produce giant circular dichroism when a vortex beam is used to excite it. These measurements can be understood by studying the symmetries of the sample and the total angular momentum that vortex beams carry. Our results show that circular dichroism can provide a wealth of information about the sample when combined with the control of the total angular momentum of the input field.

Circular dichroism from single plasmonic nanostructures with extrinsic chirality

Abstract: Circular dichroism (CD) studies on single nanostructures can yield novel insights into chiroptical physics that are not available from traditional ensemble-based measurements, yet they are challenging because of their weak signals. By introducing an oblique excitation beam, we demonstrate the observation and spectroscopic analysis of a prominent plasmonic chiroptical response from a single v-shaped gold nanorod dimer nanostructure. We show that circular differential scattering from the obliquely excited gold nanorod dimer yields a characteristic bisignate peak-dip spectral shape at hybridized energies of the dimer. This chiroptical response can be ascribed to extrinsic chirality which depends on the geometry configurations of the chiral arrangement. Due to strong near-field coupling, the dipole orientations of the hybridized resonance modes can be in favor of the incident circularly polarized light where a maximum g-factor of ∼0.4 is observed. Promising applications of this chiroptical arrangement as a key component can be in electronics, photonics, or metamaterials.

Extraction and characterization of keratin from bovine hoof: A potential material for biomedical applications

Abstract: Keratin from the hoof is a less explored source for making valuable products. In this paper we present the extraction of pure keratin from bovine hooves and characterized them to better address the possible exploitation of this bio-resource as an alternative material for tissue engineering applications. The keratin protein from the pulverized hooves was extracted by reduction, which was observed to be pure, and two polypeptide chains of molecular weight in the range of 45–50 and 55–60 KDa were determined using SDS-PAGE assay. FTIR analysis complementing circular dichroism (CD) data, established that hoof keratin predominantly adopted α-helical conformation with admixture of β-sheet. The keratin was shown to have appreciably high denaturation temperature (215°C) as indicated by differential scanning calorimetric (DSC) analysis. Thermogravimetric analysis (TGA) also showed the retention of 50% of the original weight of the sample even at a temperature of 346°C. The keratin from the hoof had been observed to be biocompatible when analyzed with MTT assay using fibroblast cells, showing more than 90% cell viability. Hence, hoof keratin would be useful for high value biomedical applications.