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Statins have displayed significant, although heterogeneous, anti-tumour activity in breast cancer disease progression and recurrence. They offer promise as a class of drugs, normally used for cardiovascular disease control, that could have a significant impact on the treatment of cancer. Understanding their mode of action and accurately assessing their efficacy on live cancer cells is an important and significant challenge. Stimulated Raman scattering (SRS) microscopy is a powerful, label-free imaging technique that can rapidly characterise the biochemical responses of live cell populations following drug treatment. Here, we demonstrate multi-wavelength SRS imaging together with spectral phasor analysis to characterise a panel of breast cancer cell lines (MCF-7, SK-BR-3 and MDA-MB-231 cells) treated with two clinically relevant statins, atorvastatin and rosuvastatin. Label-free SRS imaging within the high wavenumber region of the Raman spectrum (2800-3050 cm -1) revealed the lipid droplet distribution throughout populations of live breast cancer cells using biocompatible imaging conditions. A spectral phasor analysis of the hyperspectral dataset enables rapid differentiation of discrete cellular compartments based on their intrinsic SRS characteristics. Applying the spectral phasor method to studying statin treated cells identified a lipid accumulating phenotype in cell populations which displayed the lowest sensitivity to statin treatment, whilst a weaker lipid accumulating phenotype was associated with a potent reduction in cell viability. This study provides an insight into potential resistance mechanisms of specific cancer cells towards treatment with statins. Label-free SRS imaging provides a novel and innovative technique for phenotypic assessment of drug-induced effects across different cellular populations and enables effective analysis of drug-cell interactions at the subcellular scale.

Synthetic nanostructured materials incorporating both organic and inorganic components offer a unique, powerful, and versatile class of materials for widespread applications due to the distinct, yet complementary, nature of the intrinsic properties of the different constituents. We report a supramolecular system based on synthetic nanoclay (Laponite,bLap/b) and peptide amphiphiles (PAs,bPAH3/b) rationally designed to coassemble into nanostructured hydrogels with high structural integrity and a spectrum of bioactivities. Spectroscopic and scattering techniques and molecular dynamic simulation approaches were harnessed to confirm thatbPAH3/bnanofibers electrostatically adsorbed and conformed to the surface ofbLap/bnanodisks. Electron and atomic force microscopies also confirmed an increase in diameter and surface area ofbPAH3/bnanofibers after coassembly withbLap/b. Dynamic oscillatory rheology revealed that the coassembledbPAH3-Lap/bhydrogels displayed high stiffness and robust self-healing behavior while gas adsorption analysis confirmed a hierarchical and heterogeneous porosity. Furthermore, this distinctive structure within the three-dimensional (3D) matrix provided spatial confinement for the nucleation and hierarchical organization of high-aspect ratio hydroxyapatite nanorods into well-defined spherical clusters within the 3D matrix. Applicability of the organic-inorganicbPAH3-Lap/bhydrogels was assessediin vitro/iusing human bone marrow-derived stromal cells (hBMSCs) andiex vivo/iusing a chick chorioallantoic membrane (CAM) assay. The results demonstrated that the organic-inorganicbPAH3-Lap/bhydrogels promote human skeletal cell proliferation and, upon mineralization, integrate with the CAM, are infiltrated by blood vessels, stimulate extracellular matrix production, and facilitate extensive mineral deposition relative to the controls.

Shell-isolated nanoparticles (SHINs) have attracted increasing interest for non-interfering plasmonic enhanced sensing in fields such as materials science, biosensing, and in various electrochemical systems. The metallic core of these nanoparticles is isolated from the surrounding environment preventing direct contact or chemical interaction with the metal surface, while still being close enough to enable localized surface plasmon enhancement of the Raman scattering signal from the analyte. This concept forms the basis of the shell isolated nanoparticle-enhanced Raman spectroscopy (SHINERS) technique. To date, the vast majority of SHIN designs have focused on SiO2 shells around spherical nanoparticle cores and there has been very limited published research considering alternatives. In this article, we introduce a new polymer-based approach which provides excellent control over the layer thickness and can be applied to plasmonic metal nanoparticles of various shapes and sizes without compromising the overall nanoparticle morphology. The SHIN layers are shown to exhibit excellent passivation properties and robustness in the case of gold nanosphere (AuNP) and anisotropic gold nanostar (AuNS) core shapes. In addition, in situ SHINERS spectro-electrochemistry measurements performed on both SHIN and bare Au nanoparticles demonstrate the utility of the SHIN coatings. Correlated confocal Raman and SEM mapping was achieved to clearly establish single nanoparticle SERS sensitivity. Finally, confocal in situ SERS mapping enabled visualisation of the redox related molecular structure changes occurring on an electrode surface in the vicinity of individual SHIN-coated nanoparticles.

A fast, simple and sensitive micellar enhanced spectrofluorimetric method is performed for the determination of Daclatasvir dihydrochloride (DAC) in its pharmaceutical dosage form and in spiked human plasma. The fluorescence intensity (FI) was measured at 367 nm after excitation at 300 nm. In aqueous solution, the FI of DAC was greatly enhanced by >110% in the presence of sodium dodecyl sulphate (SDS). The detection method was linear over the range of 12.93 to 161.60 ng/mL, with a limit of detection of 1.75 ng/mL. The proposed method was successfully applied to the determination of DAC in its pharmaceutical dosage form and the mean % recovery of DAC in spiked human plasma was 95.42 ± 2.52. The developed methodology was also extended to stress studies of DAC after exposure to different forced degradation conditions including acidic, alkaline, photolytic, thermal and oxidative environments.

Analytical approaches for the quantitation of warfarin in plasma are high in demand. In this study, a novel surface enhanced Raman scattering (SERS) technique for the quantification of the widely used anticoagulant warfarin sodium in pharmaceutical dosage form and in spiked human plasma was developed. The colloidal-based SERS measurements were carefully optimized considering the laser wavelength, the type of metal nanoparticles, their surface functionalization and concentration as well as the time required for warfarin to associate with the metal surface. Poly(diallyldimethylammonium chloride) coated silver nanoparticles (PDDA-AgNPs) were established as a substrate which greatly enhanced the weak warfarin Raman signal with high reproducibility. The limit of detection was calculated in both water and human plasma to be 0.56 nM (0.17 ngmL-1) and 0.25 nM (0.08 ngmL-1) respectively, with a high degree of accuracy and reproducibility. The proposed method is simple, economical, and easily applied for routine application requiring only small plasma samples and also could be potentially useful for pharmacokinetic research on warfarin.

Covalent co-assembly holds great promise for the fabrication of hydrogels with controllable nanostructure, versatile chemical composition, and enhanced mechanical properties given its relative simplicity, high efficiency, and bond stability. This report describes our approach to designing functional multicomponent hydrogels based on photo-induced chemical interactions between an acrylamide-functionalized resilin-like polypeptide (RLP) and a peptide amphiphile (PA). Circular dichroism (CD) spectroscopy, electron microscopy, and amplitude sweep rheology were used to demonstrate that the co-assembled hydrogel systems acquired distinct structural conformations, tunable nanostructures, and enhanced elasticity in a PA concentration-dependent manner. We envisage the use of these materials in numerous biomedical applications such as controlled drug release systems, microfluidic devices, and scaffolds for tissue engineering.

The multiplexed detection of protein biomarkers in plasma present over a range of clinically relevant concentrations continues to be difficult for surface-based bioaffinity detection platforms such as surface plasmon resonance (SPR). As well as nonspecific adsorption, challenges include quantitative comparison between targets whose concentrations differ by orders of magnitude, regenerating SPR chips after plasma exposure, and the two- or four-channel limitation of many commercial SPR instruments limiting sample throughput. In this article, we explore an approach where two protein biomarkers alpha-1 antitrypsin (AAT) and Tau 381 are detected in tandem within a single SPR channel at micromolar and femtomolar concentrations, respectively. This was achieved by creating a mixed antibody (antiAAT and antiTau) monolayer on the chip surface. After the adsorption of AAT and/or Tau, further specificity was obtained via the adsorption of a DNA aptamer specific to each target. The detection range for each target was controlled via the relative surface density ratio of each antibody type as well as each aptamer concentration. Calibration measurements were performed in both buffer and spiked plasma with the detection of native concentrations of ∼39 fM (Tau) and ∼65 μM (AAT) in a human plasma sample. Finally, tandem measurements of both targets within the same SPR signal channel were demonstrated at these very different concentrations.

In this paper, we report on a new type of self-assembled plasmonic nanostructure, called gold suprashells, which are assembled around superparamagnetic iron oxide nanoparticle (SPION) cores. Gold suprashells have multiple surface plasmon resonances over a broad vis–NIR wavelength range, which makes them useful in applications where broadband absorption is required. For example, suprashells are efficient substrates that enhance surface-enhanced Raman scattering signals across multiple excitation wavelengths. This unique multiresonant character is afforded by the suprashell structure, which comprises anisotropic assemblies of nanoparticles of tunable length. Furthermore, gold suprashells generate more heat when excited with a laser compared to the nanoparticle building blocks, therefore making them promising materials for photothermal applications. The suprashells can potentially be assembled onto any negatively charged core, which opens up multiple possibilities for the development of multifunctional core/...

The potential to bioprint and study 3D bacterial biofilm constructs could have great clinical significance at a time when antimicrobial resistance is rising to dangerously high levels worldwide. In this study, clinically relevant bacterial species including Escherichia coli, Staphylococcus aureus (MSSA), Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa were 3D bioprinted using a double-crosslinked alginate bioink to form mature bacteria biofilms, characterized by confocal laser scanning microscopy (CLSM) and fluorescent staining. Solid and porous bacteria-laden constructs were reproducibly bioprinted with thicknesses ranging from 0.25 to 4 mm. We demonstrated 3D bioprinting of thicker biofilms (>4 mm) than found in currently available in vitro models. Bacterial viability was excellent in the bioprinted constructs, with CLSM observation of bacterial biofilm production and maturation possible for at least 28 d in culture. Importantly, we observed the complete five-step biofilm life cycle in vitro following 3D bioprinting for the first time, suggesting the formation of mature 3D bioprinted biofilms. Bacterial growth was faster in thinner, more porous constructs whilst constructs crosslinked with BaCl2 concentrations of above 10 mM had denser biofilm formation. 3D MRSA and MSSA biofilm constructs were found to show greater resistance to antimicrobials than corresponding two-dimensional (2D) cultures. Thicker 3D E. coli biofilms had greater resistance to tetracycline than thinner constructs over 7 d of treatment. Our methodology allowed for the precise 3D bioprinting of self-supporting 3D bacterial biofilm structures that developed biofilms during extended culture. 3D biofilm constructs containing bacterial biofilms produce a model with much greater clinical relevance compared to 2D culture models and we have demonstrated their use in antimicrobial testing.

The ability to directly detect Tau protein and other neurodegenerative biomarkers in human plasma at clinically relevant concentrations continues to be a significant hurdle for the establishment of diagnostic tests for Alzheimer's disease (AD). In this article, we introduce a new DNA aptamer/antibody sandwich assay pairing and apply it for the detection of human Tau 381 in undiluted plasma at concentrations as low as 10 fM. This was achieved on a multichannel surface plasmon resonance (SPR) platform with the challenge of working in plasma overcome through the development of a tailored mixed monolayer surface chemistry. In addition, a robust methodology was developed involving various same chip control measurements on reference channels to which the detection signal was normalized. Comparative measurements in plasma between SPR and enzyme-linked immunosorbent assay (ELISA) measurements were also performed to highlight both the 1000-fold performance enhancement of SPR and the ability to measure both spiked and native concentrations that are not achievable with ELISA.

Gold nanorods (NRs) have attracted a great deal of interest for a variety of biomedical and sensing applications. However, developing robust methods for biofunctionalizing NRs has continued to be challenging, especially for NR-DNA conjugates. This is due to the presence of cetyltrimethylammonium bromide (CTAB), which plays an essential role in controlling the anisotropic particle growth. In this article, we systematically explore the growth of a polydopamine (PDA) layer on a range of NR surfaces, comparing different polyelectrolyte and alkanethiol coatings as well as direct CTAB displacement. This revealed that the PDA layer thickness and growth rate is strongly dependent on the underlying nanorod functionalization chemistry and allowed us to establish a preferred route for the creation of stable, non-aggregated suspensions of PDA-coated NRs. The utility of this platform was then demonstrated by self-assembling packed monolayers of single-stranded DNA on the outer surface. Both the surface attachment and bioactivity of the resulting NR-DNA conjugates was then demonstrated by performing bulk solution and single nanoparticle imaging fluorescence measurements.

A new method is introduced for self-assembling citrate-capped gold nanoparticles into supraparticles with crystallographically aligned building blocks. It consists in confining gld nanoparticles inside a cellulose acetate membrane. The constituent nanoparticles are in close contact in the superstructure, and therefore generate hot spots leading to intense Surface-Enhanced Raman Scattering (SERS) signals. They also generate more plasmonic heat than the nanoparticle building blocks. The supraparticles are internalized by cells and show low cytotoxicity, but can kill cancer cells when irradiated with a laser. This, along with the improved plasmonic properties arising from their assembly, makes the gold supraparticles promising materials for applications in bioimaging and nanomedicine.

Discussions.

The zinc-bromine redox flow battery (RFB) is one of a very few commercially viable RFB energy storage system capable of integration with intermittent renewable energy sources to deliver improved energy management. However, due to the volatility of the electrogenerated bromine and potential for its crossover from positive to negative electrolytes, this system requires the use of quaternary ammonium complexes (N-methyl-N-ethylpyrrolidinium, (MEP)) to capture this bromine. This produces an immiscible phase with the Br2 which requires a complex network of pipes, pumps and automated controls to ensure access to the electroactive material during discharge. In this work, the use of novel quaternary ammonium complexes to capture the electrogenerated bromine but to keep it in the aqueous phase is examined. Three compounds, 1-(carboxymethyl) pyridine-1-ium, 1-(2-carboxymethyl)-1-methylmorpholin-1-ium and 1-(2-carboxymethyl)-1-methylpyrrolidin-1-ium, were found to successfully reduce the volume of the immiscible phase formed on complexing with the polybromide (Brx-) whilst displaying similar enthalpy of vaporisation values as that of MEP. Electrochemical analysis also revealed that these compounds did not impact on the electrode kinetics of the Br-/Brx- reaction indicating that the resulting surface film formed with these compounds behaved as a chemically modified electrode, in contrast to the surface film formed with MEP.

A novel dual nanoparticle amplification approach is introduced for the enhanced surface plasmon resonance (SPR) detection of a target protein at subattomolar concentrations. Thrombin was used as a model target protein as part of a sandwich assay involving an antithrombin (anti-Th) modified SPR chip surface and a thrombin specific DNA aptamer (Th-aptamer) whose sequence also includes a polyadenine (A30) tail. Dual nanoparticle (NP) enhancement was achieved with the controlled hybridization adsorption of first polythymine-NP conjugates (T20-NPs) followed by polyadenine-NPs (A30-NPs). Two different nanoparticle shapes (nanorod and quasi-spherical) were explored resulting in four different NP pair combinations being directly compared. It was found that both the order and NP shape were important in optimizing the assay performance. The use of real-time SPR measurements to detect target concentrations as low as 0.1 aM is a 10-fold improvement compared to single NP-enhanced SPR detection methods.

The use of a simple gel electrophoretic method to study mixtures of differently shaped biofunctionalized nanoparticles (NP's) that undergo bioaffinity interactions is demonstrated. Both gold nanorods (NR's) and quasi-spherical nanoparticles (qNS's) were functionalized with an interacting antigen and antibody pairing (alpha-1 antitrypsin (AAT) protein and antiAAT) or non-interacting antibody controls (antiBNP). Gel-based measurements were accompanied with transmission electron microscopy (TEM) and UV-vis spectroscopy analysis before and after separation. Initial measurements of NR and qNS bioconjugates suspended individually were applied to optimize the gel separation conditions and it was demonstrated that higher particle uniformities could be obtained relative to the initial stock solutions. A series of NR and qNS mixtures prepared at various stoichiometric ratios were then compared for both interacting (antiAAT–AAT) and non-interacting (antiAAT–antiBNP) particle conjugates. Both gel images and extinction measurements were utilized to demonstrate reduced NP concentrations transported along the gel due to bioaffinity-induced NP assembly. This confirmed that gel electrophoresis can be extended to identifying particle aggregation associated with protein bioaffinity interactions as well as being an established tool for separating particles based on size, shape and surface chemistry.

Enzymes are crucial for the cell metabolism and in industrial applications such as the manufacture of biosensors and medicines. A method for inactivating enzymes non-invasively and on demand could reveal the role of a biocatalyst in a particular cell function and enable fine-tuning the therapeutic dose of enzyme-based drugs. In this manuscript we introduce a universal method for inactivating enzymes with light. It consists of attaching the enzymes to photothermal nanoheaters (gold nanorods). Upon irradiation with light resonant with their LSPR the plasmonic nanoparticles generate heat, which denatures the enzymes. The resulting increase in temperature is highest in the vicinity of the nanoheaters, which allows the localized inactivation of nanorod-bound enzymes without damaging other proteins or cell membranes present in the same solution. Here we demonstrate this concept by selectively turning off an enzyme cascade via the selective inactivation of one of its components. We also prove that the target enzyme can be inactivated without unduly damaging cells under controlled irradiation conditions. The results shown here pave the way for using the proposed methodology to discern the role of target enzymes in intracellular signaling pathways.

Nanomaterials are increasingly being developed for applications in biotechnology, including the delivery of therapeutic drugs and of vaccine antigens. However, there is a lack of screening systems that can rapidly assess the dynamics of nanoparticle uptake and their consequential effects on cells. Established in vitro approaches are often carried out on a single time point, rely on time-consuming bulk measurements and are based primarily on populations of cell lines. As such, these procedures provide averaged results, do not guarantee precise control over the delivery of nanoparticles to cells and cannot easily generate information about the dynamics of nanoparticle-cell interactions and/or nanoparticle-mediated compound delivery. Combining microfluidics and nanotechnology with imaging techniques, we present a microfluidic platform to monitor nanoparticle uptake and intracellular processing in real-time and at the single-cell level. As proof-of-concept application, the potential of such a system for understanding nanovaccine delivery and processing was investigated and we demonstrate controlled delivery of ovalbumin-conjugated gold nanorods to primary dendritic cells. Using time-lapse microscopy, our approach allowed monitoring of uptake and processing of nanoparticles across a range of concentrations over several hours on hundreds of single-cells. This system represents a novel application of single-cell microfluidics for nanomaterial screening, providing a general platform for studying the dynamics of cell-nanomaterial interactions and representing a cost-saving and time-effective screening tool for many nanomaterial formulations and cell types.

Localized surface plasmons within silver nanocubes less than 50 nm in size were investigated using high-resolution cathodoluminescence hyperspectral imaging. Multivariate statistical analysis of the multidimensional luminescence data set allows both the identification of distinct spectral features in the emission and the mapping of their spatial distribution. These results show a 490-nm peak emitted from the cube faces, with shorter-wavelength luminescence coming from the vertices and edges; this provides direct experimental confirmation of theoretical predictions.

A novel wide-field approach for the real-time Surface Enhanced Raman Scattering (SERS) imaging of multiple silver nanoparticle clusters suspended in solution is described. This method enables direct correlation of the SERS activity of a single nanoparticle aggregate and its size through measurement of the cluster diffusion coefficient and can also be performed in a high-throughput basis. As a first demonstration, we investigate the salt-induced aggregation of silver nanoparticles in the presence of a reporter tag molecule, which has a high affinity for the nanoparticle surface. In addition to tracking individual particles, direct comparison of Rayleigh and SERS videos of the same colloid solution enabled measurement of the fraction of individual clusters that are SERS active and the dependence of this value on the relative concentration of the tag molecule. Furthermore, given the ability to also rapidly profile any nonuniformity in particle size distributions, we expect this approach will not only provide a new tool for the fundamental understanding of SERS but also significantly contribute to the development of an array of emerging nanoparticle-enhanced biomolecule and imaging detection platforms.

Die Entdeckung kleiner nichtcodierender RNA-Molekule (MikroRNAs; miRNAs), die in der Lage sind, die Genexpression zu steuern, hat eine neue Komplexitatsebene bei der Regulation der Zellfunktionen aufgezeigt. Seit einigen Jahren wird nun versucht, die regulatorische Funktion von hunderten bis tausenden von miRNAs zu verstehen, die in pflanzlichen und tierischen Zellen exprimiert werden. Um hier entscheidend weiterzukommen, ist die Entwicklung quantitativer bioanalytischer Verfahren erforderlich, mit denen ein schneller, paralleler Nachweis aller miRNAs in einer bestimmten Zelle oder Gewebeprobe moglich ist. In diesem Kurzaufsatz beschreiben wir einige der neuesten Verfahren zur Hochdurchsatzprofilierung von miRNAs und schildern die besonderen technischen Herausforderungen, die dabei gemeistert werden mussen.

Time and frequency resolved transient absorption measurements yield two-dimensional images that map the dynamical correlation between the center and width of the scattering function. Global analysis of such data allows unique diagnostics of the mechanics underlying the time evolution. We specialize in the case of surface plasmon resonances of optically driven nanoparticles. We present a catalog of 2D maps that can be used to fingerprint physically meaningful cases, and we provide two experimental examples to illustrate the diagnostic value of the maps and their utility in extracting the various time constants at play. In silver nanorods, the experiment shows a ﷿/2 phase shift between the oscillations of the center and the width of the plasmon resonance. Inspection of the maps allows the assignment that the center of the plasmon resonance tracks the strain in shape-oscillations, while the width tracks the strain rate. This finding is the basis of the novel mechanism of plasmon damping due to electron scattering from the electrophoretic potential generated by the motion of the interfacial double layer in colloidal nanoparticles. Measurements in gold nanoparticles show over-damped oscillations, which obscure the phase correlation between the center and width of the plasmon. The damping is dominated by inhomogeneous dephasing, and the time dependence of the width, which follows the temperature of the nanoparticles, and is diagnostic of the interband transition contribution to the plasmon resonance. I. Introduction In a recent publication, we presented an analysis of spectrally resolved transient absorption in silver nanorods and demonstrated that the observed ﷿/2 phase shift between the modulation of the center and the width of the plasmon resonance suggests a hitherto unrecognized mechanism of plasmon broadening. 1 This new mechanism is specifically related to electron scattering from the electrophoretic potential generated by the motion of interfacial double layer of colloidal nanoparticles. Here we give a more general analysis of transient absorption with all physically meaningful cases to show that the two-dimensional (I,t) image of the signal provides mechanistic diagnosis. Transient absorption is a general technique, and when performed with probe pulses that are much broader than the interrogated absorption feature (white light) they generate two-dimensional dynamical correlation maps between the width and the position of the resonance. The power of the method is fully realized by global fits to the images rather than separate time slices that present the dynamics at a particular wavelength. The discussion applies to transient absorption in any system where the spectral distribution is characterized by two moments, line center and line width. However, we take our example from surface plasmon resonance (SPR) spectroscopy of nanoparticles. Optically active collective oscillation of conduction electrons

Modulation of the surface plasmon resonance of colloidal silver nanoparticles driven by particle shape oscillations is interrogated via transient-scattering measurements using white light as the probe. The two-dimensional (λ, t) image of the spectrally resolved scattering shows π/2 phase shift between the modulation of center Ω(t) and width Γ(t) of the resonance − while Ω is modulated by the strain amplitude, the plasmon-damping rate Γ is modulated by the strain rate. We ascribe the effect to scattering from the electrophoretic potential generated by the motion of the interfacial double layer of the colloidal particles.

A sensitive method for the analysis of single nucleotide polymorphisms (SNPs) in genomic DNA that utilizes nanoparticle-enhanced surface plasmon resonance imaging (SPRI) measurements of surface enzymatic ligation reactions on DNA microarrays is demonstrated. SNP identification was achieved by using sequence-specific surface reactions of the enzyme Taq DNA ligase, and the presence of ligation products on the DNA microarray elements was detected using SPRI through the hybridization adsorption of complementary oligonucleotides attached to gold nanoparticles. The use of gold nanoparticles increases the sensitivity of the SPRI so that single bases in oligonucleotides can be successfully identified at a concentration of 1 pM. This sensitivity is amply sufficient for performing multiplexed SNP genotyping by using multiple PCR amplicons and should also allow for the direct detection and identification of SNP sequences from 1 pM unamplified genomic DNA samples with this array-based and label-free SPRI methodology. As a first example of SNP genotyping, three different human genomic DNA samples were screened for a possible point mutation in the BRCA1 gene that is associated with breast cancer.

A novel surface attachment strategy that utilizes RNA-DNA surface ligation chemistry to create renewable RNA microarrays from single-stranded DNA (ssDNA) microarrays on gold surfaces is demonstrated. The enzyme T4 DNA ligase was used to catalyze the formation of a phosphodiester bond between 5'-phosphate-modified ssDNA attached to the surface and the 3'-hydroxyl group of unlabeled RNA molecules from solution in the presence of a complementary template DNA strand. Surface plasmon resonance imaging (SPRI) measurements were performed to characterize the ligation process as well as to verify the bioactivity of the ssRNA microarray in terms of (i) the hybridization adsorption of complementary DNA onto the RNA array to form a surface RNA-DNA heteroduplex and (ii) the hydrolysis of the RNA microarrays with either ribonuclease S or ribonuclease H (RNase H). The hydrolysis of the surface-bound RNA with RNase H required the presence of a surface heteroduplex and, upon completion, regenerated the original 5'-phosphate-terminated ssDNA array elements. These ssDNA array elements could be ligated again to create a new RNA microarray. These RNA microarrays can be used in the study of RNA-protein/RNA/aptamer bioaffinity interactions and for the enzymatically amplified SPRI detection of DNA in the presence of RNase H.

The kinetics of the ribonuclease H (RNase H) surface hydrolysis of RNA-DNA heteroduplexes formed on DNA microarrays was studied using a combination of real-time surface plasmon resonance imaging (SPRI) and surface plasmon fluorescence spectroscopy (SPFS). Time-dependent SPRI and SPFS data at various enzyme concentrations were quantitatively analyzed using a simple model that couples diffusion, enzyme adsorption, and surface enzyme kinetics. This model is characterized by a set of three rate constants, enzyme adsorption (k(a)), enzyme desorption (k(d)), enzyme catalysis (k(cat)), and one dimensionless diffusion parameter (beta). Values of k(a) = 3.15 (+/-0.20) x 10(6) M(-1).s(-1), k(d) = 0.10 (+/-0.05) s(-1), and k(cat) = 0.95 (+/-0.10) s(-1) were determined from fitting all of the SPRI and SPFS data sets. One of the most interesting kinetic parameters is the surface RNase H hydrolysis reaction rate constant (k(cat)), which was found to be approximately 10 times slower than that observed in solution, but approximately 100 times faster than that recently observed for the exonuclease III surface hydrolysis of double-stranded DNA microarrays (k(cat) = 0.009 s(-1)). Moreover, the surface coverage of the intermediate enzyme-substrate complex (ES) was found to be extremely small during the course of the reaction because k(cat) is much larger than the product of k(a) and the bulk enzyme concentration.

The kinetics of protein adsorption/desorption onto peptide microarrays was studied using real-time surface plasmon resonance (SPR) imaging. S protein binding interactions were examined using an array composed of five different peptides: N terminal and C terminal immobilized wild-type S peptide (S1 and S2), an alternate binding sequence derived by phage display (LB2), an NVOC-protected S peptide, and a FLAG peptide control sequence (F). Kinetic measurements of the S protein-S1 peptide interaction were analyzed to determine a desorption rate constant (k(d)) of 1.1 (+/-0.08) x 10(-2) s(-1), an adsorption rate constant (k(a)) of 1.9 (+/-0.05) x 10(5) M(-1) s(-1), and an equilibrium adsorption constant (K(Ads)) of 1.7 (+/-0.08) x 10(7) M(-1). SPR imaging equilibrium measurements of S protein to S1 peptide were performed to independently confirm the kinetically determined value of K(Ads). Rate constants for the S2 and LB2 peptides on the array were measured as follows: 1.6 (+/-0.04) x 10(5) M(-1) s(-1) (k(a)) and 1.1 (+/-0.07) x 10(-2) s(-1) (k(d)) for S2, 1.2 (+/-0.05) x 10(5) M(-1) s(-1) (k(a)) and 1.1 (+/-0.03) x 10(-2) s(-1) (k(d)) for LB2. In addition to S protein adsorption/desorption, real-time SPR imaging of peptide arrays was applied to study the surface enzymatic activities of the protease factor Xa. Enzymatic cleavage of the substrate peptide (P1) was shown to follow first-order kinetics and proceed at a rate 10 times faster than that of the mutant peptide (P2), with cleavage velocities of 5.6 (+/-0.3) x 10(-4) s(-1) for P1 and 5.7 (+/-0.3) x 10(-5) s(-1) for P2.

The growth of anodic sulphide films on vicinal Cd x Hg 1-x Te (CMT) surfaces has been investigated as a function of sulphide layer thickness by rotation anisotropy using second harmonic generation (SHG). The presence of thin CdS films on the CMT surface does not alter the fourfold pattern observed for the bare CMT surface. However, with increasing sulphide film thickness (>100 nm) and for growth at constant potential, a drastic loss in the reflected SH intensity occurs. This reduction has been attributed to the formation and incorporation of β-HgS in the layer at the more positive potentials. This compound absorbs the SH signal generated by the composite system of CMT/sulphide layer.

The zinc-bromine hybrid redox flow battery (RFB) is one of the few flow battery systems that have been commercially implemented for medium-to-large scale energy storage applications. One of the issues identified with this flow battery is bromine crossover from the positive electrolyte to the zinc side, leading to a loss of energy efficiency. This problem can be overcome through the use of inorganic-based complexing agents that bind the bromine in an immiscible liquid phase which is pumped from the electrode surface and stored in an external reservoir. This bromine-rich phase is recirculated back to the positive electrode during discharge of the battery. Currently, N-methyl-N-ethylpyrrolidinium bromide (MEP) is the choice complexing agent for industrial batteries. However, interest lies in developing a complexing agent that will bind with the electrogenerated bromine but remain in the aqueous phase of the solution. This would improve the charged materials ability to disperse throughout the solution and reduce the complexity of the pumping requirements currently used. A number of novel additives (based on specific quaternary ammonium salts, QBr) have been synthesized and characterized by focusing on both their impact on the Br2/Br− electrochemical kinetics and on their physical properties. Initial observations indicate that the amount of the immiscible liquid phase had been drastically reduced with the novel additives. Electrochemical techniques (including AC impedance spectroscopy and potentiodynamic polarisation) show that MEP and QBr have similar R CT values with a depressed semi circular profile (Figure 1). However, cyclic voltammetry shows a reduced reversibility of the Br2/Br− reaction with QBr present compared to MEP. The data from Tafel extrapolation show a 10-fold reduction in the exchange current density (j 0) between QBr and MEP. However, for the various solutions containing the QBr, MEP and no additive, the same Tafel slope was evaluated (B a ≈ 88 mV) as well as the same equilibrium potential (E 0 ≈ 0.88 V vs. SCE). This difference in the electrochemical kinetics between no additive/QBr and MEP from the CV is arguably due to the fact that the MEP can capture the electrogenerated bromine at the electrode surface better than QBr. These uncertainties led to the testing of the physical properties to determine how well the bromine is binding with the additives in question. The physical properties were found to be quite similar for all the additives (including MEP). The Br2 vapour pressure measured using an isoteniscope resulted in similar enthalpies of vaporization (ΔH ≈ 30 kJ mol− 1) and the quantity of Br2 remaining within the aqueous phase from iodometric titrations. Therefore, it can be assumed that the complex additives used bind similar concentrations of Br2 in the immiscible phase. This ability to capture (and release) larger amounts of bromine, was investigated using Raman spectroscopy and the presence of Br3 − and Br5 − species could be identified. By normalizing to the peak assigned to Br3 − the effectiveness of the novel additives can be compared to MEP based on the Br5 − content (Figure 2). The concentration ratios have an apparent effect on the intensity of the complexed peaks. However, this also shows that the new QBr salt has a similar effect on the physical properties of the electrolyte as MEP. From this series of synthesised QBr compounds, the objective to reduce the immiscible liquid phase has been achieved. This is evident from both visual inspections and the effect that QBr had on the physical properties. MEP still proves to be the better additive in terms of electrochemical kinetics. This serves as a strong starting point for finding a potential candidate that is as effective as MEP with the absence of the immiscible liquid. Figure 1

A number of organisms and organelles are capable of self-propulsion at the micro- and nanoscales. Production of simple man-made mimics of biological transportation systems may prove relevant to achieving movement in artificial cells and nano/micronscale robotics that may be of biological and nanotechnological importance. We demonstrate the propulsion of particles based on catalytically controlled molecular self-assembly and fiber formation at the particle surface. Specifically, phosphatase enzymes (acting as the engine) are conjugated to a quantum dot (the vehicle), and are subsequently exposed to micellar aggregates (fuel) that upon biocatalytic dephosphorylation undergo fibrillar self-assembly, which in turn causes propulsion. The motion of individual enzyme/quantum dot conjugates is followed directly using fluorescence microscopy. While overall movement remains random, the enzyme-conjugates exhibit significantly faster transport in the presence of the fiber forming system, compared to controls without fuel, a non-self-assembling substrate, or a substrate which assembles into spherical, rather than fibrous structures upon enzymatic dephosphorylation. When increasing the concentration of the fiber-forming fuel, the speed of the conjugates increases compared to non-self-assembling substrate, although directionality remains random.

Surface-enhanced Raman scattering (SERS) is a promising imaging modality for use in a variety of multiplexed tracking and sensing applications in biological environments. However, the uniform production of SERS nanoparticle tags with high yield and brightness still remains a significant challenge. Here, we describe an approach based on the controlled co-adsorption of multiple dye species onto gold nanorods to create tags that can be detected across a much wider range of excitation wavelengths (514 – 1064 nm) compared to conventional approaches that typically focus on a single wavelength. This was achieved without the added complexity of nanoparticle aggregation or growing surrounding metallic shells to further enhance the surface-enhanced resonance Raman scattering (SERRS) signal. Correlated Raman and scanning electron microscopy mapping measurements of individual tags were used to clearly demonstrate that strong and reproducible SERRS signals at high particle yields (>92 %) were readily achievable. The polyelectrolyte-wrapped nanorod-dye conjugates were also found to be highly stable as well as non-cytotoxic. To demonstrate the use of these universal tags for the multimodal optical imaging of biological specimens, confocal Raman and fluorescence maps of stained immune cells following nanoparticle uptake were acquired at several excitation wavelengths and compared with dark-field images. The ability to colocalize and track individual optically encoded nanoparticles across a wide range of wavelengths simultaneously will enable the use of SERS alongside other imaging techniques for the real-time monitoring of cell-nanoparticle interactions.

Spherical and monodispersed silver nanoparticles (AgNPs) are ideal for fundamental research as the contribution from size and shape can be accounted for in the experimental design. In this paper a seeded growth method is presented, whereby varying the concentration of sodium borohydride-reduced silver nanoparticle seeds, different sizes of stable spherical nanoparticles with a low polydispersity nanoparticles are produced using hydroquinone as a selective reducing agent. The surface-enhanced Raman scattering (SERS) enhancement factor for each nanoparticle size produced (17, 26, 50, and 65 nm) was then assessed using three different analytes, rhodamine 6G (R6G), malachite green oxalate (MGO) and thiophenol (TP). The enhancement factor gives an indication of the Raman enhancement effect by the nanoparticle. Using non-aggregated conditions and two different laser excitation wavelength (633 nm and 785 nm) it is shown that an increase in particle size results in an increased enhancement for each analyte used.

Reflection second harmonic generation is employed to examine the surfaces of epitaxial layers of CdxHg1-xTe (CMT) grown on vicinal GaAs (100) substrates. Different vicinal tilts, with respect to the GaAs substrates, are observed in the epitaxial CMT layers. We also report here on the measurement of the second-order nonlinear coefficient (d36) of CMT. Because CMT is strongly absorbing at the 1.06 µm wavelength, this measurement was performed by comparing the second harmonic intensity reflected from the CMT surface to that measured for a quartz sample in transmission. Directly comparable expressions for the reflected and transmitted second harmonic intensities are derived from which a value of d36 = 365±15 pm V-1 is obtained. This value is much larger than those reported for similar zinc-blende-type materials and is attributed to an electronic resonance enhancement.

The second order nonlinear coefficient (d36) of the narrow band gap semiconductor, mercury cadmium telluride (MCT), is measured. Because MCT is strongly absorbing at a 1.06 μm wavelength, the measurement was performed by comparing the second harmonic intensity reflected from the material surface to the second harmonic intensity measured for a quartz sample in transmission. The analysis depends on the derivation of comparable expressions for the reflected and transmitted intensities. Using this approach a value of d36=350±40 pm/V is obtained, a value much larger than those reported for similar zinc-blende type materials. The large magnitude of the MCT d36 is attributed to an electronic resonance enhancement.

In-situ measurements of ellipsometry and second harmonic generation (SHG) were carried out to monitor the electrochemical growth of native anodic oxide films on Hg1−xCdxTe (MCT). Growth of the anodic oxide was performed using two different methods viz., by linear sweep voltammetry and by applying a constant current density. The influence of scan rate and the magnitude of the applied current density on the properties of the growing films were examined. From the ellipsometry data, we have shown that the measured refractive index value of 2.19 for the oxide film remains unchanged for moderate and high oxide growth rates. Only at very slow growth rates were significant increases in the refractive index observed (n=2.4), indicating an increase in the compactness of the layer. For film thicknesses in excess of ∼1200 A, a non-zero value for the extinction coefficient was found, indicating the incorporation of HgTe particles within the anodic oxide film. SHG rotational anisotropy measurements, performed on the MCT with and without an anodic oxide film showed only the four-fold symmetry associated with the MCT and so confirmed that the oxide was centrosymmetric. However, an increase in the SH intensity was observed in the presence of the oxide and this has been attributed to multiple reflections in the thin oxide film and also to the increase in the χ(2) non-linear susceptibility tensor as a result of charge accumulation at the MCT/anodic oxide interface.

Second harmonic generation has been employed in-situ to follow the growth of a thin anodic sulphide film on CdxHg1−xTe (CMT). The anisotropy patterns recorded for the bare CMT surface show that the sample is a vicinal {100} face. This is supported in particular by the observation of a non-vanishing pattern in the SIN–SOUT configuration. Also, the observation of a four-fold pattern in the case of the CdS film confirms that the film has retained the ccp structure of the underlying CMT substrate. The SH signal recorded during the growth of the CdS film follows an oscillatory pattern with film thickness. This has been attributed to the interference between the two dominating SH waves generated within the CdS film.

Rotation anisotropy by second harmonic generation (SHG) is carried out on epitaxial Hg1−xCdxTe (MCT) and oxide- and sulphide-covered MCT surfaces and shows the fourfold symmetry pattern expected from the {100} surface (C4v symmetry). The uneven nature of the four peaks confirm the vicinal surface obtained from the growth of the MCT on GaAs {100} substrate orientated 4° toward the 〈110〉 direction. The increase in the SH intensity observed for the oxide-covered MCT surface is associated with charge accumulation at the MCT/oxide interface since the oxide is centrosymmetric and cannot generate SH. The CdS layer on the other hand is strongly nonlinear active and generation here comes from a composite of one noncentrosymmetric layer on top of another. This leads to interactions in the observed SH arising from the coupling depths (∼40 nm) at the two interfaces and from the coherence length (∼1200 nm) in the CdS layer. The in-situ SHG measurements during the growth of the anodic oxide and sulphide layers would suggest that a species, most likely HgTe is embedded in the anodic layer during the initial stages and absorbs the SH radiation at 532 nm. The rotational anisotropy of the sulphide-covered MCT surface confirms that the CdS layer formed maintains the cubic closed pack symmetry of the underlying MCT.

Second-harmonic (SH) rotational anisotropy measurements performed on epitaxial (CMT) layers grown on CdTe B substrate exhibits an interference in the SH signal originating from the bulk and from the CMT surfaces. The threefold symmetry of the epilayer was shown to be sensitive to the nature of adsorbed species at the surface when in contact with an electrolyte solution despite the strong SH generation in the bulk of the layer. The modification of the SH response from a non-centrosymmetric semiconducting material to such an extent is unusual since bulk SH generation is considered as the dominant contributor in these instances. The case of CMT is, however, rather specific in that the observed SH signal originates from, at most, only the top 40 nm of the CMT epilayer. This important difference means that surface modification by means of electron donating or withdrawing groups will play a large role in the observed SHG signal.

Second-harmonic (SH) rotation anisotropy measurements have been performed for the first time on epitaxial CMT layers grown on CdTe〈1 1 1〉 B substrate. The CMT response shows a strong surface contribution to the SH signal. This modification of the SH response from a non-centrosymmetric II–VI material has not been previously reported since bulk SH generation has always been considered as the dominant contributor in these instances. The difference in the case of the CMT however is that the observed SH signal orginates from, at most, only the top 30 nm of the CMT epilayer.

Hyper Rayleigh Scattering (HRS) is used to determine the absolute first hyperpolarizability of gold nanorods with an aspect ratio of 2.2 and 2.7. Two different long axis lengths are used, namely 25.5 nm and 64 nm. This allows for a discussion of the size effect for these centrosymmetric nanoparticles. A comparison of the first hyperpolarizabilities obtained with that of spherical nanoparticles, also centrosymmetric particles, is then made to discuss the role of the shape of these particles on the origin of the response. For the smallest nanorods, a strong hyperpolarizability normalized per atom is determined underlining the role of the interface and the shape in determining such a large absolute value. For the larger nanorods, the first hyperpolarizability per atom is smaller than that of the smaller nanorods but remains larger than the one obtained for gold nanospheres with a similar volume indicating that the shape and the surface response still continue to play a major role. These results are in agreement with the multipolar theory for the first hyperpolarizability of centrosymmetric nanoparticles but show that for nanorods, the surface regime pertains over a longer size range.

In-situ measurements of ellipsometry and second harmonic generation (SHG) are carried out to examine the growth of thin CdS films on CdxHg1−xTe (CMT) from aqueous sulphide solutions. The change in the extinction coefficient (at λ = 632.8 nm) with film thickness indicates that impurities, notably β-HgS, become increasingly incorporated in the CdS layer. The low value for the refractive index (n = 1.73) on the other hand suggests that the film is porous. In this first reported monitoring by SHG of the growth of a non-centrosymmetric layer on another such layer, we have observed an increase of the SH response as the CdS layer was grown. From first principles of non-linear optics, this increase should be quadratic with the film thickness. However, the observed increase does not depart clearly from a linear increase owing to the very thin CdS films grown. Anisotropy measurements have been performed prior to the growth and after the growth of the thin film. A clear change of the pattern attributed to the CdS film is observed but the SH response does not arise solely from the CdS film.

One of the main challenges in the development of new analytical platforms for ultrasensitive bioaffinity detection is jointly achieving a wide dynamic range in target analyte concentration, especially for approaches that rely on multistep processes as a part of the signal amplification mechanism. In this paper, a new surface-based sandwich assay is introduced for the direct detection of B-type natriuretic peptide (BNP), an important biomarker for cardiac failure, at concentrations ranging from 1 aM to 500 nM. This was achieved using nanoparticle-enhanced surface plasmon resonance (SPR) where a DNA aptamer is immobilized on a chemically modified gold surface in conjunction with the specific adsorption of antiBNP coated gold nanocubes in the presence of the biomarker target. A concentration detection range greater than eleven orders of magnitude was achieved through dynamic control of only the secondary nanoparticle probe concentration. Furthermore, detection at low attomolar concentrations was also achieved in undiluted human serum.

The preparation and characterization of stable and non-aggregated colloidal suspensions of gold nanorod–molecular dye complexes which exhibit very bright surface-enhanced resonance Raman scattering (SERRS) signals is described. A systematic study was performed where both the localized surface plasmon resonance (LSPR) of the nanorod and the molecular resonance of dyes adsorbed onto the rod surface were selectively tuned with respect to the laser excitation wavelengths. Resonance coupling was found to be a significant factor in the overall SERRS enhancement. The polymer stabilized nanorod–dye conjugates were prepared without the added complexity of nanoparticle aggregation as well as having good control over the surface coverage and orientation of the dye molecules. Furthermore, we demonstrate that this new class of Raman nanotags greatly outperforms an approach based on quasi-spherical gold nanoparticles.

Although the application of ultrasound for the control and modulation of various chemical processes is a subject of continued interest [l], there is surprisingly little discus- sion in the recent literature of the effects of ultrasound on fundamental electrochemical processes. In early systematic studies of the hydrogen evolution reaction under acidic conditions [2,3], it was observed that, on a platinum elec- trode, the overvoltage for hydrogen evolution decreased by up to 30mV on the application of ultrasound, and that this decrease was largely independent of current density in the approximate range l-100 mA cm-‘, i.e. the Tafel slope was largely unaffected under these conditions. On removal of the ultrasound, the original polarization was regained only on continued electrolysis for up to 1 h. These results refer to electrolytes which had been pre-electrolysed for 10 to 20h. With more extensive pre-electrolysis (36h), the results were generally similar; however, the polarization decrease on the application of ultrasound was smaller (by a factor of two) under comparable conditions, and the origi- nal polarization was recovered rapidly on cessation of irradiation. On the application of ultrasound to a nickel electrode evolving hydrogen, the same general phe- nomenon was observed, i.e. a decrease in overvoltage without a significant change in Tafel slope [3]. For these experiments, the acoustic frequency was 3OOkHz and the ;icoustic intensity was estimated to be approximately I Wcmw2 of electrode area.

The application of biofunctionalized nanoparticles possessing various shapes and sizes for the enhanced surface plasmon resonance (SPR) detection of a protein biomarker at attomolar concentrations is described. Three different gold nanoparticle shapes (cubic cages, rods and quasi-spherical) with each possessing at least one dimension in the 40-50 nm range were systematically compared. Each nanoparticle (NP) was covalently functionalized with an antibody (anti-thrombin) and used as part of a sandwich assay in conjunction with a Au SPR chip modified with a DNA-aptamer probe specific to thrombin. The concentration of each NP-antibody conjugate solution was first optimized prior to establishing that the quasi-spherical nanoparticles resulted in the greatest enhancement in sensitivity with the detection of thrombin at concentrations as low as 1 aM. When nanorod and nanocage antibody conjugates were instead used, the minimum target concentrations detected were 10 aM (rods) and 1 fM (cages). This is a significant improvement (10(3)) on previous NP-enhanced SPR studies utilizing smaller (~15 nm) gold NP conjugates and is attributed to the functionalization of both the NP and chip surfaces resulting in low nonspecific adsorption as well as a combination of density increases and plasmonic coupling inducing large shifts in the local refractive index at the chip surface upon nanoparticle adsorption.

The demand for improved technologies capable of rapidly detecting pathogens with high sensitivity and selectivity in complex environments continues to be a significant challenge that helps drive the development of new analytical techniques. Surface-based detection platforms are particularly attractive as multiple bioaffinity interactions between different targets and corresponding probe molecules can be monitored simultaneously in a single measurement. Furthermore, the possibilities for developing new signal transduction mechanisms alongside novel signal amplification strategies are much more varied. In this article, we describe some of the latest advances in the use of surface bioaffinity detection of pathogens. Three major sections will be discussed: (i) a brief overview on the choice of probe molecules such as antibodies, proteins and aptamers specific to pathogens and surface attachment chemistries to immobilize those probes onto various substrates, (ii) highlighting examples among the current generation of surface biosensors, and (iii) exploring emerging technologies that are highly promising and likely to form the basis of the next generation of pathogenic sensors.

The recent discovery of short, non-protein coding RNA molecules, such as microRNA molecules (miRNAs), that can control gene expression has unveiled a whole new layer of complexity in the regulation of cell function. Since 2001, there has been a surge of interest in understanding the regulatory role of the hundreds to thousands of miRNAs expressed in both plants and animals. Significant progress in this area requires the development of quantitative bioanalytical methods for the rapid, multiplexed detection of all miRNAs that are present in a particular cell or tissue sample. In this Minireview, we discuss some of the latest methods for high-throughput miRNA profiling and the unique technological challenges that must be surmounted in this endeavor.

The recent discovery of short, non-protein coding RNA molecules, such as microRNA molecules (miRNAs), that can control gene expression has unveiled a whole new layer of complexity in the regulation of cell function. Since 2001, there has been a surge of interest in understanding the regulatory role of the hundreds to thousands of miRNAs expressed in both plants and animals. Significant progress in this area requires the development of quantitative bioanalytical methods for the rapid, multiplexed detection of all miRNAs that are present in a particular cell or tissue sample. In this Minireview, we discuss some of the latest methods for high-throughput miRNA profiling and the unique technological challenges that must be surmounted in this endeavor.