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Spatial Heterodyne Offset Raman Spectroscopy (SHORS) integrating Spatially Offset Raman Spectroscopy (SORS) with a fiber-coupled spatial heterodyne spectrometer (SHS) to enable a high sensitivity, ‘on-the-fly’ and movement-free deeper detection method for biomaterial models.

It is well known that extracellular matrix stiffness can affect cell fate and change dynamically during many biological processes. Existing experimental means for in situ matrix stiffness modulation often alters its structure, which could induce additional undesirable effects on cells. Inspired by the phenomenon of depth sensing by cells, we introduce here core–shell microfibers with a thin collagen core for cell growth and an alginate shell that can be dynamically stiffened to deliver mechanical stimuli. This allows for the maintenance of biochemical properties and structure of the surrounding microenvironment, while dynamically modulating the effective modulus “felt” by cells. We show that simple addition of Sr2+ in media can easily increase the stiffness of initially Ca2+ cross-linked alginate shells. Thus, despite the low stiffness of collagen cores (

Spatially offset Raman spectroscopy (SORS) offers the prospect of collecting spectral information detailing the molecular composition of biomaterials at greater depths below the surface layers than are normally probed by conventional Raman spectroscopy. By collecting off-axial scattered light, the technique overcomes the large background from in-line light within scattering media. In this paper we present a configuration which enables the highly efficient collection of spectral markers, indicative of bone health, including Raman signatures to assess phosphate, collagen and carbonate content, at millimeter depths. We demonstrate the effectiveness of the technique by performing spectral decompositions to analyze the molecular distribution of these markers non-invasively, using in vitro model systems, comprising bone and tissue, in situ.

The field of microfluidics promises new portable, low-cost sensing systems, as well as the capabilities to measure the physical or chemical properties of precious samples, for which only small volumes are available. However, when using microfluidic channels with millimeter to micron scale dimensions, together with optical sensing methods, these configurations result in short path lengths over which the signal can be acquired. Whilst polarimetry would greatly benefit from using small volumes, providing important information on the structure of chiral biomarkers in life sciences, the small interrogation volumes associated with the use of minute samples decreases the numbers of molecules in the light path that cause an optical rotation and reduces the sensitivity of the technique. Here, we show that when an optical beam, passing through a chiral sample, undergoes multiple reflections from suitably aligned external micromirrors, the usual cancelling out of the optical rotation, that occurs when the rotated polarized beam is passed back through a solution following reflection at a single mirror, can be negated. This enables the chirality of molecular species present in a microfluidic sample to be measured with increased sensitivity. This approach was validated experimentally using solutions of D-(+)-glucose as a model system, by investigating the effect of multiple reflections of a linearly polarized He-Ne laser beam and a 403 nm diode laser beam across the microfluidic channel. It was found that there was a 30-fold enhancement in the limit of detection with as few as 11 reflections through the sample.

Microflow cytometry has become an attractive tool for counting and analysis of complex cell population and finds many applications in life science research. However, its implementation in screening small sized targets, such as microorganisms, is limited by challenges associated with sample handling and detection in a flow. Here, we report the function of a 3D flow focusing microfluidic system as a high performance, multi-parameter flow cytometry. The system consists of a detection chamber that can precisely control a highly focused sample flow in 3D and an integrated optical fiber to collect the scattered light at the vertical plane. The ability of the system to detect small beads (1 µm), differentiate subtle differences (2 µm) and accurately profile a mixed bead population is demonstrated. Furthermore, multiple types of information about the sample, including the inherent biochemical information (e.g., fluorescence or Raman signal) and physical properties (e.g., size) are simultaneously generated, allowing sophisticated cellular analysis. Together with the facile and robust operation, this provides a versatile tool that could be used for multi-parametric analyses in a diversity of applications.

Optical-based microfluidic cell sorting has become increasingly attractive for applications in life and environmental sciences due to its ability of sophisticated cell handling in flow. The majority of these microfluidic cell sorting devices employ two-dimensional fluid flow control strategies, which lack the ability to manipulate the position of cells arbitrarily for precise optical detection, therefore resulting in reduced sorting accuracy and purity. Although three-dimensional (3D) hydrodynamic devices have better flow-focusing characteristics, most lack the flexibility to arbitrarily position the sample flow in each direction. Thus, there have been very few studies using 3D hydrodynamic flow focusing for sorting. Herein, we designed a 3D hydrodynamic focusing sorting platform based on independent sheath flow-focusing and pressure-actuated switching. This design offers many advantages in terms of reliable acquisition of weak Raman signals due to the ability to precisely control the speed and position of samples in 3D. With a proof-of-concept demonstration, we show this 3D hydrodynamic focusing-based sorting device has the potential to reach a high degree of accuracy for Raman activated sorting.

Raman activated cell sorting has emerged as a label-free technology that can link phenotypic function with genotypic properties of cells. However, its broad implementation is limited by challenges associated with throughput and the complexity of biological systems. Here, we describe a three-dimensional hydrodynamic focusing microfluidic system for a fully automated, continuous Raman activated cell sorting (3D-RACS). The system consists of a 3D printed detection chamber (1 mm3) that is integrated with a PDMS based sorting unit, optical sensors and an in-line collection module. It has the ability to precisely position cells in the detection chamber for Raman measurements, effectively eliminating spectroscopic interference from the device materials. This enables the sorting of a range of cell sizes (from 1 μm bacteria to 10's μm mammalian cells) with stable operation over >8 hours and high throughput. As a proof-of-concept demonstration, Raman-activated sorting of mixtures of Chlorella vulgaris and E. coli has demonstrated a purity level of 92.0% at a throughput of 310 cells per min. The platform employed in this demonstration features a simple “Raman window” detection system, enabling it to be built on a standard, inverted microscope. Together with its facile and robust operation, it provides a versatile tool for function-based flow cytometry and sorting applications in the fields of microbiology, biotechnology, life science and diagnostics.

Spatially Offset Raman Spectroscopy involves the collection off-axial scattered light, enabling the rapid collection of high signal-to-noise spectral information including that for phosphate, collagen and carbonate at millimeter depths, all indicative of bone health.

We present a fully automated laser system with low-intensity noise for coherent Raman scattering microscopy. The robust two-color system is pumped by a solid-state oscillator, which provides Stokes pulses fixed at 1043 nm. The tunable pump pulses of 750 to 950 nm are generated by a frequency-doubled fiber-feedback femtosecond optical parametric oscillator. The resulting pulse duration of 1.2 ps provides a viable compromise between optimal coherent Raman scattering signal and the necessary spectral resolution. Thus a spectral range of 1015 to 3695 cm − 1 with spectral resolution of

The specific and multiplexed detection of DNA underpins many analytical methods, including the detection of microorganisms that are important in the medical, veterinary, and environmental sciences. To achieve such measurements generally requires enzyme-mediated amplification of the low concentrations of the target nucleic acid sequences present, together with the precise control of temperature, as well as the use of enzyme-compatible reagents. This inevitably leads to compromises between analytical performance and the complexity of the assay. The hybridization chain reaction (HCR) provides an attractive alternative, as a route to enzyme-free DNA amplification. To date, the linear nucleic acid products, produced during amplification, have not enabled the development of efficient multiplexing strategies, nor the use of label-free analysis. Here, we show that by designing new DNA nanoconstructs, we are able, for the first time, to increase the molecular dimensionality of HCR products, creating highly branched amplification products, which can be readily detected on label-free sensors. To show that this new, branching HCR system offers a route for enzyme-free, label-free DNA detection, we demonstrate the multiplexed detection of a target sequence (as the initiator) in whole blood. In the future, this technology will enable rapid point-of-care multiplexed clinical analysis or in-the-field environmental monitoring., DNA detection: enzyme-free amplification A hybridization chain reaction permits multiplexed and label-free DNA detection. Nucleic acid detection generally requires a suitable enzyme to amplify the typically low concentration of target nucleic acid sequences, as well as careful control of temperature. However, this limits the ability for multiplexing strategies. Now, a team led by Jonathan M. Cooper at University of Glasgow in the UK report the use of DNA nanoconstructs to enhance the molecular dimensionality of hybridization chain reaction (HCR) products, enabling label-free multiplexed detection; the use of a 3-arm DNA construct creates additional sites to initiate a chain reaction with high efficiency, removing the need for otherwise complex labeling approaches.

Spatially offset Raman spectroscopy is integrated with a fiber-coupled spatial heterodyne spectrometer to collect Raman spectra from deep within opaque or scattering materials. The method, named spatial heterodyne offset Raman spectroscopy generates a wavenumber-dependent spatial phase shift of the optical signal as a "spectral" image on a charge-coupled device detector. The image can be readily processed from the spatial domain using a single, simple, and "on-the-fly" Fourier transform to generate Raman spectra, in the frequency domain. By collecting all of the spatially offset Raman scattered photons that pass through the microscope's collection objective lens, the methodology gives an improvement in the Raman sensitivity by an order of magnitude. The instrumentation is both mechanically robust and "movement-free," which when coupled with the associated advantages of highly efficient signal collection and ease of data processing, enables rapid interfacial analysis of complex constructs based on established biomaterials models.

In creating mirrored silicon structures for micro-optics, the smoothness of the surface and etch rate are crucial parameters. We demonstrate a method of creating both 45° and 90° etch-planes from monocrystalline silicon for use as retro-reflective sidewalls in a microfluidic device. The technique uses the same photolithographic pattern orientation, but with two different etchants. Etching on direction in Si(100) with potassium hydroxide (KOH) gives vertical surfaces (where e.g. the high surface tension influences etching of crystallographic silicon planes), whilst tetramethylammonium hydroxide (TMAH) gives 45° sidewalls. We illustrate the use of these fabricated structures by creating arrays of micromirrors that enable an optical beam to be reflected parallel back and forth from 45° and −45° tilted vertical structures. This device has potential uses in optofluidic spectroscopic applications, where there is a need to increase the effective pathlength of a beam through a sample whilst keeping the device as small as possible.

Single cell Raman spectroscopy measures a spectral fingerprint of the biochemistry of cells, and provides a powerful method for label-free detection of living cells without the involvement of a chemical labelling strategy. However, as the intrinsic Raman signals of cells are inherently weak, there is a significant challenge in discriminating and isolating cells in a flowing stream. Here we report an integrated Raman-microfluidic system for continuous sorting of a stream of cyanobacteria, Synechocystis sp. PCC6803. These carotenoid-containing microorganisms provide an elegant model system enabling us to determine the sorting accuracy using the subtly different resonance Raman spectra of microorganism cultured in a (12)C or (13)C carbon source. Central to the implementation of continuous flow sorting is the use of "pressure dividers" that eliminate fluctuations in flow in the detection region. This has enabled us to stabilise the flow profile sufficiently to allow automated operation with synchronisation of Raman acquisition, real-time classification and sorting at flow rates of ca.

By directly monitoring single cell growth in a microfluidic platform, we interrogated genome-deletion effects in Escherichia coli strains. We compared the growth dynamics of a wild type strain with a clean genome strain, and their derived mutants at the single-cell level. A decreased average growth rate and extended average lag time were found for the clean genome strain, compared to those of the wild type strain. Direct correlation between the growth rate and lag time of individual cells showed that the clean genome population was more heterogeneous. Cell culturability (the ratio of growing cells to the sum of growing and nongrowing cells) of the clean genome population was also lower. Interestingly, after the random mutations induced by a glucose starvation treatment, for the clean genome population mutants that had survived the competition of chemostat culture, each parameter markedly improved (i.e., the average growth rate and cell culturability increased, and the lag time and heterogeneity decreased). However, this effect was not seen in the wild type strain; the wild type mutants cultured in a chemostat retained a high diversity of growth phenotypes. These results suggest that quasi-essential genes that were deleted in the clean genome might be required to retain a diversity of growth characteristics at the individual cell level under environmental stress. These observations highlight that single-cell microfluidics can reveal subtle individual cellular responses, enabling in-depth understanding of the population.

We report on the design and fabrication of a hybrid sensor that integrates transmission-mode localized surface plasmonic resonance (LSPR) into a quartz crystal microbalance (QCM) for studying biochemical surface reactions. The coupling of LSPR nanostructures and a QCM allows optical spectra and QCM resonant frequency shifts to be recorded simultaneously and analyzed in real time for a given surface adsorption process. This integration simplifies the conventional combination of SPR and QCM and has the potential to be miniaturized for application in point-of-care (POC) diagnostics. The influence of antibody-antigen recognition effect on both the QCM and LSPR has been analyzed and discussed.

A new approach is described herein, where neutron reflectivity measurements that probe changes in the density profile of thin films as they absorb material from the gas phase have been combined with a Love wave based gravimetric assay that measures the mass of absorbed material. This combination of techniques not only determines the spatial distribution of absorbed molecules, but also reveals the amount of void space within the thin film (a quantity that can be difficult to assess using neutron reflectivity measurements alone). The uptake of organic solvent vapours into spun cast films of polystyrene has been used as a model system with a view to this method having the potential for extension to the study of other systems. These could include, for example, humidity sensors, hydrogel swelling, biomolecule adsorption or transformations of electroactive and chemically reactive thin films. This is the first ever demonstration of combined neutron reflectivity and Love wave-based gravimetry and the experimental caveats, limitations and scope of the method are explored and discussed in detail.

Biogenic calcium carbonate has come to the attention of many researchers as a promising drug delivery system due to its safety, pH sensitivity and the large volume of information already in existence on its medical use. In this study, we employed bovine serum albumin (BSA) as an additive to synthesize a series of porous calcium carbonate microspheres (CCMS). These spheres, identified as vaterite, are stable both in aqueous solutions and organic solvents. Camptothecin, an effective anticancer agent, was loaded into the CCMS by simple diffusion and adsorption. The camptothecin loaded CCMS showed sustained cell growth inhibitory activity and a pH dependent release of camptothecin. With a few hours, the release is negligible under physiological conditions (pH = 7.4) but almost complete at pH 4 to 6 (i.e. pHs found in lysosomes and solid tumor tissue respectively). These findings suggest that porous, biogenic calcium carbonate microspheres could be promising carriers for the safe and efficient delivery of anticancer drugs of low aqueous solubility.

Vesicle fusion was used to deposit mixed dimyristoyl phosphatidylethanolamine–dimyristoyl phosphatidylserine (DMPE–DMPS) phospholipid bilayers on Au electrodes. Bilayer structure and composition, when exposed to aqueous NaF and subject to an applied electrochemical potential, were studied using electrochemical, spectroscopic and neutron reflectivity (NR) techniques. Interfacial capacitance data indicate the formation of compact films. Chronocolometric data show that surface charge is significantly altered by the presence of lipid in the potential range −0.75 < E/V (Ag|AgCl) < 0.35. NR measurements were made on lipid films in which the hydrocarbon tails were either fully hydrogenous (h-DMPE–h-DMPS) or perdeuterated (d-DMPE–d-DMPS), in each case serially exposed to D2O and H2O electrolytes and subject to different applied potentials. Guided by simulations of candidate interfacial structures, these yield the spatial distributions of lipid and solvent within the layers. Adjacent to the electrode, a compact inner leaflet is formed, with potential-dependent solvent volume fraction in the range 0.09 < ϕS < 0.19; there was no evidence of an intervening water layer. The outer leaflet contains rather more solvent, 0.52 < ϕS < 0.55. NR-derived film thickness and PM-IRRAS intensity data show that the lipid molecules are tilted from the surface normal by ca. 26°. Bilayer solvation and charge data show a strong correlation for the inner leaflet and very little for the outer leaflet.

Crown ether functionalised conducting polymer films were used to complex barium ions from acetonitrile solution. It was found that fully-functionalised N-derivatized polypyrrole films do not possess adequate mechanical stability, but dilution with unfunctionalised bithiophene co-monomer leads to a series of copolymer films with excellent stability. Film reactivity, composition and structure were investigated using electrochemical, nanogravimetric, FTIR, XPS and neutron reflectivity techniques. The first three of these provided spatially integrated barium populations and neutron reflectivity provided spatially resolved compositional profiles. Measurements at various stages of film fabrication yielded spatial distributions of co-monomer, crown ether, solvent and barium (as perchlorate) components. Critically, the amount of free volume to accommodate crown motifs and barium within the film was limited by the film's internal microstructure and solvent content; the low solvent volume fraction creates a different local environment to solution.

Surface enhanced resonance Raman spectroscopy (SERRS) is a powerful molecular sensing tool that can be applied to a number of applications in the field of molecular diagnostics. We demonstrate that by using electron beam lithography to manipulate the nanoscale geometry of Ag split-ring resonators we can tune their optical properties such that they exhibit two independently addressable high frequency plasmon resonance modes for SERRS. This tailored multimodal, polarization dependent activity enables the split rings to act as discriminating sensors, with each resonance tuned for a particular sensing purpose. The structures are used as multiwavelength, multianalyte DNA SERRS sensors, with each resonance tuned to both the absorption wavelength of a differently colored Raman reporter molecule and its corresponding laser excitation wavelength. The ability of each resonance to independently sense small concentrations of a single DNA type from within a mixed population is demonstrated. Also shown is the effect of the split ring's dichroic response on the SERRS signal and the sensor's limit of detection of each resonance mode (switching its sensory reaction "on" and "off" depending on the orientation of the exciting light).

A new strategy to produce stable barcodes using biological self-assembly of streptavidin- and biotin-functionalized quantum dots is reported. Such systems are of potential use in multiplexed immunoassay and nucleic acid hybridization assays.

In this paper we demonstrate that the nucleation density of single-walled carbon nanotubes (SWNTs), formed by thermal catalytic chemical vapor deposition, strongly depends on the grain size of Al underlayers covered with a native oxide (Al/Al2O3). By varying the Substrate temperature during Al sputter deposition it was possible to investigate the effect of Al grain size on growth without inducing changes in the underlayer thickness, surface chemistry, or any other growth parameter. The resulting SWNT growth structures ranged from low-density 2D nanotube networks that lay across the surface of the substrate to high density 3D nucleation which gave rise to vertical "forest" growth. The height of the SWNT "forest" was observed to increase with increasing Al deposition temperature as follows, 200 > 100 > 60 > 20 degrees C on Si/Al but in the order 100 > 200 > 60 > 20 degrees C on SiO2/Al substrates for fixed growth conditions. The differences in the SWNT growth trends on Si and SiO2 substrates are believed to be due to the existence of an optimal Al/Al2O3 underlayer grain size for the formation of active catalytic nanoparticles, with larger Al/Al2O3 grains forming on SiO2 than Si at a fixed substrate temperature. Numerous surface analysis techniques including AFM, XPS, FESEM, TEM, and Raman spectroscopy have been employed to ascertain that the observed changes in nanotube growth for this system are related primarily to changes in underlayer morphology.

Recently, the need for super-repellent surfaces has been increasing, with applications ranging from material science to Lab-on-a-Chip diagnostics and microfluidics. We propose a novel and simple method to engineer super-hydrophobic surfaces using commercially available negative-tone resists, namely SU-8 and the photodefinable spin-on silicone elastomer, PDSE. The surface energies of fabricated materials were investigated and applied to the transition from the super-hydrophobic to hydrophilic states, occurring during liquid phase evaporation.

Time-resolved specular neutron reflectivity measurements are presented and interpreted for electroactive polyvinylferrocene (PVF) films subject to potentiodynamic electrochemical control. New data acquisition methodology allows an effective measurement time scale on the order of seconds, which is an improvement over conventional methodology by 2 to 3 orders of magnitude. Reflectivity profiles were obtained for PVF films exposed to aqueous 0.1 M NaClO4 in which PVF films are thermodynamically permselective, with contrast variation via H2O and D2O. Irrespective of any model, the raw profiles show chemically reversible film "breathing" due to redox-driven solvent entry and exit during polymer oxidation and reduction, respectively. Modeling reveals three compositionally distinct regions within the polymer film: interfacial regions at the electrode and solution interfaces and a "bulk" interior. The new methodology, supported by simultaneous in situ visible transmission spectroscopy, reveals an unprecedented level of insight into the temporal and spatial mechanistic details of film solvation changes, including a two-stage (de)solvation mechanism for redox switching, differences in interior (in)homogeneity for reduced and oxidized films, and permselectivity failure under dynamic electrochemical conditions for the reduced (but not oxidized) state, in contrast to static conditions that allow permselectivity for both states.

The objective of this paper is the study of the electrochemical oxidation and subsequent polymerisation of 1,3-dihydroxybenzene, 3-aminophenol and 1,3-phenylenediamine at pH 7. These related monomers were subsequently utilised by the authors in the production of pinhole free masks for the construction of features on gold surfaces of nanometer dimensions (neutral pH was required due to the use of biological templates). It is shown that whilst the electrochemical oxidation of 1,3-phenylenediamine is very similar to that of 3-aminophenol, it is quite dissimilar to that of 1,3-dihydroxybenzene and this can be attributed to the lack of interaction between the substituents on the ring due to their meta orientation. This behaviour can be correlated with differences in the HOMO values of the molecules as calculated using Crystal98. X-ray photoelectron spectroscopy was used to characterise the polymer films and their thin layer nature.

An artificial olfactory system (or “electronic nose”) has been developed to investigate the interactions between polymer-modified sensors with odorant vapors from the headspace of primary alcohol samples. Complementary pairs of polymer-coated quartz crystal microbalance sensors and polymer/carbon black-coated micro-resistance sensors have been used to produce a characteristic value for the odorants (Sfr), related to the odorant molecular density, which can be used in electronic nose applications for odor discrimination. An application-specific integrated circuit (ASIC) is also described which controls and collects data from the quartz crystal microbalance-based sensors, enabling the future development of a hand-held/miniaturized detection system utilizing these types of sensor.

This paper describes studies of a combined electrochemical/acoustic wave sensor for detection of metal ions in aqueous solutions. The sensor is based upon surface complexation of the metal ions at thiodisuccinic acid (TDS) functionalised self-assembled monolayer (SAM) modified electrodes on 10 MHz AT-cut quartz resonators, functioning as a quartz crystal microbalance (QCM). The SAM-modified electrodes were employed for the detection of Cd(II) ions in single and mixed metal ion solutions, with Pb(II) or Cu(II) as the interferants. Acoustic determination was based on the QCM frequency change associated with the binding to the surface-immobilised ligand of metal ions as a function of their concentration. Frequency response data were acquired under emersed and immersed conditions for ligand binding to the Au electrode and, subsequently, metal ion binding to the ligand. In the latter case, the data were interpreted according to a range of isotherms, of which the Temkin isotherm was found empirically best to describe the concentration dependence. The acoustic wave data yielded information on equilibrium binding stoichiometry and energetics. The extent of metal ion binding was also determined by coulometric assay (upon reduction to the zero-valent metal) after transfer to metal ion-free electrolyte solution. XPS studies of surface-bound metal-TDS ligand complexes confirmed the binding stoichiometry obtained from the electrochemical and acoustic wave responses, and provided information on the ligand–substrate binding and bound analyte oxidation state.

Background In cancer cells in vitro, the glycolytic pathway and the mitochondrial tricarboxylic acid (TCA) cycle are programmed to produce more precursor molecules, and relatively less ATP, than in differentiated cells. We address the questions of whether and where these changes occur in vivo in glioblastomas grown from C6 cells in rat brain. These gliomas show some spatial organization, notably in the upregulation of membrane proton transporters near the rim. Results We immunolabeled pairs of proteins (as well as DNA) on sections of rat brains containing gliomas, measured the profiles of fluorescence intensity on strips 200 µm wide and at least 3 mm long running perpendicular to the tumor rim, and expressed the intensity in the glioma relative to that outside. On averaged profiles, labeling of a marker of the glycolytic pathway, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), was, as expected, greater in the glioma. Over distances up to 2.5 mm into the glioma, expression of a marker of the TCA cycle, Tom20, a pre-protein receptor on the translocation complex of the mitochondrial outer membrane, was also upregulated. The ratio of upregulation of Tom20 to upregulation of GAPDH was, on average, slightly greater than one. Near the rim (0.4–0.8 mm), GAPDH was expressed less and there was a peak in the mean ratio of 1.16, SEM = 0.001, N = 16 pairs of profiles. An antibody to V-ATPase, which, by pumping protons into vacuoles contributes to cell growth, also indicated upregulation by about 40%. When compared directly with GAPDH, upregulation of V-ATPase was only 0.764, SD = 0.016 of GAPDH upregulation. Conclusions Although there was considerable variation between individual measured profiles, on average, markers of the glycolytic pathway, of mitochondria, and of cell proliferation showed coherent upregulation in C6 gliomas. There is a zone, close to the rim, where mitochondrial presence is upregulated more than the glycolytic pathway, in agreement with earlier suggestions that lactate is taken up by cells near the rim. Electronic supplementary material The online version of this article (doi:10.1186/s13104-015-1191-z) contains supplementary material, which is available to authorized users.

This paper describes the development and evaluation of a custom-built impedance analyzer, which uses a multiplexing bridge circuit to characterize an array of polymer-coated quartz crystal microbalance (QCM) sensors. The analyzer is constructed on a single printed circuit board with minimum components and is sufficiently compact for integration into a handheld format. The custom-built device is used to observe the changes that occur in QCM sensors when experimental conditions such as polymer coating film thickness, odorant vapor pressure, and relative molecular mass are varied. An equivalent electric circuit for a QCM is used to model the conductance and susceptance data captured by the analyzer. The measured response of an array of QCM sensors demonstrates that the custom-built device is a suitable instrument for detecting different gases and understanding polymer–vapor interactions.

We report here on the fabrication of a three-dimensional array of nanoparticles which bridges the gap between lithographically defined gold electrode contacts separated by 20 nm. The nanoparticle assemblies are formed from about 5 nm gold nanoparticles and benzenedimethanethiol (BDMT) bridging ligands. These assemblies are introduced between the contacts using a layer-by-layer protocol with successive BDMT self-assembly being followed by nanoparticle adsorption until the gap is bridged. The temperature dependent electrical properties of these devices are analysed to establish whether they are consistent with the notion that the networks are built up from molecularly interlinked discrete gold nanoparticles. To aid this analysis the molecular conductance of single bridging molecules is also characterized at room temperature using a recently introduced method based on the scanning tunnelling microscope (STM). From these measurements it is concluded that the room temperature electrical properties of the nanostructured networks are limited by the small interparticle connectivity and the inherent resistance of the linker molecules.

This paper describes improvements in high resolution large area e-beam lithography when a thin titanium layer is applied to substrates prior to the application of resist. The technique is particularly useful when there is a requirement to pattern long unsupported nanostructures such as lines which are more than a millimetre long but have widths of less than 20nm. Such feature sizes are now readily achieved using the most advanced e-beam lithography tools which can have patterning writing fields in excess of one millimetre and spot sizes of less than 5nm. In this paper, we describe a simple technique to improve the adhesion of different resist structures, particularly on III-V materials to enable the rapid evaluation of high resolution e-beam lithography and we show that X-ray photoelectron spectroscopy (XPS) can be useful in understanding substrate-resist interactions.

The electropolymerization of trans-[RuCl2(vpy)4] (vpy=4-vinylpyridine) on Au or Pt electrodes was studied by cyclic voltammetry, electrochemical quartz crystal microbalance (EQCM) technique, and Raman spectroscopy. Cyclic voltammetry of the monomer at a microelectrode shows the typical Ru(III/II) and Ru(IV/III) waves, together with the vinyl reduction waves at −1.5 and −2.45 V and adsorption wave at −0.8 V. Electrodeposition on EQCM technique performed under potential cycling between −0.9 and −2.0 V revealed that the polymerization proceeded well in advance of the vinyl reduction waves. At potentials more positive than −0.9 V, soluble oligomers were deposited irreversibly on the electrode during the oxidative sweep. The film also showed reversible mass changes due to the oxidation and accompanying ingress of charge-balancing anions and solvent into the film. In contrast, potentiostatic growth of the polymer at −1.6 V was slower because the oligomeric material was lost completely from the electrode. Unreacted vinyl groups were detected by in situ Raman spectroscopy for films grown at −0.7, −0.9, and −1.6 V but were absent when the polymerization was carried out at −2.9 V vs Ag/Ag+.

We present the design and fabrication of 1-to-N multimode interference (MMI) splitters, suitable for use in integrated optical fluorescence array sensing, with particular applications in lab-on-a-chip (micro-TAS) technologies. Electron beam irradiation of germanium-doped flame hydrolysis deposited silica was used to define the MMI waveguide regions. The splitters were integrated with microfluidic channels to form direct-excitation fluorescence sensor chips for use at visible wavelengths. Characterization of the waveguides shows that predictable splitting ratios can be achieved. Two devices are presented: a 1/spl times/2 splitter integrated with one analytical chamber and a 1/spl times/4 array device for multipoint excitation. A photomultiplier tube was used to assess the analytical performance of the chip, in response to standard aliquots of fluorophore (31 nM to 1.25 /spl mu/M).

The optical properties of germanium-doped silica layers deposited by flame-hydrolysis have been investigated by analyzing the effective refractive indices of the modes using an inverse-WKB (Wentzel–Kramers–Brillouin) approach followed by an optimization algorithm. The shape obtained for the refractive index profile is consistent with the compositional and density characterization, performed by X-ray photoelectron spectroscopy (XPS) and X-ray reflectivity, respectively. We believe that the origin of the non-uniformity was the high-temperatures to which the as-deposited soot is subjected in order to obtain fully consolidated, high-optical quality films.

In situ neutron reflectivity measurements have been made on spin cast polyvinylferrocene (PVF) films maintained under electrochemical potential control. The data show that the interiors of both oxidized and reduced films are close to homogeneous. The results show that neutron reflectivity measurements, including isotopic substitution, can provide novel insights into film composition and structure at the electrode/polymer/solution interface. Isotopic substitution of the solvent has allowed the contributions of the solvent and the solute to the total film composition to be separated. Responses were determined to variations in the imposed potential (a constraint at the electrode/polymer interface) and the electrolyte concentration (a constraint at the polymer/solution interface). Solvent entry occurs upon oxidation, resulting is a more diffuse polymer/solution interface. Only at very high concentrations is salt permeation into PVF films sufficiently high to be considered as a possible source of co-ions for r...

We describe a method, based on X-ray photoelectron spectroscopy (XPS) measurements, to assess the extent of protein adsorption or binding on a variety of different muTAS and biosensor interfaces. Underpinning this method is the labeling of protein molecules with either iodine- or bromine-containing motifs by using protocols previously developed for radiotracer studies. Using this method, we have examined the adsorption and binding properties of a variety of modified electrodeposited polymer interfaces as well as other materials used in muTAS device fabrication. Using polymer interfaces modified with poly(propylene glycol) (PPG) chains, our results indicate that a chain of at least approximately 30 monomer units is required to inhibit nonspecific adsorption from concentrated protein solutions. The XPS methodology was also used to probe specific binding of avidins and enzyme conjugates thereof to biotinylated and mixed biotin/PPG-modified polymer interfaces. In one example, using competitive binding, it was established that the mode of binding of a peroxidase-streptavidin conjugate to a biotinylated modified polymer interface was primarily via the streptavidin moiety (as opposed to nonspecific binding via the enzyme conjugate). XPS evaluation of nonspecific and specific peroxidase-streptavidin immobilization on various functionalized polymers has guided the design and fabrication of functionalized interdigitated electrodes in a biosensing muTAS device. Subsequent characterization of this device using scanning electrochemical microscopy (SECM) corroborated the adsorption and binding previously inferred from XPS measurements on macroscale electrodes.

In this paper, the effects of irradiation of Ge-doped FHD silica with 2 MeV Ar+ ions were optically characterized using an m-line technique based on grating couplers. An increase of refractive index as high as 1.2 × 10−2 was obtained, larger than the values normally reported for UV or electron-beam irradiation of the same material (typically of ≈10−3). Thermal annealing has been carried out and an activation energy of 0.36 eV for the recovery of the refractive index has been estimated, suggesting that a bond rearrangement mechanism could be responsible for the change in refractive index.

We describe the development of an integrated lensed Arrayed Waveguide Grating (AWG) microspectrometer for localized multiplexing fluorescence measurements. The device, which has a footprint that is only 1 mm wide and 1 cm long, is capable of spectroscopic measurements on chip. Multiple fluorescence signals were measured simultaneously based upon simple intensity readouts from a CCD camera. We also demonstrate the integration of the AWG spectrometer with a microfluidic platform using a lensing function to confine the beam shape for focused illumination. This capability enhances signal collection, gives better spatial resolution, and provides a route for the analysis of small volume samples (e.g. cells) in flow. To show these capabilities we developed a novel "bead-AWG" platform with which we demonstrate localized multiplexed fluorescence detection either simultaneously or successively. Such an integrated system provides the basis for a portable system capable of optical detection of multi-wavelength fluorescence from a single defined location.

A new method for imaging membrane viscosity is presented using molecular rotors and a combined TIRF-FLIM approach. Lifetimes of artificial membranes of pure DOPC and DPPC were measured and DOPC over a range of temperatures.

We present a straightforward method for measuring the relative viscosity of fluids via a simple graphical analysis of the normalised position autocorrelation function of an optically trapped bead, without the need of embarking on laborious calculations. The advantages of the proposed microrheology method are evident when it is adopted for measurements of materials whose availability is limited, such as those involved in biological studies. The method has been validated by direct comparison with conventional bulk rheology methods and has been applied both to characterise synthetic linear polyelectrolytes solutions and to study biomedical samples.

The fabrication of microelectrodes integrated within ultra-low-volume microtiter chambers for the amperometric determination of metabolites continues to be of interest in the subject of single-cell and high-throughput screening. The microsystem described in this paper consists of a two-microelectrode sensor with a microfluidic dispensation technology, which is able to deliver both very low titers (6.5 pL) and single heart cells into a low-volume microphotoelectrochemical cell. Devices were fabricated using photolithography and liftoff giving reproducible sensors integrated within high aspect ratio titer chambers (with a volume of 360 pL), made of the photoepoxy SU8. In this paper, the determination of lactate was optimized using an enzyme-linked assay based upon lactate oxidase, involving the amperometric determination of hydrogen peroxide at +640 mV versus an internal Ag|AgCl pseudoreference. The microsystem (including the microfluidic dispensers and structures as well as the microsensor) was subsequentl...

Dedifferentiation of chondrocytes during in vitro expansion remains an unsolved challenge for repairing serious articular cartilage defects. In this study, a novel culture system was developed to modulate single cell geometry in 3D and investigate its effects on the chondrocyte phenotype. The approach uses 2D micropatterns followed by in situ hydrogel formation to constrain single cell shape and spreading. This enables independent control of cell geometry and extracellular matrix. Using collagen I matrix, we demonstrated the formation of a biomimetic collagenous “basket” enveloping individual chondrocytes cells. By quantitatively monitoring the production by single cells of chondrogenic matrix (e.g. collagen II and aggrecan) during 21-day cultures, we found that if the cell’s volume decreases, then so does its cell resistance to dedifferentiation (even if the cells remain spherical). Conversely, if the volume of spherical cells remains constant (after an initial decrease), then not only do the cells retain their differentiated status, but previously de-differentiated redifferentiate and regain a chondrocyte phenotype. The approach described here can be readily applied to pluripotent cells, offering a versatile platform in the search for niches toward either self-renewal or targeted differentiation.

Cell proliferation and migration are fundamental processes in determining cell and tissue behaviour. In this study we show the design and fabrication of a new single cell microfluidic structure, called a “vertically integrated array” or “VIA” trap to explore quantitative functional assays including single cell attachment, proliferation and migration studies. The chip can be used in a continuous (flow-through) manner, with a continuous supply of new media, as well as in a quiescent mode. We show the fabrication of the device, together with the flow characteristics inside the network of channels and the single cell traps. The flow patterns inside the device not only facilitate cell trapping, but also protect the cells from mechanical flow-induced stress. MDA-MB-231 human breast cancer cells were used to study attachment and detachment during the cell cycle as well as explore the influences of the chemokine SDF-1 (enabling the quantification of the role of chemokine gradients both on pseudopod formation and directional cell migration).

Bacteria persistence is a well-known phenomenon, where a small fraction of cells in an isogenic population are able to survive high doses of antibiotic treatment. Since the persistence is often associated with single cell behaviour, the ability to study the dynamic response of individual cells to antibiotics is critical. In this work, we developed a gradient microfluidic system that enables long-term tracking of single cell morphology under a wide range of inhibitor concentrations. From time-lapse images, we calculated bacterial growth rates based on the variations in cell mass and in cell number. Using E. coli and Comamonas denitrificans to amoxicillin inhibition as model systems, we found the IC50 determined via both methods are in a good agreement. Importantly, the growth rates together with morphological dynamics of individual cells has led to the discovery of a new form of persistence to amoxicillin. Normal cells that are sensitive to amoxicillin gain persistence or recover from the killing process, if they have had an opportunity to utilise the cytoplasm released from lysed cells close-by. We term this acquired persistence in normal growing cells “opportunistic persistence”. This finding might shed new insights into biofilm resistance and the effect of antibiotics on environmental microbes.

During the electrochemical reduction and oxidation of polybithiophene/polyxylylviologen (PBT/PXV) bilayers, background electrolyte ions move into and out of the film in order to maintain overall charge neutrality within the film. Since the electrolyte contains two mobile ions of opposite charge (tetraethylammonium and perchlorate), in general, charge neutrality can be obtained by either the influx of one, or the efflux of the other. The comparison of electrochemical quartz crystal microbalance (EQCM) mass measurements made on fast and slow timescales, together with variation in the inner PBT and outer PXV film thicknesses, has been used to evaluate which ions transfer from the solution to the film at different stages of reduction and oxidation. These measurements show that the predominant ion motion is ClO4− in response to both reduction and reoxidation of the PXV layer. However, at both the onset of PXV2+ reduction and the onset of PXV0 reoxidation, a small number of TEA+ ions move. Furthermore, the different film transport rates of ClO4− and TEA+ mean that a rapid change in electrode potential coupled with a thick inner PBT layer can lead to the transient accumulation of electrolyte within the film. These observations lead to a mechanism accounting for charge and mass trapping and for rectification effects, and show that even minor inner layer redox conversion to a doped state can open up efficient charge transport pathways from the underlying electrode to the outer polymer layer.

Vapour sensors were produced by electrodeposition of poly(pyrrole) using different counter-ions onto both quartz crystal microbalance (QCM) electrodes and separate interdigitated microelectrodes. The ratio of the relative change of resistance to that of frequency on exposure to a vapour was measured and was found to be independent of vapour concentration over a wide range, but characteristic of the vapour for a given polymer. The multi-property measurement results have shown the feasibility of distinguishing odourants and mixtures using a significantly lower number of different types of sensing material.

In this paper we describe the use of a quartz crystal microbalance (QCM) to follow the progress of a reaction between solution species and specific electrodeposited polymers on an electrode surface, as might be appropriate during immobilisation stages in biosensor production. Two classes of modification are illustrated: firstly, the (reversible) chelation of Ni 2+ by carboxylic acid groups pendant to a poly(pyrrole) chain, which may subsequently be used to bind a second molecule; and secondly, the (irreversible) substitution reaction at a polymer bound activated ester by a bifunctional solution phase species bearing both nucleophilic amine and metal ligating thiol groups.

Research into low volume electroanalytical sensors is becoming increasingly relevant, as scientists and engineers seek to explore the analytical benefits of miniaturization. This is particularly noticeable in the fields of biotechnology and drug discovery, where in the future, decrease in size holds a number of potential benefits, including for example, increased throughput. In this article, two methods for the fabrication of such microstructures are described, both involving the integration of metal microelectrodes on glass substrates, using different polymeric systems to fabricate (or “micromachine”) the surrounding titre chamber. In this context, photopolymerizable polyimide and SU-8 were used to form chambers with volumes as small as 190 pL (with dimensions in the range 100–160 µm and depths between 20 and 25 µm). Such structures were characterized using model redox compounds including ferrocene monocarboxylic acid and hydrogen peroxide, and finally were applied to the quantitative detection of glucose, using glucose oxidase.

The effects of redox state (Os[II] vs. Os[III]) and counter ion (perchlorate vs. p -toluenesulphonate) on the polymer and solvent density distributions in films of electroactive [Os(bipy) 2 (PVP) 10 Cl] + were determined using neutron reflectivity. The changes in polymer structure upon immersion in solvent and upon redox cycling the film in these electrolytes were also investigated. The as-prepared dry polymer film swelled appreciably upon immersion in aqueous solutions. Using p -toluenesulphonate as the counter ion caused pronounced changes to film structure upon both redox cycling and holding it in a fixed oxidation state; the perchlorate anion was found to be much less disruptive. Surprisingly, the effect on film structure of changing anion was much more pronounced than the effect of changing film redox state, i.e. charge density.