A methodology to evaluate different histological preparations of soft tissues: Intervertebral disc tissues study.

A tissue preparation method will inevitably alter the tissue content. This study aims to evaluate how different common sample preparation methods will affect the tissue morphology, biomechanical properties, and chemical composition of samples. The study focuses on intervertebral disc (IVD) tissue; however, it can be applied to other soft tissues. Raman spectroscopy synchronized with nanoindentation instrumentation was employed to investigate the compositional changes of IVD, specifically, nucleus pulposus (NP) and annulus fibrosus (AF), together with their biomechanical properties of IVD. These properties were examined through the following histological specimen types: fresh cryosection (control), fixed cryosection, and paraffin-embedded. The IVD tissue could be located using an optical microscope under three different preparation methods. Paraffin-embedded samples showed the most explicit details where the lamellae structure of AF could be identified. In terms of biomechanical properties, there was no significant difference between the fresh and fixed cryosection (p > 0.05). In contrast, the fresh cryosection and paraffin-embedded samples showed a significant difference (p < 0.05). It was also found that the tissue preparations affected the chemical content of the tissues and structure of the tissue, which are expected to contribute to biomechanical properties changes. Fresh cryosection and fixed cryosection samples are more promising to work with for biomechanical assessment in histological tissues. The findings fill essential gaps in the literature by providing valuable insight into the characteristics of IVD at the microscale. This study can also become a reference for a better approach to assessing the mechanical properties and chemical content of soft tissues at the microscale.

Correlation of the degenerative stage of a disc with magnetic resonance imaging, chemical content, and biomechanical properties of the nucleus pulposus.

Intervertebral disc degeneration (IDD) is closely related to changes in the intervertebral disc (IVD) composition and the resulting viscoelastic properties. IDD is a severe condition because it decreases the disc's ability to resist mechanical loads. Our research aims to understand IDD at the cellular level, specifically the changes in the viscoelastic properties of the nucleus pulposus (NP), which are poorly understood. This study employed a system integrating nanoindentation with Raman spectrometry to correlate biomechanics with subtle changes in the biochemical makeup of the NP. The characterization was, in turn, correlated with the degenerative severity of IVD as assessed using magnetic resonance imaging (MRI) of different patients with spinal stenosis, degenerative spondylolisthesis, and degenerative scoliosis. It is shown that there is an increase in the crosslinking ratio in collagen, a reduction in proteoglycan, and a build-up of minerals upon the rise in the severity level of the disc damage in the NP. Assessment of mechanical characteristics reveals that the increasing disc degeneration makes the NP lose its elasticity, becoming more viscous. This shows that the tissue undergoes abnormalities in weight-bearing ability, which contributes to spinal instability. The correlation of the individual discs shows that grades III and IV have similarities in the changes of Amide I and III toward the storage modulus. In contrast, grades IV and V correlate with mineralization toward the storage modulus. Reduction of proteoglycan has the highest impact on the changes of the storage modulus in all grades of IDD. Connecting compositional alterations to IVD micromechanics at various degrees of degeneration expands our understanding of tissue behavior and provides critical insight into clinical diagnostics, treatment, and tissue engineering.

Dynamics and thermodynamics for competitive adsorptive removal of methylene blue and rhodamine B from binary aqueous solution onto durian rind.

Concurrent adsorptive removal of methylene blue (MB) and rhodamine B (RhB) onto durian rind (DR) agricultural waste, from an aqueous binary solution as a model of wastewater containing multiple synthetic dyes, was investigated. The concurrent adsorption of the dyes followed pseudo-second-order kinetics. The adsorption isotherm was well simulated by the Langmuir model, implying a monolayer adsorption to the surface with a homogeneous binding energy. The adsorption process was governed by external mass transfer through two-step intraparticle diffusion of the dyes onto the adsorbent surface. The adsorption efficiency of MB (96.4%) is much higher than that of RhB (56.3%). This is attributed to the higher rate constant for the adsorption of MB (0.348 g mg-1 min-1) as compared to that of RhB (0.151 g mg-1 min-1). The adsorption behavior suggested that the two cationic dyes in the binary solution diffused and adsorbed independently and randomly onto the DR surface. The adsorption capacity of MB and RhB in the binary solution (47.4 mg g-1 and 32.9 mg g-1, respectively) is lower than those of their single solute solutions (93.3 mg g-1 and 62.8 mg g-1, respectively), suggesting a competitive effect in their concurrent adsorption. This was confirmed based on the adsorption characteristics of the binary solution with different molar ratios. The competitive effect was attributed to either non-interactive or repulsive electrostatic interactions between the positively charged dyes in the binary system. The domination of MB is attributed to its smaller molecular size, higher planarity, and faster adsorption kinetics compared with RhB.

Degradation of ciprofloxacin in aqueous solution using ozone microbubbles: spectroscopic, kinetics, and antibacterial analysis.

Ciprofloxacin (CIP) has been listed in the last version of the surface water due to its ability to kill human cells by inhibiting the activity of DNA topoisomerase IV. Thus, CIP, along with other antibiotic pollution has become a serious threat to the environment and public health. Ozonation has been used as an advanced technique that is applied in wastewater treatment to remove CIP, but the primary limitation of this method is the low solubility of ozone in water. This study is the first report of CIP removal in a scale-up of its aqueous solution using a self-developed aerator pump-enhanced ozonation (APO) system, which only employs a propeller and a zigzag arrangement of meshes. This aerator pump decreased the size of ozone bubbles by 90% and increased the effective ozone solubility to 0.47 ppm. The mechanism of degradation of CIP is attributed to an oxidation reaction of the antibiotic with reactive oxygen species, such as hydroxyl, oxygen, and hydroperoxyl radicals, generated on the surface of the ozone microbubbles. It was found that the rate and efficiency of degradation of CIP using the APO system were 3.64 × 10-3/min and 83.5%, respectively, which is higher compared with those of conventional flow ozonation (FO) systems (1.47 × 10-3/min and 60.9%). The higher degradation efficiency of CIP by the APO system was also revealed by its higher electrical energy efficiency (0.146 g/kWh), compared to that of the FO system (0.106 g/kWh). The degradation of CIP was also monitored by the resulting antibacterial activity against Escherichia coli and Staphylococcus aureus.

Antimicrobial activity of silver sulfide quantum dots functionalized with highly conjugated Schiff bases in a one-step synthesis.

In the present paper, low-dimensional Ag2S QDs were fabricated for the first time, with four different dithiocarbazate derivative Schiff bases (SB) as capping agents in a one-pot synthesis. These SB-capped Ag2S QDs were almost spherical with an average size range of 4.0 to 5.6 nm, which is slightly smaller than conventional thioglycolic acid (TGA)-capped Ag2S QDs. We demonstrate that the growth of Gram-positive bacteria (Bacillus subtillus and Staphylococcus aureus), Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa), and a prevalent fungal pathogen (Candida albicans) are inhibited more when the bacterial and fungal cells were nurtured with the synthesized SB-Ag2S QDs, compared with TGA-Ag2S QDs or free unbound Schiff bases. The minimum inhibitory concentration (MIC) results confirmed that even low concentrations of SB-Ag2S QDs were able to inhibit bacterial (MIC 5-75 µg mL-1) and fungal growth (MIC 80-310 µg mL-1), and in some cases they performed better than streptomycin (8-25 µg mL-1). Lethality bioassay results confirmed that SB-Ag2S QDs were not toxic to brine shrimp (Artemia salina). The results show that capping agents are essential in the design of functional Ag2S QDs, and highlight that Schiff bases provide an excellent opportunity to optimize the biological activities of silver based QDs.

Adsorption Behavior and Dynamic Interactions of Anionic Acid Blue 25 on Agricultural Waste.

In this study, adsorption characteristics of a negatively charged dye, Acid Blue 25 (AB25), on pomelo pith (PP) was studied by varying the adsorption parameters, with the aim of evaluating the adsorption mechanism and establishing the role of hydrogen bonding interactions of AB25 on agricultural wastes. The kinetics, intraparticle diffusion, mechanism, and thermodynamics of the AB25 adsorption were systematically evaluated and analyzed by pseudo-first-order and pseudo-second-order kinetic models, the Weber-Morris intraparticle and Boyd mass transfer models, the Langmuir, Freundlich, Dubinin-Radushkevich, and Temkin isotherm models, and the Van't Hoff equation. It was found that AB25 adsorption followed pseudo-second-order kinetics, governed by a two-step pore-volume intraparticle diffusion of external mass transfer of AB25 onto the PP surface. The adsorption process occurred spontaneously. The adsorption mechanism could be explained by the Langmuir isotherm model, and the maximum adsorption capacity was estimated to be 26.9 mg g-1, which is comparable to many reported adsorbents derived from agricultural wastes. Changes in the vibrational spectra of the adsorbent before and after dye adsorption suggested that AB25 molecules are bound to the PP surface via electrostatic and hydrogen bonding interactions. The results demonstrated that the use of pomelo pith, similar to other agricultural wastes, would provide a basis to design a simple energy-saving, sustainable, and cost-effective approach to remove negatively charged synthetic dyes from wastewater.

Highly emissive, solution-processable and dynamic Eu(III)-containing coordination polymers.

A new class of soluble Eu(III) coordination polymers based on a tridentate ditopic pybox ligand has been developed displaying high metal emission quantum yields of up to 73% as well as a unique dynamic behaviour in solution.

Graphene liquid marbles as photothermal miniature reactors for reaction kinetics modulation.

We demonstrate the fabrication of graphene liquid marbles as photothermal miniature reactors with precise temperature control for reaction kinetics modulation. Graphene liquid marbles show rapid and highly reproducible photothermal behavior while maintaining their excellent mechanical robustness. By tuning the applied laser power, swift regulation of graphene liquid marble's surface temperature between 21-135 °C and its encapsulated water temperature between 21-74 °C are demonstrated. The temperature regulation modulates the reaction kinetics in our graphene liquid marble, achieving a 12-fold superior reaction rate constant for methylene blue degradation than at room temperature.

Transient absorption spectroscopy on spiropyran monolayers using nanosecond pump-probe Brewster angle reflectometry.

Self-assembled monolayers of 11-(3',3'-dimethyl-6,8-dinitrospiro[chromene-2,2'-indoline]-1'-yl) undecanoic acid (amphiphilic spiropyran) at the air-water interface are studied using Brewster angle reflectometry. Transient kinetics of the spiropyran to merocyanine conversion are recorded in a UV-pump, VIS-probe configuration. By varying the probe wavelength using an optical parametric oscillator, we are able to reconstruct absorption spectra of intermediate states with a time-resolution of 10 nanoseconds, limited by the temporal convolution of the two laser pulses. After UV irradiation, spiropyran converts to merocyanine in two stages. The first occurs within a timescale of several tens of nanoseconds and is heavily convoluted with the system response time, whereas the second stage occurs over a few hundred nanoseconds. During the rise time there is a small red shift in the transient absorption spectrum of ~20 nm. We assign the red shift and the slower kinetics to the isomerization of a merocyanine isomer cis about the central methine bond to those that are trans about the same bond.

Highly wrinkled cross-linked graphene oxide membranes for biological and charge-storage applications.

Inspired by the amphiphilicity of graphene oxide (GO), the surface of water is used as a template for the assembly of a GO film. Methacrylate-functionalized GO sheets can be cross-linked instantaneously at the water-air interface to form a highly wrinkled membrane spreading over an extended area. The multiple covalent linkages amongst the GO sheets enhances the in-plane stiffness of the film compared to noncovalently bonded GO films. The highly convoluted GO membrane can be used in two applications: the promoting of spontaneous stem-cell differentiation towards bone cell lineage without any chemical inducers, and for supercapacitor electrodes. Due to reduced van der Waals restacking, capacitance values up to 211 F g(-1) can be obtained. The scalable and inexpensive nature of this assembly route enables the engineering of membranes for applications in regenerative medicine and energy-storage devices where secondary structures like nanotopography and porosity are important performance enhancers.

Comparative study of random and oriented antibody immobilization as measured by dual polarization interferometry and surface plasmon resonance spectroscopy.

Dual polarization interferometry (DPI) is used for a detailed study of antibody immobilization with and without orientation control, using prostate specific antigen (PSA) and its antibody as model. Thiol modified DPI chips were activated by a heterobifunctional cross-linker (sulfo-GMBS). PSA antibody was either directly immobilized via covalent binding or coupled via the Fc-fragment to protein G covalently attached to the activated chip. The direct covalent binding leads to a random antibody orientation and the coupling through protein G leads to an end-on orientation. Ethanolamine (ETH) was used to block remaining active sites following the direct antibody immobilization and protein G immobilization. A homobifunctional cross-linker (BS3) was used to stabilize the antibody layer coupled on protein G. DPI provides a real-time measurement of the stepwise molecular binding processes and gives detailed geometrical and structural values of each layer, i.e., thickness, mass, and density. These values evidence the end-on orientation of closely packed antibody on protein G layer and reveal structural effects of ETH blocking/deactivation and BS3 stabilization. With the end-on immobilized antibody, PSA at 10 pg/mL can be detected by DPI through a sandwich complex that satisfies the clinical requirement (assuming <30 pg/mL as clinically safe). However, the randomly immobilized antibody failed to detect PSA at 1 ng/mL. In a parallel study using surface plasmon resonance (SPR) spectroscopy, random and end-on antibody immobilization on streptavidin-modified gold surface was evaluated to further validate the importance of antibody orientation control. With the closely packed antibody layer on protein G surface, SPR can also detect PSA at 10 pg/mL.

Wide-field single metal nanoparticle spectroscopy for high throughput localized surface plasmon resonance sensing.

Noble metal nanoparticles (mNPs) have a distinct extinction spectrum arising from their ability to support Localized Surface Plasmon Resonance (LSPR). Single-particle biosensing with LSPR is label free and offers a number of advantages, including single molecular sensitivity, multiplex detection, and in vivo quantification of chemical species etc. In this article, we introduce Single-particle LSPR Imaging (SLI), a wide-field spectral imaging method for high throughput LSPR biosensing. The SLI utilizes a transmission grating to generate the diffraction spectra from multiple mNPs, which are captured using a Charge Coupled Device (CCD). With the SLI, we are able to simultaneously image and track the spectral changes of up to 50 mNPs in a single (∼1 s) exposure and yet still retain a reasonable spectral resolution for biosensing. Using the SLI, we could observe spectral shift under different local refractive index environments and demonstrate biosensing using biotin-streptavidin as a model system. To the best of our knowledge, this is the first time a transmission grating based spectral imaging approach has been used for mNPs LSPR sensing. The higher throughput LSPR sensing, offered by SLI, opens up a new possibility of performing label-free, single-molecule experiments in a high-throughput manner.

Defining a threshold surface density of vitronectin for the stable expansion of human embryonic stem cells.

Current methodology for pluripotent human embryonic stem cells (hESCs) expansion relies on murine sarcoma basement membrane substrates (Matrigel™), which precludes the use of these cells in regenerative medicine. To realize the clinical efficacy of hESCs and their derivatives, expansion of these cells in a defined system that is free of animal components is required. This study reports the successful propagation of hESCs (HES-3 and H1) for > 20 passages on tissue culture-treated polystyrene plates, coated from 5 µg/mL of human plasma-purified vitronectin (VN) solution. Cells maintain expression of pluripotent markers Tra1-60 and OCT-4 and are karyotypically normal after 20 passages of continuous culture. In vitro and in vivo differentiation of hESC by embryoid body formation and teratoma yielded cells from the ecto-, endo-, and mesoderm lineages. VN immobilized on tissue culture polystyrene was characterized using a combination of X-ray photoemission spectroscopy, atomic force microscopy, and quantification of the VN surface density with a Bradford protein assay. Ponceau S staining was used to measure VN adsorption and desorption kinetics. Tuning the VN surface density, via the concentration of depositing solution, revealed a threshold surface density of 250 ng/cm², which is required for hESCs attachment, proliferation, and differentiation. Cell attachment and proliferation assays on VN surface densities above this threshold show the substrate properties to be equally viable.

Delayed onset of photochromism in molybdenum oxide films caused by photoinduced defect formation.

We report the photochromic properties of amorphous MoO3 films deposited by dc sputtering with different O2 flow rates. The kinetics of film coloration under UV light irradiation is determined using optical transmission spectroscopy. Changes in the absorbance and refractive index were derived from the analysis of transmittance spectra. The absorbance spectra exhibited a growing broad peak centered around 830 nm, which was induced by the UV irradiation. In the early stages of irradiation, the absorbance of the films did not change but their refractive indices did change. This induction time was correlated with the O2 partial pressure during the film deposition, which was controlled by the O2 flow rate. The origins of this observation are discussed.

Cooperative photoinduced two-dimensional condensation in Langmuir films observed using nanosecond pump-probe Brewster angle microscopy.

Two-dimensional condensation was initiated in a self-assembled mixed monolayer of spiropyran and octadecanol by a nanosecond laser pulse. The dynamics of the process were monitored using nanosecond pump-probe Brewster angle microscopy. Domain growth followed a power law with a growth exponent of 0.47 at a velocity approaching 20 ms(-1). This represents a limit for the rate of longitudinal signaling of pressure waves through a self-assembled amphiphilic layer at an interface and adds to our understanding of signal transmission rates in biomimetic membranes where morphological change in one region can be signaled to a more remote region.

Transient Brewster angle reflectometry of spiropyran monolayers.

Brewster angle reflectometry has been developed as a tool for determining the absorbance and refractive index changes in molecular monolayers containing spiropyran. The method is sensitive to changes in both the real and imaginary parts of the refractive index in the monolayers. It was used to monitor the conversion of spiropyran to merocyanine and the reversal of this reaction when the molecules were immobilised on quartz using silane coupling. An analytical solution of Fresnel formula allowed the transient reflectometry data to be converted into transient absorption information. Absorbances of transients as low as approximately 10(-6) were possible using the current apparatus with a single laser pulse transient measurement. It was found that spiropyran photoconverted to merocyanine with an efficiency of approximately 0.1. The photochemical reversion of converted merocyanine to spiropyran occurred with efficiencies of 0.03-0.2 and this was probably site dependent. It was found that the thermal conversion from merocyanine to spiropyran was slow and even after 10 min there was no significant thermal reversion. This measurement was possible because the real part of the refractive index of the monolayer could be monitored with time using an off-resonance probe at a wavelength where the merocyanine did not absorb light meaning that the probe did not photobleach the sample. Thus our method also provides a non-intrusive method for probing changes in molecules in thin films.

Practical synthesis of maleimides and coumarin-linked probes for protein and antibody labelling via reduction of native disulfides.

The cellular tracking, detection and sensing of protein or antibody movement are important aspects to advance our understanding of biomolecular interactions and activity. Antibodies modified with fluorescent dyes are also valuable tools, especially in immunology research. We describe here a proof-of-principle study of a new water-soluble coumarin probe with a maleimide thiol-reacting unit to fluorescently tag biomolecules. Highlights include: (1) a convenient water-based preparation of N-substituted maleimides, (2) a one-pot preparation of activated maleimido-esters, and (3) a bio-conjugation protocol for the selenol-promoted reduction of native disulfide bonds and the 'site-specific' labelling of antibodies with no significant loss of activity.

Time-resolved brewster angle microscopy for photochemical and photothermal studies on thin-films and monolayers.

Transient events in thin films and interfaces have been studied using the technique of time resolved pump-probe nanosecond Brewster angle microscopy. For p-polarized light there is a minimum reflectivity at the Brewster angle. When the interface is viewed with light that is both incident and reflected at the Brewster angle the resulting image is dark. Subsequent small changes is refractive index will then cause an increase in the reflectivity in affected regions providing high contrast images of an altered interface with a dark background level. This is the basis of Brewster angle microscopy. In the present work two synchronized nanosecond pulsed lasers were used in the pump-probe configuration in order to induce changes at an air-liquid interface and to monitor the resulting morphology changes over a range of time delays from nanosecond to milliseconds after laser-excitation. This method can be used to observe morphological changes in phase altering thin-films and molecular monolayers. Further it can be used to obtain information about transient photochemistry even in optically thin materials and nano-films. In the current work the method is used to monitor laser induced processes in phase separating binary liquid mixtures as well as in monolayers of photo-responsive amphiphilic molecules derived from spiropyran on water. The two systems are quite different but provide valuable comparisons.

Water expansion dynamics after pulsed IR laser heating.

A nanosecond pulsed IR (1.9 microm) laser rapidly heated water, in an open vessel, to temperatures well below the boiling point. The subsequent dynamics of volume expansion were monitored using time-resolved interferometry in order to measure the increase in the water level in the heated area. The water expanded at less than the speed of sound, taking just less than 100 ns to increase its height by approximately 500 nm at surface temperature jumps of 20 K. The initial expansion was followed by an apparent contraction and then a re-expansion. The first expansion phase occurred more slowly than the timescale for bulk H-bond re-structuring of the water, as determined from vibrational bands in the Raman spectra, and represents the limit to the rate at which the overpressure caused by sudden heating can be released. The second phase of the expansion was caused by hydrodynamic effects and is accompanied by morphological changes resulting in light scattering as well as droplet spallation.

Experimentally determined growth exponents during the late stage of spinodal demixing in binary liquid mixtures.

Spinodal demixing was initiated in two systems, with critical and off-critical compositions, using nanosecond pulsed laser-induced temperature jumps (T-jumps) of various magnitude. In this way, deep quenches could be imposed on the systems. One system was the simple triethylamine (TEA)/water mixture and the other was the ionic mixture of 2-butoxyethanol (2BE)/water/KCl. The demixing process was followed using the technique of nanosecond time-resolved microscopic shadowgraphy. The growth of the evolving phase-separated domains followed a simple power law with respect to time in every case. For a given composition, the magnitude of the T-jump had little effect on the growth exponent, however the composition was found to influence the rate of domain growth. At off-critical mole fractions of 0.2 with respect to TEA, the domains grew according to the following expression: L(t)=t(0.70) (where L(t)= the domain size) whereas at the critical TEA mole fraction of 0.08 the domains grew as L(t)=t(0.52). 2BE/water/KCl mixtures quenched at the just off-critical composition of fraction with respect to 2BE evolved as L(t)=t(0.63). These results will be compared to theoretical models and simulations and discussed in terms of estimated Reynolds numbers as well as the consumption and conversion of the available surface energy that fuels the demixing process.