Biological Microenvironments Research Articles

Development and characterization of a voltammetric carbon-fiber microelectrode pH sensor.

This work describes the development and characterization of a modified carbon-fiber microelectrode sensor capable of measuring real-time physiological pH changes in biological microenvironments. The reagentless sensor was fabricated under ambient conditions from voltammetric reduction of the diazonium salt Fast Blue RR onto a carbon-fiber surface in aprotic media. Fast-scan cyclic voltammetry was used to probe redox activity of the p-quinone moiety of the surface-bound molecule as a function of pH. In vitro calibration of the sensor in solutions ranging from pH 6.5 to 8.0 resulted in a pH-dependent anodic peak potential response. Flow-injection analysis was used to characterize the modified microelectrode, revealing sensitivity to acidic and basic changes discernible to 0.005 pH units. Furthermore, the modified electrode was used to measure dynamic in vivo pH changes evoked during neurotransmitter release in the central nervous system of the microanalytical model organism Drosophila melanogaster.

Surface Engineering and Patterning Using Parylene for Biological Applications

Parylene is a family of chemically vapour deposited polymer with material properties that are attractive for biomedicine and nanobiotechnology. Chemically inert parylene “peel-off” stencils have been demonstrated for micropatterning biomolecular arrays with high uniformity, precise spatial control down to nanoscale resolution. Such micropatterned surfaces are beneficial in engineering biosensors and biological microenvironments. A variety of substituted precursors enables direct coating of functionalised parylenes onto biomedical implants and microfluidics, providing a convenient method for designing biocompatible and bioactive surfaces. This article will review the emerging role and applications of parylene as a biomaterial for surface chemical modification and provide a future outlook.

Interplay of CCR2 signaling and local shear force determines vein graft neointimal hyperplasia in vivo

Leukocytes play a central role in vein graft neointimal hyperplasia, which is significantly augmented under low shear conditions. The current concept is that shear force regulates leukocyte adhesion predominately through up-regulation of chemokines and growth factors within the graft wall. Using rabbit and murine vein graft models, we demonstrate that CC chemokine receptor 2/monocyte chemoattractant protein-1 mediated monocyte recruitment and a low shear environment act synergistically to augment neointimal hyperplasia development and removal of either of the conditions leads to a significant reduction in neointimal thickening. We propose a novel concept that the shear stress response element phenotypically stems from the complex interplay of the biological and physical microenvironments.

Two-photon absorbing nanocrystal sensors for ratiometric detection of oxygen.

Two nanocrystal-osmium(II) polypyridyl (NC-Os(II)PP) conjugates have been designed to detect oxygen in biological environments. Polypyridines appended with a single free amine were linked with facility to a carboxylic acid functionality of a semiconductor NC overlayer to afford a biologically stable amide bond. The Os(II)PP complexes possess broad absorptions that extend into the red spectral region; this absorption feature makes them desirable acceptors of energy from NC donors. Fluorescence resonance energy transfer (FRET) from the NC to the Os(II)PP causes an enhanced Os(II)PP emission with a concomitant quenching of the NC emission. Owing to the large two-photon absorption cross-section of the NCs, FRET from the NC to the Os(II)PP can be established under two-photon excitation conditions. In this way, two-photon processes of metal polypyridyl complexes can be exploited for sensing. The emission of the NC is insensitive to oxygen, even at 1 atm, whereas excited states of both osmium complexes are quenched in the presence of oxygen. The NC emission may thus be used as an internal reference to correct for fluctuations in the photoluminescence intensity signal. These properties taken together establish NC-Os(II)PP conjugates as competent ratiometric, two-photon oxygen sensors for application in biological microenvironments.

Spaceflight-relevant types of ionizing radiation and cortical bone: Potential LET effect?

Extended exposure to microgravity conditions results in significant bone loss. Coupled with radiation exposure, this phenomenon may place astronauts at a greater risk for mission-critical fractures. In a previous study, we identified a profound and prolonged loss of trabecular bone (29–39%) in mice following exposure to an acute, 2 Gy dose of radiation simulating both solar and cosmic sources. However, because skeletal strength depends on trabecular and cortical bone, accurate assessment of strength requires analysis of both bone compartments. The objective of the present study was to examine various properties of cortical bone in mice following exposure to multiple types of spaceflight-relevant radiation. Nine-week old, female C57BL/6 mice were sacrificed 110 days after exposure to a single, whole body, 2 Gy dose of gamma, proton, carbon, or iron radiation. Femora were evaluated with biomechanical testing, microcomputed tomography, quantitative histomorphometry, percent mineral content, and micro-hardness analysis. Compared to non-irradiated controls, there were significant differences compared to carbon or iron radiation for only fracture force, medullary area and mineral content. A greater differential effect based on linear energy transfer (LET) level may be present: high-LET (carbon or iron) particle irradiation was associated with a decline in structural properties (maximum force, fracture force, medullary area, and cortical porosity) and mineral composition compared to low-LET radiation (gamma and proton). Bone loss following irradiation appears to be largely specific to trabecular bone and may indicate unique biological microenvironments and microdosimetry conditions. However, the limited time points examined and non-haversian skeletal structure of the mice employed highlight the need for further investigation.

The FARSIGHT Project: Associative 4D/5D Image Analysis Methods for Quantifying Complex and Dynamic Biological Microenvironments

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008

Cancer in Bone

Bone is one of the most preferential metastatic target sites for cancers such as breast, prostate and lung cancers. Although the precise molecular mechanism underlying this preference needs to be elucidated, bone appears to possess unique biological microenvironments that allow circulating cancer cells to home, proliferate and survive. As a consequence of cancer expansion, cellular and molecular homeostasis of bone microenvironments is disturbed and bone is destroyed, leading to the development of bone metastases. Thus, understanding of the crosstalk between cancer cells and bone is critical to design mechanism-based effective and specific therapeutic interventions for bone metastases.

Vapor‐based polymer coatings for potential biomedical applications

AbstractOver the last decade, biology and biotechnology have witnessed an extraordinary development spanning genomics, proteomics, and metabolics. This progress was so rapid and definite that it not only changed the face of modern biology, but indeed altered the way day‐to‐day business is done in biology and related fields. This scientific advancement came with a need for concurrent technological advances. In this context, the ability to interface sophisticated devices with relevant biological microenvironments has emerged as a critical challenge. Already, novel biomaterials are on the horizon that promise to fulfill the rigid criteria of being both biocompatible under the conditions of a versatile range of biological applications and compatible with the increasing demands for miniaturization, integration, and throughput of future device architectures. As currently employed solvent‐based polymer coatings are increasingly reaching their limits, a range of unconventional materials, such as vapor‐based polymer coatings, are discussed as attractive alternatives. One of the main features of vapor‐based polyreactions is their versatility in synthesizing both simple and complex polymers with relative ease and at generally low temperatures. The advantages of the chemical vapor deposition (CVD) technique also include control of the composition and architecture of the resulting materials, high accuracy, solvent‐free environments, excellent adhesion, and the ability to accommodate custom‐tailored surface modifications. For further illustration, selected examples of polymer‐based surface engineering approaches using vapor‐based polyreactions are discussed in this review. For instance, reactive coating technology uses CVD polymerization to deposit a wide range of chemically functionalized polymer coatings on various substrate materials. Its simplicity in providing chemically reactive groups and its applicability to three‐dimensional geometries (e.g. for microfluidics) enables exact tailoring of surface properties and the preparation of biologically relevant microenvironments. CVD‐based reactive coatings are compatible with soft lithographic processes allowing for patterning of proteins, DNA, cytokines, and mammalian cells. Copyright © 2006 Society of Chemical Industry

Metal-Capped Brownian and Magnetically Modulated Optical Nanoprobes (MOONs): Micromechanics in Chemical and Biological Microenvironments

Modulated optical nanoprobes (MOONs) are microscopic (spherical and aspherical) fluorescent particles designed to emit varying intensities of light in a manner that depends on particle orientation. MOONs can be prepared over a broad size range, allowing them to be tailored to applications including intracellular sensors, using submicrometer MOONs, and immunoassays, using 1−10 μm MOONs. When particle orientation is controlled remotely, using magnetic fields (MagMOONs), it allows modulation of fluorescence intensity in a selected temporal pattern. In the absence of external fields, or material that responds to external fields, the particles tumble erratically due to Brownian thermal forces (Brownian MOONs). These erratic changes in orientation cause the MOONs to blink. The temporal pattern of blinking reveals information about the local rheological environment and any forces and torques acting on the MOONs, including biomechanical forces as observed in macrophages. The rotational diffusion rate of Brownian MOONs is inversely proportional to the particle volume and hydrodynamic shape factor, for constant temperature and viscosity. Changes in the particle volume and shape due to binding, deformation, or aggregation can be studied using the temporal time pattern from the probes. The small size and the large number of MOONs that can be viewed simultaneously provide local measurements of physical properties, in both homogeneous and inhomogeneous media, as well as global statistical ensemble properties.

Electrophoresis in nanometer inner diameter capillaries with electrochemical detection.

Separations have been achieved in 770- and 430-nm-inner diameter capillaries. The extremely low sample volumes involved in the study of biological microenvironments such as single cells has led to the desire to develop separation techniques in these ultrasmall capillaries. Total sample volumes as low as 12 fL have been injected using these nanometer inner diameter capillaries. Separations of several catecholamines have been accomplished in these submicrometer capillaries using both capillary zone electrophoresis and micellar electrokinetic chromatography with end-column amperometric detection.

Oxygen microsensor and its application to single cells and mouse pancreatic islets.

An oxygen microsensor with a < 3-micron tip diameter was developed for monitoring oxygen levels at single cells and mouse pancreatic islets. The sensor was fabricated by electrochemically recessing an etched Pt wire inside a pulled glass micropipet and then coating with cellulose acetate. This fabrication process was found to be simpler than previous oxygen electrode designs of comparable size. The microsensors had a average sensitivity of 0.59 +/- 0.29 pA/mmHg (mean +/- SD, n = 42), signals that were minimally perturbed by convection, and response times of < 1 s. The electrode was used to measure the oxygen gradient around and inside single mouse islets. The measurements demonstrate that oxygen levels within even the largest islets at maximal glucose stimulation are 67 +/- 1.6 mmHg (mean +/- SD, n = 5), indicating that islets have adequate oxygen supplies by diffusion under tissue culture conditions to support insulin secretion. The electrode was also used to record the dynamics of oxygen level at single islets as a function of glucose concentration. As glucose level was changed from 3 to 10 mM, oxygen level decreased by 15.8 +/- 2.3 mmHg (mean +/- SEM, n = 6) and oscillations with a period of 3.3 +/- 0.6 min (mean +/- SEM, n = 6) appeared in the oxygen level. In islets bathed in quiescent solutions containing 10 mM glucose, similar oscillations could be observed. In addition, in the quiet solutions it was possible to detect faster oscillations with a period of 12.1 +/- 1.7 s (mean +/- SEM, n = 6) superimposed on the slower oscillations. Oxygen consumption could also be observed at single insulinoma cells using the electrode. Individual cells also showed oscillations in oxygen consumption with a period of a few seconds. The results demonstrate that the electrode can be used for dynamic oxygen level recordings in biological microenvironments.

Fine litter chemistry, early-stage decay, and nitrogen dynamics under plantations and primary forest in Lowland Amazonia

One year field exposures of leaf litter from replicated plots of Pinus caribaea var. hondurensis Barrett and Golfari, Carapa guianensis Aubl., Euxylophora paraensis Hub., a Leguminosae combination ( Dalbergia nigra Fr. All., Dinizia excelsa Ducke, Parkia multijuga Benth.), and adjacent upland ( terra firme) forest at the Curuá-Una Forest Reserve, Pará, Brazil were used to examine the factors controlling leaf litter decay and N dynamics in a lowland tropical environment. Initial leaf litter N concentrations ranged from 4.4 ( P. caribaea) to 16.3 mg g −1 dry matter (Leguminosae), and initial lignin concentrations from 190.8 (Leguminosae) to 459.3 mg g −1 dry matter (forest). Pinus caribaea leaf litter lost the least mass (28%), and the Leguminosae leaf litter the most (61%), during the year long incubations. Initial and 1-y proximate C fractions, N concentrations and polyphenol concentrations were not related to mass loss. Annual N accumulation or depletion from leaf litter under the plantations and forest was related to C loss ( R 2=0.93, P=0.007) and holocellulose loss ( R 2=0.84, P=0.02). When leaf litter was placed outside its stand of origin, there was a significant location effect on decay rates, indicating that differences in the physical and biological microenvironments under the monospecific plots affected litter decomposition.

Multiple Separations in Microfabricated Channels: From Biological Microenvironments to DNA

A continuous sample introduction and separation scheme is presented as an alternative to the current slab gel and microfabricated chip technologies for biological separations. This new technique involves continuous sample introduction via a conventional small bore capillary coupled to a small dimension rectangular channel. Both free zone and size based separations have been carried out in the rectangular channel. Laser induced fluorescence and electrochemical detection schemes have been employed with this technique. Some of the areas this technology has been used to investigate include monitoring dynamic events from microenvironments, monitoring analytes over long periods of time, and performing DNA separations.

Bridging the gap between general circulation model (GCM) output and biological microenvironments

Bridging the gap between general circulation model (GCM) output and biological microenvironments

Novel approaches towards a detailed control of the mechanism and efficiency of photosensitized processes in vivo

The photoexcitation of an endogenous or exogenous biological chromophore can result in the re-emission of light as fluorescence or phosphorescence; this may be used for photodiagnostic applications or in the conversion of at least part of the absorbed radiation into thermal or chemical energy. The last two processes generate photothermal and photochemical sensitization respectively leading to the irreversible damage of cells and tissues; hence they can be used for phototherapeutic purposes. The efficacy and scope of photodiagnostic and phototherapeutic techniques are improving as we increase our knowledge about the influence of biological microenvironments on the mechanisms of photoprocesses and as new approaches are developed for controlling the biodistribution of photosensitizing agents in vivo and the depth/diffusion of incident light into tissues. In particular, even the localized and sublethal damage of cells can often be modulated to generate intracellular signalling in order to either induce a programmed cell death (apoptosis) or an acceleration of cell metabolism (photostimulation).

A Carbon Dioxide Microelectrode for in Situ ρCO2 Measurement

A microelectrode is described which permits a nondestructive method for measuring ρCO2 in living cells or small samples. Our objective was to develop an easily fabricated ρCO2 microelectrode that would permit analysis of small samples and withstand insertion into the soil. We developed a ρCO2 microelectrode by making use of a newly developed micro-pH electrode (MEPH4, WPI) and a semiliquid chlorotrifluoroethylene oil-wax gas permeable membrane. The fabricated microelectrode has a tip diameter of ≤120 μm (o.d.) and a response time of ≤3 min with a linear (nearly Nernstian) slope (58.0 ± 2 mV/log10 ρCO2). The ρCO2 microelectrode response is independent of the test solution pH and electrolytes. Measurements of ρCO2 with the microelectrode compared favorably with those from a standard ρCO2 macroelectrode (model 95-02, Orion). and the regression line had a slope of 1.2 and r = 0.98. This microelectrode is simple to fabricate, and the tip diameter is sufficiently small (± 120 μm) to permit measurement of CO2 in small sample volumes. The first direct potentiometric determinations of rhizosphere (soil-root interface) ρCO2 were carried out to demonstrate the performance of the microelectrode. Rhizoplane (<1 mm from the root) ρCO2 values at the five leaf stage were higher for Sordan-757 (9.14 ± 0.39 and 11.33 ± 0.28 kPa) than for Sordan-333 (7.15 ± 0.57 and 9.55 ± 0.30 kPa) on low and high CaCO3 soils, respectively. The ρCO2 microelectrode is an improved technique for examining the root-soil interface and other biological microenvironments.

Glass capillary gas-chromatographic analysis of free amino acids in biological microenvironments using electron capture or selected ion-monitoring detection

Studies on analysis of free animo acids using a support-coated, open-tube capillary column, and electron-capture detection or selective ion monitoring have been performed on samples from biological microenvironments. For most amino acids the detection limit was found to be less than 1 pg. The preparation of the support-coated open-tube capillary column is described as well as the gas-chromatographic conditions for direct injection and temperature-programmed separation of the N-heptafluorobutyryl iso butyl ester derivatives. Electron-capture detection and selected ion monitoring are compared with respect to linearity and sensitivity and the bases for the greater sensitivity of electron-capture detection compared with flame-ionization detection using halogenated derivatives is discussed. Applications of the gas-chromatographic method for analysis of free amino acids in environments deliberately chosen very small are demonstrated.