Self-referencing optrodes for measuring spatially resolved, real-time metabolic oxygen flux in plant systems

The ability to non-invasively measure metabolic oxygen flux is a very important tool for physiologists interested in a variety of questions ranging from basic metabolism, growth/development, and stress adaptation. Technologies for measuring oxygen concentration near the surface of cells/tissues include electrochemical and optical techniques. A wealth of knowledge was gained using these tools for quantifying real-time physiology. Fiber-optic microprobes (optrodes) have recently been developed for measuring oxygen in a variety of biomedical and environmental applications. We have adopted the use of these optical microsensors for plant physiology applications, and used the microsensors in an advanced sensing modality known as self-referencing. Self-referencing is a non-invasive microsensor technique used for measuring real-time flux of analytes. This paper demonstrates the use of optical microsensors for non-invasively measuring rhizosphere oxygen flux associated with respiration in plant roots, as well as boundary layer oxygen flux in phytoplankton mats. Highly sensitive/selective optrodes had little to no hysteresis/calibration drift during experimentation, and an extremely high signal-to-noise ratio. We have used this new tool to compare various aspects of rhizosphere oxygen flux for roots of Glycine max, Zea mays, and Phaseolus vulgaris, and also mapped developmentally relevant profiles and distinct temporal patterns. We also characterized real-time respiratory patterns during inhibition of cytochrome and alternative oxidase pathways via pharmacology. Boundary layer oxygen flux was also measured for a phytoplankton mat during dark:light cycling and exposure to pharamacological inhibitors. This highly sensitive technology enables non-invasive study of oxygen transport in plant systems under physiologically relevant conditions.     

A self referencing platinum nanoparticle decorated enzyme-based microbiosensor for real time measurement of physiological glucose transport

Glucose is the central molecule in many biochemical pathways, and numerous approaches have been developed for fabricating micro biosensors designed to measure glucose concentration in/near cells and/or tissues. An inherent problem for microsensors used in physiological studies is a low signal-to-noise ratio, which is further complicated by concentration drift due to the metabolic activity of cells. A microsensor technique designed to filter extraneous electrical noise and provide direct quantification of active membrane transport is known as self-referencing. Self-referencing involves oscillation of a single microsensor via computer-controlled stepper motors within a stable gradient formed near cells/tissues (i.e., within the concentration boundary layer). The non-invasive technique provides direct measurement of trans-membrane (or trans-tissue) analyte flux. A glucose micro biosensor was fabricated using deposition of nanomaterials (platinum black, multiwalled carbon nanotubes, Nafion) and glucose oxidase on a platinum/iridium microelectrode. The highly sensitive/selective biosensor was used in the self-referencing modality for cell/tissue physiological transport studies. Detailed analysis of signal drift/noise filtering via phase sensitive detection (including a post-measurement analytical technique) are provided. Using this highly sensitive technique, physiological glucose uptake is demonstrated in a wide range of metabolic and pharmacological studies. Use of this technique is demonstrated for cancer cell physiology, bioenergetics, diabetes, and microbial biofilm physiology. This robust and versatile biosensor technique will provide much insight into biological transport in biomedical, environmental, and agricultural research applications.     

Quantifying biofilm structure: facts and fiction

There is no doubt among biofilm researchers that biofilm structure is important to many biofilm processes, such as the transport of nutrients to deeper layers of the biofilm. However, biofilm structure is an elusive term understood only qualitatively, and as such it cannot be directly correlated with any measurable parameters characterizing biofilm performance. To correlate biofilm structure with the parameters characterizing biofilm performance, such as the rate of nutrient transport within the space occupied by the biofilms, biofilm structure must first be quantified and expressed numerically on an appropriate scale. The task of extracting numerical parameters quantifying biofilm structure relies on using biofilm imaging and image analysis. Although defining parameters characterizing biofilm structure is relatively straightforward, and multiple parameters have been described in the computer science literature, interpreting the results of such analyses is not trivial. Existing computer software developed by several research groups, including ours, for the sole purpose of analyzing biofilm images helps quantify parameters from biofilm images but does nothing to help interpret the results of such analyses. Although computing structural parameters from biofilm images permits correlating biofilm structure with other biofilm processes, the meaning of the results is not obvious. The first step to understanding the quantification of biofilm structure, developing image analysis, methods to quantify information from biofilm images, has been made by several research groups. The next step is to explain the meaning of these analyses. This presentation explains the meaning of several parameters commonly used to characterize biofilm structure. It also reviews the authors' research and experience in quantifying biofilm structure and their attempts to quantitatively relate biofilm structure to fundamental biofilm processes.     

Mini-review: convection around biofilms

Water that flows around a biofilm influences the transport of solutes into and out of the biofilm and applies forces to the biofilm that can cause it to deform and detach. Engineering approaches to quantifying and understanding these phenomena are reviewed in the context of biofilm systems. The slow-moving fluid adjacent to the biofilm acts as an insulator for diffusive exchange. External mass transfer resistance is important because it can exacerbate oxygen or nutrient limitation in biofilms, worsen product inhibition, affect quorum sensing, and contribute to the development of tall, fingerlike biofilm clusters. Measurements of fluid motion around biofilms by particle velocimetry and magnetic resonance imaging indicate that water flows around, but not through biofilm cell clusters. Moving fluid applies forces to biofilms resulting in diverse outcomes including viscoelastic deformation, rolling, development of streamers, oscillatory movement, and material failure or detachment. The primary force applied to the biofilm is a shear force in the main direction of fluid flow, but complex hydrodynamics including eddies, vortex streets, turbulent wakes, and turbulent bursts result in additional force components.     

Analysis of Structural and Physiological Profiles To Assess the Effects of Cu on Biofilm Microbial Communities

We investigated the effects of copper on the structure and physiology of freshwater biofilm microbial communities. For this purpose, biofilms that were grown during 4 weeks in a shallow, slightly polluted ditch were exposed, in aquaria in our laboratory, to a range of copper concentrations (0, 1, 3, and 10 μM). Denaturing gradient gel electrophoresis (DGGE) revealed changes in the bacterial community in all aquaria. The extent of change was related to the concentration of copper applied, indicating that copper directly or indirectly caused the effects. Concomitantly with these changes in structure, changes in the metabolic potential of the heterotrophic bacterial community were apparent from changes in substrate use profiles as assessed on Biolog plates. The structure of the phototrophic community also changed during the experiment, as observed by microscopic analysis in combination with DGGE analysis of eukaryotic microorganisms and cyanobacteria. However, the extent of community change, as observed by DGGE, was not significantly greater in the copper treatments than in the control. Yet microscopic analysis showed a development toward a greater proportion of cyanobacteria in the treatments with the highest copper concentrations. Furthermore, copper did affect the physiology of the phototrophic community, as evidenced by the fact that a decrease in photosynthetic capacity was detected in the treatment with the highest copper concentration. Therefore, we conclude that copper affected the physiology of the biofilm and had an effect on the structure of the communities composing this biofilm.     

Effect of biofilm on the rheological properties of cohesive sediment

Biofilm, a product of metabolic activity, has an important effect on the physico-chemical properties of cohesive sediment. However, little effort has been made to determine the substantial effects of biofilm growth on specific sediment properties, for example rheological properties. Understanding the changes associated with biofilm growth and quantifying the time scales over which these changes occur are important for understanding how biofilms mediate sediment properties and processes and the development of sediment transport mechanics. The effect of biofilm on the rheological properties of cohesive sediment was investigated experimentally. The rheological properties of sediment slurries with and without biofilm at different growth phases were measured and compared. Measurement showed biofilm growth has a significant effect on the rheological properties of cohesive sediment. Rheological equations for biofilm sediment and expressions for rheological properties which change over time are proposed. These equations, and information on biofilm sediment, are important for inclusion of biosedimentological processes in models of sediment dynamics.     

Survival strategies of infectious biofilms

Modern medicine is facing the spread of biofilm-related infections. Bacterial biofilms are difficult to detect in routine diagnostics and are inherently tolerant to host defenses and antibiotic therapies. In addition, biofilms facilitate the spread of antibiotic resistance by promoting horizontal gene transfer. We review current concepts of biofilm tolerance with special emphasis on the role of the biofilm matrix and the physiology of biofilm-embedded cells. The heterogeneity in metabolic and reproductive activity within a biofilm correlates with a non-uniform susceptibility of enclosed bacteria. Recent studies have documented similar heterogeneity in planktonic cultures. Nutritional starvation and high cell density, two key characteristics of biofilm physiology, also mediate antimicrobial tolerance in stationary-phase planktonic cultures. Advances in characterizing the role of stress response genes, quorum sensing and phase variation in stationary-phase planktonic cultures have shed new light on tolerance mechanisms within biofilm communities.     

Early development and quorum sensing in bacterial biofilms

 We develop mathematical models to examine the formation, growth and quorum sensing activity of bacterial biofilms. The growth aspects of the model are based on the assumption of a continuum of bacterial cells whose growth generates movement, within the developing biofilm, described by a velocity field. A model proposed in Ward et al. (2001) to describe quorum sensing, a process by which bacteria monitor their own population density by the use of quorum sensing molecules (QSMs), is coupled with the growth model. The resulting system of nonlinear partial differential equations is solved numerically, revealing results which are qualitatively consistent with experimental ones. Analytical solutions derived by assuming uniform initial conditions demonstrate that, for large time, a biofilm grows algebraically with time; criteria for linear growth of the biofilm biomass, consistent with experimental data, are established. The analysis reveals, for a biologically realistic limit, the existence of a bifurcation between non-active and active quorum sensing in the biofilm. The model also predicts that travelling waves of quorum sensing behaviour can occur within a certain time frame; while the travelling wave analysis reveals a range of possible travelling wave speeds, numerical solutions suggest that the minimum wave speed, determined by linearisation, is realised for a wide class of initial conditions. Received: 10 February 2002 / Revised version: 29 October 2002 / Published online: 19 March 2003 Key words or phrases: Bacterial biofilm – Quorum sensing – Mathematical modelling – Numerical solution – Asymptotic analysis – Travelling wave analysis     

Analysis of structural and physiological profiles to assess the effects of Cu on biofilm microbial communities

We investigated the effects of copper on the structure and physiology of freshwater biofilm microbial communities. For this purpose, biofilms that were grown during 4 weeks in a shallow, slightly polluted ditch were exposed, in aquaria in our laboratory, to a range of copper concentrations (0, 1, 3, and 10 microM). Denaturing gradient gel electrophoresis (DGGE) revealed changes in the bacterial community in all aquaria. The extent of change was related to the concentration of copper applied, indicating that copper directly or indirectly caused the effects. Concomitantly with these changes in structure, changes in the metabolic potential of the heterotrophic bacterial community were apparent from changes in substrate use profiles as assessed on Biolog plates. The structure of the phototrophic community also changed during the experiment, as observed by microscopic analysis in combination with DGGE analysis of eukaryotic microorganisms and cyanobacteria. However, the extent of community change, as observed by DGGE, was not significantly greater in the copper treatments than in the control. Yet microscopic analysis showed a development toward a greater proportion of cyanobacteria in the treatments with the highest copper concentrations. Furthermore, copper did affect the physiology of the phototrophic community, as evidenced by the fact that a decrease in photosynthetic capacity was detected in the treatment with the highest copper concentration. Therefore, we conclude that copper affected the physiology of the biofilm and had an effect on the structure of the communities composing this biofilm.     

Inhibition of biofilm formation, quorum sensing and infection in Pseudomonas aeruginosa by natural products-inspired organosulfur compounds

Using a microplate-based screening assay, the effects on Pseudomonas aeruginosa PAO1 biofilm formation of several S-substituted cysteine sulfoxides and their corresponding disulfide derivatives were evaluated. From our library of compounds, S-phenyl-L-cysteine sulfoxide and its breakdown product, diphenyl disulfide, significantly reduced the amount of biofilm formation by P. aeruginosa at levels equivalent to the active concentration of 4-nitropyridine-N-oxide (NPO) (1 mM). Unlike NPO, which is an established inhibitor of bacterial biofilms, our active compounds did not reduce planktonic cell growth and only affected biofilm formation. When used in a Drosophila-based infection model, both S-phenyl-L-cysteine sulfoxide and diphenyl disulfide significantly reduced the P. aeruginosa recovered 18 h post infection (relative to the control), and were non-lethal to the fly hosts. The possibility that the observed biofilm inhibitory effects were related to quorum sensing inhibition (QSI) was investigated using Escherichia coli-based reporters expressing P. aeruginosa lasR or rhIR response proteins, as well as an endogenous P. aeruginosa reporter from the lasI/lasR QS system. Inhibition of quorum sensing by S-phenyl-L-cysteine sulfoxide was observed in all of the reporter systems tested, whereas diphenyl disulfide did not exhibit QSI in either of the E. coli reporters, and showed very limited inhibition in the P. aeruginosa reporter. Since both compounds inhibit biofilm formation but do not show similar QSI activity, it is concluded that they may be functioning by different pathways. The hypothesis that biofilm inhibition by the two active compounds discovered in this work occurs through QSI is discussed.     

Development and experimental evaluation of a steady-state, multispecies biofilm model

A steady-state model for quantifying the space competition in multispecies biofilms is developed. The model includes multiple active species, inert biomass, substrate utilization and diffusion within the biofilm, external mass transport, and detachment phenomena. It predicts the steady-state values of biofilm thickness, species distribution, and substrate fluxes. An experimental evaluation is carried out in completely mixed biofilm reactors in which slow-growing nitrifying bacteria compete with acetate-utilizing heterotrophs. The experimental results show that the model successfully describes the space competition. In particular, increasing acetate concentrations causes NH(4) (+)-N fluxes to decrease, because nitrifiers are forced deeper into the biofilm, where they experience greater mass-transport resistance.     

Measuring metabolism and biophysical flux in the tissue, cellular and sub-cellular domains: recent developments in self-referencing amperometry for physiological sensing

Ultimately, advances in genomics, proteomics and metabolomics will be realized by combining these approaches with biophysical sensors for understanding the functional and structural (physiological) aspects of sub-cellular systems (cytomics). Therefore, the emergence of the new fields of cytomics and physiomics will require new technologies to probe the functional realm of living cells. While amperometric sensors have been used, their sensitivity and reliability are significantly improved through the development of new strategies and data acquisition systems for the operation of the sensors. This includes the application of the principles of the vibrating or self-referencing microsensor to the operation of amperometric sensors. The development of self-referencing amperometry (SRA) is significant because it effectively converts static concentration sensors into dynamic biophysical sensors that directly monitor physiological flux. SRA has been developed for analytes such as O2, NO, H2O2 and ascorbate. These sensors have been validated against non-biological microscopic flux sources that were theoretically modeled, before being applied to biological research. This new sensor technology has been shown, through research in a wide variety of biological and biomedical research projects, to be an important new tool in the arsenal of the cell biologist. SRA technology has been adapted through SRA-H2O2 and SRA-NADH sensors, for electrochemically coupled enzyme based self-referencing biosensors (SRB) for glucose, glutamate and ethanol. These developments in self-referencing sensor technologies offer great promise in extending electroanalytical chemistry and biosensor technologies from the micro to the nanoscale where researchers can study physiology at the sub-cellular and organellar levels.     

Intestinal administration of copper and its transient release into venous rat blood serum concomitantly with metallothionein

The molecular side of copper transport in biological systems is unknown. It was attempted to examine the copper and metallothionein (MT) release into the portal blood in rats in vivo. After direct administration of Cu(II) into the jejunum the copper and MT levels were distinctively higher in the portal venous serum compared with that of the vena cava inferior. MT in gel filtrated serum samples was analyzed immunologically employing ELISA and a monoclonal antibody to rat MT-I. Affinity chromatography on Protein A-Sepharose resulted in a higher immunoreactivity in the portal compartment as deduced from an elevated MT-antibody complex. It is assumed that MT serves as a genuine transport system for cuprous copper during the mucosal-to-serosal flux of this biologically important transition metal.     

Pseudomonas aeruginosa Displays Multiple Phenotypes during Development as a Biofilm

Complementary approaches were employed to characterize transitional episodes in Pseudomonas aeruginosa biofilm development using direct observation and whole-cell protein analysis. Microscopy and in situ reporter gene analysis were used to directly observe changes in biofilm physiology and to act as signposts to standardize protein collection for two-dimensional electrophoretic analysis and protein identification in chemostat and continuous-culture biofilm-grown populations. Using these approaches, we characterized five stages of biofilm development: (i) reversible attachment, (ii) irreversible attachment, (iii) maturation-1, (iv) maturation-2, and (v) dispersion. Biofilm cells were shown to change regulation of motility, alginate production, and quorum sensing during the process of development. The average difference in detectable protein regulation between each of the five stages of development was 35% (approximately 525 proteins). When planktonic cells were compared with maturation-2 stage biofilm cells, more than 800 proteins were shown to have a sixfold or greater change in expression level (over 50% of the proteome). This difference was higher than when planktonic P. aeruginosa were compared with planktonic cultures of Pseudomonas putida. Las quorum sensing was shown to play no role in early biofilm development but was important in later stages. Biofilm cells in the dispersion stage were more similar to planktonic bacteria than to maturation-2 stage bacteria. These results demonstrate that P. aeruginosa displays multiple phenotypes during biofilm development and that knowledge of stage-specific physiology may be important in detecting and controlling biofilm growth.     

A non-invasive method for measuring preimplantation embryo physiology

The physiology of the early embryo may be indicative of embryo vitality and therefore methods for non-invasively monitoring physiological parameters from embryos could improve preimplantation diagnoses. The self-referencing electrophysiological technique is capable of non-invasive measurement of the physiology of individual cells by monitoring the movement of ions and molecules between the cell and the surrounding media. Here we use this technique to monitor gradients of calcium, potassium, oxygen and hydrogen peroxide around individual mouse preimplantation embryos. The calcium-sensitive electrode in self-referencing mode identified a region of elevated calcium concentration (approximately 0.25 pmol) surrounding each embryo. The calcium gradient surrounding embryos was relatively steep, such that the region of elevated calcium extended into the medium only 4 microns from the embryo. By contrast, using an oxygen-sensitive electrode an extensive gradient of reduced dissolved oxygen concentration was measured surrounding the embryo and extended tens of micrometres into the medium. A gradient of neither potassium nor hydrogen peroxide was observed around unperturbed embryos. We also demonstrate that monitoring the physiology of embryos using the self-referencing technique does not compromise their subsequent development. Blastocyts studied with the self-referencing technique implanted and developed to term at the same frequency as did unexamined, control embryos. Therefore, the self-referencing electrode provides a valuable non-invasive technique for studying the physiology and pathophysiology of individual embryos without hindering their subsequent development.     

Fast in vitro translation system immobilized on a surface via specific biotinylation of the ribosome

The ribosome is the macromolecular machine responsible for translating the genetic code into polypeptide chains. Despite impressive structural and kinetic studies of the translation process, a number of challenges remain with respect to understanding the dynamic properties of the translation apparatus. Single-molecule techniques hold the potential of characterizing the structural and mechanical properties of macromolecules during their functional cycles in real time. These techniques often necessitate the specific coupling of biologically active molecules to a surface. Here, we describe a procedure for such coupling of functionally active ribosomes that permits single-molecule studies of protein synthesis. Oxidation with NaIO4 at the 3' end of 23S rRNA and subsequent reaction with a biotin hydrazide produces biotinylated 70S ribosomes, which can be immobilized on a streptavidin-coated surface. The surface-attached ribosomes are fully active in poly(U) translation in vitro, synthesizing poly(Phe) at a rate of 3-6 peptide bonds/s per active ribosome at 37 degrees C. Specificity of binding of biotinylated ribosomes to a streptavidin-coated quartz surface was confirmed by observation of individual fluorescently labeled, biotinylated 70S ribosomes, using total internal reflection fluorescence microscopy. Functional interactions of the immobilized ribosomes with various components of the protein synthesis apparatus are shown by use of surface plasmon resonance.     

Magnetic resonance imaging of structure, diffusivity, and copper immobilization in a phototrophic biofilm

Magnetic resonance imaging (MRI) was used to spatially resolve structure, water diffusion, and copper transport and fate in a phototrophic biofilm [corrected]. MRI was able to resolve considerable structural heterogeneity, ranging from classical laminations approximately 500 mum thick to structures with no apparent ordering. Pulsed-field gradient (PFG) analysis spatially resolved water diffusion coefficients which exhibited relatively little or no attenuation (diffusion coefficients ranged from 1.7 x 10(-9) m(2) s(-1) to 2.2 x 10(-9) m(2) s(-1)). The biofilm was then reacted with a 10-mg liter(-1) Cu(2+) solution, and transverse relaxation time parameter maps [corrected].were used to spatially and temporally map copper immobilization within the biofilm. Significantly, a calibration protocol similar to that used in biomedical research successfully quantified copper concentrations throughout the biofilm. Variations in Cu concentrations were controlled by the biofilm structure. Copper immobilization was most rapid (approximately 5 mg Cu liter(-1) h(-1)) over the first 20 to 30 h and then much slower for the remaining 60 h of the experiment. The transport of metal within the biofilm is controlled by both diffusion and immobilization. This was explored using a Bartlett and Gardner model which examined both diffusion and adsorption through a hypothetical film exhibiting properties similar to those of the phototrophic biofilm. Higher adsorption constants (K) resulted in longer lag times until the onset of immobilization at depth but higher actual adsorption rates. MRI and reaction transport models are versatile tools which can significantly improve our understanding of heavy metal immobilization in naturally occurring biofilms.     

Surface plasmon resonance for real-time evaluation of immobilized fructosyltransferase activity

The extracellular enzyme fructosyltransferase (FTF) is considered to be a significant virulence factor in the dental biofilm. We have developed a method using surface plasmon resonance to detect the activity of immobilized FTF in situ. This real time technique provides a sensitive direct assay for characterizing functional properties of immobilized enzymes such as FTF.     

Enzyme-bound copper of dopamine β-monooxygenase.: Activation of the holoenzyme by added copper and uncoupling of electron transfer from hydroxylation by copper salicylate

Preparations of dopamine β-monooxygenase containing a full complement of copper (4.2 copper atoms per tetramer) show increased ascorbate-supported catalytic activities after addition of an excess of copper ions. The significance ot this observation on the question of the number of copper atoms per active site is discussed.Low molecular weight copper complexes such as copper salicylate cause uncoupling of electron transport from hydroxylation. This uncoupling is probably the reason for the well-known inhibition of this enzyme observed at high copper concentration.The onset of inhibition by the copper chelator bathocuproine disulfonate occurs on a faster time scale than the removal of enzyme-bound copper. Nevertheless, the copper removal is sufficiently rapid to require that it be considered in interpretation of inhibition experiments with chelators.     

H+ flux kinetics around plant roots after short-term exposure to low temperature: identifying critical temperatures for plant chilling tolerance

H⁺ flux kinetics were measured in solution around the roots of chilling-tolerant pea (Pisum sativum) and bean (Vicia faba), chilling-sensitive cucumber (Cucumis sativus) and pumpkin (Cucurbita pepo), and intermediate corn (Zea mays) species using a microelectrode technique to measure net flux. As a root warmed to room temperature alter 90 min at 4°C, at which temperature the H⁺ flux was near zero, the flux rose (influx) and then fell. These changes occurred at two apparent critical temperatures, which were higher for the more chilling-sensitive species. The First, lower, apparent critical temperature may represent the start of passive inward H⁺ transport. The higher critical temperature may represent the start of active H⁺ extrusion. From these apparent critical temperatures we have calculated the real critical temperature and the time delay of the chilling signal transduction process. Passive and active H⁺ transporters appear to have the same real critical temperature of chilling sensitivity, about 9°C, but have, respectively, 4 min and 11 min time delays. Measurement of these apparent critical temperatures may provide quick and reliable screening for chilling sensitivity in plant breeding programmes. Future ion flux studies may show the cellular location of chilling stress perception and the signal transduction pathways.