On-chip manipulation and detection of magnetic particles for functional biosensors

We demonstrate the real-time on-chip detection and manipulation of single 1 microm superparamagnetic particles in solution, with the aim to develop a biosensor that can give information on biological function. Our chip-based sensor consists of micro-fabricated current wires and giant magneto resistance (GMR) sensors. The current wires serve to apply force on the particles as well as to magnetize the particles for on-chip detection. The sensitivity profile of the sensor was reconstructed by simultaneously measuring the sensor signal and the position of an individual particle crossing the sensor. A single-dipole model reproduces the measured sensitivity curve for a 1 microm bead. For a 2.8 microm bead the model shows deviations, which we attribute to the fact that the particle size becomes comparable to the sensor width. In the range between 1 and 10 particles, we observed a linear relationship between the number of beads and the sensor signal. The real-time detection and manipulation of individual particles opens the possibility to perform on-chip high-parallel single-particle assays.     

Rapid biosensor for detection of antibiotic-selective growth of Escherichia coli

A rapid biosensor for the detection of bacterial growth was developed using micromechanical oscillators coated in common nutritive layers. The change in resonance frequency as a function of the increasing mass on a cantilever array forms the basis of the detection scheme. The calculated mass sensitivity according to the mechanical properties of the cantilever sensor is approximately 50 pg/Hz; this mass corresponds to an approximate sensitivity of approximately 100 Escherichia coli cells. The sensor is able to detect active growth of E. coli cells within 1 h. The starting number of E. coli cells initially attached to the sensor cantilever was, on average, approximately 1,000 cells. Furthermore, this method allows the detection of selective growth of E. coli within only 2 h by adding antibiotics to the nutritive layers. The growth of E. coli was confirmed by scanning electron microscopy. This new sensing method for the detection of selective bacterial growth allows future applications in, e.g., rapid antibiotic susceptibility testing.     

Modification of bacterial cell membrane dynamics and morphology upon exposure to sub inhibitory concentrations of ciprofloxacin

Ciprofloxacin (CPX), a second generation fluoroquinolone antibiotic, is used as a primary antibiotic for treatment against gastroenteritis, drug-resistant tuberculosis, and malignant otitis externa. CPX is a broad spectrum antibiotic that targets the DNA gyrase of both Gram-positive and Gram-negative bacteria. Irrational and improper usage of CPX results in emergence of CPX resistant organisms emphasizing the importance of using lethal doses of CPX. Here, we have systematically analysed the effect of CPX at sub lethal concentrations on live E. coli membrane and growth dynamics. As a result of CPX interaction at sub-lethal concentrations, we detected filamentation of the bacterial cells during cell division. Although CPX is a DNA targeting antibiotic and did not result in considerable increase of live E. coli cell surface roughness, we observed significant enhancement in the lipid diffusion coefficients possibly due to disrupted lipid packing or altered lipid composition. Interestingly, we seem to observe slightly higher extent of lipid diffusion alteration when bacterial inner membrane specific label FM4-64 was used in comparison to the non-specific membrane dye. Both these results are contrary to that observed in bacterial cells for colistin, a membrane targeting antibiotics. Our work highlights the need for using multiple, complementary surface and depth sensitive techniques to obtain information on the realistic nature of bacterial cell membrane remodelling due to non-membrane targeting antibiotics. Our work could have implications for identification of potential biomembrane markers at sub-lethal concentrations even for antibiotics which are non-membrane targeting that could help in unravelling pathways for emergence of antimicrobial resistance.     

The BARC biosensor applied to the detection of biological warfare agents

The Bead ARray Counter (BARC) is a multi-analyte biosensor that uses DNA hybridization, magnetic microbeads, and giant magnetoresistive (GMR) sensors to detect and identify biological warfare agents. The current prototype is a table-top instrument consisting of a microfabricated chip (solid substrate) with an array of GMR sensors, a chip carrier board with electronics for lock-in detection, a fluidics cell and cartridge, and an electromagnet. DNA probes are patterned onto the solid substrate chip directly above the GMR sensors, and sample analyte containing complementary DNA hybridizes with the probes on the surface. Labeled, micron-sized magnetic beads are then injected that specifically bind to the sample DNA. A magnetic field is applied, removing any beads that are not specifically bound to the surface. The beads remaining on the surface are detected by the GMR sensors, and the intensity and location of the signal indicate the concentration and identity of pathogens present in the sample. The current BARC chip contains a 64-element sensor array, however, with recent advances in magnetoresistive technology, chips with millions of these GMR sensors will soon be commercially available, allowing simultaneous detection of thousands of analytes. Because each GMR sensor is capable of detecting a single magnetic bead, in theory, the BARC biosensor should be able to detect the presence of a single analyte molecule.     

Streptococcus faecium ATCC 9790 penicillin-binding proteins and penicillin sensitivity are heavily influenced by growth conditions: proposal for an indirect mechanism of growth inhibition by beta-lactams

The effects of variations in growth conditions on the penicillin response of Streptococcus faecium ATCC 9790 were studied. Changes in the growth temperature and medium composition were found to cause striking changes in the bacterial generation time, cellular penicillin sensitivity (minimum inhibitory concentration), sensitivity of peptidoglycan synthesis to inhibition by penicillin, rate of autolysis, and labeling pattern of penicillin-binding proteins. However, no constant relationship between these parameters and the minimum inhibitory concentration could be observed. Similar electrophoretic patterns for penicillin-binding proteins were observed in cells grown in different media at the optimal growth temperature. Inhibition of cell division by penicillin in cells grown at this temperature (but not at higher or lower temperatures) caused filamentation of the bacteria. In cells grown in a chemically defined medium at the optimal temperature (but not at temperatures above or below), complete inhibition of cell division was associated with only partial inhibition (34% after 150 min) of peptidoglycan synthesis. It is suggested that the status and physiological importance of individual penicillin-binding proteins in S. faecium are heavily influenced by growth conditions. Depending on the growth conditions, different penicillin-binding proteins may perform the cellular function, indispensible for bacterial growth.     

Rapid Biosensor for Detection of Antibiotic-Selective Growth of Escherichia coli

A rapid biosensor for the detection of bacterial growth was developed using micromechanical oscillators coated in common nutritive layers. The change in resonance frequency as a function of the increasing mass on a cantilever array forms the basis of the detection scheme. The calculated mass sensitivity according to the mechanical properties of the cantilever sensor is ~50 pg/Hz; this mass corresponds to an approximate sensitivity of ~100 Escherichia coli cells. The sensor is able to detect active growth of E. coli cells within 1 h. The starting number of E. coli cells initially attached to the sensor cantilever was, on average, ~1,000 cells. Furthermore, this method allows the detection of selective growth of E. coli within only 2 h by adding antibiotics to the nutritive layers. The growth of E. coli was confirmed by scanning electron microscopy. This new sensing method for the detection of selective bacterial growth allows future applications in, e.g., rapid antibiotic susceptibility testing.     

Development of a novel, multi-analyte biosensor system for assaying cell division: identification of cell proliferation/death precursor events

A novel, miniaturized biosensor system was created by combining the electrophysiological response of immobilized cells with superoxide-sensing technology, optical and fluorescence microscopy. Vero cells were immobilized in a calcium alginate matrix (at a density of 1.7 x 10(6) cells ml(-1)). A 0.5 cm x 0.5 cm piece of cell-containing gel matrix was aseptically adhered on a glass microscope slide with a microfabricated gold electrode array, sealed with a cover slip and provided with Dulbecco's medium +10% (v/v) fetal calf serum every day by means of a capillary feeding tube. During a culture period of 7 days, the membrane potential of immobilized cells was continuously monitored, while cell division was assayed with an optical microscope. In addition, daily measurements of immobilized cell membrane potential, viability, RNA and calcium concentration, radical oxygen species (ROS) and glutathione accumulation, were conducted by fluorescence microscopy after provision of an appropriate dye. Superoxide accumulation was assayed by covering the electrodes with superoxide dismutase (SOD). Maximum cell membrane potential values and superoxide production were observed upon initiation of cell division. Using the novel biosensor, we were able to correlate seven different cell physiological parameters to each other and formulate a model for ROS-mediated signaling function on cell division and death. In addition, we were able to predict cell proliferation or death by comparing the relative response of the electrophysiological and superoxide sensor during the culture period.     

Timing the start of division in E. coli: a single-cell study

We monitor the shape dynamics of individual E. coli cells using time-lapse microscopy together with accurate image analysis. This allows measuring the dynamics of single-cell parameters throughout the cell cycle. In previous work, we have used this approach to characterize the main features of single-cell morphogenesis between successive divisions. Here, we focus on the behavior of the parameters that are related to cell division and study their variation over a population of 30 cells. In particular, we show that the single-cell data for the constriction width dynamics collapse onto a unique curve following appropriate rescaling of the corresponding variables. This suggests the presence of an underlying time scale that determines the rate at which the cell cycle advances in each individual cell. For the case of cell length dynamics a similar rescaling of variables emphasizes the presence of a breakpoint in the growth rate at the time when division starts, tau(c). We also find that the tau(c) of individual cells is correlated with their generation time, tau(g), and inversely correlated with the corresponding length at birth, L(0). Moreover, the extent of the T-period, tau(g) - tau(c), is apparently independent of tau(g). The relations between tau(c), tau(g) and L(0) indicate possible compensation mechanisms that maintain cell length variability at about 10%. Similar behavior was observed for both fast-growing cells in a rich medium (LB) and for slower growth in a minimal medium (M9-glucose). To reveal the molecular mechanisms that lead to the observed organization of the cell cycle, we should further extend our approach to monitor the formation of the divisome.     

Individual‐based and stochastic modeling of cell population dynamics considering substrate dependency

In this article, we propose an individual‐based and stochastic modeling approach that is capable of describing the bacterial cell population dynamics during a batch culture. All stochastic nature inherent in intracellular molecular level reactions and cell division processes were considered in a single model framework by embedding a sub‐model describing individual cell's growth kinetics in a discrete event simulation algorithm. The resultant unique feature of the model is that the effects of the stochasticities on the cell population dynamics can be investigated for different substrate‐dependent cell growth kinetics. When Monod kinetics was used as the sub‐model, the stochasticities only slightly affected the cell mass increase and substrate consumption profiles during the batch culture although they were still important in describing the changes of cell population distributions. When Andrews substrate inhibition kinetics was used, however, it was revealed that the overall cell population dynamics could be seriously influenced by the stochasticities. Under a critical initial substrate level, the cell population could proliferate against the substrate inhibition only when the stochasticities were considered. Biotechnol. Bioeng. 2009;103: 891–899. © 2009 Wiley Periodicals, Inc.     

Configurational Statistics of Magnetic Bead Detection with Magnetoresistive Sensors

Magnetic biosensors detect magnetic beads that, mediated by a target, have bound to a functionalized area. This area is often larger than the area of the sensor. Both the sign and magnitude of the average magnetic field experienced by the sensor from a magnetic bead depends on the location of the bead relative to the sensor. Consequently, the signal from multiple beads also depends on their locations. Thus, a given coverage of the functionalized area with magnetic beads does not result in a given detector response, except on the average, over many realizations of the same coverage. We present a systematic theoretical analysis of how this location-dependence affects the sensor response. The analysis is done for beads magnetized by a homogeneous in-plane magnetic field. We determine the expected value and standard deviation of the sensor response for a given coverage, as well as the accuracy and precision with which the coverage can be determined from a single sensor measurement. We show that statistical fluctuations between samples may reduce the sensitivity and dynamic range of a sensor significantly when the functionalized area is larger than the sensor area. Hence, the statistics of sampling is essential to sensor design. For illustration, we analyze three important published cases for which statistical fluctuations are dominant, significant, and insignificant, respectively.     

Blue Fluorescent cGMP Sensor for Multiparameter Fluorescence Imaging

Cyclic GMP (cGMP) regulates many physiological processes by cooperating with the other signaling molecules such as cyclic AMP (cAMP) and Ca²⁺. Genetically encoded sensors for cGMP have been developed based on fluorescence resonance energy transfer (FRET) between fluorescent proteins. However, to analyze the dynamic relationship among these second messengers, combined use of existing sensors in a single cell is inadequate because of the significant spectral overlaps. A single wavelength indicator is an effective alternative to avoid this problem, but color variants of a single fluorescent protein-based biosensor are limited. In this study, to construct a new color fluorescent sensor, we converted the FRET-based sensor into a single wavelength indicator using a dark FRET acceptor. We developed a blue fluorescent cGMP biosensor, which is spectrally compatible with a FRET-based cAMP sensor using cyan and yellow fluorescent proteins (CFP/YFP). We cotransfected them and loaded a red fluorescent probe for Ca²⁺ into cells, and accomplished triple-parameter fluorescence imaging of these cyclic nucleotides and Ca²⁺, confirming the applicability of this combination to individually monitor their dynamics in a single cell. This blue fluorescent sensor and the approach using this FRET pair would be useful for multiparameter fluorescence imaging to understand complex signal transduction networks.     

Fluorescence-Based Quasicontinuous and In Situ Monitoring of Biofilm Formation Dynamics in Natural Marine Environments

Analyzing the dynamics of biofilm formation helps to deepen our understanding of surface colonization in natural environments. While methods for screening biofilm formation in the laboratory are well established, studies in marine environments have so far been based upon destructive analysis of individual samples and provide only discontinuous snapshots of biofilm establishment. In order to explore the development of biofilm over time and under various biotic and abiotic conditions, we applied a recently developed optical biofilm sensor to quasicontinuously analyze marine biofilm dynamics in situ. Using this technique in combination with microscope-assisted imaging, we investigated biofilm formation from its beginning to mature multispecies biofilms. In contrast to laboratory studies on biofilm formation, a smooth transition from initial attachment to colony formation and exponential growth could not be observed in the marine environment. Instead, initial attachment was followed by an adaptation phase of low growth and homogeneously distributed solitary bacterial cells. Moreover, we observed a diurnal variation of biofilm signal intensity, suggesting a transient state of biofilm formation of bacteria. Overall, the biofilm formation dynamics could be modeled by three consecutive development stages attributed to initial bacterial attachment, bacterial growth, and attachment and growth of unicellular eukaryotic microorganisms. Additional experiments showed that the presence of seaweed considerably shortened the adaptation phase in comparison with that on control surfaces but yielded similar growth rates. The outlined examples highlight the advantages of a quasicontinuous in situ detection that enabled, for the first time, the exploration of the initial attachment phase and the diurnal variation during biofilm formation in natural ecosystems.     

Rapid, femtomolar bioassays in complex matrices combining microfluidics and magnetoelectronics

A significant challenge for all biosensor systems is to achieve high assay sensitivity and specificity while minimizing sample preparation requirements, operational complexity, and sample-to-answer time. We have achieved multiplexed, unamplified, femtomolar detection of both DNA and proteins in complex matrices (including whole blood, serum, plasma, and milk) in minutes using as few as two reagents by labeling conventional assay schemes with micrometer-scale magnetic beads, and applying fluidic force discrimination (FFD). In FFD assays, analytes captured onto a microarray surface are labeled with microbeads, and a controlled laminar flow is then used to apply microfluidic forces sufficient to preferentially remove only nonspecifically bound bead labels. The density of beads that remain bound is proportional to the analyte concentration and can be determined with either optical counting or magnetoelectronic detection of the magnetic labels. Combining FFD assays with chip-based magnetoelectronic detection enables a simple, potentially handheld, platform capable of both nucleic acid hybridization assays and immunoassays, including orthogonal detection and identification of bacterial and viral pathogens, and therefore suitable for a wide range of biosensing applications.     

Chlamydia co-opts the rod shape-determining proteins MreB and Pbp2 for cell division

Chlamydiae are obligate intracellular bacterial pathogens that have extensively reduced their genome in adapting to the intracellular environment. The chlamydial genome contains only three annotated cell division genes and lacks ftsZ. How this obligate intracellular pathogen divides is uncharacterized. Chlamydiae contain two high-molecular-weight (HMW) penicillin binding proteins (Pbp) implicated in peptidoglycan synthesis, Pbp2 and Pbp3/FtsI. We show here, using HMW Pbp-specific penicillin derivatives, that both Pbp2 and Pbp3 are essential for chlamydial cell division. Ultrastructural analyses of antibiotic-treated cultures revealed distinct phenotypes: Pbp2 inhibition induced internal cell bodies within a single outer membrane whereas Pbp3 inhibition induced elongated phenotypes with little internal division. Each HMW Pbp interacts with the Chlamydia cell division protein FtsK. Chlamydiae are coccoid yet contain MreB, a rod shape-determining protein linked to Pbp2 in bacilli. Using MreB-specific antibiotics, we show that MreB is essential for chlamydial growth and division. Importantly, co-treatment with MreB-specific and Pbp-specific antibiotics resulted in the MreB-inhibited phenotype, placing MreB upstream of Pbp function in chlamydial cell division. Finally, we showed that MreB also interacts with FtsK. We propose that, in Chlamydia, MreB acts as a central co-ordinator at the division site to substitute for the lack of FtsZ in this bacterium.     

Correlating single cell motility with population growth dynamics for flagellated bacteria

Many bacteria used for biotechnological applications are naturally motile. Their "bio-nanopropeller" driven movement allows searching for better environments in a process called chemotaxis. Since bacteria are extremely small in size compared to the bulk fluid volumes in bioreactors, single cell motility is not considered to influence bioreactor operations. However, with increasing interest in localized fluid flow inside reactors, it is important to ask whether individual motility characteristics of bacteria are important in bioreactor operations. The first step in this direction is to try to correlate single cell measurements with population data of motile bacteria in a bioreactor. Thus, we observed the motility behavior of individual bacterial cells, using video microscopy with 33 ms time resolution, as a function of population growth dynamics of batch cultures in shake flasks. While observing the motility behavior of the most intensively studied bacteria, Escherichia coli, we find that overall bacterial motility decreases with progression of the growth curve. Remarkably, this is due to a decrease in a specific motility behavior called "running". Our results not only have direct implications on biofilm formations, but also provide a new direction in bioprocess design research highlighting the role of individual bacterial cell motility as an important parameter.     

Micromechanical oscillators as rapid biosensor for the detection of active growth of Escherichia coli

A rapid biosensor for the detection of bacterial growth was developed using micromechanical oscillators coated by common nutritive layers. The change in resonance frequency as a function of the increasing mass on a cantilever array forms the basis of the detection scheme. The sensor is able to detect active growth of Escherichia coli cells within 1 h which is significantly faster than any conventional plating method which requires at least 24 h. The growth of E. coli was confirmed by scanning electron microscopy. This new sensing method for the detection of active bacterial growth allows future applications in, e.g., rapid antibiotic susceptibility testing by adding antibiotics to the nutritive layer.     

Optical imaging fiber-based live bacterial cell array biosensor

A live cell array biosensor was fabricated by immobilizing bacterial cells on the face of an optical imaging fiber containing a high-density array of microwells. Each microwell accommodates a single bacterium that was genetically engineered to respond to a specific analyte. A genetically modified Escherichia coli strain, containing the lacZ reporter gene fused to the heavy metal-responsive gene promoter zntA, was used to fabricate a mercury biosensor. A plasmid carrying the gene coding for the enhanced cyan fluorescent protein (ECFP) was also introduced into this sensing strain to identify the cell locations in the array. Single cell lacZ expression was measured when the array was exposed to mercury and a response to 100nM Hg(2+) could be detected after a 1-h incubation time. The optical imaging fiber-based single bacterial cell array is a flexible and sensitive biosensor platform that can be used to monitor the expression of different reporter genes and accommodate a variety of sensing strains.     

FtsZ Dynamics during the Division Cycle of Live Escherichia coli Cells

The dynamics and assembly of bacterial cell division protein FtsZ were monitored in individual, growing and dividing Escherichia coli cells in real time by microculture of a merodiploid strain expressing green fluorescent protein (GFP)-tagged FtsZ. Cells expressing FtsZ-GFP at levels less than or equivalent to that of wild-type FtsZ were able to grow and divide over multiple generations, with their FtsZ rings visualized by fluorescence. During the late stages of cytokinesis, which constituted the last one-fourth of the cell cycle, the lumen of the FtsZ ring disappeared as the whole structure condensed. At this time, loops of FtsZ-GFP polymers emanated outward from the condensing ring structure and other unstable fluorescent structures elsewhere in the cell were also observed. Assembly of FtsZ rings at new division sites occurred within 1 min, from what appeared to be single points. Interestingly, this nucleation often took place in the predivisional cell at the same time the central FtsZ ring was in its final contraction phase. This demonstrates directly that, at least when FtsZ-GFP is being expressed, new division sites have the capacity to become fully functional for FtsZ targeting and assembly before cell division of the mother cell is completed. The results suggest that the timing of FtsZ assembly may be normally controlled in part by cellular FtsZ concentration. The use of wide-field optical sectioning microscopy to obtain sharp fluorescence images of FtsZ structures is also discussed.     

Mechanical limitation of bacterial motility mediated by growing cell chains

Contrasting most known bacterial motility mechanisms, a bacterial sliding motility discovered in at least two gram-positive bacterial families does not depend on designated motors. Instead, the cells maintain end-to-end connections following cell divisions to form long chains and exploit cell growth and division to push the cells forward. To investigate the dynamics of this motility mechanism, we constructed a mechanical model that depicts the interplay of the forces acting on and between the cells comprising the chain. Due to the exponential growth of individual cells, the tips of the chains can, in principle, accelerate to speeds faster than any known single-cell motility mechanism can achieve. However, analysis of the mechanical model shows that the exponential acceleration comes at the cost of an exponential buildup in mechanical stress in the chain, making overly long chains prone to breakage. Additionally, the mechanical model reveals that the dynamics of the chain expansion hinges on a single non-dimensional parameter. Perturbation analysis of the mechanical model further predicts the critical stress leading to chain breakage and its dependence on the non-dimensional parameter. Finally, we developed a simplistic population-expansion model that uses the predicted breaking behavior to estimate the physical limit of chain-mediated population expansion. Predictions from the models provide critical insights into how this motility depends on key physical properties of the cell and the substrate. Overall, our models present a generically applicable theoretical framework for cell-chain-mediated bacterial sliding motility and provide guidance for future experimental studies on such motility.