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.     

A luminescent Escherichia coli biosensor for the high throughput detection of beta-lactams

A group-specific bioluminescent Escherichia coli strain for studying the action of beta-lactam antibiotics is described. The strain contains a plasmid, pBlaLux1, in which the luciferase genes from Photorhabdus luminescens are inserted under the control of the beta-lactam-responsive element ampR/ampC from Citrobacter freundii. In the presence of beta-lactams, the bacterial cells are induced to express the luciferase enzyme and three additional enzymes generating the substrate for the luciferase reaction. This biosensor for beta-lactams does not need any substrate or cofactor additions, and the bioluminescence can be measured very sensitively in real time by using a luminometer. Basic parameters affecting the light production and induction in the gram-negative model organism E. coli SNO301/pBlaLux1 by various beta-lactams were studied. The dose-response curves were bell shaped, indicating toxic effects for the sensor strain at high concentrations of beta-lactams. Various beta-lactams had fairly different assay ranges: ampicillin, 0.05-1.0 microg/ml; piperacillin, 0.0025-25 microg/ml; imipenem, 0.0025-0.25 microg/ml; cephapirin, 0.025-2.5 microg/ml; cefoxitin, 0.0025-1.5 microg/ml; and oxacillin, 25-500 microg/ml. Also, the induction coefficients (signal over background noninduced control) varied considerably from 3 to 158 in a 2-hour assay. Different non-beta-lactam antibiotics did not cause induction. Because the assay can be automated using microplate technologies, the approach may be suitable for higher throughput analysis of beta-lactam action.     

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.     

Stable-light-emitting Escherichia coli as a biosensor

We have studied possibilities for constructing Escherichia coli strains capable of producing stable light. Light production in E. coli is achieved by cloning the genes encoding bacterial luciferase from Vibrio harveyi. To gain the advantage of sensitive detection of light we transferred the genes under the control of a strong, regulatable promoter system. Stabilization of light produced by E. coli clones was accomplished by finding the optimal plasmid construction and growth conditions as well as suitable measuring buffers. The adjustment of the luciferase synthesis for bioluminescence measurements to a high but not harmful level gives healthy cells and stable luciferase. Cultivation at 30 °C in an uninduced state was found to be the most important factor in getting stable-light production. The overall cell metabolism being unstressed gives us the possibility of monitoring cell physiology and factors affecting it via bioluminescence reactions in vivo. To make the results easy to interpret the light emission has to be stable during a measurement period of one to several hours. In the case of the original light-producing bacteria, Vibrio and Photobacterium strains it has not thus far been possible to find conditions where light emission would be stable for several hours. Based on our findings an automated biosensor system can be developed to monitor the effects of biologically active compounds against stable-light-producing bacteria.     

Electrogenerated chemiluminescence biosensor incorporating ruthenium complex-labelled Concanavalin A as a probe for the detection of Escherichia coli

The interaction of riboflavin with salmon sperm double-stranded DNA based on the decreasing of the oxidation signal of guanine and adenine bases was studied electrochemically with a pencil graphite electrode (PGE) using differential pulse voltammetry. The decrease in the intensity of the guanine and adenine oxidation signals after interaction with riboflavin was used as an indicator signals for the sensitive determination of riboflavin. Under the optimum conditions, a linear dependence of the guanine and adenine oxidation signals was observed for the riboflavin concentration in the range of 0.5-70 μg mL(-1) with a detection limit of 0.34 μg mL(-1) at ds-DNA modified PGE. The reproducibility and applicability of the analysis to pharmaceutical dosage forms and urine sample were also investigated. These results showed that this DNA biosensor could be used for the sensitive, rapid, simple and cost effective detection and determination of riboflavin-ds-DNA interaction. Pretreated pencil graphite electrode (PPGE) was also used for the determination of riboflavin by differential pulse adsorptive stripping voltammetry. With PPGE, a linear relationship was obtained for riboflavin over the concentration range of 0.003-0.88 μg mL(-1) with differential pulse adsorptive stripping voltammetric signal and with a detection limit of 0.076 ng mL(-1). Both determination methods were fully validated and applied for the analysis of riboflavin.     

Development of a biosensor for on-line detection of tributyltin with a recombinant bioluminescent Escherichia coli strain

A biosensor was developed for the detection of tributyltin (TBT), using a bioluminescent recombinant Escherichia coli:: luxAB strain. Dedicated devices allowed the on-line measurement of bioluminescence, pH and dissolved oxygen values and the feed-back regulation of temperature. Bacterial physiology was monitored by the measurement of the cellular density, respiratory activity and the intracellular level of ATP, glucose and acetate levels. Our results showed that a synthetic glucose medium gave a better TBT detection limit than LB medium (respectively 0.02 micro M and 1.5 micro M TBT). High growth and dilution rates ( D=0.9 h(-1)) allowed maximum light emission from the bacterium. Moreover, simple atmospheric air bubbling was sufficient to provide oxygen for growth and the bioluminescence reaction. Real-time monitoring of bioluminescence after TBT induction occurred with continuous addition of decanal up to 300 micro M, which was not toxic throughout a 7-day experiment. The design of our biosensor and the optimization of the main parameters that influence microbial activity led to the capacity for the detection of TBT.     

Biological laser printing of genetically modified Escherichia coli for biosensor applications

One of the primary requirements of cell- or tissue-based sensors is the placement of cells and cellular material at or near the sensing elements of the device. The ability to achieve precise, reproducible and rapid placement of cells is the focus of this study. We have developed a technique, biological laser printing or BioLP, which satisfies these requirements and has advantages over current technologies. BioLP is capable of rapidly depositing patterns of active biomolecules and living cells onto a variety of material surfaces. Unlike ink jet or manual spotting techniques, this process delivers small volume (nl to fl) aliquots of biomaterials without the use of an orifice, thus eliminating potential clogging issues and enabling diverse classes of biomaterials to be deposited. This report describes the use of this laser-based printing method to transfer genetically-modified bacteria capable of responding to various chemical stressors onto agar-coated slides and into microtiter plates. The BioLP technology enables smaller spot sizes, increased resolution, and improved reproducibility compared to related technologies.     

The eclipse period of Escherichia coli

The minimal time between successive initiations on the same origin (the eclipse) in Escherichia coli was determined to be approximately 25-30 min. An inverse relationship was found between the length of the eclipse and the amount of Dam methyltransferase in the cell, indicating that the eclipse corresponds to the period of origin hemimethylation. The SeqA protein was absolutely required for the eclipse, and DnaA titration studies suggested that the SeqA protein prevented the binding of multiple DnaA molecules on oriC (initial complex formation). No correlation between the amount of SeqA and eclipse length was revealed, but increased SeqA levels affected chromosome partitioning and/or cell division. This was corroborated further by an aberrant nucleoid distribution in SeqA-deficient cells. We suggest that the SeqA protein's role in maintaining the eclipse is tied to a function in chromosome organization.     

Interdigitated array microelectrode based impedance biosensor coupled with magnetic nanoparticle-antibody conjugates for detection of Escherichia coli O157:H7 in food samples

An impedance biosensor based on interdigitated array microelectrode (IDAM) coupled with magnetic nanoparticle-antibody conjugates (MNAC) was developed and evaluated for rapid and specific detection of E. coli O157:H7 in ground beef samples. MNAC were prepared by immobilizing biotin-labeled polyclonal goat anti-E. coli antibodies onto streptavidin-coated magnetic nanoparticles, which were used to separate and concentrate E. coli O157:H7 from ground beef samples. Magnitude of impedance and phase angle were measured in a frequency range of 10 Hz to 1 MHz in the presence of 0.1M mannitol solution. The lowest detection limits of this biosensor for detection of E. coli O157:H7 in pure culture and ground beef samples were 7.4 x 10(4) and 8.0 x 10(5)CFU ml(-1), respectively. The regression equation for the normalized impedance change (NIC) versus E. coli O157:H7 concentration (N) in ground beef samples was NIC=15.55 N-71.04 with R(2)=0.95. Sensitivity of the impedance biosensor was improved by 35% by concentrating bacterial cells attached to MNAC in the active layer of IDAM above the surface of electrodes with the help of a magnetic field. Based on equivalent circuit analysis, it was observed that bulk resistance and double layer capacitance were responsible for the impedance change caused by the presence of E. coli O157:H7 on the surface of IDAM. Surface immobilization techniques, redox probes, or sample incubation were not used in this impedance biosensor. The total detection time from sampling to measurement was 35 min.     

Immobilization of enzyme on long period grating fibers for sensitive glucose detection

Glucose oxidase (GOD) immobilized long period grating (LPG) fibers have been proposed for the specific and sensitive detection of glucose. The treatment of LPG fibers with aminopropyl triethoxysilane has induced biding sites for the subsequent GOD immobilization. Field emission scanning electron microscopy, confocal laser scanning microscopy, infrared spectroscopy and Raman spectroscopy have provided detailed evidences about the effectiveness of the adopted biofunctionalization methodology. The enzyme activity is conserved during the immobilization step. Fabricated LPG sensor was tested on different glucose solutions to record the transmission spectra on an optical spectrum analyzer. The wavelength shifts in the transmission spectra are linearly correlated with the glucose concentration in the range of 10-300 mg dL(-1). The fabricated sensor gives fast response and is demonstrated to be of practical utility by determining glucose contents in blood samples. Proposed technique can further be extended to develop LPG fiber based novel, sensitive and label free nanosensors for disease diagnosis and clinical analysis.     

A DNA sequence-specific electrochemical biosensor based on alginic acid-coated cobalt magnetic beads for the detection of E. coli

A new type of DNA sequence-specific electrochemical biosensor based on magnetic beads for the detection of Escherichia coli is reported in the present work. Alginic acid-coated cobalt magnetic beads, capped with 5'-(NH(2)) oligonucleotide and employed not only for magnetic separation but also as the solid adsorbent, were used as DNA probes to hybridize with the target E. coli DNA sequence. This assay was specific for E. coli detection depending on the uid A gene, which encodes for the enzyme β-d-glucuronidase produced by E. coli strains. When daunomycin (DNR) was used as DNA hybridization indicator, the target sequences of E. coli hybridized with the probes resulted in the decrease of DNR reduction peak current, which was proportional to the E. coli concentration. The optimization of the hybridization detection was carried out and the specificity of the probes was also demonstrated. This DNA biosensor can be employed to detect a complementary target sequence for 3.0×10(-10) mol/L and denatured PCR products for 0.5 ng/μL. The linear range of the developed biosensor for the detection of E. coli cells was from 1.0×10(2) to 2.0×10(3) cells/mL with a detection limit of 50 cells/mL. After a brief enrichment process, a concentration of 10 cells/mL E. coli in real water samples was detected by the electrochemical biosensor.     

Development of SCAR primers based on a repetitive DNA fingerprint for Escherichia coli detection

The present study aimed to use enterobacterial repetitive intergenic consensus (ERIC) fingerprints to design SCAR primers for the detection of Escherichia coli. The E. coli strains were isolated from various water sources. The primary presumptive identification of E. coli was achieved using MacConkey agar. Nineteen isolates were selected and confirmed to be E. coli strains based on seven biochemical characteristics. ERIC-PCR with ERIC 1R and ERIC 2 primers were used to generate DNA fingerprints. ERIC-PCR DNA profiles showed variant DNA profiles among the tested E. coli strains and distinguished all E. coli strains from the other tested bacterial strains. A 350 bp band that predominated in five E. coli strains was used for the development of the species-specific SCAR primers EC-F1 and EC-R1. The primers showed good specificity for E. coli, with the exception of a single false positive reaction with Sh. flexneri DMST 4423. The primers were able to detect 50 pg and 10⁰ CFU/ml of genomic DNA and cells of E. coli, respectively.     

Aptamer selection for the detection of Escherichia coli K88

In this study, the first group of single-stranded DNA aptamers that are highly specific to enterotoxigenic Escherichia coli (ETEC) K88 was obtained from an enriched oligonucleotide pool by the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure, during which the K88 fimbriae protein was used as the target and bovine serum albumin as counter targets. These aptamers were applied successfully in the detection of ETEC K88. They were then grouped under different families based on the similarity of their secondary structure and the homology of their primary sequence. Four sequences from different families were deliberately chosen for further characterization by fluorescence analysis. Having the advantage of high sensitivity, fluorescence photometry was selected as single-stranded DNA quantification method during the SELEX process. Aptamers with the highest specificity and affinity were analyzed to evaluate binding ability with E. coli. Since ETEC K88 is the only type of bacterium that expressed abundant K88 fimbriae, the selected aptamers against the K88 fimbriae protein were able to specifically identify ETEC K88 among other bacteria. This method of detecting ETEC K88 by aptamers can also be applied to bacteria other than ETEC K88.     

Resonant waveguide grating biosensor for living cell sensing

This article presents theoretical analysis and experimental data for the use of resonant waveguide grating (RWG) biosensors to characterize stimulation-mediated cell responses including signaling. The biosensor is capable of detecting redistribution of cellular contents in both directions that are perpendicular and parallel to the sensor surface. This capability relies on online monitoring cell responses with multiple optical output parameters, including the changes in incident angle and the shape of the resonant peaks. Although the changes in peak shape are mainly contributed to stimulation-modulated inhomogeneous redistribution of cellular contents parallel to the sensor surface, the shift in incident angle primarily reflects the stimulation-triggered dynamic mass redistribution (DMR) perpendicular to the sensor surface. The optical signatures are obtained and used to characterize several cellular processes including cell adhesion and spreading, detachment and signaling by trypsinization, and signaling through either epidermal growth factor receptor or bradykinin B2 receptor. A mathematical model is developed to link the bradykinin-mediated DMR signals to the dynamic relocation of intracellular proteins and the receptor internalization during B2 receptor signaling cycle. This model takes the form of a set of nonlinear, ordinary differential equations that describe the changes in four different states of B2 receptors, diffusion of proteins and receptor-protein complexes, and the DMR responses. Classical analysis shows that the system converges to a unique optical signature, whose dynamics (amplitudes, transition time, and kinetics) is dependent on the bradykinin signal input, and consistent with those observed using the RWG biosensors. This study provides fundamentals for probing living cells with the RWG biosensors, in general, optical biosensors.     

A biosensor for environmental genotoxin screening based on an SOS lux assay in recombinant Escherichia coli cells.

A genetically controlled luminescent bacterial reporter assay, the SOS lux test, was developed for rapid detection of environmental genotoxins. The bioassay is based on the recombinant plasmid pPLS-1, which was constructed as a derivative of pBR322, carrying the promoterless luxCDABFE genes of Photobacterium leiognathi downstream of a truncated cda gene from ColD with a strong SOS promoter. E. coli recA+ strains containing this construction are inducible to high levels of light production in the presence of substances or agents that cause damage to the DNA of the cells. The light signal, reflecting the SOS-inducing potency, is recorded from the growing culture within 1 s, and the test results are available within 1 to 2 h. Induction of bioluminescence was demonstrated by treatment of E. coli C600(pPLS-1) with 6 genotoxic chemicals (mitomycin C, N-methyl-N'-nitro-N-nitrosoguanidine, nalidixic acid, dimethylsulfate, hydrogen peroxide, and formaldehyde) and with UV and gamma radiation. A clear dose-response relationship was established for all eight genotoxins. The sensitivity of the SOS lux test is similar to that of other bioassays for genotoxicity or mutagenicity, such as the SOS chromotest, umu test, and Ames mutatest. These results indicate that the SOS lux test is potentially useful for the in situ and continuous detection of genotoxins.     

Evanescent wave absorbance based fiber optic biosensor for label-free detection of E. coli at 280 nm wavelength

A novel label-free technique for the detection of pathogens based on evanescent wave absorbance (EWA) changes at 280 nm from a U-bent optical fiber sensor is demonstrated. Bending a decladded fiber into a U-shaped structure enhances the penetration depth of evanescent waves and hence sensitivity of the probe. We show that the enhanced EWA response from such U-bent probes, caused by the inherent optical absorbance properties of bacterial cells or biomolecules specifically bound to the sensor surface, can be exploited for the detection of pathogens. A portable optical set-up with a UV light emitting diode, a spectrometer and U-bent fiber optic probe of 200 μm core diameter, 0.75 mm bend radius and effective probe length of 1cm demonstrated an ability to detect less than 1000 cfu/ml.     

Polyaniline based catalase biosensor for the detection of hydrogen peroxide and azide

The CAT/PANi/ITO bioelectrode has been prepared as a catalase biosensor and shows response for monitoring not only of H₂O₂ but also azide. The sensor exhibited an excellent response to the H₂O₂ and azide. The linear range of H₂O₂ was 0.064∼1 mM and for azide 0.125∼4 mM, respectively. Catalase biosensor was based on the principle of the measurements as the decrease in the differentiation of oxygen level, which has been caused by the inhibition of catalase in the bioactive layer of the biosensor by azide. The repeatability experiments were done in triplicate. The logarithm response of the biosensor to H₂O₂ (r² = 0.99), as well as, for azide (r² = 0.90) were reported, respectively. The bioelectrode was characterized by CV and AFM. The proposed biosensor would be applied for the determination of H₂O₂ and azide in various biological samples.     

Eclipse period without sequestration in Escherichia coli

The classical Meselson-Stahl density shift experiment was used to determine the length of the eclipse period in Escherichia coli, the minimum time period during which no new initiation is allowed from a newly replicated origin of chromosome replication, oriC. Populations of bacteria growing exponentially in heavy ((15)NH(4)+ and (13)C(6)-glucose) medium were shifted to light ((14)NH(4)+ and (12)C(6)-glucose) medium. The HH-, HL- and LL-DNA were separated by CsCl density gradient centrifugation, and their relative amounts were determined using radioactive gene-specific probes. The eclipse period, estimated from the kinetics of conversion of HH-DNA to HL- and LL-DNA, turned out to be 0.60 generation times for the wild-type strain. This was invariable for widely varying doubling times (35, 68 and 112 min) and was independent of the chromosome locus at which the eclipse period was measured. For strains with seqA, dam and damseqA mutants, the length of the eclipse period was 0.16, 0.40 and 0.32 generation times respectively. Thus, initiations from oriC were repressed for a considerable proportion of the generation time even when the sequestration function seemed to be severely compromised. The causal relationship between the length of the eclipse period and the synchrony of initiations from oriC is discussed.