Immunobiosensor chips for detection of Escherichia coil O157:H7 using electrochemical impedance spectroscopy

Impedance biosensor chips were developed for detection of Escherichia coli O157:H7 based on the surface immobilization of affinity-purified antibodies onto indium tin oxide (ITO) electrode chips. The immobilization of antibodies onto ITO chips was carried out using an epoxysilane monolayer to serve as a template for chemical anchoring of antibodies. The surface characteristics of chips before and after the binding reaction between the antibodies and antigens were characterized by atomic force microscopy (AFM). The patterns of the epoxysilanes monolayer, antibodies, and E. coli cells were clearly observed from the AFM images. Alkaline phosphatase as the labeled enzyme to anti-E. coli O157:H7 antibody was used to amplify the binding reaction of antibody-antigen on the chips. The biocatalyzed precipitation of 5-bromo-4-chloro-3-indolyl phosphate by alkaline phosphatase on the chips in pH 10 PBS buffer containing 0.1 M MgCl2 increased the electron-transfer resistance for a redox probe of Fe(CN)6(3-/4-) at the electrode-solution interface or the electrode resistance itself. Electrochemical impedance spectroscopy and cyclic voltammetric method were employed to follow the stepwise assembly of the systems and the electronic transduction for the detection of E. coli. The biosensor could detect the target bacteria with a detection limit of 6 x 10(3) cells/mL. A linear response in the electron-transfer resistance for the concentration of E. coli cells was found between 6 x 10(4) and 6 x 10(7) cells/mL.     

Fabrication of a disposable biosensor for Escherichia Coli O157:H7 detection

As the safety in the food supply becomes critical, the demand for a rapid, low-volume, and sensitive microbial detection device has dramatically increased. A biosensor based on an electrochemical sandwich immunoassay using polyaniline has been developed for detecting foodborne pathogens, such as Escherichia coli (E. coli) O157:H7. The biosensor is comprised of two types of proteins: capture protein and reporter protein. The capture protein is immobilized on a pad between two electrodes, while the reporter protein is attached to conductive polymers. After adding the sample, the target protein binds to the reporter protein and forms a sandwich complex with the capture protein. The conductive polymer that is attached to the reporter protein serves as a messenger, reporting the amount of target protein captured in the form of an electrical signal. The architecture of the biosensor utilizes a lateral flow format, which allows the liquid sample to move from one pad to another by capillary action. Experiments to evaluate the best construction materials, the optimal polyaniline and antibody concentrations, and the distance between electrodes are highlighted in this paper. Results show that the biosensor could detect approximately 7.8/spl times/10/sup 1/ colony forming unit per milliliter of E. coli O157:H7 in 10 min.     

Quantum dot biolabeling coupled with immunomagnetic separation for detection of Escherichia coli O157:H7

A sensitive, specific, and rapid method for the detection of E. coli O157:H7 was demonstrated using quantum dots (QDs) as a fluorescence marker coupled with immunomagnetic separation. Magnetic beads coated with anti-E. coli O157 antibodies were employed to selectively capture the target bacteria, and biotin-conjugated anti-E. coli antibodies were added to form sandwich immuno complexes. After magnetic separation, the immuno complexes were labeled with QDs via biotin-streptavidin conjugation. This was followed by a fluorescence measurement using a laptop-controlled portable device, which consisted of a blue LED and a CCD-array spectrometer. The peak intensity of the fluorescence emission was proportional to the initial cell concentration of E. coli O157:H7 in the range of 10(3)-10(7) CFU/mL with a detection limit at least 100 times lower than that of the FITC-based method. The total detection time was less than 2 h. Neither E. coli K12 nor Salmonella typhimurium interfered with the detection of E. coli O157:H7.     

Interdigitated Array microelectrode-based electrochemical impedance immunosensor for detection of Escherichia coli O157:H7

A label-free electrochemical impedance immunosensor for rapid detection of Escherichia coli O157:H7 was developed by immobilizing anti-E. coli antibodies onto an indium-tin oxide interdigitated array (IDA) microelectrode. Based on the general electronic equivalent model of an electrochemical cell and the behavior of the IDA microelectrode, an equivalent circuit, consisting of an ohmic resistor of the electrolyte between two electrodes and a double layer capacitor, an electron-transfer resistor, and a Warburg impedance around each electrode, was introduced for interpretation of the impedance components of the IDA microelectrode system. The results showed that the immobilization of antibodies and the binding of E. coli cells to the IDA microelectrode surface increased the electron-transfer resistance, which was directly measured with electrochemical impedance spectroscopy in the presence of [Fe(CN)(6)](3-/4-) as a redox probe. The electron-transfer resistance was correlated with the concentration of E. coli cells in a range from 4.36 x 10(5) to 4.36 x 10(8) cfu/mL with the detection limit of 10(6) cfu/mL.     

Magnetoelastic immunosensors: amplified mass immunosorbent assay for detection of Escherichia coli O157:H7

A mass-sensitive magnetoelastic immunosensor for detection of Escherichia coli O157:H7 is described, based on immobilization of affinity-purified antibodies attached to the surface of a micrometer-scale magnetoelastic cantilever. Alkaline phosphatase is used as a labeled enzyme to the anti-E. coli O157:H7 antibody, amplifying the mass change associated with the antibody-antigen binding reaction by biocatalytic precipitation of 5-bromo-4-chloro-3-indolyl phosphate in a pH 10.0 PBS solution. The detection limit of the biosensor is 10(2) E. coli O157:H7 cells/mL. A linear change in the resonance frequency of the biosensor was found to E. coli O157:H7 concentrations ranging from 10(2) to 10(6) cells/mL.     

Carbon nanotubes for immunomagnetic separation of Escherichia coli O157: H7

Bovine serum albumin-functionalized multiple-walled carbon nanotubes with encapsulated ferromagnetic elements were conjugated with pathogen-specific antibody, and the conjugate was evaluated for immunomagnetic separation of Escherichia coli O157:H7 in pure and mixed (with Salmonella Typhimurium) cultures.     

Antibody microarray detection of Escherichia coli O157:H7: Quantification, assay limitations, and capture efficiency

A sandwich fluorescent immunoassay in a microarray format was used to capture and detect E. coli O157:H7. Here, we explored quantitative aspects, limitations, and capture efficiency of the assay. When biotinylated capture antibodies were used, the signal generated was higher (over 5-fold higher with some cell concentrations) compared to biotinylated protein G-bound capture antibodies. By adjusting the concentration of reporter antibody, a linear fluorescent response was observed from approximately 3.0 x 10(6) to approximately 9.0 x 10(7) cells/mL, and this was in agreement with the number of captured bacteria as determined by fluorescence microscopy. Capture efficiency calculations revealed that, as the number of bacteria presented for capture decreased, capture efficiency increased to near 35%. Optimization experiments, with several combinations of capture and reporter antibodies, demonstrated that the amount of bacteria available for capture (10(6) versus 10(8) cells/mL) affected the optimal combination. The findings presented here indicate that antibody microarrays, when used in sandwich assay format, may be effectively used to capture and detect E. coli O157:H7.     

Long-range surface plasmon-enhanced fluorescence spectroscopy biosensor for ultrasensitive detection of E. coli O157:H7

A new biosensor platform for the detection of bacterial pathogens based on long-range surface plasmon-enhanced fluorescence spectroscopy (LRSP-FS) is presented. The resonant excitation of LRSP modes provides an enhanced intensity of the electromagnetic field, which is directly translated to an increased strength of fluorescence signal measured upon the capture of target analyte at the sensor surface. LRSPs originate from a coupling of surface plasmons across a thin metallic film embedded in dielectrics with similar refractive indices. With respect to regular surface plasmon-enhanced fluorescence spectroscopy, the excitation of LRSPs offers the advantage of a larger enhancement of the evanescent field intensity and a micrometer probing depth that is comparable to the size of target bacterial pathogens. The potential of the developed sensor platform is demonstrated in an experiment in which the detection of E. coli O157:H7 was carried out using sandwich immunoassays. The limit of detection below 10 cfu mL(-1) and detection time of 40 min were achieved.     

Procedures for preparing Escherichia coli O157:H7 immunoliposome and its application in liposome immunoassay

Although Escherichia coli serotype O157:H7 was identified as a human pathogen in the ninth decade of the twentieth century, it has become recognized as a major foodborne pathogen. In the United States, the severity of E. coli O157:H7 infection in the young and the elderly has had a tremendous impact on human health, the food industry, and federal regulations regarding food safety. In laboratory diagnosis, most microbiologic assays rely on a single phenotype to selectively isolate this pathogen. However, the process is labor- and time-consuming. It is important eventually to develop new assay procedures to detect them. Immunoliposomes, anti-E. coli O157:H7 antibody-tagged liposomes, encapsulating a visible dye, sulforhodamine B, were used in the present study for the development of a field-portable colorimetric immunoassay to detect E. coli O157:H7. The N-succinimidyl-S-acetylthioacetate derivative of the antibodies (anti-E. coli O157: H7) was first conjugated through the reactive N-(kappa-maleimidoundecanoyloxy) sulfosuccinimide ester derivative of dipalmitoylphosphatidylethanolamine and subsequently incorported into liposomes to form the immunoliposomes. A plastic-backed nitrocellulose strip with two immobilized zones is the basis for a sandwich assay to detect E. coli O157:H7. The first zone is the antigen capture zone (AC zone), which is used in a sandwich (noncompetitive) assay format; the other is the biotin capture zone (BC zone), which is used as a positive control for the strip. During the capillary migration of the wicking reagent containing 50 microL of immunoliposomes and 90 microL of the test sample, E. coli O157:H7 with surface-bound immunoliposomes is captured at the AC zone, while the unbound immunoliposomes migrate and bind to the antibiotin antibodies coated on BC zone. The color density of the AC zone were directly proportional to the amount of E. coli O157:H7 in the test sample. The detection limit of the current assay with heat-killed E. coli O157:H7 was approximately 2500 cells. The selectivity of the newly developed biosensor system was investigated, and pathogens, including Salmonella typhimurium and Listeria genus specific, were proven to have no interference with the detection of E. coli O157:H7.     

Insights into mucosal innate responses to Escherichia coli O157 : H7 colonization of cattle by mathematical modelling of excretion dynamics

Mathematical model-based statistical inference applied to within-host dynamics of infectious diseases can help dissect complex interactions between hosts and microbes. This work has applied advances in model-based inference to understand colonization of cattle by enterohaemorrhagic Escherichia coli O157 : H7 at the terminal rectum. A mathematical model was developed based on niche replication and transition rates at this site. A nested-model comparison, applied to excretion curves from 25 calves, was used to reduce complexity while maintaining integrity. We conclude that, 5–9 days post inoculation, the innate immune response negates bacterial replication on the epithelium and either reduces attachment to or increases detachment from the epithelium of the terminal rectum. Thus, we provide a broadly applicable model that gives novel insights into bacterial replication rates in vivo and the timing and impact of host responses.     

Detection of Escherichia coli O157:H7, Salmonella typhimurium, and Legionella pneumophila in water using a flow-through chemiluminescence microarray readout system

Fast, sensitive, and especially, multianalyte test systems are currently of high interest for the monitoring and quality control of drinking water, since traditional microbiological methods are labor intensive and can take days until a response is achieved. In this study, the first flow-through chemiluminescence microarray was developed and characterized for the rapid and simultaneous detection of Escherichia coli O157:H7, Salmonella typhimurium, and Legionella pneumophila in water samples using a semiautomated readout system. Therefore, antibody microarrays were produced on poly(ethylene glycol)-modified glass substrates by means of a contact arrayer. For capturing bacteria, species-specific polyclonal antibodies were used. Cell recognition was carried out by binding of species-specific biotinylated antibodies. Chemiluminescence detection was accomplished by a streptavidin-horseradish peroxidase (HRP) catalyzed reaction of luminol and hydrogen peroxide. The chemiluminescence reaction that occurred was recorded by a sensitive charge-coupled device (CCD) camera. The overall assay time was 13 min, enabling a fast sample analysis. In multianalyte experiments, the detection limits were 3 x 10(6), 1 x 10(5), and 3 x 10(3) cells/mL for S. typhimurium, L. pneumophila, and E. coli O157:H7, respectively. Quantification of samples was possible in a wide concentration range with good recoveries. The presented system is well suited for quick and automatic water analysis.     

Immunomagnetic isolation of enterohemorrhagic Escherichia coli O157:H7 from ground beef and identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and database searches

A rapid (25 min) and facile method was developed for the isolation and identification of the enterohemorrhagic Escherichia coli (serotype O157:H7) in ground beef. The isolation method employed microscopic magnetic beads coated with antibodies covalently bonded to the surface that were specific to antigens of serotype O157. This selective preconcentration step was necessary because direct matrix-assisted laser desorption/ionization (MALDI) MS analysis of bacteria was not amenable, serving to isolate the bacteria from meat components and other nontarget bacteria. The immunomagnetic separation increased the sensitivity of the method and permitted the detection of bacteria in meat. MALDI time-of-flight MS furnished bacterial mass spectra that were useful for organism identification. Molecular weight database searches using the Expert Protein Analysis System proved useful for confirmation of the organism's identity. Bacterial biomarkers from direct MALDI analysis of pure bacterial suspensions were consistently present in bacterial suspensions of buffer/tryptic soy broth (positive controls) and meat extract samples. The detection limits were 2 x 10(6) cells/mL for the experimental approach used herein. Cross-reactivity studies performed on three nontarget bacterial strains revealed that the immunomagnetic beads are specific only to E. coli strain serotype O157:H7, and there is no cross-reactivity with the other relatively innocuous strains studied.     

Metapopulation dynamics of Escherichia coli O157 in cattle: an exploratory model.

Livestock movement is thought to be a risk factor for the transmission of infectious diseases of farm animals. Simple mathematical models were constructed for the transmission of Escherichia coli serogroup O157 between Scottish cattle farms, and the models were used in a preliminary exploration of factors contributing to the levels of infection reported in the field. The results suggest that cattle movement can make a significant contribution to the observed prevalence of E. coli O157-positive farms, but is not by itself sufficient for the persistence of E. coli O157. The results also suggest that cattle movements involving infected farms with cattle shedding an exceptional amount of E. coli O157, 'super-shedders', also make a substantial contribution to the prevalence of infected farms. Simulations indicate that E. coli O157 could have reached the currently observed prevalence levels in less than a decade. Implications and findings from our models are discussed in relation to possible control of E. coli O157 in Scottish cattle.     

Electrical Determination of E.coli O157:H7 Using Tin-Oxide Nanowire Coupled With Microfluidic Chip

Electrical determination of E.coli O157:H7 was investigated by antibody-modified individual SnO₂ nanowire (TONW) coupled with microfluidic chip. The static responses to E.coli and S.typhimurium on new device were studied. The responses to E.coli on the device that contained an unmodified or a modified TONW under continuous injection were also compared. Finally, an alternative mechanism to explain the observed behavior was proposed     

Low‐density Polyethylene Films Loaded by Titanium Dioxide and Zinc Oxide Nanoparticles as a New Active Packaging System against Escherichia coli O157:H7 in Fresh Calf Minced Meat

Antibacterial packaging is introduced as a new method to prevent microbial food spoilage. Antibacterial effects of TiO₂, ZnO and mixed TiO₂–ZnO nanoparticle‐coated low‐density polyethylene (LDPE) films on Escherichia coli PTCC1330 were investigated. Bactericidal efficiency of 0.5%, 1% and 2% TiO₂ and ZnO nanoparticles and also 1% mixed TiO₂–ZnO nanoparticles with ratios of 25/75, 50/50 and 75/25 were tested under ultraviolet (UV) and fluorescent lights at two states: films alone and fresh calf minced meat packed. Maximum colony‐forming unit (CFU) reduction of 99.8% and 99.7% were obtained using 1% and 2% ZnO nanoparticle‐coated LDPE film under fluorescent light for films alone as well as 99.8% and 99.6% for fresh calf minced meat packed. 90.3% and 51.8% CFU reduction were recorded for 1% TiO₂ nanoparticle‐coated LDPE films in the presence of UV light at direct contact with bacteria and fresh calf minced meat packed, respectively. Maximum CFU reductions of 96% and 64.1% were obtained using 50/50 ratio of TiO₂/ZnO nanoparticles at the presence of UV light for film alone and fresh calf minced meat packed, respectively. ZnO nanoparticle‐coated LDPE films were identified as the best case to improve shelf life and prevent E. coli growth in fresh calf minced meat.     

Sensitive quantification of Escherichia coli O157:H7, Salmonella enterica , and Campylobacter jejuni by combining stopped polymerase chain reaction with chemiluminescence flow-through DNA microarray analysis

Rapid analysis of pathogenic bacteria is essential for food and water control to preserve the public health. Therefore, we report on a chemiluminescence (CL) flow-through DNA microarray assay for the rapid and sensitive quantification of the pathogenic bacteria Escherichia coli O157:H7, Salmonella enterica , and Campylobacter jejuni in water. Using the stopped polymerase chain reaction (PCR) strategy, the amount of amplified target DNA was strongly dependent on the applied cell concentration. The amplification was stopped at the logarithmic phase of the PCR to quantify the DNA products on the DNA microarray chip. The generation of single-stranded DNA sequences is essential for DNA hybridization assays on microarrays. Therefore, the DNA strands of the PCR products were separated by streptavidin-conjugated magnetic nanoparticles. This was achieved by introducing a reverse primer labeled with biotin together with a digoxigenin labeled forward primer for CL microarray imaging. A conjugate of an antidigoxigenin antibody and horseradish peroxidase recognized the digoxigenin-labeled antistrands bound to the probes on the microarray surface and catalyzed the reaction of luminol and hydrogen peroxide. The generated light emission was recorded by a sensitive charge-coupled device (CCD) camera. The quantification was conducted by a flow-through CL microarray readout system. The DNA microarrays were based on an NHS-activated poly(ethylene glycol)-modified glass substrate. The DNA probes which have the same DNA sequence as the reverse primer were immobilized on this surface. The full assay was characterized by spiking experiments with heat-inactivated bacteria in water. The total assay time was 3.5 h, and the detection limits determined on CL microarrays were for E. coli O157:H7, S. enterica , and C. jejuni 136, 500, and 1 cell/mL, respectively. The results of the DNA microarray assay were comparable to the SYBR green-based assays analyzed with a real-time PCR device. The advantage of the new microarray analysis method is seen in the ability of a high multiplex degree on DNA microarrays, the high specificity of DNA hybridization on DNA microarrays, and the possibility to get quantitative results on an automated CL flow-through microarray analysis system.     

An integrated decoupler for capillary electrophoresis with electrochemical detection: application to analysis of brain microdialysate.

An approach to capillary electrophoresis with electrochemical detection (CE-EC) suitable for determination of dopamine in 1-min brain microdialysate samples is described. The CE-EC system includes an electrochemical detection cell that permits easy, precise, and permanent alignment of a carbon fiber microelectrode with a separation capillary (30-micron i.d., 75-cm length). Amperometric detection was performed at a constant applied potential of 600 mV with respect to a Ag/AgCl reference electrode. Decoupling of the electrophoretic current from the amperometric detector was accomplished with an integrated end-column decoupler prepared by etching the capillary outlet with HF. The decoupler produces baseline noise of 50 fA, or less, in the presence of 10-20-muA current in the separation capillary. The low baseline noise affords low mass (attomoles) and low concentration (nanomolar) detection limits for dopamine and 4-methylcatechol. A peak attributable to dopamine was identified in electropherograms of brain microdialysate samples obtained from anesthetized rats. Identification of the dopamine peak was confirmed by pharmacological methods. Dopamine was readily detected in 1-min brain microdialysate samples. The dopamine concentration in 1-min brain microdialysis samples was significantly altered by drug treatments and by brief electrical stimulation of dopaminergic axons.     

Single column trapping/separation and chemiluminescence detection for on-site measurement of methyl mercaptan and dimethyl sulfide

A simple, automated method for the measurement of methyl mercaptan (CH3SH) and dimethyl sulfide (DMS) has been investigated. These two sulfur gases have strong and unpleasant odors. The collection and separation are performed in sequence with a single short column packed with silica gel powder. CH3SH and DMS are separated according to their desorption temperatures and introduced into a chemiluminescence cell in this order. These two gases emit strong chemiluminescence by reaction with ozone. The calibration curves obtained are linear, which is superior to flame photometric detection of these substances. The whole system, including a small cylinder for the carrier nitrogen, can be set in a portable box. The instrument is applicable to breath odor analysis, and automated measurement of room air can also be performed. In toilet air analysis, it was observed that levels of the sulfur gases increased after dark. With this instrument, sulfur gases at a ppbv level are successfully measured by a simple procedure without much interference.     

Coupling of solid-phase microextraction and capillary isoelectric focusing with laser-induced fluorescence whole column imaging detection for protein analysis

A coupling method of solid-phase microextraction (SPME) and capillary isoelectric focusing (CIEF) with laser-induced fluorescence (LIF) whole column imaging detection (WCID) was developed for the analysis of proteins. Unlike other liquid-phase separation methods and conventional CIEF, proteins are focused into stationary bands within a pH gradient in CIEF-WCID. Thus, CIEF-WCID is the most compatible liquid-phase separation method for coupling with SPME, which can effectively resolve the problems associated with the slow desorption kinetics of SPME in a liquid phase. By combining SPME and CIEF-WCID, the desorption time can be as long as necessary, allowing complete desorption without any band broadening and analyte carryover. By using this method, R-phycoerythrin in water can be extracted by SPME in 10 min, and subsequently analyzed by CIEF-LIF-WCID within 20 min, providing a limit of detection of 3.5 x 10(-12) M (S/N = 3). The feasibility of the SPME-CIEF-LIF-WCID method was demonstrated by extracting and analyzing extracellular phycoerythrins in cultured cyanobacteria samples. Extracellular phycoerythrins at the nanomolar level were extracted and analyzed in 30 min, while avoiding the interference of the cyanobacteria cells.     

Enhanced measurement stability and selectivity for choline and acetylcholine by capillary electrophoresis with electrochemical detection at a covalently linked enzyme-modified electrode

Enzyme-modified microelectrodes were developed for the indirect amperometric detection of acetylcholine and choline following separation by capillary electrophoresis. Electrodes were prepared by first sequentially electrodepositing polypyrrole and polytyramine from the monomers on a 200-microm platinum electrode. The polymer bilayer provides enhanced selectivity and pendant amino groups for covalent coupling of acetylcholinesterase (AChE) and choline oxidase (ChO) by their reaction with glutaraldehyde. The AChE/ChO-modified electrode was then employed as an end-column detector to determine acetylcholine and choline with and without the internal standard butyrylcholine. Excellent operational stability during 2 days of continuous use was observed, with detection limits of 2 microM or 50 fmol for both acetylcholine and choline. The response from potential interferences, such as dopamine, catechol, and norepinephrine, were significantly reduced in comparison to a bare platinum electrode. The utility of this approach was demonstrated by monitoring the uptake of choline into synaptosomes.