Screen-printed electrochemical hybridization biosensor for the detection of DNA sequences from the Escherichia coli pathogen

An electrochemical biosensor for the specific detection of short DNA sequences from the E. coli pathogen is described. This hybridization device relies on the immobilization of a 25-mer oligonucleotide probe, from the E. coli lacZ gene, onto a screen-printed carbon electrode. Chronopotentiometric detection of the Co(bpy)₃⁺³ indicator is used for monitoring the hybridization event. Numerous variables of the assay protocol, including those of the probe immobilization step, the hybridization event, and the indicator association/detection, are characterized and optimized. Hybridization times of 2- and 30-min are sufficient for detecting 300- and 50 ng/mL, respectively, of the E. coli DNA target. Applicability to analysis of untreated environmental water samples is illustrated. Such single-use electrochemical sensors hold great promise for decentralized environmental and food testing for the E. coli pathogen.     

Label-free capacitive immunosensor based on quartz crystal Au electrode for rapid and sensitive detection of Escherichia coli O157:H7

A label-free capacitive immunosensor based on quartz crystal Au electrode was developed for rapid and sensitive detection of Escherichia coli O157:H7. The immunosensor was fabricated by immobilizing affinity-purified anti-E. coli O157:H7 antibodies onto self-assembled monolayers (SAMs) of 3-mercaptopropionic acid (MPA) on the surface of a quartz crystal Au electrode. Bacteria suspended in solution became attached to the immobilized antibodies when the immunosensor was tested in liquid samples. The change in capacitance caused by the bacteria was directly measured by an electrochemical detector. An equivalent circuit was introduced to simulate the capacitive immunosensor. The immunosensor was evaluated for E. coli O157:H7 detection in pure culture and inoculated food samples. The experimental results indicated that the capacitance change was linearly correlated with the cell concentration of E. coli O157:H7. The immunosensor was able to discriminate between cellular concentrations of 10²–10⁵ cfu mL⁻¹ and has applications in detecting pathogens in food samples. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were also employed to characterize the stepwise assembly of the immunosensor.     

铂纳米颗粒修饰电极对大肠杆菌的电化学快速检测

本文采用了电化学沉积法制备了铂纳米颗粒化学修饰电极（PtNP/GCE）,并将它应用于大肠杆菌的检测。原理是基于检测大肠杆菌溶液中酶与底物的反应产物,对氨基酚,实现了对大肠杆菌的快速检测。采用了铂纳米颗粒修饰电极,并对检测系统进行优化,提高大肠杆菌的检测灵敏度。大肠杆菌浓度在50—1.0×10⁵cfu/ml与响应电流成良好的线性关系,最低检测限为20 cfu/ml,检测时间在4个小时以内。与传统方法相比,该电化学方法能很好地满足食品安全、环境监控和临床医学等领域中快速检测的要求。     

Detection of E. coli using a microfluidics-based antibody biochip detection system

This work demonstrates the detection of E. coli using a 2-dimensional photosensor array biochip which is efficiently equipped with a microfluidics sample/reagent delivery system for on-chip monitoring of bioassays. The biochip features a 4 × 4 array of independently operating photodiodes that are integrated along with amplifiers, discriminators and logic circuitry on a single platform. The microfluidics system includes a single 0.4 mL reaction chamber which houses a sampling platform that selectively captures detection probes from a sample through the use of immobilized bioreceptors. The independently operating photodiodes allow simultaneous monitoring of multiple samples. In this study the sampling platform is a cellulosic membrane that is exposed to E. coli organisms and subsequently analyzed using a sandwich immunoassay involving a Cy5-labeled antibody probe. The combined effectiveness of the integrated circuit (IC) biochip and the immunoassay is evaluated for assays performed both by conventional laboratory means followed by detection with the IC biochip, and through the use of the microfluidics system for on-chip detection. Highlights of the studies show that the biochip has a linear dynamic range of three orders of magnitude observed for conventional assays, and can detect 20 E. coli organisms. Selective detection of E. coli in a complex medium, milk diluent, is also reported for both off-chip and on-chip assays. Received: 13 October 2000 / Revised: 13 November 2000 / Accepted: 13 November 2000     

An antibody microarray,in multiwell plate format,for multiplex screening of foodborne pathogenic bacteria and biomolecules

Intoxication and infection caused by foodborne pathogens are important problems worldwide, and screening tests for multiple pathogens are needed because foods may be contaminated with multiple pathogens and/or toxic metabolites. We developed a 96-well microplate, multiplex antibody microarray method to simultaneously capture and detect Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium (S. typhimurium), as well as a biomolecule (chicken immunoglobulin G or IgG employed as a proteinaceous toxin analog) in a single sample. Microarrayed spots of capture antibodies against the targeted analytes were printed within individual wells of streptavidin-coated polystyrene 96-multiwell microtiter plates and a sandwich assay with fluorescein- or Cy3-labeled reporter antibodies was used for detection. (Printing was achieved with a conventional microarray printing robot that was operated with custom-developed microplate arraying software.) Detection of the IgG was realized from ca. 5 to 25 ng/mL, and detection of E. coli O157:H7 and S. typhimurium was realized from ca. 10⁶ to 10⁹ and ca. 10⁷ to 10⁹ cells/mL, respectively. Multiplex detection of the two bacteria and the IgG in buffer and in culture-enriched ground beef filtrate was established with a total assay (including detection) time of ca. 2.5 h. Detection of S. typhimurium was largely unaffected by high concentrations of the other bacteria and IgG as well as the ground beef filtrate, whereas a small decrease in response was observed for E. coli O157:H7. The multiwell plate, multiplex antibody microarray platform developed here demonstrates a powerful approach for high-throughput screening of large numbers of food samples for multiple pathogens and toxins.     

Molecular beacons: a real-time polymerase chain reaction assay for detecting Escherichia coli from fresh produce and water

Molecular beacons (MBs) are oligonucleotide probes that fluoresce upon hybridization. The development of a real-time polymerase chain reaction (PCR) assay to detect the presence of Escherichia coli using these fluorogenic reporter molecules is reported. MBs were designed to recognize a 19-bp region of the uid A gene, coding for an enzyme β-glucuronidase. The specificity of the MB-based PCR assay was evaluated for various E. coli strains as well as bacteria species that are present in nature. The capability of the assay to detect E. coli in drinking water and produce was demonstrated. Positive detection of E. coli was demonstrated when >10¹ CFU mL⁻¹ (colony forming unit) was present in the water samples and fresh produce after 18 h of enrichment. These assays could be carried out entirely in sealed PCR tubes, enabling rapid and semiautomated detection of E. coli in food and environmental samples.     

Automatic polymerase chain reaction product detection system for food safety monitoring using zinc finger protein fused to luciferase

An automatic polymerase chain reaction (PCR) product detection system for food safety monitoring using zinc finger (ZF) protein fused to luciferase was developed. ZF protein fused to luciferase specifically binds to target double stranded DNA sequence and has luciferase enzymatic activity. Therefore, PCR products that comprise ZF protein recognition sequence can be detected by measuring the luciferase activity of the fusion protein. We previously reported that PCR products from Legionella pneumophila and Escherichia coli (E. coli) O157 genomic DNA were detected by Zif268, a natural ZF protein, fused to luciferase. In this study, Zif268–luciferase was applied to detect the presence of Salmonella and coliforms. Moreover, an artificial zinc finger protein (B2) fused to luciferase was constructed for a Norovirus detection system. In the luciferase activity detection assay, several bound/free separation process is required. Therefore, an analyzer that automatically performed the bound/free separation process was developed to detect PCR products using the ZF–luciferase fusion protein. By means of the automatic analyzer with ZF–luciferase fusion protein, target pathogenic genomes were specifically detected in the presence of other pathogenic genomes. Moreover, we succeeded in the detection of 10 copies of E. coli BL21 without extraction of genomic DNA by the automatic analyzer and E. coli was detected with a logarithmic dependency in the range of 1.0 × 10 to 1.0 × 10⁶ copies.     

Fabrication of an Electrochemical E. coli Biosensor in Biowells Using Bimetallic Nanoparticle‐Labelled Antibodies

An electrochemical biosensor was developed for the determination of Escherichia coli (E. coli) in water. For this purpose, silver‐gold core‐shell (Ag@Au) bioconjugates and anti‐E. coli modified PS‐microwells were designed in a sandwich‐type format in order to obtain higher sensitivity and selectivity. Ag@Au bimetallic nanoparticles were synthesized by co‐reduction method. The core‐shell formation was analyzed by using UV‐Vis spectroscopy and transmission electron microscopy. Biotin labeled anti‐E. coli antibodies were coupled with Ag@Au nanoparticles to form bioconjugates. The electrochemical immunosensor was prepared by immobilizing anti‐E. coli on polystyrene (PS)‐microwells via chemical bonding. These modified microwells were identified with X‐ray photoelectron spectroscopy and surface enhanced Raman spectroscopy. E. coli was sandwiched between Ag@Au bioconjugates and anti‐E. coli on PS‐microwells at different concentrations. The relationship between the E. coli concentration and stripping current of gold ions (Au³⁺) were investigated by square wave anodic stripping voltammetry at pencil graphite electrode. The proposed method can provide some advantages such as lower detection limit and shorter detection time. The electrochemical response for the immunosensor was linear with the concentration of the E. coli in the range of 10¹ and 10⁵ cfu/mL with a limit of detection 3 cfu/mL. The procedure maintains good sensitivity and repeatability and also offers utility in the fields of environmental monitoring and clinical diagnosis.     

Rapid detection of Escherichia coli O157:H7 spiked into food matrices

Food poisoning causes untold discomfort to many people each year. One of the primary culprits in food poisoning is Escherichia coli O157:H7. While most cases cause intestinal discomfort, up to 7% of the incidences lead to a severe complication called hemolytic uremic syndrome which may be fatal. The traditional method for detection of E. coli O157:H7 in cases of food poisoning is to culture the food matrices and/or human stool. Additional performance-based antibody methods are also being used. The NRL array biosensor was developed to detect multiple antigens in multiple samples with little sample pretreatment in under 30 min. An assay for the specific detection of E. coli O157:H7 was developed, optimized and tested with a variety of spiked food matrices in this study. With no sample pre-enrichment, 5 × 10³ cells mL⁻¹ were detected in buffer in less than 30 min. Slight losses of sensitivity (1-5 × 10⁻⁴ cell mL⁻¹⁾ but not specificity occur in the presence of high levels of extraneous bacteria and in various food matrices (ground beef, turkey sausage, carcass wash, and apple juice). No significant difference was observed in the detection of E. coli O157:H7 in typical culture media (Luria Broth and Tryptic Soy Broth).     

Detection of bacteria by surface-enhanced Raman spectroscopy

The detection and identification of dilute bacterial samples by surface-enhanced Raman spectroscopy has been explored by mixing aqueous suspensions of bacteria with a suspension of nanocolloidal silver particles. An estimate of the detection limit of E. coli was obtained by varying the concentration of bacteria. By correcting the Raman spectra for the broad librational OH band of water, reproducible spectra were obtained for E. coli concentrations as low as approximately 10³ cfu/mL. To aid in the assignment of Raman bands, spectra for E. coli in D₂O are also reported. Figure Light scattering apparatus used to detect bacteria     

First detection of Shiga toxin-producing Escherichia coli in shellfish and coastal environments of Morocco

Shiga toxin Escherichia coli (STEC), also called verotoxin-producing E. coli, is a major cause of food-borne illness, capable of causing hemorrhagic colitis and hemolytic–uremic syndrome (HUS). This study was carried out to evaluate the presence of (STEC) and E. coli O157:H7 in shellfish and Mediterranean coastal environments of Morocco. The contamination of shellfish and marine environment with Shiga toxin-producing E. coli (STEC) and E. coli O157:H7, was investigated during 2007 and 2008. A total of 619 samples were analyzed and 151 strains of E. coli were isolated. The presence of the stx1, stx2, and eae genes was tested in E. coli isolates strains using a triplex polymerase chain reaction. STEC was detected in three positives samples (1.9%), corresponding to the serotype O157:H7, the others Shiga toxin-producing E. coli non-O157 were also detected.     

N-Methylimidazolium modified magnetic particles-assisted highly sensitive Escherichia coli detection based on polymerase chain reaction and capillary electrophoresis

Effective bacteria detection and quantification are essential prerequisite for the prevention and treatment of infectious diseases. Herein, we report a method for the detection and quantification of Escherichia coli (E. coli).N-Methylimidazolium modified magnetic particles (MIm-MPs) are synthesized successfully and used as an efficient magnetic material for the isolation and concentration of E. coli. The factors including pH of binding buffer, concentration of elution buffer and elution time which may affect the capture and elution efficiencies are optimized. The linear correlation between bacteria concentration and peak area of polymerase chain reaction (PCR) product analyzed by capillary electrophoresis (CE) is determined. Rapid preconcentration of trace amount of E. coli (10¹ cfu mL⁻¹) in large volume of aqueous sample (500 mL) is achieved, and the capture efficiency can reach 99%. The quantification of bacteria in large volume of spiked tap water and mineral water samples is realized. The recoveries for different concentrations of E. coli in tap and mineral water samples are in the range between 83% and 93%. The results demonstrate that this MIm-MPs-PCR-CE method can be applied to detect and quantify bacteria in real samples.     

Effect of gold nanoparticles synthesized using the aqueous extract of Satureja hortensis leaf on enhancing the shelf life and removing Escherichia coli O157:H7 and Listeria monocytogenes in minced camel's meat: The role of nanotechnology in the food industry

Because herbal nanoparticles have antimicrobial properties, researchers have tried to synthesize them to aid in increasing the shelf time of food and food products. In this regard, gold nanoparticles (AuNPs) synthesized by plants are particularly important. In this study, fresh and clean leaves of Satureja hortensis were selected for the synthesis of AuNPs. We also evaluated the efficacy of these nanoparticles to increase the shelf life of and remove Escherichia coli O157:H7 and Listeria monocytogenes from minced camel's meat. The nanoparticles were analyzed by UV–visible spectroscopy, field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), Fourier-transform infrared (FT-IR) spectroscopy, energy dispersive X-ray spectroscopy, and X-ray diffraction tests. The FT-IR spectroscopy results demonstrated that the antioxidant compounds in the plant were the sources of reducing power, reducing gold ions to AuNPs. FE-SEM and TEM images revealed the size of the nanoparticles to be 22.26 nm. The 2,2-diphenyl-1-picrylhydrazyl test revealed similar antioxidant potentials for S. hortensis, AuNPs, and butylated hydroxytoluene. S. hortensis and AuNPs had high cell viability dose-dependently against the human umbilical vein endothelial cell line. At the beginning of the food industry part of this experiment, all samples of control, S. hortensis, and AuNPs were preserved at 4°C for 20 days. During these 20 days, the sensory, chemical, and microbiological parameters were assessed for all samples. AuNPs significantly inhibited the growth of E. coli and L. monocytogenes. In addition, AuNPs significantly increased the protein carbonyl content, thiobarbituric acid reactive substances, pH, peroxide value, total volatile base nitrogen, and sensory attributes (color, odor, and overall acceptability). The best results were seen in AuNPs (1%). These findings reveal that the inclusion of S. hortensis extract improves the solubility of AuNPs, which led to a notable enhancement in their preservative and antibacterial effects.     

Comparison of antibacterial ability of copper and stainless steel

In this paper, the electro-analysis and spectrophotometric analysis methods were used to study the antibacterial ability of copper and stainless steel materials. When Escherichia coli (E. coli) and photo-bacteria were used as samples, the antibacterial effect of stainless steel was very weak, while the percentage of bacteria dying from exposure to metallic copper for 30 min was over 90%. The antibacterial ability of copper has a potential application in the field of disinfection, food packaging and piping of drinking water. Translated form Huaxue Tongbao, 2006, 69(10): 772–776 (in Chinese)     

A Sensitive Nanoporous Gold-Based Electrochemical DNA Biosensor for Escherichia coli Detection

A sensitive electrochemical DNA biosensor based on a mixed monolayer structure self-assembled at nanoporous gold (NPG) electrode surface was prepared for Escherichia coli (E. coli) detection. The NPG was fabricated on gold electrode, onto which thiolated oligonucleotides (SH-DNA) and mercaptohexanol (MCH) were covalently linked forming a mixed self-assembled monolayer (SAM). The hybridization between the SH-DNA/MCH modified biosensor and E. coli DNA was monitored with differential pulse voltammetry measurement using methylene blue (MB) as the hybridization indicator. The biosensor can detect 1 × 10^?12 M DNA target and 50 cfu/μL E. coli without any nucleic acid amplification steps. The detection limit was lowered to 50 cfu/mL after 5.0 h of incubation.     

Counting of Escherichia coli by a microflow cytometer based on a photonic–microfluidic integrated device

Counting of Escherichia coli DH5α‐cell suspensions in PBS is performed using a microflow cytometer based on a photonic–microfluidic integrated device. Side‐scattered light signals are used to count the E. coli cells. A detection efficiency of 92% is achieved when compared with the expected count from a hemocytometer. The detection efficiency is correlated to the ratio of sample to sheath flow rates. It is demonstrated that E. coli can be easily distinguished from beads of similar sizes (2–4 μm) as their scattering intensities are different.     

Size‐based separations as an important discriminator in development of proximity ligation assays for protein or organism detection

Proximity ligation is a powerful technique to measure minute concentrations of target protein with high specificity, and it has been demonstrated to be effective on a wide variety of protein targets. The proximity ligation assay (PLA) technique is shown to be compromised by the amplification of a nonspecific fluorescent product that is not indicative of protein presence, which was previously unidentified in a published procedure. This result illuminates the complexity of designing the optimal PLA and the possibility of using a size‐based separation to increase the reliability of PLAs in general. Nucleic acid controls were developed to optimize the assay, which led to a novel end‐point detection method that exploits microchip electrophoresis to size the products. This method provides a greater ability to distinguish a between the target protein's signal and noise in a PLA. The utility of the PLA is demonstrated by the detection of human pathogenic Escherichia coli O157:H7 bacteria, a pathogen at the root of many recent life‐threatening food poisoning outbreaks. The results of the PLA show a detection limit of 100 E. coli O157:H7 cells with minimal cross‐reactivity with gram positive control Staphylococcus aureus bacteria. The advantages of miniaturizing this process are the 100‐fold reduction in volume, greatly reducing reagent requirements, and doubling of the thermocycling speed via noncontact infrared heating. This work, consequently, adds to the understanding of background fluorescence in PLAs, provides a method for evaluating nonspecific amplification, and shows that a qualitative PCR response indicative of the presence protein can be achieved with PLA.     

A novel fluorescence immunoassay for the sensitive detection of Escherichia coli O157:H7 in milk based on catalase-mediated fluorescence quenching of CdTe quantum dots

Immunoassay is a powerful tool for rapid detection of food borne pathogens in food safety monitoring. However, conventional immunoassay always suffers from low sensitivity when it employs enzyme-catalyzing chromogenic substrates to generate colored molecules as signal outputs. In the present study, we report a novel fluorescence immunoassay for the sensitive detection of E. coli O157:H7 through combination of the ultrahigh bioactivity of catalase to hydrogen peroxide (H₂O₂) and H₂O₂-sensitive mercaptopropionic acid modified CdTe QDs (MPA-QDs) as a signal transduction. Various parameters, including the concentrations of anti-E. coli O157:H7 polyclonal antibody and biotinylated monoclonal antibody, the amounts of H₂O₂ and streptavidin labeled catalase (CAT), the hydrolysis temperature and time of CAT to H₂O₂, as well as the incubation time between H₂O₂ and MPA-QDs, were systematically investigated and optimized. With optimal conditions, the catalase-mediated fluorescence quenching immunoassay exhibits an excellent sensitivity for E. coli O157:H7 with a detection limit of 5 × 10² CFU/mL, which was approximately 140 times lower than that of horseradish peroxidase-based colorimetric immunoassay. The reliability of the proposed method was further evaluated using E. coli O157:H7 spiked milk samples. The average recoveries of E. coli O157:H7 concentrations from 1.18 × 10³ CFU/mL to 1.18 × 10⁶ CFU/mL were in the range of 65.88%–105.6%. In brief, the proposed immunoassay offers a great potential for rapid and sensitive detection of other pathogens in food quality control.     

Analysis and classification of bacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and a chemometric approach

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a useful technique for the identification of bacteria on the basis of their characteristic protein mass spectrum fingerprint. Highly standardized instrumental analytical performance and bacterial culture conditions are required to achieve useful information. A chemometric approach based on multivariate analysis techniques was developed for the analysis of MALDI data of different bacteria to allow their identification from their fingerprint. Principal component analysis, linear discriminant analysis (LDA) and soft independent modelling of class analogy (SIMCA) were applied to the analysis of the MALDI MS mass spectra of two pathogenic bacteria, Escherichia coli O157:H7 and Yersinia enterocolitica, and the non-pathogenic E. coli MC1061. Spectra variability was assessed by growing bacteria in different media and analysing them at different culture growth times. After selection of the relevant variables, which allows the evaluation of an m/z value pattern with high discriminant power, the identification of bacteria by LDA and SIMCA was performed independently of the experimental conditions used. In order to better evaluate the analytical performance of the approach used, the ability to correctly classify different bacteria, six wild-type strains of E. coli O157:H7, was also studied and a combination of different chemometric techniques with a severe validation was developed. The analysis of spiked bovine meat samples and the agreement with an independent chemiluminescent enzyme immunoassay demonstrated the applicability of the method developed for the detection of bacteria in real samples. The easy automation of the MALDI method and the ability of multivariate techniques to reduce interlaboratory variability associated with bacterial growth time and conditions suggest the usefulness of the proposed MALDI MS approach for rapid routine food safety checks. Figure Workflow of the developed MALDI-TOF MS and chemometric approach for the analysis and classification of bacteria Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Amperometric determination of live <Emphasis Type="Italic">Escherichia coli</Emphasis> using antibody-coated paramagnetic beads

Detecting and enumerating fecal coliforms, especially Escherichia coli, as indicators of fecal contamination, are essential for the quality control of supplied and recreational waters. We have developed a sensitive, inexpensive, and small-volume amperometric detection method for E. coli -galactosidase by bead-based immunoassay. The technique uses biotin-labeled capture antibodies (Ab) immobilized on paramagnetic microbeads that have been functionalized with streptavidin (bead–Ab). The bead–Ab conjugate captures E. coli from solution. The captured E. coli is incubated in Luria Bertani (LB) broth medium with the added inducer isopropyl -D-thiogalactopyranoside (IPTG). The induced -galactosidase converts p-aminophenyl -D-galactopyranoside (PAPG) into p-aminophenol (PAP), which is measured by amperometry using a gold rotating disc electrode. A good linear correlation (R²=0.989) was obtained between log cfu mL^–1 E. coli and the time necessary to product a specific concentration of PAP. Amperometric detection enabled determination of 2×10⁶ cfu mL^–1 E. coli within a 30 min incubation period, and the total analysis time was less than 1 h. It was also possible to determine as few as 20 cfu mL^–1 E. coli under optimized conditions within 6–7 h. This process may be easily adapted as an automated portable bioanalytical device for the rapid detection of live E. coli.