Quantum dot-based array for sensitive detection of <Emphasis Type="Italic">Escherichia coli</Emphasis>

A fluorescent quantum dot-based antibody array, used in sandwich format, has been developed to detect Escherichia coli O157:H7. Numerous parameters such as solid support, optimal concentration of immunoreagents, blocking reagents, and assay time were optimized for array construction. Quantum dot-conjugated anti-IgG was used as the detecting system. The array allows the detection of E. coli O157:H7 at concentrations below 10 CFU mL⁻¹ without sample enrichment, exhibiting an increase of three orders of magnitude in the limit of detection compared to ELISA. The interference caused by Gram (+) and Gram (−) bacteria was negligible at low concentrations of bacteria.     

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).     

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.     

Chemiluminescent enzyme-linked immunosorbent assay on a strip to detect Escherichia coli O157:H7

A fast and sensitive chemiluminescent enzyme-linked immunosorbent assay method to measure pathogenic bacteria, Escherichia coli O157:H7, on immuno-chromatographic membrane was studied. Non-specific binding of proteins on membrane strip was controlled to attain the best performance of immunosensor by optimising the composition of a running buffer. The specificity of the proposed immunostrip was confirmed by conducting experiments for four different micro-organisms. A chemiluminescent signal could be successfully generated from a proposed immunostrip sensing system, and a significant change in the chemiluminescent light intensity with the concentration of target microbes was obtained. E. coli O157:H7 could be quantitatively measured in the range of 1.1?×?10^3?–1.1?×?10⁷ CFU (colony forming units) mL^?1 within 16?min by using the developed chemiluminescent immunostrip.     

A Rapid and Sensitive Aptamer‐Based Electrochemical Biosensor for Direct Detection of Escherichia Coli O111

A sensitive and specific electrochemical biosensor based on target‐induced aptamer displacement was developed for direct detection of Escherichia coli O111. The aptamer for Escherichia coli O111 was immobilized on a gold electrode by hybridization with the capture probe anchored on the electrode surface through Au‐thiol binding. In the presence of Escherichia coli O111, the aptamer was dissociated from the capture probe‐aptamer duplex due to the stronger interaction between the aptamer and the Escherichia coli O111. The consequent single‐strand capture probe could be hybridized with biotinylated detection probe and tagged with streptavidin‐alkaline phosphatase, producing sensitive enzyme‐catalyzed electrochemical response to Escherichia coli O111. The designed biosensor showed weak electrochemical signal to Salmonella typhimurium, Staphylococcus aureus and common non‐pathogenic Escherichia coli, indicating high specificity for Escherichia coli O111. Under the optimal conditions, the proposed strategy could directly detect Escherichia coli O111 with the detection limit of 112 CFU mL^?1 in phosphate buffer saline and 305 CFU mL^?1 in milk within 3.5 h, demonstrated the sensitive and accurate quantification of target pathogenic bacteria. The designed biosensor could become a powerful tool for pathogenic microorganisms screening in clinical diagnostics, food safety, biothreat detection and environmental monitoring.     

Development of a multichannel flow-through chemiluminescence microarray chip for parallel calibration and detection of pathogenic bacteria

Pathogen detection is important for health and safety reasons. Several outbreaks all over the world have shown the need for rapid, qualitative, quantitative, and, particularly, multianalyte detection systems. Hence, a multichannel flow-through chemiluminescence microarray chip for parallel detection of pathogenic bacteria was developed. The disposable chip made of acrylonitrile–butadiene–styrene (ABS) copolymer was devised as a support for a multiplexed sandwich immunoassay. Calibration and measurement was possible in one experiment, because the developed chip contains six parallel flow-through microchannels. Polyclonal antibodies against the pathogenic bacteria Escherichia coli O157:H7, Salmonella typhimurium, and Legionella pneumophila were immobilized on the chip by microcontact printing in order to use them as specific receptors. Detection of the captured bacteria was carried out by use of specific detection antibodies labelled with biotin and horseradish peroxidase (HRP)–streptavidine conjugates. The enzyme HRP generates chemiluminescence after adding luminol and hydrogen peroxide. This signal was observed by use of a sensitive CCD camera. The limits of detection are 1.8 × 10⁴ cells mL⁻¹ for E. coli O157:H7, 7.9 × 10⁴ cells mL⁻¹ for L. pneumophila, and 2.0 × 10⁷ cells mL⁻¹ for S. typhimurium. The overall assay time for measurement and calibration is 18 min, enabling very fast analysis.      

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.     

Dual gold nanoparticle lateflow immunoassay for sensitive detection of Escherichia coli O157:H7

Two patterns of signal amplification lateral flow immunoassay (LFIA), which used anti-mouse secondary antibody-linked gold nanoparticle (AuNP) for dual AuNP-LFIA were developed. Escherichia coli O157:H7 was selected as the model analyte. In the signal amplification direct LFIA method, anti-mouse secondary antibody-linked AuNP (anti-mouse-Ab-AuNP) was mixed with sample solution in an ELISA well, after which it was added to LFIA, which already contained anti-E. coli O157:H7 monoclonal antibody-AuNP (anti-E. coli O157:H7-mAb-AuNP) dispersed in the conjugate pad. Polyclonal antibody was the test line, and anti-mouse secondary antibody was the control line in nitrocellulose (NC) membrane. In the signal amplification indirect LFIA method, anti-mouse-Ab-AuNP was mixed with sample solution and anti-E. coli O157:H7-mAb-AuNP complex in ELISA well, creating a dual AuNP complex. This complex was added to LFIA, which had a polyclonal antibody as the test line and secondary antibody as the control line in NC membrane. The detection sensitivity of both LFIAs improved 100-fold and reached 1.14 × 10³ CFU mL⁻¹. The 28 nm and 45 nm AuNPs were demonstrated to be the optimal dual AuNP pairs. Signal amplification LFIA was perfectly applied to the detection of milk samples with E. coli O157:H7 via naked eye observation.     

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.     

Separation ofN-carbamoyl aspartate andl-dihydroorotate from serum by high-performance liquid chromatography with fluorimetric detection

Summary A high-performance liquid chromatographic method, with 9-anthryldiazomethane as derivatizing agent, has been developed for the simultaneous determination ofN-carbamoyl aspartate andl-dihydroorotate in serum. Sample preparation for 1 mL serum was by simple liquid-liquid extraction and then derivatization. The compounds were separated on a Luna C18(2) column by use of a gradient prepared from acetonitrile and 10 mM sodium acetate buffer, pH 6.0, and fluorimetric detection was performed at excitation and emission wavelengths of 365 nm and 412 nm, respectively. The response was found to be linearly dependent on concentration between 0.8 and 60 μg mL⁻¹ forl-dihydrooratate and between 0.9 and 90 μg mL⁻¹ forN-carbamoyl aspartate; the mean recovery rates were 50 and 51%, respectively. The limits of detection and quantification were 0.33 μg mL⁻¹ and 0.6 μg mL⁻¹, respectively, forl-dihydroorotate and 0.4 μg mL⁻¹ and 0.7 μg mL⁻¹ forN-carbamoyl aspartate. This method can be used to assess accumulation ofN-carbamoyl aspartate andl-dihydroorotate in body fluids in situations where cellular pyrimidine de novo synthesis is impaired.     

Microencapsulated Engineered <Emphasis Type="Italic">Lactococcus lactis Cells</Emphasis> for Heterologous Protein Delivery: Preparation and In Vitro Analysis

This article demonstrates the potential of encapsulated, engineered Lactococcus lactis as a vehicle for the oral delivery of therapeutic proteins. Using alginate-poly-l-lysine-alginate membrane-encapsulated L. lactis engineered to secrete the reporter protein Staphylococcal aureus nuclease, we show comparable viability and protein secretion between free and immobilized cells. After 12 h, microcapsules with a cell density of 4.8 × 10⁵ colony forming unit (CFU) ml⁻¹ grew to 2.2 × 10⁸ CFU ml⁻¹ and released 0.24 arbitrary unit (AU) ml⁻¹ of nuclease, producing similar results as free cells, which grew from 3.4 × 10⁵ to 1.9 × 10⁸ CFU ml⁻¹ and secreted 0.21 AU ml⁻¹ of nuclease. Moreover, encapsulated cells at a density of 4.4 × 10⁷ CFU ml⁻¹ grew to 2.2 × 10¹⁰ CFU ml⁻¹ in 12 h and secreted 15.3 AU ml⁻¹ of nuclease although 3.1 × 10⁷ CFU ml⁻¹ of free cells reached only 2.3 × 10⁹ CFU ml⁻¹ and released 5.6 AU ml⁻¹ of nuclease. We also show the sustained stability of the microcapsules during storage at 4°C over 8 weeks.     

Determination of viable <Emphasis Type="Italic">Escherichia coli</Emphasis> using antibody-coated paramagnetic beads with fluorescence detection

A rapid and convenient assay system was developed to detect viable Escherichia coli in water. The target bacteria were recovered from solution by immunomagnetic separation and incubated in tryptic soy broth with isopropyl-β-d-thiogalactopyranoside, which induces formation of β-galactosidase in viable bacteria. Lysozyme was used to lyse E. coli cells and release the β-galactosidase. β-Galactosidase converted 4-methylumbelliferyl-β-d-galactoside to 4-methylumbelliferone (4-MU), which was measured by fluorescence spectrophotometry using excitation and emission wavelengths of 355 and 460 nm, respectively. Calibration graphs of 4-MU fluorescence intensity versus E. coli concentration showed a detection range between 8 × 10⁴ and 1.6 × 10⁷ cfu mL⁻¹, with a total analysis time of less than 3 h. The advantage of this method is that it detects viable cells because it is based on the activity of the enzyme intrinsic to live E. coli.     

Flow injection chemiluminescence immunoassay for 17β-estradiol using an immunoaffinity column

A new flow injection chemiluminescent immunoassay was developed for the detection of 17β-estradiol (E₂). The method uses p–iodophenol (PIP) as enhancer and is based on a solid-phase immunoassay format in which an E₂–OVA immobilized immunoaffinity column inserted in the flow system is used to trap unbound horseradish peroxidase (HRP)-labeled anti-E₂ antibody after an off-line incubation of E₂ with HRP-labeled anti-E₂ antibody. The trapped enzyme conjugate was detected by injecting substrates to produce an enhanced chemiluminescence (CL) response. The linear range for E₂ was 10.0–1,000.0 ng mL⁻¹ with a correlation coefficient of 0.996 and a detection limit of 3.0 ng mL⁻¹. The sampling and chemiluminescence detection time for one sample was 400 s after a pre-incubation procedure of 30 min. Serum samples detected by this method were in good agreement with the results obtained by EIA with E₂–biotin.      

Multiplexed high-throughput electrokinetically-controlled immunoassay for the detection of specific bacterial antibodies in human serum

In previous studies we have developed a simple electrokinetically-controlled lab-on-a-chip for heterogeneous immunoassay. In that method, all the sequential operations in an immunoassay, such as reagent loading and washing, were performed automatically by electrokinetically controlling the flow in an H-shaped microchannel. Here, we demonstrated further development of a high-throughput immunoassay microfluidic chip, and the application of the new immunoassay microfluidic chip in assaying human serum. The microfluidic immunoassay analyzed ten samples in parallel in 22 min. Bacterial antibodies in samples were captured by antigens pre-patterned on the bottom wall of a microchannel and then bound with TRITC-labeled detection antibodies to generate fluorescent signals. With optimized surface concentration of antigen, the assay detected Escherichia coli O157:H7 antibody and Helicobacter pylori antibody from buffer solutions in concentration ranges of 0.02-10 μg mL⁻¹ and 0.1-50 μg mL⁻¹, respectively. Human sera that were E. coli-positive or H. pylori-positive were accurately distinguished from respective negative controls. Moreover, the two antibodies, anti-E. coli and anti- H. pylori antibodies, could be simultaneously detected from human serum. This electrokinetically-controlled immunoassay shows an excellent potential for efficiently detecting multiple pathogenic infections in clinical environments.     

Disposable Immunosensor for Escherichia Coli O157:H7 Based on a Multi-Walled Carbon Nanotube-Sodium Alginate Nanocomposite Film Modified Screen-Printed Carbon Electrode

A disposable immunosensor for the detection of Escherichia coli O157:H7 based on a multiwalled carbon nanotube–sodium alginate nanocomposite film was constructed. The nanocomposite was placed on a screen-printed carbon electrode, and horseradish peroxidase-labeled antibodies were immobilized to E. coli O157:H7 on the modified electrode to construct the immunosensor. The modification procedure was characterized by atomic force microscopy and cyclic voltammetry. Under optimal conditions, the proposed immunosensor exhibited good electrochemical sensitivity to E. coli O157:H7 in a concentration range of 10³–10¹⁰ cfu/mL, with a relatively low detection limit of 2.94 × 10² cfu/mL (S/N = 3). This immunosensor exhibited satisfactory specificity, reproducibility, stability, and accuracy, making it a potential alternative tool for early assessment of E. coli O157:H7.     

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.     

Magnetic Nanohybrids Loaded with Bimetal Core–Shell–Shell Nanorods for Bacteria Capture,Separation, and Near‐Infrared Photothermal Treatment

A novel antimicrobial nanohybrid based on near‐infrared (NIR) photothermal conversion is designed for bacteria capture, separation, and sterilization (killing). Positively charged magnetic reduced graphene oxide with modification by polyethylenimine (rGO–Fe₃O₄–PEI) is prepared and then loaded with core–shell–shell Au–Ag–Au nanorods to construct the nanohybrid rGO–Fe₃O₄–Au–Ag–Au. NIR laser irradiation melts the outer Au shell and exposes the inner Ag shell, which facilitates controlled release of the silver shell. The nanohybrids combine physical photothermal sterilization as a result of the outer Au shell with the antibacterial effect of the inner Ag shell. In addition, the nanohybrid exhibits high heat conductivity because of the rGO and rapid magnetic‐separation capability that is attributable to Fe₃O₄. The nanohybrid provides a significant improvement of bactericidal efficiency with respect to bare Au–Ag–Au nanorods and facilitates the isolation of bacteria from sample matrixes. A concentration of 25 μg mL^?1 of nanohybrid causes 100 % capture and separation of Escherichia coli O157:H7 (1×10⁸ cfu mL^?1) from an aqueous medium in 10 min. In addition, it causes a 22 °C temperature rise for the surrounding solution under NIR irradiation (785 nm, 50 mW cm^?2) for 10 min. With magnetic separation, 30 μg mL^?1 of nanohybrid results in a 100 % killing rate for E. coli O157:H7 cells. The facile bacteria separation and photothermal sterilization is potentially feasible for environmental and/or clinical treatment.     

A Validated HPLC Method for Simultaneous Determination of 2-Hydroxy-4-methoxybenzaldehyde and 2-Hydroxy-4-methoxybenzoic Acid in Root Organs of Hemidesmus indicus

A reverse phase HPLC method was developed and validated for the simultaneous determination of 2-hydroxy-4-methoxybenzaldehyde and 2-hydroxy-4-methoxybenzoic acid in the root extracts of Hemidesmus indicus. A comprehensive validation of the method including sensitivity, linearity, reproducibility, accuracy, limit of detection (LOD) and limit of quantification (LOQ) was conducted using the optimized chromatographic conditions. The method was found to be linear (r > 0.998) in the range of 5–350 μg mL⁻¹ for 2-hydroxy-4-methoxybenzaldehyde and for 2-hydroxy-4-methoxybenzoic acid (r > 0.999) in the range 10–300 μg mL⁻¹. The method was found to be precise with inter-day precision values (% RSD) in the ranges of 0.61–1.76% for 2-hydroxy-4-methoxybenzaldehyde and 1.3–2.8% for 2-hydroxy-4-ethoxybenzoic acid while intra-day precisions (% RSD) of two analytes were in the range of 0.41–1.07 and 0.95–2.5%. The limits of detection (LODs) for 2-hydroxy-4-methoxybenzaldehyde and 2-hydroxy-4-methoxybenzoic acid were 0.84 and 2.34 μg mL⁻¹. The described method was fast, sensitive and reproducible, and thus well suited for routine analysis of these two compounds from root extracts of H. indicus and other plants.     

A kinetic method for <Emphasis Type="Italic">para</Emphasis>-nitrophenol determination based on its inhibitory effect on the catalatic reaction of catalase

The inhibitory effect of para-nitrophenol on the catalytic reaction of catalase was investigated. Michaelis-Menten kinetic parameters were determined from Lineweaver-Burk plots obtained in the absence or in the presence of the inhibitor. The inhibitor pattern, revealed by the Lineweaver-Burk plots, suggested a fully mixed inhibition mechanism. Spectrophotometric monitoring of the indicator reaction: in conjunction with initial rate measurements was employed for the kinetic determination of the inhibitor. Calibration plots of initial rate vs. para-nitrophenol concentration were linear in the concentration range 0.9·10⁻⁵–2.5·10⁻⁵ mol/L and the detection limit was 3·10⁻⁶ mol/L (417 μg/L) para-nitrophenol. Interferences from other phenolic compounds like orto-cresole, meta-and orto-nitrophenol were observed.     

Raman spectroscopic study of the hydration of short-chain poly(oxyethylene)s C <Subscript>1</Subscript>E<Subscript><Emphasis Type="Italic">n</Emphasis></Subscript>C <Subscript>1</Subscript> (<Emphasis Type="Italic">n</Emphasis>=1−4)

The hypothesis that the degree of hydration of poly(oxyethylene) (POE) in aqueous solution depends on the mole ratio of water molecules to ether oxygen atoms in the molecule has been verified by studying the isotropic Raman spectra in the O−H stretching region for four short-chain POEs (C ₁E _n C ₁ withn=1−4). Excellent coincidence of the O−H stretching Raman band for all four POEs studied in the range of mole ratio H₂O/O _ether from 25 to 0.6 was observed, thus confirming the assumption stated above. A conclusion that all ether oxygen atoms in the POE molecule participate in hydrogen bonding with water molecules has been made.