An improved process using magnetic capture of antibody-conjugated bacteria for light addressable potentiometric sensor detection by the Threshold instrument was developed. Cells of Escherichia coli 0157:H7 were captured by the biotinylated anti-E. coli 0157 antibodies conjugated to streptavidin coated magnetic beads. Magnetically concentrated bacteria were further labeled with by fluorescein-conjugated anti-E. coli 0157 antibodies that were bound to urease-conjugated antifluorescein antibody. The whole bacteria-containing complex was then immobilized on 0.45? biotinylated nitro-cellulose membranes via streptavidin-biotin interactions. The rates of pH change associated with the production of NH3 by conjugated urease were measured by a LAPS technique incorporated in the Threshold instrument. This approach allowed us to detect io CFU of cultured E. coli 0157:H7 in tris-buffered saline (TBS). The same approach was applied to detect the E. coli in beef hamburger spiked with the bacteria. After a 5 to 6-hour enrichment, as low as 1 CFU/g of E. coli 0157:H7 in the hamburger could be detected. In addition, the immobilized bacterial complexes on the nitro-cellulose membranes exhibited a stability longer than 48 h at 4 and 22 °C in TBS allowing the possibility of conveniently shipping collected samples rather than raw beef to testing laboratories.

Rapid detection, identification and enumeration of pathogenic microorganisms is highly important to the food industry to address the food safety concerns. Biosensors are devices that promise to achieve these objectives, these analytical devices can accurately and selectively estimate the levels of foodborne pathogens. The most widely applied biosensors are based on the optical properties like absorption, fluorescence, reflection, refraction, dispersion etc. This review is done to provide an overview of optic based biosensor and their application in the area of food safety. Working principles of Surface plasmon resonance , resonant mirror and fiber optic based biosensor are described in the article. Resonant mirror based biosensor has been used for the estimation of S. aureus, it was proven selective for the strain being tested (Cowan-1). Using colloidal-gold conjugates in sandwich assay format increased the sensitivity of the assay. An integrated fiber optic based biosensor is used for detection of Salmonella typhimurium reducing the detection time to 30 min. Fiber optic based biosensors offer advantage of compactness, flexibility, resistance to electrical noise and small probe size.

The presence of biologically derived toxins in foods is of utmost significance to food safety and human health concerns. Biologically active amines, referred to as biogenic amines, serve as a noteworthy example, having been implicated as the causative agent in numerous food poisoning episodes. Of the various biogenic amines encountered, histamine, putrescine, cadaverine, tyramine, tryptamine, beta-phenylethylamine, spermine, and spermidine are considered to be the most significant, and can be used as hygienic-quality indicators of food. Biogenic amines can be monitored using whole-cell bioluminescent bioreporters, which represent a family of genetically engineered microorganisms that generate visible light in response to specific chemical or physical agents in their environment. The light response occurs due to transcriptional activation of a genetically incorporated lux cassette, and can be measured using standard photomultiplier devices. We have successfully engineered a lux-based bioreporter capable of detecting and monitoring the biogenic amine beta-phenylethylamine. This research represents a biologically-based sensor technology that can be readily integrated into Hazard Analysis Critical Control Point programs to provide a rugged monitoring regime that can be uniformly applied for field-based and in-house laboratory quality control analyses. Since the bioreporter and biosensing elements are completely self-contained within the sensor design, this system provides ease of use, with operational capabilities realized by simply combining the food sample with the bioreporter and allowing the sensor to process the ensuing bioluminescent signal and communicate the results. The application of this technology to the critically important issue of food safety and hygienic quality represents a novel method for detecting, monitoring, and preventing biologically active toxins in food commodities.

To address the need for a fast and sensitive method for the detection of bacterial contamination in solutions, we evaluated the use of Fourier-transform near infrared (FT-NIR) spectroscopy and multivariate pattern recognition techniques. The complex cellular composition of bacteria yields FT-NIR vibrational transitions (overtone and combination bands) that might be used for identification and sub-typing. Bacterial suspensions (E. coli HB1O1, E. coli ATCC43888, E. coli 1224, Pseudomonas aeruginosa, Bacillus cereus and Listeria innocua) were filtered to concentrate the cells and eliminate the matrix which has a strong NIR signal. FT-NIR measurements were done by using a diffuse reflection-integrating sphere. The use of a simple membrane filtration procedure to produce a thin, uniform bacterial film generated reproducible FT-NIR spectra that can be used for rapid discrimination among closely related strains. Transformation ofthe spectra with second derivatives resolved specific FT-NIR features in the information-rich spectral region of 5000-4000 cm-1 to allow principal components analysis to group the samples into different tight clusters. The use ofAnodisc membranes gave the more reproducible results. This methodology appears promising for the rapid evaluation of potential bacterial contamination in liquids with minimal sample manipulation.

Listeria monocytogenes is a deadly foodbome human pathogen. Its ubiquitous nature and its ability to grow at refrigeration temperatures makes this organism a difficult one to control. High-volume processing of food products and poor sanitary conditions of the processing plants often allow this organism to be present in processed, ready-to-eat (RTE) foods. Improved processing along with real-time detection could reduce the incidence of this pathogen. Conventional methods can detect this pathogen accurately, but take several days (2-7d) to complete, which is not practical considering the short shelf-life and cost of storage of RTE foods. Biosensor based approaches were adopted for sensitive detection of Listeria. Antibody-coupled fiber optic and microelectrical-mechanical system (MEMS) biochips were designed and examined for direct detection of L. monocytogenes from liquid samples. Also, interdigitated microsensor electrode (IME) chip and spectrofluorometer were used to measure L. monocytogenes interaction with mammalian cells (cytopathogenic activities) for indirect detection. Preliminary data generated using laboratory cultures of Listeria species indicated that L. monocytogenes could be detected in 30 mm to 1 h 30 mm depending on the techniques used.

Time-reso1ved fluorescence immunoassays using dissociation enhanced lanthanide fluorescence have been developed for detection and identification of animal and food pathogens including Clostridium botulinum toxins A and B, Staphylococcus enterotoxm B (SEB), Bacillus anthracis, E. coil 0157:H7, Salmonella, Listeria and Campylobacter. Both double antibody sandwich inimunoassays and microwell filterplate assays have been developed using polyclonal, monoclonal and phage display antibodies labeled with fluorescent europium or samarium lanthanide chelates. Multiplexed dual label eiiropium and samarium assays have been performed for Salmonella simultaneously with E. coli 0157:H7, Salmonella and Listeria. Results from different laboratories are presented to demonstrate that these time-resolved fluorescent immunoassays have high sensitivity and specificity with excellent reproducibility.

The RAPTOR is a portable, automated biosensor capable of performing rapid, ten-minute assays on a sample for four target analytes simultaneously. Samples are analyzed using a fluorescent sandwich immunoassay on the surface of short polystyrene optical probes with capture antibody adsorbed to the probe surface. Target analytes bound to the fiber by capture antibodies are detected with fluorescently labeled tracer antibodies, which are held in a separate reservoir. Since target recognition is a two-step process, selectivity is enhanced, and the optical probes can be reused up to forty times, or until a positive result is obtained. This greatly reduces the logistical burden for field operations. Numerous assays for toxins, such as SEB and ricin, and bacteria, such as Bacillus anthracis and Francisella tularensis, have been developed for the RAPTOR. An assay of particular interest for water quality monitoring and the screening of fruits and vegetables is detection of Giardia cysts. Giardia lamblia is a parasitic protozoan common in the developing world that causes severe intestinal infections. Thus, a simple field assay for screening water supplies would be highly useful. Such an assay has been developed using the RAPTOR. The detection limit for Giardia cysts was 5x104/ml for a 10-minute assay.

Multiwavelength spectroscopy is a rapid technique that provides quantitative information for the detection and identification of cells. A typical multiwavelength spectrum reflects the chemical composition, size, internal structure arid number of cells present in a sample. These properties constitute essential information for the identification and classification of cells. The multiwavelength spectrum is generated from the combined scattering and absorption characteristics of the sample. Light scattering theory is then used to deconvolute the spectrum for estimates ofthe critical parameters necessary for the detection and identification of cells. This approach has been used to determine the spectral fingerprint for blood cells, bacterial cells and protozoa. The characteristic set of optical properties for platelets, E. coil and Cryptosporidium have been determined as a function of wavelength and used for the quantitative interpretation ofUV-vis spectra within the context ofMie theory. The models developed using this approach provide reliable and accurate estimates for cell size, number, chemical composition and internal structure. Information on the chemical composition is further deconvoluted into quantitative estimates of nucleic acid and protein content. This type of detailed information is thell used for the discrimination of cell types. The technique is applicable to a wide range of cell types found in diverse environments. Advances in the development of miniaturized spectrometers increase the potential of this method as an excellent candidate for a rapid, reliable and efficient biosensor.

This paper reports on the progress made towards the implementation of a novel micron and sub-micron particle characterization technology for in-situ, continuous monitoring and detecting of microorganisms in water. The particle technology focuses on characterizing the joint particle property distribution (size, shape, and composition). The experimental set up includes two parts: a separation methodology based on Flow Field-flow Fractionation (FFFF); and a detection system consisting of a UV-vis spectrometer coupled with an optical wave guide and a multi-angle laser light scattering (MALLS) photometer. FFFF is used because it separates partiôles based on particle volume, which eliminates one variable. The UV-vis /optical waveguide constitutes the primary detection system, while the multi-angle laser light scattering photometer is used for the purpose of validation. UV-vis spectroscopy has been shown to be capable of detecting the chemical composition and size distribution for particles in the size range of 50 nm — 10 ?m. The UV-vis absorption patterns of microorganisms of interest strongly suggest the possibility of a two way (size and spectral signature) identification and classification of pathogens in water. It is evident that this methodology has the potential for continuous monitoring applications. Transmission measurements coupled with the liquid core waveguide (LCW) enables detection at very low concentration consistent with typical eluent concentration from the FFFF. In this paper it is shown that the combined system (FFFF-MALLS-UV-vis- LCW) can provide, in a matter of minutes, the spectra and size distribution of waterbome microorganisms for on-line identification and classification.

There has been increasing demand for rapid, sensitive and specific detection of Escherichia coli as an indicator of possible pathogen contamination in foods and water. Approximately 97% of E. coli strains produce ?- glucuronidase (GUS), which could permit the use of a specific microbial enzyme as an alternative approach for detection of E. coli. A procedure was developed for chemiluminometric measurement of GUS using a 1,2-dioxetene derivative as substrate, and was compared to the fluorescent assay for GUS detection. The chemiluminescent assay was found to be 10 times more sensitive than the fluorescent assay. Induction of GUS production in E. coli was maximum when p-nitropheny 1-?-D-glucuronide was used in the growth medium at 0.3 mM concentration after 8 h. GUS was isolated from the growth medium with a 30 minute immunocapture method at 37°C. Anti E. coli GUS antibodies were covalently immobilized on magnetic beads and used for the immunocapture assay. GUS from E. coli culture was captured using the prepared magnetic-beads. Compared to the chemiluminescent assay of GUS in culture filtrate, immunomagnetic capture of GUS provided signal increases up to 81x. The method permitted the detection of 1 CPU/ml of E. coli within 8 hours incubation in growth medium. The chemiluminescent enzyme capture immunoassay developed for the detection of GUS could serve as a quantitative indicator for the presence ofviable E. coli cells. The total assay time including growth, immunocapture and enzyme assay was 9 h.

Current lidar system used for bioaerosol detection utilize either elastic backscatter to detect the presence of any aerosol cloud, or use UV induced fluorescence to detect a potential bioaerosol. We are examining the possibility of using a broad bandwidth (hyperspectral) lidar system to estimate size distributions of aerosol clouds that may possess respirable biological particles. An optical parametric oscillator has been specially designed and fabricated for broad bandwidth operation in the 1.4 to 1.8 micrometer spectral region when pumped with a frequency doubled Nd:YAG laser. We have determined the spectral bandwidth, output, and pumping power characteristics of this device, and we have modeled the feasibility of using this as a source for an aerosol sizing lidar. A novel inverse Monte Carlo (IMC) technique was developed to analyze backscattering data that would result from a lidar system based on this device. Lidar simulation results show that good estimates of size distributions can be extracted for aerosols which have a 1 to 10 micrometer size range even when the signal to noise ratio is only 3:1.

Spectroscopic method of dangerous substances and microorganisms detection in water is developed. The method is based on analysis of the secondary emission spectra (Raman scattering and fluorescence spectra) of water samples. These spectra were recorded with time delay with respect to exciting light pulse. The excitation was produced by the pulsed ultraviolet radiation from copper vapor laser with non-linear optical crystal cell.

Although chemical residues do not have the same type of health consequences as microbiological pathogens, the regulation of hazardous chemicals in foods is an integral component of food safety programs worldwide. Analytical methods to monitor chemical residues are essential to help protect human health and support the compliance and enforcement oflaws and regulations, but many current analytical approaches are too time-consuming, expensive, laborious, and waste resources that could be used morejudiciously to improve food safety. Rapid and reliable approaches for the detection ofmultiple analytes at low and/or high concentrations in the field and/or laboratory are needed to reduce costs and/or increase benefits in the detection of hazardous chemicals in foods. The implementation ofsuch approaches will (a) increase productivity and/or decrease costs ofanalysis, (b) provide more statistically valid and accurate results for risk assessment and other purposes, (c) overcome trade barriers associated with the analysis of chemical residues, (d) provide more information to understand the effects and mechanisms of antimicrobial resistance and endocrine disruption, (e) allow for better verification of organic food labeling, (f) improve possible industrial HACCP programs, and (g) reduce the potential for food that has been deliberately or accidentally adulterated by toxic chemicals to reach the consumer. This paper is meant to provide an overview of current analytical capabilities and the needs for improved analytical screening methods to detect chemical residues in foods, and describes how rapid and reliable monitoring approaches can benefit regulatory agencies, industry, and consumers alike.

A new multi—channel optical biosensor instrument has been constructed for high-throughput screening analysis of drug residues in foods. The sensor uses surface plasmon resonance technology and allows either simultaneous analysis of eight samples for a single analyte or multi analyte analysis. Three muhi analyte assays kits were developed for use on the prototype instrument: clenbuterol (CBL) and ethinyl estradiol (EEO) in urine, sulfamethazine (SMT) and sulfadiazine (SDZ) in bile and SMT, SDZ and enrofloxacin (EFX) in milk. Direct analysis was performed on milk and bile samples while detection of CBL and EEO at low ppb levels required limited sample extraction. The assay/ instrument combinations were tested by end-users in laboratory and on-site environments. Sample throughput of up to 600 samples per day was achieved. Numbers of samples tested ranged from 473 samples for the urine assay to over 7500 for the bile assay. Instrument performance in the laboratory was assessed by comparison with the end-user's standard procedure. Comparison of false positive screening results and confirmed positive samples showed the results achieved by the new technique to be at least comparable with existing screening procedure with the added benefits of increased sample throughput and result generation in "real time". On site analysis showed no evidence of the technique being adversely effected by the harsher environments. The present study has clearly demonstrated that high throughput immunosensor technology can play a leading role in the detection of residues ofveterinary drugs in food.

Molecular imprinting is a useful technique for making a chemically selective binding site. The method involves building a synthetic polymeric scaffold of molecular complements containing the target molecule with subsequent removal of the target to leave a cavity with a structural memory of the target. Molecularly imprinted polymers can be employed as selective adsorbents of specific molecules or molecular functional groups. The imprinted polymers can be fashioned into membranes that can be used to form ion selective electrodes for an imprinted ion. By incorporating molecules or metal ions with useful optical properties in the binding sites of imprinted polymers, spectroscopic sensors for the imprinted molecule may be made. A variety of metal ion selective electrodes and a Pb2+ ion optrode based on imprinted polymers have been fabricated and tested.1-4 Additionally, a sensor for the hydrolysis product of the nerve agent Soman has been developed using a luminescent lanthanide ion, Eu(III), as optical transducer.5 Our research continues to explore other means to employ electrochemical and optical transductio

Pesticides are required to protect crops and provide the quantity and quality offood demanded by today's society. However, potential pesticide residue contamination ofboth food and animal feed is ofcritical concern to the food industry, and the public at large. Among the 366 pesticides that were detectable by methods used in the 1999 FDA Regulatory Monitoring Program, 90 pesticides were actually found in foods for human consumption and animal feeds. Unfortunately, rapid, chemical analysis for all of the pesticide residues is unavailable, and only a very small fraction of foods and animal feeds can be inspected. Consequently, as much as 1 % of food brought to market may contain pesticide residues exceeding FDA guidelines. In an effort to address this need, we have been developing sample vials internally coated with a surface-enhanced Raman (SER) active sol-gel material capable ofdetecting pesticides at and below regulated concentrations. The SER active material consists of a metal-doped sol-gel that provides not only a million fold increase in sensitivity but also reproducible measurements. The porous silica network offers a unique environment for stabilizing SER active metal particles and the high surface area increases the interaction between the analyte and metal particles. A sample solution is simply poured into the coated glass vial, placed in the Raman instrument, and measured. Here the potential for developing a rapid multi-residue analyzer employing the sol-gel sample vials will be presented.

There have been numerous studies on effectiveness of different sanitizers for microbial inactivation. However, results obtained from different studies indicate that microorganism cannot be easily removed from fresh cut vegetables because of puncture and cut surfaces with varying surface topographies. In this study, three step disinfection approach was evaluated for inactivation of E. coli O157:H7 on shredded lettuce leaves. Sequential application of thyme oil, ozonated water, and aqueous chlorine dioxide was evaluated in which thyme oil was applied first followed by ozonated water and aqueous chlorine dioxide. Shredded lettuce leaves inoculated with cocktail culture of E. coli O157:H7 (C7927, EDL 933 and 204 P), were washed with ozonated water (15 mg/l for 10min), aqueous chlorine dioxide (10 mg/l,for 10min) and thyme oil suspension (0.1%, v/v for 5min). Washing of lettuce leaves with ozonated water, chlorine dioxide and thyme oil suspension resulted in 0.44, 1.20, and 1.46 log reduction (log10 cfu/g), respectively. However, the sequential treatment achieved approximately 3.13 log reductions (log10 cfu/g). These results demonstrate the efficacy of sequential treatments in decontaminating shredded lettuce leaves containing E. coli O157:H7.

Hydrodynamic pressure processing (HDP) was investigated as an alternative non-thermal preservation technique to reduce microorganisms found inlon meat products. Pork /beef stew pieces and ground beef (GB) were examined. All meat samples were stored at room temperature ( 23 °C) for 22 h representing temperature abuse (TA) conditions. Following storage, samples were divided in lots for controls and HDP-treatment. Meat samples were vacuum packaged for HDP treatment (binary explosive placed 6 or 12" from meat surface; 54-L steel shock wave container). Pork/beef stew pieces were treated with 1 00 g @ 12". GB was treated with 25, 50, 75, or 100 g @ 12" and 75 @ 6". Samples were assayed for total aerobic platecounts (APC; log,0 CFU/g) after treatment. Microorganisms were reduced (2 log; P