Arthur G. Rand

Evaluation of antibody immobilization methods for piezoelectric biosensor application

The immobilization of anti-Salmonella antibodies by two methods were studied and evaluated for their potential use in a piezoelectric biosensor. The optimum temperature-time combinations for the highest immobilization yields were determined for both methods. Protein A binding was found to be 67.4+/-3.8% on the gold surface which then allowed an immobilization of 42.1+/-2.09% antibody. The degree of antibody immobilization via surface aldehyde groups of glutaraldehyde (GA) on a precoated quartz crystal with polyethylenimine (PEI) was 31.6+/-0.3%. A piezoelectric probe was designed and used in dry assays to observe the frequency change due to addition of mass by the immobilization layers. The frequency changes recorded showed a better reproducibility and less added mass for the Protein A method. The frequency decrease due to microg of added antibodies was compared to frequency decrease calculated by the Sauerbrey equation. The experimental data was found to be only approximately 8% of theoretical data. The functionality of the immobilized antibodies with the Protein A method was tested with S. typhimurium in a wet chamber and the frequency decrease was compared to results of a similar system activated with PEI-GA immobilization. The frequency decreases with S. typhimurium concentration of approximately 1.5 x 10(9) CFU/ml were 50+/-2 Hz and 44+/-3 Hz for the Protein A method and PEI-GA method, respectively. It was concluded that although both methods resulted in comparable activities in terms of % immobilized protein and frequency decreases due to Salmonella binding, the Protein A method was favorable due to stability and better reproducibility of the immobilization layers.

Hydrolytic and Transgalactosylic Activities of Commercial β-Galactosidase (Lactase) in Food Processing

Publisher Summary This chapter focuses on hydrolytic and transgalactosylic activities of commercial β -galactosidase (lactase) in food processing. Food enzyme technology is the use of commercial sources of these biocatalytic compounds in the processing of food. The enzymes used for this purpose may be derived from plant, animal, or microbial sources, but generally do not require much refining to reach food-grade status. The functionality and stability of commercial and potential commercial sources of β -galactosidases for hydrolytic processes must be improved to use the enzymes under conditions that are safe from microbial contamination. To realize this goal, research is required to develop lactases that are resistant to normal processing temperatures, such as that of pasteurization. The continuing development of new microencapsulation technologies could result in a variety of heat-resistant enzymes and more research is needed. The products formed, which are addressed in this chapter, vary with the starting substrates and microbial source of the enzyme. The complete analysis of the oligosaccharides formed requires separation procedures, such as paper chromatography (PC), column chromatography, thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and gas-liquid chromatography (GLC). In addition, the identification of new products and the glycosidic linkages formed by the enzyme requires techniques such as mass spectroscopy (MS) and nuclear magnetic resonance (NMR).

A compact fiber-optic immunosensor for Salmonella based on evanescent wave excitation

Abstract A compact fiber-optic evanescent-wave sensing system that features all-fiber optical design and red semiconductor-laser excitation has been developed and tested. A 2 × 2 fiber coupler directs the input light to the SMA-connected sensing fiber tip and the fluorescent signal back to a CCD fiber spectrophotometer. In this system, the fluorescent signal is confined in the fiber system so the signal-to-noise ratio is greatly improved and the system can be operated in ambient light conditions. A diode laser as the source has the advantages of small volume, ruggedness, low cost and stability; more importantly, since biological matrices demonstrate minimal fluorescent background at the laser wavelength of 650 nm, this system can reduce the background signal of non-essential biomolecules. To illustrate the biosensors diagnostic capabilities, a sandwich immunoassay to detect Salmonella was developed. Tapered fiber tips with different shapes and treatments were studied and optimized. The system could detect Salmonella with a concentration as low as 104 colony-forming units per milliliter (CFU ml−1).

Magnetic focusing immunosensor for the detection of Salmonella typhimurium in foods

From 1988 through 1992 Salmonellosis accounted for 27% of the total reported foodborne disease outbreaks and 57% of the outbreaks in which the pathogen was identified. The prevalence of Salmonellosis and the new requirements to monitor the organism as a marker in pathogen reduction programs will drive the need for rapid, on-site testing. A compact fiber optic fluorometer using a red diode laser as an excitation source and fiber probes for analyte detection has been constructed and used to measure Salmonella. The organisms were isolated with anti-Salmonella magnetic beads and were labeled with a secondary antibody conjugated to a red fluorescent dye. The response of the system was proportional to the concentration of Salmonella typhimurium from 3.2 X 105 colony forming units (CFU)/ml to 1.6 X 107 CFU/ml. The system was developed to utilize a fiber-optic magnetic focusing problem that attracted the magnetic microspheres to the surface of a sample chamber directly in front of the excitation and emission fibers. The signal obtained from a homogenous suspension of fluorescent magnetic microspheres was 9 to 10 picowatts. After focusing, the signal from the fluorescent labeled magnetic microspheres increased to 200 picowatts, approximately 20 times greater than the homogeneous suspension. The magnetic focusing assay detected 1.59 X 105 colony forming units/ml of Salmonella typhimurium cultured in growth media. The process of magnetic focusing in front of the fibers has the potential to reduce the background fluorescence from unbound secondary antibodies, eliminating several rinsing steps, resulting in a simple rapid assay.

Rapid detection of Staphylococcus aureus using a membrane fiber optic biosensor

A simple, sensitive and rapid chemiluminescent fiber optic biosensor utilizing monoclonal antibodies to S. aureus was developed to detect the pathogen in food. The S. aureus cells were selectively labeled with a monoclonal-horseradish peroxidase (POD) conjugate, collected by membrane filtration, and detected with a luminometer and an enhanced chemiluminescent luminol reagent. Two different diameter membranes, 25 mm and 13 mm, were first tested in a luminometer tube format assay. A hand operated syringe filtration unit was used to capture cells and the membrane was then transferred to a luminometer tube for the chemiluminescent reaction. An improved system utilized a simple but efficient microwell plate vacuum filtration unit with an 8 mm membrane sealed at the bottom of the sample well. The sample was concentrated on the membrane and positioned directly in front of a fiber optic light guide to effectively collect and transmit the signal to the luminometer. Labeling S. aureus in solution proved to be much more effective than on the membrane surface. Using the microwell plate filtration system resulted in less sample handling, better reproducibility, and dramatically reduced assay time. The variability for 25 mm and 13 mm assays were 24.7% and 13.3%, while the microwell plate assay reduced this to 4.0%. The ability of the fiber optic probe to effectively collect the signal meant the sensitivity of the assay was not compromised with smaller membrane and sample size. The sensitivity of the biosensor was 3.8 X 104 CFU/ml, adequate to detect the organism at concentrations lower than the level that could result in food poisoning. The performance of the biosensor was not effected by the food materials and by the presence of other bacteria.

Compact fiber optic immunosensor using tapered fibers and acoustic enhancement

A compact fiber-optic sensing system that features all-fiber optical design and semiconductor-laser excitation has been developed and tested. A 2X2 fiber coupler directs the input light to the SMA connected sensing fiber tip and the fluorescent signal back to a CCD fiber spectrophotometer. In this system, the fluorescent signal is confined in the fiber system so the signal-to-noise ratio is greatly improved and the system can be operate in ambient light conditions. The utilization of a red laser diode has reduced the background signal of non-essential biomolecules. The fluorescent dye used is Cy5, which has an excitation wavelength of 650 nm and a fluorescent center wavelength of 680 nm. To illustrate the biosensors diagnostic capabilities, a sandwich immunoassay to detect Salmonella is presented. Tapered fiber tips with different shapes and treatments were studied and optimized. An enhancement system employing ultrasonic concentration of target particles has also been developed and applied to the detection of Salmonella. The immunoassay was conducted in a test chamber that also serves as an ultrasonic standing-wave cell and allows microspheres to be concentrated in a column along the fiber probe. The system demonstrates broad promise in future biomedical application.

Development of a near-real-time procedure to detect Staphylococcus aureus enterotoxin A in military rations

Using a chemiluminescent fiber optic biosensor and magnetic particles, a simple, sensitive and rapid method to determine Staphylococcus aureus enterotoxin A (SEA) in military ration components was developed. Anti-staphylococcal enterotoxin A (Anti-SEA) was immobilized on magnetic particles and incubated with SEA. The beads were then collected and rinsed on a membrane filter (0.45um). The captured toxin was then selectively labeled with a monoclonal-horseradish peroxidase (POD) conjugate. SEA concentration was detected with a luminometer and a chemiluminescent enhancing reagent. Total assay time was 1.25 hours. Chemiluminescent signal due to nonspecific binding was tested with various blocking agents. Phosphate buffered saline with casein had the lowest background signal. Primary antibody concentration, secondary labeled antibody concentration and chemiluminescent substrate type were also evaluated to optimize signal intensity. The chemiluminescent fiber optic biosensor assay was compared to the Analyte 2000, a commercial fluorescent fiber optic biosensor. This assay consisted of immobilizing Anti-SEA on polystyrene waveguides, and incubating the waveguides with the toxin. The waveguide was incubated with a selectively labeled monoclonal-CY5 Dye conjugate. The sensitivity of chemiluminescent and fluorescent immunoassays were 1 ng, significantly lower than the levels needed to cause illness.

Development of a chemiluminescent enzyme capture immunoassay for the detection of Escherichia coli

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.

Modeling of a tapered tubular optical waveguide probe for fluorescent immunosensors

A tubular optical probe for fluorescent immunosensors based on magnetic focusing of paramagnetic microspheres has been developed. The optical probe features a tubular optical waveguide tapered at both ends with a tapered magnet embedded inside the waveguide. Laser light is delivered to a dye-labeled spot in the inner surface of a cuvette and the fluorescent signal is guided to an optical detection system. A 3D computer model of this system has been created and its optical properties evaluated with a commercially available non-sequential ray-trace program. Up to 10,000 rays are traced from the laser to the cuvette and again from the fluorescent source to the detector. Results lead to the optimization of the component properties.