Philip E. Pivarnik

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.

Cy5 labeled antimicrobial peptides for enhanced detection of Escherichia coli O157:H7

Fluorescently labeled antimicrobial peptides were evaluated as a potential replacement of labeled antibodies in a sandwich assay for the detection of Escherichia coli O157:H7. Antimicrobial peptides naturally bind to the lipopolysaccharide component of bacterial cell walls as part of their mode of action. Because of their small size relative to antibodies peptides can bind to cell surfaces with greater density, thereby increasing the optical signal and improving sensitivity. This method combines the specificity of a capture antibody with the increased sensitivity provided by using a labeled peptide as a detection molecule. The antimicrobial peptides cecropin P1, SMAP29, and PGQ were labeled with the fluorescent dye Cy5 via maleimide linker chemistry. Preliminary screening using a whole-cell solution binding assay revealed that Cy5 cecropin P1 enhanced the detection of E. coli O157:H7 relative to a Cy5 labeled anti-E. coli O157:H7 antibody 10-fold. Detection sensitivity of antibody and peptide were also compared with a prototype immuno-magnetic bead biosensor. Detection using Cy5 cecropin P1 resulted in a 10-fold improvement in sensitivity. Correlation of peptide antimicrobial activity with detection of E. coli O157:H7 indicated that activity was not predictive of the sensitivity of the fluorescent assay.

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).

Chemical synthesis and surface activity of lung surfactant phospholipid analogs. II. Racemic N-substituted diether phospholipids

A series of racemic 16:0 disaturated N-substituted diether phosphonolipid analogs of glycerophospholipids have been synthesized and purified. Isosteric methylene substitution at three of the four ester sites (carboxyl, phosphate) of conventional glycerophospholipids enhanced the hydrophobicity of analog compounds compared with dipalmitoyl phosphatidylcholine (DPPC), the major glycerophospholipid component of lung surfactant. Further substitutions at the nitrogen headgroup also contributed to hydrophobicity/hydrophilicity characteristics, as well as allowing graded variations in headgroup size among the members of the diether phosphonolipid analog series. Interfacial property studies showed that these compounds had significant differences in surface activity characteristics compared with DPPC, including increased adsorption and respreading facility, plus an enhanced ability to generate low surface tension (< 1 to 4 mN/m) on an oscillating bubble apparatus at 37°C. In addition, pressure-volume mechanical studies in surfactant-deficient excised rat lungs showed that the diether phosphonate analog of DPPC could partially restore pressure-volume characteristics toward normal, both as a pure component and in binary could partially restore pressure-volume characteristics toward normal, both as a pure component and in binary mixtures with palmitoyl-oleoyl phosphatidylglycerol. These findings suggest that selected analog compounds, synthesized with relatively small structural modifications from biologic glycerophospholipids, may have eventual applications as components of synthetic exogenous lung surfactants. Of more immediate importance, analog molecules with defined structural variations are convenient molecular probes for developing structure-surface activity correlates for phospholipid-like surfactants and for investigating the specificity of interactions between glycerophospholipids and other compounds such as proteins.

Chemical synthesis and surface activity of lung surfactant phospholipid analogs. III. Chiral N-substituted ether-amide phosphonolipids

A homologous series of chiral (R) ether-amide phosphonolipid analogs of naturally occurring (R) glycerophospholipids were synthesized and characterized for their interfacial behaviors. The phosphonolipids possess isoteric ether, amide, and phosphonate functions at positions corresponding to the sn-1, sn-2, and sn-3 ester functions, respectively, of naturally occurring glycerophospholipids. All compounds were synthesized with disaturated C16:0 alkyl/acyl moieties to give structural analogy with dipalmitoyl phosphatidylcholine (DPPC), the major glycerophospholipid component of lung surfactant. Further substitutions at the headgroup nitrogen were also used to generate differences in headgroup size and polarity in the synthetic compounds. The surface activity of the ether-amide phospholipids was investigated in terms of adsorption to the air-water interface, together with studies of dynamic respreading after monolayer collapse and surface tension lowering in dynamically compressed spread films and dispersions. Results showed that several ether-amide phosphonolipids had more rapid adsorption and improved dynamic respreading behavior compared to DPPC, plus the ability to lower surface tension into the range of less than 1 to 4 mN/m in spread films and in dispersions under dynamic conditions. In combination with a series of diether phosphonolipids synthetized in a companion study [1], these ether-amide compounds are useful in the development of molecular structure-surface activity correlates for lung surfactant-related materials, and should assist in investigating the specificity of interactions between phospholipids and other pulmonary biological molecules.

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.

Novel membrane technology for food and water monitoring

The need exists to improve sensitivity of detection of toxic pollutants and pathogenic microorganisms, ensuring food and water safety. Developing methods that would increase antibody binding surface area and/or improve the sampling process by specifically concentrating the analyte of interest from the diluted extracted food sample would increase the chances of finding and detecting food pathogens and their toxins. Our approach to improve sensitivity was to generate high surface nanofibrous membranes with covalently attached molecular recognition elements (MREs, e.g. antibodies and peptides) for the selective capture of target analytes through the use of electrospinning. Electrospinning is a process by which high static voltages are used to produce an interconnected membrane-like web of small fibers with diameters ranging from 50-1000 nanometers. These nanofibrous membranes can have surface areas approximately one to two orders of magnitude higher than those found in continuous films. The association of MREs with electrospun fibers presents the opportunity for developing both biosensor detection platforms with increased surface area and membrane concentrators. It is expected that the available surface area demonstrated by this technique will provide increased sensitivity, capture efficiency and fast response time in sensing applications. Antibodies and peptide-based receptors were selectively immobilized onto these nanoporous membranes for bioaffinity capture. Initial results involving fluorescent and chemiluminescent imaging for quantifying attachment and activity in association with the electrospinning process will be discussed.

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.