Stephen V. Letcher

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.

Acoustic standing-wave enhancement of a fiber-optic Salmonella biosensor

A fluorescent fiber-optic biosensor system using ultrasonic concentration of particles and cells for the detection of Salmonella typhimurium. A biosensor test chamber serves as an ultrasonic standing-wave cell that allows microspheres or cells to be concentrated in parallel layers or in a column along the axis of the cell. A fiber probe along the axis delivers laser excitation to fluorescent-labeled antibodies of Salmonella and collects the fluorescent signal. The Salmonella-antibody complexes are moved acoustically to the axis of the cell, increasing the fluorescent signal. Alternatively, the Salmonella-labelled antibody complexes attach to unlabeled antibodies that have been immobilized on the surface of polystyrene microspheres. This entire structure can be manipulated acoustically and the increase in the fluorescent signal, which can be an order of magnitude, indicates the presence of Salmonella.

Impedance characterization of a piezoelectric immunosensor part II: Salmonella typhimurium detection using magnetic enhancement.

This study investigated the usefulness and characteristics of a 5-MHz quartz crystal resonator as a sensor of biological pathogens such as Salmonella typhimurium. An impedance analyzer measured the impedance behavior of the oscillating quartz crystal exposed to various concentrations of Salmonella (10(2)-10(8) cells per ml). The Salmonella cells were captured by antibody-coated paramagnetic microspheres, and then these complexes were moved magnetically to the sensing quartz and were captured by antibodies immobilized on the crystal surface. The response of the crystal was expressed in terms of equivalent circuit parameters. The motional inductance and the motional resistance increased as a function of the concentration of Salmonella. The viscous damping was the main contributor to the resistance and the inductance in a liquid environment. The load resistance was the most effective and sensitive circuit parameter. A magnetic force was a useful method to collect the complexes of Salmonella-microspheres on the crystal surface and enhance the response of the sensor. In this system, the detection limit, based on resistance monitoring, was about 10(3) cells per ml.

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).

A wave vector, time‐domain method of forward projecting time‐dependent pressure fields

A wave vector, time‐domain (k−t) method of forward projecting time‐dependent pressure fields from complex vibrators is developed using space‐time Fourier transform methods. Axisymmetric fields are included as a special case of the general formulation in which a pressure field at one plane can be forward projected to other planes. In brief, a wave vector, time‐domain representation of the field at one plane is forward projected via a temporal convolution with a wave vector, time‐domain impulse response that is dependent on the projection distance. The projected field is then obtained via the use of an inverse spatial Fourier transform. A numerical study of the method illustrates the accuracy of the approach when implemented using fast Fourier transform (FFT) algorithms. The forward projections of simulated pressure fields from a planar ultrasonic transducer are shown to be in excellent agreement with corresponding results from the use of a time‐domain impulse response method.

Impedance characterization of a piezoelectric immunosensor. Part I: antibody coating and buffer solution.

This study investigated the impedance characteristics of a 5 MHz quartz crystal resonator oscillating in a thickness-shear mode for utilization as a biosensor. An impedance analyzer measured the impedance of the quartz crystal, which determined all mechanical properties of the oscillating quartz and its immediate environment. In this study, the impedance behavior of the oscillating crystal was characterized in air, in potassium phosphate buffer solution, and with immobilization of antibodies using protein-A. The potassium phosphate buffer behaved like a Newtonian liquid. The series resonance frequency shifted down about 900 Hz on contact with the buffer. An immobilized-antibody layer on the quartz surface behaved like a rigid mass when immersed in the buffer solution. The impedance curve following immobilization of antibodies was shifted down in frequency by about 200 Hz compared with its value when the bare crystal was immersed in the buffer solution.

Tapered tubular optical waveguide probe for magnetic focusing immunosensors

A novel fluorescence immunosensor using a tapered tubular optical waveguide probe for analyte detection has been developed based on magnetic focusing of paramagnetic microspheres. The new design features a tubular optical waveguide tapered at both ends with a tapered magnet embedded inside of the waveguide. The excitation light is injected from the middle part of the tubular waveguide and is guided to the front end where it illuminates paramagnetic particles attracted by the magnet. The associated fluorescent signal is collected by the optical probe and is guided to the distal end, where it is connected to the optical detection system. The waveguide thus serves multiple purposes: the front end of the waveguide serves as the optical probe while the rear end serves as the connector to the signal transmission fiber; the waveguide body serves as a holder for the magnet, a directional coupler for the excitation and a high split-ratio coupler for the fluorescent signal.

Forward projection of transient signals obtained from a fiber-optic pressure sensor

An analytical/experimental approach is presented to reconstruct the space‐time pressure field in a plane and forward project the resultant space‐time pressure field using tomographic and wave‐vector frequency‐domain methods. Transient pressure signals from an underwater ultrasonic planar transducer are first measured using a fiber‐optic pressure sensor which is scanned across a plane at a fixed distance from the transducer. The resulting spatial line integrals in the plane are time‐dependent signals that are first used to reconstruct the space‐time pressure field in the plane via simply implemented tomographic methods. These signals are then used to forward project the space‐time pressure field to arbitrary planes further from the transducer. Numerical results will be presented for transient signals to illustrate both the projection and the detection techniques. The results of the projected fields will be compared at various distances for synthetic signals and experimental data. [Work supported, in part, by the URI Ocean Technology Center and by ONR.]

Determination of fracture parameters using embedded fiber-optic sensors

The applicability of embedded fiber-optic sensors for the determination of fracture parameters is demonstrated. A Mach-Zehnder interferometric setup is used and mode-1 stress-intensity factors are obtained by embedding single-mode fibers in single-edge-notched specimens fabricated from Plexiglas. Optical fibers are embedded in-plane to measure axial strains at various depths and also in the transverse direction to measure the transverse strains, from which stress-intensity factors are determined. In both cases the experimental results compare well with the theoretical predictions.

The relationship between the impulse response and angular spectrum methods to evaluate acoustic transient fields

Impulse response time domain and angular spectrum or wave vector time‐domain methods can be used to investigate the spatial temporal characteristics of acoustic fields from planar ultrasonic transducers which are subjected to pulsed wideband excitations. Both methods are developed here from the time‐dependent Green’s function solution of the initial‐boundary value problem. The close relationship of the methods is made apparent by the use of spatial‐temporal Fourier transform techniques of analysis. Axisymmetric fields are then addressed as a special case via the use of Hankel and Fourier transform techniques. Finally, classical solutions to the associated harmonic boundary value problems are then shown to result from temporal Fourier transforms of the time‐dependent field solutions.