Walter F. Love

The application of evanescent wave sensing to a high-sensitivity fluoroimmunoassay

Abstract The principle of evanescent wave sensing was applied to immunological measurements of the clinically important human enzyme creatine kinase (CK; EC 2.7.3.2) isoenzyme MB form (CK-MB) with a molecular weight of about 84 000. The measurements were obtained using a green helium-neon laser based fluorimeter and the antibody conjugated fluorophor B-phycoerthrin (BPE). With conservative optical launch and collection angles of 25°, a dection limit of 0·1 ng ml −1 CK-MB was observed in less than 15 min for a fiber optic based assay in which there were no subsequent washing, handling or processing steps after exposure to the sample.

Optical Characteristics of Fiberoptic Evanescent Wave Sensors

The availability of low-loss optical fibers with the associated benefits of freedom from electromagnetic interference, small size, light weight, and wide bandwidth has led to the development of sensors, both intrinsic and extrinsic. Commercial offerings of the extrinsic type, in which the sensing element is attached to the end of the fiber, are becoming readily available for the measurement of many physical and chemical parameters, e.g., temperature, pressure, and pH. The intrinsic sensors, in which the optical fiber itself functions as the sensor, potentially offer more sensitivity and versatility. In some cases, distance or location information may be obtained with intrinsic sensors by means of optical-pulse techniques. The intrinsic sensors are usually more complex in their instrumentation or environmental requirements, and their entry into the commercial market has proceeded more slowly.

Fiber Optic Evanescent Sensor for Fluoroimmunoassay

The application of optical fibers as biosensors is a topic of increasing interest and importance. The evanescent wave technique with its short interaction distance is ideally suited to the measurement of biomolecules closely attached to the surface of a fiber.1 The use of a fluorescent tag attached to either antibody or antigen analytes thereby allows a quantitative measure of concentration from the fluorescence emanating from the fiber.2

Application of a plastic evanescent-wave sensor to immunological measurements of CKMB

Abstract The principles of evanescent-wave fluorescent spectroscopy have been applied to the development of an immunoassay for creative kinase (CK, EC 2.7.3.2) isoenzyme MB form (CKMB) with a molecular weight of about 84 000 using an injection-molded conical sensor. The sensor element is designed to: (i) provide an active surface area (157 mm2) similar to that of fiber-optic sensors; (ii) reduce the requirements for a high numerical launch aperture NA from 0.6 to approximately 0.1 or less; and (iii) provide a robust element capable of being handled by non-critical surfaces for possibble commercial applications. The resultant blunt-ended cone is constructed from a substrate of poly(methylmethacrylate) having a refractive index n1530 = 1.4952 and a propagation surface angled at 23° with respect to the cone axis. The cones are coated with mouse antibodies against CKMB and used in conjunction with a monoclonal antibody covalently coupled to the fluorescent tracer B-phycoerythrin.

Feasibility study of a plastic evanescent-wave sensor

Abstract The initial design, material and feasibility study for the application of a plastic substrate to an evanescent-wave immunosensor are described. The sensor represents a departure from the fiber-optic and planar waveguide approaches as it is of conical design. It offers possibilities for inexpensive manufacture, processing and ease of incorporation into a disposable unit, since it is injection molded from poly(methyl methacrylate). The low background characteristics and initial performance as an evanescent-wave sensing element are presented. A fluorescence-based competitive assay for digoxin is shown for the range 5–10 ng/ml, which is above the clinical range needed.

Sensitive fiber-optic immunoassay

The principles of evanescent wave theory were applied to an immunological sensor for detecting the cardiac-specific isoenzyme creatine kinase-MB (CK-MB). The detection of the CK-MB isoenzyme is used in conjunction with the total CK measurement in the diagnosis of acute myocardial infarction. The clinical range for CK-MB is from 2-100 ng/ml. Previous work which utilized the fluorophor, Fluorescein isothiocyanate (FITC), was able to discriminate between 0 and 3 ng/ml CK-MB. Use of the fluorophor B-phycoerythrin (BPE) increased the assay sensitivity to 0.1 ng/ml CK-MB. The data was collected for 15 minutes using an optical launch and collection angle of 25 degree(s). This fiber optic based system is homogeneous and requires no subsequent washing, handling, or processing steps after exposure to the sample.

Theoretical considerations for evanescent-wave immunosensors in biomedical applications

Using evanescent wave immunosensors, a fluorescent labeled analyte may be concentrated within the active surface region by a combination of diffusion to the fiber surface and trapping of the molecule or complex by an antigen-antibody affinity reaction. With the dye B- phycoerythrin, approximately 1.5 X 10-22 moles may be sensed over a 1 mm2 surface. From this number (as determined for a particular dye and instrumentation system) and antibody affinity constants, limits to assay sensitivity can be calculated and the kinetics modeled.

Semi-Automated Measurements Of The Transmission Medium

The measurement sequence for the characterization of graded-refractive-index optical waveguide fibers in a manufacturing environment is presented. The sequence has been designed for minimum human interaction and high throughput. The optical fibers, characterized by attenuation rate, bandwidth, numerical aperture, and optical core diameter, are measured on a quality assurance system driven by a mini-computer. This semi-automated system, consisting of optical and electronic hardware and software for complete characterization, is described. Finally, the measurement methods and current test conditions are presented.