<title>Fluorescent immunosensors using planar waveguides</title>

Abstract

The goal of our research program is to develop competitive and sandwich fluoroimmunoassays with high sensitivity and fast response time, that do not require external reagents. Our approach to this problem is to employ an optical immunoassay based on total internal reflection fluorescence (TIRF). Specifically, monoclonal antibodies are immobilized on a planar waveguide. Total in1ernal reflection of light in the planar waveguide sets up an evanescent field which extends about 2000 A from the interface. In the competitive immunoassay, a fluorescent label is coupled to a small synthetic antigen which is packaged with the antibody. In the absence of analyte, the fluorescently-labeled antigen binds to the antibody and is excited by the evanescent field. Upon the addition of analyte, the fluorescently-labeled antigen molecules are displaced by unlabeled antigen molecules and diffuse out of the evanescent field. In the sandwich assay, a primary or "capture" antibody is immobilized on the planar waveguide, and a secondary or tracer" antibody (which is labeled with a fluorescent dye) is added to the bulk solution. In the absence of analyte, the tracer antibody remains in solution and very little fluorescence is observed. However, upon addition of analyte, a "molecular sandwich" is formed on the waveguide, composed of: (i) the capture antibody; (ii) the analyte; and (iii) the tracer antibody. Once this sandwich forms, the tracer antibody is within the evanescent field and fluoresces. Fluorescence emission is detected by a charged-coupled device (CCD). Using this approach, we have developed a prototype immunosensor for the detection of human chorionic gonadotropin (hCG). This device meets our design goals and exhibits a sensitivity of 0. 1 -1 prnolar.

Highly sensitive optical detection of specific protein in breast cancer cells using microstructured fiber in extremely low sample volume

“…[37][38][39][40] Human chorionic gonadotropin was detected through planar waveguide fluorescent immunosensors by sandwich assay using monoclonal antibody. 41,42 Comparatively, the newly developed assay significantly detect the ER␣ protein by direct immuno binding.…”

Toward a disposable real-time DNA biosensing platform

“…The channels are 400 µm wide, 100 µm tall, and the detection zones are 1 mm in diameter. The planar waveguide system is a method of detection which has been well documented [2][3][4][5], where the evanescent field from the waveguide excites a fluorescence signal from the target molecule captured on the waveguide surface. This is done without exciting the target molecules in the bulk of the solution above the waveguide surface, so that the overall selectivity is governed primarily by the reaction occurring on the surface.…”

TIRF Array Biosensor for Environmental Monitoring

“…Planar waveguides offer a number of advantages compared to fiber optic technology, including the relative ease of preparation and integration into fluidic systems. Researchers in the field have immobilized capture biomolecules uniformly over the planar surface and monitored the fluorescent signal intensity either as a function of time or the concentration of the labeled binding partner [73,74,81,82,101].…”

“…The avidin-biotin interaction is also extensively used in the immobilization of biotinylated molecular recognition elements. This noncovalent protein-ligand interaction is commonly used either via the physical adsorption of avidin onto the surface [67,71,73,76,101) or in the production of multi-layers; often involving the use of both covalent and noncovalent interactions [34,36,84,102).…”