A fluorescence-based immunosensor using a charge-coupled device (CCD) as a detector has been developed for detecting multiple analytes within small (150 μl) sample volumes. Wells approximately 2 mm in diameter
were patterned onto glass coverslips using a photoactivated optical adhesive. In a model detection system, four different antigens were covalently attached to the bottoms of these wells, and the polymer subsequently removed to form the sensing surface. The coverslips were mounted over a scientific grade CCD operating at ambient temperature in inverted (multipin phasing) mode. A two-dimensional graded index of refraction (GRIN) lens array was used to focus the sensing surface onto the CCD. Solutions of fluorescently labeled antibodies were then placed on the coverslip, and the amount of antibody bound at each location on the coverslip was determined by quantitative image analysis. Concentrations as low as 50 ng/ml of Cy5-labeled
antibodies could be detected using the sensor. The small footprint and minimal system requirements of the design should facilitate its incorporation into portable biosensors.