Evaluation of a New CA15-3 Protein Assay Method: Optical Protein-Chip System for Clinical Application
Carbohydrate antigen 15-3 (CA15-3) is frequently measured as a breast cancer marker test. Here we describe a novel type of optical biosensor system, the optical protein chip (OPC), to detect CA15-3 in serum. The complex formed by interaction between an antibody molecule and its corresponding antigen can be detected on a silicon substrate by an optical sensor, as described in previous reports (1, 2 ). For processing and modification of the silicon substrate surface, silicon wafers were cut into
... fers were cut into ϳ2 ϫ 0.7 cm rectangles and made hydrophilic by immersion in an acidic peroxide solution (300 g/L H 2 O 2 -980 g/L H 2 SO 4 ; 1:3 by volume) and light shaking in a shaker for 30 min. The solution not only removed contaminants from the silicon surface but also increased the number of silanol groups on the surface. The hydrophilic surfaces were rinsing in distilled water 3 times and in absolute ethanol 3 times, then incubated in a mixture of 3-aminopropyltriethoxysilane and ethanol (1:15 by volume) and shaken lightly in a shaker for 2 h; The mixture liquid was then removed, and the silicon wafers were rinsed in absolute ethanol 3 times and in phosphate-buffered saline (PBS) buffer 3 times, then placed in a mixture of glutaraldehyde and PBS (1:10 by volume), shaken lightly in a shaker for 1 h, and finally, washed in PBS buffer 3 times and left in a beaker with PBS buffer until use. Through the reaction of glutaraldehyde with 3-aminopropyltriethoxysilane, Fc regions of the antibody molecules were covalently immobilized on the chip surfaces. Protein chip preparation and detection included the following steps: (a) CA15-3-specific monoclonal antibody (Biodezign) was concentrated to 0.1 g/L, and then 20 L of CA15-3 solution was delivered individually to each analytical spot on the chip by a microfluidics system (MFS) at a flow rate of 2 L/min for 10 min. (b) After the entire volume of solution flowed onto each analytical spot on the silicon surface, 40 L of diluted water was delivered individually to each spot on the chip by the MFS at a flow rate of 8 L/min for 5 min to remove all nonadsorbed CA15-3 monoclonal antibody molecules on the analytical spot surface. (c) After the entire volume of diluted water flowed onto the analytical spots, 20 L of a 1 g/L bovine serum albumin solution was delivered in the same way at a flow rate of 2 L/min for 10 min to block nonspecific binding. (d) The chip was rinsed with 50 L of diluted water in the same way at a flow rate of 10 L/min for 5 min. (e) Serum samples were diluted with equal volumes of Tween 20 (20 mL/L) to a final volume of 50 L, then the diluted samples were delivered individually to each analytical spot on the chip by the MFS at a flow rate of 2 L/min for 25 min until the entire serum solution had flowed onto the analytical areas. (f) The chip was rinsed with 100 L of diluted water in the same way at a flow rate of 20 L/min for more than 5 min. (g) The chip was removed from the MFS and dried under a stream of nitrogen. The thicknesses of layers in the analytical areas were measured with the biosensor imaging ellipsometry, which produced an ellipsometric image of a surface of each chip with a lateral resolution of 2 m.