HMDS PPF has hydrophobic properties [19,20], and other films prepared with monomers such as allylamine show hydrophilic natures [18]. microplates to approximately 25% of those obtained with the glass slides are considered to be due to the deep well structure of the microplate. Relatively large errors found in the calibration curves with the microplates may result from the disassembling process of the microplate for the thickness measurement as described in the Materials and Methods section. Open in a separate window Figure 2 Calibration curves for silver layer (a) or PPF (b) thickness as a function of deposition time obtained with glass slides (black circles) and microplates (white circles). Error bars indicate the standard deviation of five measurements. The appearance of the multilayered microplate is shown in Figure 3. It looks like a microplate made of metal because of the presence of the silver layer coated with transparent HMDS PPF. Through the deposition experiments it was shown that a polystyrene microplate was durable in the vacuum chambers during deposition by sputtering and plasma polymerization. It is also of significance that the PPF modification was possible for a complicated geometry such as that of a microplate with a deep Cephalexin monohydrate well Cephalexin monohydrate structure, which is one of the advantages of the plasma polymerization technique. Open in a separate window Figure 3 Modules of unmodified (upper) and multilayered (lower) microplates modified with a 200-nm-thick silver layer and coated with a 53-nm-thick HMDS PPF. 3.2. Homogeneous Fluorescence Detection of Cy3-Labeled Antibody in the Multilayered Microplate Before conducting the immunoassay, the basic performance of a multilayered microplate was examined by homogeneous fluorescence detection. A microplate for enzyme-linked immunosorbent assay (ELISA) was modified with a 200-nm-thick Ag layer and coated with a 53-nm-thick HMDS PPF. In our previous study the maximum fluorescence enhancement was obtained when the Cephalexin monohydrate PPF thickness on a glass slide was 63 nm [33]. However, it Rabbit Polyclonal to Actin-pan was also confirmed that almost the same degree of the enhancement effect could be observed between approximately 50C70 nm. Cy3-labeled anti-mouse IgG antibody (100 L of 1 1.6 g/mL solution) was applied to the multilayered Cephalexin monohydrate and unmodified microplates, followed by immediate fluorescence measurement by the scanner. Figure 4a shows the greatly enhanced fluorescence observed from the multilayered microplate without any immobilization step of the fluorophore-labeled protein, while the fluorescence signal was small from the unmodified microplate. This enhancement may be attributed to the fluorophore-labeled protein located approximately 60 nm above the surface of PPF. The fluorescence intensity from the multilayered microplate was approximately 18-fold compared with that from the unmodified one (Figure 4b). Open in a separate window Figure 4 Enhancement of fluorescence intensity from Cy3-labeled antibody on the multilayered microplate. Cephalexin monohydrate (a) Two-dimensional fluorescence image of the unmodified and multilayered microplate. PBS was added to wells of both microplates as a negative control; (b) Comparison of fluorescence signals. Error bars indicate the standard deviation of three measurements. Akimoto and co-workers demonstrated that a maximum 200-fold enhancement could be achieved with Al2O3 as the optical interference layer on the glass slide [12]. In this case, however, the analyte was spin-coated Rhodamine B, whereas Cy3-labeled antibody solution in PBS was applied in the modified microplate in.