Detection of Uncharged 5-Fluorouracil Exploiting Sequential Adsorption of 5-Fluorouracil-Modified Bovine Serum Albumin Using Field Effect Transistor Biosensor

Abstract

The dosage of 5-fluorouracil (5-FU), which is a widely used for cancer medication, is determined based on body surface area, although efficacy largely depends on the liver function of the individual, resulting that only 21% patients are given an optimal dose of 5-FU in these years. The measurement of the 5-FU concentration in the blood enables us to adjust individual dose adjustment. Conventional methods for 5-FU detection such as an enzyme-linked immunosorbent assay (ELISA) and liquid chromatography are not very suitable for clinical applications because they need time-consuming procedure with expensive equipments. Detection of 5-FU using a field effect transistor (FET) biosensor, which enables rapid and simple measurement, is expected to solve such problems. However, FET biosensor, which detects changes in its surface density due to the adsorption of charged molecules, was unable to detect uncharged 5-FU. In this study, a method for the FET biosensing to detect 5-FU exploiting sequential adsorption of 5-FU modified bovine serum albumin (BSA/5-FU) was proposed. By using this method, FET responses caused by the adsorption of negatively charged BSA/5-FU depending on the 5-FU concentration were detected. The SiO2 surface of the FET gate insulator was exposed to O2 plasma to introduce hydroxyl groups. After the exposure, the surface was exposed to 3-aminopropyltriethoixysilane (APTES), followed by the modification of the cross-linker, glutaraldehyde (GA). A single chain variable fragments (ScFv) and antigen binding fragments (Fab) were allowed to react with each activated GA-modified FET. After the immobilization, the residual aldehyde-groups were treated by ethanolamine to suppress the non-specific adsorption. V g-I d characteristics were measured before and after dripping of both 5-FU and BSA/5-FU on the ScFv- and Fab-immobilized FET biosensors. Finally, threshold voltage shifts (∆V g) caused by the adsorption of BSA/5-FU were obtained. To compare the capture capability of ScFv and Fab, the electrical responses of the FET biosensors functionalized with the two receptors due to the adsorption of BSA/5-FU were measured. The responses of ScFv- and Fab-immobilized FET biosensor caused by dripping of 25 μg/mL BSA/5-FU were +25 mV and +40 mV, respectively. The difference between ΔV g values for these two FET sensors using ScFv or Fab can be ascribed to the difference of affinity [1]. To verify the specificity of Fab-immobilized FET biosensor, ∆V g was measured when human serum albumin (HSA) was dropped on the FET biosensor, and the ∆V g was hardly observed. Additionally, the atomic force microscopic (AFM) images on the FET gate surface shows that the size of observed particles matches the size of BSA/5-FU, while the surface morphology and roughness are not significantly changed. These results indicated that Fab-immobilized surface specifically captured BSA/5-FU. To investigate the quantitative detectability of the Fab-immobilized FET biosensor, we measured the FET responses corresponding to the amount of adsorbed BSA/5-FU, which was related with the concentrations of 5-FU. As a result, the magnitude of ∆V g by dripping of 1000 ng/mL 5-FU and 25 μg/mL BSA/5-FU was reduced to +12 mV compared with the response of 25 µg/mL BSA/5-FU (Figure 1). These results can be attributed to that the adsorbed 5-FU inhibited the adsorption of BSA/5-FU to the Fab-immobilized surface. Therefore, we conclude that the detection of 5-FU using the FET biosensors by applying the charged BSA/5-FU is a promising simple method for monitoring the concentration of 5-FU.Reference:[1] Y. Reiter, et al., J. Biol. Chem., 269, 28, 18327-18331 (1994).Figure 1 V g-I d characteristics of Fab-immobilized FET biosensor before and after dripping of (a) 0 ng/mL 5-FU and 25 µg/mL BSA/5-FU or (b) 1000 ng/mL 5-FU and 25 µg/mL BSA/5-FU. Figure 1