Detection of tumor markers is important for cancer diagnosis. Field-effect transistors (FETs) are a promising method for the label-free detection of trace amounts of biomolecules. However, detection of electrically charged proteins using antibody-immobilized FETs is limited by ionic screening by the large probe molecules adsorbed to the transistor gate surface, reducing sensor responsiveness. Here, we investigated the effect of probe molecule size on the detection of a tumor marker, α-fetoprotein (AFP) using a FET biosensor. We demonstrated that the small receptor antigen binding fragment (Fab), immobilized on a sensing surface as small as 2–3 nm, offers a higher degree of sensitivity and a wider concentration range (100 pg/mL–1 μg/mL) for the FET detection of AFP in buffer solution, compared to the whole antibody. Therefore, the use of a small Fab probe molecule instead of a whole antibody is shown to be effective for improving the sensitivity of AFP detection in FET biosensors. Furthermore, we also demonstrated that a Fab-immobilized FET subjected to a blocking treatment, to avoid non-specific interactions, could sensitively and selectively detect AFP in human serum.

The early detection of cancer biomarker is critical for the survival of the patients. A variety of technologies have been developed for biomarker detection, such as enzyme-linked immunosorbent assay (ELISA). However, most of them suffer from a complicated labeling process, time-consuming and relatively insensitive detection. Field effect transistor (FET) has been recognized as a powerful technique, because of the prospect for sensitive, label-free, real time and multifunctional biosensing [1-3]. Yet, the detection of low-level concentrations of cancer biomarker is severely hampered by charge screening effect. The use of small receptor enables immune-binding reaction to occur within the Debye length, resulting in the enhancement of the sensitivity and detection limit of the sensing system. In this work, we developed an FET biosensor for label-free detection of alpha-fetoprotein (AFP), a liver cancer marker. In order to improve the sensitivity of FET biosensor, this biosensor was designed by immobilizing antigen binding fragment (Fab) with small size as a receptor (Figure 1). The immobilization of Fab molecules was carried out on the SiO2 gate surface of FET devices modified with monolayer of 3-aminopropylsilane by using glutaraldehyde as a cross-linker. The gate voltage (V g)-drain current (I ds) characteristics were measured before and after the reaction between cancer biomarker (antigen) in the test solution and receptor (antibody or Fab) on the gate. A shift of threshold voltage (ΔV g), evaluated from a linear region of the FET characteristics was discussed as an FET response. We have experimentally compared the sensitivity of the Fab-immobilized FET biosensors with that of antibody-immobilized one (Figure 2). Electrical measurement results showed that the use of Fab results in more than twice higher response to AFP of the same concentration than the use of antibody. It should be noted that the response of Fab-immobilized FET to 100 pg/mL AFP is still greater than the response of antibody-immobilized FET to 10 ng/mL AFP. Considering the fact that both FETs shows the response as small as 5 mV to 1 μg/mL human serum albumin (HSA) added as a negative control, it was demonstrated that, compared to the antibody-immobilized FET, the Fab-immobilized FET has similar specificity to AFP and detection ability to two-digit lower concentrations. Thus, the sensitivity of FET biosensor for label-free detection of cancer biomarker was improved by the use of Fab with small size instead of antibody. The calibration scheme of Fab-immobilized FET biosensor will be discussed at the conference. Acknowledgements This work was financially supported in part by Grants for Excellent Graduate Schools (Practical Chemical Wisdom), and by Global COE program “Center for Practical Chemical Wisdom”, both from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. References [1] D. Niwa, K. Omichi, N. Motohashi, T. Homma, T. Osaka, Sens. Actuators B, 108, 721 (2005). [2] S. Hideshima, R. Sato, S. Inoue, S. Kuroiwa, T. Osaka, Sens. Actuators B, 161, 146 (2012). [3] S. Hideshima, R. Sato, S. Kuroiwa, T. Osaka, Biosens. Bioelectron., 26, 2419 (2011).