A Reliable BioFET Immunosensor for Detection of p53 Tumour Suppressor in Physiological-Like Environment

Abstract

The concentration of wild-type tumour suppressor p53_wt in cells and blood has a clinical significance for early diagnosis of some types of cancer. We developed a disposable, label-free, field-effect transistor-based immunosensor (BioFET), able to detect p53_wt in physiological buffer solutions, over a wide concentration range. Microfabricated, high-purity gold electrodes were used as single-use extended gates (EG), which avoid direct interaction between the transistor gate and the biological solution. Debye screening, which normally hampers target charge effect on the FET gate potential and, consequently, on the registered FET drain-source current, at physiological ionic strength, was overcome by incorporating a biomolecule-permeable polymer layer on the EG electrode surface. Determination of an unknown p53_wt concentration was obtained by calibrating the variation of the FET threshold voltage versus the target molecule concentration in buffer solution, with a sensitivity of 1.5 ± 0.2 mV/decade. The BioFET specificity was assessed by control experiments with proteins that may unspecifically bind at the EG surface, while 100pM p53_wt concentration was established as limit of detection. This work paves the way for fast and highly sensitive tools for p53_wt detection in physiological fluids, which deserve much interest in early cancer diagnosis and prognosis.

Keywords: BioFET; EGFET; immunosensors; p53.

Figure 1

(a) The sensor chip (10 × 40 mm² outer dimensions), with the extended-gate gold electrode (sensing area 20 mm²) and two Ag/AgCl pseudoreferences (dark semicircular area around the gold sensing area), connected to the home-made printed circuit board, where the commercial n-type MOSFET ALD110900A is plugged in and four plugs are available to connect the printed circuit board (PCB) with the source-meter units (SMUs). (b) Sketch of the microfabrication process of the sensor chip.

Figure 2

Main steps of gold extended-gate electrode biofunctionalization procedure (not to scale).

Figure 3

BioFET average transfer curves (MOSFET I_ds as a function of the V_ref applied by the reference electrode), with standard deviations, obtained by using as extended gate (EG) a biofunctionalized gold electrode (black curve) or a bare gold one (red curve). The V_ref applied during the biosensing experiments (400 mV) is highlighted by a vertical dashed line. The range of the starting I_ds values (around ten μA) is highlighted by the grey area. Inset: both extraction of the threshold voltage (V_th) from a representative curve, by a linear extrapolation method [35], and the correspondence between I_ds and V_th changes are shown.

Figure 4

Representative behaviour of the drain-source current (I_ds at constant V_ds = 100 mV) measured by an n-type MOSFET whose gate electrode is connected with a biofunctionalized gold extended gate immersed in an electrolyte solution (PBS 100 mM solution, pH 8.0), upon the application of a constant voltage (V_ref = 400 mV) through a bulky Ag/AgCl reference electrode.

Figure 5

Real time acquisitions of the n-type MOSFET drain-source current I_ds (subtracted by the starting value) in different biosensing experiments, performed in the same conditions (V_ds = 100 mV, V_ref = 400 mV, working buffer: PBS 100 mM, pH 8.0, starting volume: 350 μL). (a–c) Single injections of 20 μL of the working buffer with diluted p53_wt, resulting in a final concentration in the fluid cell of 100 pM, 1 nM and 10 nM. (d) Single injection of 20 μL of the working buffer with diluted HSA (1 nM final concentration). Vertical bars indicate the protein injection time.

Figure 6

Calibration curve of the BioFET for the detection of p53_wt, as obtained by plotting the average values of the threshold voltage (V_th) variation as a function of the p53_wt concentration in PBS 100 mM pH 8.0, on a semilogarithmic scale. The error bars represent the standard deviation. The regression line (R² = 0.96) is reported in red. From the slope of the regression line, the sensitivity σ = 1.5 ± 0.2 mV/decade is obtained. The grey area in the bottom represents the range of V_th variations obtained by injecting the working buffer with diluted other proteins (0.6 ± 0.4 mV), which can be considered as the blank signal.

Figure 7

Mean threshold voltage (V_th) changes as a function of the p53_wt concentration in PBS 100 mM pH 8.0 solution. The error bars represent the data standard deviation. The data have been fitted by an adapted version of the Langmuir adsorption model including an offset to take into account the blank signal (see text). The parameters extracted by fitting the experimental data according to the Langmuir adsorption model (continuous red line; R² = 0.96) are: offset ΔV₀ = 1.0 ± 0.4 mV, ΔV_thmax = (10.1 ± 2.2) mV, K_D = (2.2 ± 1.3) × 10⁻⁸ M.