Measurement of Neuropeptide Y in Aptamer-Modified Planar Electrodes

Abstract

Electrochemical impedance spectroscopy (EIS) is a powerful technique for studying the interaction at electrode/solution interfaces. The adoption of EIS for obtaining analytical signals in biosensors based on aptamers is gaining popularity because of its advantageous characteristics for molecular recognition. Neuropeptide Y (NPY), the most abundant neuropeptide in the body, plays a crucial role with its stress-relieving properties. Quantitative measurement of NPY is imperative for understanding its role in these and other biological processes. Although aptamer-modified electrodes for NPY detection using EIS present a promising alternative, the correlation between the data obtained and the adsorption process on the electrodes is not fully understood. Various studies utilize the change in charge transfer resistance when employing an active redox label. In contrast, label-free measurement relies on changes in capacitance. To address these challenges, we focused on the interaction between aptamer-modified planar electrodes and their target, NPY. We proposed utilizing -ω*Z_imag as the analytical signal, which facilitated the analysis of the adsorption process using an analogous Langmuir isotherm equation. This approach differs from implantable microelectrodes, which adhere to the Freundlich adsorption isotherm. Notably, our method obviates the need for a redox label and enables the detection of NPY at concentrations as low as 20 pg/mL. This methodology demonstrated exceptional selectivity, exhibiting a signal difference of over 20-to-1 against potential interfering molecules.

Keywords: Biosensing; Electrochemical impedance spectroscopy; Langmuir isotherm; Neuropeptide Y; Single layer adsorption.

Figure 1.

Schematic for modification of the Au surface of the working electrode with ssDNA-aptamer specific for NPY. The binding of NPY to the aptamer-modified electrode shows higher adsorption compared to DA, NE, and HT.

Figure 2.

(A) AFM image of Bare Au Electrode. (B) EDS of the surface-deposited Au. Electrochemical characterization of the aptamer-modified Au electrodes using (C) CV and (D) Nyquist plot.

Figure 3.

−ω*Zimag at different potentials of NPY at aptamer-modified Au electrodes.

Figure 4.

Measurement of NPY adsorption at high frequencies at 0.4 V vs Ag|AgCl. Linear fittings show the intercept and slope, all with R² > 0.99.

Figure 5.

Langmuir adsorption isotherm of the impedimetric concentrations of NPY at (A) −0.4 V, (B) 0.0 V, (C) 0.4 V, and (D) 0.8 V vs Ag|AgCl.

Figure 6.

(A) Drift at −0.4 V (circles) and 0.8 V (squares) vs Ag|AgCl in three measurements taken 5 min apart. (B) NPY measurements at different concentrations from 1 to 1,000 pg/mL with a linear relationship between 20 and 1,000 pg/mL when plotted as −ω*Z_imag vs Log C_NPY. For comparison, measurements of 5 nM of DA, NE, and 5-HT is shown as horizontal lines.

Figure 7.

NPY measurements at different concentrations from 1 to 1,000 pg/mL in aCSF with 10% HSA showing a linear relationship between 5 and 500 pg/mL when plotted as −ω*Z_imag vs Log C_NPY.