Capacitance-Based Biosensor for the Measurement of Total Loss of L-Amino Acids in Human Serum during Hemodialysis

Abstract

In this paper, we present a biosensor based on a gold nanoparticle (AuNP)-modified Pt electrode with an adjusted membrane containing cross-linked L-amino acid oxidase for the detection and quantification of total L-amino acids. The designed biosensor was tested and characterized using the capacitance-based principle, capacitance measurements after electrode polarization, disconnection from the circuit, and addition of the respective amount of the analyte. The method was implemented using the capacitive and catalytic properties of the Pt/AuNP electrode; nanostructures were able to store electric charge while at the same time catalyzing the oxidation of the redox reaction intermediate H₂O₂. In this way, the Pt/AuNP layer was charged after the addition of analytes, allowing for much more accurate measurements for samples with low amino acid concentrations. The combined biosensor electrode with the capacitance-based measurement method resulted in high sensitivity and a low limit of detection (LOD) for hydrogen peroxide (4.15 μC/μM and 0.86 μM, respectively) and high sensitivity, a low LOD, and a wide linear range for L-amino acids (0.73 μC/μM, 5.5 μM and 25-1500 μM, respectively). The designed biosensor was applied to measure the relative loss of amino acids in patients undergoing renal replacement therapy by analyzing amino acid levels in diluted serum samples before and after entering/leaving the hemodialysis apparatus. In general, the designed biosensor in conjunction with the proposed capacitance-based method was clinically tested and could also be applied for the detection of other analytes using analyte-specific oxidases.

Keywords: L-amino acid oxidase; L-amino acids; capacitance-based biosensor; human serum; hydrogen peroxide.

Figure 1

Electrochemical analysis of Pt and Pt/AuNP electrodes. (A) CVs of Pt electrodes without (black curve) and with 1.0 mM H₂O₂ (orange curve). (B) CVs of Pt/AuNP electrodes without (black curve) and with 1.0 mM H₂O₂ (orange curve). WBS (50 mM NaH₂PO₄·2H₂O and 100 mM NaCl, pH adjusted to 7.2), potential scan rate −5 mV/s.

Figure 2

Time–current curve of the Pt/AuNP electrode used to measure the capacitance after the addition of the analyte (H₂O₂). During the polarization (WE on), the electrode was kept at 400 mV vs Ag/AgCl to discharge the AuNP layer. During the depolarization stage (WE off), H₂O₂ was added and oxidized on the electrode, transferring the charge to the Pt/AuNP electrode. The black points demonstrate the measurement points, the orange curves – the interpolating curves, and the highlighted area in orange shows the accumulated electric charge. WBS (50 mM NaH₂PO₄·2H₂O and 100 mM NaCl, pH adjusted to 7.2). The slight oscillations visible in the graphs appeared when magnetic stirring of the solution was applied.

Figure 3

Calibration curve of the Pt/AuNP electrode using the charge capacitive method for the detection of H₂O₂ in the range of 0–20 μM. A typical time–current curve used to calculate the capacitance values is shown in Figure 2. Measurements to produce error = 3. WBS (50 mM NaH₂PO₄·2H₂O and 100 mM NaCl, pH adjusted to 7.2).

Figure 4

Electrochemical analysis and calibration of Pt/AuNP/Enz electrodes. (A) CV of the electrode without (black) and with (orange) 0.1 mM H₂O₂. Potential scan rate −5 mV/s. (B) CV of the electrode without (black) and with (green) 5.0 mM L-amino acids. Potential scan rate −5 mV/s. (C) Calibration curve of the Pt/AuNP/Enz electrode using the charge capacitance-based method for the detection of amino acids in the range of 0–100 μM. Time–current curves used to calculate capacitance values are given in Supporting Information, Figure S3. (D) Calibration curve of the Pt/AuNP/Enz electrode using the charge capacitive method for L-amino acids in the range of 0–1500 μM. Time–current curves used to calculate capacitance values are given in Supporting Information, Figure S5. Measurements to produce error = 3. WBS (50 mM NaH₂PO₄·2H₂O and 100 mM NaCl, pH adjusted to 7.2).

Figure 5

Analysis of serum samples. (A) Scheme demonstrating the origin of the serum samples analyzed. Two samples of the same patient were analyzed: serum obtained from blood before entering the hemodialysis apparatus (Sample A) and serum obtained from blood leaving the hemodialysis apparatus (Sample V). (B) Sample analysis with the Pt/AuNP/Enz electrode (absolute values). (C) Sample analysis with a Pt/AuNP/Enz electrode and an alternative colorimetric kit (relative values, that is, amino acid loss, %).