Label-Free Electrochemiluminescence Nano-aptasensor for the Ultrasensitive Detection of ApoA1 in Human Serum

Abstract

A molybdenum sulfide/zirconium oxide/Nafion (MoS₂/ZrO₂/Naf) based electrochemiluminescence (ECL) aptasensor for the selective and ultrasensitive detection of ApoA1 is proposed, with Ru(bpy)₃ ²⁺ as the luminophore. The chitosan (CS) modification on the nanocomposite layer allowed glutaraldehyde (GLUT) cross-linking, resulting in the immobilization of ApoA1 aptamers. Scanning electron microscopy, tunneling electron microscopy, and energy dispersive X-ray spectroscopy were used to characterize the nanocomposite, while electrochemiluminescence (ECL), cyclic voltammetry, and electrochemical impedance spectroscopy were used to analyze the aptasensor assembly. The nanocomposite was used as an electrode modifier, which increased the intensity of the ECL signal. Due to the anionic environment produced on the sensor surface following the specific interaction of the ApoA1 biomarker with the sensor, more Ru(bpy)₃ ²⁺ were able to be electrostatically attached to the aptamer-ApoA1 complex, resulting in enhanced ECL signal. The ECL aptasensor demonstrated outstanding sensitivity for ApoA1 under optimal experimental conditions, with a detection limit of 53 fg/mL and a wide linear dynamic range of 0.1-1000 pg/mL. The potential practical applicability of this aptasensor was validated by analyzing ApoA1 in human serum samples, with recovery rates of 94-108% (n = 3). The proposed assay was found to be substantially better compared to the commercially available enzyme-linked immunosorbent assay method, as reflected from over 1500 times improvement in the detection limit for ApoA1.

Figure 1

Fabrication steps of the ApoA1 aptasensor.

Figure 2

TEM images of (A) MoS₂, (B) ZrO₂, and (C) MoS₂/ZrO₂/Naf.

Figure 3

Layer-by-layer characterization of ApoA1 aptasensor. (A) Cyclic voltammograms of (a) bare SPCE, (b) SPCE/Naf, (c) SPCE/MoS₂/ZrO₂/Naf, (d) SPCE/MoS₂/ZrO₂/Naf/CS/GLUT, (e) SPCE/MoS₂/ZrO₂/Naf/CS/GLUT/Apt, (f) SPCE/MoS₂/ZrO₂/Naf/CS/GLUT/Apt/BSA, and (g) SPCE/MoS₂/ZrO₂/Naf/CS/GLUT/Apt/BSA/ApoA1 recorded in 0.01 M PBS solution (pH 7.4) containing 5 mM [Fe(CN)₆]^3–/4– and 0.1 M KCl. (B) Cyclic voltammograms of (a) bare SPCE, (b) SPCE/MoS₂/ZrO₂/Naf, (c) SPCE/MoS₂/ZrO₂/Naf/CS/GLUT, (d) SPCE/MoS₂/ZrO₂/Naf/CS/GLUT/Apt/BSA, and (e) SPCE/MoS₂/ZrO₂/Naf/CS/GLUT/Apt/BSA/ApoA1 recorded in 0.01 M PBS solution (pH 7.4) containing 5 mM Ru[(NH₃)]₆³⁺ and 0.1 M NaCl. (C) EIS of (a) bare SPCE, (b) SPCE/MoS₂/ZrO₂/Naf, (c) SPCE/MoS₂/ZrO₂/Naf/CS/GLUT, (d) SPCE/MoS₂/ZrO₂/Naf/CS/GLUT/Apt/BS, and (e) SPCE/MoS₂/ZrO₂/Naf/CS/GLUT/Apt/BSA/ApoA1 recorded in 0.01 M PBS solution (pH 7.4) containing 5 mM [Fe(CN)₆]^3–/4– and 0.1 M KCl.

Figure 4

(A) ECL layer-by-layer characterization of ApoA1 aptasensor: (a) bare SPCE, (b) SPCE/MoS₂/ZrO₂/Naf, (c) SPCE/MoS₂/ZrO₂/Naf/CS/GLUT, (d) SPCE/MoS₂/ZrO₂/Naf/CS/GLUT/Apt/BSA, and (e) SPCE/MoS₂/ZrO₂/Naf/CS/GLUT/Apt/BSA/ApoA1. Chronocoulometry of (B) (a) aptasensor in the absence of ApoA1 and (b) aptasensor in the presence of ApoA1 recorded in 0.01 M PBS solution (pH 7.4) containing 5 mM [Fe(CN)₆]^3-/4- and 0.1 M KCl and (C) (a) aptasensor in the absence of ApoA1 and (b) aptasensor in the presence of ApoA1 recorded in 0.01 M PBS solution (pH 7.4) containing 5 mM Ru[(NH₃)]₆³⁺ and 0.1 M NaCl. The insets show the bar graph representations of the CC data.

Figure 5

Analytical performance of the aptasensor. (A) ECL response of the proposed aptasensor in detecting ApoA1 from concentrations: (a) 0.1 pg/mL, (b) 1 pg/mL, (c) 10 pg/mL, (d) 50 pg/mL, (e) 100 pg/mL, (f) 5 pg/mL, (g) 750 pg/mL, and (h) 1000 pg/mL in PBS (0.01 M, pH 7.4) containing 5 mM TPrA. (B) Calibration curve of ApoA1 aptasensor at concentrations ranging from 0.1 pg/mL to 1000 pg/mL. (C) Calibration curve of fabricated aptasensor (ECL) and ELISA (absorbance) for ApoA1 detection.