Resorc[4]arene-Modified Gold-Decorated Magnetic Nanoparticles for Immunosensor Development

Abstract

In recent years, several efforts have been made to develop selective, sensitive, fast response, and miniaturized immunosensors with improved performance for the monitoring and screening of analytes in several matrices, significantly expanding the use of this technology in a broad range of applications. However, one of the main technical challenges in developing immunosensors is overcoming the complexity of binding antibodies (Abs) to the sensor surface. Most immobilizing approaches lead to a random orientation of Abs, resulting in lower binding site density and immunoaffinity. In this context, supramolecular chemistry has emerged as a suitable surface modification tool to achieve the preorganization of artificial receptors and to improve the functional properties of self-assembled monolayers. Herein, a supramolecular chemistry/nanotechnology-based platform was conceived to develop sensitive label-free electrochemical immunosensors, by using a resorcarene macrocycle as an artificial linker for the oriented antibody immobilization. To this aim, a water-soluble bifunctional resorc[4]arene architecture (RW) was rationally designed and synthesized to anchor gold-coated magnetic nanoparticles (Au@MNPs) and to maximize the amount of the active immobilized antibody (Ab) in the proper "end-on" orientation. The resulting supramolecular chemistry-modified nanoparticles, RW@Au@MNPs, were deposited onto graphite screen printed electrodes which were then employed to immobilize three different Abs. Furthermore, an immunosensor for atrazine (ATZ) analysis was realized and characterized by the differential pulse voltammetry technique to demonstrate the validity of the developed biosensing platform as a proof of concept for electrochemical immunosensors. The RW-based immunosensor improved Ab_ATZ loading on Au@MNPs and sensitivity toward ATZ by almost 1.5 times compared to the random platform. Particularly, the electrochemical characterization of the developed immunosensor displays a linearity range toward ATZ within 0.05-1.5 ng/mL, a limit of detection of 0.011 ng/ml, and good reproducibility and stability. The immunosensor was tested by analyzing spiked fortified water samples with a mean recovery ranging from 95.7 to 108.4%. The overall good analytical performances of this immunodevice suggest its application for the screening and monitoring of ATZ in real matrices. Therefore, the results highlighted the successful application of the resorc[4]arene-based sensor design strategy for developing sensitive electrochemical immunosensors with improved analytical performance and simplifying the Ab immobilization procedure.

Scheme 1. Scheme of Immunosensor Assembly with the Resorcarene Molecules Highlighted in Light Blue

The figure shows all the stepwise modifications: functionalization, washing procedure, dropcasting, antibody immobilization, and antigen interaction.

Scheme 2. Synthesis of Resorc[4]arene-Based Linker RW

Figure 1

(A) DPV and (B) SPR signal obtained after Ab_SPS1, Ab_ATZ, and Ab_PGN absorption through RW immobilization.

Figure 2

(A) Calibration plot and (B) DPV curves obtained with increasing ATZ standard solutions (2A, in black) and spiked sample values (2B, in white).

Figure 3

(A) Comparison of adsorption isotherms of Ab_ATZ in the case of oriented (in black) and random immobilization (in white). (B) Overlap of MPA and RW-based sensors (black) signals in ATZ interaction exhibiting different sensitivity and saturation signals.