Development of polypyrrole (nano)structures decorated with gold nanoparticles toward immunosensing for COVID-19 serological diagnosis

Abstract

The rapid and reliable detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroconversion in humans is crucial for suitable infection control. In this sense, many studies have focused on increasing the sensibility, lowering the detection limits and minimizing false negative/positive results. Thus, biosensors based on nanoarchitectures of conducting polymers are promising alternatives to more traditional materials since they can hold improved surface area, higher electrical conductivity and electrochemical activity. In this work, we reported the analytical comparison of two different conducting polymers morphologies for the development of an impedimetric biosensor to monitor SARS-CoV-2 seroconversion in humans. Biosensors based on polypyrrole (PPy), synthesized in both globular and nanotubular (NT) morphology, and gold nanoparticles are reported, using a self-assembly monolayer of 3-mercaptopropionic acid and covalently linked SARS-CoV-2 Nucleocapsid protein. First, the novel hybrid materials were characterized by electron microscopy and electrochemical measurements, and the biosensor step-by-step construction was characterized by electrochemical and spectroscopic techniques. As a proof of concept, the biosensor was used for the impedimetric detection of anti-SARS-CoV-2 Nucleocapsid protein monoclonal antibodies. The results showed a linear response for different antibody concentrations, good sensibility and possibility to quantify 7.442 and 0.4 ng/mL of monoclonal antibody for PPy in the globular and NT morphology, respectively. The PPy-NTs biosensor was able to discriminate serum obtained from COVID-19 positive versus negative clinical samples and is a promising tool for COVID-19 immunodiagnostic, which can contribute to further studies concerning rapid, efficient, and reliable detections.

Keywords: COVID-19; Gold nanoparticles; Immunosensor; Impedimetric biosensor; Polypyrrole modified electrode; Polypyrrole nanotubes.

Graphical abstract

Fig. 1

Schematic representation of (i) expected PPy morphologies: (a) nanotubular and (b) globular, and (ii) biosensor construction steps for antibody detection. Steps: (a) Functionalization though SAM formation, (b) activation via EDC:NHS, (c) immobilization of N-Protein, (d) Blocking via BSA and (e) antibody detection. BSA, bovine serum albumin; EDS, energy-dispersive X-ray spectroscopy; NHS, N-hydroxysuccinimide; PPy, polypyrrole; SAM, self-assembly monolayer.

Fig. 2

Representative SEM images of (a) PPy:PSS and (b) PPy-NTs deposited on stainless-steel mesh. (c) STEM image of PPy:PSS and (d) TEM image of PPy-NTs. NT, nanotube; PPy, polypyrrole; SEM, scanning electron microscopy; STEM, TEM,

Fig. 3

CV of (a) PPy:PSS and (b) PPy-NTs at 20 mV/s in PBS. Nyquist diagrams of (c) PPy:PSS and (d) PPy-NTs at ocp in PBS. Inset: equivalent circuits used to fit the EIS data. EIS, electrochemical impedance spectroscopy; NT, nanotube; PBS, phosphate-buffered saline; PPy, polypyrrole.

Fig. 4

(a) Schematic representation of each step for the biosensor construction. FT-IR of (b) PPy:PSS and (c) PPy-NTs and Nyquist diagrams of (d) PPy:PSS and (e) PPy-NTs after each modification step. FT-IR, Fourier-transform infrared spectroscopy; NT, nanotube; PPy, polypyrrole.

Fig. 5

Nyquist diagrams of (a) PPy:PSS and (b) PPy-NTs biosensors exposed to different concentrations of anti N-protein IgG antibody obtained in PBS at OCP and the linear response between these logarithmic concentrations and the logarithmic of ΔRct for (c) PPy-NTs (d) PPy:PSS. NT, nanotube; PPy, polypyrrole.

Fig. 6

(a) Reproducibility (n = 4) of PPy-NTs/AuNPs immunosensor for an individual positive serum versus a negative response in different dilution factors (V:V) and (b) several (n = 10) confirmed positive and negative samples discriminated by their EIS response in a 1:10000 dilution factor.