key: cord-0905113-t2ulsoki
authors: Hryniewicz, Bruna M.; Volpe, Jaqueline; Bach-Toledo, Larissa; Kurpel, Kamila C.; Deller, Andrei E.; Soares, Ana Leticia; Nardin, Jeanine M.; Marchesi, Luís F.; Simas, Fernanda F.; Oliveira, Carolina C.; Huergo, Luciano; Souto, Dênio E.P.; Vidotti, Marcio
title: Development of polypyrrole (nano)structures decorated with gold nanoparticles towards immunosensing for COVID-19 serological diagnosis
date: 2022-02-07
journal: Mater Today Chem
DOI: 10.1016/j.mtchem.2022.100817
sha: 277699fd4bab13258daab1a8899123034ea03012
doc_id: 905113
cord_uid: t2ulsoki

The rapid and reliable detection of 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 (CPs) 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 CPs 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 (NTs) morphology, and gold nanoparticles (AuNPs) are reported, using a self-assembly monolayer of 3-mercaptopropionic acid and covalently linked SARS-CoV-2 Nucleocapsid protein. Firstly, 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(-1) of monoclonal antibody for PPy in the globular and nanotubular morphology, respectively. The PPy-NTs biosensor was able to discriminate serum obtained from COVID-19 positive vs negative clinical samples and is a promising tool for COVID-19 immunodiagnostic, which can contribute to further studies concerning rapid, efficient, and reliable detections.

The coronavirus disease started in late 2019 in Wuhan, China, caused by the new virus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and was rapidly transmitted through humans mostly via saliva droplets and nasal discharges from infected people. COVID-19 was classified as a global pandemic by the World Health Organization (WHO), and presents as primary symptoms fever, cough, difficulty breathing, etc. However, some patients remain asymptomatic, which raises the potential of silently sparseness of the disease [1, 2] .

The screening of new SARS-CoV-2 contaminated individuals is crucial, especially in the absence of a cure, for better isolation measures, infection control, treatment, and other epidemiological considerations [2, 3] . Pokhrel et al. have shown that lower mortality of COVID-19 can be related to early detection of infections and consequent patient isolation, comparing five countries with similar age distribution and hospital resources. 3 Nowadays, the most common tests available are the lateral flow immunochromatographic strip, realtime reverse transcriptase-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and chemiluminescence assay. However, each of the current options may provide qualities and defects that must be considered individually by case [4] . The interest of diagnostics mainly remains in a technique that supply mobility, agility in the result, ease of sampling, high detectability and minimize false positives and negatives responses. Biosensors offer an alternative sensitive method that may facilitate the diagnosis of SARS-CoV-2 infection [5] .

J o u r n a l P r e -p r o o f 4 Different materials can be used to fabricate biosensors, such as conducting polymers (CPs), molecularly imprinted polymers, metal and metal oxides nanoparticles, carbonbased materials, quantum dots, among others [6, 7] . CPs presents along their chemical structures a π-conjugated system with alternating double and single bonds, responsible for the improved movement of electrons, attributing conductivity to these materials. The use in electrochemical biosensors is compelling because of its biocompatibility, possibility of simple tunning its properties by doping and de-doping process, and the ability of couple it with several materials (gold nanoparticles, metal oxides, etc.) [8, 9] .

The performance of CPs-based electrochemical biosensors relies on changes in their electrical properties, being highly dependent on their shape, size, structure, conductivity, and morphology [10, 11] .

Among the different electrochemical biosensors, the electrochemical impedance spectroscopy (EIS) ones stand out for the possibility to detect small electrode surface variations, as the biorecognition processes, and the detection in a steady-state situation, being less destructive than other electrochemical methods, as cyclic voltammetry (CV) and differential pulse voltammetry (DPV) [12] . Usually in this type of biosensor, the charge-transfer resistance (Rct) at the electrode/electrolyte interface is measured, being sensitive to macromolecules that are recognized at the electrode surface. Usually, a redox probe is added in the electrolyte for EIS measurements since a Rct appearance is resultant of faradaic reactions at electrode/electrolyte interface [13] . However, when CPs are deposited at the electrode surface, the use of a probe becomes unnecessary because CPs themselves suffer from redox reactions and, consequently, have an associated Rct [14] .

Different polymer morphologies, such as nanoarchitectures, are able to offer material properties improvement, including a higher electroactive area and facilitated chargetransfer process, thus offering higher sensitivity, good recovery and a fast response in J o u r n a l P r e -p r o o f 5 sensing applications [8, 15] . Also, the association with gold nanoparticles (AuNPs) can   improve even more the CPs' stability, biocompatibility, sensitivity, and selectivity,   making this strategy very appealing for new COVID-19 biosensors applied as new tools for diagnosis [11, 16] .

Polypyrrole (PPy) is a well-known CP that provides a high conductivity, fair redox properties, stability, facility of synthesis, and electroactivity in phosphate buffer (pH 7.4) [17] [18] [19] [20] . In biosensors, PPy have been evaluated in different nanocomposites and morphologies, due to its promising properties in this area, achieving excellent analytical parameters, such as chitosan/PPy-NTs(polypyrrole nanotubes)/AuNPs [18] , overoxidized PPy-NTs/AuNPs [21] , polypyrrole polymer containing epoxy active side groups (PPCE) [22] and PPy/reduced graphene oxide (RGO) [23] .

In the present work, two different morphologies of PPy (globular and nanotubular), both modified with gold nanoparticles, were synthesized in stainless steel mesh electrodes. This affordable substrate allows the final product to be relatively cheap, flexible, and disposable. Firstly, the materials synthesis was characterized via scanning electron microscopy (SEM), electrochemical and spectroscopical techniques, such as EIS and CV. Later, the modified electrodes were applied to build an immunosensor for SARS-CoV-2 antibodies detection by covalently immobilization of SARS-CoV-2 Nucleocapsid protein (N) via thiol self-assembled monolayers (SAMs) methodology [24] [25] [26] , and each of the steps were characterized via EIS, CV, and Fourier-transform infrared spectroscopy (FT-IR). Finally, the materials were analytically compared by its response to purified monoclonal antibodies to the N protein and tested to identify infections in real samples. 

The electrochemical procedures were performed in an IviumStat XRe potentiostat, and a Metrohm DropSens STAT-I-400 portable potentiostat. Electropolymerization has been preferred due to its simplicity, speed, and reproducibility. It can be performed in a three-electrode conventional conformation (reference, counter, and working electrodes)

in an electrochemical solution simply containing the monomer and a supporting electrolyte, applying a constant potential and monitoring the current over time [28] . The reference electrode and counter electrode were, respectively, Ag/AgCl/KCl(sat) and a platinum spiral. The working electrode was a steel mesh with an area of 1.2 cm 2 , equally divided into four minor electrodes that were later cut to be used as our transducer in the biosensing experiments.

PPy-NTs were synthesized by the methodology previously performed by our group [17] . For the PPy:PSS synthesis, the reaction medium was composed of 50 mmol L -1 of pyrrole and 14 g L -1 NaPSS, and carried out based on previous works published elsewhere [29] [30] [31] . For the film electropolymerization it was used a constant potential of 0.8 V with a charge cutoff of 800 mC cm -2 to get similar electrochemical performance compared with PPy-NTs modified electrodes. 

For both polymers morphologies, N-Protein covalent immobilization through MPA self-assembly monolayer (SAM) formation was chosen, for future comparison. Every step for the biosensor construction is described thoroughly below. A scheme representing each step is presented in Fig. 1 30 min, and it was carefully washed by dipping it in a PBS solution for 5 min prior to EIS analysis. The biosensor was then moved for a clean PBS and the same conditions for EIS were carried out.

To analyze the biosensor ability to detect antibodies in clinical samples, the PPy-NTs biosensor was exposed to different dilutions ( 

PPy:PSS/AuNPs and PPy-NTs/AuNPs modified electrodes were characterized by SEM images, as shown if Fig. 1 (a) and (b), respectively. The images of the polymers without AuNPs can be found in Figure S1 (a, b). For PPy:PSS, the characteristic globular morphology of polypyrrole was obtained [32] , while nanotubes were randomly deposited at the stainless-steel mesh in the synthesis using MO as a template for polymerization [17] . In both cases, the presence of AuNPs is not verified in the secondary electron (SE) The modified electrodes were characterized by cyclic voltammetry, as can be seen in were fitted using a Randles-modified equivalent circuit (inset in Fig. 3 (c) ), a very wellestablished equivalent circuit for CPs modified electrodes [33, 34] . In this equivalent circuit, Rs is the series resistance, accounting for the resistance of the electrolyte, connections and electrodes, Rct is the charge-transfer resistance, related to the charge transfer processes at the material/electrolyte interface, Cdl is a constant-phase element (CPE) describing the double layer capacitance and ndl is a parameter that is related to the double layer homogeneity, thus, depicting the material morphology, it can vary from 0 to 1, being the unity a perfect flat electrode representation. Another CPE is present, Clf, that is related to the charge intercalation process at the polymeric film to maintain electroneutrality upon the redox processes and nlf, that refers to this intercalation process homogeneity. For a better fitting of PPy-NTs and PPy-NTs/AuNPs EIS data, a Warburg element was inserted at the equivalent circuit, which is related to ionic diffusion in the polymer [34, 35] . The calculated parameters are shown in Table 1 . However, it has decreased in the presence of AuNPs, probably owing to a more heterogeneous distribution of AuNPs in the film, changing the oxidated or partially oxidated areas for intercalation. For PPy-NTs, the Clf was the same with and without the AuNPs, evidencing the same number of intercalated ions, which probably is already close to the limit for this material due to the nanotube morphology, however, the decrease in the nlf shows a less homogeneous intercalation process in the presence of the AuNPs. The

Warburg coefficient (σ) decreased in the presence of AuNPs, indicating a lower resistance for the diffusion, probably due to the improvement of redox process in the presence of AuNPs and, consequently, in the ionic diffusion.

The steps of electrode modification are schematized in Fig. 4 (a) , where PPy is a representation of the two different morphologies (PPy:PSS or PPy-NTs). Each step of modification was characterized by FTIR spectra, as shown in Fig. 4 

PPy:PSS/AuNPs and (c) for PPy-NTs/AuNPs. In the spectrum of PPy:PSS/AuNPs, some pyrrole characteristic bands can be identified, as the broad band between 3000-3300 cm -1 (Fig. S4 ) related to C-H and C-H stretching vibrations, at 1448 cm -1 assigned to the conjugated C-N stretching [36] , 1168 and 894, which correspond to the breathing of pyrrole and in plane C-H deformation, respectively, at 1058 cm -1 assigned to C-C out of plane deformation and a weak absorption at 673 cm -1 [37] , related to C-H out of plane bending of pyrrole. Some PSS bands are also visible, as the band at 1635 cm -1 , attributed to the water molecules associated with PSS via hydrogen bonding [37] and at 1377 cm -1 , related to R-SO3 vibration [38] . PPy-NTs FT-IR spectrum also showed PPy characteristic bands, as the broad band from 3000-3300 cm -1 (Fig. S4) , in 1554 and 1460 cm -1 which are considered to be the result of N-H and C-H stretching vibrations, pyrrole ring stretches and conjugated C-N stretching, respectively [36] , and the bands at 1180 and 924 cm -1 , indicating the doping state of PPy [39] . Some methyl orange bands can also be observed in the spectrum of PPy-NTs, as the bands at 1400 cm -1 , which is assigned to -CH3 vibrations and at 1035 cm -1 , related to the asymmetric stretching vibration of -SO3Na group [40] .

The presence of MPA can be evidenced by the band at 3450 cm -1 (COOH vibration, Fig. S4 ), the enhancement of the band at 1635 cm -1 , assigned to C=O bonds of MPA [41] and the appearance of the bands at 1377 and 1554 cm -1 (for PPy-NTs) and 1398 and 1550 cm -1 (for PPy-PSS), for symmetric and asymmetric carboxylate stretching vibration, respectively [42] . The absence of a band at 2600-2500 cm -1 at the MPA spectrum indicates that S-H is bounded at Au surface [43] . The band at 1550 cm -1 disappeared after the N-protein immobilization, which evidences that EDC/NHS reaction and further Nprotein covalent attachment was efficient.

After the blockage step, BSA bands are not evident in the spectrum due to the overlapping with other bands, but some signals can be verified as in 1398 cm -1 related to carboxylate groups and at 1310 cm -1 assigned to amide-III vibration. The electrodes were immersed in a solution containing the mAb anti N-protein to verify the antigen/antibody interaction. In the spectrum obtained after mAb interaction, the band at 1554 cm -1 for PPy-NTs reappeared. This band was observed before for other antigen-antibody complexes [44] , indicating that the monoclonal antibody was properly attached at the biosensor surface. Also, the disappearance of the band at 1635 cm -1 may indicate that the mAb is interacting with the carbonyl group of N-protein.

The biosensor construction was electrochemically characterized by CV (Fig. S5) where a diminishment of the total current is observed after each modification step, evidencing a more difficult charge transfer and/or charge intercalation processes in the presence of macromolecules. To better understand the changes seen by the CVs, EIS was performed after each modification step for PPy:PSS/AuNPs (Figure 4 (d) ) and PPy-NTs/AuNPs (Fig. 4 (e) ). EIS data were adjusted with the equivalent circuits shown in Fig.   3 (c) and (d) for PPy:PSS/AuNPs and PPy-NTs/AuNPs, respectively, and the calculated parameters are present in Table S1 .

As commented before, Rs values cannot be properly discussed due to small variations in the constant cell in each measurement. For both PPy:PSS and PPy-NTs, a decrease in the Cdl values after each modification step is observed, indicating that the exposed electroactive area is becoming smaller with the immobilizations at the electrode surface. 

The response for both PPy morphologies to different concentrations of a monoclonal SARS-CoV-2 antibody was compared by EIS, and the respective calibration plots can be found in Fig. 5 . The PPy-NTs/AuNPs biosensor produced by this work conferred remarkable sensibility, allowing the antibodies detection in PBS, without the necessity of an electrochemical probe, using a small quantity of the bioreceptor (N-protein) and deposited in a significantly affordable material (stainless-steel mesh), which can be disposable, favoring a low-cost measurement.

However, the inaccuracy of serological tests can be a challenge [54] , requesting better sensitivity allied to an efficient selectivity to avoid false positives. Since the PPy-J o u r n a l P r e -p r o o f 23 NTs/AuNPs electrode showed improved sensibility, this morphology was selected to evaluate the device ability to separate negative from positive serum in real samples.

The PPy-NTs/AuNPs biosensor response (evaluated by ΔRct as discussed previously)

in face of different dilutions of serum samples obtained from hospitalized COVID-19

positive patients or negative samples can be found in Fig. 6 (a) . Nyquist diagrams for biosensor exposition to different dilutions of one positive and one negative serum sample is displayed in Fig. S6 . It is possible to observe a reproductible difference in response of both serums, even in highly diluted samples. SARS-CoV-2 antibodies present in the positive sample, that are absent in the negative sample, specifically interact with the bioreceptor, block the interface and consequently interfere in the charge transference process, increasing its resistance, as showed before using the monoclonal mAb solutions.

In contrast, the use of negative real samples is important to obtain the biosensor selectivity, since serum corresponds to a complex matrix composed by water, inorganic compounds and many lipids and proteins, with antibodies that recognize different antigens.

However, because serum is complex matrix, and the immunological response to SARS-CoV-2 infections varies in the population, it is important to consider that each sample may behave differently. For that reason, other serum samples of different positive and negative cases were tested (n=6 and n=4 respectively), using 1:10000 dilution factor in PBS, since at this dilution it was possible to observe on average a 3-fold increment of the ΔRct value when comparing positive/negative response. In Fig. 6 

The screening of SARS-CoV-2 antibodies is essential for the infection control. Even though many studies have been focusing on electrochemical biosensors development, the Hence, the nanostructured polymer PPy-NTs presented almost 8 times higher sensibility when compared to the globular morphology, showing an improvement of the platform when utilizing a nanostructured surface, being this platform selected to further explore its bioanalytical ability in real samples. The novel impedimetric biosensing platform rapidly (in less than one hour) and sensitively detected specific antibodies presence in human serums and showed up to be a flexible, probe-free and disposable. In addition, a small volume is used to produce the biosensor response, being promising to further finger prick blood tests development. This low-cost system can be prospected for COVID-19 immunosurveillance as well as, a small volume with further modifications, be used for infection screening or even vaccination effectivity in producing antibodies. Some limitations as the stability, which was not tested herein, the number of steps required for the bioreceptor immobilization, and the sample preparation should be explored in future works. 

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Funding: This work was suported by Alexander von Humboldt Foundation, UFPR-Proind. This study was financed in part by the Coordenação de Aperfeiçoamento