COVID-19 impedimetric biosensor based on polypyrrole nanotubes, nickel hydroxide and VHH antibody fragment: specific, sensitive, and rapid viral detection in saliva samples

https://doi.org/10.1016/j.mtchem.2023.101597Get rights and content

Highlights

  • Synthesis of COVID antibody fragments to build up a biosensor.

  • New assembly of electroactive nanomaterials to increase the sensitivity of detection.

  • Development of a direct transduction signal based on the impedimetric response of human samples.

  • Chemometric tools to explore the reliability, reproducibility, and statistical analysis of the detection results.

Abstract

SARS-CoV-2 rapid spread required urgent, accurate, and prompt diagnosis to control the virus dissemination and pandemic management. Several sensors were developed using different biorecognition elements to obtain high specificity and sensitivity. However, the task to achieve these parameters in combination with fast detection, simplicity, and portability to identify the biorecognition element even in low concentration remains a challenge. Therefore, we developed an electrochemical biosensor based on polypyrrole nanotubes coupled via Ni(OH)2 ligation to an engineered antigen-binding fragment of heavy chain-only antibodies (VHH) termed Sb#15. Herein we report Sb#15-His6 expression, purification, and characterization of its interaction with the receptor-binding domain (RBD) of SARS-CoV-2 in addition to the construction and validation of a biosensor. The recombinant Sb#15 is correctly folded and interacts with the RBD with a dissociation constant (KD) of 27.1 ± 6.4 nmol/L. The biosensing platform was developed using polypyrrole nanotubes and Ni(OH)2, which can properly orientate the immobilization of Sb#15-His6 at the electrode surface through His-tag interaction for the sensitive SARS-CoV-2 antigen detection. The quantification limit was determined as 0.01 pg/mL using recombinant RBD, which was expressively lower than commercial monoclonal antibodies. In pre-characterized saliva, both Omicron and Delta SARS-CoV-2 were accurately detected only in positive samples, meeting all the requirements recommended by the World Health Organization for in vitro diagnostics. A low sample volume of saliva is needed to perform the detection, providing results within 15 min without further sample preparations. In summary, a new perspective allying recombinant VHHs with biosensor development and real sample detection was explored, addressing the need for accurate, rapid, and sensitive biosensors.

Introduction

In December 2019, a group of patients diagnosed with severe febrile respiratory tract disease of unknown origin was reported in Wuhan (China). This disease outbreak was later associated with a previously unknown Coronavirus strain, subsequently named SARS-CoV-2, which quickly spread to all continents and the COVID-19 disease became world known [1]. After only three months, the World Health Organization (WHO) declared it a pandemic. Three years later (April/2023), more than 764,474,000 cases and 6,915,000 deaths have been confirmed worldwide (WHO, 2023). Despite high vaccination rates, COVID-19 still causes worldwide concern due to new variants emergence. The new variants, such as Omicron, can escape immunity acquired from the vaccine or previous infection [[2], [3], [4]] and cause further cases and deaths.

In addition to vaccination, one way to control the disease and the emergence of new variants is large-scale testing to identify and isolate infected patients, thus preventing virus spread. The real-time quantitative polymerase chain reaction (RT-qPCR) test is considered the gold standard assay for SARS-CoV-2 detection. However, the process complexity and equipment cost require centralized testing by highly trained technicians. In addition, the minimum time between test sample collection and result dissemination is approximately 3 h. This makes it difficult to perform tests in real-time and in locations far from diagnostic centers [[5], [6], [7]]. However, the type and quality of the patient specimen, the disease stage, and the degree of viral replication and/or clearance have an impact on test results. These factors are critical not only for PCR-based but also for other diagnostic test systems aiming to detect viral antigens [8].

The enzyme-linked immunosorbent assay and PCR techniques used in the diagnosis of viral diseases are at the forefront of well-established methods. However, these methods have deficiencies in terms of accessibility and rapidness. Concerning the technologies, there are currently more advantageous options available to be applied in the field of diagnostics [9]. Due to the limitations of traditional diagnostic techniques, antigen-detection rapid diagnostic tests have been widely used in the detection of SARS-CoV-2. Rapid tests are more practical as they can be processed outside the laboratory and provide a result within 15–20 min [10]. Although these tests are worldwide available, false positive and negative results are easily obtained, especially in the early days of infection when the viral load is small. Besides that, a nasopharyngeal sample is often collected with the use of a swab, which is a semi-invasive method with a correct collection technique causing discomfort in the testers [11]. In this context, electrochemical biosensors have the potential for the development of rapid tests for COVID-19 diagnosis. Biosensors have high sensitivity, selectivity, shorter response time, lower costs, and ease of application [[12], [13], [14]]. Several types of electrochemical biosensors have been studied to diagnose COVID-19. The mechanisms used for these detections include differential pulse voltammetry [15], square wave voltammetry [16,17], and electrochemical impedance spectroscopy (EIS) [18]. Among these detection techniques, EIS offers advantages compared to other electrochemical methods, such as high sensitivity, ease of signal quantification, minimal manipulation, and simple and rapid pretreatment [19]. In the EIS technique, biomolecule detection could be associated with differences between charge-transfer resistances, once it works by measuring the change in the whole impedance system when a specific binding event of receptors immobilized on a surface occurs. The most used biological recognition elements are antibodies, which bind specifically to an antigen to form an immune complex [20]. After the interaction between antigen and antibody, an increase in charge-transfer resistance occurs due to this interaction, which depends on the amount of immobilized antibodies and the antibody–antigen interaction [21].

Conducting polymers (CPs) are among the best materials for constructing biosensors [22]. CPs are unique materials because they are organic chains that have conjugated double bonds in the structure. In conjugation, the bonds between the carbon atoms alternate between single and double, giving the polymer semiconducting properties [23,24]. The use of CPs in biosensors has advantages such as greater sensitivity, improved conductivity, lower detection limits, cost-benefit, stability, and biocompatibility [23,24]. Polypyrrole (PPy) is one of the most used polymers in biosensor development due to its stability, biocompatibility, electrical conductivity, and good redox properties [25]. Furthermore, due to the important and attractive redox characteristics, the use of a redox probe in the electrolyte is unnecessary since the polymer presents Faradaic reactions [26]. Also, the electrochemical response can be enhanced by using nanostructures due to the larger sensor surface area, which increases sensitivity and selectivity and facilitates the charge transfer processes [27]. PPy is also promising for commercial applications due to greater environmental stability, ease of synthesis, and conductivity than other CPs [28].

Bioreceptor immobilization on the electrode is a crucial step in biosensor fabrication, success in this step ensures biosensor stability and sensitivity [22]. To create a new biosensor that can meet the current challenges, the points that must be considered include changing the surface chemistry for immobilization, improving detection limit and selectivity for better detection, and enabling data processing and analysis. Furthermore, many researchers have focused on increasing the sensitivity and sensing range of biosensors using semiconductor and metallic materials [29]. Immobilization of proteins via histidine-tag (His-tag) is an interesting and accessible method for immobilization since it focuses on protein orientation. The His-tag position on the protein can be controlled by genetic engineering, resulting in protein uniform orientation during immobilization [30,31]. His-tag proteins have a high affinity for bivalent transition metal ion complexes (e.g. Ni2+, Cu2+, Co2+) and can be immobilized on a surface through a coordination bond with the metal complex [32,33]. Nickel hydroxide is one of the materials used to construct biosensors [15]; however, the attachment of biomolecules through His-tag interaction with Ni(OH)2 was not explored so far, and this feature could be very interesting for the oriented biomolecules immobilization directly onto the material surface. In addition, Ni(OH)2 materials have some advantages, such as electrocatalytic properties, cost-effectiveness, high porosity, and electro-inactivity in physiological pH solutions [34,35].

Antibodies or immunoglobulins (Igs) are produced by vertebrate animals as part of their defensive immune response. They are heterodimeric proteins composed of two heavy and two light chains with an Fc region. The different antibody isotypes, including IgM, IgD, IgA, IgE, and IgG, are distinguished by structural differences in their heavy chain constant regions. Thanks to genetic engineering and recombinant antibody techniques, high-quality antibodies can be obtained in the laboratory, as monoclonal antibodies (mAbs) which are specific for one antigen and are produced in the laboratory by B cell hybridoma. Researchers have also created smaller antibody fragments through genetic engineering to enhance antibody performance and reduce assay costs [36,37].

Antigen-binding fragments of heavy chain-only antibodies or VHH are single-domain antibodies derived only from the heavy chain of camelid antibodies [38]. Due to the lack of glycosylation and their small size, the VHHs can be produced via Escherichia coli expression systems, being easier, faster, and with lower cost than other expression platforms, like mammalian cells [39]. Besides that, these antibodies have advantages when compared to conventional ones, such as solubility at higher concentrations, small size, greater resistance to denaturation, stability at high temperatures and high/low pH, and high specificity [[40], [41], [42], [43]]. Despite the advantages of VHHs over conventional antibodies, their use in the construction of electrochemical biosensors is still poorly reported.

In this work, we developed a biosensor based on a VHH as a biorecognition molecule for SARS-CoV-2 detection in saliva samples from SARS-CoV-2 infected patients. Saliva is also a preferable sample medium compared to nasopharyngeal samples, due to the possibility of self-collection without any discomfort, still with comparable sensitivity even with small sample quantities, which also presents high dilution capacity [44]. EIS was employed as the detection method for its advantages, such as high sensitivity, low detection limit, and rapid and non-destructive analysis, being one of the most used electrochemical techniques in biosensors [45,46]. Combining all these advantageous characteristics, in our approach, results could be readily obtained in 15 min, with a limit of quantification of 0.01 pg/mL for the proposed VHH Sb#15-His6. Accurate and sensitive detection was achieved according to WHO standards, in untreated real saliva samples, which is ideal for rapid diagnosis.

Section snippets

Plasmid constructions

The amino acid sequence of the VHH termed sybody 15 (Sb#15), which recognizes the RBD of SARS-CoV-2, was obtained from the study reported by Walter et al. [47]. Initially, a synthetic gene encoding Sb#15 optimized for E. coli expression cloned into the BglII and XhoI restriction sites of plasmid pET32a in fusion with thioredoxin A (pET32a-Sb#15, Supplementary information) was purchased from the Biomatik Corporation (Kitchener, Ontario, Canada). For expression of Sb#15 without the thioredoxin

VHH Sb#15 expression, purification, and stability analysis

Two versions of Sb#15 were produced using plasmids pETDuet1-Sb#15 and pET28a-Sb#15 containing hexa-histidine tag either at the N-(His6-Sb#15) or C-Terminal (Sb#15-His6), respectively. Both were purified from soluble E. coli extracts on immobilized nickel columns followed by size exclusion chromatography. Representative results of the purification steps are shown in (Fig. 2).

In the case of His6-Sb#15, the hexa-histidine tag was removed by TEV protease digestion before further analyses. In the

Conclusions

The development of a simple, portable, and reliable platform for rapid and sensitive SARS-CoV-2 detection has been highly challenging. To address this demand, biosensors were developed for SARS-CoV-2 spike protein RBD detection in saliva samples using PPy-NTs and Ni(OH)2 modified SPCE electrodes. The use of Ni(OH)2 is important to correctly orient an antibody immobilization on a biosensor surface so that it can avoid loss of antigen binding activity and increase the sensitivity.

Two conventional

Funding sources

This work was partially funded by CAPES (AUXPE 88881.504691/2020-01, 001), including a post-doctoral fellowship assigned to DSBR, a PhD scholarship to APMSB, and a master's scholarship to VZR. NITZ and BGG are supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) research career fellowships (303038/2019-5; 408635/2018-5; 304167/2019-3 and 304788/2018-0) and CNPq/MCTI/FNDCT Nº 18/2021 (408589/2021-3). INCT in Bioanalytics (FAPESP grant no. 2014/50867-3 and CNPq grant

Credit statement

Alecsandra Santos: Conceptualization, Methodology, Validation, Formal Analysis, Investigation, Writing – Original Draft, Visualization. Ana Paula Macedo de Souza Brandão: Conceptualization, Methodology, Validation, Formal Analysis, Investigation, Writing – Original Draft, Visualization. Bruna M. Hryniewicz: Conceptualization, Methodology, Validation, Formal Analysis, Investigation, Writing – Original Draft, Visualization. Hellen Abreu: Conceptualization, Methodology, Validation, Writing –

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

The authors acknowledge the FIOCRUZ program of technical platforms for access to the facilities for Integrated Structural Biology (RPT-15A) and Cytometry (RPT-08L). CTI Renato Archer for microscopic facilities.

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