A pencil-lead immunosensor for the rapid electrochemical measurement of anti-Diphtheria Toxin antibodies

At a Glance

Metadata	Details
Publication Date	2021-10-30
Journal	Zenodo (CERN European Organization for Nuclear Research)
Authors	Ameku W.A., Ataide V.N., Costab E.T., Gomes L.R, Paloma Napoleão-Pêgo
Institutions	Fundação Oswaldo Cruz, Universidade Federal do Rio de Janeiro

Abstract

Abstract: Diphtheria is a vaccine-preventable disease, yet immunization can wane over time to non-protective levels. We have developed a low-cost, miniaturized electroanalytical biosensor to quantify anti-diphtheria toxin (DTx) immunoglobulin G (IgG) antibody to minimize the risk for localized outbreaks. Two epitopes specific to DTx and recognized by antibodies generated post-vaccination were selected to create a bi-epitope peptide, biEP, by synthesizing the epitopes in tandem. The biEP peptide was conjugated to the surface of a pencil-lead electrode (PLE) integrated into a portable electrode holder. Captured anti-DTx IgG was measured by square wave voltammetry from the generation of hydroquinone (HQ) from the resulting immunocomplex. The performance of the biEP reagent presented high selectivity and specificity for DTx. Under the optimized working conditions, a logarithmic calibration curve showed good linearity over the concentration range of 10−5‒10−1 IU mL−1 and achieved a limit of detection of 5×10−6 IU mL−1. The final device proved suitable for interrogating the immunity level against DTx in actual serum samples with results that showed good agreement wit 1. Introduction The respiratory and cutaneous disease Diphtheria (DIPH) is caused by toxins released from the bacteria Corynebacterium diphtheriae and C. ulcerans during pharynx infections, tonsils, or skin. In severe cases, a visible pseudomembrane can develop in the upper respiratory tract along with polyneuritis and myocarditis. If not treated, the clinical presentation of the disease can quickly worsen with an overall fatality rate from 5 to 10% [1,2]. Fortunately, DIPH is a vaccine-preventable disease with the efficacy of the toxoid-based vaccine varying between 54‒87%. For herd immunity, 80‒85% of the population needs to be vaccinated [3]. A major issue is that the immunity induced by the vaccines can wane over time, and any drop in the protection levels in a population could allow for an opportunistic return of this transmissible disease leading to an outbreak [1,4]. Previous studies suggest that 49% of the French adult population presents an antibody titer below protective levels [4]. To maintain the antibody titer at a protective level, booster shots are required. The World Health Organization (WHO) recommends booster vaccinations at 10-year intervals to everyone who lives in low- or non-endemic areas to ensure life-long protection [4]. Access to a simple, rapid serological test to determine the titer of antitoxin antibodies could be highly relevant to disease control. Currently, the titer of neutralizing antibodies in the serum is determined by assays for toxin neutralization and immunoblotting, and 5]. Despite their accuracy, these diagnostics are time-consuming, require a laboratory facility and skilled personnel. The dependence on these conditions to perform the analysis is challenging, especially in resource-limited settings or out of standard laboratories [6-8]. Point-of-care (POC) technologies are urgently needed that provide a decentralized assay, fast response, and reliable results to determine anti-diphtheria toxin (anti-DTx) antibody titer. Electrochemical sensors could meet this demand through their characteristics; simple, portable, sensitive, easy-to-use, miniaturize, and operatable with a portable instrument [6,9-13]. When combined with biological recognition elements (i.e., enzymes, nucleic acid, antibodies, among others), electrochemical transducer-based POC devices have been developed to detect glucose [14-16], neurotransmitters [14], infectious agents, or their antibodies [12,17-21], pharmaceutical compounds [22-24], biomarker, [25] and DNA [26]. Furthermore, electrochemical sensors associated with biomimetic materials (i.e., nanozymes, synzymes, and metal complexes) display good robustness, long-term activity, and minimal matrix interference [9]. Numerous materials such as carbon and silver inks [25,29], tin and gold-sputtered layers [24,26], gold leaf [30], gold nanoparticles suspension [31], and microwires [32] have been used to fabricate disposable devices. However, pencil-lead electrodes (PLEs) stand out for their high electrochemical performance combined with the presence of dangling carbon bonds, carboxyl, carbonyl functional groups, and sp2-hybridized carbon atoms on the basal plane and edge [22,33,34]. These surface moieties permit a variety of different means to conjugate biological components that can be broadly applied to develop electrochemical sensors in the fields of clinical diagnosis [35], forensic [36], and environmental [37]. There are significant concerns for antibody recognition in serum when whole antigens are used in serological tests due to the presence of a variety of epitopes that can react with antibodies against pathogens [28]. Rapid, sensitive, and specific electrochemical immunosensors for infectious diseases have been successfully developed with synthetic linear peptides [19,27]. When the peptides represent epitopes, which are antibody binding sites in pathogen proteins are positioned on the molecule’s surface [28], improvements can be achieved in the sensitivity and selectivity of diagnostic assays along with the elimination of cross-reactivity. Beginning with selecting two particular and reactive epitopes identified in the diphteria toxin (DTx), this study focused on developing a low-cost and accurate electrochemical device to determine the titer of anti-DTx IgG in serum. The epitopes were synthesized in tandem and conjugated to a PLE integrated into a miniaturized three-electrode holder containing reusable reference and auxiliary electrodes using Ag/AgCl and a bare PLE, respectively. Antibodies captured by the random peptide were measured by an indirect immunoassay using a secondary antibody conjugated with alkaline phosphatase that in the presence of hydroquinone (HQ), diphosphate generates HQ electroactive-molecule detectable by square wave voltammetry (SWV). After optimization, a logarithmic calibration curve with good linearity over a wide concentration range and a low detection limit was realized. The final setup was evaluated for its capacity to measure the immunity level against diphtheria by quantifying IgG in actual serum samples and comparing it to a commercial ELISA. The high correlation in the results suggests that the peptide-modified PLE is a promising platform to assist in vaccination control programs, and the flexibility of the technology can be applied to a wide array of diseases. 2. Materials and Methods 2.1. Patients serum samples Blood samples were collected from DTP (Diphtheria/Tetanus/Pertussis)-vaccinated volunteers with no evidence of acute infection or known history of whooping cough or DIPH [38]. 2.2. Chemicals and reagents N-(3-Dimethylaminopropyl)-N’-ethyl carbodiimide hydrochloride (EDC), N-Hydroxysuccinimide (NHS), bovine serum albumin (BSA), and 2(N-morpholino) ethanesulfonic acid (MES), Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), NaH2PO4, Na2HPO4, MgCl2, and NaCl were purchased from Sigma-Merck (St Louis, MO, USA). The 0.5 mm graphite pencil lead refills (2H, H, HB, 2B, 3B, and 4B from Pentel (Tokyo, Japan) were purchased in the local stationery. Goat anti-human IgG conjugated with alkaline phosphatase (secondary IgG antibody, sec-IgG) was purchased from Thermo (Walthan, MA, USA). The manufacturer’s recommendation was restored in 1 mL of deionized water; its final concentration was 0.6 mg mL−1. A commercial ELISA kit for anti-DTx quantitative immunoassay and negative/positive standard serum samples (human serum; negative for anti-human immunodeficiency virus antibody, hepatitis B-virus surface antigen, anti-hepatitis C virus antibody) were purchased from Serion Diagnostics (Würzburg, Germany). Hydroquinone diphosphate (diPho-HQ) salt was purchased from Dropsens (Llanera, Spain). Fuming HCl was purchased from Merck (New Jersey, USA). Deionized water with a resistivity >18.1 MΩ cm was obtained from Nanopure Diamond (Barnstead, Dubuque, IA, USA) and used to prepare all solutions. 2.3. Solid-phase peptide synthesis A peptide (biEP) containing two DTx specific epitopes (GSFVMENFSSGGVDIGF) (biEP) was synthesized as a linked tandem with the insertion of two glycine residues by solid-phase chemistry using the 9-fluorenylmethoxy carbonyl (F-moc) strategy on an automated synthesizer (Multipep-1, CEM Corporation, USA) as previously described [28]. Briefly, benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) was added to the F-moc amino acid. The reaction was run in the reactor with a sintered glass filter containing Wang-Fmoc-Arg resin (Pmc). The F-moc moiety was removed with 25% 4-methylpyridine (Sigma-Merck, St Louis, MO, USA), and the F-moc amino acid coupling reagents were 0.1 mM oximes (Sigma-Merk) in dimethylformamide and 8% N-methyl morpholine. The resin-bound peptide was deprotected and cleaved using trifluoroacetic acid (Sigma-Aldrich) and triisopropylsilane (Sigma-Aldrich). The peptides were precipitated with diethyl ether and lyophilized. The concentration of the peptides was determined by measuring the optical density using the molar extinction coefficient generated by the PROTPARAM software package [http://www.expasy.ch]. The peptide sequence was confirmed by mass spectrometry (MALDI-TOF MS; Matrix-Assisted Laser Desorption Ionization Time-of-Flight). Fabrication of the electrochemical immunosensor Working PLEs were fabricated from pencil lead refills (60 mm x 0.5 mm rods). Approximately half of the lead was coated with a glaze (Metal and Wood, Sherwin-Williams, Sumaré, Brazil) that insulated the lateral surface of the rod. The unglazed portion w

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DOI: https://doi.org/10.5281/zenodo.5628477