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

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 was used for connecting electrical leads. The glaze on the end that would constitute the electrode's working surface was removed mechanically, bypassing the rod vertically on paper (Figure 1). The exposed surface was oxidized through chronoamperometry by immersion in PBS (pH 7.4) under vigorous stirring and applying +2 V (vs. Ag/AgCl) for the 50s using a CompactState portable potentiostat (Ivium Technologies B. V., Eindhoven, Netherlands). This was followed by four cycles of cyclic voltammetry (CV) with a scan rate of 100 mV/sec over a potential range +0.4 to −1.4 V. Auxiliary, and reference electrodes were bare PLE and Ag/AgCl, respectively. Next, the PLE tip was immersed in 10 µL of 0.4 M NHS prepared in 0.1 M MES and 0.5 M NaCl solution (pH 6.0) for 30 min to activate the carboxyl groups [39]. The PLE tip was then dipped for 60 min into PBS (pH 7.4) with biEP. After rinsing three times in PBS, the PLE+biEP was blocked overnight in PBS with 0.1% BSA at 4 °C. To normalize for surface variations, the electroactive area of each electrode was estimated by chronoamperometry using the applied potential of 0.35 V for 150 s and the Cottrell equation in a 5 mmol L−1 [Fe(CN)6]4− the solution was prepared in 0.1 M KCl [40]. The parameters employed in the equation were: the number of electrons (n = 1), Faraday constant (F = 96,485 C mol L-1), the concentration of [Fe(CN)6]4−= 5×10−6 mol cm-3, and the average diffusion coefficient value of the [Fe(CN)6]4− in 0.1M KCl= 5.38×10-6 cm2/sec [41]. Electrochemical assay to detect antibodies anti-diphtheria toxin Anti-DTx IgG detection was based on an indirect immunoassay wherein anti-IgG secondary antibodies conjugated with alkaline phosphatase hydrolyzed dPho-HQ to hydroquinone (HQ) resulted in changes in electrical signals, as shown schematically in Figure 1. Briefly, anti-DTx IgG was captured onto the sensitized working surface of the PLE by a 30 min incubation at 25 °C in a 10 µl solution of patient serum (1:1000 in PBS) or control antibody. After rinsing in PBS, PLEs were immersed 30 min into an anti-human IgG secondary antibody solution conjugated with alkaline phosphatase at 25 °C. Next, the PLE was inserted into a custom electrode setup that also contained reference (Ag/AgCl) and auxiliary (bare PLE) electrodes, which is described in supporting information (Figure S1). The setup permitted the insertion of all three electrodes into a solution of 0.1 M Tris-HCl and 20 mM MgCl2 (pH 9.8) with 5 mM dPho-HQ. The conversion to HQ was measured by square wave voltammetry (SWV),19, which employed 10 mV, 6.3 Hz, 10 mV, −0.6 to 0.6 V (vs. Ag/AgCl) as the values for amplitude, frequency, step, and applied potential window were, respectively. Each cycle required 19 s, and a stable measurement was observed after the fourth cycle (76 s total time) corrected by the working electrode electroactive area calculated previously. Enzyme-linked immunosorbent assay The anti-DTx IgG in sera was quantified by the indirect immunoassay using a commercial ELISA kit (Serion Diagnostics, Würzburg, Germany) following the manufacturer's instructions and solutions. Briefly, each well of a 96-well ELISA plate was sensitized with diphtheria toxin antigen followed by washing and blocking with BSA. For the assay, 100 µL of a 1:100 dilution of patient serum was added along with negative and standardized positive controls provided with the kit. After a 60 min incubation at 37 °C, wells were rinsed with PBS and then incubated with anti-human IgG secondary conjugated with alkaline phosphatase (1:15,000) for 30 min at 37 °C. Next, the wells were rinsed four times was PBS followed by the addition of p-nitrophenylphosphatase and a 30 min incubation at 37 °C in the dark. Finally, 100 µL of 0.1 N NaOH/40 mM EDTA was added as a stop solution. The optical density was measured at 405 nm wavelength using a spectrophotometer (Multiskan SkyHigh, Thermo Fisher Scientific). Analytical curve and analysis of blood serum samples To assess the analytical performance, a standard curve was generated from the positive sample by diluting 0.35 IU/ml of positive IgG from the commercial ELISA kit into a negative patient serum to prepare a range of IgG concentrations from 10−5 to 10−1 IU/ml. The indirect immunoassay was accomplished to detect IgG as described. The sera were diluted at a ratio of 1:1,000 with PBS and used 1:50,000 diluted sec-IgG solution. In both cases, the incubation time was 30 min. They were analyzed by indirect immunoassay. The intensity of current density is proportional to the IgG concentration, correlated by the analytical curve. 3. Results 3.1. Preparation of a PLE electrochemical immunosensor An electrochemical immunosensor was developed using graphite rods in pencil lead refills bound with a peptide consisting of two epitopes of DTx linked in tandem by two glycines. The epitopes chosen were previously identified by us through a SPOT synthesis analysis [38]. As shown in Figure 1A, this arrangement would allow the stepwise attachment of anti-DTx antibodies and AP-conjugated secondary antibodies that would permit the generation of HQ, a redox molecule measurable by square wave voltammetry (SWV). Each fabricated PLE was intended to be a single-use sensor. To create an electrically isolated surface, one-half of a PLE was coated with a glaze and physically manipulated on paper to expose the working area (Figure 1B). Next, it was oxidized by applying a constant potential to create graphene oxide-like structures (Figure S1), which carry oxygenated groups such as carboxyl and aldehyde [42]. These were essential chemical groups for activation with EDC/NHS that would allow the covalent attachment of biEP through an amide bond [38]. To improve the ratio between the faradaic and non-faradaic currents, and before treatment with EDC/NHS, the PLE was reduced by being subjected to cyclic voltammetry (Figure S2). The electrochemical treatment resulted in an electrode that presented a less bright and rougher appearance than before electrochemical treatment, as shown in the optical micrographs (Figure 1B), which improved device performance. Figure 1: Scheme of the process to fabricate the PLE. Optical micrographs of the side view and tip of PLE (Ø = 0.5 mm) in the following steps: (a) bare, (b) protected by glaze, (c) polished, (d) treated electrochemically followed by EDC/NHS activation. The drawings show the prepared surface (e), exposure to biEP (f), and followed by blocking with BSA (g). The choice of PLE type was critical for obtaining the desired electrochemical responses. In the market, pencil lead refills vary over a scale from 9H (harder/lighter) to 9B (blacker/softer). The H types have a high amount of wax and clay in their composition. In contrast, B types have a high content of graphite powder [43]. Here, the 4B exhibited a higher SWV readout in response to the production of HQ compared to the others (Figure 2A). In addition, its ratio for the signal to standard deviation was the highest (Figure 2A inset) and presented the most minor signal variation (0.3 ± 0.015 µA). Next, the optimal concentration of biEP to conjugate to 4B was determined over a range of concentrations combined with an assay using the positive control included with the commercial ELISA kit. The biEP concentration on the PLE surface clearly affected the current measured from the immunoreaction (Figure 2B). The ratio between SWV signals obtained after incubation in positive and negative samples only increased after exceeding 10 µg/ml of peptide (Figure 2B inset). Increasing the biEP concentration to 50 µg/ml resulted in the highest signal and the most significant ratio between positive and negative samples suggesting that more anti-DTx antibodies were captured, and non-specific interactions decreased. Interestingly, the highest biEP concentration tested, 200 µg/mL, leading to a drop in the SWV signal. One possible reason was that the higher density of biEP on the electrode blocked the surface, hindering the HQ diffusion. Therefore, the most suitable biEP concentration was determined to be 50 µg/ml and was used to produce all subsequent PLEs. Figure 2: Optimization of the PLE fabrication. (A) As described in the Materials and Methods, six commercially available pencil lead refills (2H, H, HB, 2B, 3B, and 4B) were used to prepare electrodes. Each was incubated with a control anti-DTx antibody obtained from the commercial ELISA assay (0.2 IU/ml) for 60 min. After rinsing, electrodes were incubated with anti-human IgG conjugated with alkaline phosphate diluted 1:5000 in PBS for 30 min. Lastly, electrodes were placed in a solution of 0.1 M Tris-HCl and 20mM MgCl2 (pH 9.8) with 5 mM dPho-HQ. Square wave voltammetry was performed with the parameters for amplitude, frequency, step, and applied potential window set at 10 mV, 6.3 Hz, 10 mV, −0.6 to 0.6 V (vs. Ag/AgCl), respectively. Each PLE type was assayed with three independently prepared electrodes. The inset graph represents the ratio between signal and standard deviation according to the kind of PLE. (B) The optimal concentration of the Bi-epitope peptide to sensibilize the working surface of the 4B-PLE was determined by using a range of biEP concentrations from 0.2-200 µg/ml. Next, PLEs were used to perform assays consisting of a 30 min incubation in either positive (0.2 UI/ml antibody solution from the commercial ELISA kit) or negative serum. After a 30 min incubation with an anti-human IgG antibody conjugated with alkaline phosphate (1:5000 in PBS), SWV was performed as described above. The median current densities from experiments performed in triplicate are plotted for positive (red) and negative (black) samples. The inset graph displays the ratio between positive and negative measurements according to the biEP concentration. 3.2. Optimization of experimental parameters, reproducibility, and stability To optimize the analytical signal, the level of dilution for patient serum and secondary antibodies was evaluated and the incubation times. To fix the patient dilution factor, a 1hr incubation time was chosen for both the primary and secondary antibodies, diluted 1:5000. As the dilution of the positive antibody control was increased under these conditions, there was an increase in the signal intensity up to a dilution factor of 1000, followed by a decrease at the highest dilution factor of 1:5000 (Figure 3A). The non-specific binding of antibodies to the surface of the biEP sensitized PLE showed decreasing signals with higher dilutions. However, the ratio between positive and background showed a maximum difference at a dilution of 1:1000 (Figure 3A inset), which suggested that the biEP/IgG-specific interaction was favored, and a higher dilution ratio impaired the signal due to a reduction in the availability of IgG. When using the optimal serum dilution factor at different incubation times, it was observed that 30 min was sufficient to achieve the highest signal and that a more prolonged incubation was not necessary (Figure 3B). Another critical factor was the sec-IgG concentration that identifies the biEP/IgG immunocomplexes. While a higher secondary antibody concentration leads to a higher SWV response (Figure 3C), it generates background signals with the negative control. The background signal decreased to the lowest levels at a dilution of 1:50,000, which did not impact the signal from the positive control and provided the most prominent sign-to-noise ratio (Figure 3C insert). Although longer times had a minor influence on SWV responses, a 30 min incubation was sufficient to obtain near-maximal signals (Figure 3D). The reproducibility of the PLEs was analyzed by evaluating electrodes prepared on different days using the same protocol. A relative standard deviation (RSD) of 6% in the SWV HQ response was calculated from 5 other measurements of a 10−4 IU mL−1 IgG solution, which demonstrated the practical reproducibility of the system (Figure S4, orange traces). To test stability, PLEs prepare