Electrochemical immunosensing of Crohn´s disease biomarkers using diazonium salt grafted - crystalline nanocellulose / carbon nanotubes modified electrodes

Electrochemical immunosensing of Crohn´s disease biomarkers using diazonium salt grafted - crystalline nanocellulose / carbon nanotubes modified electrodes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Electrochemical immunosensing of Crohn´s disease biomarkers using diazonium salt grafted - crystalline nanocellulose / carbon nanotubes modified electrodes Lorena García-Rodrigo, Claudia Ramos-López, Esther Sánchez-Tirado, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5259345/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Dec, 2024 Read the published version in Microchimica Acta → Version 1 posted 9 You are reading this latest preprint version Abstract This work reports the first dual immunosensor for the determination of IL-12 and IL-23, two relevant biomarkers of Crohn’s disease (CD). The strategy relies on the selective capture of the targets by the respective antibodies which were covalently immobilized onto SPCEs modified with crystalline nanocellulose (CNC) and multi-walled carbon nanotubes (MWCNTs) followed by conjugation with a detector antibody labelled with poly-HRP-Strept and amperometric transduction using the H 2 O 2 /HQ system. The developed bioplatform exhibits a high reproducibility and selectivity allowing the simultaneous determination of both biomarkers using a lower sample volume and lasting a much shorter assay time than those reported for each target with commercially available ELISA kits. It is worth highlighting the storage stability of the bioconjugates, which lasts at least 71 days. This excellent performance is probably due to the combination of the CNC properties, mainly its biocompatibility and hydrophilicity, the high surface area and open pore structure, together with the electrochemical properties of MWCNTs, which provide a nanocomposite excellent for biomolecules immobilization onto the transducer surface and electrochemical detection. The suitability and applicability of the dual immunosensor were demonstrated by analyzing raw serum and faeces spiked with IL-12 and IL-23 at the levels that can be found in samples from patients suffering severe CD. Crohn´s disease IL-12 IL-23 electrochemical immunosensor crystalline nanocellulose carbon nanotubes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION The major forms of human inflammatory bowel disease (IBD) are Crohn's disease (CD) and ulcerative colitis (UC) [1]. They arise from a complex interplay of genetics, gut microbiome dysregulation and alteration of the intestinal epithelial barrier followed by a misdirected immune response [2]. CD is an immune-mediated inflammatory disorder characterized by chronic relapsing inflammation in different segments of the gastrointestinal tract caused by a disruption in the balance among the intestinal epithelium, the commensal microbiota and the innate immune response. The presence of defects in the intestinal wall together with a dysfunction in regulatory mechanisms maintain this condition leading to the release of an array of cytokines which in turn promotes the inflammatory immune response [3]. In the intestinal wall of CD patients there are innate lymphoid cells that are stimulated by IL-12 to produce interferon gamma (IFN-γ) which in turn mediates proinflammatory functions, thus participating in the pathogenesis of the intestinal mucosa inflammation [4]. IL-23, another cytokine belonging to the IL-12 family, has also been shown to be involved in CD [5]. Indeed, the IL-12 / IL-23 system is closely connected with the appearance of inflammation in the intestinal wall and over distinct effects on the promotion of tumorigenesis, cancer progression and mechanisms of resistance related to this disease [6]. Due to this clear implication, inhibitors of IL-12/IL-23, especially the human antibody ustekitumab, have been developed for CD management. The methods used to determine these interleukins are mainly based on the ELISA technique. Commercial colorimetric ELISA kits involve specific capture antibodies for the establishment of sandwich configurations with biotinylated secondary antibodies and streptavidin- or avidin- peroxidase (HRP) conjugates. The analytical characteristics of these tests are similar. As an example, the Invitrogen Human IL-12 ELISA kit BMS238 (Thermo Fisher Scientific) provides a nonlinear semi-logarithmic calibration over the 3.1 to 200 pg mL − 1 range with a detection limit of 2.1 pg mL − 1 and an assay time slightly longer than 3 hours. In the case of IL-23, the Invitrogen Human IL-23 ELISA kit BMS2023-3 (Thermo Fisher Scientific), with a similar setup using the Avidin-HRP conjugate, provides a nonlinear semilogarithmic calibration over the 15.6 to 2000 pg mL − 1 range, with a detection limit of 4.0 pg mL − 1 and an assay time of 3.5 h. A kit for the joint determination of IL-12 and IL-23 (ELISA kit MBS2013 (Thermo Fisher Scientific) is also available in which both cytokines bind to the specific antibodies adsorbed in the microwells followed by conjugation with a biotin- anti -human IL12/IL23 antibody and the addition of Strept-HRP. Colorimetric detection provides a non-linear semi-logarithmic calibration in the 31.3 to 2000 pg mL − 1 range for both interleukins with a detection limit around 10 pg mL − 1 and an assay time of just over 4 hours. Regarding biosensors, very few examples have been found in the literature. A label-free impedimetric biosensor for IL-12 involving a disposable gold-coated silver ribbon electrode for immobilizing anti-IL-12 antibodies by covalent linking using 16-mercaptohecadecanoic (MHDA) was reported [7]. Direct measurements of charge transfer resistance (Rct) after conjugation with the target were performed proving a non-linear calibration plot. The same group developed an IL-12-immunosensor based on a printed circuit board (PCB) electrode with electroplated gold [8]. A calibration plot covering a concentration range between 0.1 and 500 pg mL-1IL-12 and a LOD value of 3.5 pg mL − 1 was obtained. Cellulose nanocrystals (CNC) is a biocompatible and sustainable biopolymer nanomaterial characterized by the high specific surface area and the presence of a large number of OH groups which can be further modified for the incorporation of binding sites for biomolecules. The porous network structure and hydrophilicity of CNC together with the film forming ability and stability [9] have converted it into a valuable nanomaterial for the preparation of electrochemical (bio)sensors. As a drawback, the electrical nonconductivity of CNC must be noted, although the transportation of ions is possible and so, ion conductivity in low-concentration solutions due to the charge mobility and porosity is remarkable [10]. In addition, combination of nanocellulose with highly conductive materials such as metal nanoparticles, graphene [11] or carbon nanotubes [12] can provide these materials with high electrical conductivity, which is necessary in some applications. Particularly, the properties of functionalized nanocellulose / multiwalled carbon nanotubes (MWCNTs) for electrochemical applications have been studied [13]. It has been shown that CNC constitutes an effective dispersing medium for carbon nanotubes, and the incorporation of the resulting hybrid nanomaterial to electrode surfaces provides improved electrical conductivity, high mechanical strength and large surface area. However, these advantages have been scarcely explored to date. A modified electrode involving nanocellulose /MWCNTs hybrids was reported for the detection of diclofenac in pharmaceutical products and biological fluids [12]. Nanocellulose and single-walled carbon nanohorns (SWCNH) were used to prepare modified electrodes for the simultaneous detection of guanine and adenine [14]. In this article, we report the development of a dual immunosensor for the simultaneous determination of the Crohn´s biomarkers IL-12 and IL-23 using screen-printed dual carbon electrodes (SPdCEs) modified with the MWCNTs/CNC nanocomposite as substrate for the immobilization of capture antibodies by electrografting of the p -aminobenzoic acid diazonium salt. Grafting methods on carbon surfaces have demonstrated to be a powerful route to incorporate linking groups in a simple and rapid way [15,16]. The immunoassay strategy involved covalent immobilization of the specific antibodies (anti-IL12 or anti-IL23) on the respective electrode surface of the modified SPdCE followed by complexation with each target and implementation of sandwich type immunoassays with biotinylated detector antibodies. The respective amperometric responses were measured after affinity binding with polymeric HRP-streptavidin conjugate (poly-HRP-Strept) using the H 2 O 2 / hydroquinone (HQ) system. The proposed multianalyte immunosensor was successfully applied to the determination of IL-12 and IL-23 biomarkers in spiked faecal samples and serum from healthy individuals. EXPERIMENTAL Apparatus and electrodes Amperometric measurements were made at room temperature using a CH1 1030B potentiostat (Chemical Instruments, Inc.) controlled by the CH1 1030B software. A µAutolab type III potentiostat (Ecochemie) controlled by FRA2 software electrochemical impedance spectroscopy (EIS) was employed for other electrochemical measurements. Screen-printed carbon electrodes both single (SPCEs, DRP-110 with a 4 mm-Ø carbon working electrode) and dual (SPdCEs, X1110 DRP with two elliptic carbon working electrodes and a surface area of 4.7 mm 2 ) including a carbon counter electrode and a silver pseudo-reference electrode were from Metrohm-DropSens. The specific DRP-CAC connection cables employed as interface between the SPCEs and the potentiostat were also from Metrohm-Dropsens. The measurements were performed in stirred solutions using 10-mL glass electrochemical cells from Pobel. A Crison model Basic 20 + pH meter, a P-Selecta Ultrasons ultrasonic bath, a Heidolph Reax Top homogenizer for small samples, and an MPW-65R centrifuge from MPW (Med. Instruments) were also used. Reagents and solutions Crystalline nanocellulose (Cellulose Nanocrystals Freeze-Dried, CNC-FD, 5–20 nm W, 100–250 nm L) with hydroxyl and sulfonic surficial groups (Figure S1 Supplementary Information) was supplied by Cellulose Lab. Inc., Fredericton, NB, Canada. Multi-walled carbon nanotubes (MWCNTs; ϕ 30 ± 15 nm, 95% purity) were from NanoLab, Brighton, MA. Before use, 25 mg of MWCNTs were suspended in 85 mL of deionized water and ultrasonically stirred for one hour. Then, they were chemically shortened and carboxylated by addition of 16 mL of 65% w/w nitric acid keeping under reflux for one hour. Once cooled at room temperature, the resulting product was centrifuged at 4000 rpm for 10 min and washed repeatedly with deionized water until washing liquids reached pH 7, and finally dried under nitrogen stream [17]. N-hydroxysulfosuccinimide (Sulfo-NHS), N-(3-dimethyl-amino-propyl)-N’-ethylcarbo-diimide (EDC), HQ, hydrogen peroxide (H 2 O 2 , 30% v/v), p -aminobenzoic acid (p-ABA), and sodium nitrite, were purchased from Sigma-Aldrich. Poly-HRP-streptavidin was from Pierce Thermo Scientific. Sodium chloride, potassium chloride, sodium di-hydrogen phosphate, di-sodium hydrogen phosphate, and tris-hydroxymethyl-aminomethane-HCl (Tris–HCl) were from Scharlab. Anti-IL-12 capture antibody (anti-IL12), human IL-12 standard (IL12) and biotin-anti-IL-12 detection antibody (Biotin-anti-IL12) were from the Human IL-12 DuoSet ELISA from R&D Systems (Cat. No. DY1270-05). Anti-IL-23 capture antibody (anti-IL23), human IL-23 standard (IL23) and biotin-anti-IL-23 detection antibody (Biotin-anti-IL23) were from the Human IL-23 DuoSet ELISA from R&D Systems (Cat. No. DY1290-05). Buffer solutions were 25 mM MES of pH 5.0 prepared from 2-(N-morpholine) ethane-sulfonic acid (Gerbu, Heidelberg, Germany), 100 mM, 50 mM phosphate buffer (PB) of pH 6.0 prepared from Na 2 HPO 4 and NaH 2 PO 4 ⋅2H 2 O (Sigma-Aldrich), and saline phosphate buffer (PBS) consisting of a 100 mM sodium phosphate buffer of pH 7.4 supplemented with 2.0 g NaCl and 50.25 mg KCl (Scharlab) in 250 mL deionized water. In all cases, the pH value was adjusted by adding the required volume of 2 M NaOH solution. Deionized water was from a Millipore Milli-Q purification system (18.2 MΩ cm). Samples The developed immunosensor was applied to the simultaneous determination of IL-12 and IL-23 in undiluted human serum and in faecal samples from healthy volunteers spiked with both cytokines at the concentration levels expected for Crohn´s disease patients. The samples were stored at -80 ºC until use. All the experiments involved in samples manipulation and analysis were performed accomplishing all the ethical issues and relevant guidelines and regulations of the implied institutions. Procedures Preparation of the MWCNTs/CNC aqueous dispersion 0.0125 g of MWCNTs previously treated with nitric acid, as described above, were added to 10 mL of a 0.25% (w/v) CNC aqueous suspension contained in a Falcon® tube. The mixture was stirred in the ultrasonic bath for 10 min under pulsed mode to avoid material overheating [18] Preparation of the dual immunsosensor The scheme displayed in Fig. 1 shows the steps involved in the modification of the SPdCEs, the preparation of the dual immunosensor and the electrochemical detection for the simultaneous determination of IL-12 and IL-23. Firstly, a 5 µL aliquot of the as prepared MWCNTs/CNC aqueous dispersion was deposited on each SPdCE surface allowing it to dry at room temperature. Next, the diazonium salt was synthesized separately by following the method previously described [19]. Briefly, a 2 mM NaNO 2 aqueous solution was added dropwise to 1 mg mL − 1 p- ABA solution prepared in 1 M HCl (38 mL NaNO 2 for each 200 mL p -ABA) and cooled with ice. The reaction was allowed proceeding during 10 min under stirring. Thereafter, the MWCNTs/ CNC/SPdCE was immersed into the diazonium salt solution and ten successive voltammetric cycles between 0 and − 1.0 V vs. Ag pseudo-reference electrode (ν = 200 mV s − 1 ) were scanned. Finally, the modified electrode was washed thoroughly with water (10 s) and methanol (10 s) and dried at room temperature. The activation of carboxylic groups was performed by the addition of 10 µL of a 0.1 mol L − 1 EDC/sulfo-NHS solution prepared in 25 mM MES buffer of pH 5.0 to each working electrode (W1 and W2) and incubated for 30 min in a humid ambient at room temperature. The electrode was then washed with the same buffer solution and the capture antibodies (anti-IL12 and anti-IL23) were immobilized on the activated Phe-(MWCNTs/CNC)/ SPdCE by dropping 2.5 µL of a 25 µg mL − 1 anti-IL12 or anti-IL23 solution on each electrode surface (W1 and W2 respectively), and incubating for 15 min at room temperature. After washing with PBS of pH 7.4, the unreacted activated groups of the electrode surfaces were blocked by adding 2.5 µL of 8% BSA in PBS pH 7.4 allowing incubation for 30 min. Thereafter, the electrode was washed with the same buffer solution. The sandwich-type dual immunosensor was implemented by dropping 2.5 µL of the IL12 and IL23 standard solutions or the sample prepared in PBS pH 7.4 on the respective anti-IL12-Phe-(MWCNTs/CNC)/SPdCE or anti-IL23-Phe-(MWCNTs/ CNC) /SPdCE allowing incubation for 15 min at room temperature. Thereafter, 5 µL of the detection biotinylated antibodies, Biotin-anti-IL12 or Biotin-anti-IL23 were dropped respectively onto the as prepared electrodes and incubated for 30 min. Finally, 5 µL of 1/1000 diluted poly-HRP-Strept in PBS pH 7.4 and 2% BSA were added allowing 20 min of incubation. The resulting dual immunosensor was washed with PBS pH 7.4 and kept with a 25 µL drop of the same buffer until the electrochemical measurements were made. All incubation steps involved in the immunosensor fabrication were performed at room temperature in a humid environment to prevent drop evaporation. HERE FIGURE 1 Amperonetric measurements The amperometric responses were measured by immersing the modified electrode into the electrochemical cell containing 10 mL of 50 mM PB pH 6.0 and 100 µL of a 100 mM HQ fresh solution prepared in the same buffer. The measurements were carried out under stirring by applying − 0.20 V vs. the Ag pseudo-reference electrode. Once the background current was stabilized (~ 50 s), 50 µL of a 100 mM H 2 O 2 solution prepared daily in 50 mM PB pH 6.0 were added and the variation in the cathodic current due to the HRP reduction of H 2 O 2 mediated by HQ, reaching the steady state in ~ 100 s, was recorded. The analytical responses are the mean values of three replicates, and the error bars displayed were estimated as three times the standard deviation of each set of replicates (α = 0.05). Samples analysis The optimized procedure was applied to the simultaneous determination of IL-12 and IL-23 in undiluted human serum and in faecal samples from a healthy volunteer. Before any pretreatment, the samples were spiked with both cytokines at the concentration levels expected for Crohn´s disease patients and stored at -80 ºC until use. Recovery studies were performed using 5 µL aliquots of raw serum spiked with IL-12 and IL-23. In the case of faecal samples, once collected and weighed accurately in a special container (OC-Auto Sampling Bottle3 Ref. V-PZ24 from Eiken Chemical Co Ltd.) the spiked sample was dispersed in 2 mL of the HEPES buffer included in the container by vigorous manual stirring. For the determination, 5 µL aliquots of this dispersion were used. All the experiments involved in the samples manipulation and the analysis were performed accomplishing all the ethical issues and relevant guidelines and regulations of the implied institutions. The standard additions method was employed for quantification by constructing a calibration plot through the addition of solutions of both targets to sample aliquots. RESULTS AND DISCUSSION As described in the Experimental section, in this work, a dual electrochemical immunosensor was prepared for the simultaneous determination of the Crohn´s disease biomarkers IL-12 and IL-23 in clinical samples. The immunosensor preparation involved the covalent immobilization of the specific anti-IL12 and anti-IL23 capture antibodies on the surface of a SPdCE modified with Phe-(MWCNTs/CNC)SPdCE (Fig. 1 ). The targets were detected using sandwich type immunoassays with the respective biotinylated secondary antibodies and affinity complexation with poly-HRP-strept conjugates. Amperometric detection was performed using H 2 O 2 as the enzymatic substrate and HQ as redox mediator. The measured cathodic current variations at -0.20 V vs Ag pseudo-reference electrode, attributed to HQ-mediated enzymatic reduction of the substrate, were directly proportional to the concentrations of IL-12 and IL-23 cytokines. Characterization studies Transmission electron microscopy (TEM) was used to characterize the structure of MWCNTs/CNC nanocomposites. Figure 2 compares the image obtained for an aqueous 0.25% wt CNC dispersion (Fig. 2 A) with that of 0.125% wt MWCNTs (Fig. 2 B) and the mixture of both nanomaterials (Fig. 2 C). As it can be seen, CNC appears in the form of rigid rodlike structures, with 5.1 ± 1.7 nm width and 100–120 nm length. Furthermore, MWCNTs appear as long and continuous tubes with 30 ± 15 nm width and 5–20 µm length. Finally, MWCNTs/CNC dispersion shows nanotubes surrounded by cellulose nanocrystals.These results agree with those reported by Durairaj et al. [18] as well as the dimensions with those claimed by the manufacturers. HERE FIGURE 2 Electrochemical characterization of the MWCNTs/CNC nanocomposites was performed using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) with 5 mM Fe(CN) 6 3−/4− solutions in 0.1 mol L − 1 PBS of pH 7.4. For comparison, SPCEs were modified by dropping 5 µL of the as prepared dispersions of MWCNTs, CNC or MWCNTs/CNC mixture onto the working electrode surface and allowing drying. Cyclic voltammograms (Fig. 3 a) showed the expected anodic and cathodic peaks of the redox probe as well as some appreciable differences in the peak currents, peak potentials, and ΔE values depending on the electrode composition. Unmodified SPCE exhibited similar anodic and cathodic currents (i pa = 22 µA and i pc = 21 µA), with an i pa / i pc ratio of 1.04. Moreover, ΔE was 320 mV. As it can be seen, slightly higher currents were obtained at the CNC/SPCE (i pa = 30 µA and i pc = 29 µA; i pa / i pc = 1.03), with a lower peak separation (ΔE = 244 mV). These results can be attributed to the high surface area of ​​the CNC and its porous structure, which allows ion transport and greater accessibility of the redox probe. [12]. Regarding the electrodes modified with MWCNTs, lower peak separation (ΔE = 175 mV) for both MWCNTs/SPCE and MWCNTs/CNC/SPCE, and higher peak currents, were observed, which was attributed to the high electrical conductivity of carbon nanotubes. However, it is worth noting the slight decrease in peak currents at the MWCNTs/CNC/SPCE ((i pa = 32 µA and i pc = 31 µA; i pa / i pc = 1.03) compared to the MWCNTs/SPCE (i pa = 35 µA and i pc = 33 µA; i pa / i pc = 1.06) probably due to the small decrease in conductivity when using the composite nanomaterial [20]. Similar conclusions arise by using EIS at the same experimental conditions (Fig. 3 b). Nyquist spectra exhibited significantly lower charge transfer resistance (R CT ) for the electrodes modified with MWCNTs or MWCNTs/CNC (R CT = 984 Ω and 1063 Ω, respectively) than that measured at the bare SPCE, R CT = 2161 Ω, and at the CNC modified electrode, 1823 Ω. This behaviour can be attributed to a better conductivity of carbon nanotubes which slightly decreases in the presence of CNC. HERE FIGURE 3 Optimization of the variables involved in the preparation of the immunosensors The variables involved in the preparation and functioning of the dual immunosensor were optimized by testing individually their effect on the amperometric response for each target biomarker. The selection criterion for the variables was the values of the specific-to-unspecific current (S/N) ratios. The optimization results for IL-12 are described below, whereas those for the individual determination of IL-23 are detailed in Supplementary Information (Figures S2–S4). Table 1 summarizes the experimental variables selected for the simultaneous determination of both biomarkers with the developed immunoplatform. The effect of the capture antibody loading (anti-IL12) over the 5 to 30 µg mL − 1 range, and of its incubation time for immobilization onto grafted MWCNTs/CNC/ SPCEs, between 15 to 60 min, were tested in the absence (N) and in the presence (S) of 2 ng mL − 1 IL-12. Figure 4 a shows as a larger S/N ratio was obtained for 25 µg mL − 1 antibody. Smaller concentrations gave rise to lower specific amperometric currents probably because of the insufficient antibody loading. Moreover, practically constant nonspecific currents were observed, thus providing lower S/N ratios. Furthermore, antibody loadings larger than 25 µg mL − 1 provoked also a decrease in the S/N ratio probably due to the hindered recognition of the target biomarker. Regarding the incubation time, the results of Fig. 4 b led us to select 15 min for further work. Bovine serum albumin (BSA) was used as the blocking agent to minimize unspecific adsorptions onto the modified anti-IL12-MWCNTs/CNC/SPCEs electrode. The effect of the blocker concentration was tested over 2 and 10% and the results were compared with those achieved by using a commercial blocking buffer (BB) consisting of 2% casein. As Fig. 4 c shows, an 8% BSA allowed obtaining a larger S/N ratio. Furthermore, a blocking time of 30 min provided also a better S/N ratio (Fig. 4 d). The effect of the incubation time of IL-12 antigen on the amperometric responses was evaluated over the 15 to 45 min. Figure 4 e shows that although the specific response is somewhat higher for 30 min, the increase in the non-specific currentwhen increasing the immobilization time provoked a worse S/N ratio compared with that achieved incubating for 15 min, which has been chosen for further work. HERE FIGURE 4 Figure 5 a shows as the specific responses increased with the biotinylated antibody loading due to the higher amount of biotin to form the affinity link with the poly-HRP-Strep conjugate for detection. Since a higher S/N ratio was obtained for 0.5 µg mL − 1 , this concentration was selected for further work. Regarding the incubation time for Biotin-anti-IL12 (Fig. 5 b) 30 min were chosen according to the same criterion. With the aim of obtaining higher specific currents and a better S/N ratio, a signal amplification strategy involving poly-HRP-Strept instead of the usual HRP-Strept conjugate was implemented. The effect of the polymer dilution on the immunosensor responses was studied over the 1/250 to 1/2000 range with the results shown in Fig. 5 c. Both specific and non-specific currents gradually decreased as the dilution increased, probably due to the lower number of detector labels on the biotinylated antibodies. However, a better S/N ratio was obtained for a 1/1000 dilution, so this value was chosen for subsequent studies. To further reduce nonspecific responses, the aqueous solutions of poly-enzyme conjugate were prepared in the presence of an optimized amount of BSA. As Fig. 5 d shows, a larger S/N ratio was achieved for 2% BSA. Finally, the effect of the incubation time of poly-HRP-Strept prepared under the previous conditions was checked (Fig. 5 e), 20 min being selected for further work. HERE FIGURE 5 In addition to these variables, the loading of MWCNTs/CNC composite on the SPCE surface was optimized by measuring the variation of the immunosensor responses over the range of 3 to 7 µL suspension. A volume of 5 µL provided a larger specific signal and a better S/N ratio. Other experimental conditions, such as the composition of the H 2 O 2 /HQ system, as well as the potential and pH for the detection, were optimized in previous works [21, 22]. Table 1 Experimental variables tested and values selected for the simultaneous determination of IL-12 and IL-23 with the developed immunosensors. Target biomarker Variable Tested range Selected value IL12 Anti-IL12 loading, µg mL − 1 Anti-IL12 incubation time, min BSA concentration, % BSA blocking time, min IL12 incubation time, min Biotin-anti-IL12 loading, µg mL − 1 Biotin-anti-IL12 incubation time, min Poly-HRP-Strept dilution BSA in poly-HRP-Strept, % Poly-HRP-Strept incubation time, min 5–30 15–60 2–10 15–45 15–45 0.1–0.75 15–45 1//2000–1/250 0–5 15–30 25 15 8 30 15 0.5 30 1/1000 2 20 IL-23 Anti-IL23 loading, µg mL − 1 Anti-IL23 incubation time, min BSA concentration, % BSA blocking time, min IL23 incubation time, min Biotin-anti-IL23 loading, µg mL − 1 Biotin-anti-IL23 incubation time, min Poly-HRP-Strept dilution BSA in poly-HRP-Strept, % Poly-HRP-Strept incubation time, min 20–50 15–45 5–10 15–45 15–45 0.05–0.75 5–45 1/1000–1/250 0–2 10–30 40 20 8 30 15 0.25 15 1/500 1 20 All the steps involved in the preparation of the dual immunosensor were monitored by CV and EIS using 5 mM Fe(CN) 6 3−/4− as the redox probe in 0.1 M PBS of pH 7.4. Similar results were obtained for both biomarkers and, therefore, only those obtained for IL-23 have been displayed in Fig. 6 . Voltammograms for the bare SPCE and MWCNTs/CNC/SPCE (Fig. 6 A curves 1 and 2) already shown in Fig. 3 were included for comparison purposes. Grafting with p -ABA (curve 3) caused a strong decrease in the peak currents which is most likely due to the electrostatic repulsion between the redox probe and the dissociated carboxyl groups on the electrode surface at the working pH. Activation with EDC/sulfo-NHS (curve 4) provoked an increase of peak currents because of the neutralization of the anionic charges. Surprisingly, conjugation with anti-IL23 capture antibody (curve 5) resulted in higher peak currents, which is contradictory to the non-conducting properties of the biomolecule. A possible explanation for this behaviour relies in the isoelectric point of the antibody, 9.3 [23], which means it is positively charged at the working pH thus reinforcing the current magnitude by electrostatic attraction of the redox probe. Thereafter, Fig. 6 B shows that the current decreased dramatically after blocking with BSA (curve 6) while successive incorporation of the other immunoreagents (curves 7 to 9) provoked slight variations in the CVs towards a less reversible behaviour, because of the presence of insulating layers with increasing thickness on the electrode surface. Similar results were obtained by EIS (Fig. 6 C, D). Apart from the large decrease in the electron transfer resistance produced by modification of SPCE with MWCNTs/CNC (Fig. 6 C, curves 1 and 2) it can be seen as grafting with p -ABA (curve 3) provoked the expected increase in the R CT value up to 4512 Ω, which then showed a further decrease to 1753 Ω after neutralization by activation with EDC/sulfo-NHS (curve 4). In addition, conjugation with anti-IL23 capture antibody (curve 5) gave rise to a lower R CT value of 1003 Ω, probably due to the reason explained above to justify the behaviour in CV [23]. Thereafter, blocking of the remaining unreacted sites with BSA produced a larger R CT value of 3276 Ω (Fig. 6 D, curve 6) in agreement with the expected decrease in the electrode conductivity at a quasi-passivated surface. The subsequent incorporation of IL-23 antigen, Biotin-anti-IL23 detection antibody and poly-HRP/Strept (curves 7 to 9) led the charge transfer resistance to values similar than that measured before the blocking. Interestingly, the presence of the biomolecules at the electrode surface provoked also the appearance of two semicircles in the EIS spectra. These results led to define the obtained Nyquist plots by two different equivalent circuits as shown in Fig. 6 . Curves 1–4 fitted well to a Randles R1(C2[R3W1]) circuit, whereas curves 5–9 should be explained by the more complex equivalent circuit depicted on the right, with at least two RC semicircuits, reflecting that some parts of the electrode are coated by the biomolecules while others remain exposed to the solution. The parallel RC circuits mean that there is a film with defects such as pinholes or a non-uniform thickness throughout the substrate [24]. HERE FIGURE 6 Analytical characteristics of the dual immunosensor for the simultaneous determination of IL-12 and IL-23 biomarkers The calibration plots constructed with the dual immunosensor for the determination of the Crohn´s disease biomarkers using the optimized working conditions are illustrated in Fig. 7 which also shows some of the amperograms registered to obtain the corresponding data. As expected, according to the sandwich-type configuration of the immunoassays, the recorded currents were directly proportional to the concentration of each target cytokine. Semilogarithmic plots with wide linear regions between 0.3 and 1000 ng mL − 1 were obtained for both interleukins. The analytical parameters of the corresponding calibration plots are summarized in Table 2 . HERE FIGURE 7 The comparison of the analytical characteristics provided by the dual immunosensor with those claimed for the ELISA kits involving the same immunoreagents allowed us to deduce some advantages when using the immunosensor. In the case of IL-12. the dynamic linear range is much wider, covering from 0.3 to 1000 ng mL − 1 whereas the ELISA kit [Human IL-12 DuoSet ELISA from R&D Systems (Cat. No. DY1270-05)] provides a non-linear semilogarithmic calibration from 0.031 to 2.0 ng mL − 1 . In addition, the ELISA method takes more than 4 hours compared to the 1h 35 min required with the immunosensor counting in both cases from the incubation of the capture antibody. Regarding IL-23, the developed method also provides a linear semi-logarithmic calibration plot ranging between 0.3 and 1000 ng mL − 1 instead of the non-linear semilogarithmic plot provided by the ELISA kit [Human IL-23 DuoSet ELISA from R&D Systems (Cat. No. DY1290-05)] covering from 0.125 to 8.0 ng mL − 1 , which requires more than 4h 40 min instead of 1h 20 min needed for the use of the immunosensor. Obviously, the greatest advantage is that the developed dual platform allows the simultaneous determination of both biomarkers in less than 2 hours, which is approximately a quarter of the time that would be required if the two ELISA kits were used separately. Table 2 Analytical characteristics of the calibration plots for IL-12 and IL-23 constructed with the developed dual immunosensor Parameter IL-12 IL-23 Slope 2.31 ± 0.04 µA/conc decade (ng mL − 1 ) 1.14 ± 0.03 µA/conc decade (ng mL − 1 ) Intercept 1.41 ± 0.07 µA 0.81 ± 0.05 nA Linear range 0.3–1000 ng mL − 1 0.3–1000 ng mL − 1 R 2 0.996 0.993 LOD 0.25 ng mL − 1 0.22 ng mL − 1 LOQ 0.26 ng mL − 1 0.29 ng mL − 1 RSD % (n = 10) (intra-day) 6.2 (0 ng mL − 1 ) 4.1 (10 ng mL − 1 ) 4.1 (0 ng mL − 1 ) 2.7 (10 ng mL − 1 ) RSD % (n = 10) (inter-day) 2.8 (0 ng mL − 1 ) 5.4 (10 ng mL − 1 ) 4.7 (0 ng mL − 1 ) 3.8 (10 ng mL − 1 ) Regarding the achieved LOD and LOQ values, it should be noted that the ELISA kits involving the same reagents report in their protocols only the lowest concentrations of the calibrations: 31.3 pg mL − 1 (IL-12) and 125 pg mL − 1 (IL-23). These values are somewhat lower or similar, respectively, to the LOQ values achieved with the developed method. There is no information about the relative standard deviation obtained with the ELISA kits, which in the case of the dual immunosensor ranged approximately between 3 and 6%. Finally, it should be noted that the sample volume required, 5 µL for both biomarkers, is twenty times smaller than that needed for one target using the ELISA test. The good analytical performance achieved is probably due to the properties of CNC, mainly its biocompatibility and hydrophilicity, as well as the high surface area and open-pore structure which facilitates the penetration of electroactive species resulting in high sensitivity and fast response [25]. These characteristics together with the electrochemical properties of carbon nanotubes provide an excellent composite nanomaterial for the immobilization of biomolecules onto the transducer surface as well as for the amperometric detection. In addition, the dual immunosensor exhibited good reproducibility and excellent stability, suggesting that it could be used for the determination of the target biomarkers in clinical samples from patients of Crohn´s disease or other IBDs. Storage stability With the aim of evaluating the storage stability of the dual immunosensor for the determination of IL-12 and IL-23 biomarkers, different immunoplatforms were prepared on the same day, stored in PBS pH 7.4 at 4 ºC, and employed to measure simultaneously both targets on different days after incubation with 10 ng mL − 1 of each target according to the procedure described in the Experimental section. The obtained results (Figure S5) indicated that the anti-IL12-MWCNTs/CNC/SPCE and anti-IL-23-MWCNTs/CNC/SPCE bioelectrodes were stable for at least 71 days (the longest storage time tested) since the current responses remained inside the \(\:\stackrel{-}{x}\) ± 3s limits, where s was the standard deviation of the measurements (n = 10) carried out on the first day. Therefore, at least during this period it is feasible to prepare the immunosensors from the stored bioconjugates, allowing the determination of IL-12 and IL-23 after incubation of the cytokines, the detection antibodies and the peroxidase conjugate, in around 60 min. This long-term stability is attributable most likely to the CNC properties, especially its biocompatibility and hydrophilicity, as well as its robustness to slight variations in pH, ionic strength or temperature [25]. Selectivity The effect of potential interfering compounds that may be present together with the target biomarkers in biological samples on the electrochemical responses obtained with the dual immunosensor was checked. The compounds tested were proteins related to inflammation disorders such as those associated with Crohn's disease, ulcerative colitis and other IBDs, as well as others usually present in human serum. Their influence was evaluated at concentration levels that correspond approximately to the normal physiological level found in healthy individuals: 5 mg mL − 1 haemoglobin (HB), 50 mg mL − 1 human serum albumin (HSA); 1 mg mL − 1 human immunoglobulin (hIgG); 100 pg mL − 1 interleukin 6 (IL-6); 200 pg mL − 1 tumour necrosis factor alpha (TNF-α) and 100 pg mL − 1 interferon gamma (INF-γ). In addition, the possible cross-interference of each biomarker in the determination of the other was evaluated at concentration levels of 2 ng mL − 1 . The results in Fig. 8 show as no significantly different responses were obtained in all cases since all the mean steady state current values remained within the ± 3 × standard deviation range of the current values measured in the absence of potential interferent. This excellent selectivity can be attributed to the practical specificity of the capture antibodies towards the target cytokines. HERE FIGURE 8 Determination of IL-12 and IL-23 in human serum and faeces The simultaneous determination of the CD biomarkers was accomplished in human serum of a healthy individual (Bio Hub Ref.100468118) and in faeces of a volunteer. In the case of serum, the procedure described in the Experimental section was applied to the determination of the two biomarkers using 5 µL aliquots of undiluted sample. For faeces, an Eiken OC-Auto Sampling Bottle3 (Ref. V-PZ25) [ https://www.eiken.co.jp/uploads/IFU/340026A-G_en_20220401.pdf ] was used for sample collection and dispersion in the appropriate medium. Specifically, 10 mg of faeces were collected each time and dispersed in 2 mL of HEPES buffer included in the container. As indicated above, all the experiments involved in samples manipulation and analysis were performed accomplishing all the ethical issues and relevant guidelines and regulations of the implied institutions. Firstly, the matrix effects from the sample solutions were evaluated by applying the Student’s t-test to compare the slope values of the calibration plots for the target standards constructed in buffer solutions with those obtained by applying the standard additions method. The results for the direct analysis of both undiluted serum and stool solution provided texp values larger than that tabulated t (ttab = 2.365), indicating that apparent matrix effects occurred in the determination of IL-12 and IL-23 under the mentioned conditions. Therefore, the standard additions method was applied for the determination of the biomarkers. This method also avoids possible variabilities in the slope value of the calibration plots constructed from serum or faeces collected from different individuals. Using this protocol, the obtained results are summarized in Table 3 . Excellent recoveries ranging between 97 ± 4% and 103 ± 6% were found in spiked serum and between 96 ± 5% and 102 ± 6% in faeces, for IL-12 and IL-23, respectively. Table 3 Determination of IL-12 and IL-23 in raw serum and faeces with the dual immunosensor. SAMPLE IL-12, ng mL − 1 IL-23, ng mL − 1 Added Found* Recovery, % Added Found* Recovery, % Serum 0.3 0.31 ± 0.02 103 0.3 0.30 ± 0.01 99 0.5 0.50 ± 0.03 100 0.5 0.49 ± 0.02 97 1.0 1.0 ± 0.3 102 1.0 1.0 ± 0.2 99 1.5 1.48 ± 0.06 99 1.5 1.51 ± 0.05 101 0.3 0.3 ± 0.1 99 0.3 0.29 ± 0.05 97 Faeces 0.5 0.51 ± 0.03 102 0.5 0.48 ± 0.04 97 1.0 1.0 ± 0.3 102 1.0 0.96 ± 0.05 96 1.5 1.5 ± 0.3 99 1.5 1.5 ± 0.2 102 * Mean value ± 2s, n = 3 CONCLUSIONS The first dual immunosensor for the simultaneous determination of IL-12 and IL-23, two relevant biomarkers of CD is reported in this work. The antibodies for the selective capture of the respective interleukins were covalently immobilized onto dual SPCEs modified with CNC and MWCNTs followed by conjugation with a detector antibody labelled with poly-HRP-Strept, and amperometric transduction using the H 2 O 2 /HQ system. The developed bioplatform provides results with a high reproducibility and selectivity using lower sample volumes and in a much shorter assay time than those reported for each target using commercially available ELISA kits. Interestingly, a long storage stability of the anti-IL12-MWCNTs/CNC/SPCE and anti-IL-23-MWCNTs/CNC/SPCE bioelectrodes of at least 71 days was observed. The good analytical performance is probably due to the combination of the CNC properties, mainly its biocompatibility and hydrophilicity, the high surface area and open pore structure, together with the electrochemical properties of MWCNTs, which provide an excellent nanocomposite for biomolecules immobilization onto the transducer surface and electrochemical detection. The suitability and applicability of the dual immunosensor were demonstrated by the analysis of raw serum and faeces spiked with IL-12 and IL-23 at the levels that can be found in samples from patients suffering severe CD obtaining excellent recoveries. Declarations Consent to participate Informed consent was obtained from all individual participants included in the study. Ethics Approval This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Hospital Clínico San Carlos, University Complutense of Madrid. Funding: The financial support of PID2021-122457OB-I00 (Spanish Ministerio de Ciencia e Innovacion) is gratefully acknowledged. Author Contribution L.G.-R.: Methodology, Investigation. C.R.-L.: Methodology, Investigation. E. S.-T.: Writing – review & editing, Investigation. L. A.: Supervision, Conceptualization. A. G.-C.: Supervision, Resources,Funding acquisition, Conceptualization. P.Y.-S.: Writing – original draft, Supervision, Resources, Funding acquisition, Conceptualization. J.M. P.: Supervision, Resources. All authors reviewed the manuscript. References Baumgart DC, Sandborn WJ (2007) Inflammatory bowel disease: clinical aspects and established and evolving therapies, Lancet 369:1641–1657 Zundler S, Neurath MF (2015) Interleukin-12: Functional activities and implications for disease, Cytokines & Growth Factor Rev. 26:559-568 Kaser A, Zeissig S, Blumberg RS (2020) Inflammatory bowel disease. Annu Rev Immunol. 28:573–576 Shida K, Kiyoshima-Shibata J, Nagaoka M, Watanabe K, Nanno M (2006) Induction of Interleukin-12 by Lactobacillus strains having a rigid cell wall resistant to intracellular digestion, J. Dairy Sci. 89:3306–3317 Aggeletopoulou I, Assimakopoulos SF, Konstantakis C, Triantos C (2018) Interleukin 12/interleukin 23 pathway: Biological basis and therapeutic effect in patients with Crohn's disease, World J. 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Supplementary Files SupplementaryInformation.docx Cite Share Download PDF Status: Published Journal Publication published 04 Dec, 2024 Read the published version in Microchimica Acta → Version 1 posted Editorial decision: Revision requested 11 Nov, 2024 Reviews received at journal 10 Nov, 2024 Reviewers agreed at journal 17 Oct, 2024 Reviews received at journal 17 Oct, 2024 Reviewers agreed at journal 17 Oct, 2024 Reviewers invited by journal 17 Oct, 2024 Editor assigned by journal 16 Oct, 2024 Submission checks completed at journal 16 Oct, 2024 First submitted to journal 14 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5259345","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":367423275,"identity":"7f8b7959-9236-45e1-a5fb-89c6f78b39b8","order_by":0,"name":"Lorena García-Rodrigo","email":"","orcid":"","institution":"University Complutense of Madrid","correspondingAuthor":false,"prefix":"","firstName":"Lorena","middleName":"","lastName":"García-Rodrigo","suffix":""},{"id":367423277,"identity":"d56fa4b8-c449-4363-b337-d09679995278","order_by":1,"name":"Claudia Ramos-López","email":"","orcid":"","institution":"University Complutense of Madrid","correspondingAuthor":false,"prefix":"","firstName":"Claudia","middleName":"","lastName":"Ramos-López","suffix":""},{"id":367423279,"identity":"8f57b7c1-9d3e-4233-9cb8-284d568d76a7","order_by":2,"name":"Esther Sánchez-Tirado","email":"","orcid":"","institution":"University Complutense of Madrid","correspondingAuthor":false,"prefix":"","firstName":"Esther","middleName":"","lastName":"Sánchez-Tirado","suffix":""},{"id":367423281,"identity":"a93d13de-5c00-4601-9b97-e47555d73d78","order_by":3,"name":"Lourdes Agüí","email":"","orcid":"","institution":"University Complutense of Madrid","correspondingAuthor":false,"prefix":"","firstName":"Lourdes","middleName":"","lastName":"Agüí","suffix":""},{"id":367423282,"identity":"bc7b416c-6678-43af-899e-203c3596d8f1","order_by":4,"name":"Araceli González-Cortés","email":"","orcid":"","institution":"University Complutense of Madrid","correspondingAuthor":false,"prefix":"","firstName":"Araceli","middleName":"","lastName":"González-Cortés","suffix":""},{"id":367423283,"identity":"3a871528-f8b5-4ced-b43c-df789625b597","order_by":5,"name":"Paloma Yáñez-Sedeño","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsUlEQVRIiWNgGAWjYLCCDwwMPKTpYJxBshZm0tQbnD988LFN2WEZ3QYew083GGzsCWu5kZZsnHPuMI/ZAR5j6RyGtMQGwlp4zKRz28BazJhzGA4nEOGw89+kLRFa/hPhsAM5bNKMCC0HGAk6TPJGmrFhz7l0HrPDbMXSOQbJhP3Cd/7wwwc/yqztzY43b/ycU2FH2GEKB0AkGxAzg91JUAMDg3wDTMsoGAWjYBSMAlwAAGkrNmafQzO0AAAAAElFTkSuQmCC","orcid":"","institution":"University Complutense of Madrid","correspondingAuthor":true,"prefix":"","firstName":"Paloma","middleName":"","lastName":"Yáñez-Sedeño","suffix":""},{"id":367423284,"identity":"c32cfb34-b382-4c1c-b77e-cddab0552b87","order_by":6,"name":"José M. Pingarrón","email":"","orcid":"","institution":"University Complutense of Madrid","correspondingAuthor":false,"prefix":"","firstName":"José","middleName":"M.","lastName":"Pingarrón","suffix":""}],"badges":[],"createdAt":"2024-10-14 08:23:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5259345/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5259345/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00604-024-06868-2","type":"published","date":"2024-12-04T15:57:32+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":67294846,"identity":"a449b303-d156-4909-81da-6e8ccdcf630f","added_by":"auto","created_at":"2024-10-23 10:49:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":263055,"visible":true,"origin":"","legend":"\u003cp\u003eSteps involved in the preparation of the dual immunosensor for the simultaneous determination of IL-12 and IL-23, and the reaction involved in the amperometric transduction.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5259345/v1/9882e01eaf00bac2f4325ca7.png"},{"id":67293070,"identity":"17267266-42e9-48a6-8144-7dd8f76bbd1c","added_by":"auto","created_at":"2024-10-23 10:33:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":307296,"visible":true,"origin":"","legend":"\u003cp\u003eTEM images of A) CNC; B) MWCNTs and C) MWCNTs/CNC dispersions.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5259345/v1/240520775429ec8c5d2dabde.png"},{"id":67293071,"identity":"48dba34f-067e-4fcd-bbb6-9a1d1ce89448","added_by":"auto","created_at":"2024-10-23 10:33:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":58031,"visible":true,"origin":"","legend":"\u003cp\u003eCyclic voltammograms (A) and Nyquist plots (B) recorded in 5 mM\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Fe(CN)\u003csub\u003e6\u003c/sub\u003e\u003csup\u003e3-/4-\u003c/sup\u003e in 0.1 mol L\u003csup\u003e-1\u003c/sup\u003e PBS of pH 7.4 at: SPCE (1, blue); CNC/SPCE (2, black); MWCNTs/SPCE (3, green); MWCNTs/CNC/SPCE (4, red).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5259345/v1/280294e38201e883e890502a.png"},{"id":67294847,"identity":"25eea9e2-84db-4a15-99f7-6cd899933ca4","added_by":"auto","created_at":"2024-10-23 10:49:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":54337,"visible":true,"origin":"","legend":"\u003cp\u003eEffect on the amperometric responses obtained for IL12 with: a) loading and b) incubation time of anti-IL12; c) concentration of BSA blocker and effect of BB; d) blocking time with BSA; e) incubation time of IL-12 antigen. Amperometric responses measured in the absence (N; light bars) and in the presence of 2 ng mL\u003csup\u003e−1\u003c/sup\u003e IL12 standard solutions (S; dark bars) as well as the resulting S/N ratio values (circles and red line). Error bars were estimated as three times the standard deviation value of three replicates.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5259345/v1/6637fe43dd9e436d50861ad4.png"},{"id":67294075,"identity":"2773bb40-0863-43dd-be1a-dbebad119a57","added_by":"auto","created_at":"2024-10-23 10:41:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":60284,"visible":true,"origin":"","legend":"\u003cp\u003eEffect on the amperometric responses obtained for IL12 with a) concentration and b) incubation time of Biotin-anti-IL12;\u003cstrong\u003e \u003c/strong\u003ec) dilution of poly-HRP-Strept; d) BSA concentration in the poly-HRP-Strept solutions, and e) incubation time of poly-HRP-Strept. Amperometric responses measured in the absence (N; light bars) and in the presence of 2 ng mL\u003csup\u003e−1\u003c/sup\u003e IL-12 standard solutions (S; dark bars) as well as the resulting S/N ratio values (circles and red lines). Error bars are estimated as three times the standard deviation value of three replicates.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5259345/v1/fac9d6aa92a2d0115d111c52.png"},{"id":67293074,"identity":"39a83dac-cf44-4a14-876c-cafd0e037e6e","added_by":"auto","created_at":"2024-10-23 10:33:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":105611,"visible":true,"origin":"","legend":"\u003cp\u003eCyclic voltammograms \u003cstrong\u003e(A,B)\u003c/strong\u003e and Nyquist plots \u003cstrong\u003e(C,D)\u003c/strong\u003e recorded for 5 mM Fe(CN)\u003csub\u003e6\u003c/sub\u003e\u003csup\u003e3-/4-\u003c/sup\u003e in 0.1 mol L\u003csup\u003e-1\u003c/sup\u003e PBS of pH 7.4 at: \u003cstrong\u003e\u0026nbsp;(A,C)\u003c/strong\u003e\u0026nbsp; SPCE (1, dark blue); MWCNTs/CNC/SPCE (2, orange); HOOC-Phe-MWCNTs/CNC/SPCE (3, green); HOOC-Phe-MWCNTs/CNC/SPCE after EDC/sulfo-NHS activation (4, light blue); anti-IL-23-MWCNTs/CNC/SPCE (5, violet). \u003cstrong\u003e(B, D)\u003c/strong\u003e blocked anti-IL23-MWCNTs/CNC/SPCE (6, pink); IL23-anti-IL23-MWCNTs/CNC/ SPCE (7, green); Biotin-anti-IL23-IL-23-anti-IL23-MWCNTs/CNC/SPCE (8, orange); poly-HRP/ Strept-Biotin-anti-IL23-IL23-MWCNTs/CNC/SPCE (9, black). The equivalent circuits used to adjust the experimental results are shown below.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5259345/v1/dd76703370ea1c2c1db60d6b.png"},{"id":67294074,"identity":"a5de6821-ddbc-48fa-9c22-13c8241d0425","added_by":"auto","created_at":"2024-10-23 10:41:27","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":51572,"visible":true,"origin":"","legend":"\u003cp\u003eCalibration plots and amperometric response obtained with the dual immunosensor for the simultaneous determination of IL-12 (1, blue) and IL-23 (2, green). Error bars are estimated as three times the standard deviation value of three replicates.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5259345/v1/ee4118eb7f40a8aa53df0f59.png"},{"id":67293078,"identity":"2cbc8318-4e45-4b93-8a19-b65b6701ff44","added_by":"auto","created_at":"2024-10-23 10:33:27","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":49973,"visible":true,"origin":"","legend":"\u003cp\u003eAmperometric responses provided by the developed immunosensor for 2 ng mL\u003csup\u003e-1 \u003c/sup\u003eIL-12 (a) or IL-23 (b) in the presence of 5 mg mL\u003csup\u003e-1\u003c/sup\u003e haemoglobin (HB), 50 mg mL\u003csup\u003e-1\u003c/sup\u003e human serum albumin (HSA); 1 mg mL\u003csup\u003e-1 \u003c/sup\u003ehuman immunoglobulin (hIgG);\u0026nbsp; 100 pg mL\u003csup\u003e-1\u003c/sup\u003e interleukin 6 (IL-6); 200 pg mL\u003csup\u003e-1 \u003c/sup\u003etumour necrosis factor alpha (TNF-α) and 100 pg mL\u003csup\u003e-1 \u003c/sup\u003einterferon gamma (INF-γ). Error bars were estimated as three times the standard deviation value of three replicates.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5259345/v1/9d71a1c6001c49eb9f8a6da0.png"},{"id":70964707,"identity":"c90a6d80-02dd-41ba-afdc-7eaec465cdc0","added_by":"auto","created_at":"2024-12-09 16:14:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1691000,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5259345/v1/03fcd294-dfe7-46fa-9242-a76f47c58d3e.pdf"},{"id":67294072,"identity":"191ff1d6-c384-4f8c-b329-513b34020ee0","added_by":"auto","created_at":"2024-10-23 10:41:27","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":543676,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5259345/v1/d43a822d374b3f8d35b423a2.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Electrochemical immunosensing of Crohn´s disease biomarkers using diazonium salt grafted - crystalline nanocellulose / carbon nanotubes modified electrodes","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe major forms of human inflammatory bowel disease (IBD) are Crohn's disease (CD) and ulcerative colitis (UC) [1]. They arise from a complex interplay of genetics, gut microbiome dysregulation and alteration of the intestinal epithelial barrier followed by a misdirected immune response [2]. CD is an immune-mediated inflammatory disorder characterized by chronic relapsing inflammation in different segments of the gastrointestinal tract caused by a disruption in the balance among the intestinal epithelium, the commensal microbiota and the innate immune response. The presence of defects in the intestinal wall together with a dysfunction in regulatory mechanisms maintain this condition leading to the release of an array of cytokines which in turn promotes the inflammatory immune response [3]. In the intestinal wall of CD patients there are innate lymphoid cells that are stimulated by IL-12 to produce interferon gamma (IFN-γ) which in turn mediates proinflammatory functions, thus participating in the pathogenesis of the intestinal mucosa inflammation [4]. IL-23, another cytokine belonging to the IL-12 family, has also been shown to be involved in CD [5]. Indeed, the IL-12 / IL-23 system is closely connected with the appearance of inflammation in the intestinal wall and over distinct effects on the promotion of tumorigenesis, cancer progression and mechanisms of resistance related to this disease [6]. Due to this clear implication, inhibitors of IL-12/IL-23, especially the human antibody ustekitumab, have been developed for CD management.\u003c/p\u003e \u003cp\u003eThe methods used to determine these interleukins are mainly based on the ELISA technique. Commercial colorimetric ELISA kits involve specific capture antibodies for the establishment of sandwich configurations with biotinylated secondary antibodies and streptavidin- or avidin- peroxidase (HRP) conjugates. The analytical characteristics of these tests are similar. As an example, the Invitrogen Human IL-12 ELISA kit BMS238 (Thermo Fisher Scientific) provides a nonlinear semi-logarithmic calibration over the 3.1 to 200 pg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e range with a detection limit of 2.1 pg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and an assay time slightly longer than 3 hours. In the case of IL-23, the Invitrogen Human IL-23 ELISA kit BMS2023-3 (Thermo Fisher Scientific), with a similar setup using the Avidin-HRP conjugate, provides a nonlinear semilogarithmic calibration over the 15.6 to 2000 pg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e range, with a detection limit of 4.0 pg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and an assay time of 3.5 h. A kit for the joint determination of IL-12 and IL-23 (ELISA kit MBS2013 (Thermo Fisher Scientific) is also available in which both cytokines bind to the specific antibodies adsorbed in the microwells followed by conjugation with a biotin- anti -human IL12/IL23 antibody and the addition of Strept-HRP. Colorimetric detection provides a non-linear semi-logarithmic calibration in the 31.3 to 2000 pg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e range for both interleukins with a detection limit around 10 pg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and an assay time of just over 4 hours. Regarding biosensors, very few examples have been found in the literature. A label-free impedimetric biosensor for IL-12 involving a disposable gold-coated silver ribbon electrode for immobilizing anti-IL-12 antibodies by covalent linking using 16-mercaptohecadecanoic (MHDA) was reported [7]. Direct measurements of charge transfer resistance (Rct) after conjugation with the target were performed proving a non-linear calibration plot. The same group developed an IL-12-immunosensor based on a printed circuit board (PCB) electrode with electroplated gold [8]. A calibration plot covering a concentration range between 0.1 and 500 pg mL-1IL-12 and a LOD value of 3.5 pg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was obtained.\u003c/p\u003e \u003cp\u003eCellulose nanocrystals (CNC) is a biocompatible and sustainable biopolymer nanomaterial characterized by the high specific surface area and the presence of a large number of OH groups which can be further modified for the incorporation of binding sites for biomolecules. The porous network structure and hydrophilicity of CNC together with the film forming ability and stability [9] have converted it into a valuable nanomaterial for the preparation of electrochemical (bio)sensors. As a drawback, the electrical nonconductivity of CNC must be noted, although the transportation of ions is possible and so, ion conductivity in low-concentration solutions due to the charge mobility and porosity is remarkable [10]. In addition, combination of nanocellulose with highly conductive materials such as metal nanoparticles, graphene [11] or carbon nanotubes [12] can provide these materials with high electrical conductivity, which is necessary in some applications. Particularly, the properties of functionalized nanocellulose / multiwalled carbon nanotubes (MWCNTs) for electrochemical applications have been studied [13]. It has been shown that CNC constitutes an effective dispersing medium for carbon nanotubes, and the incorporation of the resulting hybrid nanomaterial to electrode surfaces provides improved electrical conductivity, high mechanical strength and large surface area. However, these advantages have been scarcely explored to date. A modified electrode involving nanocellulose /MWCNTs hybrids was reported for the detection of diclofenac in pharmaceutical products and biological fluids [12]. Nanocellulose and single-walled carbon nanohorns (SWCNH) were used to prepare modified electrodes for the simultaneous detection of guanine and adenine [14].\u003c/p\u003e \u003cp\u003eIn this article, we report the development of a dual immunosensor for the simultaneous determination of the Crohn\u0026acute;s biomarkers IL-12 and IL-23 using screen-printed dual carbon electrodes (SPdCEs) modified with the MWCNTs/CNC nanocomposite as substrate for the immobilization of capture antibodies by electrografting of the \u003cem\u003ep\u003c/em\u003e-aminobenzoic acid diazonium salt. Grafting methods on carbon surfaces have demonstrated to be a powerful route to incorporate linking groups in a simple and rapid way [15,16]. The immunoassay strategy involved covalent immobilization of the specific antibodies (anti-IL12 or anti-IL23) on the respective electrode surface of the modified SPdCE followed by complexation with each target and implementation of sandwich type immunoassays with biotinylated detector antibodies. The respective amperometric responses were measured after affinity binding with polymeric HRP-streptavidin conjugate (poly-HRP-Strept) using the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/ hydroquinone (HQ) system. The proposed multianalyte immunosensor was successfully applied to the determination of IL-12 and IL-23 biomarkers in spiked faecal samples and serum from healthy individuals.\u003c/p\u003e"},{"header":"EXPERIMENTAL","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eApparatus and electrodes\u003c/h2\u003e \u003cp\u003eAmperometric measurements were made at room temperature using a CH1 1030B potentiostat (Chemical Instruments, Inc.) controlled by the CH1 1030B software. A \u0026micro;Autolab type III potentiostat (Ecochemie) controlled by FRA2 software electrochemical impedance spectroscopy (EIS) was employed for other electrochemical measurements. Screen-printed carbon electrodes both single (SPCEs, DRP-110 with a 4 mm-\u0026Oslash; carbon working electrode) and dual (SPdCEs, X1110 DRP with two elliptic carbon working electrodes and a surface area of 4.7 mm\u003csup\u003e2\u003c/sup\u003e ) including a carbon counter electrode and a silver pseudo-reference electrode were from Metrohm-DropSens. The specific DRP-CAC connection cables employed as interface between the SPCEs and the potentiostat were also from Metrohm-Dropsens. The measurements were performed in stirred solutions using 10-mL glass electrochemical cells from Pobel. A Crison model Basic 20\u0026thinsp;+\u0026thinsp;pH meter, a P-Selecta Ultrasons ultrasonic bath, a Heidolph Reax Top homogenizer for small samples, and an MPW-65R centrifuge from MPW (Med. Instruments) were also used.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eReagents and solutions\u003c/h3\u003e\n\u003cp\u003eCrystalline nanocellulose (Cellulose Nanocrystals Freeze-Dried, CNC-FD, 5\u0026ndash;20 nm W, 100\u0026ndash;250 nm L) with hydroxyl and sulfonic surficial groups (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e Supplementary Information) was supplied by Cellulose Lab. Inc., Fredericton, NB, Canada. Multi-walled carbon nanotubes (MWCNTs; ϕ 30\u0026thinsp;\u0026plusmn;\u0026thinsp;15 nm, 95% purity) were from NanoLab, Brighton, MA. Before use, 25 mg of MWCNTs were suspended in 85 mL of deionized water and ultrasonically stirred for one hour. Then, they were chemically shortened and carboxylated by addition of 16 mL of 65% w/w nitric acid keeping under reflux for one hour. Once cooled at room temperature, the resulting product was centrifuged at 4000 rpm for 10 min and washed repeatedly with deionized water until washing liquids reached pH 7, and finally dried under nitrogen stream [17]. N-hydroxysulfosuccinimide (Sulfo-NHS), N-(3-dimethyl-amino-propyl)-N\u0026rsquo;-ethylcarbo-diimide (EDC), HQ, hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 30% v/v), \u003cem\u003ep\u003c/em\u003e-aminobenzoic acid (p-ABA), and sodium nitrite, were purchased from Sigma-Aldrich. Poly-HRP-streptavidin was from Pierce Thermo Scientific. Sodium chloride, potassium chloride, sodium di-hydrogen phosphate, di-sodium hydrogen phosphate, and tris-hydroxymethyl-aminomethane-HCl (Tris\u0026ndash;HCl) were from Scharlab. Anti-IL-12 capture antibody (anti-IL12), human IL-12 standard (IL12) and biotin-anti-IL-12 detection antibody (Biotin-anti-IL12) were from the Human IL-12 DuoSet ELISA from R\u0026amp;D Systems (Cat. No. DY1270-05). Anti-IL-23 capture antibody (anti-IL23), human IL-23 standard (IL23) and biotin-anti-IL-23 detection antibody (Biotin-anti-IL23) were from the Human IL-23 DuoSet ELISA from R\u0026amp;D Systems (Cat. No. DY1290-05). Buffer solutions were 25 mM MES of pH 5.0 prepared from 2-(N-morpholine) ethane-sulfonic acid (Gerbu, Heidelberg, Germany), 100 mM, 50 mM phosphate buffer (PB) of pH 6.0 prepared from Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e and NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e\u0026sdot;2H\u003csub\u003e2\u003c/sub\u003eO (Sigma-Aldrich), and saline phosphate buffer (PBS) consisting of a 100 mM sodium phosphate buffer of pH 7.4 supplemented with 2.0 g NaCl and 50.25 mg KCl (Scharlab) in 250 mL deionized water. In all cases, the pH value was adjusted by adding the required volume of 2 M NaOH solution. Deionized water was from a Millipore Milli-Q purification system (18.2 MΩ cm).\u003c/p\u003e\n\u003ch3\u003eSamples\u003c/h3\u003e\n\u003cp\u003eThe developed immunosensor was applied to the simultaneous determination of IL-12 and IL-23 in undiluted human serum and in faecal samples from healthy volunteers spiked with both cytokines at the concentration levels expected for Crohn\u0026acute;s disease patients. The samples were stored at -80 \u0026ordm;C until use. All the experiments involved in samples manipulation and analysis were performed accomplishing all the ethical issues and relevant guidelines and regulations of the implied institutions.\u003c/p\u003e\n\u003ch3\u003eProcedures\u003c/h3\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of the MWCNTs/CNC aqueous dispersion\u003c/h2\u003e \u003cp\u003e0.0125 g of MWCNTs previously treated with nitric acid, as described above, were added to 10 mL of a 0.25% (w/v) CNC aqueous suspension contained in a Falcon\u0026reg; tube. The mixture was stirred in the ultrasonic bath for 10 min under pulsed mode to avoid material overheating [18]\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of the dual immunsosensor\u003c/h2\u003e \u003cp\u003eThe scheme displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the steps involved in the modification of the SPdCEs, the preparation of the dual immunosensor and the electrochemical detection for the simultaneous determination of IL-12 and IL-23. Firstly, a 5 \u0026micro;L aliquot of the as prepared MWCNTs/CNC aqueous dispersion was deposited on each SPdCE surface allowing it to dry at room temperature. Next, the diazonium salt was synthesized separately by following the method previously described [19]. Briefly, a 2 mM NaNO\u003csub\u003e2\u003c/sub\u003e aqueous solution was added dropwise to 1 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e \u003cem\u003ep-\u003c/em\u003eABA solution prepared in 1 M HCl (38 mL NaNO\u003csub\u003e2\u003c/sub\u003e for each 200 mL \u003cem\u003ep\u003c/em\u003e-ABA) and cooled with ice. The reaction was allowed proceeding during 10 min under stirring. Thereafter, the MWCNTs/ CNC/SPdCE was immersed into the diazonium salt solution and ten successive voltammetric cycles between 0 and \u0026minus;\u0026thinsp;1.0 V vs. Ag pseudo-reference electrode (ν\u0026thinsp;=\u0026thinsp;200 mV s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were scanned. Finally, the modified electrode was washed thoroughly with water (10 s) and methanol (10 s) and dried at room temperature. The activation of carboxylic groups was performed by the addition of 10 \u0026micro;L of a 0.1 mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e EDC/sulfo-NHS solution prepared in 25 mM MES buffer of pH 5.0 to each working electrode (W1 and W2) and incubated for 30 min in a humid ambient at room temperature. The electrode was then washed with the same buffer solution and the capture antibodies (anti-IL12 and anti-IL23) were immobilized on the activated Phe-(MWCNTs/CNC)/ SPdCE by dropping 2.5 \u0026micro;L of a 25 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e anti-IL12 or anti-IL23 solution on each electrode surface (W1 and W2 respectively), and incubating for 15 min at room temperature. After washing with PBS of pH 7.4, the unreacted activated groups of the electrode surfaces were blocked by adding 2.5 \u0026micro;L of 8% BSA in PBS pH 7.4 allowing incubation for 30 min. Thereafter, the electrode was washed with the same buffer solution.\u003c/p\u003e \u003cp\u003eThe sandwich-type dual immunosensor was implemented by dropping 2.5 \u0026micro;L of the IL12 and IL23 standard solutions or the sample prepared in PBS pH 7.4 on the respective anti-IL12-Phe-(MWCNTs/CNC)/SPdCE or anti-IL23-Phe-(MWCNTs/ CNC) /SPdCE allowing incubation for 15 min at room temperature. Thereafter, 5 \u0026micro;L of the detection biotinylated antibodies, Biotin-anti-IL12 or Biotin-anti-IL23 were dropped respectively onto the as prepared electrodes and incubated for 30 min. Finally, 5 \u0026micro;L of 1/1000 diluted poly-HRP-Strept in PBS pH 7.4 and 2% BSA were added allowing 20 min of incubation. The resulting dual immunosensor was washed with PBS pH 7.4 and kept with a 25 \u0026micro;L drop of the same buffer until the electrochemical measurements were made. All incubation steps involved in the immunosensor fabrication were performed at room temperature in a humid environment to prevent drop evaporation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHERE\u003c/b\u003e FIGURE \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAmperonetric measurements\u003c/h3\u003e\n\u003cp\u003eThe amperometric responses were measured by immersing the modified electrode into the electrochemical cell containing 10 mL of 50 mM PB pH 6.0 and 100 \u0026micro;L of a 100 mM HQ fresh solution prepared in the same buffer. The measurements were carried out under stirring by applying \u0026minus;\u0026thinsp;0.20 V vs. the Ag pseudo-reference electrode. Once the background current was stabilized (~\u0026thinsp;50 s), 50 \u0026micro;L of a 100 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution prepared daily in 50 mM PB pH 6.0 were added and the variation in the cathodic current due to the HRP reduction of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e mediated by HQ, reaching the steady state in ~\u0026thinsp;100 s, was recorded. The analytical responses are the mean values of three replicates, and the error bars displayed were estimated as three times the standard deviation of each set of replicates (α\u0026thinsp;=\u0026thinsp;0.05).\u003c/p\u003e\n\u003ch3\u003eSamples analysis\u003c/h3\u003e\n\u003cp\u003eThe optimized procedure was applied to the simultaneous determination of IL-12 and IL-23 in undiluted human serum and in faecal samples from a healthy volunteer. Before any pretreatment, the samples were spiked with both cytokines at the concentration levels expected for Crohn\u0026acute;s disease patients and stored at -80 \u0026ordm;C until use. Recovery studies were performed using 5 \u0026micro;L aliquots of raw serum spiked with IL-12 and IL-23. In the case of faecal samples, once collected and weighed accurately in a special container (OC-Auto Sampling Bottle3 Ref. V-PZ24 from Eiken Chemical Co Ltd.) the spiked sample was dispersed in 2 mL of the HEPES buffer included in the container by vigorous manual stirring. For the determination, 5 \u0026micro;L aliquots of this dispersion were used. All the experiments involved in the samples manipulation and the analysis were performed accomplishing all the ethical issues and relevant guidelines and regulations of the implied institutions. The standard additions method was employed for quantification by constructing a calibration plot through the addition of solutions of both targets to sample aliquots.\u003c/p\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cp\u003eAs described in the \u003cspan refid=\"Sec2\" class=\"InternalRef\"\u003eExperimental\u003c/span\u003e section, in this work, a dual electrochemical immunosensor was prepared for the simultaneous determination of the Crohn\u0026acute;s disease biomarkers IL-12 and IL-23 in clinical samples. The immunosensor preparation involved the covalent immobilization of the specific anti-IL12 and anti-IL23 capture antibodies on the surface of a SPdCE modified with Phe-(MWCNTs/CNC)SPdCE (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The targets were detected using sandwich type immunoassays with the respective biotinylated secondary antibodies and affinity complexation with poly-HRP-strept conjugates. Amperometric detection was performed using H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e as the enzymatic substrate and HQ as redox mediator. The measured cathodic current variations at -0.20 V vs Ag pseudo-reference electrode, attributed to HQ-mediated enzymatic reduction of the substrate, were directly proportional to the concentrations of IL-12 and IL-23 cytokines.\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization studies\u003c/h2\u003e \u003cp\u003eTransmission electron microscopy (TEM) was used to characterize the structure of MWCNTs/CNC nanocomposites. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e compares the image obtained for an aqueous 0.25% wt CNC dispersion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) with that of 0.125% wt MWCNTs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and the mixture of both nanomaterials (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). As it can be seen, CNC appears in\u003c/p\u003e \u003cp\u003ethe form of rigid rodlike structures, with 5.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7 nm width and 100\u0026ndash;120 nm length. Furthermore, MWCNTs appear as long and continuous tubes with 30\u0026thinsp;\u0026plusmn;\u0026thinsp;15 nm width and 5\u0026ndash;20 \u0026micro;m length. Finally, MWCNTs/CNC dispersion shows nanotubes surrounded by cellulose nanocrystals.These results agree with those reported by Durairaj et al. [18] as well as the dimensions with those claimed by the manufacturers.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHERE\u003c/b\u003e FIGURE \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eElectrochemical characterization of the MWCNTs/CNC nanocomposites was performed using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) with 5 mM Fe(CN)\u003csub\u003e6\u003c/sub\u003e\u003csup\u003e3\u0026minus;/4\u0026minus;\u003c/sup\u003e solutions in 0.1 mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003ePBS of pH 7.4. For comparison, SPCEs were modified by dropping 5 \u0026micro;L of the as prepared dispersions of MWCNTs, CNC or MWCNTs/CNC mixture onto the working electrode surface and allowing drying. Cyclic voltammograms (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) showed the expected anodic and cathodic peaks of the redox probe as well as some appreciable differences in the peak currents, peak potentials, and ΔE values depending on the electrode composition. Unmodified SPCE exhibited similar anodic and cathodic currents (i\u003csub\u003epa\u003c/sub\u003e= 22 \u0026micro;A and i\u003csub\u003epc\u003c/sub\u003e= 21 \u0026micro;A), with an i\u003csub\u003epa\u003c/sub\u003e/ i\u003csub\u003epc\u003c/sub\u003e ratio of 1.04. Moreover, ΔE was 320 mV. As it can be seen, slightly higher currents were obtained at the CNC/SPCE (i\u003csub\u003epa\u003c/sub\u003e= 30 \u0026micro;A and i\u003csub\u003epc\u003c/sub\u003e= 29 \u0026micro;A; i\u003csub\u003epa\u003c/sub\u003e/ i\u003csub\u003epc\u003c/sub\u003e = 1.03), with a lower peak separation (ΔE\u0026thinsp;=\u0026thinsp;244 mV). These results can be attributed to the high surface area of ​​the CNC and its porous structure, which allows ion transport and greater accessibility of the redox probe. [12]. Regarding the electrodes modified with MWCNTs, lower peak separation (ΔE\u0026thinsp;=\u0026thinsp;175 mV) for both MWCNTs/SPCE and MWCNTs/CNC/SPCE, and higher peak currents, were observed, which was attributed to the high electrical conductivity of carbon nanotubes. However, it is worth noting the slight decrease in peak currents at the MWCNTs/CNC/SPCE ((i\u003csub\u003epa\u003c/sub\u003e= 32 \u0026micro;A and i\u003csub\u003epc\u003c/sub\u003e= 31 \u0026micro;A; i\u003csub\u003epa\u003c/sub\u003e/ i\u003csub\u003epc\u003c/sub\u003e = 1.03) compared to the MWCNTs/SPCE (i\u003csub\u003epa\u003c/sub\u003e= 35 \u0026micro;A and i\u003csub\u003epc\u003c/sub\u003e= 33 \u0026micro;A; i\u003csub\u003epa\u003c/sub\u003e/ i\u003csub\u003epc\u003c/sub\u003e = 1.06) probably due to the small decrease in conductivity when using the composite nanomaterial [20].\u003c/p\u003e \u003cp\u003eSimilar conclusions arise by using EIS at the same experimental conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Nyquist spectra exhibited significantly lower charge transfer resistance (R\u003csub\u003eCT\u003c/sub\u003e) for the electrodes modified with MWCNTs or MWCNTs/CNC (R\u003csub\u003eCT\u003c/sub\u003e= 984 Ω and 1063 Ω, respectively) than that measured at the bare SPCE, R\u003csub\u003eCT\u003c/sub\u003e = 2161 Ω, and at the CNC modified electrode, 1823 Ω. This behaviour can be attributed to a better conductivity of carbon nanotubes which slightly decreases in the presence of CNC.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHERE\u003c/b\u003e FIGURE \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eOptimization of the variables involved in the preparation of the immunosensors\u003c/h2\u003e \u003cp\u003eThe variables involved in the preparation and functioning of the dual immunosensor were optimized by testing individually their effect on the amperometric response for each target biomarker. The selection criterion for the variables was the values of the specific-to-unspecific current (S/N) ratios. The optimization results for IL-12 are described below, whereas those for the individual determination of IL-23 are detailed in Supplementary Information (Figures S2\u0026ndash;S4). Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e summarizes the experimental variables selected for the simultaneous determination of both biomarkers with the developed immunoplatform.\u003c/p\u003e \u003cp\u003eThe effect of the capture antibody loading (anti-IL12) over the 5 to 30 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e range, and of its incubation time for immobilization onto grafted MWCNTs/CNC/ SPCEs, between 15 to 60 min, were tested in the absence (N) and in the presence (S) of 2 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e IL-12. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea shows as a larger S/N ratio was obtained for 25 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e antibody. Smaller concentrations gave rise to lower specific amperometric currents probably because of the insufficient antibody loading. Moreover, practically constant nonspecific currents were observed, thus providing lower S/N ratios. Furthermore, antibody loadings larger than 25 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e provoked also a decrease in the S/N ratio probably due to the hindered recognition of the target biomarker. Regarding the incubation time, the results of Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb led us to select 15 min for further work. Bovine serum albumin (BSA) was used as the blocking agent to minimize unspecific adsorptions onto the modified anti-IL12-MWCNTs/CNC/SPCEs electrode. The effect of the blocker concentration was tested over 2 and 10% and the results were compared with those achieved by using a commercial blocking buffer (BB) consisting of 2% casein. As Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec shows, an 8% BSA allowed obtaining a larger S/N ratio. Furthermore, a blocking time of 30 min provided also a better S/N ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). The effect of the incubation time of IL-12 antigen on the amperometric responses was evaluated over the 15 to 45 min. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee shows that although the specific response is somewhat higher for 30 min, the increase in the non-specific currentwhen increasing the immobilization time provoked a worse S/N ratio compared with that achieved incubating for 15 min, which has been chosen for further work.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHERE\u003c/b\u003e FIGURE \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea shows as the specific responses increased with the biotinylated antibody loading due to the higher amount of biotin to form the affinity link with the poly-HRP-Strep conjugate for detection. Since a higher S/N ratio was obtained for 0.5 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, this concentration was selected for further work. Regarding the incubation time for Biotin-anti-IL12 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb) 30 min were chosen according to the same criterion. With the aim of obtaining higher specific currents and a better S/N ratio, a signal amplification strategy involving poly-HRP-Strept instead of the usual HRP-Strept conjugate was implemented. The effect of the polymer dilution on the immunosensor responses was studied over the 1/250 to 1/2000 range with the results shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec. Both specific and non-specific currents gradually decreased as the dilution increased, probably due to the lower number of detector labels on the biotinylated antibodies. However, a better S/N ratio was obtained for a 1/1000 dilution, so this value was chosen for subsequent studies. To further reduce nonspecific responses, the aqueous solutions of poly-enzyme conjugate were prepared in the presence of an optimized amount of BSA. As Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed shows, a larger S/N ratio was achieved for 2% BSA. Finally, the effect of the incubation time of poly-HRP-Strept prepared under the previous conditions was checked (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee), 20 min being selected for further work.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHERE\u003c/b\u003e FIGURE \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition to these variables, the loading of MWCNTs/CNC composite on the SPCE surface was optimized by measuring the variation of the immunosensor responses over the range of 3 to 7 \u0026micro;L suspension. A volume of 5 \u0026micro;L provided a larger specific signal and a better S/N ratio. Other experimental conditions, such as the composition of the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/HQ system, as well as the potential and pH for the detection, were optimized in previous works [21, 22].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eExperimental variables tested and values selected for the simultaneous determination of IL-12 and IL-23 with the developed immunosensors.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTarget biomarker\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTested range\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSelected value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIL12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAnti-IL12 loading, \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAnti-IL12 incubation time, min\u003c/p\u003e \u003cp\u003eBSA concentration, %\u003c/p\u003e \u003cp\u003eBSA blocking time, min\u003c/p\u003e \u003cp\u003eIL12 incubation time, min\u003c/p\u003e \u003cp\u003eBiotin-anti-IL12 loading, \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eBiotin-anti-IL12 incubation time, min\u003c/p\u003e \u003cp\u003ePoly-HRP-Strept dilution\u003c/p\u003e \u003cp\u003eBSA in poly-HRP-Strept, %\u003c/p\u003e \u003cp\u003ePoly-HRP-Strept incubation time, min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026ndash;30\u003c/p\u003e \u003cp\u003e15\u0026ndash;60\u003c/p\u003e \u003cp\u003e2\u0026ndash;10\u003c/p\u003e \u003cp\u003e15\u0026ndash;45\u003c/p\u003e \u003cp\u003e15\u0026ndash;45\u003c/p\u003e \u003cp\u003e0.1\u0026ndash;0.75\u003c/p\u003e \u003cp\u003e15\u0026ndash;45\u003c/p\u003e \u003cp\u003e1//2000\u0026ndash;1/250\u003c/p\u003e \u003cp\u003e0\u0026ndash;5\u003c/p\u003e \u003cp\u003e15\u0026ndash;30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25\u003c/p\u003e \u003cp\u003e15\u003c/p\u003e \u003cp\u003e8\u003c/p\u003e \u003cp\u003e30\u003c/p\u003e \u003cp\u003e15\u003c/p\u003e \u003cp\u003e0.5\u003c/p\u003e \u003cp\u003e30\u003c/p\u003e \u003cp\u003e1/1000\u003c/p\u003e \u003cp\u003e2\u003c/p\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIL-23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAnti-IL23 loading, \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAnti-IL23 incubation time, min\u003c/p\u003e \u003cp\u003eBSA concentration, %\u003c/p\u003e \u003cp\u003eBSA blocking time, min\u003c/p\u003e \u003cp\u003eIL23 incubation time, min\u003c/p\u003e \u003cp\u003eBiotin-anti-IL23 loading, \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eBiotin-anti-IL23 incubation time, min\u003c/p\u003e \u003cp\u003ePoly-HRP-Strept dilution\u003c/p\u003e \u003cp\u003eBSA in poly-HRP-Strept, %\u003c/p\u003e \u003cp\u003ePoly-HRP-Strept incubation time, min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u0026ndash;50\u003c/p\u003e \u003cp\u003e15\u0026ndash;45\u003c/p\u003e \u003cp\u003e5\u0026ndash;10\u003c/p\u003e \u003cp\u003e15\u0026ndash;45\u003c/p\u003e \u003cp\u003e15\u0026ndash;45\u003c/p\u003e \u003cp\u003e0.05\u0026ndash;0.75\u003c/p\u003e \u003cp\u003e5\u0026ndash;45\u003c/p\u003e \u003cp\u003e1/1000\u0026ndash;1/250\u003c/p\u003e \u003cp\u003e0\u0026ndash;2\u003c/p\u003e \u003cp\u003e10\u0026ndash;30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e40\u003c/p\u003e \u003cp\u003e20\u003c/p\u003e \u003cp\u003e8\u003c/p\u003e \u003cp\u003e30\u003c/p\u003e \u003cp\u003e15\u003c/p\u003e \u003cp\u003e0.25\u003c/p\u003e \u003cp\u003e15\u003c/p\u003e \u003cp\u003e1/500\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAll the steps involved in the preparation of the dual immunosensor were monitored by CV and EIS using 5 mM Fe(CN)\u003csub\u003e6\u003c/sub\u003e\u003csup\u003e3\u0026minus;/4\u0026minus;\u003c/sup\u003e as the redox probe in 0.1 M PBS of pH 7.4. Similar results were obtained for both biomarkers and, therefore, only those obtained for IL-23 have been displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. Voltammograms for the bare SPCE and MWCNTs/CNC/SPCE (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA curves 1 and 2) already shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e were included for comparison purposes. Grafting with \u003cem\u003ep\u003c/em\u003e-ABA (curve 3) caused a strong decrease in the peak currents which is most likely due to the electrostatic repulsion between the redox probe and the dissociated carboxyl groups on the electrode surface at the working pH. Activation with EDC/sulfo-NHS (curve 4) provoked an increase of peak currents because of the neutralization of the anionic charges. Surprisingly, conjugation with anti-IL23 capture antibody (curve 5) resulted in higher peak currents, which is contradictory to the non-conducting properties of the biomolecule. A possible explanation for this behaviour relies in the isoelectric point of the antibody, 9.3 [23], which means it is positively charged at the working pH thus reinforcing the current magnitude by electrostatic attraction of the redox probe. Thereafter, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB shows that the current decreased dramatically after blocking with BSA (curve 6) while successive incorporation of the other immunoreagents (curves 7 to 9) provoked slight variations in the CVs towards a less reversible behaviour, because of the presence of insulating layers with increasing thickness on the electrode surface.\u003c/p\u003e \u003cp\u003eSimilar results were obtained by EIS (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, D). Apart from the large decrease in the electron transfer resistance produced by modification of SPCE with MWCNTs/CNC (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, curves 1 and 2) it can be seen as grafting with \u003cem\u003ep\u003c/em\u003e-ABA (curve 3) provoked the expected increase in the R\u003csub\u003eCT\u003c/sub\u003e value up to 4512 Ω, which then showed a further decrease to 1753 Ω after neutralization by activation with EDC/sulfo-NHS (curve 4). In addition, conjugation with anti-IL23 capture antibody (curve 5) gave rise to a lower R\u003csub\u003eCT\u003c/sub\u003e value of 1003 Ω, probably due to the reason explained above to justify the behaviour in CV [23]. Thereafter, blocking of the remaining unreacted sites with BSA produced a larger R\u003csub\u003eCT\u003c/sub\u003e value of 3276 Ω (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, curve 6) in agreement with the expected decrease in the electrode conductivity at a quasi-passivated surface. The subsequent incorporation of IL-23 antigen, Biotin-anti-IL23 detection antibody and poly-HRP/Strept (curves 7 to 9) led the charge transfer resistance to values similar than that measured before the blocking. Interestingly, the presence of the biomolecules at the electrode surface provoked also the appearance of two semicircles in the EIS spectra. These results led to define the obtained Nyquist plots by two different equivalent circuits as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. Curves 1\u0026ndash;4 fitted well to a Randles R1(C2[R3W1]) circuit, whereas curves 5\u0026ndash;9 should be explained by the more complex equivalent circuit depicted on the right, with at least two RC semicircuits, reflecting that some parts of the electrode are coated by the biomolecules while others remain exposed to the solution. The parallel RC circuits mean that there is a film with defects such as pinholes or a non-uniform thickness throughout the substrate [24].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eHERE FIGURE \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalytical characteristics of the dual immunosensor for the simultaneous determination of IL-12 and IL-23 biomarkers\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe calibration plots constructed with the dual immunosensor for the determination of the Crohn\u0026acute;s disease biomarkers using the optimized working conditions are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e which also shows some of the amperograms registered to obtain the corresponding data. As expected, according to the sandwich-type configuration of the immunoassays, the recorded currents were directly proportional to the concentration of each target cytokine. Semilogarithmic plots with wide linear regions between 0.3 and 1000 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were obtained for both interleukins. The analytical parameters of the corresponding calibration plots are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eHERE FIGURE \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe comparison of the analytical characteristics provided by the dual immunosensor with those claimed for the ELISA kits involving the same immunoreagents allowed us to deduce some advantages when using the immunosensor. In the case of IL-12. the dynamic linear range is much wider, covering from 0.3 to 1000 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e whereas the ELISA kit [Human IL-12 DuoSet ELISA from R\u0026amp;D Systems (Cat. No. DY1270-05)] provides a non-linear semilogarithmic calibration from 0.031 to 2.0 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. In addition, the ELISA method takes more than 4 hours compared to the 1h 35 min required with the immunosensor counting in both cases from the incubation of the capture antibody. Regarding IL-23, the developed method also provides a linear semi-logarithmic calibration plot ranging between 0.3 and 1000 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e instead of the non-linear semilogarithmic plot provided by the ELISA kit [Human IL-23 DuoSet ELISA from R\u0026amp;D Systems (Cat. No. DY1290-05)] covering from 0.125 to 8.0 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which requires more than 4h 40 min instead of 1h 20 min needed for the use of the immunosensor. Obviously, the greatest advantage is that the developed dual platform allows the simultaneous determination of both biomarkers in less than 2 hours, which is approximately a quarter of the time that would be required if the two ELISA kits were used separately.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAnalytical characteristics of the calibration plots for IL-12 and IL-23 constructed with the developed dual immunosensor\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIL-12\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIL-23\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSlope\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 \u0026micro;A/conc decade\u003c/p\u003e \u003cp\u003e(ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 \u0026micro;A/conc decade\u003c/p\u003e \u003cp\u003e(ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIntercept\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 \u0026micro;A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 nA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLinear range\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.3\u0026ndash;1000 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.3\u0026ndash;1000 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.996\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.993\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.25 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.22 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOQ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.26 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.29 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRSD % (n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003cp\u003e(intra-day)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.2 (0 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003cp\u003e4.1 (10 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.1 (0 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003cp\u003e2.7 (10 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRSD % (n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003cp\u003e(inter-day)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.8 (0 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003cp\u003e5.4 (10 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.7 (0 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003cp\u003e3.8 (10 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eRegarding the achieved LOD and LOQ values, it should be noted that the ELISA kits involving the same reagents report in their protocols only the lowest concentrations of the calibrations: 31.3 pg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (IL-12) and 125 pg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (IL-23). These values are somewhat lower or similar, respectively, to the LOQ values achieved with the developed method. There is no information about the relative standard deviation obtained with the ELISA kits, which in the case of the dual immunosensor ranged approximately between 3 and 6%. Finally, it should be noted that the sample volume required, 5 \u0026micro;L for both biomarkers, is twenty times smaller than that needed for one target using the ELISA test.\u003c/p\u003e \u003cp\u003eThe good analytical performance achieved is probably due to the properties of CNC, mainly its biocompatibility and hydrophilicity, as well as the high surface area and open-pore structure which facilitates the penetration of electroactive species resulting in high sensitivity and fast response [25]. These characteristics together with the electrochemical properties of carbon nanotubes provide an excellent composite nanomaterial for the immobilization of biomolecules onto the transducer surface as well as for the amperometric detection. In addition, the dual immunosensor exhibited good reproducibility and excellent stability, suggesting that it could be used for the determination of the target biomarkers in clinical samples from patients of Crohn\u0026acute;s disease or other IBDs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStorage stability\u003c/h2\u003e \u003cp\u003eWith the aim of evaluating the storage stability of the dual immunosensor for the determination of IL-12 and IL-23 biomarkers, different immunoplatforms were prepared on the same day, stored in PBS pH 7.4 at 4 \u0026ordm;C, and employed to measure simultaneously both targets on different days after incubation with 10 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of each target according to the procedure described in the \u003cspan refid=\"Sec2\" class=\"InternalRef\"\u003eExperimental\u003c/span\u003e section. The obtained results (Figure S5) indicated that the anti-IL12-MWCNTs/CNC/SPCE and anti-IL-23-MWCNTs/CNC/SPCE bioelectrodes were stable for at least 71 days (the longest storage time tested) since the current responses remained inside the \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\stackrel{-}{x}\\)\u003c/span\u003e\u003c/span\u003e \u0026plusmn; 3s limits, where s was the standard deviation of the measurements (n\u0026thinsp;=\u0026thinsp;10) carried out on the first day. Therefore, at least during this period it is feasible to prepare the immunosensors from the stored bioconjugates, allowing the determination of IL-12 and IL-23 after incubation of the cytokines, the detection antibodies and the peroxidase conjugate, in around 60 min. This long-term stability is attributable most likely to the CNC properties, especially its biocompatibility and hydrophilicity, as well as its robustness to slight variations in pH, ionic strength or temperature [25].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSelectivity\u003c/h2\u003e \u003cp\u003eThe effect of potential interfering compounds that may be present together with the target biomarkers in biological samples on the electrochemical responses obtained with the dual immunosensor was checked. The compounds tested were proteins related to inflammation disorders such as those associated with Crohn's disease, ulcerative colitis and other IBDs, as well as others usually present in human serum. Their influence was evaluated at concentration levels that correspond approximately to the normal physiological level found in healthy individuals: 5 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e haemoglobin (HB), 50 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e human serum albumin (HSA); 1 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e human immunoglobulin (hIgG); 100 pg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e interleukin 6 (IL-6); 200 pg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e tumour necrosis factor alpha (TNF-α) and 100 pg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e interferon gamma (INF-γ). In addition, the possible cross-interference of each biomarker in the determination of the other was evaluated at concentration levels of 2 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe results in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e show as no significantly different responses were obtained in all cases since all the mean steady state current values remained within the \u0026plusmn;\u0026thinsp;3 \u0026times; standard deviation range of the current values measured in the absence of potential interferent. This excellent selectivity can be attributed to the practical specificity of the capture antibodies towards the target cytokines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eHERE FIGURE \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of IL-12 and IL-23 in human serum and faeces\u003c/h2\u003e \u003cp\u003eThe simultaneous determination of the CD biomarkers was accomplished in human serum of a healthy individual (Bio Hub Ref.100468118) and in faeces of a volunteer. In the case of serum, the procedure described in the \u003cspan refid=\"Sec2\" class=\"InternalRef\"\u003eExperimental\u003c/span\u003e section was applied to the determination of the two biomarkers using 5 \u0026micro;L aliquots of undiluted sample. For faeces, an Eiken OC-Auto Sampling Bottle3 (Ref. V-PZ25) [\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.eiken.co.jp/uploads/IFU/340026A-G_en_20220401.pdf\u003c/span\u003e\u003cspan address=\"https://www.eiken.co.jp/uploads/IFU/340026A-G_en_20220401.pdf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e] was used for sample collection and dispersion in the appropriate medium. Specifically, 10 mg of faeces were collected each time and dispersed in 2 mL of HEPES buffer included in the container. As indicated above, all the experiments involved in samples manipulation and analysis were performed accomplishing all the ethical issues and relevant guidelines and regulations of the implied institutions.\u003c/p\u003e \u003cp\u003eFirstly, the matrix effects from the sample solutions were evaluated by applying the Student\u0026rsquo;s t-test to compare the slope values of the calibration plots for the target standards constructed in buffer solutions with those obtained by applying the standard additions method. The results for the direct analysis of both undiluted serum and stool solution provided texp values larger than that tabulated t (ttab\u0026thinsp;=\u0026thinsp;2.365), indicating that apparent matrix effects occurred in the determination of IL-12 and IL-23 under the mentioned conditions. Therefore, the standard additions method was applied for the determination of the biomarkers. This method also avoids possible variabilities in the slope value of the calibration plots constructed from serum or faeces collected from different individuals. Using this protocol, the obtained results are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Excellent recoveries ranging between 97\u0026thinsp;\u0026plusmn;\u0026thinsp;4% and 103\u0026thinsp;\u0026plusmn;\u0026thinsp;6% were found in spiked serum and between 96\u0026thinsp;\u0026plusmn;\u0026thinsp;5% and 102\u0026thinsp;\u0026plusmn;\u0026thinsp;6% in faeces, for IL-12 and IL-23, respectively.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDetermination of IL-12 and IL-23 in raw serum and faeces with the dual immunosensor.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSAMPLE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eIL-12, ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eIL-23, ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAdded\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFound*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRecovery, %\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAdded\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFound*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eRecovery, %\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSerum\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e103\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e102\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e1.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e101\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFaeces\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e102\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e102\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e102\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e*\u003c/b\u003e Mean value\u0026thinsp;\u0026plusmn;\u0026thinsp;2s, n\u0026thinsp;=\u0026thinsp;3\u003c/p\u003e \u003c/div\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eThe first dual immunosensor for the simultaneous determination of IL-12 and IL-23, two relevant biomarkers of CD is reported in this work. The antibodies for the selective capture of the respective interleukins were covalently immobilized onto dual SPCEs modified with CNC and MWCNTs followed by conjugation with a detector antibody labelled with poly-HRP-Strept, and amperometric transduction using the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/HQ system. The developed bioplatform provides results with a high reproducibility and selectivity using lower sample volumes and in a much shorter assay time than those reported for each target using commercially available ELISA kits. Interestingly, a long storage stability of the anti-IL12-MWCNTs/CNC/SPCE and anti-IL-23-MWCNTs/CNC/SPCE bioelectrodes of at least 71 days was observed. The good analytical performance is probably due to the combination of the CNC properties, mainly its biocompatibility and hydrophilicity, the high surface area and open pore structure, together with the electrochemical properties of MWCNTs, which provide an excellent nanocomposite for biomolecules immobilization onto the transducer surface and electrochemical detection. The suitability and applicability of the dual immunosensor were demonstrated by the analysis of raw serum and faeces spiked with IL-12 and IL-23 at the levels that can be found in samples from patients suffering severe CD obtaining excellent recoveries.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConsent to participate \u003c/h2\u003e\n\u003cp\u003eInformed consent was obtained from all individual participants included in the study.\u003c/p\u003e\n\u003ch2\u003eEthics Approval\u003c/h2\u003e\n\u003cp\u003eThis study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Hospital Cl\u0026iacute;nico San Carlos, University Complutense of Madrid.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThe financial support of PID2021-122457OB-I00 (Spanish Ministerio de Ciencia e Innovacion) is gratefully acknowledged.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eL.G.-R.: Methodology, Investigation. C.R.-L.: Methodology, Investigation. E. S.-T.: Writing \u0026ndash; review \u0026amp; editing, Investigation. L. A.: Supervision, Conceptualization. A. G.-C.: Supervision, Resources,Funding acquisition, Conceptualization. P.Y.-S.: Writing \u0026ndash; original draft, Supervision, Resources, Funding acquisition, Conceptualization. J.M. P.: Supervision, Resources. All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBaumgart DC, Sandborn WJ (2007) Inflammatory bowel disease: clinical aspects and established and evolving therapies, Lancet 369:1641\u0026ndash;1657\u003c/li\u003e\n\u003cli\u003eZundler S, Neurath MF (2015) Interleukin-12: Functional activities and implications for disease, Cytokines \u0026amp; Growth Factor Rev. 26:559-568\u003c/li\u003e\n\u003cli\u003eKaser A, Zeissig S, Blumberg RS (2020) Inflammatory bowel disease. Annu Rev Immunol. 28:573\u0026ndash;576\u003c/li\u003e\n\u003cli\u003eShida K, Kiyoshima-Shibata J, Nagaoka M, Watanabe K, Nanno M (2006) Induction of Interleukin-12 by Lactobacillus strains having a rigid cell wall resistant to intracellular digestion, J. Dairy Sci. 89:3306\u0026ndash;3317\u003c/li\u003e\n\u003cli\u003eAggeletopoulou I, Assimakopoulos SF, Konstantakis C, Triantos C (2018) Interleukin 12/interleukin 23 pathway: Biological basis and therapeutic effect in patients with Crohn\u0026apos;s disease, World J. Gastroenterol. 24: 4093-4103\u003c/li\u003e\n\u003cli\u003eYan J, Smyth MJ, Teng MWL (2018) Interleukin (IL)-12 and IL-23 and their conflicting roles in cancer, Cold Spring Harb Perspect. Biol. 10:q028530\u003c/li\u003e\n\u003cli\u003eLa Belle JT, Bhavsar K, Fairchild A, Das A, Sweeney J, Alford TL, Wang J, Bhavanandan VP, Joshi L (2007) A cytokine immunosensor for multiple sclerosis detection based upon label-free electrochemical impedance spectroscopy, Biosens. 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Macromol. 166:587\u0026ndash;600\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microchimica-acta","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"miac","sideBox":"Learn more about [Microchimica Acta](https://link.springer.com/journal/604)","snPcode":"604","submissionUrl":"https://submission.springernature.com/new-submission/604/3","title":"Microchimica Acta","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Crohn´s disease, IL-12, IL-23, electrochemical immunosensor, crystalline nanocellulose, carbon nanotubes","lastPublishedDoi":"10.21203/rs.3.rs-5259345/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5259345/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis work reports the first dual immunosensor for the determination of IL-12 and IL-23, two relevant biomarkers of Crohn\u0026rsquo;s disease (CD). The strategy relies on the selective capture of the targets by the respective antibodies which were covalently immobilized onto SPCEs modified with crystalline nanocellulose (CNC) and multi-walled carbon nanotubes (MWCNTs) followed by conjugation with a detector antibody labelled with poly-HRP-Strept and amperometric transduction using the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/HQ system. The developed bioplatform exhibits a high reproducibility and selectivity allowing the simultaneous determination of both biomarkers using a lower sample volume and lasting a much shorter assay time than those reported for each target with commercially available ELISA kits. It is worth highlighting the storage stability of the bioconjugates, which lasts at least 71 days. This excellent performance is probably due to the combination of the CNC properties, mainly its biocompatibility and hydrophilicity, the high surface area and open pore structure, together with the electrochemical properties of MWCNTs, which provide a nanocomposite excellent for biomolecules immobilization onto the transducer surface and electrochemical detection. The suitability and applicability of the dual immunosensor were demonstrated by analyzing raw serum and faeces spiked with IL-12 and IL-23 at the levels that can be found in samples from patients suffering severe CD.\u003c/p\u003e","manuscriptTitle":"Electrochemical immunosensing of Crohn´s disease biomarkers using diazonium salt grafted - crystalline nanocellulose / carbon nanotubes modified electrodes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-23 10:33:22","doi":"10.21203/rs.3.rs-5259345/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-11T13:44:13+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-10T17:24:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"246728858339030456756319323993460357288","date":"2024-10-17T23:41:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-17T21:44:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"18637838227290874835035033952776711286","date":"2024-10-17T19:35:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-17T19:30:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-16T10:29:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-16T10:28:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microchimica Acta","date":"2024-10-14T08:19:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microchimica-acta","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"miac","sideBox":"Learn more about [Microchimica Acta](https://link.springer.com/journal/604)","snPcode":"604","submissionUrl":"https://submission.springernature.com/new-submission/604/3","title":"Microchimica Acta","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"bd0e5149-6a64-46a4-80b1-434c2fe7adca","owner":[],"postedDate":"October 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-09T16:02:13+00:00","versionOfRecord":{"articleIdentity":"rs-5259345","link":"https://doi.org/10.1007/s00604-024-06868-2","journal":{"identity":"microchimica-acta","isVorOnly":false,"title":"Microchimica Acta"},"publishedOn":"2024-12-04 15:57:32","publishedOnDateReadable":"December 4th, 2024"},"versionCreatedAt":"2024-10-23 10:33:22","video":"","vorDoi":"10.1007/s00604-024-06868-2","vorDoiUrl":"https://doi.org/10.1007/s00604-024-06868-2","workflowStages":[]},"version":"v1","identity":"rs-5259345","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5259345","identity":"rs-5259345","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}