Nanoelectrode ensemble immunosensor platform for the anodic detection of anti-tissue transglutaminase isotype IgA

Highlights

• Immunosensor for IgA anti-tTG is developed using nanoelectrode ensemble.

• Sec-antibody with glucose oxidase and ferrocenyl mediator allow anodic detection.

• Detection limit is 3 times lower than previous electrochemical immunosensors.

• Immunosensor and fluroenzyme assay in celiac patients show satisfactory agreement.

Abstract

A nanostructured electrochemical sensors is developed to detect IgA isotypes of anti-tissue transglutaminase (anti-tTG) by recording an anodic electrocatalytic current. To this aim, nanoelectrode ensembles (NEEs) prepared by electroless gold deposition on track-etched polycarbonate, are at first functionalized with tissue transglutaminase (tTG) to capture anti-tTG from serum samples. In the following step, the selective detection of the IgA isotype of anti-tTG is performed by reacting with anti-IgA secondary antibody labelled with glucose oxidase. In case of positive response, the addition of glucose (substrate) and (ferrocenylmethyl)trimethylammonium (redox mediator), produces an electrocatalytic anodic current which scales with the IgA anti-tTG concentration. The optimized immunosensor provides low detection limit (0.7 U mL⁻¹), can be applied in serum samples diluted with buffer solution and show to be suitable for quantifying IgA isotype of anti-tTG in human serum samples from celiac patients.

Introduction

Celiac disease (CD) is a systemic autoimmune disease characterized by damage of the small intestinal mucosa caused by gluten ingestion in genetically predisposed patients with a prevalence of 1% of the general population [1], [2]. Early diagnosis followed by treatment with a gluten-free diet reduce morbidity and mortality associated to the disease [3]. Up to a few years ago, the gold standard for the diagnosis of CD was based on the intestinal biopsy aimed to establish the presence of lesions characteristic of the disease in the duodenum. Nowadays, new pediatric guidelines indicate that the diagnosis of CD can be assessed also by performing less invasive serological tests aimed to detect and quantify reliable biomarkers of CD in genetically predisposed subjects showing CD symptoms [4], [5]. Very recently a multicenter study supports a no-biopsy strategy among adult patients suffering from CD symptoms and with high serum concentrations of anti-tissue transglutaminase (anti-tTG) antibodies [6]. Anti-tTG immunoglobulin A is present in the 98% of the celiac patients on a gluten-containing diet [7], therefore it has been identified as an effective biomarker of CD. The diagnosis of CD is made on the basis of combined diagnostic evidences which include symptomatology, genetic test and the quantification of the serological level of anti-tTG antibodies, which in celiac patients should be 10 times higher than the cut-off of the performed test, that is the concentration value that divides negative from positive for the condition of interest. Moreover, since the level of anti-tTG in celiac patients who undergo a gluten-free diet usually decreases [8], the level of these antibodies in the serum is a reliable indicator not only for the diagnosis, but also for the follow-up of the disease [9]. Anti-tTG can be present under the form of two isotypes [10], [11], immunoglobulin IgA (mainly in dimeric form) and immunoglobulin IgG, with some category of patients however presenting IgA deficiency [12], [13]. Recently, different electrochemical biosensors for the detection of CD biomarkers have been proposed [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], which are characterized by low detection limits, high analytical sensitivity and specificity, being at the same time cost-effective, user-friendly, and suitable to provide a quick response.

The application of nanotechnology to the engineering of biosensing devices has recently acquired great interest, since the clever use of nanomaterials allows one to improve the analytical performances of biosensors, in terms of selectivity, sensitivity, detection limit and response time [25], [26], [27], [28]. As far as celiac CD diagnostics is concerned, improved analytical performances were demonstrated for immunosensors based on nanoelectrode ensemble (NEEs), used for the sensitive electrochemical [29] and electrochemiluminescent [30] detection of IgG anti-tTG. NEEs are random arrays of nanodisk electrodes, typically prepared by the electroless template deposition of gold within the nanopores of track-etch polycarbonate (PC) membranes [31], [32], [33], [34]. Fig. 1A shows the typical SEM image of a NEE. Because of their geometric features, NEE typically operate under total overlap diffusion regime [35], [36], [37], [38], [39], [40]. Under these conditions, the faradic current is proportional to the overall geometric area of the NEE while the double layer charging current is proportional only to the active area, that is the overall area of the metal nanoelectrodes exposed to the electrolyte solution [35]. For this reason, NEE provide highly improved signal-to-noise ratio allowing to reach detection limits 2–3 orders of magnitude lower than those achievable with conventional electrodes [35], [41], [42].

By exploiting the affinity of protein for the polycarbonate membrane of the NEE, it is possible to immobilize large amount of antibodies or antigens to be used as biorecognition elements without any preliminary modification of the electrode surface, at the same time maintaining the native antigenicity [43], [44], [45], [46]. If redox enzymes are used as labels, the electrochemical signal from redox mediators diffusing from the nanoelectrodes to the label can be used to detect the target analyte [29], [43], [45], [46].

In the present work we study a novel NEE based anti-tTG sensor able to detect selectively the isotype IgA of anti-tTG by exploiting the above described advantages of NEEs to IgA detection. As schematized in Fig. 1-B, in our NEE-based sensor, tissue transglutaminase (tTG) is at first immobilized on the PC of the NEE to be used as capture element which binds the target anti-tTG. The voltammetric detection is performed after incubation with an anti-IgA secondary antibody which binds specifically to the IgA isotype [1], [19]. This secondary antibody is labelled with glucose oxidase (GOx) so that, by adding to the electrolyte glucose, as GOx substrate, and (ferrocenylmethyl)trimethylammonium (FA⁺), as redox mediator [34], [47], when an anodic potential is applied, the electrocatalytic cycle shown in Fig. 1-B is produced. Hereafter, the fully functionalized immunosensors will be named IgA-NEE.

The results achieved have been compared with those obtained by “classical” fluorenzyme immunoassay (FEIA) [1]. Note that he cut-off level for anti-tTG level in serum that distinguishes healthy individuals from celiac patients is methodology dependent and for the FEIA method for IgA anti-tTG it is below 7 Unit/mL for negative and >10 Unit/mL for positive tests, as detailed below in Section 2.5.

With respect to the previous NEE-based sensor for IgG anti-tTG recently proposed by us, [29] the present one differs, first of all, for the target analyte. Moreover the choice of performing the anodic detection of IgA anti-TG with GOx as enzyme label and FA⁺ as redox mediator instead of cathodic detection with horse radish peroxidase and hydroquinone [29] has the following advantages: i) GOx is a stable, widely used enzyme and FA⁺ is an efficient mediator for this enzyme; ii) FA⁺ undergoes highly reversible electrochemical oxidation at moderately positive potential values, with fast heterogeneous electron transfer kinetics, therefore providing the best signal/noise ratios achievable with NEEs [34], [35], [36], [41]; iii) from a practical viewpoint, operating at anodic potentials avoids the time consuming degassing of the samples.