Amperometric immunosensor for the diagnosis of Celiac Disease

Abstract

newline ABSTRACT newlineThe present study was aimed at developing a quick, sensitive, specific and affordable immunosensor for diagnosis of Celiac disease. Celiac disease (CD), is an autoimmune disorder of the upper small intestine triggered from the gluten ingestion (cereal protein that can be found in wheat, rye and barley). The current diagnostic tests for CD include a series of blood tests called the Coeliac panel screening, and a small bowl biopsy. These methods however, suffer from distinct disadvantages which include being very complex, extremely tedious and require skilled operators. Furthermore, these methods are expensive and are not designed to give rapid analysis, thus inevitably lowering the quality of life for the patient. newlineThe present study was undertaken for development of a gliadin and tissue transglutaminase (tTG) fabricated Multi Walled Carbon Nano-Tube (MWCNT) based immunosensor for the rapid quantification and accurate detection of anti-gliadin and anti-transglutaminase antibodies in low concentrations to aid in point-of-care analysis. The sensing mechanism relied on electrochemical measurements in the form of Cyclic Voltammetry (CV) and Differential Pulse Voltammetry (DPV) in the presence of redox indicator. The sensitivity of the gliadin based immunosensor was 119.2 (and#956;A/cm2)/ng and lower limit of detection (LOD) was 0.78ng/6and#956;l (130ng/ml) using CV. However, a sensitivity of 22.2 (and#956;A/cm2)/ng and LOD of 4.2ng/6and#956;l (700ng/ml) using CV was recorded with tissue transglutaminase based immunosensor. The sensor was highly specific, simple and took only 30 min for detection of anti-gliadin and anti-transglutaminase antibodies respectively. newlineFurther, to enhance the sensitivity of MWCNT based immunosensor, various nanocomposite were developed and fabricated on the surface of MWCNT/Au-nanoparticles (MWCNT/Au-NPs) screen printed electrode for quantifying specific antibodies in low concentrations. For this, a Mercaptopropionic acid (MPA)/Polyamidoamine (PAMAM) modified nanohybrid gliadin based immunosensor was developed for electrochemical detection of anti-gliadin antibodies. The electrode surface was fabricated with mercaptopropionic acid for functionalization of the AuNP surface. In order to enhance the sensitivity of the sensor, polyamidoamine (PAMAM) dendrimer was successively embedded on MPA through carbodiimide coupling. The antigen (gliadin) was immobilized on PAMAM to form nanoprobe for sensing specific anti-gliadin antibodies of IgG isotype in newlinexii newlinehuman serum. The electrochemical detection of the anti-gliadin antibodies (ranging from 0.001-1000ng per 6and#956;l) present in the serum sample was carried out through the antigen antibody interaction and recorded using 50mM of K3Fe(CN)6 that served as a redox indicator. The sensitivity of the sensor was 130.63 and#956;Acm-2ng-1 using DPV with a regression coefficient (R2) 0.98 and LOD of 0.09ng/6and#956;l. The modified surface morphology was characterized by scanning electron microscopy (SEM). The sensor was highly specific for anti-gliadin antibody and can detect the Celiac disease within 20 min. newlineNext, a GQD/PAMAM nanohybrid modified multiwalled carbon nanotubes gold nanoparticles (MWCNT-AuNP) based immunosensor was proposed for detection of Celiac disease in human. The electrode surface was fabricated with cysteine for AuNP functionalization. The amine groups were further coupled with carboxyl moieties of graphene quantum dots (GQDs) using EDC-NHS cross linking chemistry. To enhance the sensitivity of the sensor, polyamidoamine G4 (PAMAM) dendrimer was embedded on GQDs through carbodiimide coupling. The antigen (tissue transglutaminase) was immobilized on GQDs to form nanoprobe for sensing specific anti-tissue transglutaminase antibodies of IgA isotype in human serum. The electrochemical detection of the anti-tTG antibodies (ranging from 0.0001pg to 10000pg per 6and#956;l) present in the serum samples was carried out through the antigen antibody interaction and recorded using 50mM of K3Fe(CN)6 that served as a redox indicator. The sensitivity of the sensor was 1297.94 and#956;A cm-2 pg-1 using DPV with a regression coefficient (R2) 0.9717 and LOD of 0.12fg/6and#956;l. The modified surface morphology was characterized by FE-SEM. The sensor was again highly specific for anti-tTG antibody and can detect the Celiac disease within 10 min. newlineValidation studies of the developed nanohybrid sensor in terms sensitivity and accuracy was performed using serum samples of the suspected Celiac patients and compared with standard method of diagnosis i.e. ELISA. The comparative analysis of the developed method (MWCNT-AuNP/GQD/PAMAM/tTG based composite sensor) with earlier reported sensors for the detection of celiac disease was also studied. The nanohybrid sensor was found to be better than earlier reported methods due to higher sensitivity (1297.94 and#956;Acm-2pg-1), specificity, lower LOD (0.12 fg/6and#956;l). The developed nanocomposite based sensor can detect Celiac disease within 10 min. newlinexiii newlineTherefore, it is found to be a new innovative device for the diagnosis of Celiac disease. newlineKeywords: Autoimmunity, Celiac disease, Graphene Quantum dot, Immunosensor, Nanocomposite, PAMAM dendrimer, Tissue transglutaminase sensor, gliadin sensor.