Development of electrochemical immunosensors for detection of insulin antibodies using Indolepyrazole nanoparticles

Abstract

Insulin antibodies have shown to be a strong predictor of diabetes development in genetically susceptible individuals and the development of type 1 diabetes is strongly associated to the presence of antibodies that attack beta cells (islet cells), such as insulin. The development of a sensitive, selective, efficient, and economical insulin detection system is crucial for the diagnosis and management of diabetes. The existing methods can produce results that are very specific, highly sensitive, and dependable. They include notable disadvantages, including high costs, extensive time requirements, the necessity for substantial sample preparation, and the emission of hazardous radiation. Electrochemical assays, characterised by their high sensitivity and selectivity, rapid reaction time, straightforward automation, dependable results, and relatively low cost, can address the drawbacks of conventional approaches. Furthermore, electrochemical sensors utilising biosensor technology provide the most efficacious approach for the detection of antibodies to antigens. Electrochemical immunosensors have emerged as powerful tools for the rapid and sensitive detection of biomarkers. The detection of insulin antibodies is critical for the management of autoimmune responses in diabetic patients. This study aimed to develop and optimise an electrochemical immunosensors for the detection of insulin antibodies using indole-pyrazole capped cobalt or gold nanoparticles. Nanoparticles (cobalt or gold) capped with novel indole-pyrazole derivatives (bis indole-pyrazole or chromone indole-pyrazole) were synthesised and immobilised onto the surface of a bare platinum electrode. Indole-pyrazole derivatives were selected due to their electron-rich, conjugated structures and potential to enhance charge transfer and sensor sensitivity. This modification enhanced the specific surface area and stability of the sensor platform. Insulin antigen was then introduced to promote antibody binding, followed by blocking with bovine serum albumin (BSA) to prevent non-specific binding and improve the accuracy, sensitivity, and reliability of the immunosensor. Each metal nanoparticle had two sensors: one capped with bis indole-pyrazole and the other with chromone-indole-pyrazole. All sensors demonstrated high detection ability for insulin antibodies, with a low detection limit, good selectivity, sufficient stability, and excellent recovery rates. This demonstrates the potential of the developed immunosensors for quantifying insulin antibodies. However, sensors incorporating the chromone-indole-pyrazole derivative outperformed those with the bis indole-pyrazole derivative, exhibiting lower limits of detection (LODs) and higher recovery rates for both metals. The green synthesis of cobalt and gold nanoparticles (CoNPs and AuNPs) using lemon peel extract was successfully achieved, with indole-pyrazole derivatives (BIP and CIP) acting as additional capping agents. Phytochemical screening was conducted to identify the compounds in the lemon peel extract, such as phenols, flavonoids, tannins, and alkaloids, which played a role in nanoparticle stabilisation and reduction. Characterisation techniques such as UV-Vis, FTIR, SEM, and EDS confirmed the successful formation of CoNPs and AuNPs with well-defined morphological and elemental properties. The UV-Vis spectra displayed characteristic surface plasmon resonance peaks, indicating nanoparticle formation. SEM analysis showed that the nanoparticles were predominantly spherical, with some aggregation due to phytochemical interactions. EDS data further confirmed the elemental composition, demonstrating the presence of cobalt and gold within the nanoparticles The fused indole-pyrazole derivatives viz bis indole-pyrazole 4a (BIP) and chromone indole-pyrazole 4b (CIP) were synthesised by reacting aldehyde derivatives, thiosemicarbizide and an indole through a one-pot synthesis via 2+3 annulation. Then, characterised using fourier transform infrared (FTIR), nuclear magnetic resonance (NMR), and time-of-flight mass spectrometry (TOF-MS). To better understand their properties since they are newly synthesised compounds, biological evaluations were performed. Initially, a mutagenicity test was performed, and the compounds showed no significant increase in revertant colonies against S. typhimurium TA 98 and TA 100 strains. In the MTT assay for cytotoxicity against two human cancer cell lines, A549 and HepG-2; and one normal, HEK 293. Compound 4b showed high potency against the cancer cell lines, with IC50 values of 18.70 and 50.07 μg/mL, respectively. Whilst, both compounds showed low inhibition level against HEK 293 at 100 μg/mL. The in vitro inhibition of α-amylase and α-glucosidase, compound 4a demonstrated excellent in vitro inhibition of α-amylase and α-glucosidase, with the IC50 values of 3.9 μg/mL and 12.1 μg/mL, respectively. Compound 4a exhibited strong inhibitory activity against α-amylase and α-glucosidase, for the anticancer activity only compound 4b showed promising results.