My Ssec Capstone Project Interleukine-10


Interleukine-10 (IL-10) is the prototype of the anti-inflammatory cytokines, which are secreted during the pro-inflammatory process and during the acute stage of inflammation from T-cells, macrophages, B cells and keratinocytes1. It was firstly discovered in 1989 as a cytokine synthesis inhibitory factor (CSIF) produced by T helper 2 (Th2) cells. Its inhibitory action is exerted primarily towards the most typical markers of inflammation, such as IL-1, IL-6, IL-8, and anti-human tumor necrosis factor-alpha (TNF-?) cytokine that causes cell apoptosis. Thus, IL-10 is considered a potential therapeutic tool for various diseases especially for autoimmune diseases associated with inflammatory components. This can be achieved by inhibiting the pro-inflammatory function of various innate immune cells and adapting endothelial cells 2-4.
For early diagnosis and treatment of inflammatory-related diseases, it is urgent to detect and quantify these biomarkers at early stages of inflammation. In this regard, the recombinant human IL-10 (rh IL-10) antigen has been tested in healthy volunteers, patients with Crohn’s disease, rheumatoid arthritis, psoriasis, hepatitis C, and HIV using various immunodiagnostic techniques such as enzyme-linked immunosorbent assay (ELISA) and flow cytometry5-8. However, these technologies are time-consuming and cost-effective. In addition, their applications require the use of labels for detection and need specialized personnel. Therefore, in order to detect these biomarkers at minute concentrations and predict the first signs of inflammation, there is an increasing interest for using other techniques, which provide fast, real-time, relatively cheap and reliable medical diagnosis that could be utilized at the patient’s bedside using point-of-care testing (POCT).
Nowadays, the progress in the field of microelectronics and biosensors led to the creation of a new generation of electrochemical biosensors which fulfill these requirements 9-11. Recently, Baraket et al. have reported the detection of Interleukine-10 cytokine within the range 1-15pg/mL by using a fully integrated micro-electro-mechanical system (BioMEMS) based on electrochemical impedance spectroscopy measurements (EIS) 9. The human IL-10 monoclonal antibody (IL-10 mAb) was immobilized onto gold microelectrodes by using carboxyl diazonium, which was used as the bio-recognition element for its recombinant (rhIL-10) antigen. For enhancement of detection limit and immunosensor sensitivity, Lee et al. have proposed a new capacitance material based on hafnium dioxide for IL-10 detection within the linear range of 0.1–20 pg/mL using PBS buffer as electrolyte 10. Here, the authors have used aldehyde–silane (11-(triethoxysilyl) undecanal (TESUD)) self-assembled monolayer (SAMs) to directly immobilize the IL-10 mAb via covalent binding. More recently, Baraket et al. have developed new biosensors based on immobilized anti-IL-10 mAb’s onto gold microelectrodes through functionalization with self-assembled monolayers (SAMs) of 16-Mercaptohexadecanoic acid (MHDA) 11. The developed biosensor was used for detection of the cytokine biomarker rhIL-10 antigens that are secreted in human plasma during acute stages of inflammation after left ventricle assisted device (LVAD) implantation for patients suffering from heart failure (HF). Based on EIS measurements, the biosensor showed high selectivity and sensitivity toward the corresponding cytokines IL-10 before and after 24 hrs and 72 hrs of LVADs implantation at 16.9 pg/mL, 62.4 pg/ mL, and 37.6 pg/mL, respectively. Moreover, Garcia-Cruz et al have developed an impedimetric immunosensor using a poly(pyrrole) nanowires deposited on flexible thermoplastic poly (ethylene terephthalate) and polyether ether ketone substrates using innovative nanocontact printing technique 12. After functionalization with diazonium and immobilization with Interleukin 6 antibody (IL6, Ab), the immunosensor was tested for the quantification of Interleukin 6 recombinant human antigen (IL6, Ag) using EIS measurements. The immunosensor showed high sensitivity of 0.013 (pg/mL)?1 (linear fitting at R2 = 0.99) and limit of detection (LOD) of 0.36 pg/mL in a linear range of 1–50 pg/mL for Ag IL-6.
Other researchers have used silicon nitride (Si3N4)-based semiconductors as basic substrates in electrochemical biosensors due to its high mechanical, thermal and chemical stability toward attacked salt ions. However, few works of electrochemical impedance measurements on insulating surfaces such as Si3N4 have been reported 13-18. The functionalization of the insulating semiconductor (IS) surface with compact and dense SAM of covalently attached biomolecules is the key issue in the development of robust and stable IS-based immunosensors 19-21. Therefore, to enhance the conducting properties of Si3N4 layer and to improve performance of the biosensors (specificity, stability, sensitivity, and detection limit), recent studies have focused on the use of conducting polymers (CPs) as tools (transducers) for amplification of the signal response arising from the antibody–antigen interactions22. Due to their fascinating electrical, electronic, magnetic, and optical properties, conducting polymers are of great importance in various technological applications 23-25. These intrinsic properties have been exploited for the preparation of CP nanocomposites with entrapped nano-scaled biomolecules (e.g. proteins and single-stranded DNA oligomers) 26,27. In addition, CPs modified with biomaterials exhibit unique catalytic/affinity properties that can be easily applied in the design of bioanalytical sensors 28.
Among CPs, polypyrrole (PPy) was extensively studied for the design of bioanalytical sensors, due to its good biocompatibility, high electrical conductivity, good redox properties and long-term environmental stability 22. In this regard, PPy is widely considered as an effective material for immobilization of bioactive materials and for transduction/amplification of analytical signal within immunosensing devices 29. The immobilization of bioactive molecules could be achieved by various physical (e.g. electrostatic) and chemical covalent attachment to conducting polymer films carrying reactive functional groups such as amino (–NH2) or carboxylic (–COOH) groups 30-32. Chemical immobilization is considered the most favorable and efficient technique because it can be performed under controlled experimental conditions. In addition, this will solve the problem of detachment and/or denaturation of the immobilized biomolecules that occur when physical methods are used. Moreover, covalent immobilization maintains the activity of the biomolecules, increases stability, and ensures accessibility of the analyte to detect a specific biological event (e.g. antigen–antibody coupling, hybridization of complementary oligonucleotides, or enzyme-catalyzed reactions) 33, 34.