Design and Validation of Point-of-care Immune-sensor for Measurements of Ferritin Blood Levels in COVID-19 Patients using Carbon Based Electrodes modified with Nanostructures
Authors: Prof. Ass. Valbona Aliko, Prof. Dr. Majlinda Vasjari , Prof. Ass. Caterina Faggio, MSc. Gerta Hajdaraj
Ferritin is a major intracellular iron storage protein present in all cells, tissues and tissue fluids of the organism. A small amount is found in plasma and serum, which is a reflection of iron stores in healthy individuals. It binds free ions of the trace element, neutralizing its toxic properties and increasing its solubility. In the soluble form, the body is able to expend iron as needed, in particular for regulation of cellular oxygen metabolism. Low ferritin levels result in lower iron concentrations and iron deficiency anemia. Elevated levels of ferritin, or hyperferritinemia, indicate the presence of viruses and bacteria into the body. Hyperferritinemia can also be caused by a genetic mutation. In this case, it leads to neurological disorders and vision problems. WHO is updating its global guidelines on the use of serum and plasma ferritin thresholds for diagnosis of iron deficiency and risk of iron overload. WHO recognizes that ferritin is typically assessed in serum or plasma with enzyme immunoassays, after venous blood collection. Clinical observations on Covid-19 patients have been reported cases accompanied by elevated levels of ferritin in blood and a lot of causes for this are hypothesized. Additionally, recent studies from Italian scientists have shown that ferritin is able to activate macrophages, which are a type of white blood cell of the immune system, with a critical role in innate immunity of being the body’s first line of defense (Colafrancesco et al., 2014). Evidence of hyperferritinemia in patients with septic shock, catastrophic antiphospholipid syndrome, and other medical conditions characterised by macrophage activation, are also reported (Mehta et al., 2020). Similar observations have been reported by scientists from China and the United States. It is well-known that when activated, macrophages begin to secrete cytokines. These are a category of signaling molecules that mediate and regulate immunity. At low concentrations, they are safe for the body and help to protect it against viruses and bacteria. At high levels, the so called "cytokine storm" develops, which can be lethal for half of the patients, especially for the elderly (Mehta et al., 2020; Shoenfeld, 2020). Thus, hyperferritinemia has been associated with increased illness severity and adverse outcomes, including COVID-19 (Ruscitti et al., 2020). Actually, three methods of ferritin levels measured in blood consists exists: radiometric [radioimmunoassays (RIA) and immunoradiometric assay (IRMA)], nonradiometric [enzyme linked immuno sorbent assay (ELISA) and chemoluminisence] and agglutination (turbidimetric and nephelometric), although the risk for radioactive contamination and expensive equipment are important drawbacks of RIA and IRMA. Current ferritin detection relies completely on clinical laboratories and requires trained technicians and costly equipment for the analysis. Furthermore, existing methods require venous blood. To date, point-of-care nano-biosensors for the ferritin blood level measurement are not in use in Albanian clinical-biochemical laboratories. Their introduction will be time and cost-saving of comparable accuracy and reliability to existing equipment. This project aims to study the possibilities for the development of new voltametric immunosensor for determination of ferritin in blood. In comparison with other immunological methods based on fluorescence, chemiluminescence, surface-plasmon resonance, or quartz crystal microbalance, electrochemical immunoassay has attracted considerable interest because of the advantages of simple instrumentation, easy signal quantification, low cost of the entire assay, and potential ability for real-time and on-site detection (Chikkaveeraiah 2012, Ronkainen 2014). The various forms of voltammetry (linear sweep, differential pulse, square-wave, stripping) and amperometry are the most widely used electrochemical methods for detection of protein biomarkers (Chikkaveeraiah 2012). Electrochemical detection strategies employing nanostructured surfaces, nanoparticle labels, have proved to offer new opportunities for highly sensitive protein detection. In this proposal the principles of biological recognition, antibody-antigen reaction will be combined with the advantages of electrochemical detection strategies. Due to the previous experience on sensor and biosensors development based using modified CPE (Broli, PhD 2014) with crude tissue, pure enzyme, Au-np, natural np, etc., the new ferritin immune-sensor will be experimented in terms of a point-of-care testing device. Additionally, designing a low cost point-of-care device will help in having a fast, reliable real-time measurement of ferritin, using capillary blood which make it home-usable and field settings or at community health clinic, bypassing the need for a cold-chain, expensive laboratory equipment and trained personnel. Shortening the time of measurement and bypassing the long lasting steps in blood samples preparation, ferritin immune-sensor opens great possibilities toward early detection of COVID-19 presence and on-time successful treatment intervention.