Screen‐printed Gold Electrodes Functionalized with Grafted p‐Aminobenzoic Acid for the Construction of Electrochemical Immunosensors. Determination of TGF‐β1 Cytokine in Human Plasma

Abstract

Screen‐printed gold electrodes were modified with grafted p‐aminobezoic acid to provide scaffolds with high loading of surface confined carboxyl groups that can be used to construct electrochemical immunosensors. As a specific application, an immunosensor for the determination of the cytokine transforming growth factor beta 1 (TGF‐β1) is reported. The capture antibody (anti‐TGF) was covalently immobilized on the surface of the prepared disposable scaffold and a sandwich‐type immunoassay was implemented with biotinylated detector antibody (Biotin‐anti‐TGF) and streptavidin labeled with alkaline phosphatase (AP‐Strept). The transduction event was monitored by differential pulse voltammetry after addition of 1‐naphthyl phosphate (1‐NPP) as the enzyme substrate. The different variables affecting the preparation and the analytical behavior of the AP‐Strept‐Biotin‐anti‐TGF‐TGF‐β1‐anti‐TGF‐SPAuE immunosensor were optimized, and the functionalized gold electrodes and the prepared immunosensor were characterized using several techniques. A linear range extending between 0.05 and 3.0 ng/mL TGF‐β1, adequate for the determination of the cytokine in human plasma, and a LOD of 10 pg/mL, better than that claimed for commercial ELISA kits, were achieved. The immunosensor exhibits also a great selectivity against other proteins and an excellent storage ability. The usefulness of the immunosensor was demonstrated by analyzing human plasma spiked with TGF‐β1.

Revisiting Electrochemical Biosensing in the 21st Century Society for Inflammatory Cytokines Involved in Autoimmune, Neurodegenerative, Cardiac, Viral and Cancer Diseases

“…Table 2 shows that both diazonium salts-based electrografting and click and electroclick chemistries have been used for the construction of electrochemical biosensors for inflammatory cytokines. These methods involved the functionalization of carbon nanomaterials [ 27 , 41 , 42 , 43 , 52 ] and electrode surfaces [ 11 , 13 , 14 , 26 , 36 , 43 , 46 , 52 , 54 , 55 , 57 , 58 , 59 , 60 , 61 ] or the incorporation of ethynyl-functionalized antibodies [ 41 , 57 , 59 ].…”

“…Therefore, there is increasing evidence that quantification of cytokine-based immune fingerprints provides an accurate way to characterize the immune function and to stratify and diagnose autoimmune diseases, infection, cancer and other immune-related deficiencies, as well as to monitor their evolution and treatments [ 1 , 10 ]. However, since the expected levels of cytokines in the extracellular milieu are at a pM concentration range, their determination requires the availability of highly sensitive analytical methods, able to perform the determination in a small number of samples to also fit children and infants [ 1 , 11 ]. Moreover, being aware of the dynamic transition of the immune status of sepsis patients from an initial pro-inflammatory phase (“cytokine storm”) to an anti-inflammatory phase within a short period of time (several hours to a few days), the development of accurate methodologies able to provide near real-time data of cytokines patients’ status is urgently needed to overcome the highly heterogeneous patient cohorts during the course of disease development and improve the therapy efficiency through personalized selection [ 1 ].…”

Novel Sensitive Immunosensor for the Selective Detection of Engrailed 2 Urinary Prostate Cancer Biomarker

“…Here, 4-aminobenzoic acid (4-ABA) was electropolymerized on the surface of a gold electrode to form a thin film of poly-ABA (PABA). PABA offers several advantages, mainly i) interesting conductive and self-doping properties (Sriwichai et al 2010), stability and compatibility with biological molecules (Guerrero et al 2018(Guerrero et al , 2019, and iii) possibility of presenting activated carboxyl groups which can interact with biomolecules' functional groups (Ferreira et al 2015;Mattos et al 2013;Santos et al 2019). In this work, anti-EN2 antibodies were covalently anchored due to the formation of an amide bond with the activated carboxyl groups of the PABA film.…”