Ultrasensitive Immunosensor Array for TNF-α Detection in Artificial Saliva using Polymer-Coated Magnetic Microparticles onto Screen-Printed Gold Electrode

Tumor necrosis factor-α (TNF-α) is a biomarker of inflammation that occurs in patients suffering from heart failure (HF). Saliva can be sampled in a non-invasive way, and it is currently gaining importance as matrix alternative to blood in diagnostic and therapy monitoring. This work presents the development of an immunosensor array based on eight screen-printed gold electrodes to detect TNF-α in saliva samples. Two different functionalization strategies of electrodes were compared. In the first, anti-TNF-α antibodies were chemically bonded onto the electrode by functionalization with 4-carboxymethylaniline. The other functionalization procedure involved the binding of antibodies onto polymer-coated magnetic microparticles, which were then deposited onto the electrode by pulsed chronoamperometry. Finally, the chronoamperometry technique was applied to characterize the modified SPEAu. The use of a secondary antibody anti-TNF-α (Ab-TNF-α-HRP) labelled with horseradish peroxidase (HRP, 2 µg·mL−1) was investigated using tetramethylbenzidine (TMB, pH = 3.75) as electrochemical substrate containing 0.2 mM of H2O2. A sandwich-type detection strategy with a secondary antibody anti-TNF-α provided chronoamperometric analyses in 10 s for each sample. Linearity, precision, limit of detection, and selectivity of devices were investigated. Interferences were evaluated by analyzing solutions containing other cytokine produced during the acute stage of inflammation. The immunosensor showed good performance within the clinically relevant concentration range, with a precision of 8%, and a limit of detection of 0.3 pg/mL. Therefore, it may represent a promising tool for monitoring HF in a non-invasive way.

[1]  P. Gisondi,et al.  Targeting tumor necrosis factor α in psoriasis and psoriatic arthritis , 2008 .

[2]  T. McDonagh,et al.  Heart Failure Epidemiology: European Perspective , 2013, Current cardiology reviews.

[3]  F. Di Francesco,et al.  The effect of sampling procedures on the urate and lactate concentration in oral fluid , 2018 .

[4]  Biykem Bozkurt,et al.  Activation of cytokines as a mechanism of disease progression in heart failure , 2000, Annals of the rheumatic diseases.

[5]  Hojatollah Yousefimanesh,et al.  Evaluation of salivary tumor necrosis factor-alpha in patients with the chronic periodontitis: A case-control study , 2013, Journal of Indian Society of Periodontology.

[6]  Abdelhamid Elaissari,et al.  Novel strategy for sulfapyridine detection using a fully integrated electrochemical Bio-MEMS: Application to honey analysis. , 2017, Biosensors & bioelectronics.

[7]  Song Wei Loo,et al.  Cytokine and Cancer Biomarkers Detection: The Dawn of Electrochemical Paper-Based Biosensor , 2020, Sensors.

[8]  L. Ivashkiv,et al.  Inhibition of IL-6 and IL-10 Signaling and Stat Activation by Inflammatory and Stress Pathways1 , 2000, The Journal of Immunology.

[9]  Abdelhamid Errachid,et al.  A fully integrated electrochemical biosensor platform fabrication process for cytokines detection. , 2017, Biosensors & bioelectronics.

[10]  Gordana Brumini,et al.  Salivary levels of tumor necrosis factor-alpha in oral lichen planus. , 2004, Mediators of inflammation.

[11]  Maria Giovanna Trivella,et al.  Diazonium modified gold microelectrodes onto polyimide substrates for impedimetric cytokine detection with an integrated Ag/AgCl reference electrode , 2013 .

[12]  P. Yáñez‐Sedeño,et al.  Electrochemical immunosensor for simultaneous determination of interleukin-1 beta and tumor necrosis factor alpha in serum and saliva using dual screen printed electrodes modified with functionalized double-walled carbon nanotubes. , 2017, Analytica chimica acta.

[13]  D. Fotiadis,et al.  Electrochemical biosensor platform for TNF-α cytokines detection in both artificial and human saliva: Heart failure , 2017 .

[14]  L. Hofman,et al.  Human saliva as a diagnostic specimen. , 2001, The Journal of nutrition.

[15]  David F. Lappin,et al.  Saliva and serum levels of B-cell activating factors and tumor necrosis factor-α in patients with periodontitis. , 2014, Journal of periodontology.

[16]  Michael Thompson,et al.  Harmonized guidelines for single-laboratory validation of methods of analysis (IUPAC Technical Report) , 2002 .

[17]  Dimitrios I. Fotiadis,et al.  A novel chronoamperometric immunosensor for rapid detection of TNF-α in human saliva , 2018, Sensors and Actuators B: Chemical.

[18]  Hatem Fessi,et al.  Submicron magnetic core conducting polypyrrole polymer shell: Preparation and characterization. , 2016, Materials science & engineering. C, Materials for biological applications.

[19]  N. Herrmann,et al.  A Meta-Analysis of Cytokines in Alzheimer's Disease , 2010, Biological Psychiatry.

[20]  Pedro Estrela,et al.  Electrochemical ELISA Protein Biosensing in Undiluted Serum Using a Polypyrrole-Based Platform , 2020, Sensors.

[21]  N. Herrmann,et al.  A Meta-Analysis of Cytokines in Major Depression , 2010, Biological Psychiatry.

[22]  G. Rogler,et al.  TNF-alpha blockers in inflammatory bowel diseases: practical consensus recommendations and a user's guide. , 2009, Swiss medical weekly.

[23]  F. Di Francesco,et al.  Determination of salivary α-amylase and cortisol in psoriatic subjects undergoing the Trier Social Stress Test , 2018 .

[24]  Vijay Viswanathan,et al.  Increased levels of circulating (TNF-α) is associated with (-308G/A) promoter polymorphism of TNF-α gene in Diabetic Nephropathy. , 2018, International journal of biological macromolecules.

[25]  M. K. Sezgintürk,et al.  A highly sensitive immunosensor based on ITO thin films covered by a new semi-conductive conjugated polymer for the determination of TNFα in human saliva and serum samples. , 2017, Biosensors & bioelectronics.

[26]  Carlos Orrego,et al.  Cytokines and acute heart failure , 2008, Critical care medicine.

[27]  R. Locksley,et al.  The TNF and TNF Receptor Superfamilies Integrating Mammalian Biology , 2001, Cell.

[28]  Yorgos Goletsis,et al.  A computational approach for the estimation of heart failure patients status using saliva biomarkers , 2017, 2017 39th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC).

[29]  Pål Aukrust,et al.  Systemic inflammation in heart failure – The whys and wherefores , 2006, Heart Failure Reviews.

[30]  R Thorpe,et al.  Immunoassays for detecting cytokines: what are they really measuring? , 1995, Journal of immunological methods.