Abstract
Kelvin probe force microscopy (KPFM) allows the detection of single binding events between immunoglobulins (IgM, IgG) and their cognate antibodies (anti-IgM, anti-IgG). Here an insight into the reliability and robustness of the methodology is provided. Our method is based on imaging the surface potential shift occurring on a dense layer of ∼5 × 107 antibodies physisorbed on a 50 μm × 90 μm area when assayed with increasing concentrations of antigens in phosphate buffer saline (PBS) standard solutions, in air and at a fixed scanning location. A comprehensive investigation of the influence of the main experimental parameters that may interfere with the outcomes of KPFM immune-assay is provided, showing the robustness and reliability of our approach. The data are supported also by a thorough polarization modulation infrared reflection-absorption spectroscopy (PM-IRRAS) analysis of the physisorbed biolayer, in the spectral region of the amide I, amide II and amide A bands. Our findings demonstrate that a 10 min incubation in 500 μL PBS encompassing ≈ 30 antigens (100 zM) triggers an extended surface potential shift that involves the whole investigated area. Such a shift quickly saturates at increasing ligand concentration, showing that the developed sensing platform works as an OFF/ON detector, capable of assessing the presence of a few specific biomarkers in a given assay volume. The reliability of the developed methodology KPFM is an important asset in single molecule detections at a wide electrode interface.
| Original language | English |
|---|---|
| Pages (from-to) | 73-79 |
| Number of pages | 7 |
| Journal | Journal of Materials Chemistry C |
| Volume | 12 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - 20 Nov 2023 |
| MoE publication type | A1 Journal article-refereed |
Funding
The following projects are acknowledged for partial financial support: “A binary sensor with single-molecule digit to discriminate biofluids enclosing zero or at least one biomarker (NoOne)” ERC Starting Grant 2021 (GA 101040383); “Tecnologie portatili e protocolli innovativi per la diagnosi ultrasensibile di Xylella fastidiosa direttamente in piante e vettori” (1LIVEXYLELLA) Ministero dell’agricoltura, della sovranità alimentare e delle foreste – MIPAAF D.M. n.419161 del 13/09/2022; Centro di Innovazione Regionale Digital Assay, Regione PUGLIA DR n.702 del 08/11/2022; Italian network of excellence for advanced diagnosis (INNOVA), Ministero della Salute -code PNC-E3-2022-23683266 PNC-HLS-DA, CUP: C43C22001630001; Complementary National Plan PNC-I.1 “Research initiatives for innovative technologies and pathways in the health and welfare sector” D.D. 931 of 06/06/2022, DARE - DigitAl lifelong pRevEntion initiative, code PNC0000002, CUP: B53C22006420001, PNRR MUR project PE0000023-NQSTI; MUR – Dipartimenti di Eccellenza 2023–2027 – Quantum Sensing and Modelling for One-Health (QuaSiModO).