Leveraging Design of Experiments to Unravel the Amplification Mechanism of Single‐Molecule Wide‐Field Biosensors
Abstract Detecting single molecules on large interfaces, spanning several square micrometers, is often considered unfeasible due to the minimal perturbation individual molecules exert on the sensing surface. However, biological systems, such as cellular membranes, demonstrate remarkable sensitivity,...
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| Format: | Article |
| Language: | English |
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Wiley-VCH
2025-07-01
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| Series: | Advanced Materials Interfaces |
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| Online Access: | https://doi.org/10.1002/admi.202500090 |
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| author | Michele Catacchio Mariapia Caputo Lucia Sarcina Cinzia Di Franco Matteo Piscitelli Erika Castrignanò Paolo Bollella Gaetano Scamarcio Eleonora Macchia Luisa Torsi |
| author_facet | Michele Catacchio Mariapia Caputo Lucia Sarcina Cinzia Di Franco Matteo Piscitelli Erika Castrignanò Paolo Bollella Gaetano Scamarcio Eleonora Macchia Luisa Torsi |
| author_sort | Michele Catacchio |
| collection | DOAJ |
| description | Abstract Detecting single molecules on large interfaces, spanning several square micrometers, is often considered unfeasible due to the minimal perturbation individual molecules exert on the sensing surface. However, biological systems, such as cellular membranes, demonstrate remarkable sensitivity, achieving single‐molecule detection on interfaces as large as 103 µm2, despite the stark mismatch between molecular footprints and surface areas. While these amplification mechanisms are well‐documented, their molecular and biophysical foundations remain poorly understood. To contribute to probing these phenomena, a Design of Experiments (DoE) approach explores how pH and ionic strength in conditioning solutions influence Surface Plasmon Resonance (SPR) detection in the single‐molecule regime. Conditioning a physisorbed layer of capturing antibodies at low pH emerges as the key strategy, enabling the reliable detection of only 6 ± 2 IgG molecules with a significant SPR signal. The analysis further reveals that pH conditioning induces a refractive index shift within the antibody layer, which is quantitatively correlated with changes in zeta potential (ζ‐potential). These findings provide critical insights into the mechanisms driving ultrasensitive SPR detection and establish a data‐driven framework for advancing biosensing technologies. |
| format | Article |
| id | doaj-art-dd8d70c84a5144ae9dcc8a1a4938d1b4 |
| institution | Matheson Library |
| issn | 2196-7350 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | Wiley-VCH |
| record_format | Article |
| series | Advanced Materials Interfaces |
| spelling | doaj-art-dd8d70c84a5144ae9dcc8a1a4938d1b42025-07-31T05:08:29ZengWiley-VCHAdvanced Materials Interfaces2196-73502025-07-011214n/an/a10.1002/admi.202500090Leveraging Design of Experiments to Unravel the Amplification Mechanism of Single‐Molecule Wide‐Field BiosensorsMichele Catacchio0Mariapia Caputo1Lucia Sarcina2Cinzia Di Franco3Matteo Piscitelli4Erika Castrignanò5Paolo Bollella6Gaetano Scamarcio7Eleonora Macchia8Luisa Torsi9Dipartimento di Farmacia‐Scienze del Farmaco Università degli Studi di Bari Aldo Moro Bari 70125 ItalyDipartimento di Farmacia‐Scienze del Farmaco Università degli Studi di Bari Aldo Moro Bari 70125 ItalyDipartimento di Chimica Università degli Studi di Bari Aldo Moro Bari 70125 ItalyCNR IFN Bari 70125 ItalyDipartimento Interateneo di Fisica Università degli Studi di Bari Aldo Moro Bari 70125 ItalyDipartimento di Farmacia‐Scienze del Farmaco Università degli Studi di Bari Aldo Moro Bari 70125 ItalyDipartimento di Chimica Università degli Studi di Bari Aldo Moro Bari 70125 ItalyNEST Istituto Nanoscienze ‐ CNR and Scuola Normale Superiore Pisa I‐56127 ItalyCentre for Colloid and Surface Science Dipartimento di Chimica Università degli Studi di Bari Aldo Moro Bari 70125 ItalyDipartimento di Chimica Università degli Studi di Bari Aldo Moro Bari 70125 ItalyAbstract Detecting single molecules on large interfaces, spanning several square micrometers, is often considered unfeasible due to the minimal perturbation individual molecules exert on the sensing surface. However, biological systems, such as cellular membranes, demonstrate remarkable sensitivity, achieving single‐molecule detection on interfaces as large as 103 µm2, despite the stark mismatch between molecular footprints and surface areas. While these amplification mechanisms are well‐documented, their molecular and biophysical foundations remain poorly understood. To contribute to probing these phenomena, a Design of Experiments (DoE) approach explores how pH and ionic strength in conditioning solutions influence Surface Plasmon Resonance (SPR) detection in the single‐molecule regime. Conditioning a physisorbed layer of capturing antibodies at low pH emerges as the key strategy, enabling the reliable detection of only 6 ± 2 IgG molecules with a significant SPR signal. The analysis further reveals that pH conditioning induces a refractive index shift within the antibody layer, which is quantitatively correlated with changes in zeta potential (ζ‐potential). These findings provide critical insights into the mechanisms driving ultrasensitive SPR detection and establish a data‐driven framework for advancing biosensing technologies.https://doi.org/10.1002/admi.202500090biochemical amplificationdesign of experimentimmunoassaysplasmonic sensorssingle‐molecule sensingsurface‐plasmon‐resonance |
| spellingShingle | Michele Catacchio Mariapia Caputo Lucia Sarcina Cinzia Di Franco Matteo Piscitelli Erika Castrignanò Paolo Bollella Gaetano Scamarcio Eleonora Macchia Luisa Torsi Leveraging Design of Experiments to Unravel the Amplification Mechanism of Single‐Molecule Wide‐Field Biosensors Advanced Materials Interfaces biochemical amplification design of experiment immunoassays plasmonic sensors single‐molecule sensing surface‐plasmon‐resonance |
| title | Leveraging Design of Experiments to Unravel the Amplification Mechanism of Single‐Molecule Wide‐Field Biosensors |
| title_full | Leveraging Design of Experiments to Unravel the Amplification Mechanism of Single‐Molecule Wide‐Field Biosensors |
| title_fullStr | Leveraging Design of Experiments to Unravel the Amplification Mechanism of Single‐Molecule Wide‐Field Biosensors |
| title_full_unstemmed | Leveraging Design of Experiments to Unravel the Amplification Mechanism of Single‐Molecule Wide‐Field Biosensors |
| title_short | Leveraging Design of Experiments to Unravel the Amplification Mechanism of Single‐Molecule Wide‐Field Biosensors |
| title_sort | leveraging design of experiments to unravel the amplification mechanism of single molecule wide field biosensors |
| topic | biochemical amplification design of experiment immunoassays plasmonic sensors single‐molecule sensing surface‐plasmon‐resonance |
| url | https://doi.org/10.1002/admi.202500090 |
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