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Towards single molecule biosensors using super-resolution fluorescence microscopy
Xun Lu1, Philip R Nicovich2, Katharina Gaus2
1School of Chemistry, Australian Centre for NanoMedicine and the ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, University of New South Wales, Sydney 2052, Australia.
Insights
This study shows single molecule localisation microscopy (SMLM) can monitor antibody-antigen binding events on surfaces. This technique offers a new way to characterize biosensing interfaces for quantitative analysis.
Area of Science:
- Biosensing and Interface Science
- Single-molecule Biophysics
- Surface Chemistry
Background:
- Conventional immunosensors rely on bulk measurements, lacking molecular-level characterization of interfaces.
- Designing immunosensing interfaces requires precise control over antibody immobilization and nonspecific adsorption.
- Current methods lack tools to assess the molecular-level performance of these sophisticated interfaces.
Purpose of the Study:
- To investigate the feasibility of using single molecule localisation microscopy (SMLM) for monitoring antibody-antigen binding events.
- To establish indium tin oxide (ITO) surfaces as a viable platform for SMLM in biosensing applications.
- To demonstrate the potential of SMLM for characterizing biosensing interfaces at the single-molecule level.
Main Methods:
- Utilized indium tin oxide (ITO) surfaces for SMLM.
- Modified ITO surfaces with self-assembled monolayers using organophosphonic acid derivatives.
- Controlled and monitored antigen and antibody immobilization at the single-molecule level.
- Observed antibody binding to antigen-modified surfaces using SMLM.
Main Results:
- Confirmed ITO surfaces are suitable for SMLM.
- Demonstrated control over antigen and antibody density on the surface.
- Showed that antibody binding is dependent on both surface antigen concentration and solution antibody concentration.
- Validated SMLM's capability to monitor binding events at the single-molecule level.
Conclusions:
- Single molecule localisation microscopy (SMLM) can effectively monitor antibody-antigen binding events.
- SMLM provides a powerful tool for characterizing biosensing interfaces at the molecular level.
- This approach paves the way for massively parallel, single-molecule detection schemes for quantitative biosensing.
Abstract:
Conventional immunosensors require many binding events to give a single transducer output which represents the concentration of the analyte in the sample. Because of the requirements to selectively detect species in complex samples, immunosensing interfaces must allow immobilisation of antibodies while repelling nonspecific adsorption of other species. These requirements lead to quite sophisticated interfacial design, often with molecular level control, but we have no tools to characterise how well these interfaces work at the molecular level. The work reported herein is an initial feasibility study to show that antibody-antigen binding events can be monitored at the single molecule level using single molecule localisation microscopy (SMLM). The steps to achieve this first requires showing that indium tin oxide surfaces can be used for SMLM, then that these surfaces can be modified with self-assembled monolayers using organophosphonic acid derivatives, that the amount of antigens and antibodies on the surface can be controlled and monitored at the single molecule level and finally antibody binding to antigen modified surfaces can be monitored. The results show the amount of antibody that binds to an antigen modified surface is dependent on both the concentration of antigen on the surface and the concentration of antibody in solution. This study demonstrates the potential of SMLM for characterising biosensing interfaces and as the transducer in a massively parallel, wide field, single molecule detection scheme for quantitative analysis.
