Towards single molecule biosensors using super-resolution fluorescence microscopy.
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.
School 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.
Xun
X
Lu
EMBL Australia Node in Single Molecule Science, School of Medical Sciences and the ARC Centre of Excellence in Advanced Molecular Imaging, University of New South Wales, Sydney 2052, Australia.
Philip R
PR
Nicovich
EMBL Australia Node in Single Molecule Science, School of Medical Sciences and the ARC Centre of Excellence in Advanced Molecular Imaging, University of New South Wales, Sydney 2052, Australia.
Katharina
K
Gaus
School 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. Electronic address: justin.gooding@unsw.edu.au.
J Justin
JJ
Gooding
D000906:Antibodies / Q000276:immunology; D000937:Antigen-Antibody Reactions / Q000276:immunology*; D000941:Antigens / Q000276:immunology; D015374:Biosensing Techniques; D006801:Humans; D008856:Microscopy, Fluorescence; D036103:Nanotechnology; D017971:Tin Compounds / Q000737:chemistry
D000906:Antibodies; D000941:Antigens; D017971:Tin Compounds; C109984:indium tin oxide
Immunosensors; Indium tin oxide; Self-assembled monolayers; Single molecule localisation microscopy; Superresolution fluorescence microscopy