Text preview for : Attaching Antibodies to MAC Levers with the Bifunctional Amine-Amine Reactive PEG Tether - App Note part of Agilent Attaching Antibodies to MAC Levers with the Bifunctional Amine-Amine Reactive PEG Tether - App Note Agilent Attaching Antibodies to MAC Levers with the Bifunctional Amine-Amine Reactive PEG Tether - App Note 5989-8561EN c20141208 [6].pdf
Back to : Attaching Antibodies to M | Home
Keysight Technologies
Attaching Antibodies to MAC Levers with the
Bifunctional Amine-Amine Reactive PEG Tether,
Aldehyde-PEG18-NHS
Application Note
Introduction
Biological molecules can be attached to silicon and silicon nitride AFM probes,
transforming them into sensitive, nanoscale, biochemically selective sensors. The
biochemically modified AFM probes can be used to study single molecule interactions
in various AFM-based techniques. One such AFM-based method that relies on the
biochemically modified AFM probes is a force spectroscopy (FS) technique called
molecular recognition force microscopy (MRFM) [Riener 2003, Hinterdorfer 2004].
In MRFM, distinct interactions between even just one ligand molecule, which is
immobilized on the tip of the AFM probe, and complimentary receptor molecules,
which are immobilized on an opposing substrate, can be investigated one by one as
the biochemically modified AFM probe first approaches and then is subsequently
withdrawn away from the substrate. Unbinding interactions between the ligand and a
receptor can be quantified because when the AFM probe is withdrawn away from the
substrate, the molecular binding complex between the ligand and the receptor cause
the AFM probe cantilever to bend. The bending of the cantilever can be monitored
optically. It bends in relation to its inherent spring constant and the strength of
the interaction between the ligand and the receptor. As the probe is withdrawn,
the cantilever continues to bend until the ligand-receptor complex dissociates.
Consequently, AFM-based force spectroscopy experiments can give valuable
information about the structure and dynamics of molecular unbinding events at the
single molecule level [Noy 1997] and to gain an understanding of the intramolecular
forces involved in protein folding and polymer elongation [Allison 2002]. Another
single molecule AFM technique that utilizes immobilized ligands on an AFM probe
and receptor molecules immobilize on a substrate is topography-recognition imaging
(TREC). TREC is a dynamic AFM technique in which an AFM probe, which contains
Figure 1. Direct immobilization of ligands to the tip
immobilized ligand molecules on its tip, oscillates in AC mode while it is scanned of an AFM probe. This type of immobilization can
over a substrate which contains bound receptor molecules. A Keysight AFM that often lead to a high density of molecules on the
is equipped with PicoTREC can resolve the molecular recognition signals from the probe, but these ligands are generally not able to
diffuse within a defined volume of space nor are
AFM surface topography signals so that the lateral positions of functionally active they able to reorient and bind with the receptors in
receptors on the substrate can be resolved with nanometer resolution [Stroh 2004a an optimal manner.
and Stroh 2004b, Kienberger 2004b]. PicoTREC has been used to map molecular
binding interactions on a variety of samples. The list includes cadherin binding sites
on vascular endothelial cells [Chtcheglova 2007], vascular endothelial growth factor
(VEGF) receptors on endothelial cells [Lee 2007], molecular interactions between
nucleic acids - protein [Lin 2006] interactions between antibodies and antigens [Bash
2006, Lohr 2007, Marcus 2006, Stroh 2004a, Stroh 2004b], interactions between
virus particles and human receptors [Ebner 2007], and molecular interactions between
small ligands and their receptors [Ebner 2005].
The proper orientation of ligand molecules on the tip of the probe are extremely
important in MRFM. For example, as depicted in Figure 1, direct covalent
immobilization of ligands on the AFM probe tip [Chtcheglova 2004, Lee 1994, Lin 2005
Sekiguchi 2003, Vinckier 1998] and physisorbtion [Lehenkari 1999, Wojcikiewicz 2004]
may generate high ligand densities, but these ligands may not be free to diffuse within
a defined volume of space nor be able reorient and permit optimal ligand-receptor
binding to occur. Figure 2. Ligand immobilization with PEG
linkers. The PEG linker confines the ligands
within a defined volume of space and they have
Consequently, it has been demonstrated in most MRFM and TREC imaging studies enough flexibility and freedom to diffuse, then
that it is advantageous to attach ligand molecules to the end of a flexible PEG reorient and bind with they complimentary
receptors in an optimal manner.
(polyethylene glycol) tether that is in turn covalently attached to the tip of the AFM
probe (Figure 2). The flexible tether gives the ligand molecules the ability to reorient
03 | Keysight | Attaching Antibodies to MAC Levers with the Bifunctional Amine-Amine Reactive PEG Tether, Aldehyde-PEG18-NHS