Biomedical Engineering Reference
In-Depth Information
Fig. 7.6
Normalized plot of
the magnetic moment per
particle versus the applied
external magnetic field
7.4
Protein Detection Assay Using Magnetic Nanoparticles
7.4.1
Standard Protein Detection Assay
Biomarkers can be detected either by their accumulation at a stationary binding
site over a sensor surface or by the binding of biomarkers to tags in a solution
inducing aggregation of the magnetic tags. For the GMR nanosensors discussed
in this chapter, the former will be described. For NMR-based biosensors discussed
in Chaps. 9 and 10, the latter method of protein detection will be described. One of
the most effective and specific methods of detecting proteins on a sensor surface,
like a GMR nanosensor, is by means of a “sandwich assay.” Typically known for its
use in the enzyme-linked immunosorbent (ELISA), the sandwich assay involves the
formation of a three-layered structure where two antibodies (or aptamers, diabodies,
Fab fragments, etc.) form a sandwich around a protein (also called the “analyte”)
of interest (Fig.
7.7
a). One of the antibodies, generally referred to as the “capture
antibody,” is directly immobilized on the sensor surface. In order to make the
sandwich assay highly specific, a monoclonal capture antibody is traditionally used.
A solution of monoclonal antibodies means that every antibody in the solution has
the exact same Fab region and therefore will bind to only one epitope on one protein.
The capture antibody makes up the foundation of the sandwich assay and serves to
selectively immobilize a specific protein of interest directly over the sensor surface.
The second antibody, known as the detection antibody, is delivered in solution
and binds to a second epitope on the captured protein of interest. The detection anti-
body is typically polyclonal and pre-modified with a reactive chemistry, enabling
facile attachment of the detection antibody to the tag of interest. A polyclonal
antibody solution is one in which all the antibodies react with the same protein;
however, they may bind to different epitopes on that protein with varying affinities.
Therefore, the Fab region is not uniform across all the antibodies in a polyclonal
solution but recognizes different regions of the same protein. Typically the detection
