Biomedical Engineering Reference
In-Depth Information
Fig. 7.7
(
a
) General schematic of a protein sandwich assay built from bottom to top with the
capture antibody, analyte, and detection antibody attached to a magnetic nanotag. (
b
) General
schematic of the DNA sandwich assay with the capture DNA, target DNA (shown in
dark
black
), and detection DNA shown, built from bottom to top and attached to a magnetic nanotag.
(
c
) Schematic of reverse-phase protein assay with target protein spotted directly on the GMR sensor
surface and bound detection antibody with attached magnetic nanotag
antibody is modified with biotin, and the tag is modified with streptavidin since the
biotin-streptavidin interaction is one of the strongest non-covalent receptor-ligand
interactions in biochemistry (association constant, K
a
10
14
-10
15
M
1
). In the
ELISA, the tag of interest is typically colorimetric or fluorescent. However, when
using magnetically responsive biosensors, the tag of interest is magnetic. Therefore,
the more protein that is present in the system, the more detection antibodies bind
and the more magnetic tags bind. As the number of magnetic tags increases over
the sensor, the MR in the underlying magnetically responsive sensor changes
proportionally, producing larger signals. In this way, quantitative protein detection
is possible with this assay. Similar to a protein sandwich assay, it is also possible
to form a DNA or RNA sandwich structure by utilizing a capture oligonucleotide
sequence and biotinylated detection oligonucleotide sequence for highly sensitive
nucleic acid detection (Fig.
7.7
b).
A “reverse-phase” assay can be used to detect proteins of interest in many
patients' blood samples simultaneously by reorganizing the traditional sandwich
assay (Fig.
7.7
c). In the reverse-phase assay, instead of functionalizing a capture
antibody onto the sensor surface, patient samples containing proteins of interest
such as cell lysates are immobilized directly onto the GMR sensor array. Then, a
solution containing detection antibodies complementary to the protein of interest is
introduced and will bind to the immobilized protein of interest over the GMR sensor.
Since the detection antibody is biotinylated, it can then bind to magnetic nanotags
coated with streptavidin in the same way as the detection antibody and magnetic
nanotag interaction in the traditional sandwich assay. In addition, if one separates
