Biomedical Engineering Reference
In-Depth Information
6.4.2
Detection of Low-Molecular-Weight Antigen
About a decade ago, a novel immunoassay principle called open sandwich
immunoassay (OS-IA) was proposed for the detection of mainly low-molecular-
weight antigens in a noncompetitive mode [
26
-
31
]. This method is based on the
antigen-dependent interchain interaction of separated V
L
and V
H
chains from
a single antibody variable region. To perform OS-IA, for example, one of the
separated chains is immobilized on the plate. When the sample containing the
antigen and another chain that is labeled with an enzyme is added on, both chains
reassociate according to the antigen concentration in the sample, resulting in the
positive colorimetric or chemiluminescent signal on the plate after washing and
substrate addition. In this distinguished method, the detection sensitivity depends
on several parameters. One is the affinity of the antibody used, and another is
the antigen dependency of the V
H
=V
L
association. However, equally important is
the sensitivity of the detection method, and typical detection limits obtained in the
described OS enzyme-linked assays (OS-ELISA) were in the few nanomolar range
[
27
-
31
].
Since the separated variable regions are smaller than (approximately 2
3:5 nm) the original antibody to be captured within the Debye length at the gate
surface, the detection principle of bio-FET will overcome the detection limit of
OS-ELISA. Compared with the OS-ELISA, the OS-FET has the advantages of
label-free and quantitative detection. Because of the label-free measurement, we can
omit the experimental process of labeling. Moreover, we can estimate quantitatively
the number of target molecules with charges and directly transduce the charge
density changes based on the capture of target on the gate into the electrical signals
using the OS-FET. In the present study, we propose a new detection method, open
sandwich-based immunofield-effect transistor (OS-FET) for a label-free and highly
sensitive detection of low-molecular-weight antigen, which is based on the detection
of intrinsic molecular charges of one of the separated chains using the field effect.
Here, we report on the direct transduction of open sandwich immunoassay at the
gate surface into an electrical signal using the FET.
In our setup, the V
H
chain is chemically immobilized on the Si
3
N
4
gate surface.
Then, the small antigen is sandwiched with the free V
L
chain tethered with a
negatively charged protein (MBP), and the V
H
chain is immobilized on the gate
surface (Fig.
6.19
). In this study, bisphenol A (BPA) is utilized as model antigen with
a molecular mass of 228 and negligible charge at neutral pH [
32
]. The compound
is a widely used small monomer in the manufacture of polycarbonate plastics
and epoxy resins and has been reported to have estrogenic effects. While many
methods were devised to detect BPA, the detection limit attained was more than
the nanomolar range [
33
-
38
]. However, recent research suggested that the exposure
of BPA at the picomolar range changed some cell functions [
38
]. Therefore, the
development of a more sensitive method to detect BPA is urgently needed. As shown
in Fig.
6.19
, the free V
L
chain used is tethered with MBP with 13 negative charges
2
