Biomedical Engineering Reference
In-Depth Information
anti-mouse antibodies and the same dye substrate as the T strip. he immobilized enzymes
are now able to activate the dye substrate in both the test and control strips, conirming the
pregnancy.
he pregnancy strip assay has now been generalized to a variety of immunochromatographic
strip (ICS) tests. he nonproit organization Program for Appropriate Technology in Health
(PATH; Seattle, WA), which focuses on global health applications, has developed ICS tests for
diphteria toxin and a number of sexually transmitted diseases (gonorrhea, syphilis, chancroid,
and chlamydia), among others. hese devices require minimal training by health workers but
oten only provide a yes/no readout answer (rather than an analyte level) and sometimes still
need sample preconditioning.
4.5.2 Homogeneous Phase Immunoassays
Because proteins come with a wide range of surface charges and surface chemistries, they tend to
adhere more to surfaces than the relatively homogeneous nucleic acids. herefore, implementing
protein-based assays is generally more problematic, requiring eforts at nonfouling as discussed pre-
viously. However, microluidics has been applied to implementing immunoassays in several formats.
Successful CE on a chip approaches involve the electrophoretic separation of bound and unbound
labeled analytes, or monitoring the change in electrophoretic migration speed of labeled antibodies
bound or unbound to their target ligands. hese methods are sensitive and highly reproducible.
A completely diferent approach involves the use of the T-sensor to perform a “difusion
immunoassay” (DIA;
Figure 4.10
). In the original embodiment of the T-sensor, two luids
interact during parallel low until they exit the microchannel. Large particles, such as blood
cells, do not difuse signiicantly within the time the low streams are in contact. Small par-
ticles difuse rapidly between streams, whereas larger polymers difuse more slowly and equili-
brate between streams further from the point of entry to the device. As interdifusion proceeds
(
Figure 4.10a-c
), interaction zones are formed in which sample and reagents may bind and react
(
Figure 4.10d
). If an indicator solution is used in the detection solution, the difusion interac-
tion zones will be optically detectable (
Figure 4.10f
). he positional variation in intensity of
that signal is a complex function of the concentration of the indicator and analyte. However, it
is straightforward to calibrate the optical response to analyte concentration.
In the DIA, the transport of molecules perpendicular to low in a microchannel is afected by
binding between antigens and antibodies. he DIA is based on the diference in difusion coef-
icients of antigens and antigen-antibody complexes. Like other T-sensor assays, it relies entirely
on the luid dynamics of solutions and chemical interactions of components in solution—no
interactions with immobile phases such as the channel walls are required or desired. By imaging
a
e
f
Detection area
w
d
w
b
Slowly diffusing
antibody
1.2
l
Interdiffusion
zone
d
0.8
c
0.4
z
d
y
Rapidly diffusing
antigen
0
200
x
400
600
800
Position (µm)
FIGURE 4.10
The.DIA.in.a.T-sensor..(From.A..Hatch,.A..E..Kamholz,.K..R..Hawkins,.M..S..Munson,.
E.. A.,. Schilling,. B.. H.. Weigl. and. P.. Yager,. “A. rapid. diffusion. immunoassay. in. a. T-sensor,”.
Nat.
Biotechnol.
.19,.461,.2001..Reproduced.with.permission.from.the.Nature.Publishing.Group..Figure.
contributed.by.Paul.Yager.)
