Biomedical Engineering Reference
In-Depth Information
Very uniform temperature within the sample are also required to make sure that ampliication
process proceeds with predictable idelity. As it turned out, Si itself poisoned the polymerase
reaction, so some form of chemical insulator had to be interposed between the reaction solution
medium and the device itself. hermal cycling consisting of a static cell apposed to a single Peltier
heater and cooler has been used, as well as a serpentine channel that runs a sample sequentially
through zones on a chip held at three diferent temperatures, and even a system that consisted
of a single loop that caused cycling of the contents by buoyancy. Ultimately, the apposition of a
polymeric chamber to a high-powered Peltier element has proven to be one of the most eicient
strategies, allowing 35 full extension cycles for short sequences in less than 8 minutes.
In sum, there are still several practical challenges in the ield: many designs are expensive
(they are not disposable, so cross-contamination is diicult to avoid), most can only perform
DNA/RNA ampliication, and detection methods such as gel electrophoresis are not as ame-
nable to miniaturization as the device volume is shrunk further.
4.6.6 High-Throughput Protein Immunoblotting on a Chip
Given a sample of tissue extract or homogenate, speciic proteins can be detected by a technique
known as
protein immunoblot
or
Western blot
. he irst step in the technique uses electrophoresis
to separate the proteins in a gel matrix, usually a polyacrylamide gel; oten the proteins are denatured
with the addition of a detergent (such as sodium dodecyl sulfate) so that protein migration inside the
gel is only a function of its size and not its charge or shape. he second step in the technique con-
sists of transferring (or “blotting”) the proteins to an antibody-coated membrane (typically made of
nitrocellulose or polyvinylidene luoride (PVDF)) that captures the protein(s) of interest.
Until recently, Western blots have essentially been a manual, extremely slow technique that
lasts between 5 and 12 hours because of the long incubation steps. Obviously, this low through-
put precludes Western blots from being used in proteomics analysis. here are large cancer tis-
sue and sample repositories that have grown over several decades (containing tens of thousands
of frozen samples from patients whose disease outcomes are now known) which are waiting to
be analyzed with a high-throughput immunoblotting approach.
In situ fabrication
a
6%T, Ab
6%T
3%T
Glass chip
chamber
Photomask
UV
UV
UV
Define antibody functionalized
blotting region
Define separation
gel region
Define loading gel region with
stacking interface
b
1
2
3
Sample
injector
Load and stack sample
Page
Transfer
Blot
Loading
4
5
6
7
i
i
i
i
i
Chamber
Channel
array
8
300 µm
FIGURE 4.25
Microluidic.platform.that.integrates.electrophoretic.separation.and.immunoblotting.
of.proteins..(From.Mei.He.and.Amy.E..Herr,.“Automated.microluidic.protein.immunoblotting,”.
Nat.
Protoc.
.5,.1844.(2010)..Reprinted.with.permission.from.Nature.Publishing.Group.)
