Page 197 - An Introduction to Microelectromechanical Systems Engineering

176 MEMS Applications in Life Sciences

the membrane so that heat flows through the highly thermally conductive silicon
walls of the chamber, the temperature uniformity of the fluid was greatly improved.
A fan was added for more rapid cooling. These modifications have yielded much
tighter closed-loop temperature control and enabled faster cycling, from around 35s
per cycle to as little as 17s per cycle. These cycle times are far faster than the approxi-
mately 4 min per cycle needed in the industry-workhorse Applied Biosystems
®
GeneAmp PCR System 9600 [13].
The LLNL system has detection capability in addition to amplification. In a
variation of traditional PCR, the addition of TaqMan dyes (probes), which link to
certain sections of a DNA strand (just like the primers), results in fluorescence of
green light from each replicated DNA strand when excited by a blue or ultraviolet
source [13, 14]. Thus, the intensity of the fluorescence is proportional to the number
of replicated DNA strands matching the TaqMan probe in the solution. This proce-
dure has the advantage of simultaneous DNA amplification and detection but only
works when suitable primers and probe have been added to the solution for the type
of DNA under test. Thus, the number of different DNA sections potentially being
identified is equal to the number of PCR chambers that can be run simultaneously.
In demonstrations at LLNL with different cells, there was no detectable fluorescence
signal for the first 20–25 cycles, depending on the initial concentration. After cycling
on the order of 5–15 minutes, the signal appeared and rapidly grew if there was a
match.
In the LLNL system, the light source is a filtered blue LED through the silicon
nitride window. A handheld prototype, which represents the holy grail of DNA
analysis, is about the size of a one-quart milk carton, including computer, display,
and keypad, and is powered by a separate 0.5-kg battery with a run time of two
hours. Larger but still portable systems using this technology, available from Micro-
fluidic Systems, can presently identify over 10 airborne pathogens.
Electrophoresis on a Chip

Determining the sequence of nucleotides in a DNA strand involves amplification
and chemical labeling of the amplified DNA fragments with specific fluorescent
or radioactive tags and a subsequent distinct detection step that analyzes the
labeled DNA products. The entire process is called DNA sequencing. Its underlying
principles are beyond the scope of this book, but the eager reader is referred to
Stryer’s book on biochemistry [6]. One detection technique is electrophoresis, which
employs the separation of charged molecules, including DNA, in suspension under
the effect of an electric field [see Figure 6.5(a)]. In solution, a hydrogen ion dissoci-
ates from each phosphate in the DNA backbone, leaving the DNA strand with a net
negative charge [see Figure 6.3(b)]. The charge-to-mass ratio is approximately the
same for strands of different lengths, but, when driven with an electric field through
a molecular sieve, larger molecules move more slowly [9]. Thus, after a given time,
groups of small molecules move farther than larger ones. A limitation of electropho-
resis is that as the sample sits in solution, it is also diffuses both up and down the
channel. Because the diffusion distance grows with time, short electrophoretic sepa-
ration and detection times are advantageous, which implies the use of a high electric
field over a short distance. Electrophoresis can separate DNA fragments up to about
3,000 bases in length.