Download Applications Lecture 4 - Rose

MEMS applications lecture 2 (Ahmed): Biological/Biomedical Applications of
MEMS
I.
Measuring Blood Chemistry
a. Many parameters can be sensed
i. O2
ii. CO2
iii. metabolites (e.g. urea)
iv. electrolytes (e.g. K+)
b. Example: two approaches for measuring glucose
i. electrochemical sensing
ii. optical sensing
c. Blood Chemistry traditionally sensed with large blood gas analyzers (ppt
fig)
i. expensive
ii. finicky
iii. located in central hospital lab (samples had to be transported)
iv. required special training for lab techs
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d. Miniaturization has led to a major shift in the way blood chemistry is
analyzed
i. point of care blood chemistry analyzer (e.g. iSTAT, ppt fig)
1. uses disposable cartridge (ppt fig)
2. allows paramedics to make measurements on the spot.
3. all blood is contained within the cartridge—allows easy
disposal of biohazard
ii. home blood chemistry testing
1. mostly blood glucose
2. sensing technique is a variation on the optical sensor
iii. Catheter-tip monitoring systems (ppt fig)
1. can be used for continuous monitoring of blood parameters
a. useful for short term applications
i. During surgery
ii. patients in the Intensive Care Unit
b. not useful long term
i. degradation of sensor function
ii. can cause clotting of blood—requires
prophylactic anticoagulation therapy
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cDNA microarray technology
I.
The goal of cDNA microarray technology
a. Examine individual differences in the expression of genes
b. To use this information to individualize the prevention/treatment of
disease
II.
Review: Gene expression
a. The DNA strands in cells code for specific genes, which usually code for
individual proteins. (the production of these proteins is known as gene
expression)
b. Gene expression takes place in two stages
i. Transcription—DNA is turned into RNA via the enzyme RNA
polymerase.
ii. Translation—RNA is turned into Protein in the rough Endoplasmic
Reticulum found in the cytoplasm of the cell.
c. To determine which genes are being expressed in an individual, we can
look at what sequences of mRNA are present. Any mRNA molecules that
are present in high volumes would indicate genes that have been activated
in an individual.
d. It’s very difficult to detect and make copies of mRNA, but it is much
easier to make copies of DNA.
i. Turn mRNA into its component DNA (called cDNA) by using the
enzyme reverse transcriptase and amplify the number of DNA
molecules using polymerase chain reaction.
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ii. Use hybridization techniques to mark the specific DNA molecules
if they are present.
III.
Generalized theory of cDNA microarrays
a.
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a. different sets of cloned DNA sequences are attached to a microscope slide
at different spots
b. “control” and “experimental” DNA (each with a different fluorescent
label) are added to see which ones stick to the different regions of the slide
c. by examining the relative binding of the control and experimental DNA,
an individualized profile can be constructed
IV.
General overview of the use of microarrays
a. Identification of potential genes of interest (currently, there are arrays that
can assay 40,000 genes/slide)
b. Make multiple copies of cDNA from these genes using PCR
c. Print cDNA onto microscope slide
d. Labeling of experimental DNA
i. fluorescence
ii. radioactivity
e. visualization of label
V.
cDNA printing
a. printing involves repeatably placing a few nanoliters of solution onto the
microscope slide
b. types of print head
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i. Fountain-pen type print heads (A and B) above
1. problem: non-uniform distribution of dye
ii. Pin-and-Loop
1. problem: uses too much DNA, low density of spots
iii. Ink jet print head
1. seems to work best
c. steps for batch production of gene chips using printing heads
i. print heads are mounted on a high-precision arraying robot
ii. load clean print heads with cDNA (blot off excess solution)
iii. “print” small amounts of cDNA onto regions of each of the chips
in the batch (can be up to 100 at a time)
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iv. clean off old cDNA using a wash (followed by a steam dry)
v. load new cDNA molecules
VI.
Analysis of fluorescently-labeled microarrays
a. measurement of dye concentration
i. lasers used to excite fluorescence
1. quenching can be a problem
ii. can use simple CCD scanners
iii. new generation: Confocal scanners
1. only image at the surface of the slide
b. the output
i. red excitation—gene present in experimental sample only
ii. green excitation—gene present on control sample only
iii. yellow excitation—gene present on both control and experimental
VII.
Another approach to microarrays—the Affymetrix gene chip
a. Photolithography-based construction of 25-nucleotide DNA fragments
(known as oligonucleotides)
b. advantages
i. much higher densities can be placed on the chip
c. disadvantages
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i. smaller fragments mean less accuracy
1. usually use multiple sites for the same gene
ii. each chip is a 75 mask process
1. expensive
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VIII.
References
a. Figures in the notes are from “A Primer of Genome Science” by Gibson
and Muse
b. other reading
i. Winegarden, Neil, “Microarrays” in Encyclopedia of Medical
Devices and Instrumentation, Second Edition” 2006: John Wiley
and Sons
ii. Array Manufacturing Summary on the Affymetrix web page
http://www.affymetrix.com/technology/manufacturing/index.affx
*i and ii are available at http://www.rose-hulman.edu/~ahmed/mems.htm
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