BME 5110 Biomedical Microdevice

Protein / DNA Microarray

Group Members:
Lam Ka Fung Kelvin(14056303g)
Yeung Wai Lung Raylun(15124886g)
Li Wai Ming (15045969g)
Kwok Chi Wa (12128131g)
Tong Hei long (1611759g)
Lam Wai Lok (16118549g)
Outline

1. Introduction
2. Current Technology

3. Current Challenge

4. Future opportunities for overcoming

challenges

5. Conclusion
Introduction
Introduction
• a pattern of ssDNA
probes immobilized
on a slide.

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• use hybridization to
detect a specific DNA
or RNA in a sample.
DNA Microarray
Definition:
•are solid supports (glass or silicon) upon which
DNA is attached in an organized grid fashion.
•Probe, represents a single gene.
•Synonyms of DNA microarrays such as DNA chips,
gene chips, DNA arrays, gene arrays and biochips.
DNA Microarray Technology
• Defined as a high-throughput and verstaile technology
• Parallel gene expression analysis
• Detection of polymorphism and mutation in DNA
• clection of microscopic DNA spots on solid surface

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DNA Microarray - Review

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DNA Microarray Principle
• a) A DNA chip can be manufactured to contain hundreds of
thousands of synthetic single-stranded DNA sequences.
• b) The unknown DNA from a patient is separated into single
strands, and labeled with a fluorescent dye.

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• c) The unknown DNA is inserted into the chip and allowed to
hybridize with the DNA on the chip.
• d) The tagged DNA will bind only to the complementary DNA
on the chip.
• e The principle of DNA microarrays lies on the hybridization
between the nucleotide.
Principle

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Data Analysis
• Slides are dried and
scanned
• Hybridized target

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emissions
• Software analysis
• Green, yellow, red
spots
Gene Expression: Central
Dogma

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Microarry
• “A snapshot that captures the activity pattern of
thousands of genes at once”

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Manufacturing of Glass cDNA
Microarray
1. selection of the
material to spot onto
the microscope surface

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2. Prparation and
purification of DNA
sequence
3. Spotting DNA
solution onto chemcial
modified glasses slides
Current Technology
Masked photolithography
Rinse the silicon substrate with blocking agent
which can be removed when exposed to UV
light source

Silicon substrate Silicon substrate

Masked photolithography

Expose to UV light source to

UV UV remove block agent at specific
light light location on the microarray

Silicon substrate Silicon substrate

Masked photolithography
Adding the nucleotide base A to the microarray and
only the middle chain will bond to base A due to the
lack of blocking agent

Rinse the silicon

substrate with
A A A blocking agent
again

Silicon substrate Silicon substrate

Masked photolithography
Expose to UV light source to
remove block agent at specific
location on the microarray again
UV UV
light light

A A

Silicon substrate Silicon substrate

Masked photolithography
Adding the nucleotide base T to the microarray and base T
will bond to the chains except the middle one due to the
existence of blocking agent

Rinse the silicon

T T T substrate with
blocking agent again

A T A T

Silicon substrate Silicon substrate

Masked photolithography

UV UV
light light
Repeat the cycle until
the desired sequence
is obtained on
microarray substrate

A C A
G G C
T A T T A T

Silicon substrate Silicon substrate

 Mask-less photolithography

1. A digital mirror device (DMD) is used to replace the function of masks

2. A DMD contains thousand of micro-sized mirrors with each mirror placed on top
of a hinge sustained with a torsion spring
3. The UV light is directed to specific location on microarray by software
4. Works in the same principle as masked photolithography except no masks are
used
Non-Contact Ink-Jet Printing
• The DNA sample is withdrawn from the source plate up into
the print head and then moved to a location above the slides.

• The sample is then forced through a small orifice causing the

ejection of a droplet from the print head onto the surface.
Non-Contact Ink-Jet Printing
• Two types on non-contact ink-jet technology

Piezoelectric
Syringe-solenoid

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Piezoelectric Printing Technology
• Piezoelectric printing technology uses a
piezoelectric crystal

• Places the piezoelectric crystal in contact

with a glass capillary which holds the
sample fluid

• The sample is drawn up into the reservoir

and the crystal is biased with a voltage,
which causes the crystal to deform,
squeeze the capillary, and eject a small
amount of fluid from the tip
24
Rose, Don. "Microfluidic technologies and instrumentation for printing DNA microarrays." Microarray Biochip
Technology (2000): 19-38.
Piezoelectric Printing Technology
• The fast response time of the crystal
permits fast dispensing rates, on the
order of several thousand drops per
second.
• Furthermore, the small deflection of
the crystal results in drop volumes on
the order of hundreds of picoliters.

• The main difficulties in implementing

piezoelectric dispensing include air
bubbles which reduce the reliability of
the system, relatively large sample
volumes, and difficulties with sample
changing.
25
Tian, Jingdong, Kuosheng Ma, and Ishtiaq Saaem. "Advancing high-throughput gene synthesis
technology." Molecular BioSystems 5.7 (2009): 714-722.
Syringe-Solenoid Printing Technology
• Syringe-solenoid technology
combines a syringe pump with a
micro-solenoid valve to provide
quantitative dispensing in the
nanoliter range
• A high-resolution syringe pump
is connected to both a high-
speed micro-solenoid valve and
a reservoir through a switching
valve.
• For printing microarrays, the
system is filled with a system
fluid, usually water, and the
syringe is connected to the
micro-solenoid valve. 26
Rose, Don. "Microfluidic technologies and instrumentation for printing DNA microarrays." Microarray Biochip
Technology (2000): 19-38.
Syringe-Solenoid Printing Technology
• Withdrawing the syringe causes the
sample to move upward into the
tip.
• The syringe then pressurizes the
system such that opening the
micro-solenoid valve causes
droplets to be ejected onto the
surface.
• With this configuration, the
minimum dispense volume is on the
order of 4-8 nL.
• Given the positive displacement
nature of the dispensing
mechanism, the reliability of the
system is very high.
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http://www.seaviewsci.com/cartesian/synquad_technology.htm
Current Challenge
 Current Challenges
• Indirect measure of
relative
concentration thus
at high
concentrations the
array will become
saturated and at
low concentrations
equilibrium favours
no binding.

https://www.slideshare.net/gantovnik/modeling-of-competitive-kinetics-of-dna-hybridization-reactions
Curr Protoc Mol Biol. Author manuscript; available in PMC 2014 May 06
 Current Challenges
• For complex mammalian genomes it is difficult to
design arrays in which multiple related DNA/RNA
sequences will not bind to the same probe on the
array. A sequence which was designed to detect
“gene A” may also detect gene B,C and D.

Curr Protoc Mol Biol. Author manuscript; available in PMC 2014 May 06
Current Challenges
• DNA array can only detect sequences that the
array was designed to detect. That is if the
solution being hybridized to the array contains
RNA or DNA species for which there is no
complimentary sequence on the array, those
species will not be detected.

Curr Protoc Mol Biol. Author manuscript; available in PMC 2014 May 06
 Further challenges on
microarrays
• Samples used for microarray analysis contain a
mixture of different cell types. Thus, with the
exception of cell type-specific genes, the source of
mRNA is unknown and limits our ability to interpret
the cellular signatures relating to the gene
expression patterns of our data.

Frontiers in Bioscience 8, s913-923, 2013

 Further challenges on
microarrays
• The variability of microarray results can be
significant, especially for genes with low
expression levels. Replication is recommended to
establish a high degree of confidence, and to
reduce the number of potential false positive
results. However, this may be difficult due to high
cost or insufficient sample amount.

Frontiers in Bioscience 8, s913-923, 2013

 Further challenges on
microarrays
• DNA microarrays provide results on mRNA
expression levels which do not necessarily
correlate with protein expression levels or
function. Thus, these results provide only an
incomplete view of the functional significance of
differentially expressed genes in the
experiments.

Frontiers in Bioscience 8, s913-923, 2013

Future Opportunities for
Overcoming Challenge
The Future of DNA Arrays
Two critical factors to be considered for future of DNA Arrays
Unbiased method (direct measure all DNA or RNA species)
Cost Reducing
(sequencing enhance cost competitive with microarrays with
possible exception of genotyping)

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Advantage of sequencing
Direct measurement (direct measure Nucleic Acid in solution)
Need only one count the number sequencing to obtain
abundance determination
Counting sequences
 linear with concentration
Signal to noise

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Unbiased approach to measuring (Unlike DNA arrays)
Is not dependent on which nucleic present
Independently detect closely related gene sequences
Novel splice forms or RNA editing may be missed due to cross
hybridization on DNA microarrays

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Conclusion
Thank you