BIM2Met Exam December 17th 2009

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EXAMINATION FRONT COVER

Course code:
BIM2Met

Course:
Bio-MEMS

Credits:
10

Responsible teacher:
Frank Karlsen (tel: 40403480)
Lars Roseng (tel: 92091594)

Responsible department:
Faculty of Science and Engineering

Classe(s):
MMT-2

Date:
December 17th 2009

Examination time, from - to:

From 9:00 14:00

The examination
question consist of
the following :

Number of pages, with Number of tasks:

enclosures:
11
5

Allowed
remedy:

Information of the

Number of
enclosures:
0

Each sub problem counts equally

enclosures:
Notes:

THE CANDIDATE HAVE TO CHECK THAT THE EXAMINATION SET IS COMPLETE

1. DNA, RNA and proteins (9,09%)

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a. Describe cell replication (2,27%)

b. Describe the different types and functions of RNA (2,27%)
c. Describe the steps from DNA to protein (2,27%)
d. What are the functions of molecular chaperones and mention
three and their corresponding functions (2,27%)
a. solution
The cell cycle in eukaryotic cells is much more complex than that in
prokaryotic cells such as the bacteria. This is a result of a number of
factors unique to eukaryotic cells: they are much larger than
prokaryotic cells, they have a more complex armada of intracellular
components (golgi bodies, endoplasmic reticulum, etc.), they have a
nucleus and nucleolus, they have multiple chromosomes containing
the cellular genome, and they have additional genetic material
inside mitochondria (and inside chloroplasts if plant cells). In order
for a eukaryotic cell to successfully complete a cell cycle, it will need
to conduct a complex cell symphony of a sort, coordinating the
replication and sorting of many different cell organelles and
chromosomes.
Eukaryotic cells undergo a cell cycle that can be divided into the
following stages as seen in figure ___ : Interphase (subdivided into
G1(Gap1), S(Synthesis), and G2(Gap2) phases), mitosis (subdivided
into prophase, metaphase, anaphase, and telophase), and cytokinesis
(cell splitting and the distribution of cellular components). Mitosis and
cytokinesis constitute the M phase of the cell cycle.
Mitosis is the common method of cell replication for tissue growth and
regeneration among all multi-cellular organisms. Typically it may last
only an hour, with most of a cell's activity spent in interphase, the time
between M phases (mitotic cell divisions).
Mitosis is also the method of cell replication used for growing an adult
multicelled organism from a primitive fertilized egg, or zygote, as cells
of an embryo or larvae divide to increase the cell numbers of the
developing organism, the new cells formed then differentiating into
specialized structures that help define the adult organism.
During mitosis, replication of cell genetic and cytoplasmic material
occurs, followed by a highly organized splitting of cell contents, that is
cytokinesis. The two cells formed following mitosis, called daughter
cells, are genetically identical, and since a cell approximately doubles
its volume and mass prior to division, each daughter cell has
approximately the cell mass of the original interphase parent cell.
For human convenience and for communication among scientists,
mitosis is artificially divided into discrete stages or phases known as
prophase, metaphase, anaphase, and telophase. Each of these
phases can also be divided into early, middle, and late stages or
phases. Remember though, that mitosis is a continuous process, but
with visibly unique phases.
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b. solution
Ribonucleic acid (RNA) is a biologically important type of molecule that
consists of a long chain of nucleotide units. Each nucleotide consists of
a nitrogenous base, a ribose sugar, and a phosphate. RNA is very
similar to DNA, but differs in a few important structural details: in the
cell, RNA is usually single-stranded, while DNA is usually doublestranded; RNA nucleotides contain ribose while DNA contains
deoxyribose (a type of ribose that lacks one oxygen atom); and RNA
has the base uracil rather than thymine that is present in DNA.
RNA is transcribed from DNA by enzymes called RNA polymerase and is
generally further processed by other enzymes. RNA is central to protein
synthesis. Here, a type of RNA called messenger RNA carries
information from DNA to structures called ribosomes. These ribosomes
are made from proteins and ribosomal RNAs, which come together to
form a molecular machine that can read messenger RNAs and translate
the information they carry into proteins. There are many RNAs with
other roles in particular regulating which genes are expressed, but
also as the genomes of most viruses.
Each nucleotide in RNA contains a ribose sugar, with carbons numbered
1' through 5'. A base is attached to the 1' position, generally adenine
(A), cytosine (C), guanine (G) or uracil (U). Adenine and guanine are
purines, cytosine and uracil are pyrimidines. A phosphate group is
attached to the 3' position of one ribose and the 5' position of the next.
The phosphate groups have a negative charge each at physiological pH,
making RNA a charged molecule (polyanion). The bases may form
hydrogen bonds between cytosine and guanine, between adenine and
uracil and between guanine and uracil. However other interactions are
possible, such as a group of adenine bases binding to each other in a
bulge, or the GNRA tetraloop that has a guanineadenine base-pair.
Chemical structure of RNA
An important structural feature of RNA that distinguishes it from DNA is
the presence of a hydroxyl group at the 2' position of the ribose sugar.
The presence of this functional group causes the helix to adopt the Aform geometry rather than the B-form most commonly observed in
DNA. This results in a very deep and narrow major groove and a shallow
and wide minor groove. A second consequence of the presence of the
2'-hydroxyl group is that in conformationally flexible regions of an RNA
molecule (that is, not involved in formation of a double helix), it can
chemically attack the adjacent phosphodiester bond to cleave the
backbone.
Secondary structure of a telomerase RNA.

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RNA is transcribed with only four bases (adenine, cytosine, guanine and
uracil), but there are numerous modified bases and sugars in mature
RNAs. Pseudouridine (), in which the linkage between uracil and ribose
is changed from a CN bond to a CC bond, and ribothymidine (T), are
found in various places (most notably in the TC loop of tRNA).[7]
Another notable modified base is hypoxanthine, a deaminated adenine
base whose nucleoside is called inosine (I). Inosine plays a key role in
the wobble hypothesis of the genetic code. There are nearly 100 other
naturally occurring modified nucleosides,of which pseudouridine and
nucleosides with 2'-O-methylribose are the most common. The specific
roles of many of these modifications in RNA are not fully understood.
However, it is notable that in ribosomal RNA, many of the posttranscriptional modifications occur in highly functional regions, such as
the peptidyl transferase center and the subunit interface, implying that
they are important for normal function.
The functional form of single stranded RNA molecules, just like proteins,
frequently requires a specific tertiary structure. The scaffold for this
structure is provided by secondary structural elements which are
hydrogen bonds within the molecule. This leads to several recognizable
"domains" of secondary structure like hairpin loops, bulges and internal
loops. Since RNA is charged, metal ions such as Mg2+ are needed to
stabilise many secondary structures.

c. solution
Protein synthesis requires two steps: transcription and translation.

It is like DNA replication in that a DNA strand is used to synthesize a strand of mRNA.
Only one strand of DNA is copied.
A single gene may be transcribed thousands of times.
After transcription, the DNA strands rejoin.

Steps involved in transcription

DNA unwinds.
RNA polymerase recognizes a specific base sequence in the DNA called a promoter and binds
to it. The promoter identifies the start of a gene, which strand is to be copied, and the direction
that it is to be copied.
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Complementary bases are assembled (U instead of T).

A termination code in the DNA indicates where transcription will stop.
The mRNA produced is called a mRNA transcript.

Processing the mRNA Transcript

In eukaryotic cells, the newly-formed mRNA transcript (also called heterogenous nuclear
RNA or hnRNA) must be further modified before it can be used.
A cap is added to the 5 end and a poly-A tail (150 to 200 Adenines) is added to the 3end of
the molecule.
The newly-formed mRNA has regions that do not contain a genetic message. These regions
are called introns and must be removed. Their function is unknown.
The remaining portions of mRNA are called exons. They are spliced together to form a
mature mRNA transcript.
d. Solution
Chaperones: A large group of unrelated protein families, whose role is to stabilize
unfolded proteins, unfold them for translocation across membranes or for degradation,
and/ or to assist in their correct folding and assembly.
In molecular biology, chaperones are proteins that assist the non-covalent folding/unfolding
and the assembly/disassembly of other macromolecular structures, but do not occur in these
structures when the latter are performing their normal biological functions. The common
perception that chaperones are primarily concerned with protein folding is incorrect. The first
protein to be called a chaperone assists the assembly of nucleosomes from folded histones and
DNA and such assembly chaperones, especially in the nucleus, are concerned with the
assembly of folded subunits into oligomeric structures.
Chaperones do not necessarily convey steric information required for proteins to fold: thus
statements of the form `chaperones fold proteins` can be misleading. One major function of
chaperones is to prevent both newly synthesized polypeptide chains and assembled subunits
from aggregating into nonfunctional structures. It is for this reason that many chaperones, but
by no means all, are also heat shock proteins because the tendency to aggregate increases as
proteins are denatured by stress. However, 'steric chaperones' directly assist in the folding of
specific proteins by providing essential steric information, e.g. prodomains of bacterial
proteases, lipase-specific foldases, chaperones in fimbrial adhesion systems...
Many chaperones are heat shock proteins, that is, proteins
expressed in response to elevated temperatures or other
cellular stresses. The reason for this behavior is that protein
folding is severely affected by heat and, therefore, some
chaperones act to repair the potential damage caused by
misfolding. Other chaperones are involved in folding newly
made proteins as they are extruded from the ribosome.

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Although most newly synthesized proteins can fold in absence

of chaperones, a minority strictly requires them.
Macromolecular crowding may be important in chaperone
function. The crowded environment of the cytosol can
accelerate the folding process, since a compact folded protein
will occupy less volume than an unfolded protein chain.
However, crowding can reduce the yield of correctly-folded
protein by increasing protein aggregation. Crowding may also
increase the effectiveness of the chaperone proteins such as
GroEL, which could counteract this reduction in folding
efficiency.
More information on the various types and mechanisms of a
subset of chaperones which encapsulate their folding
substrates can be found in the article for chaperonins.
Chaperonins are characterized by a stacked double-ring
structure and are found in prokaryotes, in the cytosol of
eukaryotes, and in mitochondria.
Other types of chaperones are involved in transport across
membranes, for example in the mitochondria and endoplasmic
reticulum (ER) in eukaryotes. Bacterial translocationspecific
chaperone maintains newly synthesized precursor polypeptide
chains in a translocation-competent (generally unfolded) state
and guides them to the translocon.
New functions for chaperones continue to be discovered, such
as assistance in protein degradation, bacterial adhesin activity,
and in responding to diseases linked to protein aggregation
(e.g. see prion).
2. Different definitions (9,09%)
a. Please define: (3,03%)
DNA:
RNA:
mRNA:
Nucleotide:
Template:
Primer:
Amplicon:
Transcription:
Complementary:
DNA: (= Deoxyribonucleic acid). Nucleic acid that contains the genetic instructions for the
development and function of organisms and life.

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(Two strands, the coding strand and the template strand; the template strand is what is used as a
template in the synthesis of mRNA; the coding strand is not used as a template, but is identical in
sequence to the mRNA except that all the U's are T's)
RNA: (= Ribonucleic acid). Single-stranded molecule that contains ribose rather than the deoxyribose
found in DNA. Several types of RNA exist, i.e. mRNA, tRNA, and rRNA.
mRNA: (= Messenger RNA). The RNA that serves as a template for the synthesis of proteins (the RNA
product of transcription).
Nucleotide: A chemical compound that consists of a heterocyclic base, a sugar, and one or more
phosphate groups. In the most common nucleotides the base is a derivative of purine (adenine or
guanine) or pyrimidine (cytosine, uracil, thymine), and the sugar is the pentose (five-carbon sugar)
deoxyribose or ribose. Nucleotides are the structural units of DNA and RNA.
Template: Is the molecule, gene or piece of RNA and DNA that is used for amplification or
hybridization.
Primer: Is a piece of DNA or RNA or an oligo that used for indicating the start of artificial genetic
processes e.g. to indicate where the DNA polymerase or Reverse Transcriptase should start its
copying
Transcription: The process through which a DNA sequence is enzymatically copied to produce a
complementary RNA which most often serves as a template for the production of a protein.
Amplicon: The product given after exponential amplification of a give template or target.
Complementary: The DNA consists of two different strands. These two strands are complementary to
each other by A-T and C-G. It means that one A always have a T on the other strand while and G
always have a C on the other strand and the opposite.

b. What are the main differences between RT-PCR and NASBA?

(3,03%)
The main difference is the enzymes involved and the temperature cycles.
Both amplify and detect mRNA but one with temperature cycles from
annealing temperature to denaturating, the other only with one isothermal
method performing the whole amplification at 41oC. RT-PCR is mainly
using poly-T primers while NASBA can be used independent of poly-T
primers. The analytical sensitivity of RT-PCR is dependent on the length of
the mRNA amplified while NASBA is not dependent.
c. How can you study gene expressions and function: mention
two approaches? (3,03% )
You can make a mutation inside the gene and study how this
influences the gene expression.
It is possible to clone a gene into a vector that can be
transferred into a host that could be a bacteria or yeast. Inside
the vector different kinds of transcription and translations can
be studied by including the needed information into the
artificial vector.
3. Describe the principle of osmotic pressure and how it can be utilised
to measure glucose (blood sugar)? (9,09%)

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Osmotic pressure results from the diffusion of water down its own
concentration gradient. Water (solvent) will move (diffuse) to regions
with a higher concentration of dissolved foreign components
(solutes), whereas the solutes would move towards the region of
higher concentration of pure water. However, if one separates two
different compartments through a partition that selectively blocks
certain molecules in the volume (solutes) while others are allowed to
pass through unhindered (such as the solvent water) the natural
diffusion down the concentration gradients of the hindered
components are disturbed. Water would seek to neutralise the
concentration difference across the partition, put since the solutes
are not permitted to escape, there will always be a net diffusion of
water across the partition. The water will seek to increase the
volume until a pressure builds up on the side of the partition that
stops the net influx of water. The magnitude of the pressure is
proportional to the number of solutes trapped in the compartment,
which in turn reflects the concentration difference of water across
the partition. Such a partition is called a semi-permeable membrane,
and in the simplest description of the sensor, the solute represents
glucose.
4. There are two methods of detecting confluence (leakage) of albumin
through a nanoporous membrane. Please describe one of them?
(9,09%)
A) Method 1: Spectrophotometer: Considering a leakage of albumin across a nanoporous
membrane, the concentration of albumin will increase in the compartment outside the
membrane with time. By sampling this solution at certain time intervals, the concentration
change of albumin can be detected through the absorbance of light using a
spectrophotometer. By consulting a calibration chart, one can translate this absorbance to a
known concentration value of albumin.
Method 2: Osmotic pressure: Considering a leakage of albumin across a nanoporous
membrane, the concentration of albumin will decrease in the compartment inside the
membrane with time. By tracking the osmotic pressure inside this compartment at certain time
intervals, the concentration change can be detected through the corresponding decrease in
osmotic pressure. By consulting the expression for osmotic pressure:
Equation 1

ici RT

where = osmotic pressure (bar), i = Vant Hoff factor, ci = molar concentration (M), R =
0.08314 bar mol-1 K-1 (universal gas constant) and T = absolute temperature (K) the osmotic
pressure can be translated into the correct concentration value of albumin.

5. (9,09%)
a. Describe the following lab-on-a-chip detection schemes: (6%)
i. electrochemical detection
ii. fluorescence detection
iii. chemiluminescence and bioluminescence detection
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Solution:

Electrochemical detection: Electrochemical analysis in liquid

solutions is concerned with the measurement of electrical quantities, such
as current, and charge in order to gain information about the composition
of the solution and the reaction kinetics of it components. For all
electrochemical detection methods the chemical signal is directly
converted into the electronic domain via simple electrodes, therefore the
overall system can be more compact and the goal of miniaturization can
be driven further. As the analyte is detected by small and compact
electrodes, and detection is dependent on electrode surface area rather
than on available detection volume. Electrochemical detection can easily
be incorporated into microchips during fabrication and are less expensive
than fluorescent methods. Electrochemical methods are often used as
detection method for capillary electrophoresis on chip. Examples of
various methods for electrochemical detection:
o
Amperiometric detection: Oxidation or reduction currents of analytes
at the working electrode
o
Conductimetric detection: Determination of changes in the electrical
conductivity of the solution
o
Potentiometric detection:Potential difference (voltage) between
working electrode and reference electrode. The potential determines the
analytical concentration

Fluorescence: When illuminated at different wavelengths, some

molecules emit light of a different color. For example, fluorescein dye
emits green light when illuminated with blue light. The molecules absorb
the higher energy blue light then lose some of the energy in the form of
lower-energy photons here, green light. Dyes, excitation energy, and
attachment to the target molecule can all be modified for specific analysis.
Epifluorescence involves illuminating the specimen from above while in
trans-fluorescence the excitation light comes from below the sample.
Optical detection requires an optical geometry consisting of a light source,
lenses, filters, diffractive elements and detectors, making the instrument
bulky. Consequently, efforts have been made to miniaturize this equipment
and integrate micro-optical elements into microfluidic devices associated
with optical detection. For fluorescent measurements, the fluorescent
signal of a single molecule is independent of the dimensions of the
detection volume and remains constant. However, the background signal
that is generated by impurities in the sample, stray light, and scattering
scales linearly with the size of the detection volume. Molecular beacons
are an example of a fluorescent probe used for detection of nucleic acids.
Aptamers is another fluorescent probe for the detection of amino acids,
drugs, proteins and other molecules.

Chemiluminescence and bioluminescence: Chemiluminescence is

the generation of light (visible, ultraviolet, and infrared) caused by the
release of energy from a chemical reaction. Unlike fluorescence, where an
excitation source is needed and non-specific radiation can be produced,
chemiluminescence occurs only when the reactants are present, and noise
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is low. For lab-on-a-chip devices, enzyme-catalyzed chemilumincescence

reactions are used to detect e.g. hybridizations. These reactions can be
grouped into three types:
o
Chemiluminescent, which are chemically induced through synthetic
compounds and usually involving z highly oxidized species e.g.
electrochemical reaction
o
Bioluminescent, which arise from a living organism (many marine
animals - e.g. squid, fish; some terrestrial e.g. fireflies; some fungi and
bacteria)
o
Electrochemiluminescent, which take place by the use of electrical
current. The material emits light in response to an electric current passed
through it, or to a strong electric field.
b. Explain the differences between static coatings and dynamic
coatings (3,09%)
solution:
i. Coatings for surface modification of lab-on-a-chip
devices are characterized as static or dynamic.
ii. Static coatings: Covalently bonded to the surface, or
physically adsorbed relying on either hydrophobic
interaction or hydrogen bonding or combinations thereof
before use
iii. Dynamic coatings: Introduced with the sample in the
microsystem and will spontaneously migrate and adsorb
to the inner surface of the microchip and prevent binding
by components of the sample or reagent mixture

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6. (9,09%) Sepsis or blood positioning is a very problematic blood

infection. Sepsis is an infection that very often attack very old or
very young people and cause their death. Normally bacteria, fungi
and virus cannot exist in the blood stream. But when something is
wrong with the immune system the blood may be totally infected
within hours and may cause a rapid death.
Sepsis is caused by 15 different kinds of gram+ and gram- bacteria,
fungi as well as a number of viruses. However, the main cause of
Sepsis is by different bacterial infections. The most important
bacteria is listed here: Escherichia Coli, Klebsiella, Serratia
marcescens,
Enterobacter,
Proteus
mirabilis,
Pseudomonas
aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltiphilia,
Staphylococcus aureus, Streprococus pneumonia, Entercoccous
faecium, Entercoccus faecalis.
When a person got Sepsis the blood has a high concentration of
bacteria. The bacteria are producing a lot of endotoxins and
exotoxins that may cause the severe disease within an organism.
A company has a list of fully validated primer and probes against all
the bacteria listed above and the company knows that you are an
expert in MEMS and BioMEMS. They would like to develop a chip for
the detection of Sepsis that would work hands-free at bed-side or in
a clinical laboratory with a time-to-the-results between one to two
hours. Can you give an overview of how this company could make
such a chip?
Solution: Only a small amount of blood is necessary for the detection
of one of the bacteria above. This depends of course on the
sensitivity of the method implemented on the chip. Many of the
bacteria above are gram+ bacteria with a rather thick cell wall. The
whole blood has to be treated with an anticoagulation chemical. The
chip has to include a filter that would collect the bacteria directly
from the whole blood. When the bacteria is collected inside the chip
the bacteria has to be lysed by ultrasound, by shear forces or by
enzymatic treatment. The best would probably be to combine
ultrasound and shear forces with lysis buffer. The buffer may be
added from chambers within the chip or from storage facilities within
the surrounding instrument. Ultrasound may be created in the
instrument above the filter inside a chip or cartridge. The shear
forces may be created by special structures within the microfluidic
device that force the bacteria particles though smaller and smaller
cavtivities. After lysis it is possible to purify, heat or treat the lysed
suspension further. Heat treatment or enzymatic treatment may
make the lysed suspension amplifiable by PCR but only on the level
of DNA. Specific beads may be used that binds the RNA or DNA of
interest. It is also at this level possible to bind different bacteria
antibodies to the lysate. However, the best process would be to
generally bind RNA and DNA to large surfaces within filters or on the
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surface of beads. These filters or beads may easily be added to

microfabricated chambers in a chip or a cartridge. After the RNA and
DNA are bound to the filter the DNA or RNA may be purified by
different washing step. But it may also be used directly for
amplification with PCR or and DNA amplification step. However, if
RNA is going to be amplified further washing of the RNA/DNA has to
be done. These washing have to be done with ethanol or acetone
before drying of the area bound by RNA and DNA. When the area is
dried properly the RNA and DNA may be released from the surface
area by elution buffer or purified water. After the DNA and RNA have
been purified it is possible to hybridize the DNA or RNA to different
kinds of surface having different kinds of compounds bound to the
probes. The end labelled probes may cause enzymatic reaction that
either give luminescence or colour reactions. These probes that will
specifically react with several of genes or one of the genes from
each of the 15 bacteria described above, may be pre-added to a
surface Other labelled probes or molecular probes may cause the
release of fluorescence or impedance. Before the binding of these
probes it is possible to perform target amplification of the one or
several genes within specific reaction chambers. The chip must
contain the right number of reaction chambers according to how
many targets that may simultaneously be detected within each
chamber. Every primer-set and probe will if bound to a bacteria
sequence report the presence or absence of the bacteria. Primerpairs or probes may also be bound to active mRNA that is reporting
the activity of each bacteria that may cause sepsis. These activity
may be related to toxin, pathogenic activity or resistance activity
that may give very valuable information to the doctors that should
treat the disease. Each reaction chamber would include a predefined
number of primer-sets or probes that would be translated by the
software into specific identity or activity of bacteria sequence. This
would detect the presence and nature of any sepsis within a
suspected patience at any site of interest. It is also possible to
include primers that specifically react with consensus sequences
within all the bacteria that have to be detected. However, these
primers are limited to some few 23S or 16 S RNA that is included
within all these bacteria. The primers may undergo a sequencing
process that specifically identify each of the. After sequencing
probes has started it is possible to evaluate each target in a special
electrophoresis chip. This may be done by the integration of a matrix
or polymer within microfabricated channels. Exposing these matrix
or polymer with an electric field would cause a separation of all the
sequenced oligoes. Following a software program evaluation would
identify the right and specific sequence of the bacteria.
Injection molding may be used for the microfabrication of the e.g. a
plastic chip. If the candidate is describing different ways to
microfabricate this chip will add up scores of the candidate. It the
candidate also are able to make different drawings of the device
would give the candidate even higher scores.
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7. Developing more gentle protocols and affordable instrument for

specific blood analysis tasks is becoming possible through the recent
progress in the area of microfluidics and lab-on-a-chip-type devices.
Precise control over the cell microenvironment during separation
procedures and the ability to scale down the analysis to very small
volumes of blood are among the most attractive capabilities of the
new approaches. (9,09%)
a. Give a short overview of the main challenges for blood sample
preparation (3,03%)
b. Can you suggest four ways to perform on-chip separation of
cells from blood in microscale fields? Please also explain how
this separation could be done? (3,03%)
c. Can you suggest two different ways to extract RNA and DNA
within a Lab-on-a-chip device? (3,03%)
The solution: is given in the Blood on the chip paper: Annu. Rev. Biomed
Eng 2005 7:77-103 (see attachment). a: p79-81. b:p82-88. c: 1:
Described in solution to question 6. 2: Binding of bloods cells to beads
containing probes or antibodies. This is done easily by binding of the
anticoagulated blood to beads, washing and release of the cells after
washing.
8. Explain how the following techniques used for Lab-on-a-Chip
Application works and what kind of steps that are involved: (9,09%)
a. UV Lithography (3,03%)
b. The LIGA Process (3,03%)
c. Hot embossing (3,03%)
The solution to this question is been giving in chapter 2,3 and 5 from the
Book Bio-MEMS: Technology and Applications (see attachment). The
candidates should only give an overview of the techniques and a short
description of the possible steps involved. The candidate should be able to
show that he or she is able to use the techniques properly related to any
project that may be given to them.

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9. Protein microarrays (also called biochips, protein chips) are

measurement devices in (biomedical applications to determine the
presence and/or amount of proteins in biological samples, e.g. blood.
They are tools that can be used in many different areas of research.
Usually a multitude of different capture agents, more frequently
monoclonal antibodies, are deposited on a chip surface in a
miniature array.
a. A challenge with the microarray platform is the surface
chemistry between the biological macromolecules and the
substrate. Give examples on various schemes for immobilizing
proteins on a solid glass surface. (4,54%)
b. What is an enzyme-linked immunosorbant assay (ELISA)?
(4,54%)
Solution: Microarraying is the printing of ordered arrays of biomolecules
onto a solid surface in miniaturized format. The success of DNA
microarrays in advancing the development of high-throughput screening
has led to a wealth of valuable information. Considerable effort is now
being focused on the transfer of this technology into protein microarrays.
Proteins, however, are more complex and diverse than DNA, and there are
many challenges to be overcome.
The way in which proteins are immobilized on solid substrates will
determine the functional properties of a protein microarray. While protein
interactions are currently conducted on a variety of formats, it is still a
major challenge to predict how proteins behave in their biological
environments.
Different approaches are currently underway. Irrespective of which
approach is chosen for a particular application, fundamental challenges
still remain when it comes to interpreting the data. Extrapolation of protein
immobilization strategies from one system to another for different classes
of proteins and applications is difficult since proteins have a wide range of
structural, chemical, and biological properties.
A one-approach-fit-all scheme for screening all the various types of
proteins is improbable. The current (and the most practical) approach is to
generate surfaces that can be used for broad subsets of protein families
without compromising their functional properties.
Derivatized glass slides (substrate random).
These surfaces are appropriate for immobilization of untagged proteins.
The proteins are bound through multiple interacting groups on their
surface. Attachment occurs in a variety of orientations such that different
faces of the protein can interact with proteins or other molecules in
solution. This random orientation reduces the number of protein
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interaction sites of any particular protein through either inappropriate

orientation or inactivation due to conformational change.
The table shows examples of common available glass-based microarray
platforms:

Amine-coated or polylysine-coated slides (silanized slides):

Proteins are bound to the positively charged slide through
electrostatic interavtions to form salt bridges. They are easy to
prepare manually and reproducibility are good.
Aldehyde-coated slides (silylated slides):
Proteins are attached through a base reaction via their side chain
amino groups and the more reactive amino group at their N-termini.
The resulting covalent bonds are stronger than the salt-bridge
interactions of amine-coated slides. The slides are relatively easy to
prepare and give good signal-to-noise ratios.
Epoxy-coated slides:
Proteins are covalently attached through an epoxide ring-opening
reaction primarily via their surface amino, hydroxyl and thiol groups,
giving potentially higher binding affinity than amine-coated slides.
These slides need to be stored in a moisture-free environment prior
to use.
Oriented surfaces for tagged proteins (substrate oriented)
To optimize protein interactions, expressed proteins can be tagged at their
N- or C-termini to enable site-specific attachment. This encourages all
molecules of a protein species to be oriented in a common direction away
from the support surface, and reduces structural distortion of the proteins.
It has recently been reported up to 10-fold better target binding to
oriented capyure antibodies than randomly bound antibodies.
Streptavidin-coated slides:
The biotin-avidin reaction is one of the strongest and most stable
non-covalent interactions knwn. Proteins are tagged by biotin and
the glass surface coated with streptavidin (a non-glycosylated
avidin). The directed orientation of biotin tagged proteins on
straptavidin-coated slides does appear to be a good choice for
maintaining protein functionality.
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BIM2Met Exam December 17th 2009

c.

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What is an enzyme-linked immunosorbant assay (ELISA)?

Enzyme-linked immunosorbant assay (ELISA) is a biochemical technique used mainly in

immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has
been used as a diagnostic tool in medicine.
There are different types of ELISA assays:
-

"Indirect" ELISA
Sandwich ELISA
Competitive ELISA
Reverse ELISA

Performing an ELISA involves at least one antibody with specificity for a particular antigen.
The sample with an unknown amount of antigen is immobilized on a solid support (usually a
polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or
specifically (via capture by another antibody specific to the same antigen, in a "sandwich"
ELISA).
After the antigen is immobilized the detection antibody is added, forming a complex with the
antigen. The detection antibody can be covalently linked to an enzyme, or can itself be
detected by a secondary antibody which is linked to an enzyme through bioconjugation.
Between each step the plate is typically washed with a mild detergent solution to remove any
proteins or antibodies that are not specifically bound. After the final wash step the plate is
developed by adding an enzymatic substrate to produce a visible signal, which indicates the
quantity of antigen in the sample.

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BIM2Met Exam December 17th 2009

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10.
Consider the deionized water laminar flow in a Microchannel
with the constant circular cross-section, as shown in Figure 1. The
length of the channel-segment is 1000 m. Suppose the flow is the
steady flow; the body force inside could be neglected; and the flow
velocity at any cross-section here is uniform. However, the flow
velocity along the X axis direction, is changing with the coordinates,
the relative equation is U(x)= - x2+1. (9,09%)
Question: According to the Navier-Stokes Equations, calculate the
total pressure drop of the fluid through the Microchannel.
Some parameters may be needed for the aforementioned question:
The inlet velocity, uo, is 0.3 m/s;
The density of the water, = 1103 kg/m3;
The kinematic viscosity of water =1.00610-6 m2/s.

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BIM2Met Exam December 17th 2009

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Figure 1

11.

Within a micro-concentrating unit, the deionized water from

the upper layer AB channel laminarly flows down to the bottom layer
CD channel, through a hole BC channel. A cross-section view of
these 3D micro-channels is shown in Figure 2. (9,09%)

Figure 2
The geometric dimensions of these rectangular microchannels are listed as
below,
Section AB [WAB (Width) HAB (Depth) LAB (Length) ]: 300 m 100
m 3200 m.
Section CD [WCD (Width) HCD (Depth) LCD (Length) ]: 300 m 100
m 4000 m.

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The inlet velocity, uA, is 0.4m/s; and the local pressure at inlet (crosssection A-A'), PA, is
120kPa. The height difference between the center line of channel AB and
the center line of channel CD is 400 m. The outlet velocity at D-D', uD is
0.2m/s. The local pressure at outlet (cross-section D-D'), PD, is 90kPa.
a. Determine the hydraulic diameter of microchannel AB, and calculate
the Reynolds number at the inlet; (3,03%)
b. Evaluate the pressure loss due to walls friction in micro-channel AB;
(3,03%)
c. When the water flows down through hole (BC channel), there are two
bends at B-B' and C-C', the real pressure drop PAD does not simply
equal to the value (PA-PD), but the Bernoullis equation based on
energy approach still works. Determine the total pressure drop PAD
due to the friction of the wall.
Some parameters may be needed for the aforementioned
three questions:
The density of the water, = 1103 kg/m3;
The kinematic viscosity of water =1.00610-6 m2/s. (3,03%)

10.
The N-S equation is given as following:

u
0
For the steady flow, t
.

The body force inside could be neglected, Fx 0

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BIM2Met Exam December 17th 2009

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The flow velocity at any cross-section here is uniform, then

u u 2 u 2 u

0
y z 2 y 2 z

So the 1-D N-S equation could be simplified as:

2u
u
P
u
2
x
x
x

2
Because, u ( x) x 1 .

The N-S equation will be,

2u
u
P
( x 1)
2

x
x

x
P
( x 2 1)( 2 x)
2
x
P

(2 x 2 x 3 ) 2
x

0.001
0

2
(2 x 2 x ) 2 dx x x 4
4

997987.5 10 ( Pa)

11.
a
The hydraulic diameter of microchannel AB , (3,03%)

4S
2ab 2 100 10 6 300 10 6
DH

150 10 6 (m) 150m

6
P ab
400 10
uDH 0.4 150 10 6
Re DH

59.64

1.006 10 6
b
The pressure loss due to walls friction in micro-channel AB, (3,03%)

For the square duct,

20

0.001

2 x 0

0.001

BIM2Met Exam December 17th 2009

f

14.2
14.2

Re DH 59.64

The pressure loss due to walls friction in micro-channel AB,

PAB f

4L
DH

1
2
U
2

14.2 4 3200 10 6 1
3
2

10 0.4
6
59.64
150 10
2

1.625 10 3 ( Pa)

c
According to the Bernoullis equation, (3,03%)

U A2
U D2
gZ A PA
gZ D PD PAD
2
2

PAD (U A2 U D2 ) g ( Z A Z D ) ( PA PD )
2
3
10

(0.4 2 0.2 2 ) 10 3 10 400 10 6 30 10 3

2
30.064 10 3 ( Pa)

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