Sk microfluidics and lab on-a-chip-ch3

Microfluidics
and Lab-on-a-Chip
for biomedical
applications
Chapter 3 : Molecular biology
and selected analytical tools.

By Stanislas
CNRS
Université de Lyon, FRANCE
Stansan International Group

CONTEN
T

Chapter 1: Introduction.

Chapter 2 : Basic principles of Microfluidics.
Chapter 3 : Basis of molecular biology and analytical tools.
Chapter 4 : Micromanufacturing.
Chapter 5 : Lab-on-a-Chip & applications.
Chapter 6 : Cancer diagnostics and monitoring.

There is a wide range of applications of Lab-on-aChip in the field of chemistry, biochemistry,
environmental control, bio defense….
Our objective today is to speak about applications in
the field of medical diagnostics and monitoring.
We will choose the field of the cancer.
Thus, a little bit of (molecular) biology….

Biological cell
The cell is the basic unit of life. It was discovered by Robert Hooke (1665)
and is the functional unit of all known living organisms. It is classified as a
living thing, and is often called the building block of life.
There are two types of cells: Prokaryotic cells are usually independent,
while Eukaryotic cells are often found in multicellular organisms.
Some organisms, such as most bacteria consist of a single cell.
Other organisms, such as humans, are multicellular. Humans have about
100 trillion or 1014 cells ; a typical cell size is 10 µm; a typical cell mass is 1
nanogram. The largest cells are about 135 µm, the smallest, can be some 4
µm. The largest known cells are unfertilised ostrich egg cells which weigh
3.3 pounds.
The word cell comes from the Latin cellula, meaning, a small room.
The human body contains many different organs, such as the heart, lung,
and kidney... Cells also have a set of "little organs," called organelles.

Typical Eukaryotic Cell
1) Nucleolus
2) Nucleus
3) Ribosome
4) Vesicle
5) Rough
endoplasmic
reticulum (ER)
6) Golgi apparatus
7) Microtubule
8) Smooth ER
9) Mitochondria
10) Vacuole
11) Cytoplasm
12) Lysosome
13) Centrioles

DN
A the genetic instructions used
DNA is a nucleic acid that contains
in the development and functioning of all known living organisms
and some viruses. The main role of DNA molecules is the longterm storage of information.
Chemically, DNA consists of two long polymers of simple units
called nucleotides, with backbones made of sugars and
phosphate groups joined by ester bonds. These two strands run
in opposite directions to each other and are therefore antiparallel.
Attached to each sugar is one of four types of molecules called
bases. It is the sequence of these four bases along the backbone
that encodes information. This information is read using the
genetic code, which specifies the sequence of the amino acids
within proteins.

Chromosome
A chromosome is an organized structure of DNA and protein that is
found in cells. It is a single piece of coiled DNA containing many
genes, regulatory elements and other nucleotide sequences.
Chromosomes also contain DNA-bound proteins, which serve to
package the DNA and control its functions.
Scheme of a Chromosome :
(1) Chromatid. One of the two
identical parts of the chromosome
after S phase.
(2) Centromere. The point where the
two chromatids touch, and where
the microtubules attach.
(3) Short arm
(4) Long arm. In accordance with
the display rules in Cytogenetics,
the short arm is on top.

A gene is a unit of heredity in a living
organism. It is normally a stretch of
DNA that codes for a type of protein
or for an RNA chain that has a
function in the organism.
All proteins and functional RNA
chains are specified by genes. All
living things depend on genes.
Genes hold the information to build
and maintain an organism's cells
and pass genetic traits to offspring.
A modern working definition of a
gene is "a locatable region of
genomic sequence, corresponding
to a unit of inheritance, which is
associated with regulatory regions,
transcribed regions, and or other
functional sequence regions ".

GENE
S

Telomere
A telomere is a region of repetitive DNA at the end of a chromosome, which
protects the end of the chromosome from deterioration.
If cells divided without telomeres, they would lose the ends of their
chromosomes, and the necessary information they contain.
The telomeres protect the chromosomes and are consumed during cell
division due to an enzyme, the telomerase reverse transcriptase.
The telomere shortening mechanism normally limits cells to a fixed number
of divisions, and this is responsible for aging on the cellular level.
Most cancers are "immortal" cells which have ways of evading this
programmed destruction. Activation of the Alternative Lengthening of
Telomeres (ALT) pathway which involves p53 and pRb may lead to the arrest
of cell proliferation.
Elizabeth Blackburn, Carol Greider, and Jack Szostak were awarded the
2009 Nobel Prize in Physiology or Medicine for the discovery of how
chromosomes are protected by telomeres and the enzyme telomerase.

Telomere

Three-dimensional
molecular structure
of a telomere.

Human chromosomes (grey)
capped by telomeres (white).
Telomeric repeat (human) :
TTAGGG.....

Genome
Projects

The Human Genome sequence is
complete
approximately 3.2 billion base pairs

Genomics Technologies
• Next-Generation DNA sequencing
• Automated annotation of sequences
• DNA microarrays
– gene expression (measure RNA levels)
– single nucleotide polymorphisms (SNPs)
– ChIP-chip, genomic tiling, etc

• Proteomics (mass-spec)
• Protein chips
• Protein-protein interactions

Single Molecular DNA manipulation
At Humboldt University of Berlin the new approach was developed
for DNA manipulation of single macromolecules on solid substrate.
No one other known approach allows that.

The picture
demonstrates
ability of
assembling of
the word
"SCIENCE"
on atomically
flat substrate
from DNA
molecules

Cycle cellulaire
Phase M : mitose,
séparation d’une
cellule mère en 2
cellules filles.
Phase G1: phase de
croissance et contrôle
avant réplication.
Phase S : synthèse de
l’ADN, réplication des
chromosomes.
Phase G2 : Contrôle de
la qualité de réplication
avant division.

Proteins are organic compounds
made of amino acids arranged in a
linear chain and folded into a
globular form.

Proteins

The amino acids in a polymer are
joined together by the peptide bonds
between the carboxyl and amino
groups of adjacent amino acid
residues.
The sequence of amino acids in a
protein is defined by the sequence of
a gene, which is encoded in the
genetic code. In general, the genetic
code specifies 20 standard amino
acids
Proteins are a primary constituent of
living things and one of the chief
classes of molecules studied in
biochemistry.

3D structure of myoglobin
showing coloured alpha helices.
This protein was the first to have
its structure solved by X-ray
crystallography.

Types of Microarrys (BioChips)

Oligonucleotide Array: a "DNA Chip"
A collection of
microscopic DNA
spots attached to a
solid surface forming
an array; used to
measure the
expression levels of
large numbers of
genes simultaneously.
An oligonucleotide array consists of a series of short (typically
20~30 bases) single-stranded DNA sequences (oligonucleotides,
or "oligos") attached to a glass chip about the size of a
microscope cover slip. In the arrangement shown here, each
adjacent oligo differs from its neighbor only at the last base. In
the example, the first four oligos in block 1 begin with
GAGCCAAGCTG and end with A, G, C, or T, respectively.

Fluorescence Detection DNA Chip

Exemple of a DNA Chip

Called the "GreeneChip," this device consists of a glass slide
onto which are attached nearly 30,000 pieces of genetic material
taken from thousands of different viruses, bacteria, fungi and
parasites. When human fluid and tissue samples are applied to
the chip, these probes will stick to any closely related genetic
material in the samples. This allows the rapid and specific
identification of any pathogens therein-even those related to but
genetically distinct from the ones represented on the chip.

Example of a System for Biochips Fabrication

SonoPlot provides innovative fluid dispensers and chemical surface
treatments for microelectronics and the life sciences. The
Microplotter line of dispensers can print microcircuitry on a desktop
for research and development or deposit high-density grids of
biomolecules such as DNA or proteins for fabrication of microarrays.
A wide variety of materials can be treated with the SonoCoat surface
modification process, producing ideal surfaces for adhering
molecules in a microarray.

DNA Chip Reader
A DNA chip reader is used to analyze colossal amounts of
genetic information. On the DNA chip, hybridization is performed
on the DNA labeled by a fluorescent dye. The DNA chip is then
scanned by laser beam and by measuring the fluorescent
intensity of the hybridized DNA spot, the genetic information is
acquired from among the targeted DNA. (Hybridization is process
to link 2 chains of DNA each having a complementary base.)

In the 1990s, DNA arrays provided the means to
analyze patterns of gene expression in a living cell.
DNA microarrays often consist of glass slides with
spots of attached DNA fragments. The DNA
fragments act as probes for specific sequences in a
sample.

In the early 1990s, Stephen Fodor and his team
developed a technique to produce miniature arrays of
biological molecules. Their work led to the first DNA
chip, and became the basis of techniques for largescale genomic studies. The company Affymetrix was
spun off in the early 1990s to focus on DNA
GeneChips®.

Examples of DNA Microarrys
(BioChips)

Application of BioChips
DNA chips have been extensively used for research
applications in academia and in industrial laboratories.
A big progress in bioinformatics is still needed in order to
be able to explore in a reliable way the DNA data in the field
of medicine.
It is expected that many DNA chip for medical diagnostics
and monitoring will be developed in the next few years.
One of challenges for the Biochip industry is also the
integration of microarrays with microfluidics, in order to
achieve Microsystems, which include the extraction of the
genetic material (e.g. DNA or RNA from white blood cells or
from rare circulating tumor cells), purification and
amplification of the extracted material and, finally, the
analysis of this material with DNA chips.

Protein biochip
and other microarray technologies
Microarrays are not limited to DNA analysis; protein microarrays,
antibody microarray, chemical compound microarray can also be
produced using biochips. Randox Laboratories Ltd. launched
Evidence, the first protein Biochip Array Technology analyzer in
2003. In protein Biochip Array Technology, the biochip replaces the
ELISA plate or cuvette as the reaction platform. The biochip is used
to simultaneously analyze a panel of related tests in a single
sample, producing a patient profile. The patient profile can be used
in disease screening, diagnosis, monitoring disease progression or
monitoring treatment.

Antibody Microarrays for Biomarker Discovery
Antibodies have the potential to identify biomarkers that are novel,
unusually spliced or modified or are present in a differential
concentration in cancer serum samples with respect to normal
samples. They combine the advantages of an unbiased discovery
approach (as is the case for Mass Spectrometry techniques) with
the sensitivity of an immunoassay for detecting low abundance
serum proteins (such as ELISA). As an added advantage, the
antibodies can be used for discovery, purification, identification and
characterization of the novel biomarker molecules.

Schematic description
of protein immobilization

Antibody microarray
An antibody microarray is a
specific form of protein
microarrays, a collection of
capture
antibodies
are
spotted and fixed on a solid
surface, such as glass,
plastic and silicon chip for
the purpose of detecting
antigens.
Antibody
microarray is often used for
detecting
protein
expressions from cell lysates
in general research and
special
biomarkers
from
serum or urine for diagnostic
applications.

ELISA - immunoassay
Enzyme-linked immunosorbent assay (ELISA), also known as an
enzyme immunoassay (EIA), 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 and plant
pathology, as well as a quality control check in various industries.
In ELISA, an antigen is affixed to a surface, and then a specific
antibody is applied over the surface so that it can bind to the antigen.
This antibody is linked to an enzyme, and in the final step a substance
is added that the enzyme can convert to some detectable signal. Thus
in the case of fluorescence ELISA, when light of the appropriate
wavelength is shone upon the sample, any antigen/antibody
complexes will fluoresce so that the amount of antigen in the sample
can be inferred through the magnitude of the fluorescence.

ELISA - immunoassay

i) Test serum is incubated with antigen immobilised on a 96-well
plate or microscope slide
ii) Secondary antibodies labelled with an enzyme are added
Iii) After washing, any bound secondary antibodies can be
detected using the marker. The label used is an enzyme which
induces a colour change when the substrate is added.

Capillary Electrophoresis
(CE)

Schematic representation of the arrangement
of the main components of a
typical Capillary Electrophoresis (CE) instrument.

Capillary Electrophoresis
(CE)

Capilary Electrophoresis - Design Consideration

Gel Electrophoresis
Gel Electrophoresis is a technique used to separate
macromolecules - especially proteins and nucleic acids - that
differ in size, charge or conformation. As such, it is one of the
most widely-used technique in Molecular Biology.
When charged molecules are placed in
an electric field, they migrate toward either
the positive or negative pole according to
their charge. Proteins, which can have either
a net positive or net negative charge Nucleic
acids have a consistent negative charge
and migrate toward the anode.
Proteins and nucleic acids are electrophoresed within a matrix or
"gel". Most commonly, the gel is cast in the shape of a thin slab..
The gel is immersed within an electrophoresis buffer.
The gel itself is composed of either agarose or polyacrylamide,
each of which have attributes suitable to particular tasks.

Agarose Concentration
Agarose Concentration: By using
gels with different concentrations of
agarose, one can resolve different
sizes of DNA fragments. Higher
concentrations of agarose facilite
separation of small DNAs, while low
agarose concentrations allow
resolution of larger DNAs.
The image to the right shows
migration of a set of DNA fragments
in three concentrations of agarose,
all of which were in the same gel
tray and electrophoresed at the
same voltage and for identical times.
Notice how the larger fragments are
much better resolved in the 0.7%
gel, while the small fragments
separated best in 1.5% agarose. The
1000 bp fragment is indicated in
each lane.

Gel Electrophoresis Apparatus

(e.g. forensic
investigations)

Gel Electrophoresis
In the early days of DNA manipulation, DNA fragments were
laboriously separated by gravity. In the 1970s, the powerful
tool of DNA gel electrophoresis was developed. This
process uses electricity to separate DNA fragments by size
as they migrate through a gel matrix.

First commercial system :
Agilent 2100 Bioanalyzer
Agilent Technologies,
Waldbronn, Germany
Caliper Technologies,
Mountain View, CA

Lab-on-a-Chip Products
Cutting edge Lab-on-a-Chip Products
Agilent Technologies is the leader in
commercial microfluidic Lab-on-a-Chip
technology.
This technology utilizes a network of
channels and wells that are etched onto
glass or polymer chips to build mini-labs.
Pressure or electrokinetic forces move pico-liter volumes in finely
controlled manner through the channels.
Lab-on-a-Chip enables sample handling, mixing, dilution,
electrophoresis and chromatographic separation, staining and detection
on single integrated systems.
The main advantages of Lab-on-a-Chip are ease-of-use, speed of
analysis, low sample and reagent consumption and high reproducibility
due to standardization and automation.

Applications of Electrophoresis

Chromatography
Family of laboratory techniques for the separation of mixtures. It
involves passing a mixture dissolved in a "mobile phase" through a
“stationary phase”, which separates the analyte to be measured from
other molecules in the mixture and allows it to be isolated
Preparative chromatography seeks to separate the components of a
mixture for further use (and is thus a form of purification).
Analytical chromatography operates with smaller amounts of material
and seeks to measure the relative proportions of analytes in a
mixture.
Column chromatography is a
separation technique in which the
stationary bed is within a tube. The
particles of the solid stationary
phase or the support coated with a
liquid stationary phase may fill the
whole inside volume of the tube
(packed column).

Schematic Diagram of Liquid
Chromatography

echniques by physical state of mobile phase
Affinity chromatography
It is often used in biochemistry in the purification of proteins
bound to tags. These fusion proteins are labelled with
compounds such as biotin or antigens, which bind to the
stationary phase specifically. After purification, some of these
tags are usually removed and the pure protein is obtained.

Size exclusion chromatography
Size exclusion chromatography separates molecules according to
their size (or more accurately according to their hydrodynamic
diameter or hydrodynamic volume). Smaller molecules are able to
enter the pores of the media and, therefore, take longer to elute,
whereas larger molecules are excluded from the pores and elute
faster.

Schematic of pore vs. analyte size

Chromatogram of Orange
Juice Compounds

High Performance Liquid
Chromatography

Electrophoresis vs. Chromatography

Polymerase Chain Reaction (PCR)

How PCR is done ?
Liquid-Phase PCR Reactors :
Three different phases: denaturing, hybridization, extension
(1)Denaturing : A heating temperature above 90°- 95°C breaks
adouble-stranded DNA molecule into two complementary singlestranded DNA molecules.
(2) Hybridization : The single-stranded DNA molecules is cooled at a
lower temperature 50°C - 60°C and seek their complementary
strands to create double-stranded DNA molecules.
(3) Extension : The incomplete double-stranded DNA molecules are
extended with the help of an enzyme called DNA polymerase.
It occurs at a temperature of 70° - 75°C.

Polymerase Chain Reaction
Polymerase chain reaction (PCR) enables researchers to
produce millions of copies of a specific DNA sequence in
approximately two hours. This automated process
bypasses the need to use bacteria for amplifying DNA.

Integrated PCR and detection microsystem

Channel size: 40 μm×90
μm×2.2 m
⇒ The sample is forced to
through three temperature
zones with a constant velocity.
⇒ The cycle time is proportional
to the capillary length.
⇒ The process only depends on
the speed of the fluid flow and
not on the thermal constant of
the system.
⇒ Relatively fast cycling.

Continuous Flow
- PCR Miniaturisation

Continuous Flow PCR - Miniaturisation

Schematic of a modern conventional FACS

icrofabricated fluorescence-activate
cell sorter (µFACS)

Principle of confocal Microscopy

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Sk microfluidics and lab on-a-chip-ch3

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