BME3103

Bio-sensors and Bio-devices

Spring 2020

This material was prepared by Dr. Raymond H. W. Lam and Dr. Cecil Chen.
Nucleic acids
• Nucleic acid sequence represents unique signature for living organism
including mammals, microorganisms, and virus.
– Deoxyribonucleic acid (DNA)
– Ribonucleic acid (RNA)
– MicroRNA
• It is of particular interests because it often serves as the biomarkers
associated with diseases or pathogens.
• For example, emerging evidences showed that there are seven signature
mRNAs in saliva elevated due to oral cancer. MicroRNAs, small (~22 nt)
regulatory RNAs present in blood stream due to dysfunction of cancer, was
recently found its promise as tissue-based markers for cancer classification
and prognostication.
• In addition, for analysis of pathogenicity, chromosomal markers with
species-specific sequences can be used to differentiate pathogenic strains.
 Avian influenza
• H7N9 virus is in bird markets as human cases rise rapidly.
• In early 2014, the inefficient pathogen detection had forced the
government to have no choice but culled about 20,000 poultry after a
sample imported from mainland China was detected positive for H7N9.
• This policy affected many Hong Kong citizens because it halted poultry
sales right before the eve of the Chinese New Year.
• H7N9 is a RNA virus. Currently, PCR is the major tool to evaluate and
diagnose the infection.

http://www.virology.ws/ ChinaFotoPress via Getty Images

RNA biomarker
• DNA transcription is
essential for gene
expression, which
provides important RNA
biomarkers for disease or
pathogen identification.
• However, the ‘larger’
RNAs are mostly used
inside cells. Cell lysis
(break down of cell
membrane) is required,
which complicates the
experimental procedure.

pixgood.com
Circulating microRNAs
• MicroRNA, small (~22 nt) regulatory RNAs present in blood stream due to
dysfunction of cancer, was recently found its promise as tissue-based
markers for cancer classification and prognostication
• Compared with many type of biomarkers for disease diagnosis, microRNA is
particular interesting because it circulates in blood stream with remarkable
stability that protects them from endogenous RNase activity. Moreover,
microRNA is released by tumor and directly
entering in blood serum, so it eases the need of biopsy collection for
extracting nucleic acid based biomarker.
Thus, an affective detection for microRNA
offers unprecedented approach to identify
and evaluate the risk of cancer at early stage, and has been shown with
clinical potential for many cancers such as
non-small cell lung cancer, prostate cancer,
breast cancer, and gastric cancer

nfo.agscientific.com
Lecture
Genetic Hybridization
DNA basepairs
• A nucleic acid composed of two polynucleotide strands wound
around a central axis to form a double helix
• The molecule is double-stranded and composed of two strands
in an antiparallel and complementary arrangement
A DNA strand

The other DNA strand

Nucleotides The subunits of nucleic acids; composed of a

phosphate, a sugar, and a nitrogen-containing base. The
fundamental structural unit of the nucleic acid group of organic
macromolecules.
DNA basepairs

Note that ‘A’ binds to ‘T’, and ‘C’ binds to ‘G’. This affinity is very specific.
Phosphodiester bond formation
5' end to 3' end, dehydration
process: bio.miami.edu

As long as we can provide one type of the nucleotides in each time of the
repeated condensation reactions, we can designate a DNA single-strand in
any desired sequence.
 Nearest-neighbor ΔG°37
Hybridization free energy sequence (5'-3'/3'- (kJ/mol)
5’)
• Hybridization is a phenomenon in which AA/TT -4.26
single-stranded DNA or RNA molecules
anneal to complementary DNA or RNA. AT/TA -3.67
• The free energy can describe much strong TA/AT -2.5
the binding between DNA/RNA strands, or CA/GT -6.12
how much energy is required to ‘melt’ the GT/CA -6.09
DNA/RNA molecules.
CT/GA -5.4
• The required free energy can be calculated
by the nearest-neighboring method GA/CT -5.51
– This method treats DNA as a string of CG/GC -9.07
interactions between 'neighboring' GC/CG -9.36
base pairs.
GG/CC -7.66
– This energy difference provide the
affinity of DNA hybridization and how Terminal A-T base 4.31
stable the hybridized double helix is, i.e. pair
more negative, more stable Terminal G-C base 4.05
pair
 Nearest-neighbor ΔG°37
Example sequence (5'-3'/3'- (kJ/mol)
• Calculate the free energy of hybridization 5’)
5' C-G-T-T-G-A 3' AA/TT -4.26
3' G-C-A-A-C-T 5' AT/TA -3.67
TA/AT -2.5
ΔG°37 (predicted) = ΔG°37 (CG
initiation) + ΔG°37 (CG/GC) + CA/GT -6.12
ΔG°37(GT/CA) + ΔG°37 (TT/AA) + GT/CA -6.09
ΔG°37 (TG/AC) + ΔG°37 (GA/CT) + CT/GA -5.4
ΔG°37 (AT initiation)
GA/CT -5.51
= 4.05 + (-9.07) + (-6.09) + (-4.26) + CG/GC -9.07
(-6.12) + (-5.51) + (4.31) GC/CG -9.36
GG/CC -7.66
= -22.69 kJ/mol
Terminal A-T base 4.31
pair
Terminal G-C base 4.05
pair
Hybridization free energy (RNA/RNA)
• The DNA/RNA free energy is not provided here as there are too many
cases.
• Unit conversion: 1 cal = 4.1868 J
Hybridization thermodynamics

• In a simplistic view the formation of an oligonucleotide duplex via

hybridization can be viewed as a two state model. If enough time is
allowed for the reaction to occur the forward and the reverse reaction
rates will be equal and equilibrium is achieved. The reaction equilibrium
can be described as follows:

• where A and B imply strands A and B in the random coil state and AB
implies the ordered AB duplex state.
• The equilibrium constant, K, for the reaction is given by the law of mass
action:
Hybridization thermodynamics
• According to the Van’t Hoff equation, the relation between free energy,
ΔG, and K is ΔG°(37) = -RTln K, where R = 8.314 J / (mol·K) is the ideal
gas law constant, and T is the temperature of the reaction deviated from
37 °C . This gives, for the nucleic acid system,

• The melting temperature, Tm, occurs when half of the double-stranded

nucleic acid has dissociated.
• If no additional nucleic acids are present, then [A], [B], and [AB] will be
equal, and equal to half the initial concentration of double-stranded
nucleic acid, [AB]initial (unit: M). This gives an expression for the melting
point of a nucleic acid duplex of

+ 37 °C
Lecture
Microarrays
 experimental design
DNA microarrays
• DNA microarray is an array of DNA spots
that contains pmol of a specific DNA probes. statistical processing and analysis
• DNA probes are oligonucleotides that has
complementary sequence to different
mRNAs of detection. With the labeled target condition 1 condition 2
(with fluorescence tag), the hybridization
allows the inspection of large number of
genes simultaneously. condition 3
• Hybridization temperature is a key factor.
• DNA microarray enables large scale
screening.

genes
conditions

http://en.wikipedia.org/wiki/DNA_microarray
DNA microarray for mRNA identification
• mRNAs have to be reverse transcribed to cDNA, followed by attachment of
specific dyes before applying to DNA microarray

http://en.wikipedia.org/
 DNA spotting
 DNA spotting usually uses
multiple pins
 DNA in microtiter plate
 DNA usually PCR amplified
 Oligonucleotides can also be
spotted

Benfey and Protopapas, "Genomics" © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper
Saddle River, New Jersey 07458

For slide-based microarrays, the probe DNA is affixed directly to the surface of a
glass microscope slide. The probe DNA can range from a medium-length
oligonucleotide (usually 20–25 bases in length) to an entire cDNA clone or larger.
Oligonucleotide arrays have become more common and can be obtained from
several different commercial vendors.
 General usage
In general, slide-based arrays are used to make a direct comparison between
two different RNA samples. These can be a tissue sample vs. a reference, mutant
vs. wild type, treated vs. control, etc. The microarray provides a readout of the
relative differences in abundance of the RNAs present in each sample.

cell type A
make
Wild type extract hybridize to
labeled
mRNA microarray
cDNA

more in “A”
cell type B more in “B”
Mutant equal in A & B
cDNA microarrays: key points
 Hybridize two samples/chip (i.e., direct comparison of
samples)
 Non-standardized production can affect reproducibility
 Longer sequences can have cross-hybridization with other
genes
 Don’t necessarily need to know all genes in genome
 Only qualitative measures
 There’s no way that all of your microarray data can be
validated.
It’s strongly recommended that any key findings be
verified by independent means.
Northern blots and quantitative RT-PCR are the typical ways of doing
this; real-time, quantitative RT-PCR is generally the method of choice.
Lecture
Hairpin
Hairpin
• Hybridization free energy of DNA has inspired many types of sensing
mechanism.
• Hairpin formation:
– Hairpin is a formation that two regions of the single strand DNA or
RNA has complementary sequences and form double helix partially.
– Stem: double helix region
– Loop: unpaired sequence left as single strand loop
– Temperature can determine the
opening/closing state of a hairpin
molecule.
• Both end units can be modified to be with
a quencher and a fluorophore (reporter).
 Hairpin-based molecular beacon
• A quencher can deactivate the fluorescence via efficient energy
transmission when their separation distance is short.

• The hairpin molecule can be

designed with a free energy
level for binding to a target
DNA ‘higher’ (more negative)
than that for its self strand-
looping.
• The operation temperature
can be set slightly below the
temperature for the strand-
looping, for a better sensitivity.

Tyagi, Nature Biotechnology, 1998, 16, page 49

Lecture
Polymerase Chain Reaction (PCR)
 Polymerase chain reaction (PCR)
Devised by Kary Mullis in 1985 – produces enormous numbers of copies of DNAs
Cycles of temperature steps
• Template – Completely denature at 94 °C.
• Primers – Anneal to denatured template at lower temperature (just below the
melting temperature).
• Polymerase – Carry out templated DNA synthesis starting with the primers at
an
• Product – Serves as template for the next round of DNA synthesis

en.wikipedia.org
Miniaturized PCR
• PCR required thermal cycler to complete the amplification cycles, which
make the miniaturization very difficult.
• Many microfabricated PCR devices actually requires additional thermal
controller.
• We have learnt the piezoresistive temperature sensing.

(silicon containing microchannels

flowing with liquid samples)
Heater: discovery of Joule heating

• First experimentally proven by

James Prescott Joule in 1841
– Hence the name: ‘Joule Heating’
• What did he do?
– Passed a current through a wire
of fixed length,
– Immersed in a bath of water of
fixed volume/mass
– Observed the temperature in
the water varried with current,
length of wire, and time.
Heater: thermal energy generation

where:
Q  I 2  R t
Q = amount of Energy produced (in the form of heat)
UNIT: joule; 1 joule = kg m /s2
I = Current UNIT: ampere
R = Resistance UNIT: ohm
t = Time UNIT: second
Heater: specific heat of capacity
Q
C
where:
M T
C = Specific heat of capacity UNIT: Joule / kg / K
M = Mass UNIT: kg
ΔT = Temperature change UNIT: K
• Ideally, the temperature of the material rises continuously while
we apply electrical current.
• However, there are always heat losses!
– Radiation: dQ/dt = - φrad. ΔT4
– Convection: dQ/dt = - φconv. ΔT
– Conduction: dQ/dt = - φcond. ΔT = - Atotal ĸ ΔT
where Atotal = (Total) area attaching with nearby bodies UNIT: Joule / kg / K
ĸ = Thermal conductivity UNIT: kg
ΔT = Temperature difference relative to the nearby material UNIT: K
Heater: Joule heating
• Heat losses increase with the level of the material
temperature higher than its environment.
• In the steady state, we can always find:
dQ/dt = CM× dT/dt =Heat generation + Heat loss = 0
CM× dT/dt = I2R - φcond. ΔT - φconv. ΔT - φrad. ΔT4 = 0

• When ΔT is small enough such that radiation effect is

negligible:
I2R = (φcond. + φconv.) ΔT
• Therefore, the Joule Heating will generate an increment of the
bulk material temperature. In this course, we may consider
only the conduction effect; and hence:

I2R = Atotal ĸ ΔT
Lecture
Sandwich Hybridization
 Sandwich hybridization based
fluorescence detection
• Multiplexed Hybridization Detection with Multicolor Co-localization of
Quantum Dot Nanoprobes
• Target DNA links quantum dots such that the co-localization blends the
color.

When target DNA is present, the green

and red particles co-localized at the same
spot, yielding a mixed yellow color
Yi-Ping Ho, et al, Nano letter, 2005
 Sandwich hybridization based
magnetophoretic effect
• Magnetic microspheres (MMPs) were designed to bind with color particles
when target (DNA) molecules are present. Thus, with an externally applied
magnetic field, the MMPs along with the liked color particles would be
attracted to the sidewall, leading to a change of solution color due to the
reduction of color particles suspended in the solution.

Chem. Commun., 2012, 48, 3164–3166

Sandwich hybridization based
magnetophoretic effect A Magneticm
i cropar
l ticl s (M
MP s)
Polystyrene micropartices (PMP s )
P1rpoB
P2rpoB
Auxiliary blocker pagA
Magnet
TrpoB
Two types of probes, P1 and P2,
w/ target TrpoB w/o target TrpoB
were designed to hybridize in
juxtaposition with a target DNA. As
such, using magnetic microspheres
(MMPs) attached with P1 and
polystyrene microspheres (PMPs)
attached with P2, the present
target DNA leads to the
sandwiched structure, MMPs-
target -PMPs. When an external
magnet is attached, the
sandwiched structure can be
B
attracted to the sidewall, leaving
the solution color changed from
opaque to transparent.
Exercise Nearest-neighbor ΔG°37
M1 (5’-CCCTATCAC -3’) is a microRNA- sequence (5'-3'/3'- (kJ/mol)
transcription DNA sequence representing a 5’)
type of cancer. You want to design a pair of AA/TT -4.26
probes via sandwich hybridization to
accomplish a colorimetric detection for M1. AT/TA -3.67
Thus, the probes P1 and P2 need to be able to TA/AT -2.5
hybridize with M1 in juxtapositions. You have CA/GT -6.12
come out with these two designs:
GT/CA -6.09
• In design 1, you design P1 as a probe with
the complementary sequence for the first 5 CT/GA -5.4
bases of M1, while P2 is with the GA/CT -5.51
complementary sequence for the following CG/GC -9.07
4 bases. What are P1 and P2? (from 5’ to 3’)
GC/CG -9.36
• In design 2, you design P1 as a probe with
the complementary sequence for the first 4 GG/CC -7.66
bases of M1, while P2 is with the Terminal A-T base 4.31
complementary sequence for the following pair
5 bases. What are P1 and P2? (from 5’ to 3’) Terminal G-C base 4.05
• Which design yields a more stable pair
hybridization? (calculate the free energy) (-24.22 kJ/mole; -25.39 kJ/mole)
Comparison
– Polymerase chain reaction (PCR)
• Gold standard for nucleic acid detection
• Needs primer, thermal cycler, and complicate protocols
• The sensitivity is boosted by the powerful chain reaction (aM level)
– Hybridization based detection
• DNA microarray
– Rapid and simple operation.
– Fluorescence reader is required.
• Hairpin based detection
– The noise is significantly reduced
– Combined with fluorophore and quencher, the fluorescence
signal can be shown without pre-staining
• Sandwich hybridization based detection
– Varied approaches available
– Sensitivity can be optimized to pM level
Lecture
Microcantilever DNA-Sensor
Working principle
• From the view of chemistry, surface-stress is the reversible work per unit area
required to elastically strain a surface and to increase the surface area.
• For typical micro-cantilever structures, the contribution from the surface-
strain can be neglected and the surface energy change directly equates to the
variation in surface stress. Thus, the surface stress can be expressed as

• where ΔσS is the surface stress, and ΔG is the effective surface energy. For an
extreme case that the amount of DNA in the liquid sample being tested is high
enough, ΔG can have an expression of
ΔG = ξ × ρS1/2 × ΔG°37
where ξ is a correction factor, ρS is the surface coverage of DNA molecules
over the upper surface of the micro-cantilever beam, i.e. average number of
DNA molecule per unit area (unit: mole/m2).
Fabrication