DNA in Sensing

Sensors
Prepared by;

Serhenk ÇELİK 20519613

Gözde ERGİN 20519698
B.Didem KABAKÇI 20519737
Necdet DALGIÇ 20519627

Materials Science and Technology-III

Chemical Engineering Department of Hacettepe University
21th April 2010
OUTLINE
 DNA Structure
 What is biosensor
 DNA Biosensor
 Types of DNA Biosensor;
 Optical Biosensor
 Optic Fiber
 Evanescent Waves(Surface Plasmon Resonance)
 Gold Nanoparticles

 Quantum Dots
 Electrochemical Biosensor
 Piezoelectric Biosensor
 Advantages&Disadvantages
 New Trends
DNA Structure
• Contains genetic material for all living organisms

• Double helix structure; composed of two

strands held together by hydrogen bonds

• Made of four different nucleotides(bases)

Adenine , Thymine, Guanine, Cytosine

• Unique complementary structure of DNA

between the base pairs
What is a biosensor?

• Biosensors are analytical devices which use biological interactions to

provide either qualitative or quantitative results
There are 4 types biosensor;
• Enzymesensors
• Immunosensors
• Microbial
• DNA
• Biosensors convert a biochemical reaction or interaction into an
analytical signal that can be further amplified, processed and
recorded.
DNA Biosensor

 DNA is especially well suited for biosensing applications, base-pairing

interactions between complementary sequences are specific.
Nucleotide bases will re-form hydrogen bonds only with specific bases:
adenine pairs with thymine, and cytosine pairs with guanine.

 The single stranded DNA (ssDNA) is relatively stable, the DNA molecule
will re-form into the double stranded configuration. Re-annealing between
the ssDNAs from different sources is called hybridization.
Working Principle of a DNA Biosensor

 In biosensors for DNA sequence detection, the molecular recognition event

is commonly hybridization of a known probe to an unknown target
sequence. In biosensors, this event typically occurs directly on the surface
of a signal transducer.
Applications
 Diagnostic of;

 Bacterial food contamination

 Genetically modified organisms
 Biological agents.

 Molecular diagnostics;

 Genome sequencing detecting

 Inherited disease
 Human pathogens
 Drug screening
Optical Fibers

• Hybridization event is detected by fluorescence, by measuring the total internal

reflection in the fiber
DNA probe is placed in the end of the fiber
After hybridization, changes in the fluorescence intensity resultant
from association between the DNA duplex and the label is measured

• Fluorescent dyes bind to DNA making them fluorescent and readily detectable
(Ethidium bromide commonly used )
Optical fibers
 By transmitting light for very long distances without signal lost, allow
detection of inaccessible or dangerous samples

 In a fiber-optical array biosensor;

simultaneous detection of multiple nucleotide sequences using
combinations of different fluorescent labels

 poor stability
 interference from environmental light
 high cost of quartz optical fibers for UV light transmission
Evanescent and Acoustic waves

 Evanescent wave biosensors such as surface plasmon resonance (SPR)

Indirectly detect DNA hybridization by measuring variations in the
surface optical parameters (interfacial refractive index)

 Acoustic wave sensors used with a liquid sample detect changes in physical
properties such as mass, viscosity, charge density

 Notable applications in detecting human genetic mutations and genetically

modified organisms
Evanescent and Acoustic waves

Both evanescent and acoustic waves are attractive sensors

 real-time label-free optical detection

 the hybridization reaction can be resolved within a few minutes
 the probe surface can be re-used over 100 times

- these are among the least sensitive DNA biosensors

Gold Nanoparticles as DNA Biosensors
 Colorimetric method.

 High stability, less expensive and complex.

 Built as microparticles/nanoparticles conjugates.

 Color of GNP based biosensor changes when there is a binding between

microparticle and GNP including target DNA.

 Spectrophotometric method is used to determine the the change of

color.
• Layout for DNA colorimetric detection with GNPs and latex microspheres
Quantum Dots as Biosensors
 Colorimetric method.

 A type of nanoparticle, based on fluorescense emission of detection.

 Much brighter and stable, direct proportionality between size of a dot and
color.

 Wide absorption and narrow emission spectrum.(easy to excite&detect)

 When a “target” DNA is present, a bridge-like structure is formed.

Illumination occurs.
 An Example of GNP based Biosensor
• Gold nanoparticle is used as DNA biosensor.

• It detects the specific products of genetically modified parts of the most

transgenic plants and it provides visual detection of them.

• Rapid, simple and reliable method for detection of GMOs.

• Schematic illustration of the principle of the nanoparticle-based DNA biosensor for

visual detection of GMO.
Electrochemical DNA Sensors
 Well suited for DNA analysis, electrochemical reaction give an electronic
signal directly,there is no need expensive signal transduction element.

o Potentiometric

o Amperometric

o Voltametric
 Electrochemistry based sensors offer sensitivity,selectivity and low cost for
the detection of selected DNA sequences or mutated genes associated
with human disease.
 Direct electrochemistry of DNA
 Electrochemistry at polymer-modified electrodes
 Electrochemistry of DNA-specific redox reporters
 Electrochemical amplifications with nanoparticles
 Electrochemical devices based on DNA-mediated change transport
chemistry.
Electrochemical DNA biosensor for the
detection of Hepatitis B virus
 DNA sensors have the potential application in diagnosis of diseases
 An electrochemical DNA biosensor was developed based on the
recognition of target DNA by hybridization detection.

 After covalent immobilization of DNA related to hepatitis B virus on

glassy carbon electrode, electrode was immersed in solution containing
ssDNA to form double strand DNA at the electrode surface.

 Hybridization was detected by using the electrochemical indicator (Cu

complex) where electro activity and strong association with the double
strained DNA lead to voltammetric signal.

 HBV could be quantified with a detection limit of 7.0 × 10−8 M.

Piezoelectric Mass Readout
Piezoelectric DNA Biosensors
Immobilized DNA probe
Target DNA
Form duplex---mass increase

Cantilever sensors
• Chemical vapors at very low
concentrations can be detected based
on the surface stress changes generated
by the interactions between probe and
target molecules on their surfaces

• The magnitude of the surface stress

Decrease in crystal’s resonance frequency change depends on the type of
interaction taking place which includes

• Hydrogen bonding
• Electrostatic,
• van der Waals forces, etc.

The increase in mass that can accompanies hybridization

is detected by the deflection of a laser beam reflected
from the cantilever surface.
Advantages
 Easily synthesized in the laboratory, regenerated for multiple use

 High detection sensitivity and physico-chemical stability relative to other

recognition elements (enzymes, antibodies etc. )

 Earlier diagnosis of infectious diseases

 Rapid detection of trace DNA levels

Disadvantages

• Difficulties in the transition to clinical use

• Lack of affinity and stability of DNA chains in solid surfaces

• Few technology for manufacturing at a competitive cost

• None of DNA biosensor type could fulfill all needs for a given application

• Very low levels of nucleic acids in biological fluids, require previous

amplification
 What is New Trend?
 Development of a method carried out in volume not on surface.

 Using nanotechnology such as quantum dot nanoparticles, nanowires or

nanotubes.
 Development of artificial antibodies such as aptamer or peptide selected by

phage display method.

 Development of Lab-on-a-chip for detection of antigen.
Lab-On-Chip
Aim of the development of Lab-on-a-chip system is
performing all stages on a chip.
Lab-on-a system consists of;
• Microchanells and micropumps
• Sample pretreatment units
• Reservoir for probes, substrates, buffers and others,
• Detectors,
• Sampling more then one analyte or sample at the
same time
REFERENCES

Trends in DNA biosensors (F.R.R. Teles, L.P.

Fonseca)

Nanoparticle-based DNA biosensor for visual

detection of genetically modified organisms (Despina
P. Kalogianni, Theodora Koraki,Theodore K. Christopoulos
Penelope C. Ioannou)
OUTLINE

 Overview

 Electrochemical Biosensor

 Bio-analytical applications of gold

nanoparticles in DNA sensing
DNA Biosensors; hybridization
 DNA is well suited for biosensing applications, base-pairing
interactions between complementary sequences are specific.

Nucleotides will re-form hydrogen bonds only with specific bases:

adenine pairs with thymine, cytosine pairs with guanine.

DNA biosensors relies on specific

hybridization of probe to an unknown target
sequence directly on the surface of a signal
transducer
Electrochemical DNA biosensor for the detection of
Hepatitis B virus
DNA sensors have the potential application in diagnosis of diseases
The detection basically consists of three steps;
1.Probe immobilization
The immobilization of single stranded DNA (probe) related to
hepatitis B virus on electrode

2.Hybridization
Hybridization of ssDNA with their complementary sequences
(target) at electrode surface

3.Electrochemical measurements
Electochemical detection was performed by voltammetry
Hybridization indicator where electroactivity and association with
the immobilized double stranded DNA lead to significantly
enhanced voltammetric signal
The preparation of surface of biosensor &
modification with DNA
Covalent immobilization of DNA on glassy carbon electrode(GCE)
GCE was first oxidized at +0.50 V
Modification of the electrode ;by dropping chemical solutions
ssDNA solution was dropped on the modified GCE surface electrode
was rinsed with water to eliminate the DNA adsorbed

Hybridization on electrode
The modified electrode was immersed in buffer containing target
ssDNA for 1 h at 42 ◦C with shaking to form dsDNA at the electrode

The detection of hybridization

Hybridization was detected by using Cu complex as indicator
Hepatitis B virus could be quantified with a detection limit of
7.0 × 10−8 M
Gold Nanoparticles
• DNA detection based upon Au- NPs with immobilized DNA ‘‘probe’’
(DNA-Au-NP) that recognize complementary DNA targets of interest
• DNA target as a linking molecule to aggregate Au-NPs with
complementary probe allows DNA detection
• The colorimetric hybridization signal is governed by the difference in
optical properties of dispersed and aggregated gold nanoparticles

Mixing two probes with a solution of DNA

target ; formation of a polymeric network
of DNA-Au-NPs with a red-to-purple color
change
Au NPs ;optic properties
The surface plasmon resonance (SPR) of Au-NPs is responsible for their
intense colors

 Monodisperse 13-nm diameter Au-NPs appear red and exhibit a

relatively narrow absorption band centered at 520 nm

 Aggregated Au-NPs appears purple in color, red shift in the surface

plasmon resonance of the particles from 520 to 574 nm

 When the interparticle distance is greater than the average particle

diameter, the suspension appears red
as the interparticle distance decreases to less than the average particle
diameter, the color shifts to blue or purple, depending on the level of
flocculation and the particle concentration.
Gold nanoparticle Hepatitis B virus DNA probes
 Alkanethiol modified ssDNA was bound with Au NPs to form HBV DNA
gene probes, through covalent binding of Au-S
 Modified DNA immobilized on a nylon membrane surface acting as
capturing probes
 HBV DNA was detected visually by hybridization based on highly
sensitive aggregation

 Au nanoparticle gene probes could detect as low as 10-11 M

HBV DNA molecules on a nylon membrane
Evident by transmission electron microscopy, the nanoparticles
assembled into large network aggregates when nanoparticle HBV
DNA gene probes were applied to detect HBV DNA molecules in
liquid .
TEM images of DNA-linked Au nanoparticle
Composite targets HBV DNA extracted from serum of patient were
added to system composed of Au NP supportesd probes.
TEM showed the nanoparticles self- assembled into massive aggregates

a:An assembly formed from Au nanoparticle HBV DNA probes with composite DNA;
b:Control of a, irrelevant DNA was added

ref:Journal of Nanjing Medical University, The detection of HBV DNA with gold nanoparticle gene probes
REFERENCES
 Xue-Mei Li, Heng-Qiang Ju, Nucleic acid biosensor for detection of hepatitis B
virus using copper complex as electrochemical indicator, Analytica Chimica
Acta, 2007
 Dong Xia, Xiaoping Luob, The detection of HBV DNA with gold nanoparticle
gene probes, Journal of Nanjing Medical University,2007
 Chad A. Mirkin, Gold nanoparticle probes for the detection of nucleic acid, 2006
 Chad A. Mirkin, Selective Colorimetric Detection of Polynucleotides Based on
the Distance-Dependent Optical Properties of Gold, 1997
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