dna in sensing sensors

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DNA in Sensing

Sensors PreparePreparedd byby;;

SSerhenkerhenk ÇELKÇELK 2051961320519613Gözde ERGN20519698Gözde ERGN20519698

B.Didem KABAKÇIB.Didem KABAKÇI 2051973720519737Necdet DALGIÇNecdet DALGIÇ 2051962720519627

MaterialsMaterials Science and TechnologyScience and Technology--IIIIIIChemical Engineering Department of Chemical Engineering Department of HacettepeUniverHacettepeUniverssitityy

21t21th April 2010h April 2010



DNAStructure

Contains genetic material for all living organisms

Double helix structure; composed of twostrands held together by hydrogen bonds

Made of four different nucleotides(bases)Adenine , Thymine, Guanine, Cytosine

Unique complementary structure of DNAbetween the base pairs



What is a biosensor?

Biosensors are analytical devices which use biological interactions toprovide either qualitative or quantitative resultsThere are 4 types biosensor;

EnzymesensorsImmunosensors

MicrobialDNA

Biosensors convert a biochemical reaction or interaction into ananalytical signal that can be further amplified, processed andrecorded.



DNA Biosensor

y DNA is especially well suited for biosensing applications, base-pairinginteractions between complementary sequences are specific.

Nucleotide bases will re-form hydrogen bonds onlywith specific bases:

adenine pairs with thymine, and cytosine pairs with guanine.

y The single stranded DNA (ssDNA) is relatively stable, the DNA molecule willre-form into the double stranded configuration.Re-annealing between thessDNAs from different sources is called hybridization.



Working Principle of a DNA Biosensor

y

In biosensors for DNA sequence detection, the molecular recognition eventis commonly hybridization of a known probe to an unknown targetsequence. In biosensors, this event typically occurs directly on the surfaceof a signal transducer.



Applications

y Diagnostic of;

y Bacterial food contamination

y Genetically modified organisms

y Biological agents.

y Molecular diagnostics;

y Genome sequencing detecting

y Inherited disease

y Human pathogens

y Drug screening



Optical Fibers

Hybridization event is detected by fluorescence, by measuring the totalinternal reflection in the fiberDNA probe is placed in the end of the fiber

After hybridization, changes in the fluorescence intensity resultantfrom association between the DNA duplex and the label is measured

Fluorescent dyes bind to DNA making them fluorescent and readilydetectable (Ethidium bromide commonly used )



Optical fibers

By transmitting light for very long distances without signal lost, allowdetection of inaccessible or dangerous samples

In a fiber-optical array biosensor;

simultaneous detection of multiple nucleotide sequences usingcombinations of different fluorescent labels

poor stability

interference from environmental light

high cost of quartz optical fibers for UV light transmission



Evanescent and Acoustic waves

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

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

y Acoustic wave sensors used with a liquid sample detect changes in physicalproperties such as mass, viscosity, charge density

y 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

y Colorimetric method.

y High stability, less expensive and complex.

y

Built as microparticles/nanoparticles conjugates.

y Color of GNP based biosensor changes when there is a binding betweenmicroparticle and GNP including target DNA.

y

Spectrophotometric method is used to determine the the change of color.



Layout for DNA colorimetric detection with GNPs and latex microspheres



Gol a oparticl is s as ios sor.

It t cts t specific products of genetically odified parts of t e osttransgenic plants and it provides visual detection of t em.

apid, simple and relia le met od f or detection of GM s.

Schematic illustration of the principle of the nanoparticle-basedDNA biosensor for

visual detection of GMO.

An Example of GNP based Biosensor



Electroc emical Sensors

y Well suited f or analysis, electroc emical reaction give an electronicsignal directly,t ere is no need expensive signal transduction element.

y Electroc emistry based sensors off er sensitivity,selectivity and low cost f ort e detection of selected sequences or mutated genes associated

wit uman 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 transportchemistry.

oPotentiometric

oAmperometric

oVoltametric



Electrochemical DNA biosensor for thedetection of Hepatitis B virus

y DNA sensors have the potential application in diagnosis of diseases

y An electrochemical DNA biosensor was developed based on therecognitionof target DNA by hybridization detection.

y After covalent immobilization of DNA related to hepatitis B virus onglassy carbon electrode, electrode was immersed in solution containingssDNA to form double strand DNA at the electrode surface.

y Hybridization was detected by using the electrochemical indicator (Cu

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

y HBV could be quantified with a detection limit of 7.0 × 108 M.



Piezoelectric Mass Readout

The increase in mass that can accompanies hybridization

is detected by the deflection of a laser beam reflectedfrom the cantilever surface.

Immobilized DNA probe

Target DNA

Form duplex---mass increase

Decrease in crystals resonance frequency

Chemical vapors at very lowconcentrations can be detected based onthe surface stress changes generated bythe interactions between probe and targetmolecules on their surfaces

The magnitude of the surface stresschange depends on the type of interactiontaking place which includes

Hydrogen bonding Electrostatic, van der Waals forces, etc.

yCantilever sensorsPiezoelectric DNA Biosensors



Advantages

y Easily synthesized in the laboratory, regenerated for multiple use

y High detection sensitivity and physico-chemical stability relative to otherrecognition elements (enzymes, antibodies etc. )

y Earlier diagnosis of infectious diseases

y 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 previousamplification



What is ew Trend?y Development of a method carried out in volume not on surface.

y Using nanotechnology such as quantum dot nanoparticles, nanowires or

nanotubes.

y Development of artificial antibodies such asaptamer or peptideselected by

phage display method.

y Development of Lab-on-a-chip for detection of antigen.

Lab-On-Chip

Aim of the development of Lab-on-a-chipsystem is

performing all stages on a chip.Lab-on-a system consists of; Microchanells and micropumps Sample pretreatment unitsReservoir for probes, substrates, buffers and others, Detectors, Sampling more then one analyteor sample at the sametime



REFERENCES

y Trends in DNA biosensors (F.R.R. Teles, L.P. Fonseca)

y Nanoparticle-based DNA biosensor for visualdetection of genetically modified organisms (DespinaP. Kalogianni, Theodora Koraki,Theodore K. ChristopoulosPenelope C. Ioannou)



OUTLINE

Overview

Electrochemical Biosensor

Bio-analytical applications of gold

nanoparticles in DNA sensing



DNABiosensors; hybridization

yDNA is well suited for biosensing applications, base-pairing interactionsbetween complementary sequences are specific.

Nucleotides will re-form hydrogen bonds onlywith 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 virusDNA 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 DNAC ovalent immobilization of DNA on glassy carbon electrode(GC E)

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 electrodewas 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

T he detection of hybridizationHybridization was detected by using Cu complex as indicator

Hepatitis B virus could be quantified with a detection limit of

7.0 × 108 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 withcomplementary 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 theirintense colors

y M onodisperse 13-nm diameter Au-NPs appear red and exhibit a

relatively narrow absorption band centered at 520 nm

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

plasmon resonance of the particles from 520 to 574 nm

y 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 ascapturing 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

E vident by transmission electron microscopy, the nanoparticlesassembled into large network aggregates when nanoparticle HBV DNA gene probes were applied to detect HBV DNA molecules inliquid .



TEM images of DNA-linked Au nanoparticle

Composite targets HBV DNA extracted from serum of patient wereadded 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

y 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

y Dong Xia, Xiaoping Luob, The detection of HBV DNA with gold nanoparticle

gene probes, Journal of Nanjing Medical University,2007

y Chad A. Mirkin, Gold nanoparticle probes for the detection of nucleic acid, 2006

y Chad A. Mirkin, Selective Colorimetric Detection of Polynucleotides Based on

the Distance-Dependent Optical Properties of Gold, 1997

dna in sensing sensors

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