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DNA Based Biosensors

Yingli Fu

Biological Resources EngineeringUniversity of Maryland, College Park

December 10, 2003

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Outline

Introduction

Principles of DNA biosensors

Types of DNA biosensors

Improvement of DNA biosensors

DNA biosensor miniaturization

References

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Introduction

Biosensor:

DNA biosensor:Motivated by the application to clinical diagnosis

and genome mutation detection

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DNA Structure

DNA structures---doublehelix (complementary)

4 bases:Adenine (A), Guanine (G),

Thymine (T), and Cytosine (C)

sugar (deoxyribose)

phosphate group

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DNA Stability

Hydrogen bonding between base pairs

Stacking interaction between bases along axis ofdouble-helix

Size and base content and sequence

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Principles of DNA biosensors

Nucleic acid hybridization

---rennealing b/w the ssDNAs from different sources

Perfect match---stable dsDNA, strong

hybridization

One or more basemismatches----weak hybridization

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Forms of DNA BiosensorsElectrodes

Chips

Crystals

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Immobilization of DNA Probe onto

Transducer SurfaceThiolated DNA for self assembly onto gold transducers

Covalent linkage to the gold surface via functional

alkanethiol-based monolayers

Use of biotylated DNA for complex formation with asurface-confinedavidin or strepavidin

Covalent (carbodiimide) coupling to functional groupson carbon electrodes

simple adsorption onto carbon surfaces

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Types of DNA Based Biosensors

Optical, Electrochemical and Piezoelectric

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Molecular Beacon Based Optical

Fiber DNA Biosensors

Ligate and light. Schematics diagram ofreal-time monitoring

of the nucleic acid ligation process by a MB.

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Piezoelectric DNA Biosensors

quartz crystal microbalance (QCM) transducers

Immobilized DNA robe

Target DNA

Form du lex---mass increase

Decrease in crystals resonance frequency

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Piezoelectric DNA Biosensors (conts)

Frequencytime response of a PNA/QCM to additions of the target (T) and mismatch (M)

oligonucleotides. The hybridization event results in decreased frequency,reflecting the

increased mass of the crystal.

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Electrochemical DNA Biosensor

DNA-immobilized electrodes, based on detectionof hybridization redox intercalators to recognize dsDNA

DNA-mediated electron transferusing mediators Use of ferrocene-labeled oligonucleotide probes that

hybrize to immobilized DNA

Enzyme labels were used to amplify the signaland improve the sensitivity Peroxidase

Glucose dehydrogenase (GDH)

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Electrochemical DNA Biosensor---An Example

Amperometric DNA sensor using thepyrroquinoline quinone glucose dehydrogenase-

avidin conjugate

Kazunori Ikebukuro, Yumiko Kohiki, Koji Sode *

Biosensors and Bioelectronics 17 (2002) 1075--1080

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Material and Methods

pyrroquinoline quinone-dependent glucosedehydrogenase ((PQQ)GDH) for DNAhybridization labeling

Detection via biotin-avidin binding

Target and probe DNA sequence: Target DNA: 5-bio-TCGGCATCAATACTCATC-3.

Probe DNA: 5-bio-GATGAGTATTGATGCCGA-3 Control DNA: 5-bio-CTGATGAACATACTATCT-3

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Material and Methods (conts)

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Results

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Conclusions

The (PQQ)GDH/avidin conjugate based DNA

biosensor is highly sensitive and selective to the

target Salmonla invA virulence gene

The sensor response increased with the addition

of glucose and in the presence of 6.3 mM

glucose the response increased with increasing

DNA in the range 5.0x10^8-1.0x10^5

This DNA biosensor would be applicable for

single nuleotide polymorphism detection

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Improvement

Fluorescent Bioconjugated Nanoparticles

DNA dendrimers

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Fluorescent BioconjugatedNanoparticles---signal amplification

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Results

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DNA dendrimers---increase sensitivity

Schematic drawing showing the hybridization detection at the dendrimer/QCM

biosensor. The 38-merprobe is attached to the core dendrimerby complementary

oligonucleotide (a(-)) binding on one (a(+)) of the outerarms. The probe sequence

for target hybridization is 5d-GGG GAT CGAAGA CGA TCA GAT ACC GTC

GTA GTC TTAAC-3d.

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DNA biosensor miniaturization

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Concept of DNA microarray

Figure 2

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DNA microarray

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DNA Microarray (conts)

The fluorescence intensities foreach spot is indicative of the

relative aboundance of the corresponding DNA probe in the

Nucleic acid target samples

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The light directed probe array synthesis process used for the preparation of

Affymetrixs Gene Chip

Affymetrixs Gene Chip

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DNA biosensor not limited to DNA detection, butmore

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References

Kazunori Ikebukuro, Yumiko Kohiki,Koji Sode 2002. Amperometric DNA sensor

using the pyrroquinoline quinone glucose dehydrogenase-avidin conjugate.

Biosens. Bioelectron. 17,10751080

Wang, J. 2000. SURVEY AND SUMMARY From DNA biosensors to gene chips.

Nucleic Acids Res. 28(16),3011-3016

Wang, J., M. Jiang. T. W. Nilsen, R. C. Getts.1998. Dendritic nucleic acid probes

forDNA Biosensors.J. AM. CHEM. SOC. 120,8281-8282

Zhao, X., R. Tapec-Dytioco, and W. Tan. 2003. Ultrasensitive DNA Detection

Using highly fluorescent bioconjugated nanoparticles.J. AM. CHEM. SOC. 125,

11474-11475

Zhai, J., H. Cui, and R.Yang. 1997. DNA based biosensors.Biotechnol. Adv.

15(1),43-58

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