DNA Based Biosensors

DNA Based Biosensors DNA Based Biosensors Yingli Fu Biological Resources Engineering University of Maryland, College Park December 10, 2003 Outline Introduction Principles of DNA biosensors Types of D[.]

DNA Based Biosensors

Yingli Fu

Biological Resources Engineering University of Maryland, College Park

December 10, 2003

Introduction

Principles of DNA biosensors

Types of DNA biosensors

Improvement of DNA biosensorsDNA biosensor miniaturization

References

DNA Structure

DNA structures -double

 4 bases:

Adenine (A), Guanine (G),

Thymine (T), and Cytosine (C)

 sugar (deoxyribose)

 phosphate group

DNA Stability

Hydrogen bonding between base pairs

Stacking interaction between bases along axis of double-helix

Size and base content and sequence

Principles of DNA biosensors

Nucleic acid hybridization

-rennealing b/w the ssDNAs from different sources

Forms of DNA Biosensors

Electrodes

Chips

Crystals

Immobilization of DNA Probe

onto Transducer Surface

alkanethiol-based monolayers

Use of biotylated DNA for complex formation with a

carbon electrodes

Types of DNA Based

Biosensors

Optical, Electrochemical and Piezoelectric

Molecular Beacon Based

Optical Fiber DNA Biosensors

‘Ligate and light’ Schematics diagram of real-time monitoring

Piezoelectric DNA Biosensors

quartz crystal microbalance (QCM) transducers

Immobilized DNA probe

Target DNA Form duplex -mass increase

Decrease in crystal’s resonance frequency

Piezoelectric DNA Biosensors (cont’s)

Frequency–time 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.

Electrochemical DNA

Biosensor DNA-immobilized electrodes, based on detection of

hybridization

 redox intercalators to recognize dsDNA

 DNA-mediated electron transfer using mediators

 Use of ferrocene-labeled oligonucleotide probes that hybrize to immobilized DNA

Enzyme labels were used to amplify the signal and improve the sensitivity

 Peroxidase

 Glucose dehydrogenase (GDH)

Kazunori Ikebukuro, Yumiko Kohiki, Koji Sode *

Biosensors and Bioelectronics 17 (2002) 1075 1080

Material and Methods

pyrroquinoline quinone-dependent glucose

dehydrogenase ((PQQ)GDH) for DNA hybridization labeling

Detection via biotin-avidin binding

Target and probe DNA sequence:

 Probe DNA: 5’-bio-GATGAGTATTGATGCCGA-3’

Material and Methods (cont’s)

Results

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

Fluorescent Bioconjugated NanoparticlesDNA dendrimers

Fluorescent Bioconjugated Nanoparticles -signal

amplification

Results

DNA dendrimers -increase

sensitivity

Schematic drawing showing the hybridization detection at the dendrimer/QCM biosensor The 38-mer probe is attached to the core dendrimer by complementary oligonucleotide (a(-)) binding on one (a(+)) of the outer arms The probe sequence

DNA biosensor miniaturization

Concept of DNA microarray

       Figure 2

DNA microarray

DNA Microarray (cont’s)

The fluorescence intensities for each spot is indicative of the relative aboundance of the corresponding DNA probe in the Nucleic acid target samples

The light directed probe array synthesis process used for the preparation of Affymetrix’s Gene Chip

Affymetrix’s Gene Chip

DNA biosensor not limited to DNA detection, but more…

Kazunori Ikebukuro, Yumiko Kohiki, Koji Sode 2002 Amperometric DNA sensor using the pyrroquinoline quinone glucose dehydrogenase-avidin conjugate.

Biosens Bioelectron 17,1075—1080

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

for DNA 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