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 DNA biosensors

Improvement of DNA biosensors

DNA biosensor miniaturization

References

IntroductionBiosensor:

DNA biosensor:

Motivated by the application to clinical diagnosis and genome mutation detection

DNA Structure

DNA structures---double helix (complementary) 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-helixSize and base content and sequence

Principles of DNA biosensors

Nucleic acid hybridization ---rennealing b/w the ssDNAs from different sources

Perfect match ---stable dsDNA, strong hybridization

One or more base mismatches ----weak hybridization

Forms of DNA Biosensors

Electrodes

Chips

Crystals

Immobilization of DNA Probe onto Transducer Surface

Thiolated 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 a surface-confined avidin or strepavidin

Covalent (carbodiimide) coupling to functional

groups on carbon electrodes

simple adsorption onto carbon surfaces

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 of the nucleic acid ligation process by a MB.

Piezoelectric DNA Biosensorsquartz 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)

Electrochemical DNA Biosensor---An Example

Amperometric DNA sensor using the pyrroquinoline quinone glucose dehydrogenase-avidin conjugate

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: Target DNA: 5’-bio-TCGGCATCAATACTCATC-3’. Probe DNA: 5’-bio-GATGAGTATTGATGCCGA-3’ Control DNA: 5’-bio-CTGATGAACATACTATCT-3’

Material and Methods (cont’s)

Results

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

Improvement

Fluorescent Bioconjugated Nanoparticles

DNA 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 for target hybridization is 5-GGG GAT CGA AGA CGA TCA GAT ACC GTC GTA GTC TTA AC-3.

DNA biosensor miniaturization

Concept of DNA microarray

                          Figure 2

DNA microarray

DNA Microarray (cont’s)

The fluorescence intensities for each spot is indicative of therelative 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…

References

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