Introduction to 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
Introduction
Biosensor:




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-helix
Size 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 Biosensors
quartz crystal microbalance (QCM) transducers

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 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…
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
Thank you!