DNA Based Biosensors

1. DNA Based Biosensors Yingli Fu Biological Resources Engineering University of Maryland, College Park December 10, 2003

2. Outline • Introduction • Principles of DNA biosensors • Types of DNA biosensors • Improvement of DNA biosensors • DNA biosensor miniaturization • References

3. Introduction • Biosensor: • DNA biosensor: Motivated by the application to clinical diagnosis and genome mutation detection

4. DNA Structure • DNA structures---double helix (complementary) • 4 bases: Adenine (A), Guanine (G), Thymine (T), and Cytosine (C) • sugar (deoxyribose) • phosphate group

5. DNA Stability • Hydrogen bonding between base pairs • Stacking interaction between bases along axis of double-helix • Size and base content and sequence

6. 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

7. Forms of DNA Biosensors • Electrodes • Chips • Crystals

8. Immobilization of DNA Probe onto Transducer Surface • Thiolated DNAfor self assembly onto gold transducers • Covalent linkage to thegold surface via functional alkanethiol-based monolayers • Use of biotylated DNA for complex formation with a surface-confinedavidin or strepavidin • Covalent (carbodiimide) coupling to functionalgroups on carbon electrodes • simple adsorption onto carbonsurfaces

9. Types of DNA Based Biosensors • Optical, Electrochemical and Piezoelectric

10. 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.

12. Immobilized DNA probe Target DNA Form duplex---mass increase Decrease in crystal’s resonance frequency Piezoelectric DNA Biosensors • quartz crystal microbalance (QCM) transducers

13. 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.

14. 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)

15. 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

16. 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’

17. Material and Methods (cont’s)

18. Results

19. 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

20. Improvement • Fluorescent Bioconjugated Nanoparticles • DNA dendrimers

21. Fluorescent Bioconjugated Nanoparticles---signal amplification

22. Results

23. 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¢.

24. DNA biosensor miniaturization

25. Concept of DNA microarray Figure 2

26. DNA microarray

27. 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

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

29. DNA biosensor not limited to DNA detection, but more…

30. 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

31. Thank you!