1. New approach for the determination of the hybridization efficiency of ssDNA nanopatches 1. CNR-INFM, Laboratorio Nazionale TASC, Trieste, Italy 2. SISSA, Trieste Italy 3. Sincrotrone Trieste SCpA, Trieste Italy Mauro Melli 1,2, Marco Lazzarino 1, Matteo Castronovo 2,3, Loredana Casalis 3 and Giacinto Scoles 2,3

2. Outline ssDNA hybridation Density The nanografting on a cantilever Cantilever as balance Experimental Setup Conclusion and Outlook

3. ssDNA hybridization Monolayer of single-stranded DNA (ss-DNA) have an important role in many biotechnology applications The hybridization of self assembled monolayers (SAM) of ssDNA was found to be inversely proportional to the molecular density of the probes on the surface [Tarlov et al. J. Am. Chem. Soc. 119, 8916 (1997) ] Recently our group ( E. Mirmomtaz, M. Castronovo, F.Bano, L. Casalis ) has observed that grafted-ssDNA nanostructures even at high density hybridize Disorder vs. density effects

4. Density DNA monolayer Actually, in most cases nobody knows the REAL density of both a SAM and a grafted monolayer!! The used technics are all indirect and different High and low density have no quantitative meaning Which is the most incontestable and direct way to misure the density ? weight directly the molecules

5. Cantilever as balance Dinamic Resonance Mode For the first oscillation mode (vertical) a cantilever can be approximated as a mass linked to a spring where m* is the “effective mass” of the cantilever ad k is given by (Y is the Young’s modulus) k m*

6. Cantilever as balance The resonance frequency is If a mass is added to the free end of cantilever the resonance frequency changes according to where  is a geometric parameter <1

7. Dna-patch by grafting ssDNA patch are produced with alkanethiol-DNA molecules on very flat gold substrate Using nanografting an atomic force microscope (AFM) based lithography The hybridization is monitored by measurements of heigth and compressibility with the AFM

8. The Hybridization on a cantilever Sensitive to DNA mass Geometry parameter (length, width,thickness) minimize the mass and maximize the resonance frequency of cantiliver The oscillation frequency should be in a accessible range Allow grafting Stiff enough for grafting: k » k grafting (~ 0.6N/m) Ultra flat gold surface (RMS < 1nm)

9. The Hybridization on a cantilever Sensitivity 1 base ssDNA -> 330 Da = 5.4810 -22 g 20 base ssDNA -> 6600 Da = 1.110 -20 g Surface 910 -8 cm 2 (3x3  m 2 ) Max density 2.5 10 13 molecules/cm 2 2.25 10 6 molecules 2.4810 -14 g 10%-hybridation => 2.4810 -15 g

10. The Hybridization on a cantilever Cantilever 25x5x2.5(lwt(  m)) f 0 = 5,519,907 Hz k = 213 N/m  ·m * /f 0  6.40·10 -17 g/Hz = 5819 molecules/Hz Cantilever 15x5x2.5 f 0 = 15,333,075 Hz k = 984 N/m  ·m * /f 0  1.38·10 -17 g/Hz = 1257 molecules/Hz

11. The fabrication of cantilever Cantilever fabrication is the results of several process steps (i.e., Lithographical process, Etching, Sacrifical layer release Superficial treatment) Problems: – Microfabrication damages surfaces – Gold grows by islands on silicon

12. The fabrication of cantilever Cantilever fabrication is the results of several process steps (i.e., Lithographical process, Etching, Sacrifical layer release Superficial treatment) Problems: – Microfabrication damages surfaces – Gold grows by islands on silicon Solutions: – 200 nm of silicon oxide is grown on the starting wafer – Interface of palladium (10nm) between gold and silicon

13. Grafting on cantiliver

15. Experimental Setup Optical microscope to align the laser s R T Turbo Pump x-y stage laser CCD microscope Network Analyzer 4-quad photodiode Vibration actuated by a piezo element

16. Experimental Setup Optical microscope to align the laser s R T Turbo Pump x-y stage laser CCD microscope Network Analyzer 4-quad photodiode Vibration actuated by a piezo element

17. Experimental Setup Optical microscope to align the laser s R T Turbo Pump x-y stage laser CCD microscope Network Analyzer 4-quad photodiode Vibration actuated by a piezo element

18. Experimental Setup Optical microscope to align the laser s R T Turbo Pump x-y stage laser CCD microscope Network Analyzer 4-quad photodiode Vibration actuated by a piezo element

19. Experimental Setup Optical microscope to align the laser s R T Turbo Pump x-y stage laser CCD microscope Network Analyzer 4-quad photodiode Vibration actuated by a piezo element

20. Experimental Setup Optical microscope to align the laser s R T Turbo Pump x-y stage laser CCD microscope Network Analyzer 4-quad photodiode Vibration actuated by a piezo element

21. First Test 1st peak2nd peak3rd peak Position (Hz)425921042608504261990 Width (Hz)9661620 Frequency

22. Outlook Characterization of reproducibility Calibration with known mass Start a systematic analysis of DNA hybridization

23. Bridge  Reduce mass without reducing significantly k  Sensible increase of sensitivity  Heat and set the temperature of the surface