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

Outline

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

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

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

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*

3

3

4 l

wtYk

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

*0

2

1

m

kf

ff

mm

0

*2

mm

kmf

*2

1)(

Dna-patch by grafting

ssDNA patch are produced with alkanethiol-DNAmolecules 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

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)

The Hybridization on a cantilever

Sensitivity

1 base ssDNA -> 330 Da = 5.48•10-22 g 20 base ssDNA -> 6600 Da = 1.1•10-20 g

  Surface 9•10-8 cm2 (3x3m2) Max density 2.5 1013 molecules/cm2 2.25 106 molecules 2.48•10-14 g

10%-hybridation => 2.48•10-15 g

The Hybridization on a cantilever

Cantilever 25x5x2.5(l•w•t(m))

f0 = 5,519,907 Hz

k = 213 N/m ·m*/f06.40·10-17 g/Hz =

5819 molecules/Hz

Cantilever 15x5x2.5

f0 = 15,333,075 Hz

k = 984 N/m ·m*/f01.38·10-17 g/Hz

= 1257 molecules/Hz

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

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

Grafting on cantiliver

Grafting on cantiliver

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

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

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

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

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

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

First Test

1st peak 2nd peak 3rd peak

Position (Hz) 4259210 4260850 4261990

Width (Hz) 966 1620 1620

Frequency

Outlook

Characterization of reproducibility Calibration with known mass Start a systematic analysis of DNA

hybridization

Bridge

Reduce mass without reducing significantly k

Sensible increase of sensitivity

Heat and set the temperature of the surface