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New approach for the determination of the hybridation efficiency

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					       New approach for the
determination of the hybridization
efficiency of ssDNA nanopatches

                Mauro Melli 1,2 , Marco Lazzarino 1, Matteo
                  Castronovo 2,3, Loredana Casalis 3 and
                                         Giacinto Scoles 2,3




                l    CNR-INFM, Laboratorio Nazionale TASC,
                     Trieste, Italy
                l    SISSA, Trieste Italy
                l    Sincrotrone Trieste SCpA, Trieste Italy
Outline


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


l   Monolayer of single-stranded DNA (ss-DNA) have an important 
    role in many  biotechnology applications 
l   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) ]
l   Recently our group (E. Mirmomtaz, M. Castronovo, F.Bano, L. Casalis) 
    has observed that  grafted-ssDNA nanostructures even at high 
    density hybridize
l   Disorder vs. density effects 
Density DNA monolayer

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


           weight directly the molecules
Cantilever as balance

l   Dinamic Resonance Mode
l   For the first oscillation mode (vertical) a cantilever can 
    be approximated as a mass linked to a spring

                                                    k

                                                    m*
    where m* is the “effective mass” of the cantilever ad

    k is given by                        (Y is the Young’s modulus)
Cantilever as balance

l   The resonance frequency is




l   If a mass is added to the free end of cantilever the 
    resonance frequency changes according to
                                 

    where b is a geometric parameter <1
Dna-patch by grafting

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

l   Sensitive to DNA mass
      l   Geometry parameter (length, width,thickness) minimize
          the mass and maximize the resonance frequency of 
          cantiliver
      l   The oscillation frequency should be in a accessible range


l   Allow grafting
      l   Stiff enough for grafting: k » k grafting (~ 0.6N/m)
      l   Ultra flat gold surface (RMS < 1nm)
The Hybridization on a cantilever

Sensitivity

l    1  base ssDNA ->  330 Da = 5.48•10-22 g 
l   20 base ssDNA -> 6600 Da = 1.1•10-20 g
     
l   Surface 9•10-8 cm2  (3x3mm2)
l   Max density 2.5 1013 molecules/cm2  
l   2.25 106 molecules    2.48•10-14 g    
l   10%-hybridation => 2.48•10-15 g 
The Hybridization on a cantilever

l   Cantilever 25x5x2.5(l•w•t(mm))   l   Cantilever 15x5x2.5 

l   f0  = 5,519,907 Hz               l   f0  = 15,333,075 Hz
l   k  =  213 N/m                    l   k  =  984 N/m
l   a = 2·m*/f0=6.40·10-17 g/Hz =    l   a = 2·m*/f0=1.38·10-17 g/Hz = 
    5819 molecules/Hz                    1257 molecules/Hz
The fabrication of cantilever
l   Cantilever fabrication is the results of several process steps (i.e.,
    Lithographical process, Etching, Sacrifical layer release Superficial
    treatment)
l   Problems:
     –   Microfabrication damages surfaces
     –   Gold grows by islands on silicon
The fabrication of cantilever
l   Cantilever fabrication is the results of several process steps (i.e.,
    Lithographical process, Etching, Sacrifical layer release Superficial
    treatment)
l   Problems:
     –   Microfabrication damages surfaces
     –   Gold grows by islands on silicon
l   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 
                  CCD
                                align the laser

                 microscope



       laser


Vibration                       4-quad       s     RT
actuated by a                   photodiode
piezo element
    Turbo Pump

                                                 Network
                              x-y stage          Analyzer
        Experimental Setup
                                Optical 
                                microscope to 
                  CCD
                                align the laser

                 microscope



       laser


Vibration                       4-quad       s     RT
actuated by a                   photodiode
piezo element
    Turbo Pump

                                                 Network
                              x-y stage          Analyzer
        Experimental Setup
                                Optical 
                                microscope to 
                  CCD
                                align the laser

                 microscope



       laser


Vibration                       4-quad       s     RT
actuated by a                   photodiode
piezo element
    Turbo Pump

                                                 Network
                              x-y stage          Analyzer
        Experimental Setup
                                Optical 
                                microscope to 
                  CCD
                                align the laser

                 microscope



       laser


Vibration                       4-quad       s     RT
actuated by a                   photodiode
piezo element
    Turbo Pump

                                                 Network
                              x-y stage          Analyzer
        Experimental Setup
                                Optical 
                                microscope to 
                  CCD
                                align the laser

                 microscope



       laser


Vibration                       4-quad       s     RT
actuated by a                   photodiode
piezo element
    Turbo Pump

                                                 Network
                              x-y stage          Analyzer
        Experimental Setup
                                Optical 
                                microscope to 
                  CCD
                                align the laser

                 microscope



       laser


Vibration                       4-quad       s     RT
actuated by a                   photodiode
piezo element
    Turbo Pump

                                                 Network
                              x-y stage          Analyzer
First Test




        Frequency                1st peak   2nd peak   3rd peak
                    Position (Hz) 4259210   4260850    4261990
                    Width (Hz)   966        1620       1620
Outlook


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




q Reduce mass without reducing significantly k
qSensible increase of sensitivity 
qHeat and set the temperature of the surface

				
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posted:7/19/2013
language:English
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