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  • SELF-ASSEMBLED MONOLAYERS OF PEPTIDE NUCLEIC ACIDS (PNA):

    FROM THE MOLECULAR STRUCTURE TO BIOSENSOR APPLICATIONS.

    C. Briones , E. Mateo-Martí , V. Parro, C. Rogero Centro de Astrobiología (CSIC-INTA). Madrid, Spain

    J.A. Martín-Gago, C. Gómez-Navarro (UAM) , J. Mendez, E. Román, Instituto de Ciencia de Materiales de Madrid (CSIC). Madrid, Spain

    Victor Fernandez, Marcos Pita Instituto de Catalisis (CSIC). Madrid, Spain

    http://www.icmm.csic.es/esisna/

    Interdisciplinary work: biology, chemistry, Physics

  • Motivation of the research project:

    To investigate the interaction of nucleic acids with solid surfaces

    Origin of life on earth could be a catalytic process Metallic oxides, pyrite, clay minerals, silicates

    Surface science can model all these interactions

    XPS,AFM, NEXAFS, IRS Gold, pyrite, silicon, nanoparticles

    1.- Nanoscience:

    To build new biosensors from the bases

    3S Specific Sensitive Simple

    2.-Nanotechnology:

  • ssDNA: (single stranded) single chain

    dsDNA double stranded in double chain (helicoidal : Every nucleo-base recognaice by H- bridges a complementary one

    Sugar (deoxyribose)

    Phosphate group

    Nitrogeneous bases (nucleo-bases)

    DNA (deoxyribonucleic acid): polynucleotide

    nucleotide

    Rapid bio-background for physicists

    backbone

  • ?? ??labelling

    Two unknown nucleotides Which is the sequence

    DNA- microaarrays, how does it work?

    AG AT AC

    GT GC TC

    Robot imprinting

    Inkjet or piezo actuation technology. Spots of 50-150 μm in diameter, and low-density microarrays with no more than 5,000 spots/cm2

    Target

    Probe

  • TC

    ?? ??labelling

    Robot imprinting

    ?? = AG

    Fluoresce Scanner

    DNA- microaarrays, how does it work? Two unknown nucleotides

  • In every spot a sequence

    DNA

    Spacer

    Surface

    DNA- microaarrays, how does it work?

    DNA-chip pattern

  • Drawbacks of the current technology: Nanotechnology comes to help…

    1.- Fluorescence molecular label of the target

    2.- Expensive equipments

    3.- No quantitative information, lack of specificity, not very sensitive

  • -Alkanethiol layers have been extensively studied due to their important technological properties and their outstanding capability to form self-

    assembled monolayers (SAMs).

    AFM From E.Barrena and C.Ocal

    Alkanethiols self-assembled monolayers

    gold

    S-head

    Alkyl chain

    Functional group

  • IDEA!!!

    Let us add a S atom at the end of a DNA to recover the alkanethiol ideas and made a solid-state biosensor

  • Clean Au Immobilized DNA Hybridized DNA

    More details: E. Casero, M. Darder, D. J. Díaz, F. Pariente, J. A.Martín-Gago,H. Abruña and E. Lorenzo

    Lamgmuir 2003,19, 6230-6235

    strong surface-molecule molecule-molecule interactions

    -Thiolated DNA also immobilized on gold surfaces, but most of the results have been disappointing: DNA fold into itself leading to a formless globular structure, with a very reduced bioactivity.

  • PNA is an achiral and uncharged DNA mimic

    the sugar-phosphate backbone has been replaced with a peptide-

    like N-(2-aminoethyl)glycine polyamide structure. The nucleobases are connected by methylenecarbonyl linkages.

    PEPTIDE NUCLEIC ACID (PNA)

  • Menchise et al., PNAS 2003

    PNA recognizes complementary DNA with stronger affinity than DNA-DNA:

    (5’)

    (3’)

    O N N

    NH2

    O

    O O

    O OO P

    O O

    O OO P

    O O

    O OO P

    O

    O

    O OO P

    N NH

    O

    O

    NH2 N

    N N N

    NH2

    N

    N N NH

    O

    (N)

    O

    N

    NH

    O

    O

    N

    NH

    O

    O

    N

    NH

    O

    O

    N

    NH

    O

    (C)

    NNH

    O

    O

    N

    N NN

    H2N

    NN

    O

    H2N

    N

    NH2N

    HN

    O

    N

    Can be used as probe for DNA biosensors Absence of P in the backbone:

    Specific signature: no fluorescence

  • EXPERIMENTAL PROCEDURE

    Cysteine

    Spacer

    ssPNA

  • Au

    H2O H2OTarget NANA

    Immobilization (22ºC; 4 h)

    Washing (22ºC; 15 min)

    Hybridization (53-58ºC; 1 h)

    Washing (50-58ºC; 15 min)

    XPS, AFM,XANES,IRS

    ss-PNA Immobilized: Structural characterization

    Ordered SAM

    PNA-DNA duplex Functional characterization

    biosensor

    EXPERIMENTAL PROCEDURE

    XPS, AFM,XANES,IRS

  • H2O H2OTarget NANA

    Immobilization (22ºC; 4 h)

    Washing (22ºC; 15 min)

    Hybridization (53-58ºC; 1 h)

    Washing (50-58ºC; 15 min)

    ss-PNA Immobilized: Structural characterization

    Ordered SAM

    PNA-DNA duplex Functional characterization

    biosensor

    EXPERIMENTAL PROCEDURE

    XPS, AFM,XANES,IRS XPS, AFM,XANES,IRS

  • H2O H2OTarget NANA

    Immobilization (22ºC; 4 h)

    Washing (22ºC; 15 min)

    Hybridization (53-58ºC; 1 h)

    Washing (50-58ºC; 15 min)

    ss-PNA Immobilized: Structural characterization

    Ordered SAM

    PNA-DNA duplex Functional characterization

    biosensor

    EXPERIMENTAL PROCEDURE

    XPS, AFM,XANES,IRS XPS, AFM,XANES,IRS

  • H2O H2OTarget NANA

    Immobilization (22ºC; 4 h)

    Washing (22ºC; 15 min)

    Hybridization (53-58ºC; 1 h)

    Washing (50-58ºC; 15 min)

    ss-PNA Immobilized: Structural characterization

    Ordered SAM

    PNA-DNA duplex Functional characterization

    biosensor

    EXPERIMENTAL PROCEDURE

    XPS, AFM,XANES,IRS XPS, AFM,XANES,IRS

  • 49nm

    50nm

    RESULTS : Structure of the layers

    AFM images: • ordered arrangement of the molecules, with reproducible aligned and meandering patterns.

    • The ordered protrusions are 6 to 7 nm high from the bare surface.

    • Width: 10 to 30 nm.

    The ssPNA molecules stand-up on the surface with a small tilt.

  • RESULTS : Structure of the layers

    - XANES spectra at the N-edge at grazing and normal emission indicate a preferential orientation of the molecule with the nucleo- bases nearly- paralel to the surface plane.

    In te

    ns ity

    (a rb

    .u ni

    ts )

    420415410405400 Photon energy (eV)

    Normal incidence

    70º off normal incidence

    Most of the π* orbitals lie along the backbone of the PNA and the σ* are parallel to the nucleobases plane,

  • 49nm

    Proposed Structural model

    - Self-assembling of ssPNA molecules

    promoted by non- complementary

    H-bonding between nucleobases

  • RESULTS : Concentration dependence

    41nm

    [ssPNA] = 0.1μM

    - Linear features following crys- tallographic directions

    - Height: ~ 1 nm - Length: ~ 10 nm - Individual mole- cules lying on the surface

    200nm

    [ssPNA] = 0.5μM

    - Layer not com- plete

    - Groups of mo- lecules stand up as islands anchored to the upper part of the step edges

    50nm

    [ssPNA] ≥ 10μM

    - Ordered features are lost

    - Surface saturated of amorphous groups of mole- cules

    50nm

    [ssPNA] = 1.0μM

    - Aligned and meandering patterns

    - Height: 6-7 nm - Width: 10-30 nm - Groups of mole- cules stand up on the whole surface

    - Optimal coverage

    With increasing PNA concentration

  • Advantage of ssPNA over ssDNA in the formation of bioSAMs

    i) the lack of charged groups in the PNA backbone avoids electrostatic repulsions either among neighbouring molecules or among the solvent counterions.

    ii) although relatively flexible, the PNA molecule is more rigid than DNA due to the planar amide

    Now, let us see if they work as biosensors…

  • RESULTS : Concentration dependence

    41nm

    [ssPNA] = 0.1μM

    200nm

    [ssPNA] = 0.5μM

    50nm

    [ssPNA] ≥ 10μM[ssPNA] = 1.0μM

    With increasing PNA concentration

    PNA molecules standing up on the surface

    PNA molecules lying on

    the surface

    PNA molecules intermixed one with each other

  • RESULTS : Hybridization to DNA

    The hybridization of complementary DNA is detected by XPS: - Increase of N1s/Au4f ratio in a factor of 2.8 to 3.

    - Detection of a clear P2p signal.

    130 132 134 136 138

    396 398 400 402 404

    1.4

    1.2

    1.0

    0.8

    0.6

    0.4

    0.2

    0.0

    In te

    ns ity

    (a rb

    . U ni

    ts )

    600 500 400 300 200 100 0 Binding energy (eV)

    O 2p N 1s

    C 1s Au 4f

    Au

    Immob. PNA

    PNA/DNA

    XPS overview

    P 2p

  • RESULTS : Characterization of the biosensor

    0,001

    0,01

    0,1

    1

    10

    0,1 1 10 100

    Immobilized Hybridized

    N 1s

    /A u4

    f r at

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