Biological Nanostructures and NEXAFS
Connecting a patterned silicon substrate with bio-molecules opens many
new possiblities for assembling nanostructures. For example,
snippets of DNA molecules can be used as "rubber bands" for
making connections between nano-objects. DNA chips involve a patterned substrate
that acts as a collection miniature chemical beakers. Biosensors are being developed
that provide a field test for the presence of viruses, using an electrical or an optical
readout (via liquid crystals or fluorescence). An interdisciplinary
group of researchers at the UW-Madison has formed a center for exploring the
interface between structured
surfaces and living systems.
Organic and biological molecules can be attached to silicon via an intermediary gold layer and a carbon-sulfur-gold bond (see the next two panels). Another method uses siloxane chemistry directly on oxidized silicon (third panel).
By building up a scaffolding of multiple molecular layers it is possible to mimic the surface
surface of a living object.
To find out about the chemical bonds and the corresponding molecular orbitals in such layers we use
NEXAFS (near-edge x-ray absorption spectroscopy), a spectroscopic technique using
synchrotron radiation. Below are a few examples
of organic and biological molecules at surfacec, all the way to DNA and proteins.
For an introduction see: Xiaosong Liu et al.,
Can J. Chem. 85, 793 (2007).
STM images of various gold coatings on a stepped silicon surface (70 nm between step bunches).
The coating needs to be as smooth as possible to preserve the step pattern.
A. Kirakosian et al.,
J. Appl. Phys. 90, 3286 (2001).
Alkanethiols Adsorbed on Gold-Coated Silicon
Alkane chains passivate the surface and prevent biomolecules from denaturing.
The orientation of the chains is obtained by polarized synchrotron radiation
using dipole selection rules. These are rather simple for transitions from the
C 1s core level to the C 2p valence orbitals: The intensity is highest when the
polarization points parallel to the orbital, and it follows a cos2(theta) pattern.
The polarization angle theta relative to the sample surface is indicated in the spectra below.
The C-H orbitals lie nearly parallel to the surface, whereas the C-C orbitals of the backbone
lie nearly perpendicular. That implies a perpendicular orientation of the molecules.
A more quantitative analysis shows that there is a range of tilt angles around this
average orientation.
Self-Assembled Monolayers of Siloxanes
Attaching alkane chains by siloxane chemistry is more direct, but also more delicate.
The role of humidity and settling time in preparing a well-ordered self-assembled monolayer (SAM)
is demonstrated here for octadecyltrichlorosilane (OTS).
The set of polarization-dependent NEXAFS spectra on the left is from a
SAM that had enough humidity and time to order itself, while the spectra on the right
are from a quickly-dried layer. The polarization dependence has disappeared on the right.
(The spectra are offset from each other, because otherwise they would collapse to the same curve.)
The lack of order is connected with a reduced coverage, as can be seen from the intensity
scale. This is consistent with a model of crowded molecules forming an ordered SAM
to increase their packing density.
R. Peters et al.,
Langmuir 18, 1250 (2002).
Oriented DNA Snippets at a Surface
The same type of spectroscopy can be applied to more complex molecules,
such as DNA segments. The nitrogen 1s core level is chosen in order to focus
onto the nucleotide bases, the only part of DNA containing nitrogen.
The base pairs have characteristic pi* orbitals that point perpendicular to the bases
and parallel to the axis of the double-helix. From the polarization dependence, one can tell
that the DNA molecules are partially oriented and that the axis of the double-helix
lies perpendicular to the surface on average.
Systematic work with single-stranded DNA shows how the degree of orientation depends
on the length of the DNA molecules and on the bases (collaboration with NRL and NIST).
The orientation of DNA immobilized at a surface is important for designing DNA microarrays.
A single-stranded DNA molecule attached to a surface
needs to stick out
to allow a complementary strand to bind to it.
D. Y. Petrovykh et al.,
J. Am. Chem. Soc. 128, 2 (2006) , supporting information.
Oriented Proteins at a Surface
Going farther up in complexity we have tackled small proteins, such as RNase A. It consists of 124 amino acids (about a thousand atoms not counting H). In NEXAFS, the characteristic orbital of proteins is the pi* orbital of the peptide bond, which connects the amino acids to form the backbone of the protein. As with DNA, the average orientation of the peptide bonds can be determined from the polarization dependence of the NEXAFS intensity at the N 1s edge. A modulation of 20% was found for this orbital when RNase A was immobilized in oriented fashion. That is consistent with a calculation based on the crystallographic data in the protein data base.
When the protein is immobilized in random orientation, the polarization dependence vanishes. Oriented immobilization of proteins is essential for the functioning of biosensors, because the docking site has to point outwards in order to link to a target virus.
Xiaosong Liu et al.,
Langmuir 22, 7719 (2006).
Supported by NSF-DMR
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