Multiphoton polymerization technique constructs biomolecular structures in 3-D
Method may help create new sensors
Researchers have developed a procedure
for using the multiphoton polymerization effect to harden biocompatible polymers
into three-dimensional structures that may help create biosensors with greater surface
area. The group — from the Foundation for Research and Technology in Iraklion,
Greece — recently tested the method by binding biotin to the surface of a
structure and studying the result with fluorescence microscopy and a surface acoustic
According to Maria Farsari, the principal investigator,
most micropatterned biological sensors — many of which draw on techniques
developed in the semiconductor industry — are strictly two-dimensional. “By
going from 2-D to 3-D, one can greatly increase the active surface area and, thus,
the sensitivity of the system without sacrifices to the size of the sensor,”
Using the multiphoton effect to harden the biocompatible polymer Ormocer, researchers
created 3-D structures that could be useful in developing lab-on-a-chip-type analytical
devices because they significantly increase the active surface area. Images courtesy
of Maria Farsari.
The group starts with a UV-photocurable
organic-inorganic polymer called Ormocer, from Micro Resist Technology of Berlin,
that is transparent in the visible and near-infrared. The polymer also can be cross-linked
and is mechanically and thermally stable. It takes both UV radiation and the presence
of a photoinitiator to cause the polymer to set. Farsari and her colleagues first
used the three-photon effect to create a 3-D structure from the polymer in 2005.
As described in the Oct. 2 issue of
Applied Physics Letters, to activate the polymer, they first used Amplitude
Systemes’ laser femtosecond oscillator operating at 1028 nm with an average
power of 1 W, a pulse duration < 200 fs and a repetition rate of 50 MHz. For
the recent research, they used an ytterbium laser with similar characteristics.
To control the laser beam, they used an X-Y galvanometric mirror digital scanner
from Scanlabs that they modified for a high-numerical-aperture microscope objective.
A mechanical shutter turns the beam off, and a piezoelectric microscope stage moves
The researchers built several microscopic
components that consist of a latticework of step-in squares, looking something like
After creating the structures, they attached biotin
to them using UV-activated cross-linking of photobiotin. They chose biotin because,
once it has been immobilized, it readily reacts with avidin, which reacts again
with biotin or biotinylated molecules. A wide range of macromolecules, including
proteins, polysaccha-rides and nucleic acids, can be linked readily to biotin without
serious effect on their biological, chemical or physical characteristics. “The
strength of the avidin-biotin bond and the generic nature of the biotinylation process
make avidin-biotin technology highly amenable to selective patterning of various
properties,” Farsari said.
To adhere the biotin to the component
surface, the researchers covered the components with photobiotin, dried the samples
in the dark and exposed them to UV radiation from a KrF laser made by Tui-Laser
AG of Munich, Germany. Then they covered them for at least an hour with Atto 565
streptavidin, a fluorophore that binds strongly to biotin.
After rinsing the excess fluorophore
off the components, the scientists watched the structures’ fluorescence under
a Nikon microscope equipped with a camera and green filter. They observed very strong
fluorescence from each part of the component. Neither Ormocer nor photobiotin fluoresces
when excited by green light. The group saw no fluorescence from components that
had been treated only with biotin. This helped confirm that the fluorescences came
from the bound biotin and not from biotin somehow trapped in the pores of the material.
To determine how much biotin was cross-linked
to the component, they employed a Love wave device. This instrument, which uses
a shear horizontal-surface acoustic wave device and a thin polymer layer as a waveguide,
can monitor the adsorption process because mass adsorbed to the polymer surface
modifies the phase change of the surface acoustic wave.
This was the most challenging part
of the experiment, Farsari said. “The materials system is very complex, and
the fluorescence could originate from other sources,” she said. “The
acoustic wave sensor proved very useful.”
The investigators spin-coated the Ormocer
onto a specially prepared piezoelectric crystal and tested several concentrations
of photobiotin. Each concentration was then cross-linked. Once completed in this
way, the crystals were exposed to an avidin solution, and the binding was monitored
in real time using the device.
The results demonstrated more dramatic
phase changes for higher concentrations of photobiotin. They also showed that the
photobiotin in concentrations exceeding 100 mg/ml achieves saturation of the Ormocer
Farsari said that he and his colleaques
are planning to combine the technique with other methods, with an eye toward creating
patterns of biomolecules by binding them to the immobilized biotin.
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