Università degli studi di Roma La Sapienza
Dottorato di Ricerca in Scienza dei Materiali XIX Ciclo
Synthesis and characterization
of nanostructured polymers
for optical, electronic, and
biological applications
Dott. Iole Venditti
Supervisor:
Prof. Maria Vittoria Russo
i
Index
Chapter 1 Introduction ......................................................................................................................1
1.1 Nanotechnologies ...................................................................................................................1
1.2 Nanostructured materials: bottom up and top down approach ...............................................4
1.3 Nanostructured polymers and their applications ....................................................................7
1.3.1 Optical application: photonic crystals.............................................................................7
1.3.2 Biochemical applications: bioconjugates........................................................................9
1.3.3 Sensors..........................................................................................................................10
Chapter 2 Aim of the thesis.............................................................................................................11
Chapter 3 Materials and instruments...............................................................................................14
3.1 Materials...............................................................................................................................14
3.1.1 Monomers, and catalysts ..............................................................................................14
3.1.2 Enzymes and chemicals for bioactivity tests ................................................................14
3.1.3 Amorphous polymers....................................................................................................15
3.2 Instruments ...........................................................................................................................15
3.2.1 Gravimetric measures ...................................................................................................15
3.2.2 Criostat, centrifuges, and stove.....................................................................................15
3.2.3 Climatic chamber and spinner ......................................................................................15
3.2.4 IR spectroscopy ............................................................................................................15
3.2.5 UV-vis spectroscopy.....................................................................................................16
3.2.6 NMR spectroscopy .......................................................................................................16
3.2.7 GPC measures...............................................................................................................16
3.2.8 DLS (Dynamic Ligth Scattering)..................................................................................16
3.2.9 SEM (Scanning Electron Microscopy) .........................................................................17
3.2.10 Conductometric titrations ...........................................................................................17
3.2.11 Sensoristic measures...................................................................................................17
Chapter 4 Synthesis and characterization of nanostructured polymers ...........................................19
4.1 Nanostructured polymers prepared by modified emulsion polymerization..........................19
4.1.1. PPA nanobeads obtained by Rh(cod)Cl]2tmeda catalyst .............................................19
4.1.2 PPA nanobeads prepared by KPS initiator ...................................................................19
4.1.3 PMMA nanobeads ........................................................................................................21
4.1.4 PMMA Photonic crystal deposition..............................................................................22
4.1.5 PS nanobeads................................................................................................................22
4.1.6 P(S/HEMA) co polymer nanobeads .............................................................................23
4.1.7 P(PA/HEMA), P(PA/AA) and P(PA/DMPA) co-polymers nanobeads........................23
4.2 Preparation of bioconjugates from nanostructured polymers and enzymes: test of
biocatalytic activity ....................................................................................................................25
i
4.2.1 PMMA and PS nanospheres .........................................................................................25
4.2.2 Adsorption and desorption experiments .......................................................................25
4.2.3 Lipolytic assay of free and immobilized lipases...........................................................26
4.2.4 Esterase activity measurements ....................................................................................26
4.2.5 Lipase adsorption kinetics ............................................................................................26
4.2.6 Evaluation of the adsorption isotherms.........................................................................27
4.2.7 pH and thermal stability measurements........................................................................27
4.3 Sensors .................................................................................................................................27
4.4 Nanostructured polymers obtained by a new methodology (European patent) ....................29
4.3.1 PPA...............................................................................................................................31
4.3.2 PMMA..........................................................................................................................31
4.3.3 PS..................................................................................................................................32
Chapter 5 Results and discussion ....................................................................................................33
5.1 Bottom up approach: modified emulsion synthesis ..............................................................33
5.1.1 New polymeric materials for PCs.................................................................................35
5.1.1.1 Synthesis of PPA nanospheres using Rh catalyst..................................................37
5.1.1.2 Synthesis of PPA nanospheres using KPS initiator...............................................40
5.1.1.3 Synthesis of PMMA nanospheres .........................................................................44
5.1.1.4 PMMA PCs deposition .........................................................................................47
5.1.1.5 Synthesis of PS and P(S/HEMA) copolymers ......................................................52
5.1.1.6 Synthesis of P(PA/HEMA), P(PA/AA) and P(PA/DMPA) copolymers...............53
5.2 New polymeric materials for the preparation of Bioconjugates ...........................................59
5.3 Nanostructured polymers applied to Sensors........................................................................66
5.4 Top down approach ..............................................................................................................72
5.4.1 New methodology.........................................................................................................72
Chapter 6 MIT Photonic Bands gap................................................................................................74
6.1 Introduction ..........................................................................................................................74
6.2 The Maxwell Equations in periodic media ...........................................................................76
6.2.1 Bloch waves and Brillouin zones..................................................................................77
6.2.2 The origin of the photonic band gap.............................................................................79
6.2.3 Computational techniques ............................................................................................83
6.3 Three-dimensional Photonic Crystals: Bands Structures calculation ...................................84
6.3.1 User Reference .............................................................................................................84
6.3.2 A Few Words on Units .................................................................................................85
6.3.3 Model of Diamond Lattice of Spheres..........................................................................85
6.3.4 Model applied and modified to PS, PPA, and PMMA Spheres....................................87
Chapter 7 Conclusions ....................................................................................................................90
Summary .........................................................................................................................................92
ii
Tables..............................................................................................................................................96
Table 1........................................................................................................................................96
Table 2a. .....................................................................................................................................97
Table 2b......................................................................................................................................98
Table 3........................................................................................................................................98
Table 4........................................................................................................................................99
Table 5........................................................................................................................................99
Table 6......................................................................................................................................100
Table 7......................................................................................................................................101
Acknowledgments......................................................................................................................... 102
References..................................................................................................................................... 103
iii
Chapter 1 Introduction
1.1 Nanotechnologies
The scientific work on the study of nanoscopic world started at the end of the
fifties, ideally inspired by the Richard Feynman’s talk in 1959 at the California
Institute of Technology (Caltech). Fundamentally, Feynman asserted that “we can
not manipulate the atoms one at a time only because nobody studied the problem
sufficiently; in fact physic principles that hinder this possibility were not know”.
Nanotechnologies are devoted to the manipulation and manufacture of materials
and devices on the nanometric dimensions. The materials structured on
nanometric or subnanometric scale show new properties and functionalities that
can be modulated by selective control of morphology, dimensions and assembling
of particles.
There are two principal reasons for qualitative differences in material behaviour at
the nanoscale (traditionally defined as less than 100 nanometres). First, quantum
mechanical effects come into play at very small dimensions and lead to new
physics and chemistry. Second, a defining feature at the nanoscale is the very
large surface-to-volume ratio of these structures. This means that no atom is very
far from a surface or interface, and the behaviour of atoms at these higher-energy
sites affects significantly the properties of the material.
Quantized effects arise in the nanometre regime because the overall dimensions of
objects are comparable to the characteristic wavelength for fundamental
excitations in materials. For example, electron wave functions (de Broglie wave)
in semiconductors have λ typically on the order of 10 to 100 nanometres. Such
excitations include the wavelength of electrons, photons, phonons, and magnons,
to name a few. These excitations carry the quanta of energy through materials and
thus determine the dynamics of their propagation and transformation from one
form to another. When the size of structures is comparable to the quanta
themselves, it influences how these excitations move through and interact in the
material. Small structures may limit flow, may create wave interference effects,
and may otherwise bring into play quantum mechanical selection rules not
apparent at larger dimensions.
1
Quantum mechanical properties for confinement of electrons in one dimension
have long been exploited in solid-state electronics. Semiconductor devices are
grown with thin layers of differing compositions so that electrons (or “holes” in
the case of missing electron charges) can be confined in specific regions of the
structure (known as quantum wells). Thin layers with larger energy band gaps can
serve as barriers that restrict the flow of charges to certain conditions under which
they can “tunnel” through these barriers (resonant tunnelling diodes) [1-3].
Superlattices are periodic structures of repeating wells that set up a new set of
selection rules which affect the conditions for charges to flow through the
structure. Superlattices have been exploited in cascade lasers to achieve far
infrared wavelengths. The propagation of photons is altered dramatically when the
size and periodicity of the transient structure approach the wavelength of visible
light (400 to 800 nanometres). When photons propagate through a periodically
varying dielectric constant, quantum mechanical rules define and limit the
propagation of the photons depending on their energy (wavelength). This new
behaviour is analogous to the quantum mechanical rules that define the motion of
electrons through crystals, giving band gaps for semiconductors. In one
dimension, compound semiconductor superlattices can be grown epitaxially with
the alternating layers having different dielectric constants, thus providing highly
reflective mirrors for specific wavelengths as determined by the repeat distance of
layers in the superlattice. These structures are used to provide “built-in” mirrors
for vertical-cavity surface-emitting lasers, which are used in communications
applications. In two and three dimensions, periodic structures known as photonic
crystals offer additional control over photon propagation. Photonic crystals are
being explored in a variety of materials and periodicities, such as two-dimensional
hexagonal arrays of posts fabricated in compound semiconductors or stacked
loglike arrays of silicon bars in three dimensions. The dimensions of these
structures depend on the wavelength of light being propagated and are typically in
the range of a few hundred nanometres for wavelengths in the visible and near
infrared. Photonic crystal properties based on nanostructured materials offer the
possibility of confining, steering, and separating light by wavelength on
unprecedented small scales and of creating new devices such as lasers that require
2
very low currents to initiate lasing (called near-thresholdless lasers). These
structures are being extensively investigated as the tools for nanostructuring
materials are steadily advancing. Researchers are particularly interested in the
infrared wavelengths, where dimensional control is not as stringent as at the
shorter visible wavelengths and where optical communications and chemical
sensing provide motivation for potential new applications [4-7].
The preponderance of surfaces is a major reason for the change in behaviour of
materials at the nanoscale. Since up to half of all the atoms in nanoparticles are
surface atoms, properties such as electrical transport are no longer determined by
solid-state bulk phenomena. Likewise, the atoms in nanostructures have a higher
average energy than atoms in larger structures, because of the large proportion of
surface atoms. For example, catalytic materials have a greater chemical activity
per atom of exposed surface as the catalyst is reduced in size at the nanoscale.
Defects and impurities may be attracted to surfaces and interfaces, and
interactions between particles at these small dimensions can depend on the
structure and nature of chemical bonding at the surface. Molecular monolayer
may be used to change or control surface properties and to mediate the interaction
between nanoparticles. Surfaces and their interactions with molecular structures
are basic to all biology. The intersection of nanotechnology and biotechnology
offers the possibility of achieving new functions and properties with
nanostructured surfaces [8-9].
The most part of industries that invest in nanotechnologies come from materials
areas and produce organic, inorganic and metallic nanomaterials. They have
applications as polymers, batteries, electronic devices, cosmetics, sensors, fuel
cells, catalysts, covering of metals. In this context, polymers are potentially ideal
nanoscale building blocks because of their length scale, well-defined architecture,
controlled synthesis, ease of processing and wide range of chemical functionality
that can be incorporated [9-12].
3
1.2 Nanostructured materials: bottom up and top down approach
Two very different paths exist to realize nanostructured materials: the top-down
strategy of miniaturizing current technologies and the bottom-up strategy of
building ever-more-complex molecular devices atom by atom.
Top-down approaches are good for producing structures with long-range order
and for making macroscopic connections, while bottom-up approaches are best
suited for assembly and establishing short-range order at nanoscale dimensions.
The most common top-down approach to fabrication involves lithographic
patterning techniques using short-wavelength optical sources [13,14]. A key
advantage of the top-down approach, as developed in the fabrication of integrated
circuits, is that the parts are both patterned and built in place, so that no assembly
step is needed. Optical lithography is a relatively mature field because of the high
degree of refinement in microelectronic chip manufacturing, with current shortwavelength optical lithography techniques reaching dimensions just below 100
nanometres (the traditional threshold definition of the nanoscale). Shorterwavelength sources, such as extreme ultraviolet and X-ray, are being developed to
allow lithographic printing techniques to reach dimensions from 10 to 100
nanometres. Scanning beam techniques such as electron-beam lithography
provide patterns down to about 20 nanometres [15,16]. Here the pattern is written
by sweeping a finely focused electron beam across the surface. Focused ion
beams are also used for direct processing and patterning of wafers, although with
somewhat less resolution than in electron-beam lithography. Still-smaller features
are obtained by using scanning probes to deposit or remove thin layers.
Mechanical printing techniques (nanoscale imprinting, stamping, and moulding)
have been extended to the surprisingly small dimensions of about 20 to 40
nanometres [17]. The details of these techniques vary, but they are all based on
making a master “stamp” by a high-resolution technique such as electron-beam
lithography and then applying this stamp, or subsequent generations of it, to a
surface to create the pattern. In one variation a stamp's surface is coated with a
very thin layer of material (the “ink”) that can then be deposited (“inked”) directly
onto the surface to reproduce the stamp's pattern. For example, the controlled
patterning of a molecular monolayer on a surface can be achieved by stamping an
4
ink of thiol functionalized organic molecules directly onto a gold-coated surface
(molecules that contain a sulfur end group, called a thiol, bond strongly to gold).
In another approach the stamp is used mechanically to press the pattern into a thin
layer of material. This surface layer is typically a polymeric material that has been
made pliable for the moulding process by being heated during the stamping
procedure. Plasma etching can then be used to remove the thin layer of the
masking material under the stamped regions; any residual polymer is thus
removed, and a nanoscale lithographic pattern is left on the surface. Still another
variation is to make the relief pattern out of photoresist on a silicon wafer by
optical or electron-beam lithography then pour a liquid precursor over the pattern,
and finally cure it. The result is a rubbery solid that can be peeled off and used as
a stamp. These stamps can be inked and printed; they can also be pressed to the
surface then a liquid polymer allowed to flow into the raised regions of the mask
by capillary action and cured in place. A characteristic of this latter approach is
that the stamp is flexible and can thus be used to print nanoscale features on
curved surfaces. These nanoscale printing techniques offer several advantages
beyond the ability to use a wider variety of materials with curved surfaces. In
particular, such approaches can be carried out in ordinary laboratories with farless-expensive equipment than that needed for conventional submicron
lithography. The challenge for all top-down techniques is that, while they work
well at the microscale (at millionths of a metre), it becomes increasingly difficult
to apply them at nanoscale dimensions. A second disadvantage is that they
involve planar techniques, which means that structures are created by the addition
and subtraction of patterned layers (deposition and etching), so arbitrary threedimensional objects are difficult to construct.
Bottom-up, or self-assembly, approaches to nanofabrication use chemical or
physical forces operating at the nanoscale to assemble basic units into larger
structures. As component size decreases in nanofabrication, bottom-up approaches
provide an increasingly important complement to top-down techniques.
Inspiration for bottom-up approaches comes from biological systems, where
nature has harnessed chemical forces to create essentially all the structures needed
5
by life. Researchers hope to replicate nature's ability to produce small clusters of
specific atoms, which can then self-assemble into more-elaborate structures.
A number of bottom-up approaches have been developed for producing
nanoparticles, ranging from condensation of atomic vapours on surfaces to
coalescence of atoms or molecules in liquids [18-20]. Emulsion synthesis is the
main technique developed to produce polymeric or composite nanobeads with
defined size and containing specific functional groups [21-25].
Emulsion synthesis polymerization can produce highly monodispersed spherical
particles from polymers such as polystyrene, polymethylmethacrylate, etc. If the
particles are highly charged, because they have covalently attached ionizing
groups, they will self-assemble in solution into highly ordered, non-close-packed
three-dimensional arrays, known as crystalline colloidal arrays (CCAs), in which
the lattice spacing are much greater than the particle diameters.
In fact, once obtained the nanostructured particles, their self-assembling has yet to
be realised. Low polidispersity is fundamental to obtain low defect density and
long-range ordering of self-assembling nanospheres. For example, a recent study
on silica nanoparticles concerning the effect of polidispersity on the dimension of
well ordered domain of self-assembled nanospheres reports that the presence of
spheres with different dimensions is directly responsible for line defects [26].
Production of accurately size-controlled nanosphere with predictable and
reproducible characteristics requires in turn thorough control of the synthesis
procedure, conditions and parameters.
The conditions of self-assembled nanoparticles deposition must be selected in
order to enhance the ordering action arising from the particles interactions, which
are due to interparticle forces, such as lateral capillary forces (LCF), flotation
forces, convection forces, and electrostatic forces when the particles are
electrically charged. Control on the action of these forces is also important
because they were shown to influence the crystal domain size and particle
orientation
for
nanospheres
of
different
materials
(silica,
polystyrene/polymethylmethacrylate/polyacrylate copolymers) [27-32].
A different example of self-assembly that achieves a limited degree of control
over both formation and organization is the growth of quantum dots. Indium
6
gallium arsenide (InGaAs) dots can be formed by growing thin layers of InGaAs
on GaAs in such a manner that repulsive forces caused by compressive strain in
the InGaAs layer results in the formation of isolated quantum dots [33]. After the
growth of multiple layer pairs, a fairly uniform spacing of the dots can be
achieved. Another example of self-assembly of an intricate structure is the
formation of carbon nanotubes under the right set of chemical and temperature
conditions [34].
1.3 Nanostructured polymers and their applications
Nanotechnology may allow to manufacture lighter, stronger, and programmable
materials that require less energy to produce than conventional materials, that
produce less waste than with conventional manufacturing, and that promise
greater fuel efficiency in land transportation, ships, aircraft, and space vehicles.
Nanocoatings for both opaque and translucent surfaces may render them resistant
to corrosion, scratches, and radiation. Nanoscale electronic, magnetic, and
mechanical devices and systems with unprecedented levels of information
processing may be fabricated, as may chemical, photochemical, and biological
sensors for protection, health care, manufacturing, and the environment; new
photoelectric materials that will enable the manufacture of cost-efficient solarenergy panels; and molecular-semiconductor hybrid devices that may become
engines for the next revolution in the information age. The potential for
improvements in health, safety, quality of life, and conservation of the
environment are vast.
Thereafter specific application areas of nanostructured polymers are discussed.
1.3.1 Optical application: photonic crystals
The terms “photonic crystal” and “photonic band gap” are now widely used by
research workers in the field of optoelectronics. For people concerned with the
possibility of applying new physical concepts to technological developments, the
first challenge was to identify possible ways to the fabrication of structures with a
significant fraction of the properties of the ideal photonic band gap structure, as
initially set out by Yablonovitch and John [35,36]. Photonic band structures occur
7
if light travels through a three dimensional dielectric lattice with a refractive index
that varies periodically on length scales comparable to the wavelength; they are
analogous to electronic band structures in atomic crystals. If, for some frequency
range, a photonic crystal reflects light of any polarisation totally at any incident
angle, the crystal is said to have an absolute photonic band gap (PBG). In such a
photonic crystal, no electromagnetic wave can propagate if the frequency is within
that PBG range.
Several materials are used as basic building blocks for assembling ordered
systems: inorganic materials (e.g., silica, titania), and organic materials (e.g.,
polystyrene, polyacrylates), the latter ones having the advantages of greater
synthetic flexibility. Among them, polymethylmethacrylate (PMMA) and
polymethylmethacrylate/polystyrene (PMMA/PS) copolymers are of particular
interest, because they can be readily synthesized in the form of nanospheres as
reported by Muller, Asher and co-workers [37-40] and by Underwood et al. [41].
Successful preparation of PCs depends on the strict control of the chemical and
physical characteristics of the nanospheres and on the implementation of
appropriate deposition techniques. In fact, the photonic band structure of the
material and thus its interaction with light are determined by the chemical
structure and properties of the materials, particles dimensions, and packing order.
Low polidispersity is fundamental to obtain low defect density and long-range
ordering of the nanospheres, which is necessary to produce good quality photonic
crystals. Studies on silica nanoparticles [26] concerning the effect of
polidispersity on the presence of stacking faults, vacancies, and interstitials in
hard-spheres photonic crystals, report that the presence of spheres with different
dimensions is responsible for line dislocations propagating in the crystals;
moreover, when the polydispersity is high, the quantity of interstitial sites
increases dramatically, while the vacancy concentration remains relatively
constant. Production of accurately size-controlled nanosphere with predictable
and reproducible characteristics requires in turn thorough control of the synthesis
procedure, conditions, and parameters.
As for the PCs deposition conditions, these must be selected in order to enhance
the ordering action arising from the particles interactions, which are due to
8
interparticle forces, such as lateral capillary forces (LCF), flotation forces,
convection forces, and electrostatic forces when the particles are electrically
charged. Control on the action of these forces is also important because they were
shown to influence the crystal domain size and particle orientation [28-32] for
nanospheres of different materials (silica, polystyrene / polymethylmethacrylate /
polyacrylate copolymers).
1.3.2 Biochemical applications: bioconjugates
Nanostructured biopolymers and biopolymer/inorganic hybrids have the potential
to become important industrial materials because of their biofunctionality, tunable
surface functionalities, relative cheapness, food and drug compatibility and benign
environmental profiles. So far the understanding of the dissolution of several
naturally biopolymers, their solution behaviour, and their colloidal properties in
solution have been studied. This information, in combination with nanostructuring
inorganic systems has allowed making initial progress towards novel
nanostructured architectures across a range of biopolymer systems. There is
strong evidence that this approaches will have potential applications in functional
films, packaging, and delivery systems across a range of several business sectors.
For example the enzyme immobilization problem is an attractive application area.
Enzymes are interesting compounds, commonly used for the synthesis of
enantioenriched compounds of industrial applications, for clinical analysis and for
polymerization reactions [42-45]. For the improvement of their stability,
separation and reuse, and continuous operation, their immobilization has been
studied extensively. Often the low catalytic efficiency of these biocatalysts can
limit their use in the development of large scale bioprocessing and cannot
compete with traditional chemical processing. [46-49]. A novel approach to
improve the efficiency of immobilized lipases is to manipulate the structure of
carrier materials.
To date, extensive efforts have been conducted to optimize the structure of the
carrier’s materials to make more efficient biocatalysts [50-55]. Nonporous carrier
materials, to which surfaces the enzymes are attached, are subject to minimum
diffusion limitation. However, enzyme loading per unit mass of support is usually
9
considerably low. Alternatively, high enzyme loading can be achieved with
porous materials. Porous materials, on the other hand, suffer a much greater
diffusion limitation.
Nanostructured materials will provide the upper limits in terms of balancing the
contradictory issues including surface area, mass transfer resistance, and effective
enzyme loading. Recently reported work in this area has revealed the great
potential for the use of nonporous, nanofibrous and nanoparticle materials as a
new class of carriers for biocatalysts [56-59]. The effective enzyme loading on
nanomaterials can be very high and a large surface area per unit mass is also
provided to facilitate reaction kinetics. Moreover, the nanoparticle hybridization
with biomolecules is expected to modify the nanoparticles properties and their
interaction with the environment.
1.3.3 Sensors
Sensors are central to almost all modern control systems. For example, multiple
sensors are used in automobiles for such diverse tasks as engine management,
emission control, security, safety, comfort, vehicle monitoring, and diagnostics.
While such traditional applications for physical sensing generally rely on
microscale sensing devices, the advent of nanoscale materials and structures has
led to new electronic, photonic, and magnetic nanosensors, sometimes known as
“smart dust” [60,61]. Because of their small size, nanosensors exhibit
unprecedented speed and sensitivity, extending in some cases down to the
detection of single molecules. For example, nanowires made of carbon nanotubes,
silicon, or other semiconductor materials exhibit exceptional sensitivity to
chemical species or biological agents. Electrical current through nanowires can be
altered by having molecules attached to their surface that locally perturb their
electronic band structure. By means of nanowire surfaces coated with sensor
molecules that selectively attach particular species, charge-induced changes in
current can be used to detect the presence of those species. This same strategy is
adopted for many classes of sensing systems [62-65].
10
Chapter 2 Aim of the thesis
For this PhD thesis the research activity was focused on the synthesis and
characterization of nanostructured polymers for applications in optics (photonic
crystals), sensors and biocatalysis (enzymes-polymeric nanospheres hybrid).
The aim was to realise nanostructured polymers by adopting both the bottom up
approach (modified emulsion synthesis technique) and the top down approach
(Method for controlling the dimensions and the morphology of nanostructued
polymeric materials, PCT Int. Appl. (2006) CODEN: PIXXD2 WO 2006051572
A2 20060518). All new materials were characterized by spectroscopic techniques
(UV-vis, IR, 1H and 13C NMR), gel permeation chromatography (GPC), dynamic
light scattering (DLS), and scanning electron microscopy (SEM).
In particular, by the bottom up approach the synthesis of monodispersed
nanospheres
of
polymethylmethacrylate
(PMMA),
polystyrene
(PS),
polyphenylacetylene (PPA) will be studied. Moreover, the novel synthesis of
copolymers
(S=styrene,
nanobeads
PS/HEMA,
PPA/HEMA,
PA=phenylacetylene,
PPA/AA,
PPA/DMPA
HEMA=2-hydroxyetylmethacrylate,
AA=acrylic acid, DMPA=N,N-dimethylpropargylamine) will be also investigated
by a modified emulsion polymerization. A systematic study of the influence of the
reaction conditions on the chemico-physical characteristics of the nanobeads with
the aim of achieving their self-assembling will be considered. In particular the
role of the cosolvent and time reaction on the particles size, and the effect of the
surface-charge in the copolymers on ordered domain formation are important
topics to be investigated.
Attempts to use several polymeric nanospheres for preparation of bioconjugates
(enzymes-polymeric nanobeads hybrids) and gas sensors are foreseen. As a first
approach nanostructured polystyrene (PS) and polymethylmethacrylate (PMMA)
will be used as carriers for the preparation of bioconjugates with lipolytic
enzymes and studies on the activity of the bioconjugates will be carried out.
The applications of nanostructured polymers and copolymers in resistive sensors
for the detection of gases will be investigated.
11
Theoretical studies will be devoted to understand the photonic crystals properties
of the synthesised polymeric materials.
12
Experimental Section
13
Chapter 3 Materials and instruments
3.1 Materials
3.1.1 Monomers, and catalysts
Deionized water is obtained by Millipore-Q RG (CPMQ004R1); the PA
(phenylacetylene)
monomer
(Aldrich
99%
pure)
and
DMPA
(N,N-
dimethylpropargyl amine) monomer (Aldrich 97% pure) were distilled under
reduced pressure before use. MMA (methylmethacrylate) monomer (Aldrich 99%
pure),
S
(styrene)
monomer
(Aldrich
99%
pure),
HEMA
(2,hydroxyethylmethacrylate) monomer (Aldrich 98% pure), AA (acrylic acid)
monomer (Aldrich 99% pure).
KPS (potassium persulfate) initiator, (Aldrich 99,99% pure), Rh(I) catalyst
(chloro(1,5-cyclooctadiene)rhodium(I) dimer), (Aldrich 98% pure), SDS (sodium
dodecyl sulphate) emulsifier (Aldrich 99% pure) and the other organic solvents
(Aldrich reagent grade) were used as received.
The catalyst [Rh(COD)Cl]2t-meda (COD = cis,cis-1,5-cyclooctadiene, t-meda =
N,N,N’,N’-tetramethylethylendiamine) was synthesized according to a published
method [66].
3.1.2 Enzymes and chemicals for bioactivity tests
Water was distilled and the others solvent were Aldrich (reagent grade). Lipase
(EC 3.1.1.3) from Candida rugosa (CRL), type VII 890 U/mg, 1(±)phenylethanol, R(+) phenylethanol, S(-) phenylethanol, phenylethylacetate,
vinylacetate, tributyrin, organic solvents analytical grade, bovin serum albumin
(BSA, molecular mass: 67,000 Da), 2-morpholino-ethane sulfonic acid (MES),
used as received, were purchased from Sigma-Aldrich. Lipase from Pseudomonas
cepacia (PCL) was kindly provided by Amano Pharmaceuticals Co. Ltd. (Nagoya,
Japan).
14
3.1.3 Amorphous polymers
The amorphous PPA was synthesised as reported in literature [66]. The
commercial PMMA and PS were Goodfellow, (analysis pure). Water was distilled
and the others solvent were Aldrich (reagent grade). Cellulose membranes were
Sigma (citocromo retention C – 12400 >90% in 10 h).
3.2 Instruments
3.2.1 Gravimetric measures
The gravimetric measures of solid products and materials were carried out on
analytic scales Gibertini Mod.E50S.
3.2.2 Criostat, centrifuges, and stove
Cryostat Haake GH (Fisons) and refrigerated ultracentrifuga ALC PK121 R were
used to obatained nanostructured polymers by European Patent method. To
recuperate the washed nanostructured polymers obtained by modified emulsion
synthesis was used Centrifuge 4226. The stove used was Selecte Digitheat.
3.2.3 Climatic chamber and spinner
Climatic chamber used for controlled deposition of polymeric nanospheres for
photonic crystals was Angelantoni UY150. The spinner was Electron-Mec—PRS5V-Spinner.
3.2.4 IR spectroscopy
FTIR spectra were recorded as nujol mulls or as films deposited by CHCl3
solutions by using CsI cells, on a Perkin Elmer 1700X Fourier Transform
spectrometer and Bruker Vertex70.
15
3.2.5 UV-vis spectroscopy
Uv-vis spectra were carried out on Varian Cary100Scan UV-visible
spectrophotometer; the samples were analysed as solutions of common organic
solvents.
3.2.6 NMR spectroscopy
1
H and
13
C NMR spectra were recorded on Varian Mercury 300 spectrometer in
CDCl3 solution. The calibration was performed by internal reference: chemical
shifts (ppm) were referenced to TMS for 1H NMR, assigning the residual 1H
impurity signal in the solvent at 7.24 ppm (CDCl3).
3.2.7 GPC measures
Molecular weights were determined by Gel Permeation Chromatography by
(GPC) using a Perkin-Elmer GPC-HPLC apparatus with a LC250 pump, LC oven,
PL GEL 10 µm MIX analytical column, UV-VIS LC 90 J detector. Measurements
were performed in THF and CHCl3 (HPLC grade) as eluent, using suitable wave
length for the detection, flow rate 1mL/min; the calibration was performed using
polystyrene standards.
The progress of the reaction, for ester production, was followed by GC using a
capillary column of polyphenyl-methylsiloxane (MEGA 25 mx0.53 mmID) and a
flame ionization detector, nitrogen as carrier gas, at pressure of 0.8 bar and 473°K
for the detector. The oven gradient temperature was set in the range from 70°C to
130°C with a rate of 40°C/min. The enantiomeric resolution was obtained
employing two serial capillary columns: the first one achiral (OV-1:10mx0.53 ID)
the other one chiral (2,3 0-dimethyl-6-penthylBeta, ciclodextrin, 30% OV
1701,25mx0.25 ID).
3.2.8 DLS (Dynamic Ligth Scattering)
Nanobeads diameters and polidispersity were analyzed by DLS technique. DLS
measurements were performed using a Malvern PCS 4800 apparatus on samples
diluted in deionised water and the experimental data were analyzed by the
16
cumulants method in order to obtain the first (Z average) and second moment
(polidispersity) of the particle distribution function.[67]
3.2.9 SEM (Scanning Electron Microscopy)
The diameter of the beads and their polidispersity were determined by SEM; in
particular we obtained the polidispersity index (PI) using the formula:
PI = (dmax-dmin)/daverage
where d is the particles diameter in nm. SEM imaging was carried out using a
SEM-LEO1450VP on metalled samples and SEM-FEG LEO1530 for samples
deposited on graphite. Films of polymer nanobeads were deposited by casting or
spinning of the aqueous emulsion on glass probes; the dimensions of crystalline
domains were determined from the SEM images by using an image analysis
software tool (Scion Image for Windows, Scion Corp, Beta 4.0.2). We considered
domains limited by extended defects (grain boundaries, stacking faults,
dislocations) and also cracks were considered as domain-delineating defects,
while we did not consider point defects as domain limits; for each sample 10
images at about 8000 times magnification (the best condition to discriminate the
domains and their defects) were examined and then we considered the best
measurement result.
3.2.10 Conductometric titrations
The particle surface charge was measured by conductometric titration against
NaOH [68].
3.2.11 Sensoristic measures
The electrical characteristics of the systems were measured by specific apparatus:
a Multi Gas Controller 147 MKS, to controll the nitrogen stream passed through a
water reservoir, an alimetator Thandar TS3022S, for sensor of reference humidity
and temperature, Keithlei 595 Quasistatic CV Meters, for current measures, an
Keithlei 590 Analyser, for capacity measures, an Keithlei 2001 Multimeter, for
voltage measures of references sensors. The reference commercial hygrometer
17
was Honeywell HIH3610. All measures were realised by using two software
applications (Vier-TH023C and QB-BIGT).
18
Chapter 4 Synthesis and characterization of nanostructured
polymers
4.1 Nanostructured polymers prepared by modified emulsion polymerization
4.1.1. PPA nanobeads obtained by Rh(cod)Cl]2tmeda catalyst
PPA nanospheres were synthesised by using a modified emulsion polymerization
in aqueous dispersion (see Figure 1 and Table 1) as reported in our work [69]. In a
typical procedure, 50 mL of deionised water was degassed under a moderate flow
of Ar, for 30 min; then the calculated amounts of phenylacetylene and toluene (if
appropriate) were added and the mixture was stirred at 80 °C for 2 h in order to
make a homogeneous suspension.
Solution of [Rh(cod)Cl]2tmeda ([catalyst] = 2.0x10-4 M, satured liquid) was added
under Ar flow and the reaction was refluxed under vehement stirring in Ar
atmosphere. The polymerisation was stopped with a cold ice bath (yield ~60%)
and the emulsion of polymer nanospheres was separated from unreacted monomer
and toluene by settling and from catalyst and bulk polymer (side reaction products
always present) by filtration, centrifugation, and washing with deionised water.
Films of PPA were prepared by dropping a small amount of the emulsion onto
glass substrates and subsequent drying at room temperature and normal pressure
or by spinning or casting. The diameters of the beads were determined directly
from SEM images. The obtained materials were characterised by means of
conventional techniques as UV-vis, IR, spectroscopy, Gel Permeation
Chromatography (GPC). UV-VIS spectra in CHCl3 solution showed continuous
absorption from λ≤400 nm. IR (cm-1): 3050 (νaromatic
CH),
1597 (νaromatic
C=C).
Molecular weights of the samples, measured by GPC, resulted Mw =3-10 x 103
amu with Mw/Mn = 1.8-2.3.
4.1.2 PPA nanobeads prepared by KPS initiator
PPA nanospheres were synthesised by using a modified emulsion polymerization
with KPS initiator as reported in our work [70] (see Figure 1 and Table 1). In a
19
Figure 1. Reaction schemes of nanostructured PPA (a) and PMMA (b) synthesis;
(c) structure formula of nanostructured synthesised copolymers P(S/HEMA),
P(PA/HEMA), P(PA/AA), P(PA/DMPA).
*
Ar,90°C
(a)
n
*
Rh(I) or KPS
PPA
CH3
CH3
Ar,90°C
C
HC
(b)
CH3
KPS
CH3
CH3
*
CH3
C
*
CH2 n
C
O
O
m
O
O
O
(c)
PMMA
m
*
P(PA/HEMA)
P(S/HEMA)
H
C
m
HO
CH3
C
*
CH2 n
C
O
O
CH2CH2OH
CH2CH2OH
*
n
C
C
O
*
CH2
*
CH2 n
O
H
C
*
*
m
C
n
*
CH2
N(CH3)2
P(PA/AA)
P(PA/DMPA)
20
typical procedure 20 mL of deionised water, 0.936 g (0.01 mol) of PA and the
proper quantity of toluene were degassed for 15 minutes and then stirred in Ar
atmosphere, at 80°C for 1 hour; then KPS solution was added and the reaction
was refluxed under vehement stirring. The polymerization was stopped by
opening the flask and the light yellow emulsion (yield < 10%) was filtered
through a standard paper filter to get rid of large agglomerates, and then
centrifuged and re-dispersed with deionised water several times, in order to
remove the unreacted monomers and toluene.
Films of PPA were prepared by spinning or casting a drop of suspension at room
temperature and normal pressure. The diameters of the beads were determined
directly from SEM image. The obtained materials were characterised by means of
conventional techniques: UV-vis, IR, 1H 13C NMR spectroscopy, Gel Permeation
Chromatography (GPC). UV-VIS spectra in CHCl3 solution showed continuous
absorption from λ≤400 nm. IR (cm-1): 3050 (νaromatic
1
CH),
1597 (νaromatic
C=C).
HNMR (δ ppm): 5.84 (s H polymeric chain), 6.6 (d H phenyl ring) 6.9 (d H
phenyl ring);
13
CNMR (ppm): 127.7, 127.5, 126.5 (C m o p benzene ring),
131.7(C vinylic carrying H of the main chain), 142.8, 139.3 (C quaternary of
chain and benzene ring). Molecular weights of the samples, measured by GPC,
resulted Mw = 1-10 x 103 amu with Mw/Mn =1.6-2.0.
4.1.3 PMMA nanobeads
PMMA nanobeads were synthesised by using a modified emulsion polymerization
in aqueous dispersion (see Figure 1 and Table 2a) as reported in our work [27]; in
a typical procedure, 40 mL of deionised water, 14.00 g (0.14 mol) of
methylmethacrylate and the proper quantity of toluene, used as cosolvent, were
stirred in Ar atmosphere, at 90°C for 2 hours; then 500 mg of potassium persulfate
(10 mL of 50 g/L degassed solution in deionised water) were added and the
reaction was refluxed under vehement stirring in Ar atmosphere. The
polymerization was stopped by opening the flask; the milky white and opalescent
emulsion (yield ~80%) was filtered through a standard paper filter to get rid of
large agglomerates, and then centrifuged and re-dispersed with deionised water
(two to five times) in order to remove the unreacted monomer and toluene. The
21
reaction kinetic was studied by withdrawal of emulsion (10 mL) from the batch
reaction by a cannula at fixed times. IR (cm-1): 3000, 2950 (νC-H); 1735 (νC=O),
1484, 1380 (νCH); 1270, 1240, 1191, 1149 (νC-O). 1HNMR (δ ppm): 3.57 (s, 3H,
OCH3), 1.8 (m, 2H, CH2), 1.18-0.99-0.81 (s, CH3).
13
CNMR (ppm): 177.8
(CO2Me), 54.4 (CH2), 51.8 (OCH3), 44.9 (C chain), 16.5 (CH3). Molecular
weights of the samples, measured by GPC, resulted Mw = 1-5 ×103 amu with
MW/Mn = 1.5-1.8.
4.1.4 PMMA Photonic crystal deposition
Films of PMMA nanobeads with diameter of 250 nm were prepared by casting a
drop of emulsion onto different substrates under controlled conditions and were
characterized by SEM technique. Depositions of PMMA suspension at different
relative humidity values (RH%) were performed at room temperature in climatic
chamber in the presence of a saturated solution of different salts (NaOH·H2O:
RH= 6%; KOH·2H2O: RH= 9%; CaCl2·6H2O: RH= 29%; Ca(NO3)2·6H2O: RH=
51%; NH4NO3: RH=62%; NaCl: RH= 75%; KCl: RH= 84%). Depositions at
selected temperature (in the range T=2-50°C) were made inside a thermostatic
chamber at RH = 45- 55%. Depositions from suspensions with different ionic
strength were carried out at room temperature and humidity, by using NaCl or
MgSO4 solutions at different concentrations that were added to the emulsion and
shaken for 10 min. The different substrates used for the deposition of polymeric
films were: cover glass, silica, gold, and graphite.
4.1.5 PS nanobeads
PS nanobeads were realised by using a modified emulsion polymerization in
aqueous dispersion (see Table 3); in a typical procedure, 9 mL of deionised water,
0.40 g (0.004 mol) of styrene and the proper quantity of SDS, used as emulsifier,
were stirred in Ar atmosphere, at 90°C for 2 hours; then 180 mg of KPS
(dissolved in 2 mL of deionised water and then degassed for 5 minutes) were
added and the reaction was refluxed under vehement stirring in Ar atmosphere.
The polymerization was stopped by opening the flask; the milky white emulsion
was filtered through a standard paper filter to get rid of large agglomerates, and
22
then centrifuged and re-dispersed with deionised water several times in order to
remove the unreacted monomer, toluene, and the emulsifier.
4.1.6 P(S/HEMA) co polymer nanobeads
The copolymer P(S/HEMA) nanobeads (see Figure 1) were synthesized by using
a modified emulsion polymerization in aqueous dispersion, using the reaction
conditions (time, toluene, KPS and HEMA amount) reported in Table 3. In a
typical procedure 120 mL of deionised water, 35 g (0.336 mol) of S and the
proper quantity of toluene and HEMA, were degassed for 30 minutes with Ar and
then stirred in Ar atmosphere, at 80°C for 1 hour; then KPS solution was added
and the reaction was refluxed under vehement stirring in Ar atmosphere. The
polymerization was stopped by opening the flask (yield ~80%)and the emulsion
was filtered through a standard paper filter to get rid of large agglomerates, and
then centrifuged and re-dispersed with deionised water four times, in order to
remove the unreacted monomers and toluene.
IR (cm-1): 3450 (νOH), 3050 (νaromatic CH), 2950 (νC-H), 1725 (νC=O), 1597 (νaromatic
C=C),
1484 (νCH); 1025 (νC-O). Molecular weights of the samples, measured by
GPC, resulted Mw = 5-330 ×103 amu with MW / Mn = 1.2-2.1.
4.1.7 P(PA/HEMA), P(PA/AA) and P(PA/DMPA) co-polymers nanobeads
The P(PA/HEMA) P(PA/AA) and P(PA/DMPA) copolymers nanobeads were
realised by modified emulsion synthesis, using procedure reported in 4.1.6
paragraph (see Figure 1 and Table 4).
For P(PA/HEMA) nanospheres synthesis the typical procedure used 20 mL of
deionised water, 0.936 g (0.01 mol) of PA and the proper quantity of toluene and
HEMA, degassed for 15 minutes with Ar and then stirred in Ar atmosphere, at
80°C for 1 hour; then KPS solution was added and the reaction was refluxed
under vehement stirring in Ar atmosphere. Besides the addition of the KPS
solution, also 1 ml of aqueous solution of [Rh(COD)Cl]2tmeda (2 g/L) was poured
into the reaction mixture. The polymerization was stopped by opening the flask
and the light yellow emulsion (yield < 10%) was filtered through a standard paper
23
filter to get rid of large agglomerates, and then centrifuged and re-dispersed with
deionised water several times, in order to remove the unreacted chemicals.
UV-vis spectra in CHCl3 solution showed continuous absorption from λ≤400 nm.
IR (cm-1): 3450 (νOH), 3050 (νaromatic CH), 2950 (νC-H), 1725 (νC=O), 1597 (νaromatic
C=C),
1484 (νCH); 1025 (νC-O), 755 (δCH). 1HNMR (δ ppm): 1.18-0.99-0.81 (s,
CH3), 1.8 (m, CH2), 3.4 (s, OCH2), 5.84 (s, H polymeric chain), 6.9, 7.2 (m, H
phenyl ring); 13CNMR (ppm): 177.8 (CO2), 127.7, 127.5, 126.5 (C m o p benzene
ring), 131.7(C vinylic carrying H of the main chain), 142.8, 139.3 (C quaternary
of chain and benzene ring), 78-76 (m, OCH2), 16.5 (CH3). Molecular weights of
the samples, measured by GPC, resulted Mw = 10-30 ×103 amu with MW / Mn =
1.8-2.3.
P(PA/AA) nanobeads were prepared by this specific procedure: a 50 mL of
deionised water, 0.936 g (0.01 mol) of PA and the proper quantity of toluene and
AA, were degassed for 15 minutes and then stirred in Ar atmosphere, at 80°C for
1 hour; then KPS and APS solution was added and the reaction was refluxed
under vehement stirring in Ar atmosphere; polymerization was stopped by
opening the flask (yield ~50%) and the light yellow emulsion was filtered,
centrifuged and re-dispersed with deionised water seven times, in order to remove
the unreacted chemicals.
UV-vis spectra in CHCl3 solution showed continuous absorption from λ≤400 nm.
IR (cm-1): 3050 (νaromatic CH), 1670 (ν COOH), 1597 (νaromatic C=C), 770 (δCH).
The typical procedure for P(PA/DMPA) nanospheres synthesis was: 100 mL of
deionised water, 0.936 g (0.01 mol) of PA and the proper quantity of toluene and
DMPA, were degassed for 15 minutes and then stirred in Ar atmosphere, at 80°C
for 1 hour; then KPS and Rh (I) catalyst solution was added and the reaction was
refluxed under vehement stirring in Ar atmosphere; polymerization was stopped
by opening the flask (yield ~40%)and the light yellow emulsion was filtered,
centrifuged and re-dispersed with deionised water seven times, in order to remove
the unreacted chemicals.
UV-vis spectra in CHCl3 solution showed continuous absorption from λ≤400 nm.
IR (cm-1): 3400 (νaminoCH), 3050 (νaromatic CH), 1597 (νaromatic C=C), 770(δCH).
24
4.2 Preparation of bioconjugates from nanostructured polymers and
enzymes: test of biocatalytic activity
4.2.1 PMMA and PS nanospheres
Monodispersed nanoparticles of PMMA and PS were synthesized in four different
sizes according to literature report [27,71], and used as support for the
immobilization of enzymes. The dimension and monodispersity of the
bioconjugates were determinated with SEM measurements. Dried nanoparticles
on silica support were analyzed at 15-20 kV by a scanning electron microscope
after metallization by gold coating.
4.2.2 Adsorption and desorption experiments
CRL and PCL were adsorbed onto PS and PMMA nanoparticles via physical
adsorption and control experiments were carried out to confirm that the enzymes
were firmly attached to the particles and no free enzymes leached off the particles
during the reaction. These experiments were based on the routine evaluation of
the enzymatic activity.
A typical adsorption experiments of CRL and PCL onto PMMA and PS
nanoparticles were performed in pyrex tubes with polymer dispersion of 50 mg/ml
(w/v) by using buffer solutions of lipase at constant pH in a total volume of 2 ml,
under gentle shaking at room temperature (25°C). The time course of lipase
adsorption was followed during 3 hours and the samples were filtrated through a
cellulose nitrate filter membrane (Whatman, pore diameter 0.01-0.2 um) to
separate the particles completely.
The amount of immobilized enzyme was obtained by the standard Bradford assay
[72] of the original lipase solutions, the supernatants and the washing solutions
after
the
immobilization.
The
protein
concentration
was
determined
spectrophotometrically measuring the adsorption peak at 595 nm.
The immobilization of lipases on PMMA and PS particles was tested by surface
desorption experiments. The enzyme-covered particles were redispersed in a fixed
amount of 20 mM phosphate buffer solutions, equilibrated for 24 h to ensure a
steady state and filtered again by proper cellulose filters. The desorbed protein
25
was examined by UV measurements on the supernatant. Starting from the redispersion step, this procedure was executed at various temperatures to check for
heat-induced desorption.
4.2.3 Lipolytic assay of free and immobilized lipases
The activity of free and immobilized lipase preparations was determined
according to the standard assay of hydrolysis. In the standard conditions, the
reaction mixture composed by 2.5 ml of PBS, 0.5 ml of tributyrin and 0.1 ml of
enzyme solution (50mg/ml) was incubated at 37°C under magnetic stirring
(300rpm) for 30 min. The reaction was stopped with 3.5 ml of acetone/ethanol
mixture 1:1 and the reaction mixture titrated with 0.1 M NaOH in the presence of
phenolphthalein as indicator using an automatic burette (Methrom).
4.2.4 Esterase activity measurements
Esterase activities of CRL and PCL free and immobilised on nanostructured
polymers were derived from the rate of transesterification reaction of 1phenylethanol (0.25mM) with vinylacetate (1.25 mM) in organic solvents with an
amount of lipase of 5mg/ml. The mixture was incubated at different temperatures
for 6 h under magnetic stirring. Samples were taken for analysis at regular
intervals. The progress of the reaction, for ester production, was followed by GC.
The oven gradient temperature was set in the range from 70°C to 130°C with a
rate of 40°C/min. The enantiomeric resolution was obtained employing two serial
capillary columns: the first one achiral (OV-1:10mx0.53 ID) the other one chiral
(2,3 0-dimethyl-6-penthylBeta, ciclodextrin, 30% OV 1701,25mx0.25 ID).
4.2.5 Lipase adsorption kinetics
The adsorption of CRL and PCL onto nanostructured PMMA and PS was
performed with polymer dispersions of 50 mg/ml (w/v).
Adsorption kinetics were carried out in 10 ml buffer solutions at constant pH and
at the room temperature (25°C) by using a fixed lipase concentration. The time
course of CRL and PCL adsorptions on nanostructured polymers were followed
26
during 4 h. The polymer/enzyme dispersions were centrifuged and the amount of
lipase in the supernatant was quantified by the Bradford method so as described
above. The amount of adsorbed lipase was then calculated by the difference
between the amount of lipase in the solution before adsorption and the lipase
remaining in the supernatant after the adsorption.
4.2.6 Evaluation of the adsorption isotherms
Adsorption isotherms were established at room temperature (22°C) in 10 mM
phosphate buffer, pH 7.6. Known amounts of proteins and sorbent were mixed
and incubated for 3 h . Thereafter the mixture was filtered and the protein
concentration in the clear supernatant was measured by using the Bradford assay.
The amount of adsorbed protein was determined from the difference between the
protein concentration in solution before and after adsorption.
4.2.7 pH and thermal stability measurements
The thermal stabilities of bound and free lipase were investigated by measuring
their residual activities at 37°C after being incubated in buffer for 30 min at the
desired temperature in the range 30-70 °C at a fixed pH value.
The pH stability of the free and immobilized lipases was assayed by immersing
them in Phosphate Buffer Solution (PBS) in the pH range 6-8 for 1h at different
temperatures and then determining their activities.
4.3 Sensors
Nanostructured polymers PPA (I2 doped or not), P(PA/HEMA), P(PA/AA),
P(PA/DMPA), obtained by modified emulsion synthesis technique as in previous
paragraph reported, were deposited by spinning at 2000 rpm on silica substrate (5
x 5 mm) where 20 pairs of interdigitate chromium (200 nm thick and 5 µm wide)
had been evaporated and photolithographically defined (see Figure 2).
The measurements apparatus to study the electrical characteristics of
nanostructured polymers and copolymers was constituted by several instruments
represented in Figure 3: Multi Gas Controller, to control the nitrogen stream
27
passed through a water reservoir, an alimetator for sensor of reference humidity
and temperature, CV Meters for current measures, an Analyser for capacity
measures, an Multimeter for voltage measures of references sensors. The I/V
characteristics of the systems were measured with Keithlei 595 Quasistatic CV
meter instrument at applied voltage ranging from -0.5 to +0.5 V. The devices was
placed into a stainless chamber (volume 60 cm3) where nitrogen stream, passing
through a water reservoir was blown; the relative humidity (RH) for five
subsequent cycles performed on each samples was 30-50-85% as measured with
reference commercial hygrometer (Honeywell HIH3610).
Figure 2. Interdigitated substrate with nanostructured PPA film.
Figure 3. Scheme of sensors measurements apparatus.
28
4.4 Nanostructured polymers obtained by a new methodology (European
patent)
The invention proposes s a new method to realize nanostructured polymers. The
general method is based on the use of dialysis membranes as physical barriers to
slow down the mixing of a polymer solution, filling the membrane outfit, with a
non solvent of the polymer surrounding the membrane apparatus, while a few
parameters, such as temperature, stirring, polymer concentration, nature of the
solvent - non solvent couple, solvent - non solvent volume ratio and membrane
type are kept under control.
The polymer, dissolved in suitable solvent, was closed in dialysis membrane and
then immersed in a non solvent where it slowly precipitated. The solid was
analyzed by chemical-physical analysis (IR, UV-vis). Morphology and size of
samples were estimated by Scanning Electron Microscopy investigations on films
of polymer.
Different morphologies were obtained: amorphous, spongeus, reticular, spherical,
by changing the process conditions. Several trials are reported in Table 5 and
different morphologies are illustrated in Figure 4 as examples.
Figure 4. Example of PPA morphology by new metodology: n°1: cavernous;
n°2:spongeus; n°3: reticular; n°4: spheroidal agglomerates; n°5: spheres.
1
29
2
3
4
30
5
4.3.1 PPA
Amorphous PPA synthesised according [66] was dissolved in different solvents at
different concentrations to obtain specific morphologies:
ƒ
THF solvent, that permits concentration of 5 mg/ml; 5 ml of solution were
kept in dialysis membrane then immersed in beker with 100ml of distilled water
(solvent non solvent ratio = 1/40, room temperature); other samples were prepared
at lower concentration (0,5 mg/ml) and with solvent/non solvent ratio = 1/20.
ƒ
DMF solvent realized different concentrations (0.5mg/ml, 5mg/ml,
10mg/ml). Several conditions were used: different solvent- non solvent ratios,
diverse non solvent, at changed temperature and/or with particular kind of contact
between solvent- non solvent (dropping or immersion) were used; another
experimental parameter is the number of dialysis membrane.
ƒ
DMSO solvent with concentrations 0.5 and 5 mg/ml; samples were
realised with water as non solvent and different solvent-non solvent ratio.
4.3.2 PMMA
Commercial PMMA was dissolved in:
31
ƒ
THF, at concentrations 5 and 10 mg/ml, using single dialysis membrane
and water as non-solvent, with solvent/non-solvent ratio= 1/20 or 1/40;samples
were prepared at room temperature and at 40°C - 50 °C, using immersion or
dropping ( dropping rate = 1 ml/min) method.
ƒ
DMF, concentrations 5-10 mg/ml, using single dialysis membrane and
different solvent-non solvent ratios; water and methanol were used as non
solvents.
4.3.3 PS
Commercial PS was dissolved in:
ƒ
DMF, at two different concentrations, 5 and 10 mg/ml, using single
dialysis membrane. Water ethanol and methanol were used as non solvent with
different solvent-non solvent ratios.
32
Chapter 5 Results and discussion
5.1 Bottom up approach: modified emulsion synthesis
The emulsion polymerization is a classical method which allows to prepare
polymer lattices with spherical particles and a narrow particle size distribution. In
spite of the similarities between electrostatically and sterically stabilized emulsion
polymerizations, there are large differences in the polymerization rate, particle
size and nucleation mode due to the change of solubility of emulsifiers in oil and
water phases, micelle sizes and thickness of the interfacial layer at the particle
surface.
Emulsion polymerization involves dispersion of a relatively water-insoluble
monomer (e.g. styrenes, alkyl methacrylates, etc.) in water with the aid of
emulsifiers, followed by the addition of the water-soluble (ammonium
peroxodisulfate (APS), potassium persulfate (KPS)) or oil-soluble (dibenzoyl
peroxide (DBP)) initiators.
In general emulsion polymerization involves free radical polymerization in a
heterogeneous reaction system. Batch emulsion polymerization consists of three
phases: (1) particle nucleation and polymerization with (2) and without (3)
monomer droplets. In phase 1, the particle nucleation occurs until all the
monomer-swollen micelles are consumed. In phase 2, the particles grow steadily
until all monomer droplets disappear. In this interval, the total particle number and
the monomer concentration into the particles are usually considered to be
constant. In phase 3, the monomer concentration decreases with increasing
conversion and the polymerization proceeds under the monomer-starved
condition.
The whole process of emulsion polymerization involves the radical formation in
the aqueous or monomer phase, the transformation of water-soluble primary
radicals into the surface active ones, the entry of surface active radicals into the
polymer particles, desorption of transferred monomeric radicals from particles
into the aqueous phase, propagation and termination of radicals.
33
In particular the initiation of emulsion polymerization is a two-step process: (1)
the first step includes the formation of primary radicals and their transformation to
the surface active oligomeric radicals. through the addition of monomer units to
the growing radical. (2) The second step involves the entry of oligomeric (surface
active) radicals into the monomer-swollen micelles (micellar nucleation) or the
precipitation of growing radicals (homogeneous nucleation) from the aqueous
phase [73-74]. The steps of the emulsion polymerization are thereafter described:
decomposition of initiator in the aqueous phase:
I → 2 R•
(1)
water-phase propagation:
R• + M → RM• → RM•n → RM•z
(2)
entry of surface active oligomeric radical (RM•z) into the monomer-swollen
micelle or polymer particle:
RM•z → particle micelle (active particle)
(3)
precipitation of growing radical from the aqueous phase:
RM•n + M → RM•z → RM•j (primary particle)
(4)
Here, R• is the charged primary radical derived from initiator (I), M = monomer in
the water phase, RM• and RM•n are growing radicals, RM•z the surface active
radical with a high degree of hydrophobicity and RM•j the j primary particle. The
surface active radical enters the polymer particle or monomer swollen micelles,
and starts the polymerization. The restricted termination of growing radicals
within the monomer/polymer particle leads to very rapid polymerization and the
formation of polymers whit high molecular weight. The rate of emulsion
polymerization (Rp) is described by Eq.5 :
Rp = KpNpnCp/NA
(5)
where Kp is the propagation rate constant, Np the total number of polymer
particles, n the average number of radicals per particle, Cp monomer concentration
in the polymer particles and NA Avogardo’s number. Eq. 5 is used to obtain n
from experimental rate data, for systems where N remains constant.
The radical entry and exit rate coefficients depend on the particle size and
structure of the interfacial layer. The propagation rate constant is a function of
34
temperature only and can be a function of the weight fraction of the polymer
below Tg.
The presence of non-ionic emulsifier in the monomer phase and its release from
the monomer droplets during the polymerization can fulfill the condition for the
continuous secondary particle nucleation. The increased stability of monomer
droplets saturated with the non-ionic emulsifier was assumed to be a result of a
number of possible processes (formation of colloids, multilayers around the
monomer droplets, action as hydrophobe, increasing of surface areas, synergistic
effects, and adsorbtion of the monomer). The time evolution of monomer droplets
during polymerization stabilized by ionic emulsifiers and sparse water soluble
monomers, usually follow the micellar model: the monomer-swollen micelles are
the principal locus of polymerization. This model predicts that the number of
micelles transformed into the latex particles increase as the emulsifier
concentration increase [75].
In this part of my work the emulsion polymerization approach was used and
evolution of the polymeric nanobeads dimensions during the synthesis, for
different reaction conditions (time reaction, monomers ratios, catalyst or initiator
concentrations), using non ionic (co-solvent) or ionic emulsifier (SDS and/or
radical initiators) were investigated. The aim was to find the most convenient
synthesis ruotes to achieve polymers and copolymers with different functionalities
and chemical properties in nano-size.
5.1.1 New polymeric materials for PCs
Photonic crystals (PCs) are composite materials constituted by regularly spaced
assemblies of monomeric units [35,36] whose interaction with electromagnetic
radiation produces a control of the field modes in a spectral region which depends
on the particles array spacing. Several effects, like confinement of light, control of
spontaneous emission, Bragg diffraction and beam bending can be obtained using
purposely designed structures. A large effort is currently devoted to develop
chemical approaches which allow to prepare such materials by self assembling of
elements on the mesoscale dimensions, which is necessary to obtain effects in the
visible or near infrared spectral region.
35
Organic materials (e.g. polystyrene, polyacrylates) are widely used for the
preparation of the constituent units since they have the advantage of greater
synthetic flexibility in comparison with inorganic ones (e.g. titania, silica) and
because of the existence of well-known chemical synthesis routes capable of
producing monodisperse nanospheres with controlled dimensions [38,75].
Furthermore, some polymers show properties in the visible spectral region as
luminescence emission or absorption edge which can be tuned by control of the
chemical composition and/or synthesis method. These properties could produce
new effects in the photonic crystal environment, or could be enhanced or
controlled. We recently succeeded in preparing a luminescent, semiconducting
polymer, namely polyphenylacetylene (PPA), in the form of nanospheres with a
reduced dispersion, and obtained preliminary results on the formation of ordered
domains [69]. PPA photonic crystals could also find application in optical
devices, sensors and bio-sensors since PPA is a nonlinear optical material [76]
and can be used as chemical sensor for vapors [77], and also because it is a
candidate for cell and protein immobilization because of its biocompatibility [78].
Our first attempts to obtain PPA PCs were based on the preparation of polymeric
nanobeads by emulsion polymerization, using [Rh(COD)Cl]2tmeda (COD =
cis,cis-1,5-cyclooctadiene, tmeda = N,N,N’,N’-tetramethylethylendiamine) as
catalyst. However, although in that case some ordered domains were formed, their
area was limited because of the high polidispersity of the nanobeads. In fact, low
polydispersity is a necessary condition for the long-range ordering of the
nanospheres and for the minimization of the density of defects in the PC structure
[79].
In my work, with the aim of improving the control on the dimension,
polidispersity and self assembling of the nanospheres, two different routes were
explored: the first one is based on the kinetic studies with achievement of droplets
by modified emulsion synthesis, improving the colloidal stability and
consequently the nanospheres monodispersity with the presence of a co-solvent or
charged species in the reaction mixture [80]. This was achieved for example, for
PMMA and PS synthesis or by changing the PPA synthesis with the use of a
radical initiator such as potassium persulfate (KPS) instead of Rh catalyst: KPS is
36
a very common radical polymerization initiator, but it was never used for PPA or
its derivatives. For the achievement of PMMA monodispersed nanospheres a
systematic study about the deposition conditions to maximize the long ordered
crystalline area was performed.
The second method was based on the preparation of new charged copolymers,
P(S/HEMA) (poly[styrene-(co-2-hydroxyethyl methacrylate)]), P(PA/HEMA)
(poly[phenylacetylene-(co-2-hydroxyethyl
methacrylate)]),
P(PA/AA)
(poly[phenylacetylene-(co-acrylic acid)]) P(PA/DMPA) (poly[phenylacetylene(co-N’N’-dimetylpropargyl amine)]). P(S/HEMA) was prepared as a model for
the comparison of its properties with those of the new synthesised ones. HEMA is
a polar co-monomer which can site on the surface of polymeric particles because
of its hydrophilic nature, as shown by Galembeck et al. for poly(styrene-co-2hydroxyethyl methacrylate) [81,82]. The resulting particles have not a strictly
core-and-shell structure but a higher presence of hydrophilic components at the
outermost layer; therefore the nanobeads surfaces are charged. This characteristic
enhances the colloidal stability in aqueous suspensions and can induce long range
ordering in the colloidal phase, which can be used for the development of optical
sensors [6,83] based on crystalline colloidal arrays (CCA). Furthermore, the
hydroxyl groups on the particles surface improve the adhesion properties in the
coating and adhesive applications. Similar performance is expected for DMPA
and AA copolymers and moreover several applications for optical biosensors are
available because of amino and acid groups are suitable sites for attaching
biological materials such as enzymes [78].
5.1.1.1 Synthesis of PPA nanospheres using Rh catalyst
Polyphenylacetylene was synthesised by using a catalytic polymerisation in
aqueous dispersion for producing nanobeads of polymer (see Figure 5). The
catalyst [Rh(cod)Cl]2tmeda was used because Rh(I) complexes were very active
in catalytic polymerisation of phenylacetylene and for its solubility in water. The
monomer, insoluble in water, is mainly contained in droplets, even if some
monomer molecules are still present in water. The reaction starts in the aqueous
phase immediately after the addition of the catalyst, then the oligomers form and
37
when they are large enough, they tend to segregate, resulting in the formation of
colloidal particles.
Toluene was added to the reaction batch in order to help the formation of the
emulsion and to vary the size of the droplets. The uniformity of size of the
particles produced by this method depends on the physicochemical conditions of
the reaction and on the concentration of the reactants. Low polydispersity is the
most important factor for obtaining a long range ordered packed materials, above
all for noninteracting particles, as PPA beads are. Moreover, it is important that
the diameters of the beads are smaller than 800 nm for applications in optical
devices.
The polymerisations were carried out by using different conditions (see Table 1),
i.e. we varied the concentration of the monomer (H2O/PA ratio from 10:1 to 40:1)
and the reaction time (from 10 to 120 min); in particular, we investigated the
influence of the co-solvent performing reactions without toluene and with
different toluene/PA ratio, from 1:1 to 8:1. Polyphenylacetylene was also
synthesised
in
toluene,
without
water,
in
an
homogeneous
catalytic
polymerisation, in order to notice likenesses and differences with emulsion
technique: this reaction condition did not give rise to spheres, but to polymer in
bulk.
All the materials obtained have the typical polyphenylacetylene trans structure as
we proved by different spectroscopic investigations; elemental analysis and
determination of molecular weights was also used for the characterization.
SEM investigations show films consisting of layers of regular round shape beads;
the diameters of the spheres and their dispersion were determined directly from
these images (see Table 1). The concentration of phenylacetylene in water was
found to have a great influence both on dispersion and on the beads diameters,
while reaction time is of importance above all for the control of the dimensions.
However, if the reaction time is very short, i.e. 10 min, it gives rise to very high
polydispersity: this is probably due to a low conversion of the monomer, so that
when the reaction is stopped, PA is still present in the reaction mixture together
with the catalyst, and the polymerisation still continues in cold ice bath and during
the purification of the materials and no control of the emulsion homogeneity can
38
be performed in these conditions. To confirm this hypothesis, we can note that the
polydispersion is still high when a longer time was used, i.e. 30 min, with high
concentration
of
phenylacetylene,
for
example
H2O/PA=
10:1.
High
polydispersion was also found when toluene/PA ratio is high (8:1), probably
because it is more difficult to preserve the uniformity of the droplets size. We
widely investigated the reactions with toluene/PA= 4:1, and we note that the
reaction time has a great influence on the diameters of the spheres, in particular
longer time means bigger size and fairly narrow dispersion. The best results were
obtained for reaction N° 6, 7, 8 (see Table 1): the sizes of the beads cover a
narrow range and are suitable for photonic crystals application; they are quite
monodisperse and in addition give rise to well-ordered films within an area of 5x5
µm2.
Figure 5. SEM images of nanostructured PPA by modified emulsion synthesis:
(a) with Rh(I) catalyst; (b) with KPS.
a
39
b
5.1.1.2 Synthesis of PPA nanospheres using KPS initiator
In the novel PPA nanosphere synthesis using KPS initiator (Figure 5), the
PA/toluene and PA/initiator ratios and the reaction time were varied and their
effect on the particles dimension and polydispersity index (PI) was investigated as
summarized in Table 1.
Regarding the presence of co-solvent, the particles diameter obtained without
toluene and with two different toluene/PA ratios, namely 2:1 and 10:1 are
reported in Figure 6. The particles are bigger in absence of toluene and their
dimensions decrease as toluene quantity increases. This behavior is consistent
with our previous investigation about emulsion polymerization kinetics [27] in
presence of a cosolvent. In fact toluene forms a layer around the monomer
droplets that stabilizes them and controls their sizes, and moreover determines the
probability of collision, which controls the growth mechanism based on
coalescence. The increase of the amount of toluene results in smaller monomer
droplets and reduced coalescence probability, therefore the dimensions and the
polydispersity of the nanospheres decrease. The effect of KPS concentration on
the PPA nanobeads diameter is shown in Figure 7: the increase of initiator amount
produces larger particles. In this situation the polymerization rate is high and the
40
polymer chain growth is completed in short times so that the growth rate of the
particles is controlled by coalescence. Since the density of sites of nucleation is
high, this produces large and polydisperse particles. KPS could also have a role in
emulsion stabilization, because it is a salt and the ionic charges stabilize the latex
[25].
In a previous work [27] we showed that low polydispersity is the most important
factor for obtaining long range ordered packed materials. In the present reserch,
the reaction conditions (PA/toluene ratio and KPS concentration) can be set at the
values producing the lower polydispersity as reported in Table 1. In these
conditions the polydispersity is improved in comparison with the former PPA
nanobeads synthesis method [69]. The superior monodispersity and the presence
of charges on the particles surface, due to the bonding of the persulfate radicals
[79], allow for the deposition of ordered layers of nanospheres with very large
crystalline domain dimensions equal to about 12 x12 µm2 instead of 5 x 5 µm2 as
reported in our previous investigations about nanostructured PPA synthesis using
Rh catalyst[69].
Once the PA/toluene ratio and the KPS concentration are fixed, the synthesis can
be performed at different reaction times in order to explore the possibility of
controlling the dimensions of the nanobeads. The experimental results are
reported in Figure 8, showing an effective diameter control in the range of few
hundred nanometers.
The spectroscopic characterization of PPA nanobeads confirms that we obtained a
conjugated polymer with phenyls as pendant groups, according to PPA literature
data. IR spectra show the band at 3050 cm-1 (ν aromatic CH), 1597 cm-1 (ν aromatic C=C),
while the band at 740 cm-1 is absent, pointing out for a predominant “trans”
structure of PPA, as expected from radical synthesis methods. UV-vis spectra
were performed on CHCl3 solution and show continuous absorption from λ≤400
nm (Figure 9).
41
Figure 6. PPA … and P(PA/HEMA)z particles diameters obtained without
toluene and with two different toluene/PA ratios, namely 2:1 and 10:1.
500
450
diameter (nm)
400
350
300
250
200
150
100
0/1
2/1
10/1
toluene/PA (vol)
Figure 7. Effect of KPS concentration on the PPA … and P(PA/HEMA)z
nanobeads diameters.
600
diameter (nm)
500
400
300
200
100
0,00
0,02
0,04
0,06
0,08
KPS (g)
42
Figure 8. Effect of reaction time on the PPA … and P(PA/HEMA)z nanobeads
diameters.
diameter (nm)
400
300
200
100
0
30
60
90
120
150
time (min)
Figure 9. UV-vis spectrum of nanostructured PPA obtained by modified emulsion
synthesis with KPS as initiator.
2,5
2,0
ABS
1,5
1,0
0,5
0,0
300
400
500
600
700
-1
λ (cm )
43
5.1.1.3 Synthesis of PMMA nanospheres
The evolution of the PMMA nanobeads dimensions during the synthesis, for
different concentrations of the co-solvent toluene, was investigated by DLS and
SEM as a function of reaction time in the range 10-240 minutes (see Table 2a and
Figure 10 a,b).
Figure10. Diameter of PMMA nano-beads size as a function of reaction time with
different ratio of toluene/monomer (■: 0/1; □: 1/1; ●: 2/1; ○: 4/1) as obtained by
SEM (a) and DLS (b). The solid line in (a) is the fit obtained using equation (1).
diameter (nm)
400
300
200
a
100
0
diameter (nm)
400
300
200
100
0
b
0
60
120
180
240
time (min)
The dimensions obtained from DLS measurements are actually the Z average
value, which is the mean diameter weighted over the scattered light intensity, and
thus it is not directly comparable with SEM data; however the growth trend of the
nanoparticles is similar for both methods. We can see that in the presence of
toluene the growth rate is slower; the size increases initially from 100 to 250 nm;
after 60 minutes the growth slows down, with final dimensions smaller than 350
nm. The interpretation of this result is based on the assumption that the
44
polymerization without toluene shows a different behavior: the initial growth is
faster (350 nm in 60 minutes) and the final dimensions of the particles are larger,
up to 420 nm. This behavior is consistent with the study by Tanrisever et al. about
PMMA polymerization kinetics [84]: during the first 30 minutes the particles
growth is due to the polymer chain growth; after this time, different polymer
chains aggregate to each other and also agglomeration and particle coalescence
occur.
In order to model the kinetics of the particles growth, we fitted the SEM
experimental values of the particles dimensions as function of the reaction time
with an expression containing parameters useful for the discussion. In particular
we adopted an empirical formula which accurately reproduces our data for the
whole range of experimental parameters:
d = d∞ (d∞ - d0) exp (-t/τ)
(1)
Expression (1) describes an exponential growth of the particles with time constant
equal to τ, and initial (t = 0) and final (t = R) dimensions equal to d0 and d∞
respectively. Fits of experimental data obtained using equation (1) are reported in
Figure 10a and the fit parameters are shown in Table 2b for the different reaction
conditions. The presence of toluene clearly increases the reaction time constant τ
of about a factor of two. The particles initial size d0 increases with toluene volume
because in this case the co-solvent drops are bigger and contain a larger quantity
of monomer, this interpretation being in agreement with Lopez-Quintela work
[85] about the micro emulsions synthesis of different nanostructured materials.
The co-solvent forms a layer around the monomer droplets; this layer controls the
transfer of monomer molecules from inside the droplets to the surrounding
aqueous phase and can absorb the monomer molecules that are dispersed in the
solvent. Considering the long time behavior for the achievement of large
nanobeads, this is dominated by the coalescence among particles. In absence of
toluene the final size d∞ is larger because the coalescence occurs among relatively
large particles, since in this condition τ is short. In the presence of toluene, the
size of the drop limits the dimension of the particle and also determines the
probability of collision which is necessary for the coalescence, both mechanisms
45
resulting in the moderate increase of d∞ with the toluene volume fraction shown in
Table 2b.
DLS measurements of the samples prepared without toluene gave polydispersity
values close to 10-1; however, a small quantity of toluene (e.g. toluene/monomer
ratio ≥1) in the synthesis suspension is sufficient to achieve a polydispersity ≤10-2.
The samples were examined also by SEM measurements that confirm these
outcomes: high polydispersity and irregular particles shape were found for those
samples prepared in absence of toluene, while very low polydispersity and regular
round shape were observed for the other samples. These results again point out for
the role of the co-solvent. In fact, the co-solvent acts as a hydrophobic layer and
decreases the potential energy of the interface leading to an overall stabilization of
the emulsion and hence to regular shape particles [31]. These results show
analogy with the work of Capek [75] about the role of non ionic and ionic/non
ionic emulsifier for the radical polymerization in aqueous emulsions of
unsaturated monomers (styrene, alkyl(methacrylates), etc.), where the increased
stability of the emulsions containing a non-ionic emulsifier was attributed to the
effects produced by the presence of a layer of emulsifier around the monomer
droplets.
A number of experiments were performed in order to test the reproducibility of
the shape and dimensions of the polymer nanobeads and the results show that
effective control of these parameters in the range useful for PCs deposition can be
achieved through accurate control of the reaction time and of the concentration of
the co-solvent. The spectroscopic characterization performed on all the
synthesized materials gave information about their tacticity, according to literature
reports [86,87]. IR spectra showed the typical absorption bands of
polymethylmethacrylate and in particular in the region 1300-1100 cm-1 which
gives information on the tacticity of PMMA: four high-intensity absorption bands
at 1270, 1240, 1192, 1149 cm-1 are typical for a polymer rich in syndiotactic
content; 1HNMR spectra show the peaks of α-methyl protons of syndiotactic and
heterotactic PMMA at 0.81 and 0.99 ppm respectively, with intensity ratio 2/1,
but also a less intense peak at 1,18 ppm, due to isotactic chain conformation;
46
13
CNMR spectra present the peak relative to C=O2Me at 177.8, confirming the
high percentage of syndiotactic conformation in our samples.
5.1.1.4 PMMA PCs deposition
When PMMA PCs were deposited under room conditions, the resulting crystals
were characterized by relatively large ordered domains with defects consisting
mainly in stacking faults together with some pores associated with missing
particles. Our samples present a brilliant iridescence, index of the presence and
quality of the photonic bandgap of the ordered PMMA nanospheres. The three
dimensional ordering was confirmed by the SEM image. The crystalline structure
was determined using the procedure outlined by Koh et al. [88] and resulted to be
fcc. We have then investigated the variation of the temperature and of the relative
humidity to vary the solvent evaporation rate, which has a strong impact on the
dynamics of self-assembling of the PMMA suspension. Besides, the solution ionic
strength and the substrate material were also varied to study their influence on the
PCs deposition.
Effect of evaporation rate.
The effect of the relative humidity and of the temperature on the PCs domains
dimensions is reported in Figure 11 and Figure 12, respectively. Small domains
were obtained when the polymer dispersions were dried at low RH% or at high
temperature. In these conditions, the formation of large ordered arrays is
prevented because of the fast evaporation of the solvent, which limits the time
allowed for the particles to find their minimum energy position. From Figure 11 it
is apparent that larger domains are formed at intermediate evaporation rate, thus
the probability of introducing defects into the growing crystal is lower in this
condition. At an optimum evaporation rate the particle flux leading to the growing
crystals will be sufficiently high to dominate over the particle diffusion but slow
enough to allow the particle rearrangement and the elimination of defects in
crystal sites as shown also for silica monolayers [28]. The temperature effect, i.e.
the existence of an optimal temperature already investigated [29], which
maximizes the dimensions of domains, is reported for our samples in Figure 12.
This effect is clearly understood when considering that low temperatures freeze
47
the particles, while high temperatures enhance the evaporation rate of the solvent,
both cases limiting the possibility to find the minimum energy positions.
Temperatures higher than 50°C could not be used because of the polymer beads
softening.
Figure 11. Size of crystal domains vs RH%; films obtained by casting at room
temperature (the solid line is only a guide to the eye).
400
2
domain size (µm )
350
300
250
200
150
100
50
0
20
40
60
80
100
RH%
Figure 12. Size of crystal domains vs temperature; films obtained by casting at
RH = 30-60% (the solid line is only a guide to the eye).
120
2
domain size (µm )
100
80
60
40
20
0
10
20
30
40
50
Temperature (°C)
48
Experimentally, the largest domain size of 390 µm2, corresponding to a minimum
density of extended defects, was obtained at intermediate deposition conditions
(RH = 50% and T = 25°C).
Effect of ionic strength.
The intensity of the particles interactions arising from electrostatic and capillary
forces can be modulated by varying the electrolyte concentration inside the
suspension. Addition of salts was shown [28-31] to reduce electrostatic screening
length, leading to exponential decreasing of the crystalline domain dimensions in
silica PCs. We systematically changed the ionic strength (µ) of the suspension by
adding NaCl or MgSO4 and we characterized the domain dimensions as
previously described. Since high salt concentration causes salt precipitation which
is deleterious for PC formation [28,29], we used MgSO4 to obtain a higher ionic
strength using a smaller amount of salt in comparison with NaCl. Figure 13 shows
the relationship between the ionic strength of PMMA suspensions and the
obtained crystal domains.
Figure 13. Size of crystal domains vs ionic strength (µ) of the suspension; films
obtained by casting at room temperature and RH = 30-60%.
500
2
domain size (µm )
450
400
350
300
250
200
150
1E-6
1E-5
1E-4
1E-3
0,01
0,1
1
logµ
It can be seen that in our experimental conditions the domain dimensions increase
with the ionic strength of the suspension. This behavior is different from that
reported in the literature for silica PCs; however it is not clear whether in that case
49
PCs formation was limited by salt precipitation [28-31]. Using NaCl, in most
cases salt co-crystallization occurred and only in salt-free areas of the film there
were small ordered domains. Moreover, cocrystallization prevented accurate study
of the domains dimensions.
In general a trend can be outlined: the increase in ionic strength of the PMMA
suspension improves the domains dimension and the quality of the PCs. The
addition of electrolytes induces changes in the colloidal stability and in the
particles interaction during the drying procedures. Upon increasing the ionic
strength, crystals domains rapidly assemble because the electrostatic repulsion
among polymer particles and between polymer particles and substrate are reduced
and an increase of Brownian motion of particles occurs, so that they easily adopt
their lowest free energy configuration. However at high salt concentration the coprecipitation of salt affects the system and long range order is suppressed. These
results point out for the importance of capillary forces in assisting ordered
deposition of PCs [88] as compared to electrostatic forces; this topic will be
discussed in the next paragraph.
Effect of substrate.
The substrate suitability for polymer PCs deposition from aqueous suspensions is
mainly related to its wettability, since this property influences essentially the
liquid-solid interface region where the capillary forces are dominant. Obviously a
good substrate must possess also other characteristics, such as for instance low
roughness and good chemical affinity for the polymer material.
In order to explore the substrate effect on the PCs quality, we chose to cast films
of PMMA on different substrates representative of important classes of materials
(e.g. oxides, semiconductors and metals) and of different areas of applications,
from optical devices to electronics. In particular we selected cover glass, graphite,
gold, and silicon. A recent work [88] on the deposition of polymeric PCs onto
silicon and aluminum substrates showed that, in order to exploit the ordering
action of capillary forces, a low contact angle is desirable. Our experimental
evidence confirms this mechanism, as it is shown in Figure 14. In fact, larger
ordered domains were detected on wettable surfaces, evidencing an inverse
relationship between the contact angle and the size of ordered domains. A
50
possible explanation is related to the thinner meniscus region which limits the
number of colloids layers: thinner colloidal crystals can cover larger areas with
few extended defects.
Figure 14. Size of crystal domains as a function of the suspension–substrate
contact angle as produced by deposition on different substrates at room
temperature and RH = 30-60%.
1000
Si
2
size domain (µm )
800
600
400
Au
glass
200
0
0
20
40
graphyte
60
80
100
contact angle (deg.)
Thus, an accurate selection of substrate material can substantially improve the
PCs quality. As a matter of fact, the largest crystal domains of about 900 µm2
were obtained after deposition of PMMA nanospheres on the silicon substrate
(Figure 15).
51
Figure 15. SEM image of a film of PMMA deposited on Si at RH = 30-60% and
T=25°C. The crystal domain size is 900 µm2.
5.1.1.5 Synthesis of PS and P(S/HEMA) copolymers
The PS synthesis was performed by using KPS as initiator and SDS as emulsifier,
while for P(S/HEMA) synthesis no emulsifier was necessary. The synthesis
produced regularly shaped nanospheres whose diameter and polydispersity index
are reported in Table 3 with the specific reaction conditions
The dimensions of the P(S/HEMA) particles produced in a series of reactions for
different reaction time and different S/HEMA ratios (other parameters constant)
are presented in Figure16 .
At increasing of HEMA amount the particles are always smaller considering the
same reaction time: this is due to the presence of HEMA and to its typical
polymerization reaction. In fact the addition of HEMA enhances the particles
nucleation in the aqueous phase and can promote homogeneous polymerization
with tiny particle nuclei formation, besides nucleation in monomer droplets [80];
52
moreover, HEMA can form a charged screen around the nanoparticles that
controls the particles collision, and prevents particles coagulation at high
concentration.
The surface charge was measured by titration of the samples obtained with
different P(S/HEMA) content, and, as expected, the surface charge increases with
increasing HEMA content, with values comparable to those reported in the
literature for HEMA-containing polymer nanobeads [79] for the realization of
CCAs. Thus, incorporation of HEMA is an effective mean to produce charged
nanobeads, and allows to obtain long range ordering in the water medium:
characteristic iridescent suspensions were obtained by P(S/HEMA)-water
suspension when the concentration was about 18 mg/mL and nanospheres had
diameter of 200 nm.
Figure 16. The dimensions of the P(S/HEMA) particles produced at different
reaction times and different S/HEMA ratios (○: 10/1; ■: 20/1; ●: 40/1).
550
500
diameter (nm)
450
400
350
300
250
200
150
0
5
10
15
20
25
time (h)
5.1.1.6 Synthesis of P(PA/HEMA), P(PA/AA) and P(PA/DMPA) copolymers
P(PA/HEMA)
The
P(PA/HEMA)
synthesis
was
performed
by
using
KPS
and
[Rh(COD)Cl]2tmeda as initiator and catalyst; addition of the Rh catalyst was
always necessary to start the reaction. The synthesis with different reaction
53
conditions produced regularly shaped nanospheres whose diameter and
polydispersity index are reported in Table 4.
The dimensions of the P(PA/HEMA) particles produced in a series of reactions
for different synthesis parameters at constant PA/HEMA ratio equal to 10/1,
together with PPA data, are reported in Figures 6, 7, 8. The copolymer particles
are always smaller than PPA ones when considering the same reaction time: this
is due to presence of HEMA that, like in P(S/HEMA) copolymerizations,
enhances the particles nucleation in the aqueous phase, promotes homogeneous
polymerization with tiny particle nuclei formation and forms a charged screen
around the nanoparticles that controls the particles collision preventing the
particles coagulation at high concentration. As shown in Figure 6, the presence of
toluene induces the same effect on the particle dimensions for both the copolymer
and PPA, as expected from our previous study [27].
In P(PA/HEMA) polymerization, the KPS/monomer ratio has a different influence
on the particles size with respect to PPA, as shown in Figure 7: when the initiator
concentration increases, the particles dimension decreases, because the density of
polymerization sites of nucleation increases, in agreement with the report by Kang
[25] on the role of ammonium persulfate in poly(stirene-co-2-hydroxyetyl
methacrylate) synthesis. Unlike PPA, in this case the polymerization rate is slow,
so the particles growth is limited by the polymer chain growth. The possibility of
controlling the P(PA/HEMA) particle diameter by changing the reaction time is
shown in Figure 8. In the case of PPA the growth of the particles is not linear,
while it is linear for P(PA/HEMA).
As previously mentioned, the introduction of HEMA into the PPA chain results in
the production of nanobeads with surface charge. In principle, the surface charge
density depends on the HEMA content, which can be varied using different values
of the PA/HEMA monomers ratio in the synthesis mixture. According to these
considerations we changed the PA/HEMA ratio from 10/1 to 20/1 and to 2/1,
producing polymer nanobeads with different HEMA content as verified by IR
spectroscopy investigation. We report in Figure 19 the particle dimensions for the
three values of the investigated PA/HEMA ratio and for the pure PPA polymer.
By increasing the HEMA content the polymerization rate increases, producing
54
larger particles because of faster polymer chain growth. In absence of HEMA the
polymerization mechanism changes, and the pure PPA particles are larger because
their growth mechanism is different, being based on particle coalescence.
Figure 17. Effect of HEMA concentration on P(PA/HEMA)z nanobeads
diameters.
300
diameter (nm)
280
260
240
220
200
180
1/0
2/1
10/1
20/1
PA/HEMA (vol)
The surface charge was measured by titration on the samples obtained with
different PA/HEMA content, and the corresponding charge per particle is reported
in Figure 18.
As expected, the surface charge increases with increasing HEMA content, with
values comparable to those reported in the literature [79]. Thus, incorporation of
HEMA is an effective mean to produce charged nanobeads, and work is in
progress to obtain long range ordering in the liquid medium.
55
Figure 18. The surface charge measured by titration on the samples obtained with
different PA/HEMA content, and the corresponding charge per particle
(□ζ potenzial, ○superficial charge).
0
6
-10
5
2
3
-30
-
ζ (mV)
σ (e /nm )
4
-20
2
-40
1
-50
2/1
5/1
10/1
20/1
0
PA/HEMA (vol)
For the realization of solid PCs, the most critical synthesis parameters affecting
the shape and polydispersion of P(PA/HEMA) nanobeads are the PA/HEMA
monomers ratio and the KPS initiator concentration; in the best reaction
conditions
tested
in
our
experiments
(H2O/PA=20/1,
toluene/PA=2/1,
PA/HEMA=2/1, KPS=0.020g, reaction time=1.5h) we obtained regular shape
nanospheres with average diameter 270 nm and PI = 0.3 (by SEM image), but
small dimensions of the ordered domain. Besides improvement of the
polydispersity index, also selection of the best preparation conditions for the solid
PC is in progress. The spectroscopic characterization performed on the new
synthesized copolymer further confirms the presence of PPA units by its typical
IR signals (see Figure 19), in particular the band at 755 cm-1 which gives
information on the trans structure of PPA sequences in the ionic copolymer; the
presence of HEMA is detected from IR spectra that exhibit the typical bands
νC=O=1725 cm-1, νC-O=1025 cm-1 and in particular a broad intense band νO-H=3500
cm-1; the relative intensities of the PPA and HEMA bands are different for the
different PA/HEMA ratios, confirming a larger presence of HEMA for low
PA/HEMA ratio.
56
Figure 19. Typical IR spectrum of nanostructured P(PA/HEMA).
70
65
60
55
T
50
45
40
35
30
4000
3500
3000
2500
cm
2000
1500
1000
500
-1
UV-vis spectra in CHCl3 solution showed continuous absorption from λ≤400 nm,
with a peculiar slope that confirms the polyacetylene-like structure (see Figure
20).
Figure 20. Typical UV-vis spectrum of P(PA/HEMA) in CHCl3.
2,0
1,8
1,6
1,4
ABS
1,2
1,0
0,8
0,6
0,4
0,2
0,0
300
400
500
600
700
λ (nm)
57
P(PA/AA)
P(PA/AA) nanospheres were realised by modified emulsion synthesis using KPS
and APS as initiators and toluene as cosolvent. Preliminary studies showed that
the synthesis produced regularly shaped nanospheres whose diameters and
polydispersity index are reported in Table 4, with the specific reaction conditions
(see Figure 21). The evolution of the nanobeads dimensions during the synthesis,
for different water-phenylacetylene ratios were investigated by SEM as a function
of reaction time in the range 1-24 hours: the particles dimensions growth and the
polidispersity decrease for long reaction times, as aspected in analogy with
previous studies on P(PA/HEMA).
The studies in progress are devoted to analize the roles of toluene and initiators
(KPS and APS) and AA monomer amount on the dimensions and monodispersion
of the beads and the effects about the superficial charge and their self-assembling
in liquid phases.
P(PA/DMPA)
The amino copolymers P(PA/DMPA) were synthesised in water-toluene emulsion
by using KPS initiator and Rh(I) dimer catalyst in order to obtain spherical
nanoparticles (Figure 22).
Table 4 shows preliminary investigations about the influence of different
synthesis conditions on the nanoparticles diameters and polydispersity index.
We can see that the particles growth is affected mainly by the cosolvent (toluene)
presence, monomer (PPA) ratios and by the reaction time .
To analyze the roles of the synthesis conditions, such as toluene/PA ratios,
initiator/catalyst (KPS/Rh dimer) concentrations, and DMPA monomer amount,
on the dimensions and monodispersity of the beads, systematic studies are in
progress; moreover the effects about the superficial charge and their selfassembling in liquid phases will be investigated.
58
Figure 21. SEM image of P(PA/AA) with PA/AA=10/1 and reaction time=24h.
Figure 22. SEM image of P(PA/DMPA) with PA/DMPA=10/1 and time reaction
22h.
5.2 New polymeric materials for the preparation of Bioconjugates
The development of multifunctional nanostructured polymers and biopolymer
based nanocomposites represent a key issue for the advanced technology
59
(optoelettronic, sensors) and environment-friendly materials to be used as carries
for enzyme immobilisation and for advanced biomedical applications .
High biological molecules loading can be achieved with porous materials, that
however, suffer a much greater diffusional limitation. Nanostructured materials
will overcame the problems related to the use of amorphous materials for
biological materials immobilisation in terms of :
•
rapid mass transfer;
•
large surface and interface area;
•
high bioactivity recover of the enzymatic proteins;
•
higher reaction efficiency of the catalytic processes.
As it is well known, protein adsorption onto solid surfaces is controlled by the
properties of the support surface, the nature of the protein molecule and the
solution conditions. The surfaces of CRL and PCL are complex in nature, with
differences in characteristics such as hydrophobicity of the different CRL
isoenzymes. The absence of titrable groups on the surface of PS and PMMA
makes the adsorption mechanism of CRL controlled by hydrophobic and Van der
Waals bond interactions, while the contribution of electrostatic interactions is
likely to be very small. Figure 23 shows a scheme of the adsorption process.
Figure 23. Schematic representation for the preparation of the support and
enzyme immobilization.
Lipase
Polymer nanoparticles
Bioconjugate
The enzymes loading does not seem to be influenced by the dimensions of the
nanoparticles in the range 150-400 nm and we choose to carry out all the
experiments with the particles average diameters of 300 nm. The time needed for
the adsorption of CRL and PCL onto PMMA and PS nanoparticles was evaluated.
More than 80% of lipase molecules were adsorbed after 25 minutes of reaction
60
time and Figure 24 reports as an example the results which refer to the
bioconjugate CRL adsorbed onto PMMA which is representative of all the
investigated bioconjugates.
Figure 24. Adsorption kinetic of CRL on nanostructured PMMA at T=25°C
(Reaction conditions: enzyme, 50 mg/ml, reaction volume 2ml, polymer =100 mg,
phosphate buffer 0.1 M, pH 7.6, T=25°C, average diameter of nanoparticles 300
nm).
adsorbed amount of CRL (mg of enzyme/100 mg of
carrier)
100
90
80
70
60
50
40
30
20
10
0
0
20
40
60
80
100
120
140
Time (min)
Adsorption of lipases onto the selected polymers is a fast process, owing to the
high affinity of lipase molecules to the hydrophobic surfaces as demonstrated in a
previous work [89].
The most important aspect in lipase-nanoparticle bioconjugates systems is the
retention of the biocatalytic activity after adsorption onto nanoparticles surfaces.
In our study, the specific activity was calculated using the amount of enzyme
estimated from the calibration curve and compared with that of the free enzyme in
solution under identical conditions. The specific activity of free and immobilized
lipases are reported in Table 6. It is noteworthy that, in comparison to the
behavior of the free or immobilized enzymes on amorphous supports, CRL and
PCL lipases exhibit a significant increase of their activity when adsorbed on
nanostructured polymers. CRL and PCL show retention activity values from 60 up
61
to 74% when adsorbed on nanostruttured PMMA and PS, respectively, while
lipases adsorption on amorphous polymers gives retention activity value up to
about 20-30%. These results can be explained by the fact that the remarkable high
specific area of the nanostructured polymers can provide more potential reaction
sites for lipase adsorption.
It is also note worthy that the activity of bioconjugates was not affected by
temperature and pH variations in the range useful for applications (see Figure 25).
Figure 25. Thermal and pH stabilities of the free and immobilized CRL on
nanostructured PMMA and PS (Reaction conditions: enzyme, 50 mg/ml, reaction
volume 2ml, polymer =100 mg, phosphate buffer 0.1 M, pH 7.6, T=25°C, average
diameter of nanoparticles 300 nm).
4,5
Specific activity (UI/mg of protein)
4
3,5
3
CRL 50 mg/ml
CRL su PMMA
CRL su PS
2,5
2
1,5
1
0,5
0
0
10
20
30
40
50
60
70
T(°C)
62
4,5
Specific activity (UI/mg of protein)
4
3,5
3
2,5
2
CRL 50 mg/ml
CRL su PMMA
CRL su PS
1,5
1
0,5
0
5,5
6
6,5
7
7,5
8
pH
These results indicate that the thermal stability of the immobilized lipases on
nanostructured polymers is much higher than that of the free ones, owing to the
formation of physical interactions of the lipases with the polymeric carriers, which
prevents the conformation transition of the enzyme at high temperature. As far as
pH stability is concerned, there was no loss of activity for the immobilized lipases
in the pH range 6 to 8, while the free enzymes lost rapidly their activity up to 80%
when the solutions reached pH ≥ 8.5.
Adsorption isotherms, in which the amount of proteins adsorbed per unit weight
of sorbent is plotted against the protein concentration in solution, display different
characteristics of the protein-sorbent interaction, such as the adsorption affinity of
the proteins for the nanostructured carriers.
Adsorption isotherms at 25°C and at constant pH values, were obtained after 1h of
incubation time of CRL and PCL with the nanostructured PMMA and PS. Figure
26 shows, as an example, CRL adsorption mass and activity isotherms with a
well-defined plateaus of adsorption onto nanostructured PMMA for equilibrium
lipase concentrations in solutions with Ceq = 2mg/ml. A Langmuir type model
fitted both the mass and activity trend of adsorbed CRL and PCL onto PMMA and
PS nanoparticles.
Figure 26. Adsorption isotherms of CRL immobilised on nanostructured PMMA
at different temperatures (Reaction conditions: enzyme, 50 mg/ml, reaction
63
volume 2ml, polymer =100 mg, phosphate buffer 0.1 M, pH 7.6, T=25°C, average
diameter of nanoparticles 300 nm).
amount of adsorbed enzyme (mg of enzyme/100 mg
of support)
100
90
80
70
T=25°C
T=50°C
60
50
40
30
20
10
0
0
1
2
3
4
5
6
7
enzyme (mg/ml)
Redispersion of the bioconjugates in the phosphate buffer (0.2M, pH7,6) did not
result in changes of the surface concentration of CRL and PCL. In the case of
CRL, adsorbed on noanostructured PMMA and PS, no lipase was detected in the
supernatant as a function of reaction time and at different temperature (20 and
40°C). The strong hydrophobic character of the carriers and the presence of large
hydrophobic domains on the surface of the CRL molecules probably hinders the
desorption. The time of PCL desorption from nanostructured polymers is also
very slow and the bioconjugate retains up to 80% of the initial enzyme activity
after 8 hours. In figure 27 the desorption kinetic of CRL and PCL immobilised on
nanonstructured PMMA as a function of the temperature are reported as an
example. These experimental results are in good agreement with the adsorption
isotherms obtained for CRL and PCL at T=25°C above reported.
Figure 27. Desorption kinetics of CRL(a) and PCL(b) immobilised on
nanostructured PMMA at different temperatures (Reaction conditions: enzyme, 50
64
mg/ml, reaction volume 2ml, polymer =100 mg, phosphate buffer 0.1 M, pH 7.6,
T=25°C, average diameter of nanoparticles 300 nm)
100
90
% of residual activity
80
70
T=20°C
T=40°C
60
50
a
40
30
20
10
0
0
1
2
3
4
5
6
7
8
9
Time (h)
100
90
% of residual activity
80
70
60
20°C
40°C
50
40
b
30
20
10
0
0
1
2
3
4
5
6
7
8
9
Time (h)
From an industrial point of view, one of the most important features concerning
immobilized CRL and PCL preparations, is their reuse stability, which can really
65
reduce the process costs. In order to evaluate the reuse stability, the immobilized
lipases were washed in PBS after every run and reintroduced into fresh solutions.
This process was repeated up to 10 cycles and no loss of activity was observed for
the immobilized enzymes within the employed conditions.
Table 7 reports the results of esterase activity and selectivity in the
transesterification reaction of 1-phenylethanol with vinylacetate in organic
solvents employing free and immobilised CRL and PCL preparations. The
reaction media were selected on the basis of the polymers solubility and solvent
logP (the logarithmic ratio of the concentrations of the solute in the solvent is
called log P, where P, the partition coefficient, is a measure of differential
solubility of a compound in two solvents).
Moreover, a remarkable feature, observed for both lipases, is that the activity of
adsorbed lipases onto the nanostructured polymers is substantially higher than that
of free lipases in solution, with conversion values that from 10% (free CRL) rise
up to 50% for the immobilised one in n-hexane. As far as CRL is concerned, also
their enantioselectivity seems to be influenced by the nature of the immobilising
support. A comparison of the enantiomeric excess (e.e.) values, for the free and
bioconiugates CRL preparation shows that there is an increase of the selectivity
upon the adsorption onto the polymeric nanostructured supports. This result
highlights the possibility that CRL molecules have retained their catalytic activity
also in organic medium and that is also possible to obtain new protein conformers
stabilised after adsorption on nanoparticles with improved enantioselectivity.
5.3 Nanostructured polymers applied to Sensors
Preliminary investigations on humidity sensors based on nanostructured PPA (I2
doped or pristine) and its copolymers P(PA/HEMA) membranes showed fast and
reproducible current intensity variations in the range RH 5–90%. The sensor
geometry allows its application in miniature devices and the nanostructure
enhances the response to humidity variations with respect to previous studies
based on amorphous I2-doped phenylacetylene [77].
In Figure 28, the calibration curves sensors based on of nanostructured PPA (I2
doped or undoped) and P(PA/HEMA) exposed to water vapors are reported.
66
Figure 28. Calibration curves of sensors based on nanostructured PPA (I2 doped
and undoped) and P(PA/HEMA).
1E-9
Vpol=1Volt
RH step: 5% ogni 300 sec; RH stop: 1000sec W/D
undoped PPA
I2 doped PPA
I(A)
1E-10
P(PA/HEMA)
1E-11
1E-12
0
20
40
60
80
100
RH(%)
We observed a sharp increase of the current intensity for a humidity sensor based
on nanostructured PPA with respect to data of non nanostructured I2 doped PPA
reported in literature [77]: the best results are obtained for nanostructured PPA
that shows an current intensity variation in the range 10-11-10-9A vs RH=5-90%.
The work in progress is dedicated to investigate the response in the low RH range
and to different gases: low levels of water vapors are in fact critical for
applications in microelectronics or semiconductor industries, where gases of
ultrahigh purity are used. The investigated materials can be sensible also to CO, or
CO2 or alchools, so as reported in the literature for similar non nanostructured
polymers [90].
The reproducibility of the response of nanostructured PPA and P(PA/HEMA)
sensors was tested; current intensity values were reproduced when recorded in
different cycles of measurements, in which the same RH percentages (5%) were
reached in the test chamber (Figure29). The sensors were also tested in
subsequent cycles, and in these preliminary studies no significantly ageing or
degradation of its performance for working times as long as three days were
observed.
67
Figure29. The time response and stability of resistive sensor based on
nanostructured PPA (I2 doped or undoped) and PPA/HEMA.
The mechanism of interaction between PPA, P(PA/HEMA) and H2O molecules
may be interpreted on the basis of previous XPS studies performed on
polyphenylacetylene and polymonosubstituted acetylenes [91]. The polymer
surface of each nanobead covalently links OH groups deriving from H2O
dissociation and the OH groups are able to promote the hopping of H3O+ from a
site to another, thus suggesting a ionic conduction, which may be due also to K+
residual from the KPS initiator, used for the modified emulsion synthesis of
nanostructured polymers. Electrochemical dissociation of water occurs upon
applying a voltage, so as suggested by investigations performed on similar
materials [90]; for these materials which behave as a sponge for humidity, the
interpretation of the electrical response is based on diffusion processes of water in
the vapor phase inside the nanostructured material. Moreover, measures in
alternated current point out the important ionic contribute to the conduction; in
Figure 30 a,b and 31 a,b we report preliminary results: the most interesting
responses were obtained by sensors based on nanostructered undoped PPA and
nanostructured P(PA/HEMA) that showed reproducible signals during the time
68
(Figure 32 a,b); the I vs RH curve for these polymers showed a sigmoid curve that
confirms the hypothesis of a main ionic contribute to the conductivity of these
materials; it is just important to note that a good response in the whole range low
high RH values is observed. Further investigations will be devoted to test
nanostructured PPA (I2 doped or undoped ) and its copolymers in resistive sensors
for the detection of several gases, because of the nanostucured morphology
enhances the sensibility.
Figure 30: I vs RH curves at 100 KHz for sensor based on nanostructured
undoped PPA (a) and nanostructured PPA/HEMA (b).
(a)
-4
6.6x10
Vpol=10Vpp AC @ 100KHz
-4
6.4x10
-4
6.2x10
-4
I(A)
6.0x10
-4
5.8x10
-4
5.6x10
-4
5.4x10
-4
5.2x10
-4
5.0x10
0
20
40
60
80
100
80
100
RH(%)
(b)
-4
8.6x10
Vpol=10Vpp AC @ 100KHz
-4
8.6x10
-4
I(A)
8.5x10
-4
8.5x10
-4
8.4x10
-4
8.4x10
0
20
40
60
RH(%)
69
Figure31. I vs RH curves at 10 KHz for sensor based on nanostructured undoped
PPA (a) and nanostructured PPA/HEMA (b).
(a)
-5
3.7x10
Vpol=10Vpp @ 10KHz
-5
3.7x10
-5
I(A)
3.6x10
-5
3.6x10
-5
3.5x10
-5
3.5x10
-5
3.4x10
0
20
40
60
80
100
80
100
RH%
(b)
-4
1.6x10
Vpol=10Vpp @ 10KHz
-4
1.6x10
-4
1.5x10
-4
I(A)
1.5x10
-4
1.4x10
-4
1.4x10
-4
1.3x10
-4
1.3x10
0
20
40
60
RH%
70
Figure 32. The time response and stability of sensor based on nanostructured
undoped PPA and nanostructured PPA/HEMA: (a) measures at 100 KHz (b)
measures at 10 KHz..
(a)
-4
9,0x10
Vpol=10Vpp AC @ 100KHz
-4
8,5x10
-4
8,0x10
-4
7,5x10
I2 doped PPA
P(PA/HEMA)
I(A)
-4
7,0x10
undoped PPA
-4
6,5x10
-4
6,0x10
-4
5,5x10
-4
5,0x10
0
30000
60000
90000
t(sec.)
(b)
-4
Vpol=10Vpp @ 10KHz
2,5x10
-4
2,0x10
-4
I(A)
1,5x10
-4
1,0x10
-5
5,0x10
I2 doped PPA
P(PA/HEMA)
PPA undoped
0,0
0
20000
40000
60000
80000
time(sec.)
71
5.4 Top down approach
In these recent years the nanoscience has developed new features in dramatically
increasing challenges in science of materials. The study of new materials with
controllable dimensions and shape at sub micrometric scale is producing
applications and new perspectives in several fields such as electronics,
photoelectronics, textile, pharmaceutical, medicine, energy.
High cost, often due to the processing scale-up, are mainly responsible of difficult
tranfert of nanotechnology from scientific laboratories to industrial productions.
In this situation the interest about polymeric materials is increasing: the easy
functionalization, the solubility in common solvents, the optical-electrical
properties in some cases, promise several appreciable opportunities of application;
in fact this materials join low costs, easy processing, and lightness.
In general, nanostructured polymers were realized by modified emulsion synthesis
using emulsifiers, solvents, and catalyst that represents additional costs; attempts
to renounce to some of these chemicals were investigated; however a significant
lowering of yields was found [92-94].
We have developed a new method that is easy and cheap; it permits to realize
nanostucture from natural or /and synthetic polymers.
5.4.1 New methodology
With the aim to producing polymers with the shape of spherical nanoparticles
through easier way than emulsion synthesis, first we considered the novel topdown approach to polyphenylacetilene (PPA), polymethylmethacrylate (PMMA),
and polystyrene (PSt). We used a new process of precipitation post-synthesis,
based on interfacial phenomena connected to the polymer solution contact with a
non solvent, using dialysis membrane as physical barrier to slow down the
process. To slow down the process the dialysis bag was immersed in solvent and
the non solvent was added by dropping; the gradual blending into the bag
increases slowly the interfacial tension that permits the aggregation of the
polymer molecules and minimization of free energy by achieving a spherical
shape of the particles. The blending rate is mainly factor that influenced the
process and the final morphology, and it is determined by several parameters:
72
couple solvent-non solvent, temperature, type of contact (immersion-dropping),
numbers of dialysis membrane.
In general, the obtained morphology is defined by kinetics and thermodynamic
factors [95]: the spherical morphology is the equilibrium morphology determined
by prevalence of thermodynamics factors. Morphologies different from the sphere
are main favored by kinetics factors. The main thermodynamic parameter is the
free interfacial energy: when the precipitation process is slow enough the
equilibrium spherical morphology is achieved and permits the minimization of
energy. About kinetics parameters the principal one is the diffusion motions of
polymer chains in solvent and non solvent: the mobility of polymer chains is
abated during the dialysis process and then the separation and rearrangement can
be slowed down with respect to the aggregation rate.
The different morphologies so far obtained were classified in five typologies:
•
n°1: cavernous;
•
n°2:spongeus;
•
n°3: reticular;
•
n°4: spheroidal agglomerates;
•
n°5: spheres (dimensions in the range 4 µm -100 nm).
In Figure 4 there are some examples of different PPA morphology:
Obviously our research was focused on the realization of spheres by strict control
of the dimensions and of polidispersity which are essential topics to be reached
for applications in opto-electronics. In fact, optical properties depend on
dimensions and monodispersion of particles and on their lattice [39,75].
The advantages of the new method are several: low cost, general application of
the process, optimizable for every polymer, natural and/or synthetic, soft work
condition, without use of emulsifier and stabilizing, obtaining immediately pure
products and realizing easy recovery of solvents.
Table 5 points out several results about PPA, PMMA and PS nanostructerd
polymers obtained by this new methodology: each polymer realizes different
morphologies and moreover produces nanospheres (morphology 5) using DMF as
solvent: a strict control of the nanobeads dimensions depend on the polymer
concentration and/or on the number of membrane used.
73
Chapter 6 MIT Photonic Bands gap
6.1 Introduction
Optical systems have been the subject of enormous practical and theoretical
interest in recent years, with a corresponding need for mathematical and
computational tools.
In particular three-dimensional photonic crystals are intensively studied with the
goal of achieving control of light propagation and, especially, a complete photonic
band gap in all directions[96-98]. Among the possible structures, fcc-based
systems like opals structure (e.g., polystyrene or silica spheres in air) and inverse
opals are of great interest as they can be produced with bottom-up approaches
based on casting from colloidal solutions, possibly followed by infiltration and
template removal [99,100].
Therefore, besides synthesis of new polymeric materials, with controlled shape
and dimensions and able to self-assembling realizing photonic crystals, a
framework of my research was devoted to study a possible modeling of optical
properties of new systems, attempting to define their bands structure, using
analysis tools known for analogous systems.
One fundamental approach in optical systems analysis is eigenmode
decomposition: the possible forms of electromagnetic propagation are expressed
as a set of definite-frequency (time-harmonic) modes. In the absence of nonlinear
effects, all optical phenomena can then be understood in terms of a superposition
of these modes, and many forms of analytical study are possible once the modes
are known. Of special interest are periodic (or translationally-symmetric) systems,
such as photonic crystals (or waveguides), which give rise to many novel and
interesting optical effects [96]. Another important basic system is that of resonant
cavities, which confine light to a point-like region. There, the boundary conditions
are, in principle, irrelevant if the mode is sufficiently confined, so they can be
treated under the rubric of periodic structures as well via the “super-cell”
technique.
74
S. G. Johnson and J. D. Joannopoulos proposed a fully-vectorial, threedimensional method for computing general eigenmodes of arbitrary periodic
dielectric systems, including anisotropy, based on the preconditioned blockiterative solution of Maxwell's equations in a planewave basis [101]. The result of
this work is available as a free and flexible computer program downloadable from
the Web [102], and in the framework of my research, I studied and used this
method for the theoretical calculation of the photonic bands gap of my new
polymeric nanopheres.
There are a few common approaches to eigen-decomposition of electromagnetic
systems: frequency-domain, time-domain and transfer-matrix methods.
The first, which I employed for my calculation using MIT Photonic Bands gap
(MPB) package, is to expand the fields as definite-frequency states in some
truncation of a complete basis (e.g. planewaves with a finite cutoff) and then to
solve the resulting linear eigenproblem. Such methods have seen widespread use,
with many variations distinguished by the critical choices of basis and eigensolver
algorithm [98-100]. This is the frequency-domain method that will be discussed in
the following paragraphs.
Another common technique involves the direct simulation of Maxwell's equations
over time on a discrete grid by definite-difference time-domain (FDTD)
algorithms; the time-varying response of the system to some input is Fourier
transformed and the peaks in the resulting spectrum correspond to the
eigenfrequencies
[103].
This
has
the
unique
feature
of
finding
the
eigenfrequencies only to compute the associated fields; then the simulation is reruns with a narrow-band filter for each state. Time-domain calculations can also
address problems of a dynamic nature, such as transmission processes, that are not
so amenable to eigenmethods.
A third class of techniques are referred to as “transfer-matrix” methods: at a fixed
frequency, one computes the transfer matrix relating field amplitudes at one end
of a unit cell with those at the other end (via finite-difference, analytical, or other
methods) [104]. This yields the transmission spectrum directly, and mode
wavevectors via the eigenvalues of the matrix; in some sense, this is a hybrid of
time- and frequency-domain. Transfer-matrix methods may be especially
75
attractive when the structure is decomposable into a few more-easily solvable
components, and also for other cases such as frequency-dependent dielectrics.
In any method, the computation is characterized by some number N of degrees of
freedom (e.g., the number of planewaves or grid points), and one might be
interested to compare how the number of operations (the complexity) in each
algorithm scales with N, but unfortunately, there is no simple answer. The
difficulty in time and frequency-domain cases comes from the number of
iterations, which scales in different ways depending upon how the problem size is
increased. One traditional advantage of time-domain has been its ability to extract
modes in the interior of the spectrum without computing the lower-frequency
states, but we will show that this is feasible in frequency-domain as well. Both
time- and frequency-domain methods remain useful tools to extract eigenmodes
from many structures.
In the following paragraphs, I shall describe the method for obtaining the
eigenmodes of Maxwell's equations using MPB, software based on frequency–
domain methods. The discussion was divided into two parts: first, I shall review
how Maxwell's equations can be cast as an eigenproblem for the frequencies;
second, I shall illustrate the methods with numerical results applied to known
systems (model) and to my new polymeric systems.
6.2 The Maxwell Equations in periodic media
The study of wave propagation in three-dimensionally periodic media was
pioneered by Felix Bloch in 1928, unknowingly extending an 1883 theorem in
one dimension by G. Floquet. Bloch proved that waves in such a medium can
propagate without scattering, their behavior governed by a periodic envelope
function multiplied by a planewave. Although Bloch studied quantum mechanics,
leading to the surprising result that electrons in a conductor scatter only from
imperfections and not from the periodic ions, the same techniques can be applied
to electromagnetism by casting Maxwell’s equations as an eigenproblem in
analogue with Schrödinger’s equation. By combining the source-free Faraday’s
and Ampere’s laws at a fixed frequency ω, i.e. time dependence e
r
obtain an equation in only the magnetic field H :
-iωt
, one can
76
r
r v ⎛ ω ⎞2 r
1
∇×
ε ∇ × H = ⎜⎝ c ⎟⎠ H
(1)
where ε is the dielectric function ε (x, y, z) and c is the speed of light. This is an
r
r
eigenvalue equation, with eigenvalue (ω/c)2 and an eigen-operator ∇ × 1 ∇ ×
ε
that is Hermitian (acts the same to the left and right). under the inner product
r v
r
r
H
∫ * ⋅H ' between two fields H and H ' . The two curls correspond roughly to the
“kinetic energy” and 1/ε to the “potential” compared to the Schrödinger
Hamiltonian ∇2 + V. It is sometimes more convenient to instead write a
r r r r
r
2
generalized Hermitian eigenproblem in the electric field Ε, ∇ × ∇ × Ε = (ω / c ) εΕ ,
which separates the kinetic and potential terms. Electric fields that lie in higher ε,
i.e. lower potential, will have lower ω; this is discussed further in the context of
the variational theorem of Eq. (3). Thus, the same linear-algebric theorems as
those in quantum mechanics can be applied to the electromagnetic wave solutions.
The fact that the eigenoperator is Hermitian and positive-definite (for real ε > 0)
implies that the eigenfrequencies ω are real, for example, and also leads to
orthogonality, variational formulations, and perturbation-theory relations. An
important difference compared to quantum mechanics is that there is a
r r
r r
transversality constraint: one typically excludes ∇ ⋅ H ≠ 0 (or ∇ ⋅ εΕ ≠ 0 )
eigensolutions, which lie at ω = 0; i.e. static-field solutions with free magnetic (or
electric) charge are forbidden.
6.2.1 Bloch waves and Brillouin zones
A photonic crystal corresponds to a periodic dielectric function ε(x) = ε (x + Ri)
for some primitive lattice vectors Ri (i = 1, 2, 3 for a crystal periodic in all three
dimensions). In this case, the Bloch-Floquet theorem for periodic eigenproblems
rr
r r
r
r
states that the solutions to Eq. (1) can be chosen of the form H ( x ) = e ikx ⋅ H n , kr ( x )
()
r
r
with eigenvalues ω n k , where H n ,kr is a periodic envelope function satisfying:
77
( )⎞⎟
r
r 1 r
r
r
r
⎛ ωn k
∇ + i k × ∇ + ik × H n, kr = ⎜
⎜ c
ε
⎝
(
) (
)
⎟
⎠
2
r
H n, kr
(2)
yielding a different Hermitian eigenproblem over the primitive cell of the lattice at
r
each Bloch wavevector k . This primitive cell is a finite domain if the structure is
periodic in all directions, leading to discrete eigenvalues labelled by n = 1, 2, · · ·.
r
r
These eigenvalues ω n k are continuous functions of k , forming discrete
()
“bands” when plotted versus the latter, in a “band structure” or dispersion
r
diagram; both ω and k are conserved quantities, meaning that a band diagram
r
maps out all possible interactions in the system. (Note also that k is not required
r
to be real; complex k gives evanescent modes that can exponentially decay from
the boundaries of a finite crystal, but which cannot exist in the bulk.)
Moreover, the eigensolutions are periodic functions of k as well: the solution at k
r
is the same as the solution at k + Gj , where Gj is a primitive reciprocal lattice
vector defined by Ri Gj = 2πδ
i,j
. Thanks to this periodicity, one need only
compute the eigensolutions for k within the primitive cell of this reciprocal lattice
or, more conventionally, one considers the set of inequivalent wavevectors closest
r
to the k = 0 origin, a region called the first Brillouin zone. For example, in a onedimensional system, where R1 = a for some periodicity a and G1 = 2π /a, the first
Brillouin zone is the region k = −π/a · · · π/a ; all other wavevectors are equivalent
to some point in this zone under translation by a multiple of G1. Furthermore, the
first Brillouin zone may itself be redundant if the crystal possesses additional
symmetries such as mirror planes; by eliminating these redundant regions, one
obtains the irreducible Brillouin zone, a convex polyhedron that can be found
tabulated for most crystalline structures. In the preceding one-dimensional
example, since most systems will have time-reversal symmetry (k → −k), the
irreducible Brillouin zone would be k = 0 · · −π/a .
The familar dispersion relations of uniform waveguides arise as a special case of
the loch formalism: such translational symmetry corresponds to a period a → 0. In
78
this case, the Brillouin zone of the wavevector k (also called β) is unbounded, and
r
the envelope function H n, kr is a function only of the transverse coordinates.
6.2.2 The origin of the photonic band gap
A complete photonic band gap is a range of ω in which there are no propagating
r
r
(real k ) solutions of Maxwell’s equations (2) for any k , surrounded by
propagating states above and below the gap. There are also incomplete gaps,
which only exist over a subset of all possible wavevectors, polarizations, and/or
symmetries.
For both sorts of gaps the origins are the same, and can be understood by
examining the consequences of periodicity for a simple one-dimensional system.
Let us consider a one-dimensional system with uniform ε = 1, which has
planewave eigensolutions ω(k) = ck, as depicted in Figure 33 (left). This ε has
trivial periodicity a for any a ≥ 0, with a = 0 giving the usual unbounded
dispersion relation. We are free, however, to label the states in terms of Bloch
envelope functions and wavevectors for some a ≠ 0, in which case the bands for
|k| > π/a are translated (“folded”) into the first Brillouin zone, as shown by the
dashed lines in Figure 33 (left). In particular, the k = −π/a mode in this description
now lies at an equivalent wavevector to the k = π /a mode, and at the same
frequency; this accidental degeneracy is an artifact of the “artificial” period we
have chosen. Instead of writing these wave solutions with electric fields
r
Ε(x ) ~ e ± iπx / a , we can equivalently write linear combinations e(x) = cos(πx/a) and
o(x) = sin((πx/a) as shown in Figure 34, both at ω = cπ/a. Now, however, suppose
that we perturb ε so that it is nontrivially periodic with period a; for example, a
sinusoid ε (x) = 1+∆·cos(2πx/a), or a square wave as in the inset of Figure 33. In
the presence of such an oscillating “potential,” the accidental degeneracy between
e(x) and o(x) is broken: supposing ∆ > 0, then the field e(x) is more concentrated
in the higher- ε regions than o(x), and so lies at a lower frequency. This opposite
shifting of the bands creates a band gap, as depicted in Figure 33 (right). (In fact,
79
from the perturbation theory described subsequently, one can show that for ∆ << 1
the band gap, as a fraction of mid-gap frequency, is ∆ω/ω ~∆/2.)
Figure 33. Left: Dispersion relation (band diagram), frequency ω versus
wavenumber k, of a uniform one-dimensional medium, where the dashed lines
show the “folding” effect of applying Bloch’s theorem with an artificial
periodicity a. Right: Schematic effect on the bands of a physical periodic
dielectric variation (inset), where a gap has been opened by splitting the
degeneracy at the k = ±π/a Brillouin-zone boundaries (as well as a higher-order
gap at k = 0).
Figure 34. Schematic origin of the band gap in one dimension. The degenerate k
= ±π/a planewaves of a uniform medium are split into cos(π x/a) and sin(π x/a)
standing waves by a dielectric periodicity, forming the lower and upper edges of
the band gap, respectively; the former has electric-field peaks in the high
dielectric (nhigh) and so will lie at a lower frequency than the latter (which peaks
lie in the low dielectric).
80
By the same arguments, it follows that any periodic dielectric variation in one
dimension will lead to a band gap, albeit a small gap for a small variation; a
similar result was identified by Lord Rayleigh in 1887.
More generally, it follows immediately from the properties of Hermitian
eigensystems that the eigenvalues minimize a variational problem:
ω n2, kr = min
r
r r
r
(
)× Ε
i
k
∇
+
∫
Ε n , kr
r
ε
Ε
∫ n,kr
2
2
r
n,k
c2
(3)
r
in terms of the periodic electric field envelope Ε n , kr , where the numerator
minimizes the “kinetic energy” and the denominator minimizes the “potential
energy”. Here, the n > 1 bands are additionally constrained to be orthogonal to the
lower bands:
r
r
r
r
r ⋅ H r = εΕ * r ⋅ Ε r = 0
H
*
∫ m,k n,k ∫ m,k n,k
(4)
r
for m < n. Thus, at each k , there will be a gap between the lower “dielectric”
bands that are less concentrated in the high dielectric: the air bands are forced out
by the orthogonality condition, or otherwise must have fast oscillations that
81
increase their kinetic energy. (The dielectric/air bands are analogous to the
valence/conduction bands in a semiconductor.)
In order for a complete band gap to arise in two or three dimensions, two
additional hurdles must be overcome. First, although in each symmetry direction
r
of the crystal (and each k point) there will be a band gap by the one-dimensional
argument, these band gaps will not necessarily overlap in frequency (or even lie
between the same bands). In order to get their overlapping, the gaps must be
sufficiently large, which implies a minimum ε contrast (typically at least 4/1 in
3D).
Since the 1d mid-gap frequency ~( cπ a ε ) varies inversely with the period a, it
is also helpful if the periodicity is nearly the same in different directions; thus, the
largest gaps typically arise for hexagonal lattices in 2d and fcc lattices in 3d,
which have the most nearly circular/spherical Brillouin zones. Second, one must
take into account the vectorial boundary conditions on the electric field: moving
r2
across a dielectric boundary from ε to some ε’<ε, the inverse “potential” ε Ε
r
r
will decrease discontinuously if Ε is parallel to the interface ( Ε is continuous)
r
r
and will increase discontinuously if Ε is perpendicular to the interface ( Ε ┴ is
continuous). This means that, whenever the electric field lines cross a dielectric
boundary, it is much harder to strongly contain the field energy within the high
dielectric, and the converse is true when the field lines are parallel to a boundary.
Thus, in order to obtain a large band gap, a dielectric structure should consist of
thin, continuous veins/membranes along which the electric field lines can run; this
way, the lowest band(s) can be strongly confined, while the upper bands are
forced to a much higher frequency because the thin veins cannot support multiple
modes (except for two orthogonal polarizations). The veins must also run in all
r
directions, so that this confinement can occur for all k and polarizations,
necessitating a complex topology in the crystal.
Ultimately, however, in two or three dimensions we can only suggest rules of
thumb for the existence of a band gap in a periodic structure, since no rigorous
criteria have yet been determined. This made the design of 3d photonic crystals a
82
trial and error process, with the first example by Ho et al. [98] of a complete 3d
gap coming three years after the initial 1987 concept. As is discussed by the final
section below, a small number of families of 3d photonic crystals have since been
identified, with many variations thereof explored for fabrication.
6.2.3 Computational techniques
Because photonic crystals are generally complex, high index-contrast, two- and
three-dimensional vectorial systems, numerical computations are a crucial part of
most theoretical analyses. Such computations typically fall into three categories:
time-domain “numerical experiments” that model the time-evolution of the fields
with arbitrary starting conditions in a discretized system (e.g. finite difference);
definite-frequency transfer matrices wherein the scattering matrices are computed
in some basis to extract transmission/reflection through the structure; and
frequency-domain methods to directly extract the Bloch fields and frequencies by
diagonalizing the eigenoperator.
Any frequency-domain method begins by expanding the fields in some complete
r r
r r
basis, H kr ( x ) = ∑ hnbn ( x ) , transforming the partial differential equation (2) into a
n
discrete matrix eigenvalue problem for the coefficients hn. Truncating the basis to
N elements leads to N × N matrices, which could be diagonalized in O(N3) time
by standard methods. This is impractical for large 3d systems, however, and is
also unnecessary; typically, one only wants the few lowest eigenfrequencies, in
which case one can use iterative eigensolver methods requiring only ~ O(N) time.
Perhaps the simplest method is based directly on the variational theorem (3):
given some starting coefficients hn, the variational (“Rayleigh”) quotient can be
iteratively minimized using e.g. preconditioned conjugate-gradient descent. This
yields the lowest band’s eigenvalue and field, and upper bands are found by the
same minimization while orthogonalizing against the lower bands (“deflation”).
There is one additional difficulty, however, and that is that one must at the same
r r
r
time enforce the (∇ + ik ) ⋅ H kr = 0 transversality constraint, which is nontrivial in
three dimensions. The simplest way to maintain this constraint is to employ a
r rr
basis that is already transverse, for example planewaves hGr e iG⋅ x with transverse
83
(
)
r
r r
amplitudes hGr ⋅ G + k = 0 . (In such a planewave basis, the action of the eigenoperator can be computer via a fast Fourier transform in O(N log N) time.).
6.3 Three-dimensional Photonic Crystals: Bands Structures calculation
There are three general dielectric topologies that have been identified to support
complete 3d gaps: diamond-like arrangements of high dielectric “atoms”
surrounded by low dielectric, which can lead to 20%+ gaps between the 2nd and
3rd bands for ε=12/1 (Si:air) contrast; fcc “inverse opal” lattices of nearlytangent
low dielectric spheres (or similar) surrounded by high dielectric, giving gaps
around 10% between the 9th and 10th bands for ε =12/1; and cubic “scaffold”
lattices of rods along the cube edges, giving ~ 7% gaps between the 2nd and 3rd
bands for ε=12/1. It is notable that the first two topologies correspond to fcc (facecentered cubic) lattices, which have the most nearly spherical Brillouin zones.
As a model case we use a 3d diamond lattice of dielectric spheres in air, which
has a gap between its second and third bands [98]. Except where otherwise noted,
we solve for the first five bands at the L wavevector point with a 16 x 16x 16 grid
resolution in the (affine) primitive cell (~ 22:6 grid points per lattice constant).
6.3.1 User Reference
The use of the Photonic−Bands package turns around the control file, abbreviated
“.ctl”. The ctl file specifies the geometry you wish to study, the number of
eigenvectors to compute, what to output, and everything else specific to your
calculation. Rather than a flat, inflexible file format, however, the ctl file is
actually written in a scripting language. This means that it can be everything from
a simple sequence of commands setting the geometry, etcetera, to a full−fledged
program with user input, loops, and anything else that you might need.
In the MPB Manual the process of computing the band structure and outputting
some fields for a two and three dimensional photonic crystal using the MIT
Photonic−Bands package were described. The User Reference section and the
data analysis tutorial give a complete description of the features supported by
84
Photonic−Crystals and focused on analyzing and visualizing the results of MPB
calculations.
6.3.2 A Few Words on Units
Maxwell's equations possess an important property: they are scale-invariant.
If you multiply all of your sizes by 10, the solution scales are simply multiplied by
10 likewise (while the frequencies are divided by 10): we can solve a problem
once and apply that solution to all length−scales.
For this reason, MPB usually pick some fundamental length-scale a of a structure,
such as its lattice constant (unit of periodicity), and write all distances in terms of
that. That is, we choose units so that a is unity. Then, to apply to any physical
system, one simply scales all distances by a. This is the default behavior of
Photonic−Bands: the lattice constant is one, and all coordinates are scaled
accordingly. The frequency eigenvalues returned by the program are in units of
c/a, where c is the speed of light and a is the unit of distance. (Thus, the
corresponding vacuum wavelength is a over the frequency eigenvalue.)
6.3.3 Model of Diamond Lattice of Spheres
A diamond lattice of dielectric spheres in air is a three−dimensional structure. It's
harder to find a structure with a complete gap, the modes are no longer polarized,
the computations are far bigger, and visualization is much more difficult.
The band gap of the diamond structure was first identified and reported in: K. M.
Ho, C. T. Chan, and C. M. Soukoulis, “Existence of a photonic gap in periodic
dielectric structures”[98].
Using MPB it is possible to achieved a theoretical calculation of bands defining
the dielectric constant of materials (ε = 11.56 for example), geometry (sphere),
and dimensions (radius r=0,25 µm).
The resulting bands diagram for the diamond lattice model, is shown in Figure 35,
with the complete bands gap shaded yellow; the bands are shown in dimensionless
units c/a (ordinate), where a is the lattice constant, and along symmetry directions
in the whole Brillouin zone (ascisse).
85
Figure 35. Bands diagram for diamond lattice model.
Note that in this case for diamond model we only computed 5 bands, so in reality
the upper portion of the plot would contain a lot more bands (which are of less
interest than the bands adjoining the gap).
Now we can use Vis5D to play around with
various plots of the data (see Figure 36-37)
and can convince our self that it is a diamond
lattice.
Figure 36. First plot for visualization of fields
of diamond lattices spheres.
Figure37. Second plot for
visualization of fields of diamond
lattices spheres: isosurface at the
boundary of the dielectric
superimposed.
86
The lowest two bands have their fields concentrated within the spheres as you
might expect, flowing along more−or−less linear paths. The second band differs
from the first mainly by the orientation of its field paths. The fields for the first
band at U are depicted below, with the strongest fields (highest energy density)
shown as the most opaque, blue pixels. Next to it (see Figure 37) is the same plot
but with an isosurface at the boundary of the dielectric superimposed, so you can
see that the energy is concentrated inside the dielectric.
6.3.4 Model applied and modified to PS, PPA, and PMMA Spheres
To compute theoretical photonic bands for our polymeric crystal arrays we
adopted a 3-d structure with constant lattice a= 315 nm (spheres of diameter = 250
nm and just touching each other) in air: first we applied and compared the
diamond lattice model to PS, PPA, and PMMA nanospheres and then we modified
the lattice from diamond to fcc (face centered cubic lattice) and discussed the
results.
For the same geometry (spheres of diameter 250 nm), in diamond lattice the
results are not significantly different by changing the materials from PS (ε=2.53)
to PPA (ε=2.0) and PMMA (ε=3.3), because of the similar difference between the
dielectric constant of the materials (polymers) and media (air); but for PPA and
PMMA the bands gap presents in PS model disappears.
For the fcc lattice the system is quite complicated. Figure 38 shows the symmetry
points in the Brillouin zone for fcc lattice of PS, PPA, and PMMA.
Figure 38. Symmetry points in the Brillouin zone for a fcc lattice.
87
The bands diagrams of polymeric materials for fcc lattice are reported in Figure
39.
Figure 39. Bands diagrams for fcc lattice of PS (a), PPA (b), and PMMA (c)
nanospheres with diameter 250 nm and just touching each other.
(a)
1,2
frequency (c/a)
1
0,8
band 1
band 2
band 3
band 4
band 5
0,6
0,4
0,2
0
X
U
Γ
L
X
W
K
(b)
1,4
1,2
frequency (c/a)
1
band 1
band 2
0,8
band 3
0,6
band 4
band 5
0,4
0,2
0
X
U
L
Γ
X
W
K
88
(c)
1,2
frequency (c/a)
1
0,8
band 1
band 2
0,6
band 3
band 4
0,4
band 5
0,2
0
X
U
L
Γ
X
W
K
The bands diagrams (see Figure 39) show that there are two symmetry points (W
and U) and also a crossing of band along the symmetry line: these two features of
the band structures prevent a gap. There is a region where a band gap exists over
most, but not all, of Brillouin zone, resulting in a region of low density of states
than a forbidden frequency gap.
Further investigations will be devoted to better tune the theoretical calculation by
changing lattice structure (bcc and hpc lattices) and to apply this tool on new copolymers synthesised, that request dielectric constants measures.
89
Chapter 7 Conclusions
Using the bottom up approach (modified emulsion synthesis technique) and the
top down approach (Method for controlling the dimensions and the morphology
of nanostructural polymeric materials, PCT Int. Appl. (2006) CODEN: PIXXD2
WO 2006051572 A2 20060518 ) the novel nanostructured polymers were realised
with strict control of shape, dimensions, and polidispersity.
In particular, monodispersed nanospheres of polymethylmethacrylate (PMMA),
polystyrene (PS), polyphenylacetylene (PPA) were synthesised by a modified
emulsion
PS/HEMA,
polymerization
technique;
PPA/HEMA,
PA=phenylacetylene,
moreover,
PPA/AA,
copolymers
PPA/DMPA
HEMA=2-hydroxyetylmethacrylate,
nanobeads
(S=styrene,
AA=acrylic
acid,
DMPA=N,N-dimethylpropargylamine) were produced.
In order to optimize the production of the self assembling of polymeric
nanospheres a systematic study of the influence of the reaction conditions on the
chemico-physical characteristics of the nanobeads was performed. In particular,
the role of the cosolvent and time reaction on the particles size, and the effect of
the surface-charge in the copolymers on ordered domain formation were
investigated. The modified emulsion polymerization conditions were optimized
for each system and strict control of shape and size of the nanoparticles were
obtained. Systematic studies about the self-assembling and deposition conditions
of polymeric nanobeads were performed to investigate the photonic crystals and
colloidal crystals realization.
The new nanostructured polymers and copolymers, obtained by modified
emulsion synthesis, have been used for biotechnological applications and sensors.
Commercial lypases (CRL and PCL) were immobilised onto nanostructured
PMMA and PS: the established adsorption protocols permitted to prepare stable
bioconjugates with an activity retention up to 70%. The easy method clearly
indicates that nanostructured synthetic and biocompatible polymers can be useful
support in the enzyme immobilization technology for industrial applications.
Further work is in progress in order to investigate in depth the conformational
90
structure of the enzyme molecules on the surface of nanostructured polymers and
to realize bioconjugates using P(PA/AA) and P(PA/DMPA) nanospheres
covalently bound to enzyme.
Miniaturized humidity sensors have been prepared by coating interdigitated
electrodes with thin films of nanostructured polymers (PPA and P(PA/HEMA).
The devices were tested at different RH values, and the I/time curve of these
sensors has been investigated.
The top down approach resulted in a European patent that consists in a novel
physical processing of the pristine polymers to obtain different nanostructured
morphologies. The described method allows a controlling of the dimensions and
the morphology of the natural and synthetic polymers by means of the dialysis
technique.
The bottom-up and top-down strategies were both successful used to obtain
polymeric nanostructured materials with different chemicals structures.
The first approach, bottom up, is best suited for synthesis of nanoparticles, which
are obtained with strict control of dimensions and polydispersity that promote
self-assembly and long-range order at the nanoscale dimensions.
The second approach, top down, is advantageous for producing structures with
very large surface-to-volume ratio and for interdigitate connections in electronic
devices, without need of strict control of monodispersity and long-range order.
Theoretical investigations about photonic bands gap of nanostructured polymers
were performed by MPB software to know the photonic properties of the
synthesised polymeric nanospheres self-assebling.
91
Summary
The research activity concerning this PhD thesis was focused on the synthesis and
characterization of nanostructured polymers for applications in optics (photonic
crystals), sensors and biocatalysis (enzymes-polymeric nanospheres hybrid).
Novel nanostructured polymers were realized achieving control of shape,
dimensions and polydispersity by using the bottom up approach (modified
emulsion synthesis technique) and the top down approach (PCT Int. Appl. 2006).
All new materials were characterized by spectroscopic techniques (UV-vis, IR,
1H and 13C NMR), gel permeation chromatography (GPC), dynamic light
scattering (DLS) and scanning electron microscopy (SEM).
In particular, for optics applications the realization of polymeric photonic crystals,
PCs, were performed: PCs are composite materials constituted by regularly
spaced assemblies of units whose interaction with electromagnetic radiation
produces a control of the field modes in a spectral region which depends on the
particles dimensions and array spacing.
In this framework, several polymeric nanospheres were synthesized: PMMA
(polymethylmethacrylate), PS (polystyrene), PPA (polyphenylacetylene) and the
copolymers P(S/HEMA), P(PA/HEMA), P(PA/AA), P(PA/DMPA) (S=styrene,
PA=phenylacetylene, HEMA=2-hydroxyethylmethacrylate, AA=acrylic acid,
DMPA=N,N-dimethylpropargylamine).
In particular, nanobeads of PPA, a luminescent and semiconducting polymer,
were prepared and interesting results on the formation of ordered domains were
obtained. Nanostructured PPA is expected to find applications in optical and
electronics devices. The studies so far carried out indicate that PPA can be used as
sensitive membrane in chemical sensor for the detection of humidity showing
improved response with respect to the non-nanostructured polymer. PPA is also a
candidate for cell and protein immobilization because of its biocompatibility.
My first attempts to obtain polymeric PCs were based on the preparation of
PMMA and PS nanobeads by emulsion polymerization, using KPS (potassium
persulfate) initiator: a systematic studies about synthesis conditions, such as
effects of time reaction, cosolvent/monomer ratio and initiator/ monomer ratio, led
92
to the formation of monodispersed nanospheres that self assembly in large ordered
domains. Systematic studies about the self-assembling and deposition conditions
of polymeric nanoparticles were performed to investigate the photonic crystals
and colloidal crystals realization.
In the first approach for the synthesis of PPA nanobeads the [Rh(COD)Cl]2tmeda
(COD=cis,cis-1,5-cyclooctadiene, tmeda=N,N,N’,N’-tetramethylethylendiamine)
catalyst was used. However, although in that case some ordered domains were
formed, their area was limited because of the high polydispersity of the nanobeads
(low polydispersity is a necessary condition for the long-range ordering of
particles and for the minimization of the density of defects in the PC structure).
With the aim of improving the control on the dimension, polydispersity and self
assembling of the nanospheres, two different routes were explored.
The first one is based on studies of modified emulsion synthesis. The colloidal
stability and consequently the nanospheres monodispersity were improved with
the presence of a co-solvent (toluene) and charged species (K+) in the reaction
mixture; by changing the polymerization conditions with the use of a radical
initiator such as potassium persulfate (KPS) charged species were introduced into
the reaction; KPS, radical polymerization initiator, was never used for PPA
synthesis.
The second approach was based on the preparation of charged copolymers
obtained by modified emulsion synthesis which introduces the charge by proper
choice of the comonomer. Nanobeads of P(S/HEMA) (poly[styrene-(co-2hydroxyethyl
methacrylate)]),
P(PA/HEMA)
hydroxyethyl
methacrylate)]),
P(PA/AA)
(poly[phenylacetylene-(co-2-
(poly[phenylacetylene-(co-acrylic
acid)]) P(PA/DMPA) (poly[phenylacetylene-(co-N’N’-dimetylpropargyl amine)])
were prepared. P(S/HEMA) was a model for the comparison of its properties with
those of the new synthesized materials. The charged surface of these copolymers
enhances the colloidal stability in aqueous suspensions and induces long range
ordering in the colloidal phase, which can be used for the development of optical
sensors based on crystalline colloidal arrays (CCA). Furthermore, since it is
known that the hydroxyl groups on the particles surface improve the adhesion
properties for coating and adhesive applications, similar performance is expected
93
for P(PA/DMPA) and P(PA/AA) copolymers. Several applications for optical
biosensors are also foreseen because amino and acid groups, which are functional
moieties of our copolymers, are suitable sites for attaching biological materials
such as enzymes.
The top down route consists in a novel approach of application of a physical
processing, dialysis, to obtain different nanostructured morphologies (reticular
and spherical shape) by pristine non-nanostructured polymers. The method allows
a control of the dimensions and the morphology of natural and synthetic polymers
by means of the dialysis technique. The advantages are several: low cost, general
application and soft work conditions, without use of emulsifier and stabilizer,
which allow to achieve pure products and easy recovery of solvents.
The bottom-up and top-down strategies were both successful used to obtain
polymeric nanostructured materials with different chemicals structures.
The first approach, bottom up, is best suited for synthesis of nanoparticles, which
are obtained with strict control of dimensions and polydispersity that promote
self-assembly and long-range order at the nanoscale dimensions.
The second approach, top down, is advantageous for producing structures with
very large surface-to-volume ratio and for interdigitate connections in electronic
devices, without need of strict control of monodispersity and long-range order.
Considering the above cited features, the new nanostructured polymers and
copolymers have been used for biotechnological and sensors applications.
As a first approach commercial lipases (CRL and PCL) were immobilized onto
nanostructured PMMA and PS: the established adsorption protocols permitted to
prepare stable bioconjugates with an activity retention up to 70%. These studies
clearly indicated that nanostructured synthetic and biocompatible polymers can be
a useful support in the enzyme immobilization technology, showing an enhanced
performance with respect to that of the same lipases immobilized on the nonnanostructured polymers. Further work is in progress in order to elucidate the
interaction at the enzyme/polymer interface. The conformational structure of the
enzyme molecules on the surface of nanostructured polymers will be investigated
in depth. Bioconjugates using P(PA/AA) and P(PA/DMPA) nanospheres
covalently bound to enzyme will be further object of our studies.
94
Some of our materials have been tested for sensing response to relative humidity
(RH%). Miniaturized humidity sensors have been prepared by coating
interdigitated electrodes with thin films of nanostructured polymers (PPA and
P(PA/HEMA)). The devices were exposed to different RH % values, and the
I/time curve of these sensors has been investigated. Preliminary results suggest
that improved responses are achievable by sensors based on nanostructered
undoped PPA and P(PA/HEMA): the I vs RH curve for these polymers showed a
hysteresis curve that may be attributed to main ionic contribute to the conductivity
(ionic and electronic) of these materials.
Further investigations will be devoted to test nanostructured PPA (I2 doped or
undoped ) and its copolymers P(PA/HEMA), P(PA/DMPA) and P(PA/AA) in
resistive sensors for the detection of several gases, since it has been established
that the nanostucured morphology enhances the sensitivity of these devices.
As a completion of the experimental research, theoretical investigations about
photonic bands gap of nanostructured polymers were performed by MPB (MIT
Photonic Band gap) software to predict and understand the expected properties of
the self-assembling polymeric nanospheres as photonic crystals.
95
Tables
Table 1.
The reaction conditions of PPA nanobeads synthesis with [Rh(cod)Cl]2tmeda
catalyst (always 2.0x10-4 M for samples ID 1-17) or KPS initiator (different
amount for samples ID 18-25), their SEM diameter (diameters range for ID 1-17
and average diameters for ID 18-25), and PI.
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
H2O/PA
(% vol.)
10/1
20/1
25/1
10/1
10/1
20/1
20/1
20/1
20/1
20/1
40/1
40/1
40/1
40/1
20/1
40/1
0/1
10/1
10/1
10/1
10/1
10/1
10/1
10/1
10/1
tol/PA
T
KPS or Rh (I)
(% vol.) (min)
(g)
0/1
60
0
0/1
60
0
0/1
30
0
1/1
60
0
1/1
30
0
1.6/1
60
0
2/1
60
0
4/1
60
0
4/1
30
0
4/1
10
0
4/1
120
0
4/1
60
0
4/1
30
0
4/1
10
0
8/1
60
0
8/1
60
0
10/1
1440
0
0/1
90
0.04
2/1
90
0.008
2/1
90
0.02
2/1
90
0.04
2/1
90
0.08
10/1
30
0.04
10/1
60
0.04
10/1
90
0.04
SEM diameter
(nm)
180-500
450-900
400-1000
900-1500
200-750
400-600
560-800
430-560
500-2500
250-2000
550-1200
380-750
280-650
400-2500
300-2500
350-3600
-450
280
310
400
530
190
250
300
SEM PI
0.94
0.67
0.86
0.50
1.16
0.40
0.35
0.20
1.33
1.59
0.74
0.65
0.80
1.45
1.57
1.65
-0.62
0.14
0.31
0.50
0.67
0.84
0.52
0.33
96
Table 2a.
Reaction conditions, average diameter, and PI, obtained by SEM and DLS
measurements of PMMA nanobeads synthesis.
ID tol/MMA
(% vol.)
1
0/1
2
0/1
3
0/1
4
0/1
5
0/1
6
0/1
7
0/1
8
0/1
9
1/1
10
1/1
11
1/1
12
1/1
13
1/1
14
1/1
15
1/1
16
1/1
17
1/1
18
2/1
19
2/1
20
2/1
21
2/1
22
2/1
23
2/1
24
2/1
25
2/1
26
2/1
27
2/1
28
4/1
29
4/1
30
4/1
31
4/1
32
4/1
33
4/1
34
4/1
T
SEM diameter
(min)
(nm)
10
126
30
245
50
320
60
345
90
370
120
385
180
405
240
420
10
103
20
132
30
153
40
183
50
205
60
222
90
266
120
289
240
315
10
118
20
144
30
170
40
200
50
217
60
245
90
272
120
298
180
309
240
328
30
193
40
205
50
224
60
255
90
280
120
319
180
347
SEM PI DLS diameter
(nm)
0.05
-0.034
-0.04
-0.14
-0.14
-0.13
-0.12
-0.14
-0.005
120
0.34
157
0.24
184
0.34
209
0.29
229
0.31
243
0.07
300
0.25
315
0.27
340
0.05
137
0.07
161
0.12
193
0.11
235
0.16
-0.04
272
0.10
-0.05
335
0.06
-0.02
357
0.10
217
0.09
242
0.03
263
0.05
282
0.03
315
0.04
365
0.11
--
DLS PI
--------0.028
0.033
0.042
0.033
0.020
0.028
0.003
0.038
0.028
0.003
0.006
0.015
0.002
-0.008
-0.006
-0.002
0.015
0.014
0.035
0.012
0.017
0.005
--
97
35
4/1
240
363
0.06
390
0.040
Table 2b.
Best fitting parameters obtained from SEM experimental data using
expression (1).
τ
(s)
36.3
62.4
59.4
72.6
toluene/MMA
(%vol)
0/1
1/1
2/1
4/1
d∞
(nm)
409±6
324±5
328±5
371±8
d0
(nm)
37±2
59±2
74±3
86±4
Table 3.
PS and P(S/HEMA) nanobeads synthesis conditions, their SEM average diameter
and PI.
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
H2O/S
(% vol.)
1/1
1/1
1/1
1/1
1/1
3/1
3/1
3/1
3/1
3/1
3/1
3/1
3/1
3/1
3/1
3/1
3/1
SDS
(g)
0.25
0.25
0.025
0.025
0.025
-
KPS
(g)
0.020
0.020
0.020
0.020
0.020
0.055
0.055
0.055
0.055
0.055
0.055
0.055
0.055
0.055
0.055
0.055
0.055
S/HEMA
(% vol.)
40/1
40/1
40/1
40/1
20/1
20/1
20/1
20/1
10/1
10/1
10/1
10/1
Time
(h)
1.5
7
7
8
22
24
17
5
2.5
17
20
22
24
17
20
22
24
SEM diameter
(nm)
80
365
60
150
250
465
220
200
165
343
435
504
514
477
498
500
516
SEM PI
0.3
0.10
0.40
0.42
0.45
0.11
0.15
0.25
0.42
0.20
0.25
0.41
0.35
0.25
0.18
0.16
0.18
98
Table 4.
Reaction conditions for P(PA/HEMA), P(PA/DMPA) and P(PA/AA) nanospheres
synthesis (Rh(I) dimer used in P(PA/DMPA) synthesis, ID 11-18, APS used in
P(PA/AA) synthesis, ID 19-22), their SEM average diameter and PI (M = comonomer).
ID
M=
HEMA
M=
DMPA
M=
AA
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
tol/PA
(% vol.)
2/1
0/1
2/1
2/1
2/1
2/1
2/1
2/1
10/1
2/1
0/1
2/1
2/1
2/1
2/1
2/1
2/1
4/1
2/1
1/1
1/1
1/1
PA/M
T
KPS Rh dimer or APS dav
(% vol.) (min) (g)
(g)
(nm)
-2/1
90
0.02
270
-10/1
90
0.02
405
-10/1
30
0.02
70
-10/1
60
0.02
110
-10/1
90
0.02
220
-10/1
150 0.02
400
-10/1
150 0.055
195
-10/1
150 0.08
60
-10/1
90
0.02
175
-20/1
90
0.02
200
10/1
1440 0.010
0.010
289
10/1
60 0.010
0.024
368
10/1
120 0.010
0.024
396
10/1
150 0.010
0.024
517
10/1
180 0.010
0.024
658
10/1
1320 0.010
0.024
805
10/1
1440 0.010
0.024
1043
10/1
1440 0.010
0.010
-10/1
1200 0.010
0.140
500
10/1
120 0.010
0.035
945
10/1
1200 0.010
0.035
650
10/1
1440 0.010
0.035
650
PI
0.15
0.46
0.86
0.55
0.34
0.20
0.36
0.33
0.23
0.20
0,97
1,58
1,55
1,37
1,29
0,80
0,72
-1,3
1,2
0,88
0,86
Table 5.
New methodology to obtain nanostructured polymers: selected trials to obtain
different morphologies.
ID
D2
Solvent /
non solvent [PPA] mg/ml Membrane Solv / non solv
THF
0.5
single
1/20
Polymer Morphology
PPA
1
99
H2O
D3
THF
5
single
1/40
PPA
2
D4
H2O
DMF
H2O.
0.5
single
1/20
PPA
3
D 20
DMF
5
double
1/20
PPA
4
D7
hexane
DMF
H2O
10 (satured)
double
1/20
PPA
5
D 25
DMF
H2O
10
single
1/20
PMMA
3
D 26
DMF
MeOH
THF
H2O
10
single
1/60
PMMA
4
10
single
1/40
PMMA
5
D 33
DMF
H2O
5
single
1/20
PMMA
2
D 34
DMF
EtOH
DMF
H2O
5
single
1/20
PMMA
3
10
single
1/20
PS
4
10
single
1/20
PS
5
10
single
1/20
PS
4
D 27
D 41
D 44
D 45
DMF
MeOH
DMF
EtOH
Table 6.
The specific activity of free and immobilized lipases under standard reaction
conditions
Samples
Free CRL
CRL on
amorphous
PMMA
CRL on
nanostructured
PMMA
CRL on
amorphous PS
CRL on
nanostructured
PS
Free PCL
Bound
protein
(mg/g)
-
-
Specific
activity
(U/mg)
3,85
0,77
Activity
retention
(%)
100
20
2,31
60
1,12
29
2,83
74
7,66
100
100
PCL on
amorphous
PMMA
2,46
32
PCL on
nanostructured
PMMA
PCL on
amorphous PS
PCL on
nanostructured
PS
5,13
67
2,24
29
5,62
73
Table 7.
Esterase activity and selectivity of CRL and PCL free and immobilised on
nanostructured polymers employed in the transesterification reaction of (±)-1phenylethanol with vinylacetate in different organic solvents (Reaction
conditions: enzyme 5 mg, reaction volume=1 ml, reaction temperature T=40°C,
reaction time = 6h).
ENZYMES
free CRL
CRL on amorphous
PMMA
CRL on nanostructured
PMMA
CRL on amorphous PS
CRL on nanostructured
PS
free PCL
PCL on amorphous
PMMA
PCL on nanostructured
PMMA
PCL on amorphous PS
PCL on nanostructured
PS
yield
acetonitrile
logP=0,03
yield
eep%
t-buthylmethyl-etere
logP=0,94
yield
eep%
9,7%
13,2%
52,73
57,45
21,5%
23,6%
97,51
96,40
≤1
≤1
-
23,0%
65,92
30,5%
97,82
≤1
-
10,8%
16,7%
55,83
68,21
24,2%
28,7%
95,81
97,38
<10
<10
-
28,5%
40,2%
100
99,87
14,2%
23,6%
100
100
<10
<10
-
54,3%
100
50,4%
100
<10
-
32,4%
42,0%
100
100
21,8%
43,2%
100
100
<10
<10
-
yield eep%
101
Acknowledgments
I am very grateful to Dr. Mauro Falconieri: he introduced me to the study of
photonic crystals and optical properties of materials; I am indebted to him for his
advice, and encouragement.
I would like to thank my advisor, Prof. M. V. Russo for her assistance, and
encouragement: she gave me the possibility to carry on my research work.
Thanks to dr. Dr. Ferro for SEM images, to Dr C. Palocci for strict collaboration
about bioconjugates research and to dr. A. Bearzotti for the measurements on
resistive sensors.
My acknowledgments goes also to laboratory friends, Dr. Ilaria Fratoddi, Rosa
Vitaliano, Floriana Vitale, and Ilio Foglietta, for their kindness and
encouragement. I would like to tank my friend and colleague Dr. Rosaria
D’Amato.
Finally I am very grateful to my family, Marco and Elena, for their love,
encouragement, and patience, and to my parents and my sister Ilaria, for their
advice.
Part of this work was supported by Ministero dell’Università e della Ricerca
Scientifica
e
Tecnologica
through
project
NANOFASI,
FISR
2003.
102
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