Microfluidics Laboratory
For Scientific and Technological Applications
Project duration: 36 months from July 2008
Project coordinator: prof. Michele Maggini
Dipartimento di Scienze Chimiche
Università di Padova
Via Marzolo, 1 35131 – Padova
Phone: 049-8275662, fax: 049-8275792
e-mail: [email protected]
project webpage:
www.chimica.unipd.it/mischa
Second scientific report
month XXVI
(September 2009 – September 2010)
Research funded by
Fondazione CARIPARO
Bando Progetti di Eccellenza 2007-2008
Padova October 6, 2010
Riassunto/Summary
MISCHA è un progetto che riunisce ricercatori dei Dipartimenti di Scienze Chimiche (DISC), di Fisica (DIP),
di Ingegneria dell’Informazione (DEI) e di Principi e Impianti di Ingegneria Chimica (DIPIC) dell’Università di
Padova allo scopo di creare un laboratorio integrato per la modellizzazione, fabbricazione e
caratterizzazione di dispositivi per la microfluidica (MF). MISCHA intende valorizzare la sinergia fra
competenze diverse come impegno strategico per procedere rapidamente verso risultati anche di rilevanza
applicativa. Priorità del progetto è inoltre quella di formare giovani ricercatori in un contesto multidisciplinare
come efficace e duraturo investimento in un’area emergente di progresso tecnico-scientifico. Il progetto è
partito regolarmente nel luglio 2008 e nel corso di questo secondo anno l’attività di ricerca si è sviluppata
secondo le linee indicate nel Work Programme. Sono attivi sei assegni di ricerca su tematiche inerenti la MF
di cui quattro a totale carico del progetto e due cofinanziati. E’ attivo un sito web di progetto
(www.chimica.unipd.it/mischa) che contiene, oltre alle informazioni sulle competenze dei partecipanti e sugli
obiettivi del progetto, la documentazione relativaall’avanzamento della ricerca (documentation:
username:mischa, passwd: reserved). Il 5 luglio 2010, dopo 20 mesi dalla partenza del progetto, si è tenuto
presso il Dipartimento DIPIC il MIDTERM meeting di revisione tecnica in cui sono stati presentati e discussi i
risultati della ricerca. Il giorno 8 luglio 2010 è stata inaugurata la clean room (ISO 7, classe 10000) che
costituisce parte integrante del laboratorio di microfluidica (µLAB) in corso di realizzazione presso il DISC
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dove è a disposizione uno spazio di 50m . Nel corso della cerimonia di inaugurazione sono stati presentati i
risultati più salienti della ricerca MISCHA ed è stata apposta una targa in cui è menzionato il contributo
finanziario della Fondazione CARIPARO alla realizzazione della clean room in cui si fabbricheranno
microreattori (MR) e circuiti MF impiegando tecniche di litografia soft in ambiente controllato. Sono stati
acquisiti inoltre due reattori commerciali per lo sviluppo di sintesi in flusso continuo nell’ambito di
collaborazioni con l’industria. I ricercatori MISCHA, nel corso del secondo anno di attività, hanno
perfezionato la tecnologia per fabbricare MR da impiegare sia per sintesi chimiche sia per condurre
esperimenti di fluidodinamica ed elettroporazione di materiale cellulare (WP1). Sono stati inoltre fabbricati
alcuni prototipi di MR per la crescita di linee cellulari in collaborazione con ricercatori dell’area biomedica. Il
gruppo di caratterizzazione (WP2) ha impiegato le strumentazioni, acquisite ed implementate nella prima
fase del progetto, per misure di fluidodinamica attraverso le tecniche PIV (particle image velocimetry) e FCS
(fluorescence correlation spectroscopy) e per studiare l’elettroporazione cellulare attraverso la spettroscopia
di impedenza. Il gruppo di ricerca teorica (WP3) ha studiato i fenomeni di mescolamento nei MR, la
modellazione della fluidodinamica di fasi eterogenee e la realizzazione di geometrie di MR per
l’ottimizzazione di reazioni chimiche e lo studio cinetico comparativo flask versus flow. Nel 2010 la ricerca si
è concretizzata in due lavori a stampa e uno inviato per la pubblicazione su riviste ISI, tre comunicazioni in
atti di congressi inviate per la pubblicazione (proceedings con referee) ed è stata presentata, in forma di
comunicazioni orali o poster, in 14 convegni nazionali e internazionali o seminari. Nel secondo anno di
attività sono state compilate 7 tesi di laurea a tema microfluidico e coltivate collaborazioni sia con istituzioni
accademiche sia con aziende. La realizzazione della clean room e l’acquisizione di reattori per processi in
flusso di interesse industriale costituiscono due elementi importanti per realizzare il laboratorio integrato di
microfluidica citato sopra. D’altro canto i risultati scientifici ottenuti nel secondo anno possono essere
considerati soddisfacenti anche se il numero di pubblicazioni risulta inferiore alle aspettative, in
considerazione del numero di persone coinvolte nel progetto e dell’attività svolta. E’ tuttavia emerso, nel
corso del midterm meeting, che numerosi lavori sono prossimi ad essere inviati per la pubblicazione per cui
è ragionevole aspettarsi per il prossimo anno una produttività più rilevante.
MISCHA second year of activity has been characterized by the completion of the clean room facility within DISC for the
fabrication of MR through soft-lithography methods. The MISCHA activity was also devoted to seeding methods and
applications of modern microfluidics through the formation of students during their thesis stage, the collaboration with
industry and the involvement of participants in adjacent projects. Six young project co-workers are currently sponsored
by MISCHA. Several scientific accomplishments have been achieved, and interdisciplinary activities, among working
groups, emerged. An internet website is active to improve the online information exchange among MISCHA partners
(www.chimica.unipd.it/mischa). Scientific achievements during the second year have been quite satisfactory. In particular
the fabrication team (WP1) is now able to fabricate generic MR and microchannel networks made of different matrices,
such as NOA-81, SU-8 and PDMS. Characterization efforts (WP2) were devoted to integrate Particle Image Velocimetry
and Fluorescence Correlation Spectroscopy to MR technology for fluid motion studies and electrical measurement
protocols to model microelectrodes behaviour and carry out cells electroporation experiments. Theoretical studies (WP3)
were concerned with (i) simulation of micro- and nanoflows, (ii) characterization of mixing phenomena in MR, (iii)
segregation of suspensions in microflow (iv) optimization of MR via genetic algorithm. Collaborations with academic
institutions and industrial partners were strengthned during the second year (Universities of Trento, Roma Tor Vergata,
Milano Bicocca, Abo Akademi-Finland, Centro Ricerche Danieli-Udine; QID-Trieste, Fabbrica Italiana Sintetici,
Montecchio Maggiore, Corning Inc.-USA). In particular, an activity related to microfludic research was recognized by
MIUR, for the project "Carbon nanostructures in microfluidic reactors" (PRIN 20085M27SS_005). The results of the
research have been presented in 14 communications at national and international conferences/seminars and published
in two ISI papers. A further paper and three conference proceedings have been also submitted for publications.
2
Microfluidics laboratory for scientific and technological applications
Foreword
This report covers the MISCHA activities of seven groups belonging to four Departments of the University of
Padova, from October 2009 through September 2010. Overall performance of the second year is quite
satisfactory. However, we have to improve productivity in terms of publications and, hopefully, patents. The
communication among MISCHA participants have been improved in more working group meetings, apart
from the official meetings, expecially after the creation of a common space adjacent to the clean room facility
which has been officially opened on July 8, 2010 with a public ceremony. An internet project website is active
to stimulate the online information exchange. Collaborations, at national and international level, with groups
active in microfluidics have been strengthned. This project continued its role for "Seeding Project" through (a)
the formation of young students on microfluidics during their thesis stage; (b) the collaboration with industry
and (c) the involvement of some participants in adjacent projects. MISCHA partners clearly state that, in view
of the achieved results to date and the potential for exploitation, they wish to continue the project. The Work
Programme for the third year will not be altered and will continue towards innovative microfluidic devices for
chemical synthesis and biomedical applications.
1. Introduction
The main objectives of MISCHA are setting up an unified scientific framework for fabrication, characterization
and modelling of microfluidic devices (MFDs) through the housing of a microfluidic laboratory (MFL) in
Padova in which theoretical studies and testing of innovative MFDs for chemical syntheses and applications
in the biomedical fields are carried out. The strength of the project lies in joining together the needed
expertises available in several groups from the Departments of Chemical Sciences (DISC), Physics (DIP),
Chemical Engineering Principles and Practice (DIPIC) and Information Engineering (DEI).The Work
Programme includes three Work Packages (fabrication and functionalization of MFDs, characterization and
development of MFDs, description and modelling of MFD) that, in turn, include eleven tasks whose titles are
as follows:
Task 1.1: Fabrication of MFDs and micro-electroporation devices
Taks 1.2: Fabrication of glass MFDs and SERS studies
Task 1.3: surface inertization and functionalization
Task 1.4: nanofabrication and testing of MFDs
Task 2.1: Implementation of spectroscopic techniques
Task 2.2: Electrical characterization and modelling of microelectrodes
Task 2.3: Manipulation of molecular systems and aggregates inside MFDs
Task 3.1: Determination of solid-liquid structural interfaces properties
Task 3.2: Description of nanoflows
Task 3.3: Description of microflows
Task 3.4: Definition of multiscale approaches
1.1 MISCHA coordination activities.
The Project Management Committee, which is composed by the Project coordinator, the Work Package
managers and the webpage manager, met regularly for the high level decision making as well as for
managing the R&D activities. The MIDTERM meeting was held on July 5, 2010 at DIPIC. Most of the
meeting was devoted to technical presentations, in the form of oral and poster presentations, and to a
general discussion of the main achievements of the project and planning of future project activities. A written
reports for the meeting has been up-loaded in the webpage reserved area (www.chimica.unipd.it/mischa,
documentation: username: mischa, passwd: reserved).
1.2 List of partner groups. Fabrication of microreactors (contact person: prof. G. Mistura, DIP), synthesis of
molecules and materials (CP: prof. M. Maggini, DISC), optical characterization (CP: prof. C. Ferrante, DISC),
theoretical modelling (CP: prof. A. Polimeno, DISC), microelectronics (CP: prof. S. Paccagnella, DEI),
multiphase systems (CP: prof. P. Canu, DIPIC).
1.3 Fellowships. MISCHA has funded and co-funded the grants of 10 collaborators investing 62% of the
budget in the formation of young scientists in the field of microfluidics (Table 1). Presently, 6 contracts are
active.
Table 1. List of personnel funded by MISCHA
Prasenjit Maity
ADR, 2 years
DISC
Chemical synthesis in MR
A
Ilaria Fortunati
ADR, 2 years
DISC
optical spectroscopies
A
Silvia Carlotto
ADR, 2 years
DISC
Theoretical modelling
A
Simone Gerardin
cofin ADR, 1 year
DEI
Microelectronics
A
Alessandro De Toni
cofin ADR, 2 years
DEI
Microelectronics
A
Tamara Toth
ADR, 1 year
DIP
Fabrication of MR
A
Giorgio Marinaro
contract, 1 year
DEI
Microelectronics
-
Giorgio Cellere
contract, 4 months
DEI
Microelectronics
-
Sara Pizzato
contract, 1 year
DIPIC
Multiphase systems
-
Sebastiano Valenti
contract, 1 year
DIPIC
Multiphase systems
-
cofin: matching funds by MISCHA; ADR: assegno di ricerca paid by MISCHA; contract: borsa di studio paid by MISCHA; A: active (October 1st, 2010)
1.4 MF laboratory. The Department of Chemical Sciences agreed to host the MF laboratory and assigned
two rooms (02-082 and 02-083) for this purpose. In this connection, the U. of Padova Central Administration
allocated resources to cover the cost for renovation of the mentioned rooms to accommodate a medium-size
Clean Room (CR, ISO 7, class 10000, 25 m2; dressing room, ISO 8, 6 m2) for MR fabrication through softlithography methods, and a series of instruments for the characterization and study of MRs, besides those
already available within the participants laboratories. The cost of the clean room has been covered by
MISCHA and by another project (Helios – 2008) funded by the U. of Padova that, among its objectives, has
the synthesis of functional molecular structures with MRs. In May 2010, the CR has been completed by
Galvani Srl. Next to the CR, a furnished room has been also set up as a place to meet, discuss and
exchange ideas within the MISCHA participants.
Cleanroon technicalities. A CR has a controlled level of contamination that is determined by the number of
particles per cubic meter at a specified particle size. The air entering the CR is filtered to exclude dust,
whereas the air inside is constantly re-circulated through high efficiency particulate air (HEPA) filters to
remove internally generated contaminants. Temperature and humidity are controlled by a
compression/heating system that maintains the T at 22 ± 2 °C with a relative umidity of 50 ± 10 %. The CR,
which is kept at a positive pressure, uses a laminar air flow regime to direct the filtered air downward in a
constant stream (air velocity lower than 0.45 m/s; total volume of air passing through the filters equal to
10000 m3/h). Our CR is ISO 7 for the laboratory area and ISO 8 for the dressing room: the Table reported
below shows the particle specifications for the CR according to ISO and FED standards.
Class (ISO)
ISO 7
ISO 8
ISO 9
≥0.1 µm
-
Maximum particles / m3
≥0.2 µm
≥0.3 µm
≥0.5 µm
352000
3520000
35200000
≥1.0 µm
83000
830000
8300000
≥5.0 µm
2930
29300
293000
Class (FED
STD 209E)
Class 10000
Class 100000
Room air
Inside the CR. A chemical fume hood, a ventilated cabinet and a fridge have been placed inside the CR for
the safe handling and storing of chemicals. A series of small instruments are also available in the CR as
follows: (1) a programmable spin coater (speeds up to 5000 rpm) that uses a
pneumatic system to spin the samples; (2) a collimated UV system equipped
with a 300 W mercury lamp, a mirror and a quartz lens for photolithography; (3) a
UV-O3 cleaner in which the UV radiation is employed to generate ozone for
surfaces cleaning and activation; (4) hot plates, an analytical balance, a PC
station and a set of dedicated glassware and accessories complete the CR
equipment. In the last four months, the CR has been used mainly for replica
molding experiments to produce nanostructured PDMS surfaces (DIP) and for
the fabrication of microchannel networks and MF devices using NOA-81 and SU8 resins (DiSC, DIP, DIPIC). Access to the CR is granted to all MISCHA participants, and in principle also to
other external scientists, provided that they operate under the standard rules of safety and good laboratory
practice. An opening ceremony was held on July 8th, 2010 in which the mission of the MISCHA project and
the potential uses of the CR were presented to the public by the coordinator and by two young MISCHA
participants (S. Silvestrini and I. Fortunati). A plaque was placed outside the CR indicating the financial
support of Fondazione CARIPARO to the MF laboratory. In the room next to the CR, simple experiments
were carried out showing some applications of prototype MF devices fabricated during the early stages of
the MISCHA project. The ceremony was also an unique opportunity for young MISCHA participants to show
4
their research work to the visitors in the form of poster presentations on microfabrication, surface
characterization, applications in chemical synthesis, fundamental flow dynamics and theoretical modeling.
2. Overview of the Technical Progress
2.1 WP1 (coordinator prof. Giampaolo Mistura)
Figure 1 shows an abstract of the MISCHA network Gannt chart in which the Tasks of WP1 are considered.
Figure 1. Abstract of the MISCHA Gannt chart showing the Tasks of WP1.
According to the Work Programme, deliverable D1-2 is the functionalization of MFDs with functional
molecular structures.
Task 1-fabrication (DISC, DIP). A microfluidic chip comprising only microfluidic channels in the range of
hundreds of micrometers, can hardly be competitive with devices made with teflon tubing and T- or Yjunctions, in the field of chemical synthesis. For this reason different elements were integrated in MISCHA
custom chips, such as optical fibers and electrodes (S. Silvestrini). Optical fibers were readily inserted into
NOA-81 chips, since the resin was first developed as an optical glue. Moreover, NOA-81 itself was used to
fabricate optical components such as mirrors and lenses to guide a LASER light through the chip. Electrodes
were also added to our design for carrying out electrochemical impedence spectroscopy measurements and
electroporation of live cells (in collaboration with DEI – A. De Toni). Gold, indium-tin oxide (ITO) and carbon
nanotubes (CNT) were all deemed interesting materials for this purpose. Gold electrodes were either
sputtered on a masked glass slide (30 Å thickness) or etched from a pre-deposited 50 Å layer on
polycarbonate (supplied by DEI) using aqua regia. ITO has the important advantage of transparency over
metal electrodes. ITO-on-glass chips were masked with vinyl stickers and etched with diluted HCl to fabricate
devices for electroporation where the flow inside the channel has to be monitored by optical microscopy.
CNT were tested to print circuits on filter paper for paper-based microfluidic applications similar to those
developed by G. Whitesides. CNT were suspended in water with sodium dodecylbenzosulphate, to obtain an
ink that was used to draw circuit tracks on filter paper. The paper was then soaked in liquid NOA precursor
and polymerized, yielding a hard surface with conductive properties on the drawn pattern. Patterned
surfaces and microchannels for wettability studies. Patterned surfaces were produced by a double-replica
molding of stainless steel masters made by surface micro electrical discharge machining (micro EDM)
operated in milling mode. A sequence of pillars of micrometric size and different cross-sections (circular,
square and rectangular) were fabricated to study the anisotropy of water droplets that were grown above
them (T. Toth/D. Ferraro). Also, patterned microchannels, bearing singular or sequence of microasperities of
controlled shape, were produced by micro EDM as well, to study the motional dynamic of a meniscus at low
capillary numbers. SU-8 master production. Masters have been produced in the clean room with the epoxybased negative photoresist SU-8, using lithographic techniques that allowed to manufacture features, within
the microchannels, with dimensions in the
range of tens of microns. Direct laser
writing. The fabrication of micrometric and
sub-micrometric structures through Direct
Laser Writing (DLW) has been used to
modify MF channels or to generate a
master for a MF device (I. Fortunati/N.
Figure 2. Y-shaper MFD and variable width microchannels
Rossetto).
To
this
end
DLW
is
made of PDMS replica molding of a SU-8 master
accomplished through one- or two-photon
induced photopolymerization of i) the commercial photoresist SU-8 or ii) hybrid organic-inorganic sol-gel
precursors prepared in collaborations with G. Brusatin/M. Guglielmi of the Dept. of Mechanical Engineering,
of the U. of Padova and with the group of S. Dirè at the University of Trento. DLW at 400 nm gave SU-8
masters that were replicated by means of PDMS. Examples of the 3D reconstruction of a Y-shaped MFD
and 2D section of a degrading width MF channel from fluorescence confocal images are shown in Figure 2.
50 µm
5
Lines and voxels, from hybrid organic-inorganic sol-gel precursors based on acrylic and methacrylic
photopolymerizable units, were prepared through one- and two-photon induced polymerization at 400 and
800 nm, respectively, with fs laser pulses at 76 MHz repetition rate. Two-photon irradiation (800 nm) gave
structures with hundreds of nanometer resolution, whereas with one-photon irradiation (400 nm) structures
with dimensions ranging from some micrometers to tens of micrometer were readily obtained.
Task 3-surface modification (DISC). In order to increase the compatibility of NOA-81 with alogenated
solvents, sol gel coatings were applied to the inner walls of MF channels with tetraethylorthosilane as the
precursor for a silica layer (S. Silvestrini). The resulting channels withstand acetone and CH2Cl2 to a greater
extent than their untreated counterpars, although not indefinitely. For CH2Cl2, in particular, silica-inertized
microchannels resisted for about 4 hours under constant flow, while untreated microchannels failed after just
5-10 minutes. Further studies were carried out to develop a general method for the modification of the
surface of NOA-81 resins based on chlorosilane derivatives. Hydroxide groups, exposed on the resin
surface, were silanized and characterized by IR and water contact angle measurement. We consider this
task to be completed as far as NOA-81 resins are concerned. Devices were fabricated to prove the
applicability of the surface modification protocols and a draft article is being prepared for submission. The
know-how developed in this field will be shared with the group of DIP (T. Toth/ D. Ferraro) that will use
channel walls with controlled wettability to test fluid-dynamic models.
Task 4-testing of microfluidic devices (DISC). A microfluidic approach to the optimization of the
cyclopropanation to [60]fullerene was studied (S. Silvestrini). The reaction is very fast and produces
functional fullerenes for materials science applications. Cyclopropanation experiments were performed both
in batch and microfluidic conditions, by using different settings, such as Micronit’s glass microreactors and
an IMM micromixer. The kinetic profile was determined by quenching the reaction at different times and
analyzing the crude products via HPLC. Mass transfer resistance and slow mixing phenomena were
modeled in collaboration DIPIC (D. Dalle Nogare) using differential reactor models. Notably, the calculated
kinetic constants agreed well with the experimental data. This study demonstrated how microreactors can be
used to monitor fast, single-phase homogeneous reactions with good accuracy and reproducibility and how
this can be exploited to optimize yields and selectivities in a way that would be difficult to achieve under
conventional batch conditions. A draft paper has been prepared and submitted to the 2010 European
conference on microfluidics. Fulleropyrrolidines (P. Maity). In another flask-versus-flow comparison, the
cycloaddition to [60]fullerene of azomethine ylides was assayed using a tubular reactor. Starting materials
were prepared from different precursors such as imines, aziridines or oxazolidinones. A comparative
datasheet was generated, which reflects the amount of product formed per unit of time in batch vs flow
process. Experimental data proved the superiority of flow process compared to batch as the former is 10 to
100 times more productive for various fulleropyrrolidine derivatives. Synthesis of oncological active
pharmaceutical ingredients (E. Rossi). Mesostructured flow systems were used for an efficient synthesis of
pyrimidinamino derivatives that are key components in the preparation of kinase inhibitors of the imatinib
family. In particular, a commercial microfluidic station has been used in collaboration with the pharma
company Fabbrica Italiana Sintetici SpA
(Montecchio Maggiore, Vicenza) in the
frame of a project funded by Regione
Veneto through Fondo Sociale Europeo.
Methanofullerenes for organic solar
cells. An integrated flow reactor, made
of NOA-81, MF units, teflon tubings
parts and flow cartridges, has been used
to develop a multi-step process to make
a methanofullerene compound that has
been used as active component in
polymer-fullerene organic solar cells.
The synthesis involves the generation of
a labile diazocompound on a flow
cartridge loaded with the renewable
oxidant NiO2, its immediate addition to [60]fullerene (MR-1 in the above picture) followed by a photochemical
isomerization in a NOA-81 photoreactor (MR-2) containing an array of white LED as light source. The
synthesis has worked quite nicely and is currently under examination for scaling up into a larger flow system.
Comparison of the planned activities for WP1 and actual work accomplished during the XXVI months
period. The Work Programme has been closely followed and carried out by the groups with no major
deviations. At this stage of WP1 development, further advances in the fabrication procedures require a
feedback from the users who are currently testing the chips already produced. The fabrication activity will be
carried out in the clean room which allows the production of masters with SU-8 photoresist and the controlled
production of chips with a variety of photo and soft-lithographic techniques.
6
2.2 WP2 (coordinator prof. Camilla Ferrante)
Figure 3 shows an abstract of the MISCHA network Gannt chart in which the Tasks of WP2 are considered.
Figure 3. Abstract of the MISCHA Gannt chart showing the Tasks of WP2.
According to the Work Programme, deliverable D2-2 is the implementation of the SERS technique in MFD,
measurements of FCS on simple MR with fluorescent labels, electrical manipulation through the first
(simplified) version of MF electroporation device.
Task 2.1-Characterization of the motion and chemical nature of fluids (DISC, DIP). The optical set-up
for Fluorescence Correlation Spectroscopy (FCS) has been implemented and tested. In particular, flow
velocity measurement in a microfluidic channel, using
Alexa488 dye in water, agree nicely with those recorded by
I. Fortunati for a similar chip in the laboratory of P. Schwille
at the U. of Dresden. Also, the velocity profiles of the fluid
along the channels correlates quite well with the values
predicted with the help of numerical simulations by S.
Carlotto. FCS has been also used to trace the concentration
profile of fluorescent molecules along the MFD channels.
Two prototypes of passive PDMS micromixers (Figure 4)
were fabricated and tested with FCS and fluorescence
imaging as well. Flow velocity, concentration profiles and
mixing efficiency along the channel for these prototype
Figure 4. Upper: Scheme for a micromixer exploiting
mixers were evaluated through FCS experiments, or by
the focusing effect; Lower: Fluorescence images of
imaging of the fluorescence intensity profiles along the
two micromixers exploiting lateral obstructions in the
channel. In collaboration with S. Carlotto, these systems
channel.
were modelled numerically via Navier-Stokes equations
coupled to convection-diffusion equations, showing a very
good agreement between the numerical simulation and the experimental findings.
LaPIV platform. The platform has been implemented by a new correlation method for particle image
velocimetry (PIV) yielding velocity data at single-pixel spatial resolution. This method, which is essentially an
extension of the ensemble correlation method for PIV, is particularly suited for (quasi-) stationary and
periodic flows, which are typically encountered in microfluidics and micro-scale biological flows. The main
advantage of the new method is that it can resolve steep velocity gradients and obtain unbiased
measurements of the velocity in the vicinity of flow boundaries (such as channel walls). Microrheology
investigation of submicron-sized crosslinked polymer particles (microgels). Particles in the tens of nm range
were produced by A. Biffis (DISC) through the so-called micro- or mini-emulsion polymerisation. No
macroscopic gel phase was formed; instead, soluble crosslinked polymer particles (“microgels”) were
isolated. The measure microdevice is a glass cross-rectangular channel of large aspect ratio (50 micron of
height, 1 mm of width, a few cm). We measured the velocity profile by imposing a driven pressure flow with a
microfuidic flow controller (Fluigent) From the data recorded, we measured the stress as a function of the
shear rate in different flow conditions, and for different microgels concentrations. Presently the rheological
behavior is basically still linear but the velocity profiles are expected to reveal a non Newtonian behavior at
higher concentrations combined with a lower viscosity second phase at the walls, the so-called shear
banding regime. We then characterize the wall slip which is usually much higher than simple liquids, by
extrapolating the velocity profiles obtained over a few microns above the glass wall to the z value giving a
zero velocity. Study of slip length in asymmetrically micro-patterned hydrophobic surfaces. The slip boundary
condition for single-phase flow past a chemically patterned surface has been studied. It has been shown that
modulation of fluid-solid interaction along a chemically patterned surface induces a lateral structure in the
fluid molecular organization near the surface. Consequently, various forces and stresses in the fluid vary
along the patterned surface. Each stripe is of type A or B, and the periodicity along the x (flow) direction is
realized by arranging the stripes according to ABABAB. The validity of the Navier slip boundary condition is
verified where a local slip length can be defined varying along the patterned surface.
7
Task 2.2-Electrical characterization of MFDs with integrated gold electrodes (DEI, DIP, DISC). As a first
step towards the production of a microfluidic electroporator, we focused on the simple detection of
macroscopic biological samples in a fluidic channel by means of impedance spectroscopy (EIS) measures.
For this scope, devices were built using sheets of vinyl monomer as material for the channel and gold-plated
surfaces (from common CD-ROM media) as sensing electrodes. EIS measurements inside the channel were
performed on saline solutions with different concentrations, to assay the electrochemical behaviour of the
device. The distribution of the electric field as a function of channel and electrodes geometries was studied
with Finite Elements Method simulation program (COMSOL Multiphysics) in order to maximize the field
inside the channel. As final test, biological samples were introduced in the channel, suspended in
physiological saline solution and the variation of the impedance proved that the biological samples could be
monitored by this method. ITO/ITO and Au/ITO devices. Different microfluidic chips with two integrated ITO
(ITO/ITO) or one gold and one ITO (Au/ITO) electrodes, one on the top and one on the bottom of the
channel, were characterized by EIS techniques. Data were analyzed by the equivalent electrical circuit
approach, using a Randles-like equivalent circuit. In-situ electroporation tests using both ITO/ITO and
Au/ITO devices and Chinese Hamster Ovary (CHO) cells, grown with a standard process, were carried out.
Cell were allowed to adhere to the electrode in a wet environment, then pulse trains at 1 kHz frequency were
used as electroporation stimuli. A fluorescent marker (lucifer yellow) was transferred into the cell membrane
in the process, so that the electroporation results could be observed using a UV-suitable microscope. The
experiments showed very few electroporated cells and very poor repeatability of the results. Multichambers
MISCHA Electroporator – M2E. With the know-how developed in the characterization and use of the devices
so far described, a device that is able to simultaneously electroporate the same cell population using four
different dyes in three independent chambers was developed. Such devices, referred to as M2E, were
manufacted using gold electrodes onto a polymeric substrate. In this case the transfectant was injected in
the chamber by just communicating vessels principle, speeding up the CHO electroporation protocol. M2E
devices were used for electroporation experiments using different layout for the channels and electrodes.
The electroporation protocol underwent optimization by tunig the stimulating signal. Thanks to the features of
M2E devices we were able to use different fluorescent markers like Lucifer yellow and Trypan blue at the
same time and on the same cell population. While the design of the device appears interesting, the
experiments still show very poor reproducibility.
Comparison of the planned activities for WP2 and actual work accomplished during the XXVI months
period. The activity related to the WP2 is almost in line with the timetable planned initially. The PIV and FCS
instruments have been optimized and used for measurements. The SERS analysis has been implemented in
glass capillary; but the use of SERS to detect chemical species within MFDs is still undeveloped. Fabrication
(DIP, DISC) and electrical characterization/modelling (DEI) of micro-electroporation devices (our second
pilot-research project) have been also carried out with novel electrode arrangements. Electroporation results,
however, are still poorly reproducible.
2.3 WP3 (coordinator prof. Antonino Polimeno)
Figure 5 shows an abstract of the MISCHA network Gannt chart in which the Tasks of WP3 have been
considered. The timetable and the deliverable in the Work Programme during the second year of activity has
been mainly concerned with Tasks 3.2 and 3.3.
Figure 5. Abstract of the MISCHA Gannt chart showing the Tasks of WP3.
Task 3.1 and its related deliverable D3-1 (silico evaluation of interface properties) were addressed during the
first year, and applications based on the derived methodology will be employed for activities planned at the
end of Task 3.4 for completion of deliverable D3-4 (definition of multiscale approaches) during the third year
of activity.
8
Task 3.2- molecular and coarse-grained simulations of nano-flows. Hybrid methods for nanoflows. The
study of flow properties at nanoscale has been at the center of a reassessment activity, mainly in terms of
methods employed. A clear understanding has risen from studies based on standard tools from molecular
dynamics approaches (molecular limit) to hydrodynamic descriptions (continuum limit) the nanosized flows
require hybrid methods, based on combined molecular/continuum methods. Therefore, a full analysis and
implementation of coarse-grained treatment of stochastic Navier-Stokes equations has been started aimed
at the treatment of simple model systems. The basic methodological procedure for defining non-linear
fluctuating hydrodynamic equations has been based on a set of deterministic hydrodynamics equations for
the field variables describing the fluid. Then a proper partition of deterministic drives into conservative and
dissipative contributions has been introduced. Finally standard fluctuations-dissipation arguments have been
employed to introduce multiplicative noise into the deterministic equations. Currently, numerical
implementation is being carried out based on a finite volume spatial discretization in Lagrangian formulation.
Planned developments: this activity is aimed at completion of deliverable D3.2 and will be also partially
related to deliverable D3-4; in particular we plan to address nanoflows of ordered fluids and particle
suspensions/emulsions. A specific research activity has been activated in the framework of a new thesis
degree project “Implementation of coarse-grained treatments of flow properties” (student: Nicola Fortunati).
Task 3.3-continuum models of micro-flows. Interpretation of time correlated fluorescence of an
asymmetrically focused flow in a microfluidic device We have modeled the behavior of a prototype
microfluidic device which employs two non- mixing fluids (sheath and inlet fluids) displaying an asymmetric
focused flow, in the presence of a fluorescent dye. This activity has been carried out in tight collaboration
with WP2 (Ferrante’s group), which employed fluorescence correlation spectroscopy, allowing the precise
measure of flow speeds across the channels and of the concentration profile of the central focused flux along
the flow direction. The system has been modeled via a standard Navier–Stokes finite-element approach,
coupled to convection–diffusion equations for the solute. Simulations have reproduced accurately the shape,
the position, and the width of the velocity and concentration profiles along the central channel and across the
transversal and vertical sections of the microfluidic device. The observed asymmetric flow with respect to the
center of the channel has been reproduced numerically with an error in the position determination smaller
than 1%.
Strategy for the improvement of mixing in microdevices. Many different strategies can be proposed to
enhance mixing. We have explored and modeled the behavior of microreactor prototypes having different
geometry and aimed at the improvement of mixing efficiency. These prototypes exploit either the
hydrodynamic focusing effect or the presence of microstructuration inside the microchannel. In the first case,
the focusing effect is used to reduce the diffusion distance between the molecules flowing in the reactors. In
the second approach, we have explored the influence of lateral steps fabricated inside the channels on the
mixing efficiency. The behavior of fluid features has been numerically modeled via Navier-Stokes equations
coupled to convection-diffusion equations. This work has been carried out in collaboration with WP2
(Ferrante’s group) which has employed fluorescence based techniques to characterize the mixing efficiency
in the experimental devices.
Computational optimization of microfluidic devices via genetic algorithms. We have completed the
development of a new computational strategies based on the application of evolutionary computing
algorithms for the in-silico design and optimization of MicroFluidic Devices (MFD) aimed at chemical
synthesis, in simplified geometries and for a model reaction. Evolutionary algorithms are stochastic global
optimization methods inspired by the biological mechanisms of evolution and heredity. In particular we have
employed an optmization procedure of prototypical microfluidic device which is host to a generic second
order chemical reaction A + B → C. A heuristic method has been applied to the search of the best
performing geometry, which assures a high yield in terms of product C ad a fixed exit point of the
microcircuit, given initial concentrations and flows of reactants A and B at two inlet points. The microfluidic
circuit has been represented by a binary code as a succession of empty and filled elementary cells and
artificial evolution, based on a standard genetic algorithm, has been employed to select the best performing
specimen.
Segregation of suspensions in microflow. With the aim of investigating multiphase flows within
microchannels, both experimental studies and in silico modeling are being carried out. The targeted
application is the rheological behavior of blood, with particular attention to erythrocytes and platelets motion
within blood microvessels, of crucial importance for physiological and pathological conditions such as
thrombosis or atherosclerosis. A model system that rules out biological interactions, while looking at the pure
mechanical and flow dynamics aspects, was thus envisaged. A process for synthesizing polymeric
microparticles, with sizes mimicking those of both red blood cells and platelets, was developed, via
dispersion and seeded polymerization. For imaging reasons, the microparticles needed to be fluorescent,
therefore a protocol for labeling them with a fluorescent dye was developed, obtaining stable bright beads.
Glass capillaries and microfluidic devices are useful tools for investigating flowing suspensions under the
conditions of interest. Photolithographic and replica molding techniques are widely used in fabricating such
chips, and some microfluidic devices were fabricated in the new clean room facility. Conventional optical
9
microscopy, although fast and inexpensive, is not able to distinguish between in- and out-of focus planes,
making it not suitable for the study of dense suspensions. Recent advances in imaging capabilities are
represented by Laser Scanning Confocal Microscopy (LSCM), which offers the possibility of investigating
thick samples by excluding out-of focus information through a pinhole, thus obtaining a sharp image of the
focal plane only. This technique, widely used in the biological field, is only recently being explored for
rheology applications. In collaboration with DISC, LSCM studies were carried out, enabling the visualization
inside the microchannels of the fluorescently labeled microparticles previously synthesized. A tracking
algorithm was also developed in order to follow the particle motion, and studies on the effects of flow rate
(Reynolds numbers are accurately controlled through a syringe pump) and particle concentration (which
correlates to blood hematocrit concentration) are underway.
Planned developments: 1) as a further development we plan to extend the same treatment to the in silico
design of 3D microdevices hosting complex reaction mechanisms. A new integrated software will be created
based on a C++ code employing open source libraries (integrating fluid dynamics treatment, chemical
kinetics, euristic search). A twelve-month activity, supported by other funds but in connection with MISCHA
activities has been started (recipient of grant: Lucas Stefanutti). 2) A specific research activity has been
activated in the framework of a new thesis degree project “Validation of genetic algorithms as a tools for
optimizing microdevices” (student: Matteo Montagna). This activity will be developed in collaboration with
WP1 (Maggini’s group) for testing and validation of microdevices for chemical reactivity. 3) A further novel
development will be the optimization of microfluidic devices for the separation of atomic isotopes; this new
collaboration, to be carried out with dr. Alberto Garfagnini of the Department of Physics, U. of Padova, is
aimed at exploring the use of micro-flows for isolating/enriching different isotopes in gas and liquid phases.
Comparison of the planned activities for WP3 and actual work accomplished during the XXVI months
period. As initially planned, WP3 activities in the second year months have been centered mostly Tasks 3-2
and 3-3 (models of nano-flows and micro-flows). Collaborations have been established with the other WPs,
in particular among people of DISC in WP2 and WP3. Novel collaborations outside MISCHA team have been
started, in particular with the Department of Physics (DIP) of the University of Padova.
3. Collaborations
3.1 Academic Institutions. New collaborations on MF topics have been established with colleague as follows: G.
Bissacco (Mechanical Engineering, U. of Padova) for the preparation of metal stencils through electrical erosion; A. Biffis
(DISC), E. Franceschinis, (Pharmaceutical Sciences, U. of Padova) for preparation of microemulsions and gels; M.
Bucolo (Engineering, U. of Catania); G. Paradossi (Chemistry, U. of Roma Tor Vergata); P. Schwille (U. of Dresden) for
FCS; T. Salmi (Abo Akademi, Turku, Finland), A. Garfagnini (DIP, U. of Padova) for modeling microfluidic-based
techniques for separation of isotopes, G. Albertin, (Dip. di Anatomia e Fisiologia Umana, U. of Padova) for cell coltures,
G. Brusatin, M. Guglielmi (Mechanical Engineering, U. of Padova), S. Dirè (Materials Engineering U. of Trento).
3.2 Industry. Centro Ricerche Danieli, Udine; QID – Trieste; Fabbrica Italiana Sintetici, Montecchio Maggiore for the
synthesis of active pharmaceutical ingredients with MR in the frame of a project sponsored by Regione Veneto (FSE);
Corning Inc. – USA for testing new lab-scale MRs prototypes for industrial applications: a new low-flow prototype reactor
has been given to DISC for sperimentation.
4. MISCHA as a platform for other projects
A synergistic approach to the prepararation of functionalized carbon nanostructures (fullerenes and carbon nanotubes)
under flow conditions is operative within the project sponsored by MUR, for the project "Carbon nanostructures in
microfluidic reactors" (PRIN 20085M27SS_005) coordinated by M. Maggini.
5. Publications
1.
2.
3.
4.
5.
6.
S. Carlotto, I. Fortunati, C. Ferrante, P. Schwille, A. Polimeno, Time correlated fluorescence characterization of an
asymmetrically focused flow in a microfluidic device, Microfluid Nanofluid 2010, DOI 10.1007/s10404-010-0689-x, in
press.
R. Po, M. Maggini, N. Camaioni, Polymer Solar Cells: Recent Approaches and Achievements, J. Phys. Chem. C
2010, 114, 695.
E. Rossi, T. Carofiglio, A. Venturi, A. Ndobe, M. Muccini, M. Maggini Continuous-flow synthesis of an efficient
methanofullerene acceptor for bulk-heterojunction solar cells, Energy & Envirnmental Science 2010, submitted.
S. Carlotto, A. Polimeno, Computational optimization of microfluidic devices via genetic algorithms, Proceedings of
the 2nd European Conference on Microfluidics - Microfluidics 2010 - Toulouse, December 8-10, 2010, submitted.
I. Fortunati, N. Rossetto, R. Signorini, C. Ferrante, S. Carlotto, A. Polimeno, Strategy for the improvement of mixing
in microdevices, Proceedings of the 2nd European Conference on Microfluidics - Microfluidics 2010 - Toulouse,
December 8-10, 2010, submitted.
S. Silvestrini, T. Carofiglio, M. Maggini, D. Dalle Nogare, P. Canu, Synthesis of Methanofullerenes in Microreactors:
Kinetic Analysis and Potential Applications, Proceedings of the 2nd European Conference on Microfluidics Microfluidics 2010 - Toulouse, December 8-10, 2010, submitted.
6. Communications to conferences and seminars
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1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
E. Rossi, A. Castellin, T. Carofiglio, M. Maggini – “Synthesis of Oncological Drugs by Flow Techniques” poster
presentation by E Rossi, 3° meeting nazionale dei giovani ricercatori INSTM (Padova, 22-24/03/2010)
E. Rossi, M. Maggini, T. Carofiglio, A. Castellin – “Micro/meso-structured reactors for chemical synthesis:
applications in materials science and medicinal chemistry” poster and oral presentation by E Rossi, Ischia Advanced
School or Organic Chemistry - IASOC 2010 (Ischia, 25-29/09/2010).
D. Ferraro, T. Tóth, M. Pierno, G. Mistura and G. Bissacco, “Capillary filling in patterned microchannels”,
International Soft Matter Conference 2010, 5-8 July 2010, Granada, Spain (poster).
T. Tòth, D. Ferraro, M.Pierno, G.Mistura, E. Orlandini, G.Bissacco and C.Semprebon, “Anisotropy of water
droplets on individual pillars”, International Soft Matter Conference 2010, 5-8 July 2010, Granada, Spain
(oral).
G. Mistura, “Anisotropy of water droplets on individual pillars”, Max-Planck-Institut fur Dynamik und
Selbstorganisation, Gottinga (Germania), 16 luglio 2010.
P. Donnola, T. Carofiglio, A. Castellin, M. Maggini – Padova, SISOC; Spanish Italian Symposium on Organic
Chemistry (Padova, July 2010), poster presentation, “Dioxiranes in microreactors”.
P. Maity, T. Carofiglio, M. Maggini – Padova, SISOC; Spanish Italian Symposium on Organic Chemistry (Padova,
July 2010), poster presentation, “Continuous-flow stnthesis of fulleropyrrolidines in microchannel reactors”
S. Silvestrini, T. Carofiglio, M. Maggini – Padova, 3° meeting nazionale dei giovani ricercatori INSTM (March 2010),
oral presentation.
S. Silvestrini, T. Carofiglio, M. Maggini – Bibione, Workshop “molecular functional materials” (May 2010), oral
presentation.
S. Silvestrini, D. Dalle Nogare, T. Carofiglio, P. Canu, M. Maggini – Padova, SISOC; Spanish Italian Symposium on
Organic Chemistry (Padova, July 2010), poster presentation “Principles and applications of chemical microreactors”.
S. Silvestrini, D. Dalle Nogare, T. Carofiglio, P. Canu, M. Maggini – S. Benedetto del Tronto, “XXXIII Convegno
Nazionale della Divisione di Chimica Organica” (September 2010), poster presentation “Principles and applications
of chemical microreactors”.
M. Maggini, Organic Synthesis in Microreactors, lecture to the students of the PhD School in Materials Science, U.
nd
of Milano Bicocca, April 22 2010.
N. Rossetto, I. Fortunati, R. Signorini, C. Ferrante, “Realization and characterization through fluorescence based
techniques of microfluidic devices”, Poster presentation at 3° Forum Nazionale Giovani Ricercatori INSTM (22-24
March 2010 – Padova).
N. Rossetto, I. Fortunati, R. Signorini, C. Ferrante, G. Della Giustina, G. Brusatin, M. Guglielmi, V. Tagliazucca, S.
Dirè, E. Sorato, D. Guidolin, G. Albertin, “Direct laser writing for the realization of micro- and nano-structures in
microfluidic Devices” Oral Presentation at Nano2010, (Roma 13-17 Settembre 2010).
7. Formation activity
Dissemination of research and knowledge on microfluidics by MISCHA participants has been carried out with thesis
works at the level of bachelor degree (laurea triennale) and master degree (laurea magistrale) in chemistry, physics,
science and engineering of materials.
Years 2009/10
1. Tobia Mancabelli, Microfabbricazione attraverso
processi di assorbimento a due fotoni (tutor: C.
Ferrante)
2. L. Porcelluzzi, Tecniche di velocimetria in
dispositivi microfluidici, Laurea V.O. in Fisica (tutor:
M. Pierno)
3. P. Fantinel, Sviluppo di superfici superidrofobiche
con tecniche litografiche, Laurea Triennale in Fisica
(tutor: G. Mistura)
4. Michele Gintoli (Scuola Galileiana), Studio
dell’evoluzione
del
menisco
all’interno
di
microcanali strutturati, Laurea triennale in Fisica
(tutor: M. Pierno)
5. Marco Grison (Scuola Galileiana), Fotolitografia
mediante resist SU8, Laurea triennale in Fisica
(tutor: M. Pierno)
6. Silvia Varagnolo, Caratterizzazione dello strato
limite in dispositivi microfluidici strutturati, Laurea
triennale in Fisica (tutor: M. Pierno)
7. Daniele Filippi, Fabbricazione di microstrutture
mediante fotoresist SU8, Laurea Triennale in
Scienza dei Materiali (tutor: G. Mistura).
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