£
The authors wish to be known that, in their opinion the first two
authors should be regarded as joint First Author.
PROGRAMMABLE CYTOGENETIC SUBMICROLITRE
LAB-ON-A-CHIP FOR
MOLECULAR DIAGNOSTIC APPLICATIONS
Daniela Woide, Veronika Schlentner, Teresa Neumaier, Thorsten Wachtmeister
Herwig G. Paretzke, Zeno von Guttenberg
1
, Achim Wixforth
2
and Stefan Thalhammer
*
GSF, National Research Center for Environment and Health, Institute of Radiation Protection
Ingolstädter Landstrasse 1, 85764 Neuherberg, Germany
1
Advalytix AG, Sauerbruchstrasse 50, 81377 Munich, Germany
2
University Augsburg, Chair for Experimental Physics I, Universitätsstrasse 1, 86135 Augsburg, Germany
Keywords: Nanobiotechnology, lab-on-a-chip, cytogenetics, microfluidic PCR, surface acoustic waves,
laser-based microdissection.
Abstract: This project focuses on the development of an acoustic driven, freely programmable multifunctional
biochemical lab-on-a-chip. By combining different platform elements, like microdissection-, nanofluidic-
and detection-modules, the lab-on-a-chip can be adapted to question- and patient-specific cytogenetic and
forensic applications. In contrast to many common lab-on-a-chip approaches presently available, the fluidic
handling is done on a planar surface of the lab-on-a-chip. Minute amounts of biochemical fluids are
confined in ‘virtual’ reaction chambers and ‘virtual’ test tubes in the form of free droplets. The droplets,
fluidic tracks and reaction sites are defined at the chip surface by a monolayer chemical modification of the
chip surface. Surface acoustic waves are employed to agitate and actuate these little ‘virtual’ test tubes along
predetermined trajectories. Well-defined investigations, controlled in the submicrolitre regime, can be
conducted quickly and gently on the lab-on-a-chip.
1 INTRODUCTION
Over the past decade, advances in molecular biology
have helped to enhance understanding of the
complex interplay between genetic, transcriptional
and translational alterations in, e.g., human cancers.
These molecular changes are the basis for an
evolving field of high-throughput cancer discovery
techniques using microscopic amounts of patient-
based material to detect genetic changes such as
mutations, insertions, deletions or imbalances.
To be able to reproducibly and reliably handle,
process and analyse such small samples, many
laboratories all over the world are intensively
investigating the applicability of biochips for this
purpose. Biochips are small sample carriers, where
biological material is attached for analysis. In
dependence of the kinds of molecules attached to the
surface analytical biochips are divided into DNA-
chips, protein-chips, cell-chips and lab-on-a-chip
systems. Most progress in this field occurs
especially in the area of DNA-chips (Schneegass et
al., 2001; Lagally et al., 2001; Ng and Ilag, 2003).
Existing gene chips developed by the Affymetrix
Company are a new approach in microarray
technology. Oligonucleotide arrays (e.g. genome-
wide human SNP array as well as human gene array)
are based on hybridisation to small, high-density
arrays containing tens of thousands of synthetic
oligonucleotides. The arrays are designed based on
sequence information alone and are synthesized in
situ using a combination of photolithography and
oligonucleotide chemistry. Chromatin immuno-
precipitation (ChiP) coupled with whole-genome
DNA-microarrays allows determination of the entire
spectrum of in vivo DNA binding sites for any given
protein (Buck and Lieb, 2004).
In parallel, another kind of test systems was
developed, i.e. bead-technologies (Edelmann et al.,
2004) and microfluidic systems (Harrison et al.,
256
Woide D., Schlentner V., Neumaier T., Wachtmeister T., G. Paretzke H., von Guttenberg Z., Wixforth A. and Thalhammer S. (2008).
PROGRAMMABLE CYTOGENETIC SUBMICROLITRE LAB-ON-A-CHIP FOR MOLECULAR DIAGNOSTIC APPLICATIONS.
In Proceedings of the First International Conference on Biomedical Electronics and Devices, pages 256-261
DOI: 10.5220/0001048002560261
Copyright
c
SciTePress
1993; Thorsen et al., 2002; Kwakye and Baeumner,
2003). There is growing interest in performing
chemical reactions in microfluidic devices as they
offer a variety of significant advantages over
macroscopic reactors, such as high surface-area-to-
volume ratios and improved control over mass and
heat transfer.
Several companies and universities are working
on programs employing new electronic DNA
analysis technologies. These automated techniques
are often developed in non-forensic fields, such as
medical research, genetics or biochemistry. In
genetic forensics, nucleic acid is usually extracted
from saliva, blood, semen, bone, hair and dried skin;
these are the sources for most crime scene DNA
isolations. Chemical kits for DNA isolation,
amplification and detection are available today. The
future of forensic testing will follow the path of
greater automation e.g. of the DNA fingerprinting
process. If a particular kind of polymorphism can be
detected through automation, reducing the analysis
time and expense, it may be of interest to the
forensic community. Developments, however, for
forensic applications are rare. Only Nanogen Inc.
has developed this technology with applications to
STR analysis termed APEX
®
- automated
programmable electronic matrix, by using an electric
chip as heart of the analysis (Ibrahim et al., 1998;
Rau, 1997).
While DNA-chips become commercially
important, scientific and technical development in
the last years generated different approaches of
multiparameter tests particular for medical
applications, so-called ‘lab-on-a-chip’ systems
(LOCs). Miniaturisation of analysis systems will
yield in an enormous cost-saving in regard to
materials like test tubes or microtitre plates as well
as biochemical reagents. Furthermore, a smaller
sample volume implies in the end a higher
sensitivity and homogeneity of detection.
Additionally, in comparison to serial single analyses,
parallelisation of analyses enables an enormous time
saving due to automation. These micro- and
nanolaboratories on the scale of a computer chip are
equipped with all components necessary for
cytogenetic analysis, they are portable, easy to use,
flexible, inexpensive, biocompatible and, like
computer chips, full programmable.
Here, we present an acoustic driven lab-on-a-
chip for cytogenetic and forensic applications
(Thalhammer et al., 2007). In contrast to many other
lab-on-a-chip approaches, the fluidic handling is
done on the planar surface of this chip, the fluids
being confined in ‘virtual’ reaction chambers and
‘virtual’ test tubes in form of free droplets. The
droplets, fluidic tracks and reaction sites are defined
at the chip surface by a monolayer chemical
modification of the chip surface. In comparison to
conventional closed microfluidic systems with
external pumping, afflicted with the difficulty to
further miniaturize, surface acoustic waves are
employed to agitate and actuate these little ‘virtual’
test tubes along predetermined trajectories. These
surface acoustic waves propagate on a substrate
surface, to move and mix smallest fluidic volumina.
Liquid amounts in the range from 1 micro- down to
100 picolitre are precisely moved on monolayers of
thin, chemical processed fluidic ‘tracks’ without any
tubing system. The surface acoustic waves are
generated by high frequency electrical impulses on
microstructured interdigital transducers embedded
into the lab-on-a-chip.
Minute amount of sample material is extracted
by laser-based microdissection out of e.g.
histological sections (Thalhammer et al., 2003;
Thalhammer et al., 2004). A few picogram of
genetic material are isolated and transferred via a
low-pressure transfer system onto the lab-on-a-chip.
Subsequently the genetic material inside single
droplets, which behave like ‘virtual’ beakers, is
transported to the reaction and analysis centres on
the chip surface via surface acoustic waves,
probably best known from their use as high
frequency filters in mobile phones. At these
‘biological reactors’ the genetic material is
processed, e.g. amplified via polymerase chain
reaction methods, and genetically characterized
(Guttenberg et al., 2005).
Well-defined analyses, controlled in the
submicrolitre regime, can be quickly and gently
conducted on the lab-on-a-chip. Apart from its
nearly unlimited applicability for many different
biological assays, its programmability and extremely
low manufacturing costs are another definite
advantage of this 'cytochip’. In fact, those LOCs can
be made so cheap that their use as disposables in
many areas of diagnostics can be envisioned.
2 MATERIAL & METHODS
In most microfluidic systems liquids are confined
and moved in tubes or capillaries. Usually, the
application of such systems is restricted to
continuous flow processes. However, when carefully
looking at a microscale fluid, one realizes that the
effects of e.g. surface tension by far exceed those of
gravity. The shape of a droplet on a surface is given
PROGRAMMABLE CYTOGENETIC SUBMICROLITRE LAB-ON-A-CHIP FOR MOLECULAR DIAGNOSTIC
APPLICATIONS
257
by the properties of the substrate. It either remains a
droplet or it wets the surface, depending on whether
the substrate is hydrophobic or hydrophilic. The
technology to create such fluidic tracks (fig. 1) is
very much similar to define conducting paths on an
electronic semiconductor device.
Figure 1: Chemical functionalization of the chip surface
creating a hydro-philic/hydrophobic structure. Electron
microscopy of a) the hydrophilic reaction centre on the
hydrophobic LOC surface, scale bar 10 µm, and b) the
arrangement of interdigital transducers (IDT) and electric
conduction to control the SAW, scale bar 1 mm.
Enlargement in b), electron microscopy of the interdigital
transducer in detail with comb periodicity of the double-
headed arrow, scale bar 10 µm.
Small amounts of liquids do not really need to be
confined in tubes and trenches. They form their own
test tubes, held together by surface tension effects.
These micro volumina, due to the fact that in small
droplets the effect of surface tension dominates
gravity, do not need any reservoir as surface tension
keeps the droplets in shape. Visualizing dewdrops
hanging on a spider’s web, one can observe that
average droplet size obviously depends on the
thickness of the strand: smaller droplets are attached
to finer fibres and bigger ones to thicker threads.
Apparently droplet shape conforms to the geometry
as well as the wettability of the subsurface.
A ‘lab-on-a-chip’, however, requires more than
just test tubes. More important, their cargo has to be
moved around, mixed, stirred or processed in
general.
2.1 Lab-on-a-Chip Design
The layout of the lab-on-a-chip is shown layer by
layer in a schematic drawing (fig. 2). The basic
material of the lab-on-a-chip is a lithium niobate
(LiNbO
3
) single crystal wafer polished on both
sides. The first metal layer is platinum (Pt) or nickel
(Ni) for the heater and sensor structure, followed by
a gold layer for the SAW transducer and the contact
wires. The complete chip is protected with sputter
oxide, which is removed above the contact pads. All
structures are patterned by photolithography.
A chemical functionalization of the surface or
parts thereof can be employed to laterally define a
modulation of the wetting properties, thus creating
fluidic pathways or tracks forming virtual potential
wells for a fluid on the flat surface of a chip (fig. 3).
To form a high contact angle of the oil on the chips,
the surface has to be lipophilic. However, the
hybridization array has to be wetted easily and needs
active coupling groups for the oligo DNA spots.
Therefore a chemically heterogeneous surface
modification is needed achieved by photo-
lithography. The tracking system for biochemical
reactants and oil droplet movement and heaters on
the chip is patterned with photoresist. An organic
layer of a hydrophobic silane is bound to the whole
surface. After removing the photoresist, epoxysilane
is grafted from an organic solution.
Figure 2: Design of LOC functionality. The ground
substrate (LiNbO
3
) is covered by a layer of Pt, Ni and Au
for transducers and sensor metallization. Subsequent
silanisation of the surface accounts for a
hydrophilic/hydrophobic surface chemistry, facilitating a
planar tracking system, which could be further
functionalised.
2.2 Actuation
Actuation of single droplets or closed loops of liquid
on a fluidic track is achieved by so-called surface
acoustic waves, which have been widely used in the
completely different field of radio frequency signal
processing over the last twenty years or so. Each cell
phone, for instance, contains two or more devices
operating on SAW.
Actuation of small droplets on the surface of a
SAW chip is caused by the effect of acoustic
streaming. This phenomenon appears when intensive
sound fields are travelling through a liquid. Two
major actuation forms can be described: internal
flow inside the droplet versus transport of the
droplet.
2.2.1 Interdigital Transducer
Electronic devices employing the SAW normally
utilize one or more interdigital transducers (IDTs) to
convert acoustic waves to electrical signal and vice
versa utilizing the piezo-electric effect of certain
materials (i.e. LiNbO
3
) (fig. 1). These devices are
BIODEVICES 2008 - International Conference on Biomedical Electronics and Devices
258
fabricated utilizing common processes used in the
manufacture of silicon integrated circuits. Piezo-
electricity is the ability of crystals to generate a
voltage in response to applied mechanical stress.
Depending on their design, the interdigital
transducers produce a special type of acoustic
surface wave, which can efficiently transfer energy
into liquids. Typical SAW frequencies for the fluidic
application presented here range from 100 to
200 MHz, the wavelengths are then around
20 micrometers. Transducers are copied from a
transducer mask (Advalytix); a distance of 26.5 µm
between two combs results in a resonance frequency
on the LiNbO
3
of 150.6 MHz (fig. 1).
Figure 3: a) Side-view of the lab-on-a-chip amplification
unit displaying single droplet PCR. The PCR reaction mix
(1 µl) on the hydrophilic reaction centre (40 µm in
diameter) is covered with mineral oil to avoid evaporation.
The hydrophobic area around the reaction centre holds
reaction mix and cover oil in place. b) Gel electrophoresis
of 1 µl β-Actin PCR. WM: weight marker, PeqGold 100
bp DNA ladder; lane 1+2: positive control; lane 3:
negative probe; lane 4: LOC-PCR, 500 pg target DNA.
2.2.2 Surface Acoustic Wave
Surface waves, so-called Rayleigh-waves, are
applied on the piezo-electric system without any
mechanical contact to realize actuation of the
reactants on the LOC with interdigital transducers.
A surface acoustic wave is an acoustic wave
travelling along the surface of a material having
some elasticity, with amplitude that typically decays
exponentially with the depth of the substrate. It is
the nanometre analogue of an earthquake. This kind
of wave is commonly used in piezo-electric devices
called SAW devices in electronic circuits. Its
amplitude and wavelength, however, can be
controlled by an electrical signal applied to an
appropriate transducer.
At low amplitudes, e.g. below one nanometre, a
striking SAW pulse creates internal streaming within
the fluid. Its energy is strongly absorbed and
radiated into the fluid under the Rayleigh angle. At
larger amplitudes, the internal streaming becomes a
movement of the whole droplet into the desired
direction on the chip with a desired speed (Wixforth
et al., 2004). Velocities close to one m/sec can be
achieved in this way. In this sense, the transducer
generating the surface acoustic waves can be
regarded as pump without moving parts that may be
remotely operated to control the position of one or
more single droplets on the planar fluidic network
on a LOC system.
3 PROTOTYPE
Here, we present a multifunctional lab-on-a-chip
combining different platform elements like
microdissection-, nanofluidic- and detection-
modules (fig. 4) with the aim of providing a new
platform for fast, cheap and easy investigation of
genetic material in patient or forensic samples.
Without any mechanical structuring the lab-on-a-
chip exhibits ‘virtual’ tracks, whereon samples and
reagents are acoustically driven, actuated by
electrical nanopumps (fig. 5). For the LOC, specific
reaction-predefined ‘spots’ can be generated for total
genome amplification via PCR, labelling of the
amplified material and detection.
The recent developed lab-on-a-chip system
combines serial processing with parallel downstream
applications by using a minimum amount of genetic
material as source for further investigation.
Amplification, labelling and detection of the isolated
genetic material are subsequently carried out on the
chip surface driven by surface acoustic waves.
Each lab-on-a-chip (fig. 5) has two areas
operating as biochemical reaction points, controlled
by the temperature sensor. The sensors of the chip
are calibrated by a thermoplate and resistance
measurements controlled by a LabView program.
The chip has 10 separately addressable SAW
transducers, two on each side for aligning the
reactant droplet on the heaters and for mixing during
the biochemical reactions. Opposing transducers
have different spatial periods to avoid crosstalk.
Extraction of sample material is performed via
laser-based microdissection providing the possibility
to isolate samples in the range from several cells
down to a single chromosomal band with minimum
risk of contamination. These small amounts of
genetic material, which lay in the range of several
picogram, are then transferred via a low-pressure
transfer system onto the lab-on-a-chip. This newly
developed transfer system (publication in
preparation) extracts microdissected material using
PROGRAMMABLE CYTOGENETIC SUBMICROLITRE LAB-ON-A-CHIP FOR MOLECULAR DIAGNOSTIC
APPLICATIONS
259
Figure 4: The modular lab-on-a-chip system consists of
several units for isolating, processing and analysing
minute amounts of sample material: laser-based
microdissection is followed by processing of the extracted
material and detection of hybridized probes or amplified
material by a fluorescence reader. All operations on the
LOC are controlled by SAW actuated microfluidics.
low-pressure and transfers it to the reaction centre
on the LOC. The patented transfer device allows the
precise positioning of the isolated material on top of
the LOC.
Subsequently, the sample material inside single
droplets is transported very efficiently and contact-
less to the reaction and analysis centres on the chip
surface via surface acoustic waves. Processing of
isolated genetic material like specific or unspecific
amplification is conducted on the nanofluidic device
via polymerase chain reaction methods followed by
labelling with fluorochromes.
Qualitative as well as quantitative analyses such
as real-time PCR or microarray will be carried out
using a novel ‘fluorescence reader’, especially
designed for the LOC system and forming the
principal component of the detection unit. Thus
different applications for point-of-care diagnostics
are practicable on one single lab-on-a-chip.
4 POSSIBLE APPLICATIONS
This freely programmable lab-on-a-chip system will
open new potentials in research and development in
different fields of applications ranging from
cytogenetics to pathology and forensics.
Apart from its nearly unlimited applicability for
many different biological assays, possible
applications of this system in cytogenetics are e.g.
detection of chromosomal imbalances and detection
of genomic imbalances in solid tumour tissue. After
isolation of the genetic material by laser-based
microdissection the sample is transferred to the lab-
on-a-chip using a low- pressure transfer system.
Furthermore, in a first biochemical reaction the
extracted material is enzymatically digested and
prepared for subsequent amplification e.g. Alu PCR
for SNP analyses. Whole genome amplification of
e.g. individually isolated single cells and
chromosomes followed by labelling the material
with fluorochromes can be used for e.g. fluorescence
in situ hybridization (FISH) experiments. After
mixing with different-labelled reference-DNA the
mixture can be transferred to the specific CGH array
via wetting modulated surface chemistry, hybridized
and finally detected. To make sure that
fluorochromes are incorporated uniformly into the
sample DNA as well as the reference DNA, the
labelling process can be monitored using online PCR
detection. In the same way the amount of DNA
product after amplification can be determined
exactly. This provides the possibility to mix equal
amounts of sample and reference DNA for the
hybridization mixture. The detection array,
microarray on the lab-on-a-chip, will be question-
and patient-specific spotted by dot blot technology.
Again acoustic actuation will be adopted to solve
e.g. lyophilized reagents in a specific buffer.
With regard to the emerging field of forensics,
this LOC system can also be applied to genomic
sample material as blood cells, buccal swaps or other
human cell material performing DNA fingerprint,
paternity tests or SNP analysis. As forensic analysis
should be cost and time saving, the development of a
new miniature analytical lab-on-a-chip system could
serve the market in a novel and promising way.
Real-time PCR and STR analysis could not only be
applied to the novel LOC system separately but also
be combined on one single lab-on-a-chip in a
modular way.
Figure 5: SAW driven lab-on-a-chip system with 10
interdigital transducers, two heaters and biochemical
reactant containers. Transport of minute amounts of
sample material in ‘virtual’ beakers is actuated by surface
acoustic waves generated via interdigital transducers. The
liquid phase comprising the genetic material (red) is
covered by a thin layer of mineral oil avoiding
evaporation. A load resistor heating and a peltier element
provide for precise temperature profiles required for
molecular biological methods.
BIODEVICES 2008 - International Conference on Biomedical Electronics and Devices
260
A further application in the field of systems
radiation biology is to investigate the influence of
low-dose irradiation on cell-cell interactions and
possible bystander effects. The medical use of
ionizing radiation contributes the largest fraction to
the population’s anthropogenic radiation exposure.
Thus, biopsies of suspicious diagnostic findings,
which were irradiated with standard low-dose, will
be extracted for histological examination and
isolated cell clusters will be analysed on the LOC.
By spotting a specific protein array on one part of
the LOC and moving cells and media via SAW onto
this particular array, it should be possible to detect
cancer cascades and involved proteins. This method
can be further used to determine the relation and
interaction between cancer associated proteins i.e.
p53, TGF-β and caspase.
5 OUTLOOK
This system, the acoustically driven, freely
programmable multifunctional biochemical lab-on-
a-chip system, will be
applied on different
diagnostic approaches at the single cell or single
chromosome level e.g. cytogenetics, tumour genetics
and genetic forensics.
Competitive LOCs, combining these techniques,
are worldwide not on the market. An essential
advantage of the LOC system is the modular set-up,
which allows reacting to different diagnostic
questions in a preventive medical check-up. This
implements the rapid adaptation to patient-specific
point-of-care diagnostics as well as the operator-
specific development of new molecular markers for
imaging techniques. Fields of applications for the
newly developed LOC system range from the
analysis of cell compartments and single cells in
tumour diagnosis to chromosomal imbalances in the
human genome.
ACKNOWLEDGEMENTS
The authors would like to thank Klaus Macknapp,
Deutsches Museum Munich, for providing the
electron microscopy images.
Financial support of the Bavarian Research
Foundation, Deutsche Forschungsgemeinschaft
(DFG) SFB 486 grant and German Excellence
Initiative via the ‘Nanosystems Initiative Munich
(NIM)’ is gratefully acknowledged.
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