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

, Achim Wixforth

and Stefan Thalhammer

GSF, National Research Center for Environment and Health, Institute of Radiation Protection

Ingolstädter Landstrasse 1, 85764 Neuherberg, Germany

Advalytix AG, Sauerbruchstrasse 50, 81377 Munich, Germany

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

 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

) 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

) 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

) (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

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

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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.

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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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