Nederlandse Vereniging voor Klinische Chemie en Laboratoriumgeneeskunde
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Project 46 Intracellular Detection using nano-Electrode Array (IDEA)
Onderzoekslijn Lab-on-a-Chip
Omschrijving In the past few decades, developments in microfabrication have led to the successful so-called
?lab-on-a-chip? concept (LOC) in which fluidic components, micro-reactors and micro-physical
and chemical transducers have been integrated into unique miniaturized analytical systems.
Recently, with the integration of nano-size components, LOC has also become a powerful tool for
cellular studies in biomedical applications. In this project, we propose the development of a LOC
for measurement of oxidative stress in single cells in real time.
Oxidative stress is a process related to the excessive generation of reactive oxidative species
(ROS) in cytoplasm. This is often related to mitochondrial dysfunctioning or due to an excessive
stimulation of nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase activity. The
physiological meaning of oxidative stress is illustrated by a number of human diseases, including
malignant diseases, cancer, diabetes mellitus, human immunodeficiency virus (HIV) infection,
neurodegenerative diseases (e.g., Down?s syndrome, Parkinson and Alzheimer diseases),
neuromuscular diseases, specific cardiac states (ischemia, reperfusion injury, ischemic
preconditioning, and cardiomyopathy), obstructive sleep apnea and aging. A microsystem
enabling the on-line measurement of intracellular ROS of individual cells is of great benefit for a
better understanding and treatment of the above diseases.
The ultimate goal of this proposal is to develop a tool (electrochemical sensor system which
contains micro/nano-electrodes or micro/nano-electrode arrays integrated on micro-needles) on
chip and a methodology for in-vivo and real-time cell analyses (see Fig. 1).
The intracellular electrochemical detection using the micro/nano-electrode arrays especially
shows an advantage in the direct analysis of solid tissue cells. Due to the small dimension of the
electrodes in comparison with the cellular size, the micro-needle with the micro/electrode arrays
can puncture into the cell and locally and continuously monitor a change in concentration of ROS
in specific positions within the cell. This research can lead to better understanding of
pathophysiology, diagnosis and monitoring diseases related to oxidative stress such as oxidative
phosphorylation (OXPHOS) disorders, chronic inflammation, ischemia and malignant diseases.
In addition, the proposed technique can also be an universal tool for in-vivo and continuously
monitoring oxidative stress during clinical episodes where muscular conditioning and
deconditioning have relevance for the fitness of patients.
Subsequently, this knowledge of this research is important for pharmaceutical developments:
validation of the antioxidant drugs and further in-situ manipulating and performing biochemical
experiments in a single cell (Lab-in-a-cell concept) that can avoid testing drugs and drug delivery
on animals.
Targeted measurement species in this research are intracellular reactive oxidative species caused
by internal damage to the cell such as hydrogen peroxide, hydroxyl radicals and superoxide
anions.
The working principle of the proposed sensor is based on electrochemistry including
amperometry, potentiometry or fast scan cyclic voltammetry. The sensor has a short response
time (~ 100 ms) and ultra-low detection limit. For every species, a single electrochemical
technique or a combination of these mentioned techniques will be applied to obtain a good
sensitivity, detection limit and selectivity. In addition, extra selectivity can be achieved by
modification of the electrode surface.
3
The first approach will be to measure the redox potential inside a cell as an indication for the total
concentration of ROS. The designed electrode or electrode array of the sensor can be miniaturised
and fabricated in mass production using a combination of micro- and nanotechnologies (a.o.
lithography, thin film techniques, focused ion-beam (FIB)). The miniaturization of the sensor
allows intracellular analysis with minimal disruption of the cell.
When the size of the sensor is scaled down, electrochemical phenomena related to nanodimensions
(length and width) of the electrode array such as redox cycling on interdigitated
electrodes are expected to increase dramatically. This will allow us in the second phase to also
look at individual redox species. In redox cycling, different potentials are applied to nanointerdigitated
electrodes allowing multiple oxidative/reductive conversions of the same molecule
resulting in strong amplification of the current. Redox cycling may also be used to selectively
increase the detection for one particular redox species, depending on the choice of the generator
and re-generator electrode potentials. This leads to a better detection limit, sensitivity and
selectivity.
The most important benefit of the proposed sensor is that it allows continuous, in-situ, on-line
measurement of redox species. After having made the nanoelectrode sensor, it will subsequently
be integrated in a microfluidic system containing channels for cell-transportation and structures
allowing automatic ?docking? of the cell onto micro/nanoneedles containing the electrodes. This
will enable screening of large numbers of individual cells.
Micro-needle with an
integrated electrode array
Mitochondrion
Nucleus
CELL
Cytoplasm
Figure 1: Illustration of the intracellular analysis using a micro-needle with an integrated
electrode array.
In conclusion, this project aims at:
? Determining the optimal electrochemical technique for measurement of intracellular
ROS.
? Design and realise a microdevice or system comprising nanoelectrodes for cell docking
and ROS measurement
? Investigate and analyse the relationship between observed ROS variations and a variety
of diseases.
? Evaluate the potential for use of the device in rapid, real time cell analysis used in
pharmaceutical development and drug testing.
4
? Development of cross-knowledge between nanotechnology, electrochemistry, biology
and medicine.
Projectleider Prof.dr. I.(Istvan) Vermes
Instituut Medlon BV Locatie Medisch Spectrum Twente Enschede
Trefwoorden reactive oxygen species, electrode array, apoptosis
Status Lopend
Periode 1-2004 - 1-2008
Partners Prof. dr. ir. A. van den Berg, University of Twente
Medewerkers Dr. W.A.T. Dam, dr. ir. W. Olthuis, Dr. L.L.J. van der Maas, dr. C.J.A. Doelman
Financiering 2e Geldstroom - ZON, NWO

Technologischestichting STW
Publicaties Geen publicaties gevonden.