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APTE
Association – Science Forum
Towards
an optoelectronic future: Photosynthetic fluorescence enables simple
environmental assays
Pascal
M. Baillod and Gary O. Martini (Director Science Forum) A
major issue in biosensor technologies is the development of quick and
cheap reproducible assays that can be used to determine environmental
changes. It is essential that such assays should be miniaturized in order
to allow the required mobility of the measurements. A very original
approach to this problem has been developed over the past 15 years at the
laboratory of bioenergetics of the University of Geneva. The idea is to
determine the “health” or condition of plants through a simple
spectrophotometric measure of the fluorescence resulting from
photosynthesis. Knowledge of
Photosynthesis enables the assay
Like
most biological systems, photosynthesis has a low yield that could be a
liability to its very big flexibility. Thus, of the total energy getting
to the square section of a leaf, parts
will be lost by heat dissipation, be reflected (or non absorbed, giving
the vegetation a green color) or just
pass through the leaf. The remaining energy,
absorbed by the photochemical units will permeate the biochemical
“pipelines” of photosynthesis, which can be imagined as a series of
steps, all provoking a loss of the energy in transit. One can imagine a
pipe with holes leaking every x meters, that is the idea of the
Bioenergetics lab model depicted on fig 1. Biosensors developed in
collaboration with Professor Strasser’s team measure energy losses
occurring at initial steps of the photosynthesis pipeline, the
photo-systems. These protein complexes use solar energy (photons) to
excite electrons so that they are catapulted and freed of their original
molecule of chlorophyll. The freed electrons reach the energy carriers
while flowing down the pipeline and finally join the carbon of the
atmospheric CO2, enabling it to go into the biomass (first as
glucose molecules). This process (depicted on fig 2)
generates sufficient energy and glucose
to drive the plant’s metabolism. Energy
losses of the photosystems appear under the forms of heat and
fluorescence. The latter is a radiation of energy at a wavelength of 735
nm. This knowledge comes from the biochemical purification of the
photosystems (PS) and enables the very simple assay of the bioenergetics
lab: A biosensor (fig 3) first excites the cross section of a leaf (by
illuminating it for a short time with red light - the wavelength used by
plants ). Then it measures, at the known PS wavelengths, the consequent
fluorescence. This measuring technique has been developed , the initial
difficulties have beem worked out to give the very easy current
“JIP-test”. Models for the
interpretation of the measurements
Measuring
is easy – now comes the difficult part. The measures show part of the
losses from a single step of a complicated multi-step process, an open
system under many influences. This complex problem is impossible to solve,
the only solution is to work out models that approach reality. These
models are based on the membrane flux theory (fig 1), best suited to
represent the reality of many linked energy and matter fluxes. Are
calculated with the model not only the losses at every step but also the
fraction of active photochemical units (with reaction center and
photosystems). This fraction as well as the total flux will change as a
measure of the plant’s requirements in energy and biomass and are highly
influenced by external conditions (light, temperature, humidity,
fertilizer quantity changes, etc.) and internal programmed life cycle
(growth, reproduction, hibernation). The
fraction of active photochemical units (see fig 1) gives a good idea of
the plant´s actual behavior. There are many possibilities of regulation
spanning a range with the extremes of output 0% or 100% of photochemical
activity. Regulation can be made on the number of photosynthetic complexes
switched on and off (digital regulation) or on their output (analogic
regulation - between 0 and 100%). “One can imagine a factory and its
director having to decide whether to have all the machines running at
reduced speed (analogic) or part of the
machines running at full speed (digital).“ explains Professor Strasser. But
the comparison to a factory stops here because the logic of photosynthesis
regulation seems to be closer to human nature: Just do the required
minimum so you can follow your biological program. Thus, it has been shown
that at midday when the sunshine reaches its maximum, plants will lower
the photosynthetic output by inactivating a certain number of
photochemical units. The transiting energy will still be
sufficient. Professor Strasser calls this the “siesta syndrome”, a
concept that he finds more accurate than the idea of “photoinhibition”
found in some textbooks: When a mediterranean factory closes from 1 to 5
P.M., during summer, the workers do not go to sleep, they are not
inhibited, their activity just changes. Now
to the idea of stress, that has also been redefined with a novel approach
at the Laboratory of Bioenergetics. The idea is to consider stress like a
deviation from optimality. Plants (and biological systems) are in this
approach always in a relative stress situation - they have to create a new
optimum for ever-changing environmental conditions. A plant submitted to a
sub-optimal situation creates a driving force (i.e. activation of special
metabolic or reproduction pathways) that will enable it to return to an
optimal situation. The point is to know whether the deviation from an
optimum (pollution, drought, etc.) is small enough so that the plant can
return to it. They can encounter positive stressors, as for humans (i.e.
sports, mental efforts). The
modeling and simulations behind these theories require an excellent
knowledge of computer science. Ronald Maldonado-Rodriguez (one of Prof.
Strasser’s PhD students) has developed the necessary software. His
backround of chemical engineering studies was of great help and highlight
the necessity of collaborations between different fields. Applications
The
possible applications are innumerable. First, let us mention agriculture,
which needs to undergo adaptations to sustain an ever-growing population.
Experiments have been carried out by C. Hermans, at the laboratory of
Bioenergetics, to get the fluorescence profiles of sugar beets grown under
a given stressor. Figure 4
shows that the fluorescence profiles of magnesium deficient plants
are different to those of control plants. It is therefore
possible in some cases to determine which nutritional element
(phosphate, magnesium, etc.) is deficient before the plants lose their
normal aspect. This could make it possible to add the deficient
nutritional element before the ruination of a field. There
has also been collaboration with the Medical Faculty, involving the
testing of bacteria originating photosystems that could be used as
specific target destruction drugs in disease therapy. In this case,
injected photosystems, present in the whole body, can be activated through
light excitation at a very specific location where a target (i.e.
cancerous tissue) is to be destroyed. This method presents fewer risks for
a patient than traditional chemotherapy Fluorescence
measures are already used
in the following situations. In collaboration of the Université
Libre de Bruxelles, the Laboratory of Bioenergetics “measures” the
health of urban trees in the European capital. It has been shown that high
concentrations of CO2 can diminish photosynthetic activity by
up to 20%, which relativises the idea of counting on forests as
"green lungs” cleaning up the air (since CO2 is a
substrate for photosynthesis). At the moment, Professor Strasser’s team
is also phenotyping transgenic rice in Hyderabad/India. Future: Optoelectronic
assays are to replace time-consuming chemistry analysis
Professor
Strasser believes that optoelectronic techniques will replace 80% of the
current chemical analysis: “It is like astrophysics, the difference is
that salads, trees, fields and fruits will be used as targets instead of
galaxies.” Collaborations between microelectronics and biology are not
always easy to establish. Developing the technique presented here, for
instance, required collaboration between mathematicians, computer and
microelectronics scientists at a time when this subject was not at all in
fashion. Projects promoting innovation in this particular field seldom get
a financial support, environmental or other causes have to be invoked
instead. But this situation is changing; the first “consumers” are
here, mostly natural parks and environmental government services.
Plant selection biotechnologies (with or without GMOs) are now
starting to use this at first “exotic” science. “To promote such
technologies, specific projects have to be imagined, for instance the
development of a tool to respond to one specific question“ says
Professor Strasser. With
the optoelectronic revolution, microtechnology has great development
potential in the area of applied biology. Multi-field collaborations are
the best way to enhance such progress, which partly explains the interest
of the APTE technology network with whom we have collaborated on this
article. APTE network members (companies mostly) often work in many areas.
For instance, the CSEM (Centre Suisse d’électronique et de
Microtechnologies, in
majority financed by private money) decided five years ago to launch their
first research team in biology. Current projects involve biosensor surface
passivation and modification as well as optoelectronic measures, two
fields that strongly benefit from CSEM’s long experience in
microtechnology. Many
strategies have been applied to promote networking and collaborations. At
Greifswald, Germany, for instance, several small companies who couldn’t
afford their own research invested in a university research center on
biosensors. In Switzerland, the CSEM hired an entire university research
team who had finished a project; the labs and equipment were just moved
from one site to another. A truck was sufficient for this technology
transfer! The
possibilities are infinite and all the new creative solutions will never
be enough compared to the mountain of discoveries still to be made in the
field of biology. But clearly, all the new communication technologies can
powerfully accelerate networking activities and help avoid that good ideas
remain in the closet. Overview
of some current biosensor technologies For
further details or more companies see www.apte.net
and www.apte.net/biotech
*
Members of the APTE Association
Figure
1
Figure
1: Models of photosynthetic fluxes. On
the right hand, the leaf model represents the fluxes through the
cross-section of a leaf, containing a certain calculated quantity of
photochemical units (with reaction centre, PSI and PSII) depicted with
circles. A low yield of photosynthesis can be interpreted with a model in
which a fraction of the photochemical units are inactive (dark circles).
On the left hand, the membrane model represents the fluxes through
a single photochemical unit (embedded in a chloroplast membrane). For both
models: ABS= Energy getting to the photochemical unit(s), TR= fraction of
ABS trapped by the photochemical unit(s), ET= fraction of TR that will be used in the
photochemical processes, DI= fraction of TR that is lost by fluorescence
and heat. Figure
2:
Energy cascade of photosynthesis from solar energy to
biomass formation
Figure
3
Figure 4
See
Word file for figure 4
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Copyright ©
1997-2007, APTE Association and Gary Martini. All rights reserved. |
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