A
biosensor utilizes the principle that certain biomolecules suspended in a
complex solution (e.g. blood, semen, blood plasma or serum, sputum etc.)
react specifically with another biomolecule used for detection.
For example there are many different antibodies available that bind to
one specific disease marker, antigen, only. There are also many other
proteins that react with only one specific biomolecule, for example a
particular glycolipid embedded into a cell wall (cell membrane). The
chemical reaction between the detecting molecule and the molecule in the
sample solution causes physical and/or chemical changes of reagents, or
of the nearby environment. These changes can be for example heat release,
altered dielectric constants, viscosities or pH values. These changes are
transformed in the sensor into an electrical signal, which can be related
to the total number of chemical reactions and the concentration of the
analyte in the sample solution.
Another possibility to quantify chemical reactions is to let the system
undergo additional tailored chemical modifications that enable
quantification. There are two modifications commonly used to monitor
biological reactions: one modification causes changes of opacity of a
solution proportional to the number of chemical reactions of interest.
The other modification involves introduction of fluorescent molecules by
binding them to, usually, one of the reagents. Such fluorescent
molecules, when illuminated, emit light of a slightly different color
than the excitation light, they fluoresce. Ideally, the fluorescence
intensity is proportional to the number of chemical reactions of
interest. At Imego we develop systems that measure the fluorescence
intensity to quantify the occurrence of certain reagents, e.g., disease
markers. We have focused mainly on antibody based medical
diagnostics.
Heterogeneous assays
Biochemical reactions often occur with one or more reagents being
adsorbed on a substrate surface. For example a typical antibody assay is
based on the availability of two monoclonal antibodies. One is bound to
the surface and is called the capturing antibody while the other one
serves as a detecting antibody. The detecting antibody is either modified
by binding of small fluorescent molecules to it or is itself bound to a
fluorescent particle. The disease marker that you want to detect (the
antigen) binds to the capturing antibody on the surface and in turn
captures the detection antibody from the solution phase. An
antibodyantigen-antibody “sandwich” is formed on the substrate surface.
The detection antibodies that has not bound are washed away and the
fluorescence signal from the sandwich complexes on the surface is
recorded. The signal is related to the amount of disease markers in your
sample.
For medical diagnostic applications the substrate is often flat. The
substrate may also be a particle in which case the adsorption takes place
onto a mobile and curved surface. The latter enhances the kinetics of
uptake since substrate mobility increases, and therefore speeds up the
detection. We work extensively on optimization of different parameters
that govern the sensitivity and the precision of heterogeneous assays.
These include both the optimization of functionalisation of surfaces with
antibodies by novel substrate binding and by tailoring chemical
modifications of antibodies.
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Homogeneous assay
Homogeneous assays require no separation steps. One simply mixes two
solutions of known volume: a sample solution and a solution containing
known (and high) amount of antibodies marked with for example
fluorophores. The reaction between the marked antibody and the antigen in
the sample solution changes the color characteristics of the solution.
This enables one to distinguish the intensity of light from fluorophores
bound to antibodies that have reacted with a disease marker from those
bound to free antibodies. The resulting assay is very simple indeed and
allows for a very simple fluidics and system design. At Imego we develop
homogeneous assay based on the above mentioned ability of certain
fluorophores to be sensitive to their close environment.
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Non specific adsorption
It is very important to suppress all non specific reactions that may
occur between the surface or the surface bound molecules and the
proteins (or DNA strand) in the sample solution. You only want the
molecules of interest, the target molecules, to bind to the sensor
substrate. To block all non specific reactions is far from trivial.
Usually the abundance of a protein of interest in a solution is very low,
often well below ppb, which implies that the surface is bombarded
predominantly by parasitic molecules while the incidence rate for
molecules of interest is very rare.
Bad blocking may lead to adsorption of fluorescently labeled molecules to
sites that do not signal the existence of the target molecule. This leads
to increased background fluorescence and thus decreased overall assay
sensitivity, or (in worst case) to a false positive signal. At Imego we
are putting much effort in minimising the non specific adsorption in
order to optimise the immuno assay performance.
Substrates as waveguides
An important practical limitation imposed by fluorescent probes if one
strives for ultimate sensitivity is the necessity to filter away the
exciting light whose intensity is usually many orders of magnitude higher
than the fluorescent signal intensity. One possibility to deal with this
issue is to use an optical waveguide as the substrate. The phenomenon
behind the propagation of light inside the waveguide is called total
internal reflection. It occurs when light hits the interface from an
optically dense medium towards a less optically dense medium above a
certain well defined angle. The light is reflected back into the
optically dense medium and stays inside the waveguide. A small fraction
of the light leaks out and can be used to illuminate fluorophores that
are within a couple of hundred nm from the interface.
We have investigated the performance of optical waveguides in biosensor
applications using both flat surfaces and polymer optical fibers as
substrates. The surfaces are coated with cathing antibodies that bind to
the molecules of interest when in contact with a sample solution.
Fluorescently marked antibodies that bind to the catched molecules of
interest are brought in such close vicinity to the waveguide that they
can be excited by the light inside the guide. This approach decreases the
intensity of the excitation light that reaches the fluorescence detector
by several orders of magnitude. Using this excitation technique we have
been able to achieve a tenfold decrease of the detection limit for
prostate specific antigen (PSA) detection.
Another great advantage of the waveguide approach is that it virtually
eliminates the need to wash away fluorescently marked antibodies that has
not bound to the molecule of interest. They normally contribute to an
unwanted, non specific part of the signal. Since the excitation light is
confined to a zone within few hundreds of nanometers away from the
waveguide surface, the fluorophores in the solution that has not bound to
the molecule of interest on the surface remain unexcited and do not
contribute to fluorescence signal. This simplifies the design of the
system considerably.
Simultaneous detection of several markers
We investigate the use of newly developed fluorescent probes, the so
called Quantum Dots. From the point of view of their fluorescent
properties, the Quantum Dots are convenient in use since their emission
and excitation wavelengths are well separated, and their emission spectra
appear at different wavelengths (at different colors) depending on the
particle material and size.
It is possible to excite Quantum Dots emittinging light of different
colors using a single excitation source. Their emission spectra are
narrow, much narrower than the emission spectra of conventional molecular
fluorophores. Therefore they should be ideal candidates for use in assays
which aim at simultaneous detection of several markers.
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