 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.
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. READ MORE
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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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.
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.
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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. READ MORE |