Lines Of Work
Molecular affinity BIOSENSORS
The affinity biosensor technology, based on specific molecular interaction (e.g.: antigen-antibody, DNA -DNA, enzymes-substrates, receptors-specific molecules, etc), allows detection and quantification of analytes with high sensitivity present in a sample and automatization and monitorization of the process.
The chart below shows the key elements of a molecular affinity biosensor.
For the quantification of the analyte of interest present in a liquid sample, the biosensor has a receptor that interacts specifically with the analyte, such as anti-bodies, DNA, enzymes, metabolic substrate, etc. The interaction between the analyte and the receptor generates physical-chemical changes (change of pH, mass variation, optical properties modification, generation of electrons, etc.) that is recognize by the appropriate transductor generating an electrical signal proportional to the analyte concentration within certain range. Their electrical amplification (Amplifier), simultaneous to the detection, allows quantification of the analyte concentration in the sample within the “linear proportional zone”.
This automation allows, among other advantages, reducing the time of analysis, several samples could be analysed in a short time and reporting “online” results.
Within the affinity biosensors area, Biosensores SL has been focusing its research on the use of magnetic beads. For the process of sample preparations, the magnetic beads do allow analyte preconcentration, reducing non-specific molecules and undesired molecules interfering with a specific signal. Moreover, the magnetic beads can be easily applied for various kinds of transducers, such as optical, electrochemical, potentiometric, etc. and be used with microfluidic systems.
Biosensores SL / WOLA have developed fluidic strategies of magnetic beads to allow the preconcentration in an ordered way on the highly sensitive points of the transducer. Using this technology, it is possible to amplify the analytical signals and reduce the quantity of magnetic beads used in each analysis.
Affinity magnetic particles can be used for affinity purification and separation of antibody, antigen, proteins and DNA/RNA, etc. Affinity magnetic beads have high selectivity and specificity which make them ideal tools for the separation of various biomolecules.
Example of transducer developed by BIOSENSORES in collaboration with the National Microelectronics Centre of Barcelona (CNM) for a molecular affinity biosensor. In the figure below it is shown an ISFET for a potentiometric transducer.
MOLECULAR AFFINITY BIOSENSORS Applications
MOLECULAR AFFINITY BIOSENSOR WITH MAGNETIC BEADS: analytical process
Specific antibodies have been bound to the magnetic beads for fixing the analyte through an epitope of the same (antibody B). A second labelling antibody – which will reveal the presence of the desired analyte- (antibody A) is covalently linked, in this example with the enzyme galactosidase. Both antibodies interact with the target analyte through their corresponding epitopes. In the measurement stage, through an electrochemical transducer, the interaction between the enzyme substrate (PAPG) and the enzyme generates electrons that are measured by the transducer.
ANALYTICAL STEPS IN THE use of MAGNETIC beads in affinity BIOSENSORs
An aliquot containing the analyte (green triangles) in an unknown concentration along with other unwanted molecules, interacts with an aliquot of the capture antibody (Ab1) plus another aliquot of the labelled antibody (Ab2). Both antibody concentrations (Ab1 & Ab2) are known, the concentration of the antibodies is higher than the concentration of the analyte. The only unknown is the concentration of the analyte that we want to quantify.
The mixture of the molecules that interact between them, present in the different aliquots, reach a molecular equilibrium, (where all combinations are possible between Ab1, the analyte and Ab2). Depending on the concentration of analytes and antibodies, a specific molecular equilibrium will be achieved.
The mixture of molecules and substances formed after the interaction of the antibodies with the analyte flow to the measurement cell, where the magnetic beads are trapped. And the rest of molecules are washed and removed from the measurement cell, thus avoid interference from other possible molecules when the measurement is carried by the transducer.
Step 4 & 5
Once the rest of the molecules not retained by the magnetic beads have been removed, it takes place the measuring of the concentration of the labelled antibodies through a specific procedure. In this case, since it is an oxide-reduction enzyme (marked in red), the substrate for this enzyme will be introduced, which will trigger the production of electrons that will be quantified by the electrochemical transducer.
USE OF A ION SELECTIVE FIELD EFFECT TRANSISTOR (ISFET) AS TRANSDUCER FOR MOLECULAR AFFINITY BIOSENSORS
“Basic Research on the use of magnetic fluids in microsystems” (MICROMAG) (1998-2000). https://cordis.europa.eu/project/id/BRPR970598