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Ultra-sensitive multianalyte immunoassay based on dna-senor

Deliverables

It was one of the objectives in the project to develop an immuno-PCR procedure for the determination of the tumour markers CEA and CA19-9. Immuno-PCR combines the selectivity of the classical ELISA technology with the enormous amplification power of the polymerase chain reaction, PCR. The development comprises several steps: Fist, an ELISA procedure has to be developed. In parallel, a method for antibody labelling with DNA reporter molecules has to be established. The ELISA in a microtiter plate is then followed by a PCR amplification of the DNA label and a quantitation of the amplified label by gelelectrophoresis or PCR-ELISA. Immuno-PCR is method with an enormous amplification power and allows for analyte determination in the trace level concentration range. A crucial step in the immuno-PCR procedure is the synthesis of suitable antibody-DNA conjugates. With these conjugates lower limits of detection for the target analytes CEA and CA19-9 were obtained that were 3 to 4 orders of magnitude lower than those of the corresponding ELISA. The method is thus extremely sensitive. The method can thus be readily applied where such a high sensitivity is required, e.g. in the determination of metabolites in blood.
So far, Inventus BioTec has developed a DNA-sensor based on screen printing technique and electrochemical sensing detection. The generation of a generic surface as well as the development of an electrochemical have been the main topics within this project. The generation of the generic surface has been achieved by adsorption of deglycosylated avidin to the surface of screen printed carbon electrodes. Onto this specific surfaces, biotinylated biomolecules (ssDNA, antibodies) can be immobilized in a simple way. The electrochemical detection occurs by using an enzyme catalyzed reaction. These key technologies have been successfully elaborated and tested. Within other on-going projects, these methods have been used for the development of an immuno-biosensor. It has been clearly demonstrated that these methods provide a platform for the future development of immuno-biochips as well as DNA-biochips. The working principle-generation of a generic surface as well as the electrochemical detection system-established in the amperometric DNA-sensor could be successfully transferred to the development of an immuno-biosensor. The generic surface-adsorption of deglycosylated avidin onto the surface of screen printed carbon electrodes-was used for the immobilization of biotinylated anti-Interleukin-4. The electrochemical method established within this EC-project and within on-going projects has been used for the detection of Interleukin-4 in the nanomolar range.
One of the tasks of Biosensores S.L. (BSL) during the development of the ULISA project was the design of a FIA-type system for the use of magnetic particles (MP) covered with antibodies or DNA. The peculiarity of the design permits small quantities of MPs to be deposited over the transducer in the measuring cell. Once completed the measurement, these MPs could be eliminated from the cell leaving the FIA system ready for new measurement with fresh MPs. Within the frames of the ULISA project, BSL has developed this specific technology which, being improved and modified for its specific use, could be used in viable automatic biosensing systems manipulating MPs. BSL already has a semiautomatic prototype of the FIA for biosensors with MP and with a multi-channel wet cell comprising three ISFET transducers. A specially designed miniaturised platinum electrode is used as a reference electrode for these ISFETs. This design could be considered as a first step that will yield a MP biosensor with an array of transducers. The automated FIA-type system developed by Biosensores S:L. comprises the following sub-systems: A) an injection unit for MP aliquots and reactives. This unit has a Peltier-refrigerated wet cell and a µ-syringe actuated with linear stepping motor. The µ-syringe permits injections of MP suspension liquid of volumes as small as 5 µl. B) A fluidic circuit with peristaltic pumps and with elements for mixing and temperature stabilising of reactives. C) Software both for the realisation of the analytical process and control of the system components, and for data acquisition and treatment of the signals generated by ISFETs; the two parts of software are developed by BSL.
It was one of the objectives in the project to develop methods for coupling a DNA-label to antibody molecules. These conjugates were subsequently used in immuno-PCR procedures. Two strategies were investigated. First, we took advantage of the streptavidin/biotin system. An antibody was first biotinylated and subsequently incubated with streptavidin. In a last step the biotinylated DNA-label was added. With these conjugates immuno-PCR could be successfully demonstrated. It is one disadvantage of the method, however, that the stochiometry of the ligand during coupling is crucial. Moreover, this approach can not be used for the simultaneous detection of several analytes. Consequently, we elaborated an additional coupling chemistry. Here, a heterobifunctional cross linker such as sulfo-SMCC were used. After purification these conjugates could readily be used in immuno-PCR. Due to the covalent coupling chemistry these conjugates allow for use in multianalyt determinations. The antibody-DNA conjugates synthesised by covalent chemical coupling can readily be applied in immuno-PCR. Immuno-PCR is method with an enormous amplification power and allows for analyte determination in the trace level concentration range. A crucial step in the immuno-PCR procedure is the synthesis of suitable antibody-DNA conjugates. The method developed within the project will improve the reproducibility and robustness of the method.
A new protocol for effective passive adsorption of deglycosylated avidin onto the surface of printed carbon electrodes for use as transducers for biosensors has been elaborated and tested. This protocol allows facile, specific immobilisation of biotin-labelled biomolecules (e.g. DNA probes and antibodies) resulting in the generation of generic surfaces for sensor application. By judicious choice of the correct biotin-labelled species one can produce a large variety of sensor formats suitable for, but not limited to, the fields of environmental, clinical, veterinary, defence and agricultural analysis. Screen printed carbon electrodes are widely used as transducers for biosensor application in the clinical and environmental fields. In order to introduce specificity into these systems, the usual procedure is to immobilise a specific individual biological recognition component at the surface of the electrode. For example, this could be an enzyme in the case of sensors designed to measure glucose or an antibody in the case where one wishes to carry out an immunoassay. However, the same basic electrode structure could not be used for both species. The technology described in this result allows one to very simply modify the surface of a carbon electrode with avidin (more specifically deglycosylated avidin) thus allowing the same electrode to be used in combination with any biological recognition component which had biotin incorporated into its structure. Biotinylation of biomolecules is a simple procedure for which many commercial kits are available. By use of avidin coated electrodes in combination with biotin-labelled species one can produce a generic sensor transducer platform suitable for exploitation in a number of fields using a variety of detection technologies and no longer has to prepare individual electrodes for each species one wishes to measure.