The Development Of An Immuno-electrochemical Biosensor And The Research On Its Detection Of Anthrax Spores

The development of an immuno-electrochemical biosensor and the research on its detection of anthrax sporesIn this article, we report the design, manufacture and application of an electrochemical immunosensor system that could be used for rapid pathogens detection. A series of theoretical and technical problems involved have been solved and thus its practicability has been greatly pushed forward.The detection principle of this electrochemical immunosensor system is based on the sandwich method of immunoassay. It can be regarded as an automation process of the later.In designing the detecting system, we adopted a new way different from that of the traditional biosensor. In our design, the biological recognition element and signal transducer was arranged without direct spatial contact, different from the 'in direct spatial contact' design of the conventional biosensor according to the definition of IUPAC (International Union of Pure and Applied Chemistry). This small structural change has greatly promoted the improvement of the characteristics of the detecting system.As a key part of the preparation of the biosensitive element, which is the core of a biosensor, the immobilization methods of antibody on two supporting matrix (nylon membranes and glass beads) have been studied extensively. On the surface of nylon membrane B, antibodies have been covalently linked by means of glutaraldehyde method. On the surface of nylon membrane C, antibodies have been linked through EDC method. In the case of ABC membrane, antibodies have been covalently linked to the membrane's surface spontaneously on which the antibodies containing solution was dropped directly. As to the glass beads, both glutaraldehyde and EDC methods have been used successfully for linking antibodies on its surface.The electrochemical detector (transducer) was designed in the form of three-electrode system, including working electrode, reference electrode and counterelectrode. The way they were prepared is as follows: three hollow organic glass rods of the same size were made beforehand. The inner diameter of the hole is 1mm. Two of the organic glass rods were coated with carbon ink on their entire surface according to the recommended method along with it to serve as the working and counter electrodes respectively. Another was coated with ilver/silver chloride ink in the same way to serve as the reference electrode. Then they were assembled with other auxiliary organic glass parts to form a flow electrochemical cell. Three holes in its outer wall benefit the electric contact between the three electrodes and the potentiostat, through which the conducting wires get an access to the surface of the electrodes. A real-time and dynamic measurement can be carried out on the flow electrochemical cell.The one-step glutaraldehyde protocol has been adopted in preparing the conjugate to be used in the immunoassay. Rabbit anti anthrax spores IgG and Alkaline phosphatase were linked covalently together by this method.Before the prototype was made, we set up a detecting platform operated manually, in which two peristaltic pumps, three-way valves and some silicone tubes are included. We tried to design this detecting platform in such a way that it could be transformed to the prototype to be made to fulfil a detecting task automatically. In order to achieve this goal, a series of experiments have been carried out to optimize the experimental conditions and design scheme. On this detecting apparatus, samples containing anthrax spores and two kinds of anti-antibodies were tested and an ideal result has been obtained. Base on the above work, we set out to design the prototype.Automation is one of the main targets of this work, so we designed a flexible operation program for solutions manipulation, such as the control of flowing rate, flowing direction, and the turns of them into the fluidic system, etc. The automation of this detecting system has been achieved mainly by the use of a PLC (programmable logic controller). All the valves and pumps are u...