Study On The Immunological Hirudin Capacitive Biosensors Fabricated On Self-assembled Monolayers (SAMs)

Hirudin, one kind of direct thrombin inhibitors, is a low-molecular weight protein. Recently, it has been reported that hirudin can inhibit tumor implantation and metastasis. Among all kinds of hirudins, recombinant hirudin plays a definite role in patients suffering from heparin-induced thrombocytopenia, and prevents coronary thrombosis recurrences. However, the most important adverse effects of hirudins are haemorrhage and the induction of antihirudin antibodies. Pursuing a fast and easy analytical method with high sensitivity and high selectivity to detect these low-molecular weight proteins has been put on the focus of our study.Immunosensors have many promising applications in different researching fields for their high specificity, specific selectivity and fast response time. Here, capacitive immunosensors based on self-assembled monolayers (SAMs) technology were developed to detect recombinant hirudin directly and sensitively by immobilizing polyclonal recombinant hirudin antibodies on gold electrodes.The thesis includes four parts as follows:1. IntroductionThis part contains some information related to the task divided into four main aspects: (1) the principle, the fabrication and the category of immunosensors, (2) the principle of capacitive immunosensors and their recent applications, (3) the researching development of hirudin, and (4)the purpose of this study.2. An immunological hirudin capacitive biosensor fabricated on self-assembled monolayers (SAMs) derived from thioctic acid (TA), termed as (TA-SAMs)In the experiment, we attempted to fabricate the hirudin capacitive immunosensor based on the TA-SAMs. A fast electrochemical measurement, potentiostatic step method, was applied to measure the capacitance changes caused by the interaction between the recombinant hirudin (RH) and its antibodies. The insulating properties of the immunosensors were investigated by cyclic voltammetry in Fe2+/Fe3+ solution. The immunosensor could exactly detect RH in the range of 100pg/ml ~ 5ng/ml, with a detection limit of 50pg/ml. No obvious response to the unspecific interaction was observed. The success of detecting RH showed the capacitive immunoassay might be employed as a new measurement for more practical applications.3. An immunological hirudin capacitive biosensor fabricated on SAMs derived from mercaptoacetic acid (MAA), termed as MAA-SAMsTo improve the sensitivity of the capacitive immunosensor mentioned in part 2, we selected MAA, a shorter alkanethiol chain than TA, to form the SAMs. For the MAA-SAMs modified immunosensor, a linear detection range from 5pg/ml to 1ng/ml was obtained, with a detection limit of 2.5pg/ml. This indicated capacitive immunosensors with thinner SAMs could detect the antigens with higher sensitivity. At the same time, we also studied the coverage rates of both SAMs by means of electrochemical impedance spectroscopy, and investigated the capacitance changes of the fabricating process, the effects of the ionic intensity and pH on both SAMs with potentiostatic step method. In the experiment, we also monitored the fabricating process of SAMs by quartz crystal microbalance(QCM). The result testifies the larger initial capacitance originated from thinner SAMs can ameliorate the sensitivity of capacitive biosensor. 4. The regeneration research of capacitive immunosensors Because of the adverse effects of high ionic intensity and low pH on the SAMs, the generally experimental results from measuring the interaction in-between antigen and antibody can't be obtained in the regenerated capacitive immunosensors. So we used cyclic voltammetry scanning technology by which the SAMs attached RH and its antibodies were completely removed. When the regenerating procedures were repeated, the response of the immunosensor was similar to that of immunosensors mentioned above. What's more, we investigated electrochemical behaviors of the regenerated interface, compared with that of bare gold electrode polished with alumina slurries. The experimental results prove that the electropolishing method is one of promising options to regenerate capacitive biosensors.