Evaluation Of Medicine Effects On The Interaction Of Myoglobin And Its Recognition Probes Using Atomic Force Microscopy

The study on the the biomolecular interaction is a basic research subject in biology, chemistry, medical science and so on. However, many researches focus on exploring the biomolecular interaction under basal conditions without the interference of external factors. The environment in vivo is complex especially for the patients who require long-term medication, such as hypertension patients, heart disease patients, diabetics, etc. Therefore, it is very important and meaningful to study the effects of medicines on the biomolecular interaction.In this thesis, myoglobin, which is a useful biomarker for the early assessment of acute myocardial infarction(AMI), was used as a model. A comprehensive study in regard to the effects of medicines(include promazine, amoxicillin, aspirin, sodium penicillin) on the interactions of myoglobin and its recognition probes were investigated using AFM force measurement. First, after myoglobin and recognition probes were treated by medicine, respectively, the interactions between myoglobin and recognition probes were investigated. Second, the interactions between myoglobin and recognition probes were determined in the presence of medicine using AFM force spectroscopy technique. The main content were as follows:1. Evaluation of medicine effects on myoglobin using AFM force spectroscopy measurement.The myoglobin-modified substrates were treated by promazine, amoxicillin, aspirin, and sodium penicillin, respectively. Then, the interaction between aptamer or antibody functionalized AFM tip and the treated substrate was determined using AFM force spectroscopy measurement. The results showed that the average binding force and the binding probability of myoglobin/aptamer were unchanged after myoglobin was treated by medicine. It indicated that this treatment had no effect on the interaction of myoglobin/aptamer. While the average binding force and the binding probability of myoglobin/antibody were changed after myoglobin was treated by medicine, And produced three kinds of different phenomena.(1) For promazine and amoxicillin, they resulted in the change of binding force distribution of myoglobin/antibody(i.e., from unimodal distribution to bimodal distribution) and the increase of binding probability;(2) for aspirin, it resulted in the change of the binding force distribution, while it had no influence on the binding probability of myoglobin/antibody;(3) for sodium penicillin, it resulted in the increase of the average binding force and the binding probability. These results may be attributed to the different interaction modes and binding sites between myoglobin/aptamer and myoglobin/antibody, the effects of medicines on the conformations of myoglobin, and the different structures between aptamer and antibody.2. Evaluation of medicine effects on recognition probes using AFM force spectroscopy measurement.Aptamer or antibody functionalized AFM tip was treated by promazine, amoxicillin, aspirin, and sodium penicillin, respectively. Then, the interaction between the treated functionalized AFM tip and myoglobin-modified gold substrate was determined. The results showed that the average binding force and the binding probability of myoglobin/aptamer were unchanged after aptamer was treated by medicine. It indicated that this treatment had no effect on the interaction of myoglobin/aptamer. Two kinds of different phenomena of the interaction between myoglobin and antibody were appeared after antibody was treated by medicine. In detail, for amoxicillin and sodium penicillin, they resulted in the decrease of the binding probability of myoglobin/antibody, but they had no effect on the average binding force of myoglobin/antibody; for promazine and aspirin, they had no effects on the interaction of myoglobin/antibody. This might be due to that compare with aptamer, the comformation of antibody was changed more obviously under the effects of medicine, and this possibility was also established by the experiment of circular dichroism spectrum.3. Evaluation of medicine effects on the interactions of myoglobin/aptamer or myoglobin/antibody using AFM force measurement.In the previous works, myoglobin and recognition probes were treated by medicine for a period of time, respectively, then the redundant medicine was washed by the binding buffer. In view of the actual system, the medicines often accompanied by target or recognition probe. Therefore, in order to evaluated the medicine effects on the interactions between myoglobin and its recognition probe, we investigated the interactions between myoglobin and recognition probes by AFM force measurement in the presence of medicine. The result showed that the average binding force and the binding probability of myoglobin/aptamer were unchanged, while the interaction of myoglobin/antibody were changed and produced three kinds of different phenomena.(1) for promazine, it resulted in the change of binding force distribution of myoglobin/antibody(i.e., from unimodal distribution to bimodal distribution) and the increase of the binding probability of myoglobin/antibody;(2) for aspirin, it resulted in the change of binding force distribution of myoglobin/antibody(i.e., from unimodal distribution to bimodal distribution) and decrease of the binding probability of myoglobin/antibody;(3) For amoxicillin and sodium penicillin, they resulted the decrease of the binding probability of myoglobin/antibody, and had no influence on the average binding force and binding force distribution of myoglobin/antibody. According to the previous work, the comformation of aptamer was unchanged, while the comformation of myoglobin and antibody was changed obviously under the effects of medicine; the different interaction modes and binding sites between myoglobin/aptamer and myoglobin/antibody, and the different structures between aptamer and antibody. Therefore, different results were produced between myoglobin/aptamer and myoglobin/antibody in the presence of medicine.These findings could provide informations to analyze the effect of medicine on the biological molecules, thus enrich our understanding of medicine effects on the interactions of protein and its recognition probe, and will provide the theories basis for the design of the sensor in complex system.