The Research Of Microcantilever Sensing Technology In Detection Of Aflatoxin And Characterization Of Cell Viability

The microcantilever sensors with increasingly smaller size and higher sensitivity are achieved due to the advances in micro and nanofabrication technology. The main principle of microcantilever sensor is to detect the deflection of cantilever due to the stress on cantilever surface or the resonant frequency shift due to mass adsorption or desorption when the target molecules bind to the recptors. The microcantilever sensor is highly sensitive, label free and real time in detection, and become increasingly advantageous in many fields including fundamental research, health sciences, drug development and clinical diagnosis. Based on the previous word of our group, the author developed a method to detect the highly toxic substances aflatoxin B1 using microcantilever, and study the means to quantify the cell activity based on microcantilever sensor. The main results are shown below.(1) The performance of microcantilever array sensing system was analyzed. The precondition of microcantilever array sensing is that each cantilever should be the same with other identical cantilevers under the excitation.The response of microcantilever array to temperature change were measured, and it’s found that the responses of different microcantilevers within an array were consistent with each other. The nonspecific response induced by the solution replacement and the specific response induced by binding between antigen and antibody were also detected, and the responses of different microcantilevers within an array were still consistent with each other.(2) The microcantilever sensing technology was developed to detect aflatoxin B1 (AFB1). AFB1 is a hypertoxic hepatocarcinogen, and has been reproted in a variety of food and food materials, which threatens human health. The anti-AFB1 antibodies was sulfhydrylated by sulfhydrylation reagent and then immobilized on the microcantilever gold surface, and ELISA showed that the antibodies on microcantilever surface remained active. The results showed that microcantilever array can quantificationally detect AFB1, has a detection limit of 0.03 ng/mL for AFB1, which is an improvement than icELISA and the microcantilever sensor in dynamic mode. The AFB1 dissolved in peanut solution was also successfully detected by microcantilever sensor.(3) A method to detect viability of breast cancer cell MCF-7 using microcantilever sensing technology was proposed. The MCF-7 cell was adsorbed on the microcantilever surface through (3-aminopropyl)triethoxysilane (APTES), and then the deflection curves of microcantilever was monitored. The results showed that the deflection fluctuation amplitude of living MCF-7 cell adsorbed cantilever was the largest, the deflection fluctuation amplitude of dead cell adsorbed cantilever and bare cantilever were very small, and the deflection fluctuation amplitude of starved cell adsorbed cantilever was between them. This indicated that the cantilever fluctuation was related to cell viability. The other assay about human umbilical vein endothelial cells (HUVECs) validated this conclusion. Calculating the variance of microcantilever deflection, and the cell viability was quantified by the deflection variance. On the basis, for the purpose of screening anti-cancer drug, we used microcantilever sensor to quantify the effect of paclitaxel on MCF-7 cell. When different concentration of paclitaxel was added in cell incubation process, the deflection variance of MCF-7 cell adsorbed cantilever was decreased as the paclitaxel concentration increased. This phenomenon was also observed when paclitaxel was added in the process of monitoring cantilever deflection. These indicated that the microcantilever fluctuation can be used for quantification of cell viability and rapid screening of anti-cancer drug. This method which can quantify cancer cell viability with low cost is significant for basic science and clinical treatment.(4) The mechanism of cantilever fluctuation caused by the cells was explained. CCK-8 assay showed that biological activity of MCF-7 cell was almost unchanged after the cells were treated by paclitaxel, thus our interpretation of the origin of these fluctuations is that the nanomechanical sensor (microcantilever) can transduce small movements which are driven by the force generated from the cytoskeleton, using cell metabolism as its energy source. Thus, we propose that cell viability consists of two parts:mechanical viability and biological viability. This new mode based on nanomechanical fluctuation cannot only determine the biological viability of cells, but can also quantify mechanical viability in the pN range, which cannot be determined by existing methods such as the CCK-8. The nanomechanical fluctuation provide a new concept and platform to evaluate cell viability and drug screening.(5) The sperm viability was quantified by microcantilever sensor and the effect of drugs and environment on sperm viability was evaluated. It’s significant for reproductive medicine to quantify sperm viability. Hyaluronic acid was selected as linkers to immobilize sperms on cantilever surface. Monitoring the deflection of sperms adsorbed microcantilever, we found that the fluctuation of living adsorbed cantilever was much greater than that of bare cantilever or dead sperms adsorbed cantilever, which indicated that the cantilever fluctuation was related to the sperm viability. To investigate the drug effect on sperm, two kind of drugs including ethanol and sperm viagra was added in the process of monitoring the deflection of sperm adsorbed cantilever. The microcantilever fluctuation results showed that ethanol inhibit sperm viability and sperm viagra enhanced sperm viability, indicating that microcantilever fluctuation can be used for screening drugs which changed sperm viability. Finally, the effect of temperature on sperm viability was evaluated, and it’s found that the microcantilever fluctuation was largest in temperature around 34 ℃, indicating that the sperm viability was best in this temperature. The microcantilever sensor provide a new tool for Reproductive Medicine.