Research Of Microcantilever Immunosensor Technique Based On Surface Stress Effect

Microcantilever, as the simplest MEMS device, offers different and complementary information of othe sensors. Compared with traditional sensors, microcantilever sensor shows very low detection limit, label-free and real-time detection. High specificity of antigen-antibody binding in an immunoassay guarantees specificity of microcantilever immunosensor. These features make it achieve and exceed the current technology, and open up huge possibilities in drug screening, food safety, environmental monitoring and medical diagnosis. On the basis of our preceding work, this dissertation has studied the mechanism of microcantilever bending and antibody immobilization. The main achievenment are as follows:An effective oriented immobilization of antibody using goat anti-mouse IgG was proposed. Microcantilever immunosensors (MCS) modified with thiolated goat antimouse immunoglobulin G (IgG), were developed to detect the content of ginsenoside Re. The results showed that this method can successfully immobilize antibody with antibody activity preserved. The detection limite is as low as0.5ng/mL The advantage of a secondary antibody thio-functionalized MCS was verified with the anti-paclitaxel mAb. The MCS immobilized with thiolated goat antimouse IgG had a better sensitivity than the MCS modified with thiolated anti-paclitaxel antibody.Explain the mechanism of of the surface stress caused by small-molecule antigen and antibody binding for the first time. Unlike a compressive surface stress caused by biomacromolecule antigen and antibody binding, a small molecule antigen and antibody binding on the surface gives rise to a tensile stress. The tensile stress is induced by antibody conformational change which manifests itself as Fab arm motion that exposes the C1q binding site of the antibody due to antigen binding.Antibodies against one antigen were immobilized on a gold (Au) surface of a microcantilever in three different methods:(A) sulfhydrylated antibody;(B) oriented immobilization of antibody using sulfhydrylated goat anti-mouse IgG; and (C) self-assembled mono-layer (SAM), based on11-mercaptoundecanoic acid. Random immobilization provides a much higher surface concentration of antibodies, whereas the oriented immobilization provides higher antigen binding capacity. The direction of the stress caused by conformational change in the antibody is the same in the oriented immobilization, thus leading to a greater resultant surface stress, which has special significance for the sensitivity of the sensor that is based on stress effect.An experiment by using n-alkanethiols with a same carboxyl group and different chain lengths (n=1,5,10and15) to immobilize a same receptor molecule on the gold surface of a microcantilever was designed, and the nanomechanical response of biochemical reaction was detected. A label-free microcantilever sensor for highly sensitive detection of Glycyrrhizic acid (GL) was developed. The sensitivity of the microcantilever was found to be greatly influenced by the chain length of linker that is between the receptor molecule and the microcantilever surface. The efficiency of stress transmission increases significantly with decreasing length of carbon chain.To further improving the microcantilever immunosensors, antibody fragments were proposed as receptor molecules.(A) F(ab’)2or Fab fragment was used as a receptor molecule. Because of samller volume and quality of F(ab’)2and Fab, they provide a much higher surface concentration of antigen-binding sites. Short distances between antigen-binding sites and microcantilever surface increases the efficiency of stress transmission.(B) Oriented immobilization of half fragment antibodies via their native thiol-groups directly coupled to the gold. To liberate these thiol groups, the antibody was chemically "divided" into two half fragments by chemical reduction with2-mercaptoethylamine (2-MEA). The detection limite of ginsenoside Re using microcantilever functionalized with half fragment antibodies was0.02ng/mL. Half fragment method is simple, and the fragment is oriented immobilization with a high surface concentration and stability. Immobilization via two sulfydryl has a higher stiffness which is good for stress transmission. Short distances. between antigen-binding sites and microcantilever surface increases the efficiency of stress transmission.This dissertation explains the mechanism of of the surface stress caused by small-molecule antigen and antibody binding for the first time. On the basis of this mechanism, antibody immobilization was study. Oriented immobilization provides higher antigen binding capacity. The direction of the stress caused by conformational change in the antibody is the same in the oriented immobilization, thus leading to a greater resultant surface stress. To reduce stress losse during its transmission, the chain length of linker that is between the receptor molecule and the microcantilever surface was reduced and the sensitivity was found to be improved. To further reducing stress losse and increasing surface concentration of antigen-binding sites, antibody fragments were proposed as receptor molecules and a higher sensitivity is obtained. These findings will provide new insights into the fundamental mechanisms of stress transmission, which may be exploited for biochemical sensor and nanoactuation applications.