Research Of Microcantilever Biochemical Sensing Technique Based On Optical Readout

In recent years,as a new technology,microcantilever biochemical sensing technique(MCS) is developing quickly.When specific bio-molecular interactions occur on one surface of microcantilever,a differential stress between the top and bottom surfaces of the cantilever will cause the microcantilever bending.The deflection can be measured by both optical and electrical readout methods.It has brought some new ideas for biochemical sensing technique and becomes a hotspot of research because of its advantages,such as large surface-area-to-volume ratio,very sensitive to surface effect,easy to realize array compact.In this paper,two core of development of MCS- improving detecting sensitivity and high-throughput detection were taken as the starting point.First,a single cantilever detection system based on optical lever technique was improved. Second,the improved experimental system was used to study self-assembly of macromolecules on gold surface and the specificity of antigen-antibody binding in the different antibody-modified methods.Finally,two readout methods based on microcantilever array were proposed.The detecting reproducibility of system can be improved by way of using the laser with the smaller focus radius and steady output intensity.Through experiments and theoretical analysis,by adding convex lens in the path of reflected light and optimizing the dimension parameters of microcantilever,the detecting sensitivity of system can be improved 4 and 20 times,separately.Through the use of high-precision thermostat,the thermal deformation of microcantilever can be eliminated,while the background noise is kept within reasonable scope,and the temperature difference between the sample and the cell will not impact on the system detection.The theoretical analysis showed that by conditioning the experimental parameter,the influence of refractive index change on the detection signal can be effectively eliminated without reducing the detecting sensitivity of the system.The theoretical results were validated by the experiments.Based on this,a method of detecting liquid concentration by detecting the refractive index of liquid was proposed.The self-assembly of different molecular weight of Poly(N-isopropylacrylamide) (HS-PNIPAM) on gold surface was investigated.The results showed that the kinetic curves can be divided into three stages corresponding to different conformations, respectively.The first stage responded to physical adsorption of HS-PNIPAM to gold-coated side.The second and third stages were responsible for chemical adsorption to gold-coated side with conformation transition.The kinetic curves fits Langmuir adsorption isotherm well.The results also show that the reaction rateκof HS-PNIPAM is far less than that of small molecules and decreases exponentially with the molecular weight;while the time of HS-PNIPAM's self-assembly is far more than that of small molecules and proportional to the molecular weight.The change of the surface stress is linear to the molecular weight of HS-PNIPAM.The specificity of antigen-antibody binding in three different antibody-modified methods was investigated.Antibody was immobilized on the gold-coated side of the microcantilever and the deflections of the microcantilever corresponding to the process of injecting different concentration of antigen were real-time monitored, including the clenbuterol was detected in the modified method of thiolated antibody, the clenbuterol and chloramphenicol were detected in the modified method of Staphylococcus aureus protein A(SPA),the artemisinin and aristolochic acid were detected in the modified method of second antibody(goat anti-mouse IgG).The activity of antibody and antigen-antibody binding on the gold-coated side of the microcantilever were confirmed by an Enzyme-Linked Immunosorbent Assay (ELISA).The results showed that antigen-antibody binding on microcantilever generates a compressive surface stress and the surface stress is linear to logarithm of concentration.The detecting sensitivity in the modified method of second antibody is the worst in three methods and the detecting limit is two orders of magnitude lower than IC₂₀.The detecting sensitivity in the modified method of SPA and thiolated antibody is the same.The detecting sensitivity in both modified methods can reach at least 1ng/ml and are equivalent with IC₂₀.By analysis,the detecting sensitivity is directly related to the activity of antibody and the distance from the position where antigen-antibody binding to gold-coated side.Two readout methods based on microcantilever array were proposed.First,the optical path of UC Berkeley was improved.The signal-to-noise ratio of system was increased by controlling polarization of the laser.The optical detection sensitivity can be increased by optimal design of microcantilever.Second,optical filter readout technique was introduced to MCS.