The Design Of Liquid-phase Scanning Tunneling Microscope And Protein Sub-molecular Features Characterization

Since the first scanning tunneling microscopy was invented in 1982 by G. Binning and H. Rohrer, subsequently multiple family members, such as fow-tempreture ultrahigh-vacuum scanning tunneling microscopy, electrochemical scanning tunneling microscopy etc. were built to break the limition of imaging under different extreme environments. Up to now, the scanning tunneling microscopy has been widely used in materials science, nano-science, etc. low-tempreture ultrahigh-vacuum scanning tunneling microscopy commonly used in investigating the electronic distribution and transportion of material but not suitable for the measurement of biological molecules under solution environment; electrochemical scanning tunneling microscopy can be used to study the chemical reactions in solutin,a while its’three-electrode system is much complex, and the required condition for working is extremely strict, imaging bio-molecules in solution conditions still remains great challenging. So far, the resolution of bio-molecules in reported STM images obtained from solution conditions was rather poor, especially for proteins. Besides, the dynamic biological process between biomolecules was never seen before, above limitions was mostly caused by the unstability of adsorption structure of biomolecules and the poor pro forme nee of scanning tunneling microscopy.In order to obtained the high resolution STM images of single bio-molecule and the dynamic biological process between bio-molecules, we have independently developed a low-leakage-current, high-rigidity and high-stability scanning tunneling microscopy working in solution conditions, the high performance enable it obtain atomic resolution images of graphite sample without sound and vibration isolations. These advantages have improved the efficiency of STM studies to a great degree.Transition metal compound is a typical layered material, having a strange surface electron state distribution, such atomic defects, charge density wave, etc. For the lively surface properties of these materials, it is easily oxidized in the air conditions. Relevant STM researchs on transition metal compound were taken out under low temperature ultra-high vacuum environment. We have directly cleavaged TiSe2, TaS2 and MoTe2 three typical transition metal compound in solution. Using our homebuilt scanning tunneling microscopy, we have obtained high-resolution atomic resolution images. We have shown the unique atomic defect and triangle defects on TiSe2 surface, charge density wave structure in near NC phase of TaS2 at room temperature and supramolecular structure of MoTe2.STM is capable of imaging individual protein molecules in vacuum or air. This requires sample dehydration, which may not reflect the native state. Extensive efforts have been made to image single protein molecules in solution using the STM and atomic force microscopy (AFM), but an imaged molecule typically show only round or oval shape with no sub-molecular details due to the unstable top surface of the protein molecule. Meanwhile, nuclear magnetic resonance (NMR) measurement collects all the signals from the sample rather than from a single molecule; besides, the protein size is also a limitation. Therefore, a method that can investigate protein conformations at the single-molecule level in real space and in physiological conditions, is highly desired. To obtain sub-molecular resolution images of a single protein, we have reduced leakage current by an improved coating technology, which coats the probe tip with an insulating material and can result in a 20 pA low background leakage current. Furthermore, a line-based constant height scan mode is adopted:within a certain line of scan, the scan height is not adjusted (remained constant), but when scanning the next line, the scan height is adjusted by calculating the average tunneling current of the previous line. This imaging mode can prevent the probe tp from colliding with the protein surface while a higher imaging speed can still be guaranteed to reduce the impact of the unstable surface of the protein. Using the L-STM, we have obtained sub-molecular resolution images of streptavidin, antibodies, and monomers and dimers of EGFR kinase domains. Dynamic interactions of the EGFR dimmers under the physiological condition have been documented as well, above work has been published on Nano Research,2016 (IF=8.893). Further more, by adding a 0.4T static magnetic field to the center of solution sample poor which was filled with EGFR molecules, we have studied the response of EGFR molecules to the static magnetic field. Under the effect of external magnetic field, the kinetic energy of EGFR molecules are weakened, most molecules are motionless in our STM images; to the contrary, without static magnetic field, EGFR molecules tend to be motion. Guohui Li group (Dalian Institute of Chemical Physics) have verified and explained this phenomenon through molecular dynamics simulations. More importantly, the activity of EGFR kinase molecules are inhibited by the magnetic field, stronger of the magnetic field intensity, the activity of the EGFR molecule was lower. With the high resolution STM images, we have given the direct explanations of the mechanism of above phenomenon:the magnetic field have changed the orientation of charged EGFR kinase molecule (along the magnetic field direction), which have prevented the formation of EGFR dimmers. However, only by forming dimmers, EGFR molecules will be active. This work has been published on Oncotarget,2016 (IF=5.008).Small molecule assembly/disassembly is a common phenomenon in nature, we can learn a lot of life-sustaining underlying mechanisms through the small molecular assembly. For the reported studies, most STM imaging process was carried out in vacuum conditions, thus the assembly structure need blowed-dry with shielding gas after the completion of the small molecular self-assembly. Direct observation of small molecules assembly/disassembly dynamic process under solution conditions was never been done before. We have studied the high resolution molecular arrangement structure of nonafibers under solution conditions with scanning tunneling microscopy. Then we studied the phosphokinase controled small molecule self-assembly process and EGFR kinase controled disassembly process, this is the first time that small molecule assembly/disassembly dynamic prcess in solution was directly imaged with scanning tunneling microscopy, and proved the correctness of small molecule assembly/disassembly mechanism. In addition. the self-healing dynamic process has been observed for the first time. This work has been published on Nanoscale,2016 (1F=7.76).