Day 2 :
Keynote Forum
A I Archakov
Institute of Biomedical Chemistry, Russia
Keynote: AFM physicochemical properties and activity of single protein molecules of CYP 102A1 (BM3)
Time : 10:00
Biography:
A I Archakov is a Full Member of the Russian Academy of Sciences and Professor & Scientific Advisor at Institute of Biomedical Chemistry. He has organized scientific school to study molecular organization and functioning of oxygenase cytochrome P450-containing systems, molecular mechanisms of the structure and function of membranes and biological oxidation. He has guided the institute’s members in developing a fundamentally new pharmaceutical composition “Phosphogliv” with antiviral activity for the treatment of liver diseases of various etiology. He is the pioneer in the development of proteomics in Russia. Currently, he is the International “Human Proteome” Project Coordinator in Russia. He is one of the Russia’s top 100 scientists with Hirsch number 27. He is the author of more than 700 scientific works including about 482 scientific articles, 6 monographs, 30 patents and author’s certificates. He was Scientific Advisor for 15 Doctors’ and more than 60 PhD theses. He is the winner of three state prizes of the USSR, the RSFSR and of the Russian Federation.
Abstract:
Atomic force microscopy (AFM) is a nanotechnological multifunctional molecular platform for measuring of physicochemical and functional properties of single proteins molecules. AFM was used for visualization of oligomeric state, activity, elasticity and electron transfer of single molecules of CYP 102A1 (BM3). It was shown that BM3 in water solution exists as monomer and different oligomers by use of sharp and super sharp AFM probes. Functional activity of single monomers and oligomers of BM3 was measured by AFM as well. The BM3 height fluctuations amplitude (HFA) during catalytic cycle is much larger than the HFA of the enzyme molecules in the resting state. It was found that an average HFA of dimers P450 BM3 during catalytic cycle increased up to 5.0±2Å·s-1 that was 2.5 times larger than a HFA of P450 BM3 in the resting state. It was obtained that the HFA of immobilized on mica cytochrome P450 BM3 depends on temperature, and 22ËšC is a peak of this temperature profile. Mass spectrometry (MS) measurements were used to obtain a time course of a hydroxylation product of lauric acid oxidation during the enzymatic reaction of P450 BM3 in two cases: when enzyme was solubilized in the volume and when it was immobilized on the mica chip. In both cases the number of enzyme molecules was ≈1010, and the kinetics was linear during the first 10 minutes. It was shown that in the case of solubilized enzyme kcat=10-3 s-1, and in the case of immobilized enzyme kcat=0.4·10-3 s-1 that was 2.5 times less than the first one. The purpose of our work was to find a relationship between enzyme HFA and its catalytic activity. Therefore, AFM data was analyzed together with MS data and the following equation was obtained: kcat= β· (exp(ΔA/ A0)-1, where kcat – is a catalytic rate constant (s-1); β is a proportionality factor (s-1); ΔA = Δ Ä€ – A0 (Å), where Ä€ and A0 are the average amplitudes of P450 BM3 height oscillations in the active and resting states, respectively. The value β = 5·10-6 s-1 was calculated from time dependence of reaction curves measured by AFM and MS. Elasticity of single protein was measured based on deformation of this protein under AFM probes with various radii of curvature. Young’s modulus of BM3 molecules depends on AFM modes. Based on the obtained data, the following conclusions may be made: the enzyme catalytic activity of single molecules can be measured as a HFA of BM3 oscillation during catalytic cycle.
Keynote Forum
Liqiu “Rick†Wang
The University of Hong Kong, Hong Kong
Keynote: Small is big: magic microfluidic droplets
Time : 10:35
Biography:
Liqiu “Rick” Wang received his PhD from University of Alberta (Canada) and is currently a Full Professor in the Department of Mechanical Engineering, the University of Hong Kong. He is also the Qianren Scholar (Zhejiang) and serves as the Director and the Chief Scientist for the Laboratory for Nanofluids and Thermal Engineering, Zhejiang Institute of Research and Innovation (HKU-ZIRI), the University of Hong Kong. He has over 30 years of university experience in thermal & power engineering, energy & environment, transport phenomena, materials, nanotechnology, biotechnology, and applied mathematics in Canada, China/Hong Kong, Singapore and the USA, and 2 years of industrial experience in thermal engineering and technology management. He has secured over 70 projects funded by diverse funding agencies and industries including the Research Grants Council of Hong Kong, the National Science Foundation of China and the Ministry of Science and Technology of China, totaling > US$ 15m (excluding US$ 2.2 billion for AMS project). He has published 10 books/monographs and 356 book chapters and technical articles, many of which have been widely used by researchers all over the world, and is ranked amongst the top 1% of most-cited scientists (ESI). He has also filed 22 patent applications and led an international team in developing a state-of-the-art thermal control system for the Alpha Magnetic Spectrometer (AMS) on the International Space Station.
Abstract:
Droplets of nanoliter and subnanoliter are useful in a wide range of applications, particularly when their size is uniform and controllable. Examples include biochemistry, biomedical engineering, food industry, pharmaceuticals, and material sciences. One example of their many fundamental medical applications is the therapeutic delivery system for delivering site-specific therapy to targeted organs in the body and as the carriers for newer therapeutic options. The size, the size distribution, the generation rate and the effective manipulation of droplets at a scale of nano, pico, femto and even atto liters are critical in all these applications. We make an overview of microfluidic droplet generation of either passive or active means and report a glass capillary microfluidic system for synthesizing precisely controlled monodisperse multiple emulsions and their applications in engineering materials, nanofluids, microfibers, embolic particles and colloidosome systems. Our review of passive approaches focuses on the characteristics and mechanisms of breakup modes of droplet generation occurring in microfluidic cross-flow, co-flow, flow-focusing, and step emulsification configurations. The review of active approaches covers the state-of-the-art techniques employing either external forces from electrical, magnetic and centrifugal fields or methods of modifying intrinsic properties of flows or fluids such as velocity, viscosity, interfacial tension, channel wettability, and fluid density, with a focus on their implementations and actuation mechanisms. Also included is the contrast among different approaches of either passive or active nature.