Periodic Reporting for period 1 - APTALAPS (Investigation of the interaction of mesenchymal stem cells with aptamer modified surfaces using Light-Addressable Potentiometric Sensors (LAPS)) Reporting period: 2015-07-20 to 2017-07-19 Summary of the context and overall objectives of the project The aim of this project was to develop a new method to immobilize aptamers of stem cells onto silicon-on-sapphire (SOS) substrates, to capture cells by affinity interaction between aptamer and target cells, and investigate two-dimensional electrochemical images of parameters such as local cell impedance, membrane surface-potential, ion channel activity, and two-photon fluorescence images with subcellular resolution using the impedance imaging technique Scanning Photo-induced Impedance Microscopy (SPIM) and the electrical potential imaging technique Light-Addressable Potentiometric Sensors (LAPS). Aptamers are artificial functional oligonucleic acids which can bind their respective targets with high affinity and specificity ranging from small inorganic or organic molecules to whole cells. The non-covalent affinity interaction between aptamer and cells support physiological integrin modulated cell attachment processes. Aptamers with specificity to target integrin heterodimers were going to be developed and patterned on LAPS substrates and the mechanism of attachment regulated stem cell differentiation studied. The proposed technique has the advantage that functional electrochemical imaging of the cell-surface interface can be carried out, which is not accessible to other electrochemical techniques. As described in the Proposal of the APTALAPS project, there are four objectives to be completed: Work Package 1: Aptamer development for the binding of mesenchymal stem cells; Work Package 2: Aptamer immobilisation and patterning on LAPS substrates; Work Package 3: Investigation of cell binding and signalling using LAPS; Work Package 4: Studying of stem cell differentiation on surfaces modified with aptamers by LAPS and SPIM imaging. The proposed research links into the Health area of Horizon 2020, and more specifically into innovative treatments and technologies such as cell therapies and tissue engineering. The single osteoblast cell LAPS imaging achieved in this project will have major benefits in the area of tissue engineering materials such as bone grafts and provide a new measurement tool for imaging of the cell attachment area. The results of the project will inform the development of tissue engineering materials and impact on the health care and biomaterials industries, which are central to Europe's ongoing and future economic success, and are directly relevant to the theme priority of personalizing health and care in Horizon 2020 - producing knowledge that will be applied in the area of health and medicine. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far (1) Worked performed and main results achieved Existing aptamers for mesenchymal stem cells and osteoblast cells have been reviewed and selected. The development of new aptamer of mesenchymal stem cells by SELEX has not been achieved. Oligonucleic acids were immobilized onto the 1,8-nonadiyne modified SOS surface by click reaction using microcontact printing (µCP) and noncovalent binding method using low gelling temperature agarose gel. The negative charge of immobilized DNA on surface was characterised by LAPS, which showed a potential shift. LAPS was developed for the image detection of yeast cells with high lateral resolution. Low gelling temperature agarose gel was shown to be an effective immobilization reagent for yeast cells in LAPS measurements without causing a background signal. The photocurrent images obtained were shown to be induced by the surface negative charge and the local impedance of the yeast cells, which was shown in the attached Figure. Indium tin oxide (ITO) coated glass was developed as a novel, low cost substrate material for LAPS and SPIM imaging compared to traditional expensive ultra-thin Si substrates. ITO showed good LAPS photocurrent and pH response without surface modification and insulator. Local photocurrents were produced by scanning a focused laser beam across the sample, which proved the light addressability of ITO coated glass. With a high-impedance PMMA dot deposited onto the ITO as a model system, a lateral resolution of about 2.3 µm was achieved. (2) Dissemination of the research results D Zhang participated in the 9th Meeting Engineering of Functional Interfaces from 3th to 5th July 2016 and gave the Oral/Poster presentation with the title: The image detection of yeast Saccharomyces cerevisiae by Light-Addressable Potentiometric Sensors (LAPS). D Zhang published the following manuscripts associated with the APTALAPS project and three more manuscripts associated with the project are in preparation:[1] D.-W. Zhang, F. Wu, J. Wang, M. Watkinson, S. Krause, Image detection of yeast Saccharomyces cerevisiae by light-addressable potentiometric sensors (LAPS), Electrochem. Commun., 72 (2016) 41-45.[2] F. Wu, D.-W. Zhang, J. Wang, M. Watkinson, S. Krause, Copper contamination of self-assembled organic monolayer modified silicon surfaces following a “click” reaction characterized with LAPS and SPIM, Langmuir, 33 (2017) 3170-3177.[3] F. Wu, I. Campos, D.-W. Zhang, S. Krause, Biological imaging using light-addressable potentiometric sensors and scanning photo-induced impedance microscopy, Proceedings of the Royal Society A: Mathematical, Physical and Engineering Science, 473 (2017).[4] D.-W. Zhang, F. Wu, S. Krause, LAPS and SPIM imaging using ITO-coated glass as the substrate material, Anal. Chem., 89 (2017) 8129-8133. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) D Zhang was an Assistant Professor before the start of the MSCA project. He was building an independent research group. As a result of the successful research carried out during his MSCA project, he has been promoted to be Associate Professor after his return to China. An important goal of this project is to create long-term collaboration between D Zhang and the host S Krause, which will be mutually beneficial based on the respective expertise of S Krause’s research group and D Zhang. Establishing a professional network of contacts through the collaborators of the scientists involved in this project is highly beneficial to the career development of D Zhang. D Zhang and S Krause have jointly applied for follow-up funding to the Biotechnology and Biological Sciences Research Council (BBSRC) in the UK. The application was successful, allowing D Zhang to carry on his work on high-resolution LAPS imaging for another 12 months. During the MSCA project, D Zhang supervised an A-level student in the UK for four weeks. The student was selected and supported to have a research experience in laboratories across the United Kingdom. D Zhang designed a research project about LAPS imaging for her. The project produced an opportunity to give a 17-year-old the chance to discover what a career in science and inspire the next generation of researchers, which greatly improved the communication and public engagement of the action. (A) Optical image, (B) LAPS image of yeast cells, (C) I-V curves, (D) normalised I-V curves
