Periodic Reporting for period 1 - H2O-SurfaceProbe (High Throughput Second Harmonic Method to Probe Biological Surfaces at Real-World Conditions)
Reporting period: 2020-03-01 to 2022-02-28
In the H2O-SurfaceProbe project, we have addressed a major problem in exploring biological surfaces. Such surfaces are omnipresent and essential for life. To date, two leading schools of thought exist for probing biologically relevant surfaces, i.e. lipid membranes, i) a bottom-up approach, achieving molecular level information with the cost of employing simple nonrealistic model membrane systems, such as lipid monolayers, employing surface sensitive (spectroscopic) techniques. ii) a top-down approach by using real-world membrane samples using in-vivo systems with the help of probing incorporated label molecules. Yet, both methods have significant disadvantages in probing the surface chemistry to understand biointerfaces. In an ideal scenario, one needs to access multiple length scales simultaneously, using non-invasive, label-free, and possibly membrane and interface-specific techniques. The H2O-SurfaceProbe project addressed this technological gap and utilized the ubiquitous interfacial water molecules as a probe by optimizing the ultrashort pulsed laser to probe the orientation of water molecules that are influenced by the surface chemistry. This technological advancement makes probing real-world membranes with label-free, non-invasive surface water molecules possible. Such an ambitious research goal gives rise to 4 collaborative journal article publications. In these studies, we demonstrated an ultralow detection of free-floating membrane interaction with membrane proteins with a picomolar^-1 (10^-12 M) dissociation constant. Such high sensitivity has not been reported with a non-labeled, non-invasive method. In another study, we have also shown the lipid gel to liquid transition again with non-resonant second harmonic photons. As can be seen from these examples, the high-throughput second harmonic method can open new avenues of research on biologically relevant surface science.
The findings of H2O-SurfaceProbe are important for society since the new technology developed to probe biointerfaces by using the surface water molecules is applicable to numerous biological scientific problems. As a timely example, it can potentially be employed to investigate the binding of virus antigens to specific cell antibodies. As in the case of a pandemic, second-harmonic photons can be utilized to run binding essays to various cell lines. Having such technology in hand can also serve as a multitasking tool to accurately probe chemical/biological processes on biological surfaces. In the short term, it can be utilized for diagnostic medical research and screening various therapeutics applications.
The overall objectives of the project are to develop a new alternative method to explore chemical processes occurring at biologically relevant aqueous interfaces, that is, free-floating lipid membranes, via label-free, non-invasive surface water molecules. Having such a unique method, another objective is to explore lipid membrane processes. Namely, the gel-to-liquid phase transition of lipid membranes was successfully probed by surface water molecules. The final aim is to demonstrate the power of the technique on a previously unachievable lipid membrane problem. Namely, focusing on the membrane protein-lipid membrane interactions, in a collaborative effort, we have demonstrated ultralow (10^-12 M) dissociation constant with Perfringolysin O (PFO) toxin free-floating membrane binding. That has been a challenging task to get down to such high sensitivity with a label-free, non-invasive methodology.
The findings of H2O-SurfaceProbe are important for society since the new technology developed to probe biointerfaces by using the surface water molecules is applicable to numerous biological scientific problems. As a timely example, it can potentially be employed to investigate the binding of virus antigens to specific cell antibodies. As in the case of a pandemic, second-harmonic photons can be utilized to run binding essays to various cell lines. Having such technology in hand can also serve as a multitasking tool to accurately probe chemical/biological processes on biological surfaces. In the short term, it can be utilized for diagnostic medical research and screening various therapeutics applications.
The overall objectives of the project are to develop a new alternative method to explore chemical processes occurring at biologically relevant aqueous interfaces, that is, free-floating lipid membranes, via label-free, non-invasive surface water molecules. Having such a unique method, another objective is to explore lipid membrane processes. Namely, the gel-to-liquid phase transition of lipid membranes was successfully probed by surface water molecules. The final aim is to demonstrate the power of the technique on a previously unachievable lipid membrane problem. Namely, focusing on the membrane protein-lipid membrane interactions, in a collaborative effort, we have demonstrated ultralow (10^-12 M) dissociation constant with Perfringolysin O (PFO) toxin free-floating membrane binding. That has been a challenging task to get down to such high sensitivity with a label-free, non-invasive methodology.
In this project, there four main results have been achieved. The first one is building a second harmonic scattering setup and finding its optimized parameters to probe surface water response, eliminating other high-order processes. Together with building and optimizing the optical design, scattering sample preparation bathed in aqueous solutions also requires optimization. The latter task has accomplished by preparing nanodroplet samples in various aqueous conditions (pH and ionic strength). The second set of results are achieved by probing the surface lipid membranes with the developed methodology. It was shown that probing lipid membrane gel-to-liquid phase transition via surface water is possible. Another set of results is to explore the potential of the new methodology on more challenging interfacial problems, i.e. probing free-floating membrane-membrane protein interaction via interfacial water response. We have focused on pore-forming toxins to liposome binding and demonstrated an ultrasensitive detection of ultra-tight binding with 10^-12 M dissociation constants only from interfacial water response in a label-free and non-invasive manner.
Beyond the scientific research and related deliverables, the results of this project were disseminated via two public popular science talks given at Bilkent University. Moreover, two scientific comminations to first-year chemistry students were also performed to provide an early perspective to the students what we can learn from surface water. Overall, this project gives rise to various scientific deliverables and public dissemination.
Beyond the scientific research and related deliverables, the results of this project were disseminated via two public popular science talks given at Bilkent University. Moreover, two scientific comminations to first-year chemistry students were also performed to provide an early perspective to the students what we can learn from surface water. Overall, this project gives rise to various scientific deliverables and public dissemination.
The H2O-Surface-Probe Project takes the state-of-the-art second harmonic interfacial water probing beyond the fundamental science level toward biological and medical applications. Such as a new label-free diagnostic tool for biointerfaces that can contribute to the diagnostics of diseases and an experimental drug screening tool that involve biochemical processes occurring at biointerfaces. This new methodology could potentially produce a unique biological and medical research tool that can lead start-up companies to produce and sell such high-technology products all over the globe. The start-up potentials are being investigated together with the Technology Transfer Office (TTO) of Bilkent University.