High Precision Charge and Size Spectrometry on Biomolecules and Molecular Complexes in Solution

The history of Science is inextricably linked to the history of measurement. As immortalised in the words of the 19th century Dutch Nobel Laureate Kamerlingh-Onnes, discoverer of superconductivity: "Through measurement to knowledge". In scarcely any field of research have these words rung more true than in the biophysical sciences of the last few decades, where the development and refinement of technologies to measure the properties of the molecular building blocks of nature have brought a wave of unprecedented knowledge and insight into the structure and mechanisms underpinning life. The suite of molecular building blocks that make up life is vast, and no single approach offers the answers to the myriad questions scientists may wish to ask. Any new technique that takes a different physical approach can shed new light on a problem, revealing important aspects of it that until then remained entirely out of view. This process of endless improvement and enhancement of measurement techniques plays a defining role in scientific development and underpins the continuous creation of new knowledge.
Size, or mass, and electrical charge are two fundamental physical properties that characterise biological molecules. While molecular mass has long been measured with atom-level precision, my laboratory recently developed a new experimental approach to measure the electrical charge of biological molecules with a precision better than a single elementary charge. Since techniques capable of delivering such measurements have been hitherto unavailable, we anticipate being able to add an important new dimension to the existing body of information on biomolecules. An important aspect of our new approach is that unlike most other measurement techniques we are able to access the properties of individual molecules in solution, and are not limited to observing an average value resulting from the sum of all responses of a multitude of molecules in sample. The reason why this matters is that contrary to common sense, a biological molecule of a certain kind is not necessarily identical to all its neighbours. In fact a given molecular species can exist in slightly different states for a number of different reasons. This multitude of states often carries an important signature of the biological function of the species, and such signatures are revealed in "single molecule" measurements performed in a highly parallel fashion. The ability to perform these sorts of measurements in a rapid and highly accurate fashion is still in its infancy.
The technology we shall develop aims to offer scientists in the biological, biophysical and biomedical sciences a measurement tool that will offer unprecedented insight into properties of biomolecules such as proteins in solution. Beyond the research laboratory, many medical diagnostic tests rely crucially on the ability to sensitively and reliably detect the presence of particular proteins, either free or bound to specific molecular partners in a patient-derived sample. For example in infectious diseases, a test for whether a patient has had exposure to a pathogen or not relies on detecting molecules called "antibodies" circulating in the bloodstream. This is done by checking whether the antibodies in the patient's serum bind in a reaction tube to antigens used as 'molecular bait'. The availability of a measurement tool to deliver high precision measurements of size and electrical charge on molecular scale matter in a sample will not only revolutionise biological research, but will also put detection tools offering unprecedented sensitivity into the hands of medical testing laboratories continuously on the look-out for faster, cheaper and more accurate diagnostic technologies. Viewed through the lens of societal needs during an outbreak of an infectious disease for example, the importance of new technologies that defend the stability of social structures and economies, upholding the social contract, cannot be overstated.

The proposal seeks to develop a new platform for high precision, high throughput, single molecule and single molecular complex measurement in the solution phase. The new method can viewed as the molecular scale equivalent of a FACS instrument (Fluorescence Activated Cell Sorting), with thousands of little optical foci (molecular traps) operating in parallel. Molecules or molecular complexes hop in and out of each trap, one at a time, and the time each entity spends in each trap reflects its size and/or electrical charge. Following each individual molecule as it hops through the lattice yields progressively greater precision in measurements of its properties. Because of the underlying physics, and the fact that we are observing an escape process where the measured quantity depends exponentially on the charge of the molecule, the measurement principle offers very high precision on molecular electrical charge. The method will examine several thousand molecules in a short span of time, on the order of minutes, permitting us to construct a histogram of measured states at a precision and resolution that is likely to be unprecedented for a solution phase technique. The result will be a platform technology for biomolecular measurement that can be used to detect and resolve heterogeneous states of biomolecules, for the detection of shifts in charge and/or size due to intermolecular binding, the measurement of binding affinities, potentially leading to the realisation of single-step, solution phase immunoassays. By individually measuring the properties of molecular entities suspended in a thin sliver of solution, the approach automatically eliminates any background signal from the bulk solution. This aspect therefore implicitly obviates the need for surface immobilisation, washing, and multiple sequential antibody binding steps, which is standard procedure in state of the art molecular binding assays such as ELISA (Enzyme Linked Immunosorbent Assay) and variants thereof.