NanoLiquid

All biochemical processes, chemical reactions and physical transitions obey the laws of thermodynamics. Therefore, calorimetry provides a universal means for monitoring the rate of any chemical, physical or biological processes. A range of applications of calorimetry exceeds that for virtually any other modern analytical technique. The general tendency in modern calorimetry and thermal analysis is the reduction of the size of sample and the increase in sensitivity and resolution of measuring time and temperature. Calorimetry has been applied to a highly diverse range of targets used at a variety of scales from whole-body calorimetry down to sub-nanoliter sample volumes. Despite the general tendency towards miniaturisation, simply scaling down traditional calorimetric techniques, although beneficial for material sciences, may not work well for biological objects, which cannot always be scaled down to suit instrument size or taken out of their natural environment. One of the key challenges is therefore to be able to probe the thermodynamics of biological objects, e.g. individual cells, cell compartments or individual organelles and, ultimately individual molecules, such as enzymes, without removing them from their macroscopic environment and preferably remotely, without using artificially introduced markers or inserting any sensors into such cells or organelles.

In this project we aim to develop a high frequency hybrid AC/DC modulated liquid nanocalorimeter instrument capable of real-time and label-free analysis and suitable for the analysis of ultra-small quantities of biological material (of the order of few nanograms and below, which is orders of magnitude improvement over currently available commercial instruments) or the material localised within 10-100 nm of the surface of sensor chip even in bulk samples. Such capability should enable studying cellular metabolic processes and thermodynamics of molecular interactions and biochemical processes at cellular and sub-cellular levels, in the locality of the sensor even in the bulk samples. Such capability has not been achieved or reported previously.

Our approach is fundamentally different from the general trend followed by many researchers and instrument manufacturers where the aim is to MINIATURISE THE SAMPLE and then to conduct bulk calorimetry measurements (of the whole of the miniaturised sample). Such approach suits well material sciences but is not suitable for liquid biological samples. Contrary to that general trend we aim to build an instrument for measuring local thermal properties in the defined vicinity of the miniaturised sensor, irrespective of the overall sample/object size. Our approach is therefore ultimately suitable for biological objects, which cannot be taken out of their native biological environment or be miniaturised to suit instrument limitations. The instrument will be suitable for a wide range of applications in fundamental and applied biosciences, biomedical research. Our measurement principle should allow the development of universal miniaturised implantable calorimetric sensors.

Fast, reliable and ultra-sensitive thermal characterisation platform suitable for both in vitro and in vivo experiments is of utmost importance for large number of applications in biology, medicine, pharmaceutical industries as well as security applications. Despite the widespread and growing use of isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) in life sciences these techniques are usually limited to in vitro measurements, employ large quantities of biological material taken out of its natural environment, and simplified model experimental reaction conditions. Only a handful of dedicated in vivo studies have been published, mostly employing macroscopic preparations of bacterial cultures, whilst commercial liquid calorimetry instruments are limited to sub-millilitre volumes.

Contrary to the common trend, we do not aim to miniaturise the sample, instead we aim to utilise localized thermal wave propagation phenomena to limit the measurement area to the defined locality of the sensor, irrespective of the overall sample size. Exciting a sample by a periodic heat source, for example using a sinusoidal power oscillation, causes temperature oscillations inside the sample, which have the same mathematical expression as highly damped waves. The finite speed of thermal signals means that the depth of penetration of such thermal waves into the medium will depend on the thermal conductivity of the medium and the frequency of such signals, hence can be controlled and monitored. Having achieved ~ 1 ng sensitivity in the dry mode (some 6 orders of magnitude improvement compared to commercial instruments), we are confident that the same measurement approach should result in an equivalent improvement when used in liquid mode. The expected reduction in sensitivity because of the dumping of AC signal will be compensated by further reduction in the thermal mass of the sensor, galvanic insulation and the adjustment of readout principles.

It is highly likely that combining new measurement approach with the improved sensor design will yield totally new nanocalorimetry instrument capable of analysing biological samples including in their natural bulk liquid environment. The new tool should boost a wide range of fundamental and applied multidisciplinary and interdisciplinary research in life sciences, but also in material sciences and physics. The NanoLiquid project is poised to further strengthen UK pharmaceutical industry, which is one of the UK's top exporters. The new measurement platform will promote our own research into protein folding, protein-protein interaction and protein engineering and will further our academic goals. Given the potential of label-free real-time localised thermodynamics analysis for huge number of biochemical, biomedical and biophysical applications, the development and implementation of high frequency hybrid AC/DC liquid nanocalorimeter should be considered a priority task.

The sensor readout principle and sensor design will be the principle exploitable intellectual property (IP) generated from this study. Royal Holloway London has a policy of actively identifying, protecting and commercialising generated IP, which it believes to be of value. The Research and Enterprise department is specifically set to undertake this activity on the College's behalf, including filing patent applications in the College's name where appropriate and deciding the best commercialisation route. To the end, it works with a variety of organisations to ensure that this IP is commercially exploited in such a way as to realise its maximum potential. For the purposes of this project, it is expected that Royal Holloway Research and Enterprise will lead on the exploitation of the IP. Recent examples of joint patent applications negotiated by the College and involving IP generated by Dr Soloviev (PI) with collaborators form MRC-LMB Cambridge are factual proofs of the robustness of the operating procedures adhered to by Royal Holloway Research and Enterprise.

The dissemination of our results will be done, in first instance, through publication in refereed journals of international prestige, after clearing with the Research and Enterprise (IP and priority issues). Because of the short duration, technology oriented nature of this project and the likelihood of generating valuable IP, we envisage exploiting direct route where our proof of principle data will be communicated directly to representatives from biopharmaceutical industry and capital investors. In the course of previous work Dr Soloviev established a number of contacts with pharma companies. This was further helped by the existence of focused funding and the expertise of the Research and Enterprise staff, who were able to quickly contact hundreds of relevant company representatives and departments offering licensing opportunities.