Periodic Reporting for period 1 - NaTOS (Nanoscale Thermo Optical Sensing)
Reporting period: 2018-03-01 to 2020-02-29
Proteins, the engines that drive and regulate almost all processes in living organisms, come in many variations of shape and size. Nonetheless they are all made from a limited set of building blocks. In consequence their identification is difficult. Current identification techniques are all either marker based - meaning a spectroscopically or otherwise reliably identifiable label molecule must be tethered to the protein - or use substrates carrying special receptors that catch only the proteins of interest. Both routes involve time and personnel intensive chemical protocols and require precise knowledge of the analyte’s structure. Moreover, these methods do not recognize non-targeted molecules - a severe obstacle for the study of metabolic processes without a priori knowledge of all involved molecular species.
This action aims to develop a novel biosensor, that does not rely on markers or analyte immobilization. It instead identifies free unmarked proteins by measuring the changes of their diffusive speed when exposed to a tuneable temperature gradient – the protein’s thermophoretic fingerprint.
This sensor will be based on single plasmonic antennas that act as nanoscale detector and nanoscale heaters: Plasmonic antennas absorb and scatter light efficiently if excited with a frequency close to their resonance. This enables the creation of steep temperature gradients over a distances of few nanometers. Such gradients alter the diffusive speed and the local concentration of the proteins – a physical effect called thermophoresis. Both the average speed and the number of proteins in close proximity to the plasmonic antenna can be quantified by observing the changes in its scattering intensity induced by the proteins’ movement. Since thermophoresis is shape and charge dependent even proteins of equal size and mass can be discerned via their thermophoretic fingerprints.
The fundamental technical basis of the action has been established. Namely the detection of small freely diffusing analyte's with diameters on the nanometer scale. It turned out that the device can recognize analytes on a single entity basis and such was more sensitive than initially planned.
This action aims to develop a novel biosensor, that does not rely on markers or analyte immobilization. It instead identifies free unmarked proteins by measuring the changes of their diffusive speed when exposed to a tuneable temperature gradient – the protein’s thermophoretic fingerprint.
This sensor will be based on single plasmonic antennas that act as nanoscale detector and nanoscale heaters: Plasmonic antennas absorb and scatter light efficiently if excited with a frequency close to their resonance. This enables the creation of steep temperature gradients over a distances of few nanometers. Such gradients alter the diffusive speed and the local concentration of the proteins – a physical effect called thermophoresis. Both the average speed and the number of proteins in close proximity to the plasmonic antenna can be quantified by observing the changes in its scattering intensity induced by the proteins’ movement. Since thermophoresis is shape and charge dependent even proteins of equal size and mass can be discerned via their thermophoretic fingerprints.
The fundamental technical basis of the action has been established. Namely the detection of small freely diffusing analyte's with diameters on the nanometer scale. It turned out that the device can recognize analytes on a single entity basis and such was more sensitive than initially planned.
I have successfully constructed a custom-made polarization-selective confocal microscope system. This also includes the development of a suited software package allowing for command & control, real time feedback to the experimentalist as well as dedicated signal analysis tool sets. With this system I have successfully obtained ensemble autocorrelation functions for 5 nm diameter gold nanoparticles as well as microemulsion nanodroplets, which are possess similar physical properties to proteins with masses of approximately 250 kDa. This is in line with the initial steps projected in the action’s proposal.
These autocorrelations exhibit a time resolution of few nanoseconds, which constitutes a more than 1000-fold improvement over the state of the art as well as an improvement of approx. 10- fold in particle size [Wulf et .al, The Journal of Physical Chemistry Letters 2016, 7, 4951-4955]. Autocorrelations curves represent an ensemble average over many single particle events. Observation of such single events is not possible with other state-of-the-art optical sensor systems.
The system constructed as result of this action, however, is capable of resolving events on a single-event basis – an example trace exhibiting such events is shown below. The events are indicated with red arrows.
The capacity to resolve single events constitutes a significant scientific breakthrough. It enables to extract information about the analyte and its behaviour which otherwise would be lost by ensemble averaging. Publication of these results in a high impact factor journal can be expected – the manuscript is in preparation.
While the technical challenges of resolving single events delayed the implementation of the initial idea of the proposal regarding thermophoretic fingerprinting – it will in the long run only enhance the initial idea and provide more solid understanding of the all observed analytes/processes.
These autocorrelations exhibit a time resolution of few nanoseconds, which constitutes a more than 1000-fold improvement over the state of the art as well as an improvement of approx. 10- fold in particle size [Wulf et .al, The Journal of Physical Chemistry Letters 2016, 7, 4951-4955]. Autocorrelations curves represent an ensemble average over many single particle events. Observation of such single events is not possible with other state-of-the-art optical sensor systems.
The system constructed as result of this action, however, is capable of resolving events on a single-event basis – an example trace exhibiting such events is shown below. The events are indicated with red arrows.
The capacity to resolve single events constitutes a significant scientific breakthrough. It enables to extract information about the analyte and its behaviour which otherwise would be lost by ensemble averaging. Publication of these results in a high impact factor journal can be expected – the manuscript is in preparation.
While the technical challenges of resolving single events delayed the implementation of the initial idea of the proposal regarding thermophoretic fingerprinting – it will in the long run only enhance the initial idea and provide more solid understanding of the all observed analytes/processes.
The optical sensor system developed during the course of this action is capable of monitoring analyte diffusion in optical near fields and without fluorescent labels with a timersolution of few nanoseconds - a 1000-fold improvement over the previous state of the art. The size of detectable analytes was also improved 10-fold. Most importantly the new method is capable of resolving single analytes - previous methods relied on ensemble measurments.