Biomolecules are fundamental building blocks of every organism. They are in charge of complex biological processes and their understanding is essential for fields such as biochemistry, biophysics or medicine.

The project Rapid Nanofluidic Valves for Single-Molecule Imaging (RAVASI) will deliver a unique bioanalytical tool that will enable confinement, manipulation, characterization of individual biomolecules. 


Nanofluidic Scattering Microscopy (NSM) is a recently developed optical method which unprecedented performance enables the imaging of individual biomolecules in free motion without any label [1]. It operates in physiologically relevant conditions, in real time, without the need for immobilization onto a surface. Moreover, this method can provide continuous quantitative weight, size, and conformation measurements of every single molecule imaged. Despite these attractive attributes, in its current form NSM does not allow for quantitative study of interaction kinetics between individual molecules. Due to the inherently fast Brownian motion, the time that a protein spends on average in the optically probed volume – a nanofluidic channel - is substantially shorter than the time required to record a statistically relevant number of association and dissociation events. Moreover, due to limited control of the fluid flow in nanochannels, it is not possible to perform basic fluidic operations, such as injection of analyzed biomolecules, their separation, or mixing. 

Hence, it is the main goal of this project to develop means for efficient sampling of individual biomolecules in order to acquire statistical information on their interaction at single-molecule level. This will be achieved by confining and releasing of interacting biomolecules to and from small nanofluidic volume by rapid nanofluidic valves integrated with the Nanofluidic Scattering Microscopy.


The channel open/close valves are among the most important fluidic components that allow integration of various operations into fluidic circuits. While many types of valves have been developed and established for microfluidics [2], integration of valves into much smaller nanofluidic channels present a considerable challenge. Passive valves for nanofluidic control have been developed but they permit only single-use [3-4]. Active valves based on glass deformation enables reversible operations [5] but it is not compatible with NSM read-out that requires rigid and mechanically stable architectures. Recently, a promising approach exploiting a thermo-responsive polymer ‘grafted to’ a predefined zone of a nanofluidic channel was reported [6]. By heating and cooling of the whole device, the polymer undergoes reversible phase transition associated with the volume change and thus enabling opening and closing the nanochannel. However, the response time is much slower (several minutes) than the response time required to efficiently “catch” the fast diffusing molecules (in the order of miliseconds). In addition, the temperature is changed in the bulk volume of the chip (using a thermoplate interfaced with the device), which is not applicable in biomolecular studies that often requires stable and specifically defined temperatures. In order to allow for rapid and spatially confined temperature control, this project proposes to employ the principles of optically controlled heating via plasmonic nanoparticles [7]. Thermo-responsive polymer architectures actuated by plasmonic heating have been actively studied and already found various applications in colloidal actuation, tunable plasmonics, or biosensing [8]. The ability to rapidly morph miniature structures prepared from thermo-responsive polymers makes them a promising candidate to achieve the ambitious goal of this project.

With respect to the above, the specific objectives are:

  • To develop rapid nanofluidic valves based on the principles of thermo-responsive polymers actuated by nanoplasmonic heating that will allow to confine and release ultra-low volumes into the nanofluidic system with response time at millisecond time range.
  • To integrate the nanofluidic system with single-molecule read-out that will allow for long-term monitoring of individual biomolecules enclosed in a probing volume.
  • To record and quantitatively evaluate the biomolecular interactions at single-molecule level in studies involving protein aggregation and formation of antibody-antigen complexes. It will be the first time we will be able to directly, without any chemical modifications, observe the formation and disassembly of biomolecular complexes at a single-molecule level that mimics an in-vivo state. The interaction analyses will provide for a rich pool of information that is currently lacking.


4 August 2023 - EBSA Congress (Stockholm)

B. Spackova has been invited to give a talk at a congress organised by European Biophysical Society's Association (EBSA). In a session "Breakthrough methods in molecular biophysics" she has participated with her talk "Label-free Microscopy for Investigation of Biomolecular Interactions at Single Molecule Level". In this talk, she has highlighted the recent advancements in NSM and its applications for investigating biomolecular interactions. It was also demonstrated how single-molecule imaging unveils intricate details of protein interactions in their native state, including assessment of binding affinity and the determination of ligand association and dissociation rate constants. link

5 July 2023
PIERS - The Electromagnetism Academy (Prague)

B. Špačková participated at the PIERS conference as an invited speaker. In her talk "Quantitative label-free imaging of protein diffusion and interaction at the single molecule level", she has presented the principles and applications of Nanofluidic Scattering Microscopy, focused on characterization of diffusive motion of biomolecules and their interactions.  link

27 June 2023
Academic mobility (UCT Prague)

What is so great about a postdoctoral stay, and what would probably annoy you? What is it like to move with your family, and what should you focus on when choosing a future research group? B. Špačková was an invited speaker and a panel discussion member at a seminar from the #SharingCzexpats series. She talked about her career path and shared her experience from a postdoctoral stay with PhD students from natural and technical sciences.  link

25 January 2023
Seminar at Heyrovsky Institute of Physical Chemistry of the Czech Academy of Sciences (Prague)

B. Špačková has been invited to give a seminar on her research in the prof. Hof Fluorescence Group.

November 13 2022
Conference Breakthrough technologies of the future (Prague)

The conference organized by the Division of Optics at the Institute of Physics of the Czech Academy of Science aimed to provide a forum for researchers to discuss topics related to sensing, artificial intelligence, metrology, and digitization. The presentation Label-free imaging of single biomolecules by B. Špačková on the development of an experimental platform for Nanofluidic Scattering Microscopy highlighted the advances in the field of single-molecule manipulation and imaging and provided insights into the potential applications of this technique. link

11 November 2022
Seminar at Charles University (Prague)

B. Špačková has been invited to give a seminar on her research in the Institute of Physics of Charles University at the Faculty of Mathematics and Physics.

September 7 – 9, 2022
27th International workshop on Single Molecule spectroscopy and Super-resolution Microscopy (Berlin, Germany)

B. Špačková has participated in the conference "PicoQuant - Single Molecule Workshop" in Berlin, which has a long-standing tradition in the scientific community in the field of ultra-sensitive optical detection at the level of a single molecule. The event provides an interdisciplinary platform for the exchange of ideas and the latest results among researchers and experts in the fields of physics, chemistry, biology, life sciences, and materials science. In her lecture "Potential of Nanofluidic Scattering Microscopy in quantitative imaging of single biomolecules", she has presented the latest results of her research and discussed the applications of the new method - Nanofluidic Scattering Microscopy - in the areas of biochemistry, biophysics, and pharmacology. link

September 11 -15 2022
Network Meeting on Biosensors, Biophotonics and Biophysics (Hirschegg, Austria) 

Attending the "2022 Network Meeting on Biosensors, Biophotonics and Biophysics" in Hirschegg, Austria, afforded B. Špačková the opportunity to participate in an interdisciplinary forum for networking and exchanging ideas with researchers and experts in the fields of biosensors, biophotonics, and biophysics. During her lecture, titled "Label-free single molecule imaging using Nanofluidic Scattering Microscopy," she presented the results of her latest research.

8 April 2022
Seminar at Palacky University (Olomouc)

B. Špačková has been invited to give a seminar on her research in the Joint Laboratory of Optics at Palacky University in Olomouc.


This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 101068106 – RAVASI

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