Polymer brushes – thin polymer films on the surface of a biochip. Only tens of nanometres thick, invisible to the naked eye. And yet they determine whether the biochip will function reliably. Even on such a seemingly perfect surface, tiny defects can be hidden. Is it possible to find out where they are and how the quality of the polymer brush is affected? A team led by Hana Lísalová from the Division of Optics of the Institute of Physics, in collaboration with scientists from the Faculty of Mathematics and Physics of Charles University (MFF UK), used surface-enhanced Raman spectroscopy (SERS) and molecular probes to map these hidden defects and show how they can be prevented in the future. The results were published in the journal Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy.
Film preparation and mapping
A team of scientists from the Laboratory of Functional Biointerfaces first prepared thin polymer films with different compositions on surfaces developed by a team from the Faculty of Mathematics and Physics, Charles University. These surfaces consist of glass covered with a thin film of gold with silver nano-islands. These surfaces amplify the Raman signal, i.e., the weak light scattered by molecules after laser impact. They make it possible to detect the response even from a few molecules that come into their vicinity. The signal from molecules that remain further away from the metal surface due to the presence of the polymer brush remains unamplified and therefore too weak to be detected. This is what enabled scientists to visualize microscopic defects in polymer brushes.
They used methylene blue as the probing molecule. It does not penetrate an intact polymer brush but can get closer to the substrate surface in places where there are cracks in the brush. A very low concentration solution of the blue was applied to the prepared films and, after the samples dried, the surface was mapped using Raman spectroscopy: a laser point by point was shone onto the surface and the spectrum of scattered light was measured. From the data obtained in this way, the scientists compiled detailed maps on which they were able to distinguish intact areas from areas with microdefects where the methylene blue had come close to the metal layer. Based on the intensity and shape of the signal, they also distinguished between shallow defects and deep defects extending to the substrate and compared their occurrence between different film compositions.
The experimental measurements were supplemented by computer simulations, which confirmed that methylene blue molecules can penetrate the structure of the polymer films at the defect site and settle inside, which corresponds to the observed spectral changes.
Composition is key
"Even though it is reasonable to assume that defects on antifouling surfaces are relatively rare – there are usually only a few such points on an area of units to tens of square millimetres – they increase the likelihood of unwanted molecules sticking, thereby reducing the reliability of biosensors. When mapping them, we also found that some films are more prone to microdefects than others. With this research, we have contributed another piece to the development of even more reliable biosensors. I consider the close interdisciplinary collaboration with the team from the Faculty of Mathematics and Physics of Charles University to be absolutely essential to the success of this study, as it allowed us to jointly apply their long-standing experience with the SERS method combined with computer simulations. I would therefore like to express my sincere thanks to Josef Štěpánek, Marek Procházka, Ivan Barvík, and Ondřej Kylián. I also view very positively the fact that this joint work drew on the previous experience of the first author of this work, Monika Spasovová, with Raman spectroscopy, which she had previously used in her diploma thesis at the Faculty of Mathematics and Physics of Charles University," says Hana Lísalová, head of the Laboratory of Functional Biointerfaces.
The results also showed that the composition of polymer brushes significantly affects their susceptibility to microdefects. Films rich in CBMAA (a derivative of methacrylamide with a carboxybetaine group, which contributes to resistance to protein adhesion) have fewer deep defects, making them more resistant and less permeable to the substrate than the other films studied. In contrast, brushes with a 2:1 ratio of HPMAA (a methacrylamide derivative with a hydroxypropyl group that contributes to the hydrophilicity and elasticity of the films) and CBMAA had the most microdefects, both deep and shallow.
"I am delighted that the direction we set out on years ago is developing successfully and bringing new collaborations. This result clearly shows how important interdisciplinary cooperation between classical optics, materials chemistry, and computer simulations is. Without linking these fields, we would have had a hard time getting to these findings," says Alexandr Dejneka, head of the Division of Optics.
The results pave the way for the design of polymer brushes that will be more reliable and less prone to failure even under demanding conditions. Such durable surfaces are crucial for biosensors, the accuracy and stability of which directly affect medical diagnostics and a number of other modern technologies.