Periodic Reporting for period 1 - OSCILLOGEL (An enzyme-based self-oscillating gel)
Reporting period: 2019-02-01 to 2021-01-31
One of the most interesting questions for chemists is how bioinspired or biomimetic properties can arise from the interactions of relatively simple molecules. Taking inspiration from Alan Turing’s pioneering computational work in 1952, chemists demonstrated in experiments that “simple” chemical systems can produce complex spatio-temporal phenomena or stationary patterns that may play a role in morphogenesis and the growth of form in living systems. This work highlighted the important role of feedback loops in the reaction networks for the emergence of periodicity that plays such an important role in biology.
More recently, the interplay between chemistry and mechanical forces in heterogeneous media including membranes, micelles, hydrogels etc is of increasing importance in this exciting research area. As was noted by Gregoire Nicolis in 2001 “elucidating the mechanisms of mechano-chemical couplings should lead not only to the elaboration of interesting new materials but also to the understanding of a number of biological processes of great concern.”
However, many of the systems under investigation suffer from drawbacks including lack of biocompatibility or unstable components that lead to degradation and loss of behaviour. While computational models are advanced, experimental realization remains challenging and necessitates solid experience in quite different fields as nonlinear science, polymer (physical) chemistry and enzyme reactions.
Therefore, the objectives of this project were to unite the host`s and the researcher`s previous experiences and to develop new biologically relevant systems capable of chemomechanical oscillations with chemistry open to diversification. The approach could thus be generalized, opening the path for the development of many new stable systems with regulatory functions, such as the ability to periodically open a valve. Such systems show features in common with biological machines and are of interest in soft robotics, with applications, for example, in drug delivery.
The original focus of the project was an enzymatic reaction – the urease reaction – that could form the basis of a self-oscillating enzyme-containing hydrogel. Another important aspect of the project was comparison with known chemical oscillators and identification of stability and toxicity issues. To this end, a chemical system was identified and modified such that its stability and general applicability were greatly improved.
Creating biocompatible (but not necessarily enzymatic) oscillatory reactions remains an interest for a much broader group within the community of nonlinear and materials scientists. The easy handling and stability of the chemical reaction has priority, and the possibility of combining with delicate biomaterials (natural polymers) widens the perspectives. Understanding biological processes (that are too complex to overview intuitively or analytically) can happen only by studying simplified artificial subsystems that reproduce certain characteristic but general behaviours. Periodicity is one of them.
More recently, the interplay between chemistry and mechanical forces in heterogeneous media including membranes, micelles, hydrogels etc is of increasing importance in this exciting research area. As was noted by Gregoire Nicolis in 2001 “elucidating the mechanisms of mechano-chemical couplings should lead not only to the elaboration of interesting new materials but also to the understanding of a number of biological processes of great concern.”
However, many of the systems under investigation suffer from drawbacks including lack of biocompatibility or unstable components that lead to degradation and loss of behaviour. While computational models are advanced, experimental realization remains challenging and necessitates solid experience in quite different fields as nonlinear science, polymer (physical) chemistry and enzyme reactions.
Therefore, the objectives of this project were to unite the host`s and the researcher`s previous experiences and to develop new biologically relevant systems capable of chemomechanical oscillations with chemistry open to diversification. The approach could thus be generalized, opening the path for the development of many new stable systems with regulatory functions, such as the ability to periodically open a valve. Such systems show features in common with biological machines and are of interest in soft robotics, with applications, for example, in drug delivery.
The original focus of the project was an enzymatic reaction – the urease reaction – that could form the basis of a self-oscillating enzyme-containing hydrogel. Another important aspect of the project was comparison with known chemical oscillators and identification of stability and toxicity issues. To this end, a chemical system was identified and modified such that its stability and general applicability were greatly improved.
Creating biocompatible (but not necessarily enzymatic) oscillatory reactions remains an interest for a much broader group within the community of nonlinear and materials scientists. The easy handling and stability of the chemical reaction has priority, and the possibility of combining with delicate biomaterials (natural polymers) widens the perspectives. Understanding biological processes (that are too complex to overview intuitively or analytically) can happen only by studying simplified artificial subsystems that reproduce certain characteristic but general behaviours. Periodicity is one of them.
The initial focus of the study was the enzyme urease, which is prevalent in nature and widely used in materials applications. Urease reacts with urea to produce ammonia, thereby raising the solution pH. The feedback in its reaction mechanism comes from the typical bell-shaped rate pH curve with a maximum at pH 7. As the pH increases, so does the rate of reaction and the pH of the reacting solution shows bistability between an acidic and basic state in a flow reactor. It was hypothesized that a synergistic effect between a pH responsive hydrogel and this biochemical reaction could be exploited for oscillations.
During the course of investigations, some important technical issues were identified with the use of the enzyme urease. In particular, it was established that its long-term instability in solution and leaching of the enzyme from the hydrogels of interest would pose a problem for this project. In order to alleviate these issues, discussions with the project collaborator led to the investigation of the use of urease-containing seeds. It was found that ground watermelon seeds might provide a natural immobilized source of urease with a greater stability in solution. In addition, the micron-sized particles could be loaded into hydrogels and reduce leaching of the enzyme.
Further research was directed towards a chemical oscillator with some similarities to the urease system – the Methylene Glycol – Sulfite – Gluconolactone (MGSG) reaction. It also displayed base-catalyzed feedback that resulted in a pH switch to higher pH and had been widely coupled with pH sensitive processes. However, the reaction had numerous issues including a toxic and carcinogenic component - formaldehyde - and an unstable component – gluconolactone - that hydrolyzed in the stock solution reducing the stability of this system. In addition, the shape of the oscillations was spiked and the timescales too short, making effective coupling with mechanical processes challenging.
We were able to replace the core components of formaldehyde and gluconolactone with improved counterparts, creating a new pH oscillator with significantly longer period and reproducible, stable oscillations. Moreover, the new oscillator was coupled with self-assembly of oleic acid creating periodic micelle/vesicle phase transitions, thus demonstrating its potential also to drive a new chemomechanical oscillatory system. These exciting new results will form the basis of a publication and have been disseminated at a number of conferences (on complex phenomena and chemical kinetics).
During the course of investigations, some important technical issues were identified with the use of the enzyme urease. In particular, it was established that its long-term instability in solution and leaching of the enzyme from the hydrogels of interest would pose a problem for this project. In order to alleviate these issues, discussions with the project collaborator led to the investigation of the use of urease-containing seeds. It was found that ground watermelon seeds might provide a natural immobilized source of urease with a greater stability in solution. In addition, the micron-sized particles could be loaded into hydrogels and reduce leaching of the enzyme.
Further research was directed towards a chemical oscillator with some similarities to the urease system – the Methylene Glycol – Sulfite – Gluconolactone (MGSG) reaction. It also displayed base-catalyzed feedback that resulted in a pH switch to higher pH and had been widely coupled with pH sensitive processes. However, the reaction had numerous issues including a toxic and carcinogenic component - formaldehyde - and an unstable component – gluconolactone - that hydrolyzed in the stock solution reducing the stability of this system. In addition, the shape of the oscillations was spiked and the timescales too short, making effective coupling with mechanical processes challenging.
We were able to replace the core components of formaldehyde and gluconolactone with improved counterparts, creating a new pH oscillator with significantly longer period and reproducible, stable oscillations. Moreover, the new oscillator was coupled with self-assembly of oleic acid creating periodic micelle/vesicle phase transitions, thus demonstrating its potential also to drive a new chemomechanical oscillatory system. These exciting new results will form the basis of a publication and have been disseminated at a number of conferences (on complex phenomena and chemical kinetics).
An overall important objective of the work was to determine the issues that limit the current chemomechanical oscillators in order to develop general strategies for the development of new systems. In particular, the aim was to move towards more biocompatible and biologically relevant systems. The previous state of the art involved the use of harsh, toxic chemicals, e.g. the bromate in the BZ reaction (widely used in BZ self-oscillating gels) or formaldehyde in the MGSG reaction. The use of enzymes in chemomechanical oscillators is not widespread, since there are few robust examples of enzymatic reactions displaying the necessary feedback.
A number of important findings were obtained during the course of the project that will impact on the field. Firstly, the preliminary investigations with the urease enzyme have demonstrated some key issues that may be overcome regarding enzyme stability and leaching which will be further investigated in future work. Secondly, analysis of the MGSG reaction has led to a breakthrough and the creation of a new chemical oscillator that is more stable and less toxic, and operates at a more appropriate timescale for gel swelling/shrinking processes. This was coupled with a pH sensitive self-assembly processes to demonstrate its applicability in driving periodic physical processes. It is hoped that these studies highlight the general approach that can be used to make new, stable chemomechanical oscillators in the future, thus greatly advancing the area.
A number of important findings were obtained during the course of the project that will impact on the field. Firstly, the preliminary investigations with the urease enzyme have demonstrated some key issues that may be overcome regarding enzyme stability and leaching which will be further investigated in future work. Secondly, analysis of the MGSG reaction has led to a breakthrough and the creation of a new chemical oscillator that is more stable and less toxic, and operates at a more appropriate timescale for gel swelling/shrinking processes. This was coupled with a pH sensitive self-assembly processes to demonstrate its applicability in driving periodic physical processes. It is hoped that these studies highlight the general approach that can be used to make new, stable chemomechanical oscillators in the future, thus greatly advancing the area.