Chemistry

E. coli colonies producing lactic acid as an attractant for mosquitos are an important aspect of the realization of our project. However, these colonies are encapsulated in alginate beads which are acid-labile and hence endangered by a pH value that is too low. In order to ensure the long-term stability of these beads therefore biosafety we investigated their stability in the presence of different concentrations of lactic acid over time. For this reason we developed a fast and easy photometric method to monitor their decomposition.

Alginate capsule production

Under stirring 12.6 mg Coomassie Brilliant Blue G-250 were dissolved in 200 mL water for 1 h. Afterwards, 2.00 g sodium alginate were added. After heating and further stirring a homogenous solution was obtained. This solution was dropped in defined amounts into a solution of 2.00 g calcium chloride dihydrate in 200 mL water. This resulted in the formation of colored beads with a similar size distribution (Fig. 1).

For the concentrations of lactic acids we decided to begin with an aqueous solution containing 40% lactic acid and half this until we reached a solution containing 0.31% lactic acid. Additionally, we set up a negative control containing just water and a positive control containing 0.5 M hydrochloric acid.

We added 50 beads to each solution and stored them for two weeks at room temperature.

Alginate capsules are stable in light acidic conditions.

After two weeks we examined the constitution of the alginate beads (Fig. 2). Since a significant discoloration occurred in the two most concentrated set ups, the beads are not as easy to identify as it is for the other concentrations. Nevertheless, the beads are still intact after two weeks even when they had been exposed to non-natural high concentrations of lactic acid. By this, the study suggests that the stability of the alginate gel and therefore biosafety can be ensured since no decomposition was found for concentrations of lactic acid within the concentration of cellular metabolites.

**Fig. 2** : Visual examination of the coomassie-stained alginate beads after storing them in different concentration of lactic acid at room temperature for 2 weeks.

Hydrogel

A key element of our trap is represented by the hydrogel embodying a border between the environment and the trap’s insides guaranteeing an undamaged surface due to its self-healing properties. Therefore, there are several requirements that need to be fulfilled by this gel. The hydrogel we chose can not only be obtained readily but can also be handled easily. Since its viscosity decreases above 55 °C it enables a casting into the desired form when it is hot resulting in a stable gel after it cooled to ambient temperature. Equipped with these characteristics the hydrogel offers a suitable surface on which mosquitos can land.

The gel is synthesized in a polymerization between the two bifunctional monomers pyromellitic dianhydride (PMDA) and 4,4’-oxydianiline (ODA) in N,N-dimethyl acetamide (DMAc) resulting in a poly(amic acid) polymer (PAA). The reaction is carried out at room temperature and occurs spontaneously without catalysis. Treating this PAA polymer with triethylamine (TEA) converts it into the corresponding a poly(amic acid) ammonium salt (PAS) (Fig. 3).

**Fig. 3** : Polymerization between the two bifunctional monomers ODA and PMDA resulting in PAS after treatment with triethylamine.

By pouring the reaction mixture into acetone the more polar environment forces the PAS polymer to agglomerate in order to cover the hydrophobic regions. After washing with acetone and drying the salt in vacuum, a yellowish powder is obtained. This PAS powder is stirred in water and gently heated resulting in the adjustable ready-to-use hydrogel. After it is poured into the desired shape (Figure 1) it is stored at 5 °C for one week1.

**Fig. 4** : PAS hydrogel after pouring it into the desired cast.

Self-healing properties

There are three non-covalent interactions that contribute to the mechanical strength, the thermosensitivity, the no maintainance aspect and are therefore crucial for the gel’s self-healing properties (Figure 2).

The amide functionality is able to serve as a hydrogen donor as well as an acceptor. For this, a hydrogen bond between an oxygen atom of one strand and a hydrogen atom of the amide functionality of another strand is formed. Due to the aromatic character in the repetetive motif of the polymer, the chains are able to interact trough π-π-stacking, in a similar fashion as the bases of DNA. Ultimately, through its ionic character polyanion-polycation interactions occur between the chains. Although all these interactions are accorded to be weak interactions, in sum they promote stability and regenerative abilities to the polymer. As a result of this a reptation and reentanglement of the polymer chains across a fracture interface causes the healing of the gel in the case of damage1.

**Fig. 5** : Non-covalent interactions between polymer chains. The combination of hydrogen bonding, π-π-stacking, and ionic polyanion-polycation interactions provides high mechanical strength and self-healing properties.

Synthetic procedure

To a solution of 2.41 g ODA (11.0 mmol, 1.00 eq.) in 43.0 mL DMAc 2.61 g PMDA (11.0 mmol, 1.00 eq.) were added stirred for 4 h at room temperature. Afterwards, 3.34 mL triethylamine (24.4 mmol, 2.03 eq.) were added and the solution stirred for additional 2 h. In the following, the reaction mixture was poured into 200 mL acetone and the precipitated solid crushed and dried in vacuum until no further change in its mass was detected. To 4.51 g of the obtained PAS powder 30.0 mL deionized water were added. The resulting mushy solid was stirred for 2 h at 60 °C and then poured into a culture dish. After additional heating for 30 min at 50 °C the gel was stored for 7 days at 5 °C.

References

↑Zhang, L., Wu, J., Sun, N., Zhang, X. & Jiang, L. A novel self-healing poly(amic acid) ammonium salt hydrogel with temperature-responsivity and robust mechanical properties. J. Mater. Chem. A 2, 7666–7668 (2014).

Team:Hamburg/Chemistry