Since we have successfully demonstrated that the bio-functional hydrogel could assemble itself quickly, capture uranyl efficiently and be enriched conveniently, we proposed to combine the features of these experiments in a single trial. We thus incubated triple SpyTag-SUP together with triple SpyTag-mSA and triple SpyCatcher, and contacted the product of crosslinking with simulated pollution. After adsorption, we used a magnet and biotin-coated beads to retrieve the hydrogel, together with its contents of adsorbed uranyl (Fig. 1).

We prepared 10μM uranyl nitrate solutions in TBS buffer, as well as in fresh water (sampled from Weiming Lake in Peking University) and seawater (sampled from Qinhuang Island) to simulate real-life pollution.

After mixing of 5mg/mL solutions of triple SpyTag-SUP, triple SpyTag-mSA and triple SpyCatcher, each, in a volume ratio of 1:1:2, and subsequent incubation for 1h, we were able to obtain the dual-function crosslinked hydrogel encompassing both uranium adsorption and hydrogel clearance functions.

The protein hydrogel was added into the polluted solution and the mixture shaken on a swing bed for 1min, after which the solution was contacted with a suspension of biotin-coated beads (10mg/ml) at a volume ratio of 5:1 (reaction system: beads), and shaken for 1h to ensure good contact between the protein and the beads. Finally, the beads were immobilized using a magnetic shelf, and the uranyl ion concentration in the supernatant was measured using the Arsenazo III assay.

Learn more:

The statistical analysis of experimental results is shown in Fig.18. Under the circumstances mentioned above, the Uranium Reaper achieved a relatively good adsorption efficiency (51.4% in TBS buffer, 55.8% in seawater and 60.1% in fresh water).

In this integrated experiment, the Uranium Reaper showed an adsorption rate of 60%, which corresponds well to the product of the uranyl adsorption rate and hydrogel recovery rate (90% × 70% = 63%). This result has reached the expectations but is still far removed from the efficiencies that would be needed for an industrial process. Consequently, both the uranyl adsorption and hydrogel recovery rate have to be increased to improve the overall efficiency of the Uranium Reaper, which, of course, requires further research and development.

Widening the Potential Applications of Uranium Reaper:

Cadmium and Lead Reaper

Due to the modularity of the SpyTag-SpyCatcher covalently cross-linked hydrogel, we could easily replace the SUP fused to the Triple SpyTag module with other proteins of interest, such as Cadmium-Binding Protein (CBP) or Lead-Binding Protein (LBP), and use it to adsorb a variety of other heavy metals. A preliminary result is shown in Fig. 3.

We simulated Cd and Pb pollution (10μM) in TBS buffer. Using the same strategy which we have used for uranyl removal, with the 3A-SUP monomer replaced by 3A-CBP or 3A-LBP, about 85% of the Cd 55% of the Pb could be adsorbed, respectively. The results demonstrated that the strategy has great potential for broader metal recovery applications. It is thus necessary to optimize the hydrogel under various conditions.

Visualization

We fused mRFP to the Triple SpyTag to make impart color to our hydrogel (Fig.4).

When the SUP module in the Triple SpyTag-SUP monomer was changed to mRFP, a red-colored gel was formed (Fig 2. left), and by crosslinking proteins at higher concentrations, we could get a relatively stable gel structure. This attribute of the hydrogel promises a literally colorful future in artistic creation, with potential applications in 3D printing.