You will be shown the title of a chemistry paper, together with sections excerpted from the body of the paper. The paper sections may include HTML tags like `<p>` and `</p>`; please ignore these. Your task is to write an abstract for the paper. Your response should include the abstract and no other text.

Title:
Co-Immobilization of Enzymes and Aptamers to create Self-Regenerating Ultrafiltration Membranes for Toxin Removal

Paper sections:

<p>Membrane-based filtration can offer a scalable and costeffective approach to water purification to remove bacteria, parasites, and other microscale contaminants. 1, 2 However, removing small-molecule contaminants requires small pore sizes, which in turn increases cost and energy needs. 3,4 We have demonstrated that this challenge can be overcome by functionalizing ultrafiltration membranes with aptamers capable of binding to small-molecule toxins, as this enables removal of both small and large contaminants without the need for energy-intensive techniques such as reverse osmosis. 5,6 While we have achieved efficient sequestration of toxin molecules using aptamer-functionalized membranes, a key limitation remains the regeneration of these systems once the aptamer binding sites are saturated. 7 Regeneration is possible by using heat to reversibly denature the aptamers, but this is technically challenging, and heat can be damaging to the membrane. 8 We recognized that dual-functionalization of membranes with an aptamer for toxin capture and an enzyme for toxin degradation could address this challenge and provide an autonomously self-regenerating membrane design.</p><p>We chose laccase as our enzyme component, as it is capable of degrading organic contaminants such as bisphenol A (BPA) and sulfamethoxazole. [9][10][11] Specifically, laccase catalyzes singleelectron oxidation, which results in bond cleavage and makes this enzyme versatile for degrading a variety of organic contaminants. 12,13 Although this enzyme has shown promise for use in the treatment of water to remove small-molecule contaminants, using enzymes alone in solution has significant limitations, including non-reusability and subsequent removal of the enzyme from the water. [14][15][16] Similar to our aptamer system, enzymes can be appended to membranes, but reaction kinetics are not sufficient to enable full degradation of the toxin molecules as a solution is passed through the membrane. 15 However, we hypothesized that aptamer capture and enzyme degradation could work in tandem to enable efficient and convenient toxin removal, as the aptamers can rapidly capture the toxin molecules, which can then be released over time for subsequent degradation by the enzyme (Scheme 1). Using laccase and the DNA aptamer for bisphenol A, we demonstrate Scheme 1 Dual-functionalized membranes are prepared by covalently attaching enzymes and aptamers via amide bond formation. Dual functionalization can enable capture and degradation of bisphenol A to create a self-regenerating filtration system.</p><p>Please do not adjust margins Please do not adjust margins efficient capture and removal of toxin molecules and autonomous membrane regeneration over multiple cycles.</p><p>Our first goal was to explore methods for enzyme attachment to the membrane and observe whether the enzyme function would be preserved upon bioconjugation. Given the high number of surface lysine residues found on laccase, we hypothesized that we could utilize a similar approach as previously employed for attaching the amine-functionalized aptamers (Scheme S1). 6 Specifically, the membranes are composed of polyethersulfone (PES) grafted with poly(methacrylic acid) (pMAA), and thus amine-containing molecules can be conjugated to the carboxylic acid groups of the pMAA via amide bond formation. Enzyme attachment was monitored using an absorbance-based protein assay kit to survey the reaction solution before and after bioconjugation. After optimization, we found that functionalization using 2 mg/mL of enzyme and 300mM coupling reagent provided an attachment yield of 65% (Figure S1).</p><p>To ensure that immobilizing the enzyme onto the membrane did not significantly alter its catalytic ability, we incubated the functionalized membrane and a grafted control in an aqueous solution of 200 nM BPA and measured depletion efficiency over time (Figure 1a). BPA degradation was quantified by HPLC, and overnight incubation yielded the near complete disappearance of the BPA peak at 4.75 min and the appearance of a new peak at shorter retention time, consistent with previous reports of BPA degradation. [17][18][19] As a control, we observed no change in the BPA peak for samples incubated with grafted membranes having no laccase enzyme attached (Figure S2). To assess the lifetime of enzyme activity for the laccaseimmobilized membranes, we repeated the incubation with a fresh stock of BPA solution (200nM) each day for up to 5 days (Figure 1b). We observed that the depletion capacity remains almost quantitative, suggesting that the enzyme is able to remain active over long time periods. These results were very encouraging for our ultimate goal of self-regeneration and longterm use. 20 Having demonstrated enzyme activity and stability, we set out to test our overarching hypothesis regarding synergistic function of co-immobilized aptamers and enzymes. As a baseline measure, we were curious to determine the degradation capability of the membrane functionalized with enzyme only, as this would indicate the relative importance of aptamer capture. We filtered 100 mL of 200nM BPA solution through the enzyme-conjugated membrane using a stirred cell filtration system. Quantification of the amount of BPA removed from the solution was measured by HPLC, using a 6-point calibration curve (Figure S3a-d). Unsurprisingly, we observed that only a minimal amount of BPA was removed from the solution, with an initial decrease in concentration in the first few fractions followed by a return to 200 nM. This membrane was then left to regenerate for 12 h, and we performed the same depletion test with a fresh 200 nM BPA solution (Figure S3e). Please do not adjust margins</p>

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<p>We observed similar quantities of BPA removal as in the first filtration, demonstrating the ability of the enzyme to degrade BPA molecules and retain its function over time. As a control, we also performed a depletion experiment using atrazinespiked water, and we observed that laccase shows little ability to degrade this contaminant, at least on the timescale on which solution is flowed through the membrane. (Figure S4).</p><p>After assessing the degradation capability of enzyme alone, we sought to generate and test dual-functionalized membranes having both enzyme and aptamer 21 attached (Figure 2a). Briefly, grafted membrane was incubated with a solution of DMTMM to activate the carboxylic acid groups on pMAA. Activated membranes were then incubated with a solution of laccase (2mg/mL) and our fluorophore-tagged aptamer (10 µM) The membrane in aptamer-enzyme mixture was agitated on an orbital mixer for 16 h. After this time, we removed an aliquot of solution from the membrane supernatant to quantify remaining enzyme and aptamer in solution. Using this measurement, we observed attachment yields of 64% and 82% for enzyme and aptamer respectively (Figure 2b and Figure S5). This yield for aptamer attachment is similar to that observed in our previous work, indicating that co-immobilization of enzyme does not significantly impact functionalization efficiency. We did reproducibly observe that aptamer attachment yield is higher than that of the enzyme, and we hypothesize that this could be a result of the lower steric hindrance of the amine on the aptamer terminus compared to lysine side chains on the surface of the enzyme. 22,23 Nevertheless, we were encouraged by our ability to generate membranes having both nucleic acid and protein functionality.</p><p>We next sought to compare our aptamer-enzyme dual functionalized membranes to those having aptamer alone. We repeated our stirred-cell filtration experiments to compare BPA depletion using both types of membranes (Figure 3a-b). We observed similar levels of depletion for the initial passing of 200 nM BPA spiked water through each membrane (Figure 3c-d). As expected, the aptamer-enzyme dual-functionalized membrane was slightly more effective at initially removing BPA, as the enzyme is able to degrade a small amount of the contaminant as it is flowed through the membrane. More interestingly, we showed that the aptamer-enzyme dual-functionalized membrane was capable of self-regeneration, as incubation for 12 h followed by filtration with a new 200 nM BPA solution provided similar levels of depletion to that achieved in the initial filtration. Moreover, we were able to repeat this autonomous regeneration cycle multiple times with similar effect (Figure 3e). In comparison, the membrane having only aptamers showed limited capacity for BPA removal after the first use (Figure 3f). These results support our hypothesis that co-immobilization of aptamers with enzymes can enable efficient capture of smallmolecule toxins followed by self-regeneration through enzymatic degradation.</p><p>In conclusion, we report a self-regenerating ultrafiltration membrane that is able to capture and degrade waterborne small-molecule toxins. This system relies on nucleic acid aptamers to rapidly bind the toxin molecules as they pass through the membrane, coupled with enzymes that catalyze degradation of the toxins over time in order to restore the aptamer binding sites and regenerate the membrane without requiring any human intervention. Given that regeneration happens on the time scale of hours, we envision that this platform could be of practical use for personal water filtration, as water could be purified throughout the day, and the membrane regenerated upon storage overnight. Given the versatility of nucleic acid SELEX 24 to generate aptamers for a wide range of small-molecule targets coupled with the ability of  Please do not adjust margins enzymes to degrade these molecules, we envision that future work will not only expand the scope of toxins that can be removed from water samples, but also enable the simultaneous capture and degradation of multiple types of these toxins.</p>
Aptamer-functionalized membranes offer a promising platform for toxin removal, but regeneration of binding capacity requires heat and washing. Moreover, bound molecules can be eluted, resulting in recontamination. Here we report the tandem use of aptamers and enzymes to trap and degrade small-molecule contaminants, resulting in an autonomously self-regenerating purification system.