Material designFigure 1 presents the strategy followed to synthesize materials inspired by biology. In plants, phytochelatins form complexes with intracellular heavy metal ions such as Cd2+, which are isolated and stored in vacuoles (Fig. 1a). Inspired by the chemical structure of phytochelatin, a series of pAA-based copolymers (pAA–Cys5) possessing –COOH as well as –SH side chains were synthesized (Fig. 1b)13,14,15. In brief: pAA–Cys5 was synthesized by reversible addition−fragmentation chain-transfer (RAFT) radical polymerization of S-trityl-cysteine acrylamide (S-Tri-Cys-AAm) and acrylic acid (AA) using a radical initiator and a chain transfer agent, followed by deprotection of trityl group with trifluoroacetic acid (TFA). More details can be found in Supplementary Figs. 1–2.The copolymers were synthesized following the reversible addition–fragmentation chain-transfer (RAFT) radical copolymerization of acrylic acid (AA) and AA coupled to cysteine. Supplementary Fig. 1 shows preparation of an S-protected cysteine monomer (S-Tri-Cys-AAm). A scheme for the synthesis of pAA–Cys5 is shown in Fig. S2a. Azobisisobutyronitrile (AIBN) was used as the radical initiator. Analysis of the 1H NMR spectra (Supplementary Fig. 2b) confirms successful synthesis. A gel permeation chromatography (GPC) chart for pAA–Cys5 (Fig. S2c) indicated a unimodal peak. The weight-average molecular weight (Mw) value was recorded to be 1.7 × 104, and the molecular weight distribution (Mw/Mn) value was 2.2.Validation of design of bio-inspired polymersTo verify the applicability of the design strategy in the synthesis of plant phytochelatin-inspired materials, we determined the binding affinity of pAA–Cys5 to Ca2+ and Cd2+ ions by measuring ITC in solution and compared the affinity with those of phytochelatin and other materials. The additional thermal power (dQ/dt) and enthalpy (ΔH) plotted against the molar ratio of the cysteine side chains are presented in Fig. 2a. The global shape of the ITC data characterized by a peak (ΔH > 0) indicated the coexistence of exothermic and endothermic processes. The exotherm can be interpreted as the binding of the divalent cations to the negatively-charged pAA–Cys5 brushes, whereas the endotherm represents the entropic changes caused by the release of counterions and/or the dehydration of pAA–Cys516,17,18. The ITC data for pAA–Cys5 were fitted using a two-site model (solid lines). The KD of pAA–Cys5 to Cd2+ ions, KD(Cd) = 2.1 × 10–9 M per molecule, is by four orders of magnitude smaller than that to Ca2+ ions, KD(Ca) = 2.5 × 10–5 M per molecule, indicating that the affinity of pAA–Cys5 for Cd2+ is significantly high. In contrast, the binding affinities of pAA (devoid of cysteine side chains) to Ca2+ and Cd2+ ions are too low to calculate the KD values from the ITC data (Supplementary Fig. 3). Although the direct comparison of the KD values determined by different techniques and/or under different measurement conditions is difficult, the binding affinity of pAA–Cys5 to Cd2+ ions is significantly higher than those of previously reported materials. For example, Chekmeneva et al. determined the KD values of phytochelatin analog ((αGlu-Cys)4-Gly) and glutathione by ITC measurements in 20 mM Tris-HCl buffer containing 0.1 M NaCl (pH 7.4), 3.2 × 10–7 M per molecule and 2.0 × 10–5 M per molecule, respectively19. Cheng et al. measured absorption spectra in 0.1 M Tris-HCl (pH 7.4) and obtained a comparable KD(Cd) = 3.2 × 10–7 M for the same phytochelatin analog, (αGlu-Cys)4-Gly20. Visvanathan et al. synthesized random peptides mimicking phytochelatin, oligo(L-Glu-co-L-Cys), and calculated a much weaker affinity to Cd2+ ions from the absorption spectra measured in 0.1 M Tris-HCl (pH 7.4), KD = 8.6 × 10–4 M per molecule21. In this study, the KD of pAA-Cys5 to Cd2+ ions (KD(Cd) = 2.1 × 10–9 M per molecule) was determined in 10 mM Tris-HCl (pH 7.4). This value is two to five orders of magnitude smaller than the other materials including phytochelatin (Supplementary Table 1), indicating that bio-inspired synthetic pAA-Cys5 can overtake the function of the naturally occurring peptides (Glu-Cys)n.Fig. 2: Selective capture of Cd2+ ions by bio-inspired pAA-Cys5.a Plots of additional thermal power (dQ/dt) and enthalpy (ΔH) versus the molar ratio of the cysteine side chain by titrating pAA–Cys5 with CdCl2 and CaCl2, respectively. The best-fit curves for ITC data using a two-site model are shown (solid lines). b ATR-IR spectra obtained by subtracting the spectrum of a buffer from that for pAA–Cys5 (4 w/v%) in the absence (black) and presence of 8 mM Ca2+ (blue) or Cd2+ (red). Inset: magnified view of the peak top for νCOO– (antisymmetric) band. c ATR-IR spectra obtained by subtracting the spectrum of pAA–Cys5 (4 w/v%) from that recorded for pAA–Cys5 (4 w/v%) in the presence of Ca2+ or Cd2+ (2, 4, 6, and 8 mM). d Peak intensity plotted against the concentration of Ca2+ or Cd2+ for (c). The data obtained in the presence of Cd2+ and Ca2+ are indicated in red and blue, respectively. Error bars corresponds to the instrumental noise determined from the baseline. e 1H NMR spectra of NAC (1 mM) in the absence and presence of 1 mM of Ca2+ or Cd2+. f Possible structures of the Cd2+–pAA–Cys5 complex deduced from the NMR and FTIR spectra.To gain further structural insights into the interaction of pAA-Cys5 with M2+, the attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectra of pAA–Cys5 (4% w/v) were recorded in 10 mM Tris-HCl buffer (pH 7.4) in the absence and presence of M2+. Figure 2b presents the FTIR spectra of the polymer solutions in the absence (black) and presence of Cd2+/Ca2+ ions (red/blue). The global shape of the spectra can be characterized by two peaks at 1550 and 1400 cm–1, and these peaks correspond to the antisymmetric and symmetric stretching of –COO– of pAA, respectively22. In Fig. 2c, the differential spectra around the antisymmetric –COO– band after the subtraction of the spectrum of “pAA-Cys5 with no M2+” are presented to highlight the changes caused by Ca2+/Cd2+ ions. The peak intensities decrease monotonically as [Cd2+] increase, whereas no clear trend can be detected with increasing [Ca2+] (Fig. 2d). Because the signals in 1H NMR spectra of pAA-Cys5 are too broad to determine how Cd2+ forms a complex with –CH2S– and –COO– groups precisely (Supplementary Fig. 2), we recorded the 1H NMR spectra of a model compound, N-acetylcysteine (NAC), in the absence and presence of Ca2+ or Cd2+. As shown in Fig. 2e, the increase in [Ca2+] does not cause shifts in the positions of 1H peaks, whereas the peaks of B and C associated with protons bound to carbon atoms adjacent to the –COO– and –CH2S– groups shift in the presence of Cd2+, indicating that Cd2+ ions interact not only with –COO– groups but also with –CH2S– groups. Notably, the diastereotopic splitting of CH2 signals (C) indicated the bidentate chelation mode23 of NAC to Cd2+. Furthermore, the FTIR (Supplementary Fig. 4) and the 1H NMR (Supplementary Fig. 5) spectra of NAC measured at different concentrations of Cd2+/Ca2+ ions provide clear evidence that Cd2+ ions interact with both –COO– and –CH2S– groups, whereas Ca2+ ions do not. The chelation mechanism for phytochelatin and Cd2+ has been investigated using the combination of different techniques, such as UV/vis, circular dichroism, NMR, mass spectroscopy, potentiometric titration, and isothermal titration calorimetry19,24,25,26. Jalilehvand et al. combined Cd K-edge spectra and 113Cd NMR of the concentrated aqueous solution of CdCl2 (0.5 M) and N-acetylcysteine (1.0 M) and proposed the multinuclear complex structures in which the position of Cd is determined by the balance of Cd–S and Cd–O bonds14. Wątły et al. investigated the complex stoichiometry of the well-defined oligomers, (γ-Glu-Cys)n-Gly (n = 1 – 6), and determined the chelate stoichiometry as a function of n. They reported the entropic stabilization of the chelate complexes contributes to decrease the total free energy27.Our NMR and FTIR data on pAA–Cys5 suggest three possible structures for the Cd2+–pAA–Cys5 complex (Fig. 2f): (i) Cd2+ forms a complex with one Cys side chain, (ii) Cd2+ forms a complex with two Cys side chains, and (iii) Cd2+ forms a complex with one Cys chain and–COO– group(s) of the pAA main chain. The formation of complexes between Cd2+ and two Cys side chains (ii) is unlikely because the separation distance between the Cys side chains (5 mol%) is non-uniform due to the statistical copolymerization of two monomers. As a rough estimate, if one assumes the size of AA monomer being 2.5 Å, it is plausible that the large average distance between Cys units (2.5/0.05 = 50 Å) hinders the formation of dimers. To verify if (i) is possible, two types of copolymers with the main chains containing monomers devoid of –COOH groups were synthesized, hydroxypropyl methacrylamide (HPMA, Supplementary Figs. 6 and 7) and poly(ethylene glycol methyl acrylate) (PEGMA, Supplementary Figs. 8 and 9). As presented in Supplementary Fig. 10, the ITC data for pHPMA-Cys5 and pPEGMA-Cys5 reveal no sign of interaction between the polymers and Cd2+/Ca2+ ions. This provides supporting evidence that Cd2+ forms a complex with –COO– and –CH2S–groups of a cysteine unit and –COO– group(s) of the pAA main chain as in (iii).Loading capacity and Cd2+ selectivity of bio-inspired polymersThe function of the bio-inspired pAA–Cys5 to treat water was tested based on two parameters: loading capacity and Cd2+ specificity. As shown in Fig. 3a, the pAA–Cys5 solution and the buffer containing Cd2+ were allowed to react overnight, followed by ultracentrifugation. Because the pAA–Cys5 polymer with Mw ≈ 1.7 × 104 Da cannot pass the filter with the cut-off level of 3 × 103 Da, only the ions that did not bind to pAA–Cys5 can pass through the filter. The [Cd2+] in the ultrafiltrated flow-through was determined using inductively coupled plasma optical emission spectrometry (ICP-OES). Figure 3b presents the [Cd2+] in the flow-through after the ultrafiltration of a mixed solution containing pAA–Cys5 (0.1 mg mL–1) and CdCl2 (100 µM), indicating the effective capture of almost all Cd2+. In contrast, the corresponding data of the blank buffer and the two non-pAA polymers (pHPMA-Cys5 and pPEGMA-Cys5) exhibit no remarkable changes in [Cd2+] after ultrafiltration. As the detected [Cd2+] in the flow-through of pAA–Cys5 (<2 µM) was close to the detection limit of Cd2+ by ICP-OES (ca. 0.1 µM), the exact value, [Cd2+] = 0.6 µM, was determined by Spectroquant® colorimetric assay. Notably, the non-cytotoxic level of [Cd2+]28 as well as the drinking water level defined by the World Health Organization (WHO), [Cd2+] ≤ 0.03 µM29, can be achieved readily either by increasing [pAA–Cys5] or by decreasing [CdCl2] in the eluent. For example, the combination of [pAA–Cys5] = 0.1 mg mL–1 and [CdCl2] = 10 µM resulted in [Cd2+] < 0.02 µM in the flow-through.Fig. 3: pAA-Cys5 is selective adsorbent for Cd2+ ions with high loading capacity.a Schematic for the experimental procedure. Only the ions that did not bind to pAA–Cys5 can pass through the filter because the ions bound to pAA–Cys5 polymer (Mw ≈ 1.7 × 104 Da) cannot pass the filter. b [Cd2+] in the flow-through in the absence (light gray) and presence (red) of pAA–Cys5 (0.1 mg mL–1). The corresponding data for pHPMA–Cys5 (gray) and pPEGMA–Cys5 (gray) are shown for comparison. The initial concentration of Cd2+ ([Cd]initial) was 100 µM. Error bars indicate the calibration accuracy of Spectroquant® colorimetric assay. c Table showing the initial concentrations of pAA–Cys5 and Cd2+ ([pAA–Cys5] and [Cd]initial, respectively) and Cd2+ in the flow-through ([Cd]flow-through). These values were used to calculate the amount of Cd2+ bound to 1 g of pAA–Cys5, whose maximum is 5.2 mmol g–1. (d) Concentrations of Cd2+ in the flow-through after mixing simulated wastewater ([Cd2+]initial = 10 µM, [Na+]initial = 1 mM, [K+]initial = 0.2 mM, [Mg2+]initial = 0.5 mM, [Ca2+]initial = 0.5 mM) with 10−4, 10−3, 10−2, or 10–1 mg mL–1 of pAA–Cys5, confirming an outstanding Cd2+ selectivity. Error bars indicate the calibration accuracy of Spectroquant® colorimetric assay. Note that [Cd2+] for the WHO’s drinking water standard (0.03 µM) corresponds to 0.003 ppm.To compare the function of pAA-Cys5 with other heavy metal ion adsorbents, we calculated the loading capacity, defined as the amount of Cd2+ captured by 1 g of material (Fig. 3c). It should be noted here that at high polymer concentrations, the amount of bound ions per polymer becomes less with increasing polymer concentrations, because the binding sites in polymers are not saturated by Cd2+. On the other hand, at low polymer concentrations, the equilibrium between bound and unbound states shifts towards the unbound state. Therefore, the loading capacity of pAA–Cys5 shows maximum at [pAA–Cys5] = 0.01 mg/mL based on the balance of these two effects. The maximum loading capacity of pAA–Cys5 (5.2 mmol g–1) is 4–50 times higher than that of organic compounds, such as pAA modified with hydroquinone30, poly(hydroxyethyl methacrylate)–cysteine conjugate31, and poly(N-isopropyl acrylamide) hydrogel crosslinked with cysteine32. More remarkably, the maximum loading capacity of pAA–Cys5 is comparable to that of zeolite nanoparticles coupled to poly(vinyl alcohol) nanofibers, 7.5 mmol g–1, which is the largest adsorption capacity to date3,33.The ITC data suggest that the affinity of pAA–Cys5 to Cd2+ is by four orders of magnitude larger than the affinity to Ca2+. To test if pAA–Cys5 can selectively capture Cd2+, we incubated pAA–Cys5 solutions (10–4, 10–3, 10–2, and 10–1 mg mL–1) with a buffer containing [Cd2+] = 0.01 mM (10 µM), [Na+] = 1 mM, [K+] = 0.2 mM, [Mg2+] = 0.5 mM, and [Ca2+] = 0.5 mM, which mimic the concentration levels in wastewater, respectively34,35,36 Fig. 3d shows [Cd2+] in flow-through, indicating that pAA–Cys5 can capture Cd2+ even in the presence of a large excess of abundant mono- and divalent metal ions in ground water. When using 0.1 mg mL–1 of pAA-Cys5, [Cd2+] in flow-through was lower than the acceptable concentration declared by WHO for drinking water (<0.03 µM). These results demonstrated that the polymer could be used to selectively remove Cd2+ but not other ions in groundwater, such as Na+, K+, Mg2+, and Ca2+. Such a high selectivity to toxic Cd2+ ions make them distinct from zeolites and ion exchange resins, because the ions possessing similar sizes and charges are equally captured. As presented in Supplementary Fig. 11, we also detected the decrease in [Ca2+] in flow-through, suggesting that Ca2+ interact with pAA–Cys5. This is reasonable at [Ca2+] = 0.5 mM (5 × 10-4 M), which is more than one order of magnitude higher than the KD value of pAA–Cys5 and Ca2+ (~10-5 M). As presented in Supplementary Fig. 12, the dissociation constant of pAA–Cys5 to Mg2+ was comparable to Ca2+ (~10-5 M), whereas the interactions with Na+ and K+ could not be detected by ITC (Supplementary Fig. 12). Notably, the interaction of pAA–Cys5 and Ca2+ could not be detected by FTIR spectra, which were measured even at higher concentrations (~10-3 M, Fig. 2c, d). This finding can be explained by the molar fraction of Cys side chains (5 mol%). Namely, only a small portion of –COO– groups contribute to the spectral signals. In fact, the change in the spectral intensity could be detected only in differential spectra even at [Cd2+] ~ 10-3 M, which is about six magnitudes larger than the KD value (~10-9 M). Therefore, it seems reasonable that we could not detect the interaction of pAA–Cys5 with Ca2+ and other cations spectroscopically.Hyperconfinement of bio-inspired polymers in column-based microreactorDespite the excellent performance of pAA–Cys5, polymers in solutions cannot be directly used for water treatment. One of the most straightforward strategies to separate Cd2+-bound pAA–Cys5 after treatment is immobilizing pAA–Cys5 chains on microparticle surfaces and packing these polymer-coated microparticles into a flow-through reactor. Supplementary Fig. 13 shows the synthetic scheme for the end-functionalized pAA–Cys5 prepared to coat the surfaces. To get the first proof of principle, we coated silica microparticles (diameter: 10 µm) with bilayer lipid membranes incorporating 2 mol% of biotinylated lipids, coupled pAA–Cys5–biotin (Supplementary Figs. 14−19) to the membrane via neutravidin crosslinker (Fig. 4a, Supplementary Fig. 20), and load them in a fast protein liquid chromatography (FPLC) column (inner diameter: 10 mm)37. Note that the grafting of pAA–Cys5 brushes on silica particles have two advantages. First, as demonstrated previously38,39,40,41, the average distance between polymer chains \(\left\langle d\right\rangle\) can be controlled by the molar fraction χ at a nm-accuracy,$$\left\langle d\right\rangle=\sqrt{{A}_{{{{{{\rm{lipid}}}}}}}/\chi }$$
(1)
where Alipid is the cross-sectional area of one lipid molecules, (Alipid ≈ 0.6 nm2)42. For example, the inter-polymer distance corresponding to χ = 0.02 can be estimated as \(\left\langle d\right\rangle\) ≈ 5.5 nm. Second, the grafting of brushes on silica particles makes the separation of the polymer-Cd2+ complex much simpler. The use of pAA–Cys5 gels / particles is not practically possible, because the density of the hydrated polymers is very close to the density of the medium. The homogeneous coating of the particle surface with pAA–Cys5 was confirmed by confocal fluorescence microscopy, using fluorescently labeled polymer (Fluor–pAA–Cys5–biotin, Supplementary Fig. 21). The scanning electron microscopy image of the particle surface and the three-dimensional image of pAA–Cys5-coated microparticles reconstructed from the confocal microscopy images are presented in Supplementary Fig. 22. The functionalized particles (0.85 g in weight) were packed in a compact, flow-through microreactor, which enabled the confinement of silica microparticles (surface area of 1.1 m2) functionalized with 0.6 µmol of pAA–Cys5 in a volume of 1.8 mL37. A buffer (10 mM Tris-HCl, pH 7.4) containing 100 µM CdCl2 was fed by a syringe pump at the flow rate of 0.02 mL min–1, led through the column, and fractionated (Fig. 4b).Fig. 4: Hyperconfinement of pAA-Cys5 into column-based microreactor prototype.a Functionalization of silica microparticles (diameter: 10 µm). The homogeneity of the surface coating was confirmed by the confocal fluorescence image of silica microparticles coated with fluorescently labeled pAA–Cys5–biotin (Fluor–pAA–Cys5–biotin, Supplementary Fig. 21). b The flow-through microreactor prototype based on an FPLC column that enables the confinement of silica microparticles (surface area of 1.1 m2) functionalized with 0.6 µmol of pAA–Cys5 in a volume of 1.8 mL. c [Cd2+] of the eluent plotted as a function of the fraction number. Inset: magnified plots near the onset. Error bars for x- and y-axes indicate the fraction volume and the calibration accuracy of Spectroquant® colorimetric assay, respectively. d Total amount of removed Cd2+ plotted as a function of elution volume. [Cd2+] satisfied the concentration criteria recommended by WHO for drinking water.[Cd2+] of each fraction was determined by conducting colorimetric assays, and the data were plotted as a function of the fraction number (Fig. 4c). As the volume of each fraction is 1 mL, the term “fraction number 10” indicates a “total eluent volume of 10 mL.” Notably, [Cd2+] of fractions 1–36 remained below the detection limit of the assay, which fulfills the WHO’s drinking water level. These data indicate that this prototype can be used to treat 36 mL of a concentrated solution of CdCl2 (100 µM) to bring down the concentration of the contaminant to a level that meets the appropriate standards for drinking water (<0.03 µM). The total amount of Cd2+ captured by the microreactor was calculated based on the cumulative summation of [Cd2+] of each fraction (Fig. 4d). Here, 8.2 × 10–4 g, corresponding to 4.9 × 10–8 mol, of polymers were grafted to 0.85 g of silica microparticles. This yields the estimated maximum loading capacity of Cd2+ per 1 g of polymer to be 11 mmol g–1. Taking the number-average molecular weight (Mn) of pAA–Cys5 (8.0 × 103) and pAA–Cys5–biotin (7.4 × 103), the difference in the maximum loading capacity of polymers with and without confinements are estimated to be 41.6 mol mol–1 and 79.8 mol mol–1, respectively. Currently, we interpret the higher loading capacity realized by the particle-grafted pAA–Cys5–biotin in terms of the neighbor effect or the multivalent binding43,44,45. The former is a well-established concept in understanding the effect of adjacent ligands on the binding to a linear lattice. The latter is the principle of a targeted strengthening of interactions between the binding partners forming cooperative, multiple interactions that are based on individually weak, noncovalent bonds. In both cases, the presence of binding partners in the vicinity strengthens the overall binding. As suggested by the NMR analysis, the determination of valency of interaction, i.e. the number of contributing moieties in the chelate formation, is difficult in our experimental systems. Nevertheless, it is plausible that Cd2+ that escapes from one complex can readily form another complex by interacting with a neighboring –CH2S– and –COO– pairs. Yet, using this microreactor prototype, it takes about 24 h to treat 36 mL of water, which is too slow for the water treatment in practice. This extremely low flow rate is caused by a high back pressure P, which scales with:$$P\propto \frac{L\times F}{{D}^{2}\times {{ID}}^{2}}$$
(2)
L is the length of the path (packed beads), F the flux, D the particle diameter and ID is the inner diameter of the column. The use of larger particles decreases the pressure if L is kept constant, e.g. the use of 20 µm-large particles reduces the P by a factor of 4. However, in order to keep the same surface area, one needs 2 times larger bed volume and hence the length L. Therefore, we concluded that the prototype was useful to demonstrate the proof of principle but not suited for achieving a realistic flow rate comparable to a fixed-bed column system46.Integrative water treatment systems for high-throughput, selective removal of Cd2+ ionsNext, we tried to increase the efficiency of our flow-through reactor towards the realistic application in water treatment by increasing the area-to-volume ratio and the flow rate. The area-to-volume ratio can be increased using small particles. For example, the decrease in the particle diameter by a factor of 8 results in the increase in the area-to-volume ratio by a factor or 512/64 = 8. However, as described above, the use of small particles results in a significant increase in the back pressure P, because$$P\propto \,{D}^{-2}$$
(3)
Therefore, to realize both a higher area-to-volume ratio and a lower back pressure, we designed a new reactor. Small silica microparticles (diameter: 1.2 µm) were used to achieve a high area-to-volume ratio, and the surface of microparticles was functionalized with pAA–Cys5 coupled to the head group of dioleoylphosphatidylethanolamine (DOPE), called pAA–Cys5–DOPE (Fig. 5a and Supplementary Figs. 23-25). The deposition of a lipid monolayer incorporating pAA–Cys5–DOPE (2 mol%) on the surface of silanized silica microparticles47 enables the one-step immobilization of pAA–Cys5 onto microparticles. We changed the coating protocol for smaller particles (diameter 1.2 µm) to minimize the risk of particle aggregation. Once the beads form aggregates, they could hardly be re-suspended as single particles. The surface functionalization with a lipid monolayer incorporating pAA–Cys5–DOPE (2 mol%) following our previous account47 prevented the undesired aggregation of particles caused by the multiple centrifugation cycles (Supplementary Fig. 20). The covalent coupling of a pAA–Cys5 chain directly to the lipid head group also ensure the stability of the polymer chains, which was another positive effect. The molecular sieve (pore diameter: 0.1 µm) used in the first prototype (Fig. 4) was replaced by a cellulose membrane coated with chitosan functionalized with N-succinimidyl (NHS)-terminated pAA–Cys5 (chitosan–g–pAA–Cys5, Fig. 5b) to reduce the back pressure and hence to increase the flow rate. The synthesis of chitosan–g–pAA–Cys5 is presented in Supplementary Figs. 26 and 27.Fig. 5: High-throughput, integrative water treatment system based on pAA–Cys5–functionalized membrane and particles.Combination of (a) silica microparticles (diameter: 1.2 µm) functionalized with pAA–Cys5 and (b) cellulose membrane coated with chitosan functionalized with pAA–Cys5. The surface of silanized silica microparticles was functionalized with a lipid monolayer incorporating 2 mol% of pAA–Cys5–DOPE. c Photograph of the device consisting of the microparticles and a membrane functionalized with pAA–Cys5. d [Cd2+] in the eluent plotted as a function of elution volume. The unmodified cellulose membrane and silica microparticles without polymers showed a significant leakage from the very first fraction (light gray). The cellulose/chitosan–g–pAA–Cys5 membrane showed a leakage of Cd2+ already at V = 60 mL (gray). In contrast, the combination of the membrane and the pAA–Cys5-functionalized microparticles (black) could remove 10 µM Cd2+ (typical industrial wastewater level) from 0.3 L in 1 h down to the level approved for drinking water. Error bars for x- and y-axes indicate the fraction volume and the calibration accuracy of Spectroquant® colorimetric assay, respectively.The water treatment capacity was tested by packing a cellulose/chitosan–pAA–Cys5 membrane (Supplementary Fig. 28) and small silica microparticles (1.2 µm) coated with pAA–Cys5–DOPE into an Amicon® chamber (inner diameter: 45 mm) (Fig. 5c). Here, 0.063 g, corresponding to 8.9 × 10-6 mol, of pAA–Cys5–DOPE deposited on 5 g of silica microparticles yielding the polymer/bead mass ratio of 0.013. We monitored how this system can treat the water containing 10 µM CdCl2, which replicated the concentration level in industrial wastewater. Figure 5d shows the [Cd2+] in the eluent plotted as a function of the total elution volume V. The open symbols are the concentrations determined only in the presence of the cellulose/chitosan–pAA–Cys5 membrane, whereas the solid symbols correspond to the data obtained in the presence of both the cellulose/chitosan–pAA–Cys5 membrane and the polymer-functionalized microparticles. Notably, the new system could be operated at a flow rate of 5 mL min–1, which is 250 times higher than the flow rate used for the previously described prototype (Fig. 4). Even though the diameter of the microparticles (1.2 µm) was almost by one order of magnitude smaller than those used for the previous prototype (10 µm), the back pressure remained <1 bar, which is well below the operational limit specified for a commercial chamber (3.8 bar). The flow rate of our system (300 mL h–1) is comparable to those of fixed-bed column systems, ~10–1000 mL h–1[ 46. The linear velocity, a system size-independent index of water permeability, of our system was 0.2 m h–1, which is also comparable to those of conventional systems, ~0.1–1 m h–1 46.In the presence of unmodified cellulose membrane and silica microparticles without polymers, the system could not remove Cd2+, showing a significant leakage from the very first fraction (Fig. 5d). Even in the absence of functionalized microparticles, the cellulose/chitosan–g–pAA–Cys5 membrane could remove [Cd2+], but a distinct leaking of Cd2+ could be detected already at V = 60 mL (open symbols, Fig. 5d). The loading of different amounts of chitosan–g–pAA–Cys5 exhibited a nonlinear dependence of the onset volume of the leakage on the amount of grafted chitosan–g–pAA–Cys5 (Supplementary Fig. 29). This suggests that the combination of the cellulose/chitosan–g–pAA–Cys5 membrane and the pAA–Cys5-coated particles is necessary, because the membrane alone is not sufficient to capture Cd2+ under the practical flow rate used for the water treatment (5 mL min–1). In contrast, in the presence of both the cellulose/chitosan–pAA–Cys5 membrane and the pAA–Cys5-coated microparticles, the [Cd2+] level remained below the detection limit of the colorimetric assay up to 300 mL at a flow rate of 5 mL min–1 (solid symbols, Fig. 5d). Therefore, the obtained data indicate that the combination of the cellulose/chitosan–pAA–Cys5 membrane and pAA–Cys5-functionalized microparticles (5 g) packed in ~3 mL volume can be used to treat 0.3 L of industrial wastewater within 1 h. The Cd2+ removal capacity of our system was further examined by treating the simulated wastewater containing [Cd2+] = 0.01 mM (10 µM), [Na+] = 1 mM, [K+] = 0.2 mM, [Mg2+] = 0.5 mM, and [Ca2+] = 0.5 mM. The [Cd2+] level remained below the detection limit of the colorimetric assay up to 135 mL at a flow rate of 5 mL min–1 (Supplementary Fig. 30).The regeneration and recovery of the materials can potentially be achieved by the following two strategies. In the first strategy, the polymers can be regenerated by cancelling the chelate complex by adding a competitor, such as ethylenediaminetetraacetic acid (EDTA). As presented in Supplementary Fig. 31, the continuous treatment with the buffer containing 10 mM EDTA for 200 min resulted in the recovery of function by 83%. Although this suggests the recyclability of the materials, this treatment is to simply concentrate Cd2+ ions from one aqueous phase (polluted water model) to another (EDTA buffer). In the second strategy, the heavy metal and silica particles can be recovered by taking the difference in temperature conditions for decomposition into account. For example, the organic compounds can be removed and the metal (Cd) can be recovered in a fluid phase at 600 °C, which is well below the melting temperature of silica (1710 °C). Although the potential recovery of heavy metal can add a unique advantage to our materials over zeolites and other porous inorganic materials, this approach might not be helpful for the minimization of global carbon footprints.The positive proof-of-concept obtained in this study suggests that the spatial confinement of bio-inspired, synthetic polymer materials realizes potentially recyclable flow-through systems for the high-throughput, selective removal of harmful substances from environmental water, exploiting specific recognition functions of biological systems.
Hyperconfined bio-inspired Polymers in Integrative Flow-Through Systems for Highly Selective Removal of Heavy Metal Ions
