Temperature-Controlled Hybrid Hydrogels for Reversible and Selective Zinc(II) Removal from Minimal Culture Media

Abstract

Zinc ions (Zn²⁺) play vital roles in living organisms, and Zn²⁺-deficient systems provide valuable platforms for studying zinc-dependent biological processes. Protein-functionalized materials offer advantages in constructing such systems by combining the physical removability of a solid-phase matrix with the high selectivity of zinc-binding proteins. Poly­(N-isopropylacrylamide) (PNIPAM) hydrogel serves as a promising matrix by enabling temperature-controlled reversible adsorption. The zinc-binding domain of the Zap1 protein (Zap1zf12) is well suited as the protein component owing to its Zn²⁺ specificity, dual binding sites, and PNIPAM-compatible structure. Here, we integrated Zap1zf12 into PNIPAM, generating a thermoresponsive hybrid hydrogel (PNIPAM-co-Zap1zf12) for selective Zn²⁺ removal. PNIPAM-co-Zap1zf12 was constructed through rational cross-linking sites design in Zap1zf12, optimized protein modification, and refined hydrogel synthesis. This material exhibited reversible adsorption in response to biocompatible temperature shifts (37 °C capture, 25 °C release), allowing regeneration without competitive chelators. It achieved a Zn²⁺ removal efficiency of 98.4 ± 7.3% (from 804.1 ± 41.9 nmol/L to 12.6 ± 5.8 nmol/L), and was successfully applied in various minimal culture media for selective Zn²⁺ depletion. Therefore, PNIPAM-co-Zap1zf12 extends the applicability of the hybrid hydrogels by integrating dual-site zinc-binding proteins and enabling selective Zn²⁺ adsorption. Moreover, it offers an effective approach for generating zinc-deficient conditions.

1. Introduction

Zinc ions (Zn²⁺) are essential trace elements in biological systems and play key roles in various physiological processes. Extensive research has focused on their biological functions and homeostatic regulation. − A common strategy in these studies is to induce Zn²⁺ deficiency conditions. − However, the ubiquitous presence of Zn²⁺ in laboratory environments and reagents introduces excess background Zn²⁺, making it challenging to establish such conditions and reducing experimental consistency and reproducibility.

To address this issue, various strategies have been developed for Zn²⁺ removal. Small-molecule chelators such as EDTA and TPEN are commonly employed owing to their high affinity for Zn²⁺. − However, the resulting Zn²⁺-chelator complexes are difficult to remove from solution, introducing additional experimental complications. In contrast, polymer-based materials, such as the Chelex 100 resin, provide a more practical approach by enabling physical removal of Zn²⁺ along with the solid support. Nevertheless, Chelex 100 nonspecifically removes other essential ions, making it necessary to quantify metal concentrations before and after treatment to ensure proper restoration.

Zn²⁺-binding proteins, which typically exhibit high affinity and selectivity, offer a promising functional complement for conventional polymer-supported chelating materials to achieve specific adsorption. Richardson et al. developed a protein-based material named A12-resin, utilizing the human S100A12 protein with subnanomolar affinity to Zn²⁺. A12-resin effectively depleted Zn²⁺ from complex biological solutions, confirming the potential of protein-based materials for selective Zn²⁺ removal.

Selecting an appropriate polymer matrix is essential for protein-based materials, as it not only provides structural support but also introduces additional functionalities. Among various candidates, hydrogels are capable of reversible deformation in response to diverse external stimuli, and have been widely applied in therapeutic delivery, tissue repair, and other biological applications. − Poly­(N-isopropylacrylamide) (PNIPAM) hydrogel is a thermoresponsive hydrogel that undergoes a volume transformation across its lower critical solution temperature (LCST). , Building on this feature, hybrid PNIPAM hydrogel systems combining PNIPAM with metal-binding peptides or proteins have been developed for temperature-regulated metal ion enrichment. For example, selective enrichment of Cd²⁺ and Pb²⁺ was realized by integrating cadmium-binding peptide CadRpep and lead-binding peptide PbrRpep into the PNIPAM network. , Moreover, immobilization of SUP protein extended these systems from peptide-based to protein-based applications, and enabled uranium adsorption. These applications highlight the potential of this platform to be further developed for thermoregulated reversible Zn²⁺ adsorption.

A suitable protein component is also required to achieve Zn²⁺-specific and controllable adsorption within the hybrid PNIPAM hydrogel system. Zap1 is a zinc-responsive transcriptional regulatory protein that mediates Zn²⁺ uptake and vacuolar transporters in Saccharomyces cerevisiae. The Zf1-Zf2 activation domain in Zap1 (hereafter referred to as Zap1zf12) consists of two zinc finger motifs and has been widely employed in Zn²⁺ sensors due to its high affinity and selectivity. − Its exceptional binding properties and relatively simple structure make it well-suited for incorporation into the PNIPAM hybrid system. With two binding sites, Zap1zf12 enhances the Zn²⁺ adsorption efficiency of the hybrid system and extends it to support dual-site protein integration. Notably, it is necessary to rationally design the cross-linking sites within Zap1zf12. Such a design facilitates the effective conformational switching of Zap1zf12 induced by PNIPAM, which is fundamental for temperature-controlled Zn²⁺ capture and release.

Here, we developed a novel protein-based adsorbent material (PNIPAM-co-Zap1zf12) for selective Zn²⁺ removal by combining the high Zn²⁺ selectivity of Zap1zf12 with the thermoresponsive behavior of PNIPAM. The construction strategy focused on optimizing the cross-linking site design in Zap1zf12, the protein modification process, and the hydrogel polymerization procedure (Figure ). The hydrogel enabled Zn²⁺ capture at 37 °C and release at 25 °C via temperature-induced shrinking and swelling, allowing for regeneration without the use of competitive agents. It achieved a 98.4 ± 7.3% removal efficiency in Zn²⁺ solutions, and selectively removed Zn²⁺ from various minimal culture media without significantly interfering with other metal ions such as Mg²⁺, Ca²⁺, and Fe³⁺. Therefore, this material expands the versatility of hybrid PNIPAM hydrogels to include selective adsorption of Zn²⁺ and integration of proteins with dual binding sites. In addition, it holds potential as a practical tool for constructing Zn²⁺-deficient culture environments.

1.

Flow diagram of the synthesis of the Zap1zf12 hybrid hydrogel (PNIPAM-co-Zap1zf12).

2. Materials and Methods

2.1. Bacterial Strains, Chemical Reagents, and Culture Media

All plasmid constructions were carried out in Escherichia coli DH5α, while expression assays were conducted in E. coli BL21 (DE3). The bacteria were cultured in Luria-Bertani (LB) broth.

Standard solutions of Mg²⁺, Ca²⁺, Fe³⁺, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, and Zn²⁺ were purchased from the National Institute of Metrology (China). N-isopropylacrylamide (NIPAM) and N-acryloxysuccinimide (NAS) were obtained from Bidepharm, with NIPAM recrystallized three times using 2:3 (v/v) hexane/methylbenzene mixture. Chelex 100 resin was sourced from Sigma-Aldrich. The compositions of the culture media used in this study are listed in Table S1.

2.2. Construction, Expression and Purification

The expression plasmid pET30a-12His-ELP-Zap1zf12 is illustrated in Figure S1. The genes encoding 12His-ELP-Zap1zf12 and its mutants were codon-optimized and synthesized by GenScript, then cloned into the expression plasmid between the EcoRI and HindIII sites. The relevant sequences are provided in Table S2.

The expression strains were cultured at 37 °C overnight, then diluted 1:100 into 1 L of LB medium. When the OD₆₀₀ reached 0.5, expression was induced with 0.05 mmol/L IPTG for 8 h at 25 °C.

Cells from 1 L of culture were resuspended in 25 mL of lysis solution [10 mmol/L Tris–HCl (pH = 7.4), 150 mmol/L NaCl, 10% (v/v) glycerol, 5 mmol/L 2-mercaptoethanol (β-Me), 0.1 mmol/L phenylmethylsulfonyl fluoride] and lysed by sonication for 20 min. The lysate was centrifuged and filtered through a 0.45 μm membrane, and the supernatant was loaded onto a HisTrap Ni-NTA HP column. After equilibration with 18% buffer B [Buffer A: 10 mmol/L Tris–HCl (pH = 7.4), 500 mmol/L NaCl; Buffer B: buffer A containing 500 mM imidazole], the target protein was eluted with 50% buffer B. The eluted fraction was then concentrated and buffer-exchanged in buffer C [50 mM Tris–HCl (pH = 7.4), 100 mM NaCl, 10 mM β-Me] for fusion tag cleavage by HRV3C protease. Following a 2 h digestion, the samples were loaded onto a HisTrap Ni-NTA HP column and washed with buffer A. The tag-cleaved Zap1zf12 protein was eluted with 15% buffer B, concentrated, and finally purified by gel filtration on a HiLoad 26/60 Superdex 200 column pre-equilibrated with buffer A.

2.3. Modification of Zap1zf12 Using N-Acryloxysuccinimide (NAS)

N-acryloxysuccinimide (NAS) was used to introduce acryloyl groups into the α-NH₂ group of the N-terminus and the ε-NH₂ group of the lysine residues in proteins. Purified Zap1zf12 and its mutants were prebound with Zn²⁺ to protect the metal-binding sites before being buffer-exchanged into the modification buffer [10 mM phosphate buffer (pH = 8.0)]. NAS dissolved in DMSO was added to the protein solution at a molar ratio of 1:1 relative to the number of modification sites. The reaction was carried out at 25 °C for 5 min and was then quenched by adding excess Tris–HCl (pH = 8.0). The modified proteins were desalted and analyzed by MALDI-TOF-MS.

2.4. Synthesis of Hybrid Hydrogels

The modified proteins were exchanged into the synthesis buffer [10 mM phosphate buffer (pH = 8.0)] in preparation for hydrogel synthesis. Hybrid hydrogels were synthesized via free radical polymerization. 220 mg of NAS was dissolved in 1.7 mL of ddH₂O and added to a 25 mL three-necked round-bottomed flask, followed by the addition of 100 μL of N,N-methylenebis­(acrylamide) (BIS) (22 mg/mL) and 100 μL of Na₂SO₃ (10 mg/mL). The mixture was gently stirred at 20 °C for 10 min with nitrogen venting. Polymerization was initiated by adding 100 μL of K₂S₂O₈ (40 mg/mL). After 1 min, 1 mL of modified protein (0.7 mg/mL) was introduced into the reaction. The hybrid hydrogels formed within approximately 20 min under continuous nitrogen flow. The resulting hydrogels were then transferred to 50 mL centrifuge tubes and subjected to at least five swelling–shrinking cycles in the washing buffer [10 mM Bistris (pH 7.0), 5 mmol/L TCEP] to remove unconjugated proteins and residual reactants.

Each swelling–shrinking cycle involved swelling the hydrogel at 25 °C for 6 h until fully swollen, followed by incubation at 37 °C for 6 h until fully shrunken.

2.5. Characterization of Hybrid Hydrogels

Protein samples were analyzed by sodium dodecyl sulfide-polyacrylamide gel electrophoresis (SDS-PAGE) using a GenScript SurePAGE Bistris Gels (4–12%) with MOPS SDS running buffer at a voltage of 180 V. The LCSTs of hybrid hydrogels containing different protein amounts (0, 0.1, 0.5, and 1.0 mg/mL) were determined using a Mettler DSC823e differential scanning calorimeter (DSC) under a nitrogen atmosphere. The scans were conducted from 29 to 41 °C at a heating rate of 2.0 °C/min The structure of lyophilized hydrogels was characterized using a Hitachi S-4800 scanning electron microscope at an accelerator voltage of 10.0 kV. The swelling ratio was calculated as the mass ratio of the hydrogel in its swollen state to that in its shrunken state.

2.6. Characterization of Hybrid Hydrogels for Metal Enrichment

The Zn²⁺ capture and release properties of the hybrid hydrogels were evaluated as follows. After a complete swelling–shrinking cycle (swelling at 25 °C for 6 h until fully swollen, then incubating at 37 °C for 6 h until fully shrunken) in 20 mL of 10 μmol/L Zn²⁺ solution (Figure S2), the hydrogels were divided equally into two portions. One portion was used to assess Zn²⁺ capture capacity, and the other to evaluate release performance after five additional swelling–shrinking cycles in the washing buffer. Zn²⁺ concentrations in both portions were measured by ICP–MS after acidification with HNO₃. Hydrogels without proteins were processed in parallel as a negative control.

The metal ion selectivity of the hybrid hydrogels was assessed by incubating them in a mixed-ion solution containing Mg²⁺, Ca²⁺, Fe³⁺, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, and Zn²⁺ (10 μmol/L each, 20 mL total volume) through a swelling–shrinking cycle. Next, the shrunken hydrogels were immersed in the washing buffer at 37 °C for 30 min, with this step repeated five times. Finally, the washed hydrogels were acidified, and the metal content was analyzed by ICP–MS. The distribution coefficient (K _d) of each metal ions was calculated as follows

$$K_{\mathrm{d}}=\frac{(C_{\mathrm{i}}-C_{\mathrm{e}})\times V}{C_{\mathrm{e}}\times m}$$

where C _i and C _e represent the initial and equilibrium concentrations of metal ions in the solution (μmol/L), respectively; V is the solution volume (mL); and m is the mass of the adsorbent (g). The masses of the fully swollen hydrogels were used to calculate the distribution coefficient.

To examine Zn²⁺ capture-release recyclability, the hydrogels underwent one swelling–shrinking cycle in Zn²⁺ solution (10 μmol/L, 20 mL) followed by five washing cycles. This process was repeated five times, and the residual Zn²⁺ concentration in the solution was measured by ICP–MS after each repetition.

The selective Zn²⁺ removal performance of the hybrid hydrogels was evaluated in several culture media (compositions listed in Table S1). The hydrogels were immersed in 20 mL of each medium at 25 °C for 12 h, followed by a shrinking phase at 37 °C for 5 h. In parallel, 1.0 g of Chelex 100 resin was incubated with 20 mL of each medium at 25 °C for 12 h. For both treatments, culture media were filtered through a 0.22 μm membrane and analyzed by ICP–MS before and after incubation.

Error values for all experimental data represent the mean ± standard deviation (SD) from three independent parallel experiments, and the combined standard deviations were estimated using uncertainty propagation methods. For data expressed as percentages, slight exceedances above 100% result from statistical variation in calculations and do not represent physically meaningful values. All data are physically limited to the range of [0–100%].

2.7. Quantitative Real-Time PCR (qPCR)

The transcription levels of ZRT1, ZRT2, and ZRT3 genes in S. cerevisiae were analyzed using quantitative real-time PCR (qPCR) assays. After overnight culture in yeast nitrogen base medium and the hydrogel-treated yeast nitrogen base medium, S. cerevisiae cells were harvested for total RNA extraction. Total RNA was isolated using the Bacterial Total RNA Rapid Extraction Kit (Sangon, China) following the manufacturer’s instructions. Reverse transcription reactions were carried out with 100 ng RNA using iScript cDNA Synthesis Kit (Bio-Rad). The resulting cDNA was used for qPCR with SsoFast EvaGreen Supermix (Bio-Rad). The primer sequences were as follows: for the control gene ACT1, ACT1-F (5′-CCAGAAGCTTTGTTCCATCC-3′) and ACT1-R (5′-ACGGACATCGACATCACACT-3′); for ZRT1, ZRT1-F (5′-CCACTTAATGGACCCTGCTT-3′) and ZRT1-R (5′-ATGGCAGGACACCATGAATA-3′); for ZRT2, ZRT2-F (5′-TCGAGGAGGAAGATAAAGAGCA-3′) and ZRT2-R (5′-CAGATAGCGAAAGACCCACAA-3′); for ZRT3, ZRT3-F (5′-TGAGCGTTACTGCAGGGTTC-3′) and ZRT3-R (5′-GTGCCTGAGCTATGGGACTG-3′). mRNA expression levels were quantified using the comparative C _T (2^{–ΔΔC _T} ) method.

3. Results and Discussion

3.1. Design of Zap1zf12 Cross-Linking Sites

To construct an applicable hybrid hydrogel, the Zap1zf12 protein was employed as a functional module and immobilized within the hydrogel matrix via cross-linking sites. When the temperature changes between 25 and 37 °C, the hydrogel undergoes reversible swelling and shrinking, which induces corresponding structural disruption and restoration of the immobilized protein through the cross-linking sites, thereby enabling reversible Zn²⁺ adsorption. Zap1zf12 contains two Cys₂His₂-type zinc finger domains, with each domain consisting of four critical residues for Zn²⁺ coordination (Figure a). The coordinating residues of the first domain are Cys5, Cys10, His23, and His28, while those of the second domain are Cys42, Cys47, His60, and His65. Careful design of cross-linking sites in each zinc finger is essential to achieve PNIPAM-driven Zn²⁺ capture and release. Previous studies, including the development of PNIPAM-co-CadRpep (a cadmium-adsorbing hydrogel) and the investigation of cross-linking sites in PNIPAM-co-SUP (a uranium-adsorbing hydrogel), have provided valuable insights into cross-linking site design. , For instance, a single cross-linking site may fail to regulate protein function effectively, whereas placing cross-linking sites near critical residues can enhance regulatory control. In addition, any lysine residues that do not participate in cross-linking should be replaced by arginine to prevent unintended modifications. Therefore, it is preferable to design cross-linking sites based on the lysine residues in Zap1zf12 (Lys6, Lys8, Lys26, and Lys34), as this approach minimizes the potential adverse effects of introducing additional mutations.

2.

Schematic diagrams of Zap1zf12 and its mutants (Zap1zf12-I to Zap1zf12-V) for cross-linking site design. (a) Protein structure of Zap1zf12 (PDB: 1ZW8). The two zinc finger domains are colored green and blue, with the cross-linking sites highlighted in orange. (b) Design strategy for cross-linking sites in Zap1zf12. The α-helices of Zap1zf12 are represented as cylinders, and the structure is simplified into a cartoon model. Cross-linking sites within proteins are shown as orange triangles. (c) Protein sequences of Zap1zf12 and its mutants. Residues designated for cross-linking (including the N-terminal residue) are marked with orange boxes, while mutated residues are shown in red. Critical residues in the two zinc fingers are indicated with green and blue boxes.

Four Zap1zf12 variants were designed to explore optimal cross-linking positions (Figure b,c). In the control mutant Zap1zf12-I (K6R, K8R, K26R, K34R), all lysine residues were mutated to arginine, leaving only the N-terminal residue available as a cross-linking site. Zap1zf12 V (K6R, K8E, E9K, K34R, D46K, A63K) was designed with two cross-linking sites for each zinc finger, yielding five sites in total. For the first zinc finger, the native Lys26 was retained for cross-linking. Glu9 was selected instead of Lys8 because it is closer to the critical residue Cys10 and exhibits a more favorable orientation for efficient stretching (Figure S3a). For the second zinc finger, Asp46 and Ala63 were chosen for cross-linking. These two residues are oriented in opposite directions and located near the critical sites Cys47 and His65, respectively (Figure S3b). Zap1zf12-III (K6R, K8E, E9K, K26R, K34R, A63K) and Zap1zf12-IV (K6R, K8E, E9K, K26R, A63K) were derived from Zap1zf12 V to investigate whether a simplified cross-linking site design could support reversible capture-release behavior in a dual-site protein hybrid hydrogel. These variants contain three or four cross-linking sites (including the N-terminal site), respectively.

Wild-type Zap1zf12 and all its variants were constructed, expressed, and purified, followed by zinc-binding affinity assays (Figure S4). All four variants (Zap1zf12-I to Zap1zf12 V) exhibited zinc-binding affinities comparable to that of the wild-type protein, with dissociation constants (K _d) in the 10^–10 M range for the first zinc fingers and 10^–6 M² for the second zinc fingers. These results indicate that the mutations in Zap1zf12 variants did not significantly affect their Zn²⁺ binding affinities.

3.2. Protein Modification Strategy

Before cross-linking into the hydrogel, the α-NH₂ groups of the N-terminal sites and the ε-NH₂ groups of lysine residues in the proteins were modified by NAS to introduce acryloyl groups (Figure a). Zn²⁺ was bound to the proteins before NAS modification to protect critical residues. ,, Without this protection, the zinc-binding affinity of the proteins decreased notably, and the specificity of cross-linking site modification was adversely affected (Figure S5a,b). NAS modification moderately affected the Zn²⁺-binding affinity of Zap1zf12 mutants. After correct modification (Figure b), the K _d values of the first zinc fingers shifted from the 10^–10 M range to the 10^–9 M range across all mutants, while the binding affinities of the second zinc fingers remained largely unaffected (Figure S5c). Although the Zn²⁺-binding affinity decreased by an order of magnitude, the nanomolar K _d values were still sufficient for Zap1zf12 mutants to efficiently capture Zn²⁺ in solution.

3.

Modification of protein with NAS. (a) Reaction mechanism of NAS-mediated protein modification. (b) MALDI-TOF spectra of Zap1zf12 mutants before (black) and after (red) modification.

3.3. Characterization of the Zap1zf12-Based Hybrid Hydrogel

In the SDS-PAGE analysis, the noticeably upshifted band of the NIPAM-polymerized protein indicated that the modified proteins were incorporated into the PNIPAM network (Figure a). Differential scanning calorimetry (DSC) results revealed that the LCST of the hybrid hydrogels increased with higher protein concentrations, further confirming the protein-hydrogel cross-linking (Figure b). Temperature switching between 25 and 37 °C had little effect on the Zn²⁺ binding ability or on the overall structure of Zap1zf12 (Figure S6). In contrast, the hydrogel matrix exhibited dramatic shape changes during these temperature changes, and the generated stretching forces could contribute to the disruption of the protein binding structure, thereby facilitating effective Zn²⁺ capture and release (Figure c). Moreover, the porous structure of the hydrogel supported ion exchange between the cross-linking proteins and the treated solution (Figure d).

4.

Characterization of the cross-linking, thermoresponsive, and structural properties of the hybrid hydrogels. (a) SDS-PAGE analysis of the cross-linking proteins. Lane 1: purified Zap1zf12 V protein, lane 2: modified Zap1zf12-V, lane 3: NIPAM polymer, lane 4: physical mixture of NIPAM and Zap1zf12 V, and lane 5: PNIPAM-Zap1zf12 V polymer (synthesized without BIS). A significant upward shift was observed for the PNIPAM–Zap1zf12 V polymer band. (b) DSC thermograms of the PNIPAM hydrogels with 0, 0.1, 0.5, and 1.0 mg/mL Zap1zf12 V. (c) Thermoresponsive behavior of the PNIPAM-co-Zap1zf12 hydrogel (the swelling ratio ∼15.0). (d) SEM images of the PNIPAM-co-Zap1zf12 hydrogel in its shrunken state at 37 °C.

Zn²⁺ release ratios (i.e., amount of released Zn²⁺/amount of captured Zn²⁺ by the hydrogels) were analyzed to determine the optimal cross-linking site design in Zap1zf12 for effective Zn²⁺ capture and release regulation. The Zap1zf12-I hybrid hydrogel exhibited a low Zn²⁺ release ratio of 14.1 ± 3.6%, indicating that a single cross-linking site can immobilize the proteins for Zn²⁺ capture but is insufficient to achieve adequate Zn²⁺ release. In contrast, PNIPAM-co-Zap1zf12 V demonstrated the best capture-release performance, with 97.1 ± 9.8% of the bound Zn²⁺ successfully desorbed. In this construct, each zinc finger of Zap1zf12 V contains two oppositely oriented cross-linking sites located near critical residues. This arrangement promoted the efficient disruption of the coordinating structure when the PNIPAM network swelled below the LCST, weakening Zn²⁺ binding. Conversely, when the network shrank above the LCST, the coordinating structure was restored, allowing reversible Zn²⁺ adsorption. The simplified designs of Zap1zf12-III and Zap1zf12-IV failed to achieve comparable efficiency, with release ratios of only 63.9 ± 2.6% and 80.9 ± 8.6%, respectively. Overall, the results demonstrate that strategic cross-linking site engineering in Zap1zf12 is essential for optimizing reversible Zn²⁺ adsorption (Figure a). Zap1zf12 V was therefore chosen for the construction of PNIPAM-co-Zap1zf12.

5.

Properties of PNIPAM-co-Zap1zf12 for Zn²⁺ adsorption. (a) Capture and release performance of hydrogels containing different cross-linking Zap1zf12 mutants. Release ratios = amount of released Zn²⁺/amount of captured Zn²⁺ by the hydrogels. (b) Zn²⁺ adsorption capacities of PNIPAM-co-Zap1zf12. “Before”: Zn²⁺ concentrations before treatment; “PNIPAM”: Zn²⁺ concentrations after treatment with PNIPAM; “PNIPAM-co-Zap1zf12”: Zn²⁺ concentrations after treatment with the hybrid hydrogel. Removal ratios = amount of Zn²⁺ removed/initial amount of Zn²⁺ in solutions. (c) Recyclability of PNIPAM-co-Zap1zf12 hydrogel for Zn²⁺ capturing, showing the amount of Zn²⁺ adsorbed in the hydrogel in each cycle. (d) Metal selectivity of PNIPAM-co-Zap1zf12, illustrating each metal’s percentage of total adsorption. Adsorption percentage = amount of each metal ion adsorbed/total amount of all metals adsorbed by the hydrogels. (e) Zn²⁺ removal efficiency (amount of Zn²⁺ remaining/initial amount of Zn²⁺ in media) of PNIPAM and PNIPAM-co-Zap1zf12 hydrogels in different culture media. Abbreviations: YNB, yeast nitrogen base medium; Richard’s, Richard’s synthetic medium; Fm NRRL 13616, Fusarium. moniliforme NRRL 13616 culture medium. (f) Relative transcription levels of ZRT1, ZRT2, and ZRT3 in S. cerevisiae cultured in YNB and hydrogel-treated YNB medium.

Reaction temperature, protein amount, and protein addition timing were further refined to optimize the hybrid hydrogel system. Performance was evaluated by analyzing the swelling ratios (Table S3) and Zn²⁺ capture-release capabilities (Figure S7). The hydrogel prepared at 25 °C exhibited the highest swelling ratio, while a 1 min delay before protein addition resulted in the greatest Zn²⁺ adsorption. Although adding 1.0 mg of protein increase Zn²⁺ capture capacity, excessive protein incorporation also raised the cross-linking density, which in turn hindered complete hydrogel shrinkage during Zn²⁺ release and reduced the overall release ratio. Therefore, the optimal conditions were finally determined as follows: a reaction temperature of 25 °C, the addition of 0.7 mg Zap1zf12 V protein per hydrogel, and a 1 min delay before protein addition.

3.4. Application of Zap1zf12-Based Hybrid Hydrogel for Zn²⁺ Removal

The optimal hybrid hydrogel was used for Zn²⁺ adsorption, allowing a reduction of Zn²⁺ concentrations from the micromolar range to the tens of nanomolar levels. PNIPAM showed no significant Zn²⁺ removal, indicating that adsorption is mediated by the cross-linked Zap1zf12 proteins. In a system with 804.1 ± 41.9 nmol/L Zn²⁺, PNIPAM-co-Zap1zf12 removed 98.4 ± 7.3% of the Zn²⁺, leaving only 12.6 ± 5.8 nmol/L in solution. In another system containing 1.4 ± 0.1 μmol/L Zn²⁺, the residual Zn²⁺ concentration was 65.5 ± 17.7 nmol/L, corresponding to a 95.2 ± 12.3% removal ratio (Figure b). Saturation binding experiments revealed that the hydrogel reached 66.2 ± 8.2% of its theoretical maximum binding capacity (Table S4), which is similar to that of the previously reported PNIPAM-co-CadRP. PNIPAM-co-Zap1zf12 maintained stable performance over multiple capture-release cycles, with consistent adsorption for four cycles and only a slight decrease in the fifth (Figure c). This hydrogel also demonstrated notable selectivity (Figure d, Table S5), primarily binding Zn²⁺ without detectable adsorption of Mg²⁺, Ca²⁺, Fe³⁺, or Mn²⁺. Slight adsorption of Co²⁺, Ni²⁺, and Cu²⁺ was observed. However, these metals are typically present at low concentrations in culture media, in contrast to the high levels of Mg²⁺, Ca²⁺, and Fe³⁺. As a result, the unintended binding had minimal impact on the Zn²⁺ removal performance of PNIPAM-co-Zap1zf12. This Zn²⁺-selective behavior was maintained during repeated capture-release cycles (Figure S8a). Furthermore, PNIPAM-co-Zap1zf12 exhibited stable Zn²⁺ adsorption over a pH range of 5–8, covering the typical pH of common culture media (Figure S8b).

We evaluated the practical application of PNIPAM-co-Zap1zf12 for selective Zn²⁺ removal in various culture media (Figure e, with metal concentrations before and after treatment provided in Table S6). To establish a zinc-depleted culture condition for S. cerevisiae, the Zn²⁺ concentration in yeast nitrogen base medium should be maintained below 100 nmol/L. However, due to the widespread presence of Zn²⁺, it often exceeds this limit (about 300 nmol/L in our system, Table S6) even without the addition of zinc salts. Traditionally, Chelex 100 resin has been used to remove the background excess Zn²⁺, followed by resupplementation of other essential metal ions. , In our experiments, Chelex 100 reduced Zn²⁺ from 306.9 ± 19.0 nmol/L to 48.9 ± 15.7 nmol/L, while also removing about 70% of other metal ions including Mn²⁺, Fe³⁺, Cu²⁺, Ca²⁺, and Mg²⁺. In contrast, PNIPAM-co-Zap1zf12 achieved comparable Zn²⁺ depletion with minimal impact on other metal ions, showing only a slight reduction in Cu²⁺ (from 704.0 ± 36.4 nmol/L to 555.1 ± 41.1 nmol/L). The remaining Zn²⁺ concentration (68.2 ± 9.5 nmol/L) was below the critical threshold of 100 nmol/L (Figure S9a). The treated media were used to culture S. cerevisiae for further evaluation. The transcription levels of ZRT1, ZRT2, and ZRT3 genes were assessed, which are known to increase in zinc-limited cells. ,− Cells cultured in hydrogel-treated media exhibited upregulation of these genes compared with those grown in standard yeast nitrogen base medium (Figure f), confirming that the hybrid hydrogel effectively removed Zn²⁺ from the media. Overall, these results reveal that PNIPAM-co-Zap1zf12 provides a more convenient and practical alternative for generating zinc-deficient conditions in S. cerevisiae .

The hydrogel further demonstrated selective Zn²⁺ depletion in other chemically defined minimal media, including F. moniliforme NRRL 13616 medium, M9 medium, Czapek-Dox medium, and Richard’s synthetic medium (Figure S9b-e). These media are widely used in Zn²⁺-dependent studies of bacteria and fungi, highlighting the potential applicability of PNIPAM-co-Zap1zf12 in such studies.

However, the Zn²⁺ removal efficiency of PNIPAM-co-Zap1zf12 remains limited in complex media, which typically contain undefined organic components. In LB medium, the hybrid hydrogel removed only about 21.1 ± 6.6% of Zn²⁺, leaving 5.4 ± 0.1 μmol/L in solution (Figure S9f). The limited performance may result from nonspecific interactions between medium components and the hydrogel matrix, thereby hindering the cross-linked Zap1zf12 proteins from accessing and competing for Zn²⁺. As a result, further optimization is needed to enhance the applicability of PNIPAM-co-Zap1zf12 in complex systems.

4. Conclusion

In this study, we developed a Zap1zf12-based hybrid PNIPAM hydrogel (PNIPAM-co-Zap1zf12) through the optimization of cross-linking site design, modification conditions, and hydrogel synthesis. This hydrogel achieved reversible Zn²⁺ adsorption in response to a mild, biocompatible temperature shift between 25 °C (swollen state) and 37 °C (shrunken state), enabling regeneration without competitive chelators. It also demonstrated a high Zn²⁺ removal efficiency of 98.4 ± 7.3%, reducing the concentration from 804.1 ± 41.9 nmol/L to 12.6 ± 5.8 nmol/L. Compared to the nonspecific binding of Chelex 100 resin, PNIPAM-co-Zap1zf12 selectively removed Zn²⁺ from minimal culture media without significantly affecting other essential metal ions such as Mg²⁺, Ca²⁺, and Fe³⁺. However, its performance in complex systems remained suboptimal, indicating the need for further improvement. Overall, PNIPAM-co-Zap1zf12 extends the family of hybrid PNIPAM hydrogels by integrating dual metal-binding site proteins and enabling selective Zn²⁺ capture. Furthermore, this hydrogel provides a promising strategy for constructing Zn²⁺-deficient systems, underscoring the versatile application potential of intelligent biohydrogel materials.

Glossary

Abbreviations

PNIPAM

Poly­(N-isopropylacrylamide)

NAS

N-acryloxysuccinimide

BIS

N,N-methylenebis­(acrylamide)

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c08458.

• The following files are available free of charge. Figure S1: schematic diagram of the expression plasmid; Figure S2: schematic diagram of the selective Zn²⁺ removal process using PNIPAM-co-Zap1zf12; Figure S3: structures of the cross-linking sites in the two zinc fingers of Zap1zf12; Figure S4: effects of cross-linking mutations on Zap1zf12; Figure S5: effects of modification on Zap1zf12 variants; Figure S6: effect of temperature change on the modified Zap1zf12 protein; Figure S7: Zn²⁺ capture-release performance of the hybrid hydrogels under different optimization conditions; Figure S8: Characterization of the PNIPAM-co-Zap1zf12 hydrogel; Figure S9: metal ion removal by PNIPAM, PNIPAM-co-Zap1zf12 and Chelex 100 in different media; Table S1: culture media compositions used in this study; Table S2: Primers and gene sequences used in this study; Table S3: The swelling ratios of the hybrid hydrogels in different synthesis conditions; Table S4: saturation binding performance of PNIPAM-co-Zap1zf12; Table S5: distribution coefficients of metal ions adsorbed by the hydrogel; Table S6: Metal content before and after hydrogel treatment in different media (PDF)

Jue Wang: Writingoriginal draft, Data curation, Formal analysis, Investigation, Methodology, Validation, Conceptualization, Project administration, Software, Visualization. Qingyuan Hu: Investigation, Methodology, Validation, Visualization. Chunhong Liu: Data curation, Validation. Yu Feng: Software, Validation. Jiang Zhu: Validation. Tongyang Zhu: Software, Methodology, Validation, Data curation. Hao Chen: Writingreview and editing, Supervision, Resources, Funding acquisition, Conceptualization.

The authors declare no competing financial interest.