Temperature responsive hydrogels combined with enzyme loaded metal organic frameworks actively move substrates and products and retain enzymes, enabling stable reusable catalysis and reliable glucose sensing in harsh environments.
(Nanowerk Spotlight) Enzymes drive many chemical reactions used in biotechnology. They work in water, target specific molecules with precision, and function at temperatures that do not damage proteins or reaction mixtures. These advantages make them useful for converting substrates in a controlled way, but free enzymes in solution are unstable. Heat, solvents, and agitation disrupt their structure. Once dispersed in a reaction, they cannot be retrieved easily and cannot be reused, which limits their value in continuous or large-scale systems.
Immobilization was introduced to keep enzymes stable while maintaining a usable form. Many immobilization methods rely on rigid supports such as silica, polymers, or porous particles. These strategies extend enzyme lifetime but often reduce effectiveness because the enzymes become fixed in place while molecules reach them only by diffusion.
Hydrogels were proposed as an alternative. They are soft, water-rich networks that protect proteins more gently and behave more like biological tissues. Hydrogels can be molded into large shapes and embedded in devices. Their weakness is transport. Molecules move through them slowly, which causes restricted passive mass transfer, delayed access to substrates, and accumulation of reaction products.
Hydrogels used in this way often require disposability and show poor reproducibility. Enlarging pore sizes improves diffusion but increases enzyme leakage, while tightening the network reduces leakage and further restricts transport.
Metal–organic frameworks (MOFs) entered this space because they maintain enzyme retention. These crystalline materials have uniform pores and high internal surface area. Enzymes can be encapsulated inside the frameworks, and small molecules can pass through the pores. The material protects enzymes and increases tolerance to harsh conditions compared with free enzyme.
However, MOF particles are powders. Recovery after catalysis typically requires centrifugation. Enzyme loss during handling is likely. Hydrogels provide a processable bulk form but lack active transport. MOFs protect enzymes but do not directly address how substrates and products move through macroscopic materials.
New work published in Advanced Science (“Smart Hydrogel Doped by Metal–Organic Frameworks for Renewable Self‐Pumping Enzymatic Reactors”) combines these two technologies. Researchers from Tianjin University of Technology designed a hydrogel that contracts and expands in response to temperature changes, and they embedded enzyme-loaded MOFs inside it. The hydrogel structure enables fluid exchange with the surrounding liquid, while the MOFs prevent leakage of enzymes. The design is inspired by the contraction and relaxation of cardiac tissue, which generates pressure gradients for pumping.
A) The diagram of the sustainable contraction/diastole of the heart muscle and blood pumping in/out based on hierarchical tissue structure. B) Schematic illustration of the passive mass exchange promoted by concentration difference (upper) and the active mass exchange promoted by thermo-stimulated cyclic contraction/expansion of MOFs-doped hydrogel (lower). (Image: Reproduced from DOI:10.1002/advs.20250788, CC BY) (click on image to enlarge)
The material is built around poly(N-isopropylacrylamide), or PNIPAM. PNIPAM absorbs water below its lower critical solution temperature of about 32.5 °C and becomes hydrophobic above that temperature. When it becomes hydrophobic, it contracts and expels water. When cooled, it absorbs water and expands. To improve mechanical stability, the researchers copolymerized PNIPAM with acrylamide to form a network referred to as PNA. A nonresponsive polyacrylamide gel, PAM, was used as a control.
The chosen enzyme was glucose oxidase. It converts glucose to gluconic acid and generates hydrogen peroxide. The researchers encapsulated glucose oxidase inside a zeolitic imidazolate framework known as ZIF-8. The composite, labeled GOx@ZIF-8, was formed through in situ crystallization. It achieved a loading of 79 mg g⁻¹. X-ray diffraction confirmed that ZIF-8 crystallinity was preserved. Fourier transform infrared spectroscopy indicated the integration of glucose oxidase. Transmission electron microscopy showed increased surface roughness relative to ZIF-8, consistent with enzyme incorporation.
The MOF particles were then embedded in the hydrogel. Oscillatory rheological measurements confirmed the formation of solid networks in both PNA and PAM composites. GOx@ZIF-8@PNA showed a thermal response, while GOx@ZIF-8@PAM did not. When heated, the PNA composite contracted to about 40 % of its original volume as incubation time increased. Cooling restored volume. The contraction–expansion response was reversible. Microscopy showed macroporous channels in the expanded state that collapsed in the contracted state. PAM retained its pore structure across temperatures. Both hydrogels distributed ZIF-8 particles uniformly.
To test whether the hydrogel could actively transport solutes, the researchers used methylene blue dye. When immersed in dye solution at equal concentration, contracted PNA showed faster and greater adsorption than PAM. When heated to 45 °C, dye release was faster in PNA. After six contraction–expansion cycles, PNA released about 80 % of its absorbed dye, while PAM released about 18 %. The dye moved visibly through the PNA hydrogel during temperature cycling. PAM remained dark blue.
Catalytic performance was measured using a cascade reaction. Glucose oxidase activity produced hydrogen peroxide, and horseradish peroxidase oxidized the substrate ABTS to ABTS⁺, which shows strong absorbance at 418 nm. GOx@ZIF-8@PNA in the contracted state exhibited higher activity than the same composite in its expanded state. Its performance did not differ significantly from GOx@ZIF-8@PAM in the expanded state. Immobilized systems showed lower absolute activity than free enzyme, which is described by the authors as a trade-off for improved recyclability and stability.
The researchers evaluated operating temperature. The bio-hydrogel reached maximum performance at 25 °C. At lower temperatures, pumping was stronger but catalytic activity was reduced. At higher temperatures, activity decreased. They also optimized product expulsion at temperatures above the hydrogel transition point. Using methylene blue dye, they found that 45 °C yielded efficient expulsion, while 50 °C caused excessive contraction that trapped residual product. Longer heating increased contraction and improved pumping strength.
Examining environmental tolerance, the team found that free glucose oxidase lost activity after treatment with acetone, dimethyl sulfoxide, methanol, or 60 °C heat. GOx@ZIF-8 and GOx@PNA provided partial protection. GOx@ZIF-8@PNA retained 98.0 % of activity in acetone, 98.8 % in dimethyl sulfoxide, 73.5 % in methanol, and 79.0 % at 60 °C. Continuous heating for 2 hours had minimal effect.
The team also tested reusability over repeated cycles. GOx@ZIF-8@PNA maintained activity for at least 20 cycles with negligible enzyme leakage. GOx@PNA without MOF leaked during heating and showed no recyclability. Free glucose oxidase could not be recovered. The authors note that MOF powders can be recovered but require centrifugation and are prone to enzyme loss.
The team applied the platform to other enzymes. Cytochrome c@ZIF-8 and laccase@ZIF-90 were prepared using in situ encapsulation and embedded in PNA. Both showed higher activity in the contracted hydrogel than in the nonresponsive PAM material. A two-enzyme system containing GOx@ZIF-8 and Cyt c@ZIF-8 displayed improved cascade reaction when operated with active transfer.
The composite was evaluated as a glucose sensor. GOx@ZIF-8@PNA showed a linear relationship between glucose concentration and absorbance from 0 to 8 mM with a detection limit of 230 μM. Measurements in artificial perspiration, pig serum, and fetal bovine serum agreed with commercial glucose kits. Other biomolecules at tenfold higher concentration produced negligible signal. The sensing ability was preserved after storage at room temperature for 40 days.
This work presents a system in which mass transfer and enzyme retention are addressed separately. The hydrogel uses temperature-induced contraction and expansion to move solutes in and out, and the MOFs prevent enzyme leakage and provide protection. The combination enables enzymatic materials to be reused repeatedly while maintaining performance.
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