Immobilized Enzyme Research Articles

Evaluation of oil transesterification in a packed‐bed reactor containing lipase immobilized in starch–alginate jet cutting beads

AbstractThere has been a growing interest in ecofriendly enzymatic processes. However, enzyme solubility limits the application of many biocatalysts in continuous systems, requiring the development of cost‐effective strategies for enzyme immobilization. Based on this premise, this study investigated the application of lipase immobilized in starch–alginate beads for oil transesterification in a tubular reactor. An economical derivative was produced by immobilizing Eversa Transform 2.0 in 50:50 (w/w) starch–alginate beads using the jet‐cutting technique. The biocatalyst had a particle size of about 500 μm and activity of 138.67 ± 18.53 U g−1. X‐ray photoelectron spectroscopy showed nitrogen content ranging from 6.38% to 7.29%, with uniform distribution of lipase throughout the beads. Nitrogen isotherms were characteristic of mesoporous materials, with an average pore diameter of 48.09 Å and low surface area (0.69 m2 g−1). A face‐centered central composite design was used to study soybean oil transesterification. In the best four runs, the process achieved a mean triglyceride conversion of 45%. High ester productivity levels (2.05 × 10−2% ester g−1 biocatalyst min−1 or 1.5 × 10−4% ester U−1 min−1) were obtained. Biocatalyst reuse led to a twofold increase in ester concentration (14.57% vs 7.7%). These findings confirm the successful development of a low‐cost biocatalyst suitable for use in continuous reactions.

Tuning the Properties of Polyvinylidene Fluoride/Alkali Lignin Membranes to Develop a Biocatalytic Membrane Reactor for an Organophosphorus Pesticide Degradation.

It has been observed that the immobilization of a phosphotriesterase enzyme (PTE) onto polyvinylidene fluoride (PVDF) membranes significantly decreased the enzyme activity, and this negative effect was attributed to the hydrophobic character of the membrane. The indirect indication of this reason was that the same enzyme immobilized on other membrane materials bearing hydrophilic character showed better performance. In this work, we provide direct evidence of the mechanism by immobilizing a PTE on a PVDF membrane hydrophilized by blending it with alkali lignin (AL). The PTE was immobilized on PVDF membrane by a covalent bond with the same procedure used in earlier studies to attribute changes in enzyme activity solely to the wettability properties (and not to the material chemistry). The activity of the PTE immobilized on the PVDF membrane hydrophilized with AL was 50% higher than that of the enzyme immobilized on the PVDF hydrophobic membrane. Further improvements of the membrane structure tailored for the development of a biocatalytic membrane reactor (BMR) were also promoted. In particular, the performance of the BMR was studied as a function of the thickness of the membrane, which allowed us to modulate the residence time into the enzyme-loaded membrane pores while maintaining the flow rate through the pores at a constant.

Enhancement of stability and activity of RSD amylase from Paenibacillus lactis OPSA3 for biotechnological applications by covalent immobilization on green silver nanoparticles

The key challenge to the biotechnological applications of amylases is achieving high activity and stability under extreme pH, temperature and often high levels of enzyme denaturants. This study immobilized a novel raw starch-digesting (RSD) amylase from Paenibacillus lactis OPSA3 on glutaraldehyde-activated silver nanoparticles. Effects of time, glutaraldehyde concentration, pH, temperature, and enzyme concentration on immobilization were studied, and the immobilized enzymes were characterized. pH 9.0 was optimum for the enzyme immobilization. The maximum immobilization efficiency of 82.23 ± 7.99 % was achieved at 25 °C for 120 min. After immobilization, the optimum pH and temperature changed from 9.0 to 11.0 and 60 to 70, respectively. Immobilization reduced the amylase's activation energy (KJ/mol) from the initial 58.862 to 45.449 following immobilization. The Km of the amylase decreased after immobilization, while the Vmax increased. The immobilized amylase showed significantly greater storage and thermal stability than the free amylase. At 80, enzyme half-life (min) and D value (min) increased from 12.33 to 179.11 and 40.94 to 594.98, respectively. The immobilized amylase (80–88 %) had more stability to the effects of the studied surfactants than the free enzyme. It also showed improved stability in the presence of commercial detergents compared to the free enzyme. The amylase's enhanced kinetic parameters and stability following successful immobilization on silver nanoparticles indicate its potential for application in the range of biotechnological processes where alkaline- and temperature-stable amylases are employed.

Integrating sodium cholate-modified MOF hybrid lipase and solubilization of hydrophobic candidates into a step for liganding fishing lipase inhibitors from Nelumbinis Folium

Enzyme immobilization by metal organic frameworks (MOFs) is an efficient way for screening active constituents in natural products. However, the enzyme’s biocatalysis activity is usually decreased due to unfavorable conformational changes during the immobilization process. In this study, sodium cholate was firstly used as the modifier for zeolitic imidazolate framework-8 (ZIF-8) immobilized lipase to increase both the stability and activity. More importantly, with the help of solubilization of sodium cholate, a total of 3 flavonoids and 6 alkaloids candidate compounds were fished out. Their structures were identified and the enzyme inhibitory activities were verified. In addition, the binding information between the candidate compound and the enzyme was displayed by molecular docking. This study provides valuable information for the improvement of immobilized enzyme activity and functional active ingredients in complicated medicinal plant extracts.

In situ immobilization multi-enzyme biocatalytic system on covalent organic frameworks for efficient conversion of lignocellulose to glucose

Efficient enzyme immobilization is crucial for addressing the resource utilization challenges associated with lignocellulose. However, the widespread application of immobilized enzyme systems faces significant obstacles, including low enzyme activity and the limited pore structure of existing carriers. To overcome these challenges, a novel multi-enzyme biocatalytic system (multi-enzymes@COF) was developed for the in situ immobilization of cellulose and β-glucosidase on covalent organic frameworks (COFs). Results showed that multi-enzyme@COF exhibits good crystallinity and a mesoporous structure, leading to an increased enzyme loading rate of 0.6 g/g and enhanced cellulose conversion efficiency of up to 78.7 %. Additionally, multi-enzymes@COF demonstrated remarkable stability a broader pH range (4−7) and temperature range (50–70 ℃), with the actively above 70 %. Moreover, the enzymes maintained approximately 74.7 % of their activity even after seven cycles. This research presents an innovative strategy for the effective utilization of lignocellulose through enzymatic processes, promoting sustainable and efficient resource utilization.

Production of γ-aminobutyric acid by immobilization of two Yarrowia lipolytica glutamate decarboxylases on electrospun nanofibrous membrane

This study harnesses glutamate decarboxylase (GAD) from Yarrowia lipolytica to improve the biosynthesis of γ-aminobutyric acid (GABA), focusing on boosting the enzyme's catalytic efficiency and stability by immobilizing it on nanofibrous membranes. Through recombinant DNA techniques, two GAD genes, YlGAD1 and YlGAD2, were cloned from Yarrowia lipolytica and then expressed in Escherichia coli. Compared to their soluble forms, the immobilized enzymes exhibited significant improvements in thermal and pH stability and increased resistance to chemical denaturants. The immobilization notably enhanced substrate affinity, as evidenced by reduced Km values and increased kcat values, indicating heightened catalytic efficiency. Additionally, the immobilized YlGAD1 and YlGAD2 enzymes showed substantial reusability, maintaining 50% and 40% of their activity, respectively, after six consecutive cycles. These results underscore the feasibility of employing immobilized YlGAD enzymes for cost-effective and environmentally sustainable GABA production. This investigation not only affirms the utility of YlGADs in GABA synthesis but also underscores the advantages of enzyme immobilization in industrial settings, paving the way for scalable biotechnological processes.

Fabricating Polymeric Micelles with Enrichment and Cavity Effect for In Situ Enzyme Imobilization from Natural Biosystems.

Metal-organic frameworks and hydrogen-organic frameworks (MOFs and HOFs) are attractive hosts for enzyme immobilization, but they are limited to immobilizing the purified enzymes, making industrial upscaling unattractive. Herein, aptamer-modified dual thermoresponsive polymeric micelles with switchable self-assembly and core-shell structure are constructed, which enable selective immobilization of trypsin directly from complex biological systems through a cascade operation of separation and immobilization. Their steric self-assembly provides a large amount of adsorption sites on the soluble micellar shell, resulting in high adsorption capacity and excellent selectivity. Meanwhile, their aptamer affinity ligand and cavity maintain the native conformations of trypsin and offer protective effects even in harsh conditions. The maximum adsorption capacity of the polymeric micelles for trypsin was determined to be 197 mg/g at 60 min, superior to those of MOFs and HOFs. 67.2 and 86.6% of its original activity was retained for trypsin immobilized in the cavity under strong alkaline and acidic conditions, respectively.

Purification and immobilization of β-glucosidase using surface modified mesoporous silica Santa Barbara Amorphous 15 for eco-friendly preparation of sagittatoside A

Functionalized mesoporous materials have become a promising carrier for enzyme immobilization. In this study, Santa Barbara Amorphous 15 (SBA-15) was modified by N-aminoethyl-γ-aminopropyl trimethoxy (R). R-SBA-15 was employed to purify and immobilize recombinant β-glucosidase from Terrabacter ginsenosidimutans (BgpA) in one step for the first time. Optimum pH of the constructed R-SBA-15@BgpA were 7.0, and it has 20 ℃ higher optimal temperature than free enzyme. Relative activity of R-SBA-15@BgpA still retained > 70% at 42 ℃ after 8-h incubation. The investigation on organic reagent resistance revealed that the immobilized enzyme can maintain strong stability in 15% DMSO. In leaching test and evaluation of storage stability, only trace amount of protein was detected in buffer of the immobilized enzyme after storage at 4 ℃ for 33 days, and the immobilized BgpA still maintained > 50% relative activity. It also demonstrated good reusability, with 76.1% relative activity remaining after fourteen successive enzymatic hydrolyses of epimedin A to sagittatoside A. The newly proposed strategy is an effective approach for the purification and immobilization of BgpA concurrently. In addition, R-SBA-15@BgpA was demonstrated to have high efficiency and stability in this application, suggesting its great feasibility and potential to produce bioactive compounds such as secondary glycosides or aglycones from natural products.Graphical

Activation of Inert Supports for Enzyme(s) Immobilization Harnessing Biocatalytic Sustainability for Perennial Utilization.

Although Nature's evolution and intelligence have gifted humankind with noteworthy enzyme candidates to simplify complex reactions with ultrafast, overselective, effortless, mild biological reactions for millions of years, their availability at minute-scale, short-range time-temperature stability, and purification costs hardly justify recycling/or reuse. Covalent immobilization, particularly via multipoint bonds, prevents denaturing, maintains activities for long-range time, pH, and temperature, and makes catalysts available for repetitive usages; which attracts researchers and industries to bring more immobilized enzyme contenders in science and commercial progressions. Inert-support activation, the most crucial step, needs appropriate activators; under mild conditions, the activator's functional group(s) still present on the activated support rapidly couples the enzyme, preventing unfolding and keeping the active site alive. This review summarizes exciting experimental advances, from the 1950s until today, in the activation strategies of various inert supports with five different surface activators, the cyanogen bromide, the isocyanate/isothiocyanate, the glutaraldehyde, the carbodiimide (with or without N-hydroxysuccinimide (NHS)), and the diazo group, for the immobilization of diverse enzymes for broader applications. These activators under mild pH (7.5 ± 0.5) and temperature (27 ± 3 °C) and ordinary stirring witnessed support activation and enzyme coupling and put off unfolding, harnessing addressable activities (CNBr: 40 ± 10%; -N═C═O/-N═C═S: 32 ± 7%; GA: 70 ± 15%; CDI: 60 ± 10%; -N+≡N: 80 ± 15%), while underprivileged stability, longevity, and reusabilities keep future investigations alive.

Revolutionizing protein hydrolysis in wastewater: Innovative immobilization of metagenome-derived protease in periodic mesoporous organosilica with imidazolium framework

This research focused on utilizing periodic mesoporous organosilica with imidazolium framework (PMO-IL), to immobilize a metagenome-sourced protease (PersiProtease1), thereby enhancing its functional efficiency and catalytic effectiveness in processing primary proteins found in tannery wastewater. The successful immobilization of enzyme was confirmed through the use of N2 adsorption-desorption experiment, XRD, FTIR, TEM, FESEM, EDS and elemental analytical techniques. The immobilized enzyme exhibited greater stability in the presence of various metal ions and inhibitors compared to its free form. Furthermore, enzyme binding to PMO-IL nanoparticles (NPs) reduced leaching, evidenced by only 11.41 % of enzyme leakage following a 120-min incubation at 80 °C and 6.99 % after 240 min at 25 °C. Additionally, PersiPro@PMO-IL maintained impressive operational consistency, preserving 62.24 % of its activity over 20 cycles. It also demonstrated notable stability under saline conditions, with an increase of 1.5 times compared to the free enzyme in the presence of 5 M NaCl. The rate of collagen hydrolysis by the immobilized protease was 46.82 % after a 15-minute incubation at 60 °C and marginally decreased to 39.02 % after 20 cycles indicative of sustained efficacy without significant leaching throughout the cycles. These findings underscore the effectiveness of PMO-IL NPs as a viable candidate for treating wastewater containing protein.

Functionalized ZnFe2O4@Mesoporous silica nano-support for lipase enzyme immobilization: Enhanced biocatalysis and antibacterial activity for food industry applications

Enzymes, as highly efficient biocatalysts, play a crucial role in diverse biotechnological applications. However, enzyme (re)purification and recovery challenges pose substantial obstacles, rendering them costly for industrial utilization. Addressing this, our study developed mesoporous zinc ferrite nanoparticles coated with amine-functionalized mesoporous silica structure (ZnFe2O4@MS) through the solvothermal method. These nanoparticles are an effective nano-support for immobilizing Candida rugosa Lipase (CRL), ensuring easy separability and recoverability through a magnetic field and providing a significant surface area for high mass transference capacity. Immobilizing the lipase on the nano-support (ZnFe2O4@MS/CRL) through covalent bonds on the surface and within the pores led to a notable increase in enzyme activity from 324 U/mg for free enzyme to 689 U/mg for immobilized enzyme. This indicates unchanged active sites post-immobilization, accompanied by enhanced catalytic activity. The immobilized lipases demonstrated prolonged stability at elevated temperatures, maintaining over 59% of initial catalytic activity through five cycles. Furthermore, ZnFe2O4@MS/CRL exhibited substantial antibacterial activity against Staphylococcus aureus, more than that observed in Gram-negative bacteria. The immobilized lipase was also effective in synthesizing banana flavor (isoamyl acetate) in n-hexane, achieving 64% esterification at 45 °C after 4 h. Overall, the study underscores the industrial promise of this immobilized enzyme system, emphasizing its potential for sustainable and cost-effective biotechnological processes.

Engineering Oxygen-Independent NADH Oxidase Integrated with Electrocatalytic FAD Cofactor Regeneration.

An electrochemically mediated enzyme process for nicotinamide adenine dinucleotide (NADH) oxidation and biosensing has been developed in which the oxygen-dependent activities of wild-type NADH oxidase are replaced by electrochemical regeneration of the flavin adenine dinucleotide (FAD) cofactor in the active site. Consequently, the present bioelectrocatalysis does not rely on a continuous oxygen supply through bubbling air or pure oxygen in biosynthetic applications, which reduces enzyme stability. The coupled electrochemical and enzymatic catalysis is achieved through a combination of enzyme immobilization on the electrode and electrochemical oxidation of FADH2 in the active site mediated by the electron transfer mediator ferrocene carboxylic acid (FcCA). Furthermore, to minimize the effect of dissolved oxygen when the electrocatalytic process is exposed to air, we successfully designed mutations at the Leu40 and Cys42 sites of Leuconostoc mesenteroides (LmNOx) to block the oxygen passage into the active site and to eliminate the native FAD cofactor regeneration half-reaction. The engineered enzymes, whose activities are significantly reduced or inactive in solution, are electrocatalytically active toward conversion of NADH to NAD+, demonstrating successful FAD cofactor regeneration in the active site via electrochemistry. Finally, we developed two highly responsive electrochemical biosensors for NADH detection which has a superior substrate specific to standard detectors using metal electrodes, and comparable detection range and detection limit (1-3 μM).

Ultrathin Film Antimony-Doped Tin Oxide Prevents [NiFe] Hydrogenase Inactivation at High Electrode Potentials.

For hydrogenases to serve as effective electrocatalysts in hydrogen biotechnological devices, such as enzymatic fuel cells, it is imperative to design electrodes that facilitate stable and functional enzyme immobilization, efficient substrate accessibility, and effective interfacial electron transfer. Recent years have seen considerable advancements in this area, particularly concerning hydrogenases. However, a significant limitation remains: the inactivation of hydrogenases at high oxidative potentials across most developed electrodes. Addressing this issue necessitates a thorough understanding of the interactions between the enzyme and the electrode surface. In this study, we employ ATR-IR spectroscopy combined with electrochemistry in situ to investigate the interaction mechanisms, electrocatalytic behavior, and stability of the oxygen-tolerant membrane-bound [NiFe] hydrogenase from Cupriavidus necator (MBH), which features a His-tag on its small subunit C-terminus. Antimony-doped tin oxide (ATO) thin films were selected as electrodes due to their protein compatibility, suitable potential window, conductivity, and transparency, making them an ideal platform for spectroelectrochemical measurements. Our comprehensive examination of the physiological and electrochemical processes of [NiFe] MBH on ATO thin film electrodes demonstrates that by tuning the electron transport properties of the ATO thin film, we can prevent MBH inactivation at extended oxidative potentials while maintaining direct electron transfer between the enzyme and the electrode.

Encapsulation of Immobilized β-Glucosidase with Calcium Metal-Organic Frameworks for Enhanced Stability in Hydrolysis of Cellobiose.

β-Glucosidase (β-G) holds promising applications in various fields, such as biomass energy, food, pharmaceuticals, and environmental protection, yet its industrial application is still limited by issues of stability and recycling. Herein, we first immobilized β-G onto the surface of magnetic chitosan nanoparticles (MCS/β-G) through adsorption methods. Subsequently, utilizing the metal-organic framework (MOF), CaBDC, which possesses good stability under acidic conditions, we encapsulated MCS/β-G. The resulting biocatalyst (MCS/β-G@CaBDC) exhibited excellent activity and recyclability. MCS/β-G@CaBDC can convert 91.5% of cellobiose to glucose in 60 min and maintained 81.9% activity after 10 cycles. The apparent Km value of MCS/β-G@CaBDC was 0.148 mM, lower than free β-G (0.166 mM) and MCS/β-G (0.173 mM). The CaBDC layer increased the mass transfer resistance of the reaction but also triggered structural rearrangement of β-G during the encapsulation process. This resulted in the β-sheet content rising to 68.4%, which, in turn, contributed to enhancing the rigidity of β-G. Moreover, the saturated magnetic strength of this biocatalyst could reach 37.3 emu/g, facilitating its magnetic recovery. The biocatalyst prepared in this study exhibits promising application prospects, and the immobilization method can provide valuable insights into the field of enzyme immobilization.

Use of tyrosinase nanobiosensor for detection of atrazine and simazine and its application in real water samples

ABSTRACT This study presents the development and characterization of cantilever nanobiosensors functionalized with the tyrosinase enzyme (0.05–0.001%) for the detection of atrazine and simazine in water. The optimization of tyrosinase activity through precise immobilization and sensitive layer adjustments was evaluated. Topographical analysis revealed the successful immobilization of the tyrosinase enzyme using the self-assembled monolayer technique, enhancing sensor sensitivity. Upon comparing the enzyme concentrations, it was found that the 0.006% exhibited remarkable results, including a limit of detection (LOD = 0.0080 ppb) and a limit of quantification (LOQ = 0.0267 ppb), along with enhanced storage stability for 15 days for atrazine detection. For simazine, the nanobiosensor exhibited high sensitivity (8.1532 nm/ppb) and LOD (0.0117 ppb). They demonstrated excellent reversibility (99%) over six cycles and selectivity against interfering pesticides commonly found in agricultural environments, indicating their suitability for complex sample matrices. Real water samples met regulatory standards for atrazine levels, further validating the efficacy of the nanobiosensors in detecting pesticide contamination. The enzymatic nanobiosensor achieved recoveries ranging from 97 to 101%, indicating high reproducibility. These findings demonstrate the potential of the developed nanobiosensors for sensitive and reliable pesticide detection in environmental monitoring, with implications for future applications.

Polydopamine-functionalized polyethersulfone membrane: A paradigm advancement in the field of α-amylase stability and immobilization

Biocatalytic membranes have great potential in various industrial sectors, with the immobilization of enzymes being a crucial stage. Immobilizing enzymes through covalent bonds is a complex and time-consuming process for large-scale applications. Polydopamine (PDA) offers a more sustainable and eco-friendly alternative for enzyme immobilization. Therefore, surface modification with polydopamine as mussel-inspired antifouling coatings has increased resistance to fouling. In this study, α-amylase enzyme was covalently bound to a bioactive PDA-coated polyethersulfone (PES) membrane surface using cyanuric chloride as a linker. The optimal activity of α-amylase enzyme immobilized on PES/PDA membrane was obtained at temperature and pH of 55°C and 6.5, respectively. The immobilized enzyme can be reused up to five reaction cycles with 55 % retention of initial activity. Besides, it maintained 60 % of its activity after being stored for five weeks at 4°C. Additionally, the immobilized enzyme demonstrated increased Michaelis constant and maximum velocity values during starch hydrolysis. The results of the biofouling experiment of various membranes in a dead-end cell demonstrated that the PES membrane’s water flux increased from 6722.7 Lmh to 7560.2 Lmh after PDA modification. Although α-amylase immobilization reduced the flux to 7458.5 Lmh due to enhanced hydrophilicity, compared to unmodified membrane. The findings of this study demonstrated that the membrane produced through co-deposition exhibited superior hydrophilicity, enhanced coating stability, and strong antifouling properties, positioning it as a promising candidate for industrial applications.

Enzymatic phenolics removal by tyrosinase-modified micromotors

In this study, self-propelled Pt/PPy-COOH micromotors were produced by using the sequential electrodeposition of Pt and PPy segments. Fabricated micromotors were modified with tyrosinase enzyme and was used to investigate the phenolic compound removal capacity. The micromotors was characterized by SEM and EDX analyses, and it was found that the shape of the motors was tubular and 10 µm long. The optimum pH values of free and immobilized enzymes were found to be 7.5, while the optimum temperature values were 45 °C. In the thermal stability study, it was determined that the immobilized enzyme showed 75 % activity after 5 h, while the activity of the free enzyme was around 21 %. Also, immobilized tyrosinase showed 55 % activity after 10 repeated uses. Finally, the phenol, p-cresol and o-phenylenediamine removal efficiencies of tyrosinase-based micromotors were found to be 54.54 %, 46.96 % and 53.87 % in 5 min, respectively. As a result, it can be concluded that it is possible to use tyrosinase-based micromotors as an alternative to traditional methods for phenol removal.

Fabrication of hierarchical 3D layered yttrium hydroxide nanocomposites: Their use for enzyme immobilization and catalysis of H2O2

3D layer metal hydroxides (LMHs) have attracted significant interest due to their distinctive structural features. Unfortunately, the limited electrical conductivity hinders the potential utilization of LMHs in electrochemical analysis. To address this issue, herein, gold nanoparticles (AuNPs) were introduced to layered yttrium hydroxides by a facile in-situ electrodeposition method. Subsequently, the hierarchical AuNPs/LYH nanocomposite was employed as solid supports for immobilization of horseradish peroxidase, generating an enzymatic biosensor for hydrogen peroxide detection. As a result, a linear range of 1–500 μM with a remarkably low detection limit of 0.33 μM (S/N=3) was achieved. Moreover, the biosensor also demonstrated excellent selectivity, reproducibility and stability. The superior electrochemical responses of the HRP/AuNPs/LYH nanocomposite towards H2O2 can be ascribed to the synergistic effect arising from the flower-like structures of LYHs and the high conductivity of AuNPs. Our study provides a significant approach for the development of enzyme immobilized supports through the construction of layer metal hydroxide nanocomposites.

Point-of-care testing device platform for the determinationof creatinine on an enzyme@CS/PB/MXene@AuNP-based screen-printed carbon electrode.

Screen-printed carbon electrodes (SPCE) functionalized with MXene-based three-dimensional nanomaterials are reportedfor rapid determinationof creatinine. Ti3C2TX MXene with in situ reduced AuNPs (MXene@AuNP) were used as a coreactant accelerator for efficient immobilization of enzymes. Creatinine could be oxidized by chitosan-embedded creatinine amidohydrolase, creatine amidinohydrolase, or sarcosine oxidase to generate H2O2, which could be electrochemically detected enhanced by Prussian blue (PB). The enzyme@CS/PB/MXene@AuNP/SPCE detected creatinine within the range 0.03-4.0mM, with a limit of detection of 0.01mM, with anaverage recovery of 96.8-103.7%. This indicates that the proposed biosensor is capable of detecting creatinine in a short amount of time (4min) within a ± 5% percentage error, in contrast with the standard clinical colorimetric method. With this approach, reproducible and stable electrochemical responses could be achieved for determinationof creatinine in serum, urine, or saliva. These results demonstrated its potential for deployment in resource-limited settings for early diagnosis and tracking the progression of chronic kidney disease (CKD).

Machine Learning Optimizing Enzyme/ZIF Biocomposites for Enhanced Encapsulation Efficiency and Bioactivity.

In this study, we present the first example of using a machine learning (ML)-assisted design strategy to optimize the synthesis formulation of enzyme/ZIFs (zeolitic imidazolate framework) for enhanced performance. Glucose oxidase (GOx) and horseradish peroxidase (HRP) were chosen as model enzymes, while Zn(eIM)2 (eIM = 2-ethylimidazolate) was selected as the model ZIF to test our ML-assisted workflow paradigm. Through an iterative ML-driven training-design-synthesis-measurement workflow, we efficiently discovered GOx/ZIF (G151) and HRP/ZIF (H150) with their overall performance index (OPI) values (OPI represents the product of encapsulation efficiency (E in %), retained enzymatic activity (A in %), and thermal stability (T in %)) at least 1.3 times higher than those in systematic seed data studies. Furthermore, advanced statistical methods derived from the trained random forest model qualitatively and quantitatively reveal the relationship among synthesis, structure, and performance in the enzyme/ZIF system, offering valuable guidance for future studies on enzyme/ZIFs. Overall, our proposed ML-assisted design strategy holds promise for accelerating the development of enzyme/ZIFs and other enzyme immobilization systems for biocatalysis applications and beyond, including drug delivery and sensing, among others.