Amino Functionality Enables Aqueous Synthesis of Carboxylic Acid-Based MOFs at Room Temperature by Biomimetic Crystallization.

Enzyme immobilization within metal-organic frameworks (MOFs) is a promising solution to avoid denaturation and thereby utilize the desirable properties of enzymes outside of their native environments. The biomimetic mineralization strategy employs biomacromolecules as nucleation agents to promote the crystallization of MOFs in water at room temperature, thus overcoming pore size limitations presented by traditional postassembly encapsulation. Most biomimetic crystallization studies reported to date have employed zeolitic imidazole frameworks (ZIFs). Herein, we expand the library of MOFs suitable for biomimetic mineralization to include zinc(II) MOFs incorporating functionalized terephthalic acid linkers and study the catalytic performance of the enzyme@MOFs. Amine functionalization of terephthalic acids is shown to accelerate the formation of crystalline MOFs enabling new enzyme@MOFs to be synthesized. The structure and morphology of the enzyme@MOFs were characterized by PXRD, FTIR, and SEM-EDX, and the catalytic potential was evaluated. Increasing the linker length while retaining the amino moiety gave rise to a family of linkers; however, MOFs generated with the 2,2'-aminoterephthalic acid linker displayed the best catalytic performance. Our data also illustrate that the pH of the reaction mixture affects the crystal structure of the MOF and that this structural transformation impacts the catalytic performance of the enzyme@MOF.

Amination of naringinase to improve citrus juice debittering using a catalyst immobilized on glyoxyl-agarose.

A naringinase complex was chemically aminated prior to its immobilization on glyoxyl-agarose to develop a robust biocatalyst for juice debittering. The effects of amination on the optimal pH and temperature, thermal stability, and debittering performance were analyzed. Concentration of amino groups on catalysts surface increased in 36 %. Amination reduced the ß-glucosidase activity of naringinase complex; however, did not affect optimal pH and temperature of the enzyme and it favored immobilization, obtaining α-l-rhamnosidase and ß-d-glucosidase activities of 1.7 and 4.2 times the values obtained when the unmodified enzymes were immobilized. Amination favored the stability of the immobilized biocatalyst, retaining 100 % of both activities after 190 h at 30 °C and pH 3, while its non-aminated counterpart retained 80 and 52 % of α-rhamnosidase and ß-glucosidase activities, respectively. The immobilized catalyst showed a better performance in grapefruit juice debittering, obtaining a naringin conversion of 7 times the value obtained with the non-aminated catalyst.

Bioremediation of Hazardous Pollutants Using Enzyme-Immobilized Reactors.

Bioremediation uses the degradation abilities of microorganisms and other organisms to remove harmful pollutants that pollute the natural environment, helping return it to a natural state that is free of harmful substances. Organism-derived enzymes can degrade and eliminate a variety of pollutants and transform them into non-toxic forms; as such, they are expected to be used in bioremediation. However, since enzymes are proteins, the low operational stability and catalytic efficiency of free enzyme-based degradation systems need improvement. Enzyme immobilization methods are often used to overcome these challenges. Several enzyme immobilization methods have been applied to improve operational stability and reduce remediation costs. Herein, we review recent advancements in immobilized enzymes for bioremediation and summarize the methods for preparing immobilized enzymes for use as catalysts and in pollutant degradation systems. Additionally, the advantages, limitations, and future perspectives of immobilized enzymes in bioremediation are discussed.

A New Approach in Lipase-Octyl-Agarose Biocatalysis of 2-Arylpropionic Acid Derivatives.

The use of lipase immobilized on an octyl-agarose support to obtain the optically pure enantiomers of chiral drugs in reactions carried out in organic solvents is a great challenge for chemical and pharmaceutical sciences. Therefore, it is extremely important to develop optimal procedures to achieve a high enantioselectivity of the biocatalysts in the organic medium. Our paper describes a new approach to biocatalysis performed in an organic solvent with the use of CALB-octyl-agarose support including the application of a polypropylene reactor, an appropriate buffer for immobilization (Tris base-pH 9, 100 mM), a drying step, and then the storage of immobilized lipases in a climatic chamber or a refrigerator. An immobilized lipase B from Candida antarctica (CALB) was used in the kinetic resolution of (R,S)-flurbiprofen by enantioselective esterification with methanol, reaching a high enantiomeric excess (eep = 89.6 ± 2.0%). As part of the immobilization optimization, the influence of different buffers was investigated. The effect of the reactor material and the reaction medium on the lipase activity was also studied. Moreover, the stability of the immobilized lipases: lipase from Candida rugosa (CRL) and CALB during storage in various temperature and humidity conditions (climatic chamber and refrigerator) was tested. The application of the immobilized CALB in a polypropylene reactor allowed for receiving over 9-fold higher conversion values compared to the results achieved when conducting the reaction in a glass reactor, as well as approximately 30-fold higher conversion values in comparison with free lipase. The good stability of the CALB-octyl-agarose support was demonstrated. After 7 days of storage in a climatic chamber or refrigerator (with protection from humidity) approximately 60% higher conversion values were obtained compared to the results observed for the immobilized form that had not been stored. The new approach involving the application of the CALB-octyl-agarose support for reactions performed in organic solvents indicates a significant role of the polymer reactor material being used in achieving high catalytic activity.

Functionalized Ionic Liquids-Modified Metal-Organic Framework Material Boosted the Enzymatic Performance of Lipase.

The development of immobilized enzymes with high activity and stability is critical. Metal-organic frameworks (MOFs) have attracted much academic and industrial interest in the field of enzyme immobilization due to their unique properties. In this study, the amino-functionalized ionic liquid (NIL)-modified metal-organic framework (UiO-66-NH2) was prepared to immobilize Candida rugosa lipase (CRL), using dialdehyde starch (DAS) as the cross-linker. The results of the Fourier transform infrared (FT-IR) spectra, X-ray powder diffraction (XRD), and scanning electronic microscopy (SEM) confirmed that the NIL was successfully grafted to UiO-66-NH2. The CRL immobilized on NIL-modified UiO-66-NH2 (UiO-66-NH2-NIL-DAS@CRL) exhibited satisfactory activity recovery (79.33%), stability, reusability, and excellent organic solvent tolerance. The research results indicated that ionic liquid-modified UiO-66-NH2 had practical potential for application in enzyme immobilization.

Cloning, Expression, Characterization and Immobilization of a Recombinant Carboxylesterase from the Halophilic Archaeon, Halobacterium salinarum NCR-1.

Only a few halophilic archaea producing carboxylesterases have been reported. The limited research on biocatalytic characteristics of archaeal esterases is primarily due to their very low production in native organisms. A gene encoding carboxylesterase from Halobacterium salinarum NRC-1 was cloned and successfully expressed in Haloferax volcanii. The recombinant carboxylesterase (rHsEst) was purified by affinity chromatography with a yield of 81%, and its molecular weight was estimated by SDS-PAGE (33 kDa). The best kinetic parameters of rHsEst were achieved using p-nitrophenyl valerate as substrate (KM = 78 µM, kcat = 0.67 s-1). rHsEst exhibited great stability to most metal ions tested and some solvents (diethyl ether, n-hexane, n-heptane). Purified rHsEst was effectively immobilized using Celite 545. Esterase activities of rHsEst were confirmed by substrate specificity studies. The presence of a serine residue in rHsEst active site was revealed through inhibition with PMSF. The pH for optimal activity of free rHsEst was 8, while for immobilized rHsEst, maximal activity was at a pH range between 8 to 10. Immobilization of rHsEst increased its thermostability, halophilicity and protection against inhibitors such as EDTA, BME and PMSF. Remarkably, immobilized rHsEst was stable and active in NaCl concentrations as high as 5M. These biochemical characteristics of immobilized rHsEst reveal its potential as a biocatalyst for industrial applications.

Optimized Fabrication of Carbon-Fiber Microbiosensors for Codetection of Glucose and Dopamine in Brain Tissue.

Dopamine (DA) signaling is critically important in striatal function, and this metabolically demanding process is fueled largely by glucose. However, DA and glucose are typically studied independently and, as such, the precise relationship between DA release and glucose availability remains unclear. Fast-scan cyclic voltammetry (FSCV) is commonly coupled with carbon-fiber microelectrodes to study DA transients. These microelectrodes can be modified with glucose oxidase (GOx) to generate microbiosensors capable of simultaneously quantifying real-time and physiologically relevant fluctuations of glucose, a nonelectrochemically active substrate, and DA, which is readily oxidized and reduced at the electrode surface. A chitosan hydrogel can be electrodeposited to entrap the oxidase enzyme on the sensor surface for stable, sensitive, and selective codetection of glucose and DA using FSCV. This strategy can also be used to entrap lactate oxidase on the carbon-fiber surface for codetection of lactate and DA. However, these custom probes are individually fabricated by hand, and performance is variable. This study characterizes the physical nature of the hydrogel and its effects on the acquired electrochemical data in the detection of glucose (2.6 mM) and DA (1 µM). The results demonstrate that the electrodeposition of the hydrogel membrane is improved using a linear potential sweep rather than a direct step to the target potential. Electrochemical impedance spectroscopy data relate information on the physical nature of the electrode/solution interface to the electrochemical performance of bare and enzyme-modified carbon-fiber microelectrodes. The electrodeposition waveform and scan rate were characterized for optimal membrane formation and performance. Finally, codetection of both DA/glucose and DA/lactate was demonstrated in intact rat striatum using probes fabricated according to the optimized protocol. Overall, this work improves the reliable fabrication of carbon-fiber microbiosensors for codetection of DA and important energetic substrates that are locally delivered to the recording site to meet metabolic demand.

Immobilization of human tyrosine hydroxylase onto magnetic nanoparticles - A novel formulation of a therapeutic enzyme.

Human tyrosine hydroxylase (hTH) has key role in the production of catecholamine neurotransmitters. The structure, function and regulation of hTH has been extensively researched area and the possibility of enzyme replacement therapy (ERT) involving hTH through nanocarriers has been raised as well. However, our understanding on how hTH may interact with nanocarriers is still lacking. In this work, we attempted to investigate the immobilization of hTH on magnetic nanoparticles (MNPs) with various surface linkers in quantitative and mechanistic detail. Our results showed that the activity of hTH was retained after immobilization via secondary and covalent interactions as well. The colloidal stability of hTH could be also enhanced proved by Dynamic light scattering and Zeta potential analysis and a homogenous enzyme layer could be achieved, which was investigated by Raman mapping. The covalent attachment of hTH on MNPs via aldehyde or epoxy linkers provide irreversible immobilization and 38.1 % and 16.5 % recovery (ER). The hTH-MNPs catalyst had 25 % ER in average in simulated nasal electrolyte solution (SNES). This outcome highlights the relevance of immobilization applying MNPs as a potential formulation tool of sensitive therapeutic enzymes offering new opportunities for ERT related to neurodegenerative disorders.

Immobilization of pancreatin based on ultrasound-assisted polydopamine functionalized magnetic porous chitosan for the detoxification of ochratoxin A in wine.

Ochratoxin A (OTA) is a mycotoxin that globally contaminates fruits and their products. Since OTA have a huge negative impact on health hazards and economic losses, it is imperative to establish an effective and safe strategy for detoxification. Here, pancreatin was immobilized on the surface of polydopamine functionalized magnetic porous chitosan (MPCTS@ PDA) for the degradation of OTA. Compared with free pancreatin, MPCTS@ PDA@ pancreatin displayed excellent thermal stability, acid resistance, storage stability and OTA detoxification in wine (>58%). Moreover, the MPCTS@ PDA@ pancreatin retained 43% initial activity after 8 reuse cycles. There was no significant change in the quality of wine after MPCTS@ PDA@ pancreatin treatment. Moreover, it did not exhibit cytotoxicity which facilitated its application in wine. These results demonstrated that MPCTS@ PDA@ pancreatin can be used as a highly effective biocatalysate for OTA detoxification in wine.

Controllable radical polymerization of TEMPO redox for stable and sensitive enzyme electrode interface.

Assembling functional molecules on the surface of an enzyme electrode is the most basic technique for constructing a biosensor. However, precise control of electron transfer interface or electron mediator on the electrode surface remains a challenge, which is a key step that affects the stability and sensitivity of enzyme-based biosensors. In this study, we propose the use of controllable free radical polymerization to grow stable 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) polymer as electron mediator on enzyme surface for the first time. Through scanning electron microscopy (SEM), Raman spectroscopy, electrode surface coverage measurement, static contact angle (SCA), and a series of electrochemical methods, it has been demonstrated that the TEMPO-based enzyme electrode exhibits a uniform hydrophilic morphology and stable electrochemical performance. Furthermore, the results show that the sensor demonstrates high sensitivity for detecting glucose biomolecules in artificial sweat and serum. Attributing to the quantitative and controllable radical polymerization of TEMPO redox assembled enzyme electrode surface, the as-proposed biosensor providing a use, storage, and inter-batch sensing stability, providing a vital platform for wearable/implantable biochemical sensors.

A Whole-Process Visible Strategy for the Preparation of Rhizomucor miehei Lipase with Escherichia coli Secretion Expression System and the Immobilization.

BACKGROUND: Rhizomucor miehei (RM) lipase is a regioselective lipase widely used in food, pharmaceutical and biofuel industries. However, the high cost and low purity of the commercial RM lipase limit its industrial applications. Therefore, it is necessary to develop cost-effective strategies for large-scale preparation of this lipase. The present study explored the high-level expression of RM lipase using superfolder green fluorescent protein (sfGFP)-mediated Escherichia coli secretion system. RESULTS: The sfGFP(-15) mutant was fused to the C-terminus of RM lipase to mediate its secretion expression. The yield of the fusion protein reached approximately 5.1 g/L with high-density fermentation in 5-L fermentors. Unlike conventional secretion expression methods, only a small portion of the target protein was secreted into the cell culture while majority of the fusion protein was still remained in the cytoplasm. However, in contrast to intracellular expression, the target protein in the cytoplasm could be transported efficiently to the supernatant through a simple washing step with equal volume of phosphate saline (PBS), without causing cell disruption. Hence, the approach facilitated the downstream purification step of the recombinant RM lipase. Moreover, contamination or decline of the engineered strain and degradation or deactivation of the target enzyme can be detected efficiently because they exhibited bright green fluorescence. Next, the target protein was immobilized with anion-exchange and macropore resins. Diethylaminoethyl sepharose (DEAE), a weak-basic anion-exchange resin, exhibited the highest bind capacity but inhibited the activity of RM lipase dramatically. On the contrary, RM lipase fixed with macropore resin D101 demonstrated the highest specific activity. Although immobilization with D101 didn't improve the activity of the enzyme, the thermostability of the immobilized enzyme elevated significantly. The immobilized RM lipase retained approximately 90% of its activity after 3-h incubation at 80 °C. Therefore, D101 was chosen as the supporting material of the target protein. CONCLUSION: The present study established a highly efficient strategy for large-scale preparation of RM lipase. This innovative technique not only provides high-purity RM lipase at a low cost but also has great potential as a platform for the preparation of lipases in the future.

Magnetic field-assisted surface engineering technology for active regulation of Fe3O4 medium to enable the laccase electrochemical biosensing of catechol with visible stripe patterns.

BACKGROUND: Catechol (CC), a prevalent phenolic compound, is a byproduct in various agricultural, chemical, and industrial processes. CC detection is crucial for safeguarding water quality and plays a pivotal role in enhancing the overall quality of life of individuals. Electrochemical biosensors exhibit rapid responses, have small sizes, and can be used for real-time monitoring. Therefore, the development of a fast and sensitive electrochemical biosensor for CC detection is crucial. RESULT: In this study, a laccase-based electrochemical biosensor for detection of CC is successfully developed using Fe3O4 nanoparticles as medium and optimized by applying a magnetic field. This research proposes a unique strategy for biosensor enhancement by actively controlling the distribution of magnetic materials on the electrode surface through the application of a magnetic field, resulting in a visibly alternating stripe pattern. This approach effectively disperses magnetic particles, preventing their aggregation and reducing the boundary layer thickness, enhancing the electrochemical response of the biosensor. After fabrication condition optimization, CC is successfully detected using this biosensor. The fabricated sensor exhibits excellent performance with a wide linear detection range of 10-1000 µM, a low detection limit of 1.25 µM, and a sensitivity of 7.9 µA/mM. The fabricated sensor exhibits good selectivity and reliable detection in real water samples. In addition, the laccase-based sensor has the potential for the fast and accurate monitoring of CC in olive oil. SIGNIFICANCE: The magnetic field optimization in this study significantly improved the performance of the electrochemical biosensor for detecting CC in environmental samples. Overall, the sensor developed in this study has the potential for fast and accurate monitoring of CC in environmental samples, highlighting the potential importance of a magnetic field environment in improving the performance of catechol electrochemical biosensors.

Immobilized Dipeptidase in Manganese Ion-Loaded Polyethylenimine-Induced Calcium Phosphate Nanocrystals for Carnosine Synthesis.

Carnosine is a natural bioactive dipeptide with important physiological functions widely used in food and medicine. Dipeptidase (PepD) from Serratia marcescens can catalyze the reverse hydrolytic reaction of ß-alanine with l-histidine to synthesize carnosine in the presence of Mn2+. However, it remains challenging to practice carnosine biosynthesis due to the low activity and high cost of the enzyme. Therefore, the development of biocatalysts with high activity and stability is of significance for carnosine synthesis. Here, we proposed to chelate Mn2+ to polyethylenimine (PEI) that induced rapid formation of calcium phosphate nanocrystals (CaP), and Mn-PEI@CaP was used for PepD immobilization via electrostatic interaction. Mn-PEI@CaP as the carrier enhanced the stability of the immobilized enzyme. Moreover, Mn2+ loaded in the carrier acted as an in situ activator of the immobilized PepD for facilitating the biocatalytic process of carnosine synthesis. The as-prepared immobilized enzyme (PepD-Mn-PEI@CaP) kept similar activity with free PepD plus Mn2+ (activity recovery, 102.5%), while exhibiting elevated thermal stability and pH tolerance. Moreover, it exhibited about two times faster carnosine synthesis than the free PepD system. PepD-Mn-PEI@CaP retained 86.8% of the original activity after eight cycles of batch catalysis without the addition of free Mn2+ ions during multiple cycles. This work provides a new strategy for the co-immobilization of PepD and Mn2+, which greatly improves the operability of the biocatalysis and demonstrates the potential of the immobilized PepD system for efficient carnosine synthesis.

Pepsin immobilization on activated carbon and functionalized with glutaraldehyde and genipin for the synthesis of antioxidant peptides of goat casein.

In this article, the synthesis of antioxidant peptides in the enzymatic hydrolysis of caprine casein was analyzed at three different time points (60 min, 90 min, and 120 min) using immobilized pepsin on activated and modified carbon (AC, ACF, ACG 50, ACG 100). The immobilization assays revealed a reduction in the biocatalysts' activity compared to the free enzyme. Among the modified ones, ACG 50 exhibited greater activity and better efficiency for reuse cycles, with superior values after 60 min and 90 min. Peptide synthesis was observed under all studied conditions. Analyses (DPPH, ß-carotene/linoleic acid, FRAP) confirmed the antioxidant potential of the peptides generated by the immobilized enzyme. However, the immobilized enzyme in ACG 50 and ACG 100, combined with longer hydrolysis times, allowed the formation of peptides with an antioxidant capacity greater than or equivalent to those generated by the free enzyme, despite reduced enzymatic activity.

Enzyme encapsulation in metal-organic frameworks using spray drying for enhanced stability and controlled release: A case study of phytase.

Encapsulating enzymes in metal-organic frameworks is a common practice to improve enzyme stability against harsh conditions. However, the synthesis of enzyme@MOFs has been primarily limited to small-scale laboratory settings, hampering their industrial applications. Spray drying is a scalable and cost-effective technology, which has been frequently used in industry for large-scale productions. Despite these advantages, its potential for encapsulating enzymes in MOFs remains largely unexplored, due to challenges such as nozzle clogging from MOF particle formation, utilization of toxic organic solvents, controlled release of encapsulated enzymes, and high temperatures that could compromise enzyme activity. Herein, we present a novel approach for preparing phytase@MIL-88 A using solvent-free spray drying. This involves atomizing two MOF precursor solutions separately using a three-fluid nozzle, with enzyme release controlled by manipulating defects within the MOFs. The physicochemical properties of the spray dried particles are characterized using X-ray diffraction, Fourier-transform infrared spectroscopy, and scanning electron microscopy. Leveraging the efficiency and scalability of spray drying in industrial production, this scalable encapsulation technique holds considerable promise for broad industrial applications.

Coupling protein scaffold and biosilicification: A sustainable and recyclable approach for d-mannitol production via one-step purification and immobilization of multienzymes.

Enzymatic synthesis of biochemicals in vitro is vital in synthetic biology for its efficiency, minimal by-products, and easy product separation. However, challenges like enzyme preparation, stability, and reusability persist. Here, we introduced a protein scaffold and biosilicification coupled system, providing a singular process for the purification and immobilization of multiple enzymes. Using d-mannitol as a model, we initially constructed a self-assembling EE/KK protein scaffold for the co-immobilization of glucose dehydrogenase and mannitol dehydrogenase. Under an enzyme-to-scaffold ratio of 1:8, a d-mannitol yield of 0.692 mol/mol was achieved within 4 h, 2.16-fold higher than the free enzymes. The immobilized enzymes retained 70.9 % of the initial joint activity while the free ones diminished nearly to inactivity after 8 h. Furthermore, we incorporated the biosilicification peptide CotB into the EE/KK scaffold, inducing silica deposition, which enabled the one-step purification and immobilization process assisted by Spy/Snoop protein-peptide pairs. The coupled system demonstrated a comparable d-mannitol yield to that of EE/KK scaffold and 1.34-fold higher remaining activities after 36 h. Following 6 cycles of reaction, the immobilized system retained the capability to synthesize 56.4 % of the initial d-mannitol titer. The self-assembly co-immobilization platform offers an effective approach for enzymatic synthesis of d-mannitol and other biochemicals.

Propitious catalytic response of immobilized α-amylase from G. thermoleovorans in modified APTES-Fe3O4 NPs for industrial bio-processing.

Challenges in enzyme and product recovery are currently intriguing in modern biotechnology. Coping enzyme stability, shelf life and efficiency, nanomaterials-based immobilization were epitomized of industrial practice. Herein, a α-amylase from Geobacillus thermoleovorans was purified and bound effectively on to a modified 3-Aminopropyltriethoxysilane (APTES)-Fe3O4 nanoparticle. It was revealed that the carrier-bound enzyme catalysis (pH 8 and 60 °C) was significant in contrast to the free enzyme (pH 7.5 and 55 °C). Furthermore, Zn2+ and Cu2+ were shown to cause inhibitory effects in both enzyme states. Unlike chloroform, toluene, benzene, and butanol, minimal effects were observed with ethanol, acetone, and hexane. The bound enzyme retained 27.4 % of its initial activity after being stored for 36 days. In addition, the reusability of the bound enzyme showed a gradual decline in activity after the first cycle; however, after 13 cycles, its residual activity at 53 % was observed. These data proved significant enough to use this enzyme for industrial starch and analogous substrate bio-processing.

An antifouling epoxy coated metal surface containing silica-immobilized carbonic anhydrase supraparticles for CO2 capture through microalgae.

Carbonic anhydrase (CA) has a promising application as a green and efficient biocatalyst for CO2 capture, and many successful cases of immobilizing CA have been reported. However, CA antifouling coatings on metal for CO2 sequestration have rarely been reported. Herein, dimeric CA from Sulfurihydrogenibium azorense (SazCA) with a ferritin tag, which was prepared by low-speed centrifugation with high yield, was adopted as a free enzyme and encapsulated in the sol-gel silica. The silica-immobilized CAs were dispersed into the commercialized metal-antifouling epoxy resin paint to obtain CA coated nickel foams, which had excellent stability, with 90 % and 67 % residual activity after 28 days of incubation at 30 °C and 60 °C, respectively. The CA coated nickel foams remained 60 % original activity after 6 cycles of use within 28 days. Then, a CA-microalgae carbon capture device was constructed using the CA coated nickel foams and Chlorella. The growth rate of Chlorella was significantly increased and the biomass of Chlorella increased by 29 % compared with control after 7 days of incubation. Due to the simple and cost-effective preparation process, sustainable and efficient CO2 absorption, this easy-to-scale up CA coated nickel foam has great potential in CA assisted microalgae-based CO2 capture and carbon neutrality.

Flow injection amperometric uric acid biosensor based on AuNPs-GO-CS porous composite cryogel coated on PB-PEDOT:PSS modified screen-printed carbon electrode.

An enzymatic amperometric uric acid (UA) biosensor was successfully developed by modifying a screen-printed carbon electrode (SPCE) with Prussian blue-poly(3,4-ethylene dioxythiophene) polystyrene sulfonate composite (PB-PEDOT:PSS). The modified SPCE was coated with gold nanoparticles-graphene oxide-chitosan composite cryogel (AuNPs-GO-CS cry). Uricase (UOx) was directly immobilized via chemisorption on AuNPs. The nanocomposite was characterized by scanning electron microscopy, transmission electron microscopy, ultraviolet-visible spectroscopy, and Fourier transform infrared spectroscopy. The electrochemical characterization of the modified electrode was performed by cyclic voltammetry and electrochemical impedance spectroscopy. UA was determined using amperometric detection based on the reduction current of PB which was correlated with the amount of H2O2 produced during the enzymatic reaction. Under optimal conditions, the fabricated UA biosensor in a flow injection analysis (FIA) system produced a linear range from 5.0 to 300 µmol L-1 with a detection limit of 1.88 µmol L-1. The proposed sensor was stable for up to 221 cycles of detection and analysis was rapid (2 min), with good reproducibility (RSDs < 2.90 %, n = 6), negligible interferences, and recoveries from 94.0 ± 3.9 to 101.1 ± 2.6 %. The results of UA detection in blood plasma were in agreement with the enzymatic colorimetric method (P > 0.05).

Enzyme-integrated metal-organic framework platform for cascade detection of α-amylase.

As one of the most important industrial enzymes, α-amylase is widely used in food processing, such as starch sugar and fermentation, bringing high added value to industry of more than a trillion dollars. We developed a multi-enzyme system (Glu&Gox@Cu-MOF-74) prepared by embedding α-glucosidase (Glu) and glucose oxidase (Gox) into the biomimetic metal-organic framework Cu-MOF-74 using in situ encapsulation within 15 min at room temperature for efficient and sensitive detection of α-amylase activity. Benefitting from the remarkable peroxidase-mimicking property and rigid skeleton of Cu-MOF-74, the biocatalytic platform exhibited excellent cascade activity and tolerance in various extremely harsh environments compared to natural enzymes. On this basis, a cascade biocatalytic platform was constructed for the detection of α-amylase activity with wide linear range (5-100 U/L) and low limit of detection (1.45 U/L). The colorimetric cascade scheme is important for the sensitive and selective determination of α-amylase in complex fermentation samples, and the detection time is short (∼0.5 h). This work provides new ideas for the detection of α-amylase based on the cascade amplification method.