Exploration of uricase-like activity in Pd@Ir nanosheets and their application in relieving acute gout using self-cascade reaction.

Gout, marked by the deposition of sodium urate crystals in joints and peripheral tissues, presents a considerable health challenge. Recent research has shown a growing interest in nanozyme-based treatments for gout. However, literature on nanozymes that combine uricase-like (UOX) activity for uric acid (UA) degradation with catalase (CAT)-like activity for H2O2 elimination through a self-cascade reaction is limited. Herein, we discovered that two-dimensional Pd@Ir nanosheets (NSs) exhibit UOX and CAT activities effectively. Notably, we observed a size-dependent effect of Pd@Ir on activation energy during UA degradation, with the larger Pd@Ir NSs demonstrating a lower energy barrier. The 46-nm Pd@Ir had activation energy as low as 35.9 kJ/mol, surpassing the efficiency of natural bacterial uricase and most reported nanozymes. Through a tandem self-cascade reaction of Pd@Ir, UA was effectively degraded via UOX activity, while the byproduct H2O2 was simultaneously eliminated by CAT-like activity. Cell experiments revealed that Pd@Ir protect normal cells from oxidative stress and promote cell proliferation, demonstrating an excellent self-cascade effect. Additionally, Pd@Ir substantially alleviated gout symptoms in monosodium urate-induced acute gout mice without causing toxic effects on biological organs and tissues. This study opens new avenues for using nanozyme-based cascade reaction systems in the treatment of metabolic diseases.

A gold nanomaterial-integrated distance-based analytical device for uric acid quantification in human urine samples.

In this article, we present the first demonstration of a distance-based paper analytical device (dPAD) for uric acid quantification in human urine samples with instrument-free readout and user-friendliness for the rapid diagnosis and prognosis of various related diseases. By employing gold nanoparticles (AuNPs) as a peroxidase-like nanozyme, our proposed technique eliminates the utilization of horseradish peroxidase (HRP), making the device cost-effective and stable. In our dPAD, uric acid in the sample is oxidized by the uricase enzyme and subsequently catalysed with AuNPs in the sample zone, generating hydroxyl radicals (˙OH). Then, the produced ˙OH reacts with 3,3'-diaminobenzidine (DAB) to form poly DAB (oxDAB), resulting in a coloured distance signal in the detection zone of the dPAD. The variation of the distance of the observed red-brown colour is directly proportional to the uric acid concentration. Our sensor exhibited a linear range from 0.50 to 6.0 mmol L-1 (R2 = 0.9922) with a detection limit (LOD) of 0.25 mmol L-1, covering the clinical range of uric acid in urine. Hence, there is no need for additional sample preparation or dilution. Additionally, this assay is highly selective, with no interferences. We also found that this approach could accurately and precisely determine uric acid in human control samples with the recovery ranging from 99.37 to 100.35 with the highest RSD of 4.05%. Our method is comparable with the use of a commercially available uric acid sensor at a 95% confidence interval. Consequently, the developed dPAD offers numerous advantages such as cost-effectiveness, simplicity, and ease of operation with unskilled individuals. Furthermore, this concept can be applied for extensive biosensing applications in monitoring other biomarkers as an alternative analytical point-of-care (POC) device.

Efficient and fast detection of uric acid based on a colorimetric sensing method.

The uric acid (UA) level is an important physiological indicator of the human body, and its abnormality can lead to a series of diseases. Therefore, the immediate detection of uric acid concentration has broad application prospects. Commonly used methods for the analysis of uric acid include chromatography, high-performance capillary electrophoresis and electrochemical methods. However, these methods have the disadvantages of cumbersome sample pre-treatment, high cost, time-consuming, and the need for experimental instruments and professional operators, which are extremely unfavorable for the detection of uric acid and the diagnosis of related diseases in resource-limited areas. In this study, a portable visualization method was developed for the detection of uric acid using hydrogen peroxide (H2O2) test strips. Uric acid enzyme specifically catalyzes the oxidation of uric acid to produce H2O2, which causes a significant change in the color of the H2O2 test strip. The response has good linearity in the range of 1 ∼ 50 µg mL-1. Thus, it provides a simple, rapid, and cost-effective visualized bioassay for uric acid.

Multimodal smart systems reprogramme macrophages and remove urate to treat gouty arthritis.

Gouty arthritis is a chronic and progressive disease characterized by high urate levels in the joints and by an inflammatory immune microenvironment. Clinical data indicate that urate reduction therapy or anti-inflammatory therapy alone often fails to deliver satisfactory outcomes. Here we have developed a smart biomimetic nanosystem featuring a 'shell' composed of a fusion membrane derived from M2 macrophages and exosomes, which encapsulates liposomes loaded with a combination of uricase, platinum-in-hyaluronan/polydopamine nanozyme and resveratrol. The nanosystem targets inflamed joints and promotes the accumulation of anti-inflammatory macrophages locally, while the uricase and the nanozyme reduce the levels of urate within the joints. Additionally, site-directed near-infrared irradiation provides localized mild thermotherapy through the action of platinum and polydopamine, initiating heat-induced tissue repair. Combined use of these components synergistically enhances overall outcomes, resulting in faster recovery of the damaged joint tissue.

Arginine-Rich Peptide-Rhodium Nanocluster@Reduced Graphene Oxide Composite as a Highly Selective and Active Uricase-like Nanozyme for the Degradation of Uric Acid and Inhibition of Urate Crystal.

Metal nanozymes have offered attractive opportunities for biocatalysis and biomedicine. However, fabricating nanozymes simultaneously possessing highly catalytic selectivity and activity remains a great challenge due to the lack of three-dimensional (3D) architecture of the catalytic pocket in natural enzymes. Here, we integrate rhodium nanocluster (RhNC), reduced graphene oxide (rGO), and protamine (PRTM, a typical arginine-rich peptide) into a composite facilely based on the single peptide. Remarkably, the PRTM-RhNC@rGO composite displays outstanding selectivity, activity, and stability for the catalytic degradation of uric acid. The reaction rate constant of the uric acid oxidation catalyzed by the PRTM-RhNC@rGO composite is about 1.88 × 10-3 s-1 (4 µg/mL), which is 37.6 times higher than that of reported RhNP (k = 5 × 10-5 s-1, 20 µg/mL). Enzyme kinetic studies reveal that the PRTM-RhNC@rGO composite exhibits a similar affinity for uric acid as natural uricase. Furthermore, the uricase-like activity of PRTM-RhNC@rGO nanozymes remains in the presence of sulfur substances and halide ions, displaying incredibly well antipoisoning abilities. The analysis of the structure-function relationship indicates the PRTM-RhNC@rGO composite features the substrate binding site near the catalytic site in a confined space contributed by 2D rGO and PRTM, resulting in the high-performance of the composite nanozyme. Based on the outstanding uricase-like activity and the interaction of PRTM and uric acid, the PRTM-RhNC@rGO composite can retard the urate crystallization significantly. The present work provides new insights into the design of metal nanozymes with suitable binding sites near catalytic sites by mimicking pocket-like structures in natural enzymes based on simple peptides, conducing to broadening the practical application of high-performance nanozymes in biomedical fields.

Benchmarking predictive methods for small-angle X-ray scattering from atomic coordinates of proteins using maximum likelihood consensus data.

Stimulated by informal conversations at the XVII International Small Angle Scattering (SAS) conference (Traverse City, 2017), an international team of experts undertook a round-robin exercise to produce a large dataset from proteins under standard solution conditions. These data were used to generate consensus SAS profiles for xylose isomerase, urate oxidase, xylanase, lysozyme and ribonuclease A. Here, we apply a new protocol using maximum likelihood with a larger number of the contributed datasets to generate improved consensus profiles. We investigate the fits of these profiles to predicted profiles from atomic coordinates that incorporate different models to account for the contribution to the scattering of water molecules of hydration surrounding proteins in solution. Programs using an implicit, shell-type hydration layer generally optimize fits to experimental data with the aid of two parameters that adjust the volume of the bulk solvent excluded by the protein and the contrast of the hydration layer. For these models, we found the error-weighted residual differences between the model and the experiment generally reflected the subsidiary maxima and minima in the consensus profiles that are determined by the size of the protein plus the hydration layer. By comparison, all-atom solute and solvent molecular dynamics (MD) simulations are without the benefit of adjustable parameters and, nonetheless, they yielded at least equally good fits with residual differences that are less reflective of the structure in the consensus profile. Further, where MD simulations accounted for the precise solvent composition of the experiment, specifically the inclusion of ions, the modelled radius of gyration values were significantly closer to the experiment. The power of adjustable parameters to mask real differences between a model and the structure present in solution is demonstrated by the results for the conformationally dynamic ribonuclease A and calculations with pseudo-experimental data. This study shows that, while methods invoking an implicit hydration layer have the unequivocal advantage of speed, care is needed to understand the influence of the adjustable parameters. All-atom solute and solvent MD simulations are slower but are less susceptible to false positives, and can account for thermal fluctuations in atomic positions, and more accurately represent the water molecules of hydration that contribute to the scattering profile.

Metal-ligand dual-site single-atom nanozyme mimicking urate oxidase with high substrates specificity.

In nature, coenzyme-independent oxidases have evolved in selective catalysis using isolated substrate-binding pockets. Single-atom nanozymes (SAzymes), an emerging type of non-protein artificial enzymes, are promising to simulate enzyme active centers, but owing to the lack of recognition sites, realizing substrate specificity is a formidable task. Here we report a metal-ligand dual-site SAzyme (Ni-DAB) that exhibited selectivity in uric acid (UA) oxidation. Ni-DAB mimics the dual-site catalytic mechanism of urate oxidase, in which the Ni metal center and the C atom in the ligand serve as the specific UA and O2 binding sites, respectively, characterized by synchrotron soft X-ray absorption spectroscopy, in situ near ambient pressure X-ray photoelectron spectroscopy, and isotope labeling. The theoretical calculations reveal the high catalytic specificity is derived from not only the delicate interaction between UA and the Ni center but also the complementary oxygen reduction at the beta C site in the ligand. As a potential application, a Ni-DAB-based biofuel cell using human urine is constructed. This work unlocks an approach of enzyme-like isolated dual sites in boosting the selectivity of non-protein artificial enzymes.

Uricase biofunctionalized plasmonic sensor for uric acid detection with APTES-modified gold nanotopping.

Current uric acid detection methodologies lack the requisite sensitivity and selectivity for point-of-care applications. Plasmonic sensors, while promising, demand refinement for improved performance. This work introduces a biofunctionalized sensor predicated on surface plasmon resonance to quantify uric acid within physiologically relevant concentration ranges. The sensor employs the covalent immobilization of uricase enzyme using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-Hydroxysuccinimide (NHS) crosslinking agents, ensuring the durable adherence of the enzyme onto the sensor probe. Characterization through atomic force microscopy and Fourier transform infrared spectroscopy validate surface alterations. The Langmuir adsorption isotherm model elucidates binding kinetics, revealing a sensor binding affinity of 298.83 (mg/dL)-1, and a maximum adsorption capacity of approximately 1.0751°. The biofunctionalized sensor exhibits a sensitivity of 0.0755°/(mg/dL), a linear correlation coefficient of 0.8313, and a limit of detection of 0.095 mg/dL. Selectivity tests against potentially competing interferents like glucose, ascorbic acid, urea, D-cystine, and creatinine showcase a significant resonance angle shift of 1.1135° for uric acid compared to 0.1853° for interferents at the same concentration. Significantly, at a low uric acid concentration of 0.5 mg/dL, a distinct shift of 0.3706° was observed, setting it apart from the lower values noticed at higher concentrations for all typical interferent samples. The uricase enzyme significantly enhances plasmonic sensors for uric acid detection, showcasing a seamless integration of optical principles and biological recognition elements. These sensors hold promise as vital tools in clinical and point-of-care settings, offering transformative potential in biosensing technologies and the potential to revolutionize healthcare outcomes in biomedicine.

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).

Maximization of uricase production in a column bioreactor through response surface methodology-based optimization.

Uricase (EC 1.7.3.3) is an oxidoreductase enzyme that is widely exploited for diagnostic and treatment purposes in medicine. This study focuses on producing recombinant uricase fromE. coliBL21 in a bubble column bioreactor (BCB) and finding the optimal conditions for maximum uricase activity. The three most effective variables on uricase activity were selected through the Plackett-Burman design from eight different variables and were further optimized by the central composite design of the response surface methodology (RSM). The selected variables included the inoculum size (%v/v), isopropylß-d-1-thiogalactopyranoside (IPTG) concentration (mM) and the initial pH of the culture medium. The activity of uricase, the final optical density at 600 nm wavelength (OD600) and the final pH were considered as the responses of this optimization and were modeled. As a result, activity of 5.84 U·ml-1and a final OD600of 3.42 were obtained at optimum conditions of 3% v/v inoculum size, an IPTG concentration of 0.54 mM and a pH of 6.0. By purifying the obtained enzyme using a Ni-NTA agarose affinity chromatography column, 165 ± 1.5 mg uricase was obtained from a 600 ml cell culture. The results of this study show that BCBs can be a highly effective option for large-scale uricase production.

Rationalization design, soluble expression and PEG modification of highly active recombinant human-porcine uricase mutant protein.

Uric acid is the end product of purine metabolism in humans due to inactivation of the uricase determined by the mutated uricase gene. Uricase catalyzes the conversion of uric acid into water-soluble allantoin that is easily excreted by the kidneys. Hyperuricemia occurs when the serum concentration of uric acid exceeds its solubility (7 mg/dL). However, modifications to improve the uricase activity is under development for treating the hyperuricemia. Here we designed 7 types of human-porcine chimeric uricase by multiple sequence comparisons and targeted mutagenesis. An optimal human-porcine chimeric uricase mutant (uricase-10) with both high activity (6.33 U/mg) and high homology (91.45 %) was determined by enzyme activity measurement. The engineering uricase was further modified with PEGylation to improve the stability of recombinant protein drugs and reduce immunogenicity, uricase-10 could be more suitable for the treatment of gout and hyperuricemia theoretically.

Crystal Facet Controlled Metal-Support Interaction in Uricase Mimics for Highly Efficient Hyperuricemia Treatment.

The effect of strong metal-support interaction (SMSI) has never been systematically studied in the field of nanozyme-based catalysis before. Herein, by coupling two different Pd crystal facets with MnO2, i.e., (100) by Pd cube (Pdc) and (111) by Pd icosahedron (Pdi), we observed the reconstruction of Pd atomic structure within the Pd-MnO2 interface, with the reconstructed Pdc (100) facet more disordered than Pdi (111), verifying the existence of SMSI in such coupled system. The rearranged Pd atoms in the interface resulted in enhanced uricase-like catalytic activity, with Pdc@MnO2 demonstrating the best catalytic performance. Theoretical calculations suggested that a more disordered Pd interface led to stronger interactions with intermediates during the uricolytic process. In vitro cell experiments and in vivo therapy results demonstrated excellent biocompatibility, therapeutic effect, and biosafety for their potential hyperuricemia treatment. Our work provides a brand-new perspective for the design of highly efficient uricase-mimic catalysts.

Colorimetric and fluorometric determination of uric acid by a suspension-based assay using enzyme-immobilized micro-sized particles.

Daily monitoring of serum uric acid levels is very important to provide appropriate treatment according to the constitution and lifestyle of individual hyperuricemic patients. We have developed a suspension-based assay to measure uric acid by adding a sample solution to the suspension containing micro-sized particles immobilized on uricase and horseradish peroxidase (HRP). In the proposed method, the mediator reaction of uricase, HRP, and uric acid produces resorufin from Amplex red. This resorufin is adsorbed onto enzyme-immobilized micro-sized particles simultaneously with its production, resulting in the red color of the micro-sized particles. The concentration of resorufin on the small surface area of the microscopic particles achieves a colorimetric analysis of uric acid with superior visibility. In addition, ethanol-induced desorption of resorufin allowed quantitative measurement of uric acid using a 96-well fluorescent microplate reader. The limit of detection (3σ) and RSD (n = 3) were estimated to be 2.2 × 10-2 µg/mL and ≤ 12.1%, respectively. This approach could also be applied to a portable fluorometer.

Site-Specific Conjugation of Bottlebrush Polymers to Therapeutic Protein via Bioorthogonal Chemistry.

Achieving efficient and site-specific conjugation of therapeutic protein to polymer is crucial to augment their applicability in the realms of biomedicine by improving their stability and enzymatic activity. In this study, we exploited tetrazine bioorthogonal chemistry to achieve the site-specific conjugation of bottlebrush polymers to urate oxidase (UOX), a therapeutic protein for gout treatment. An azido-functionalized zwitterionic bottlebrush polymer (N3-ZBP) using a "grafting-from" strategy involving RAFT and ATRP methods was synthesized, and a trans-cyclooctene (TCO) moiety was introduced at the polymer end through the strain-promoted azide-alkyne click (SPAAC) reaction. The subsequent coupling between TCO-incorporated bottlebrush polymer and tetrazine-labeled UOX using a fast and safe bioorthogonal reaction, inverse electron demand Diels-Alder (IEDDA), led to the formation of UOX-ZBP conjugates with a 52% yield. Importantly, the enzymatic activity of UOX remained unaffected following polymer conjugation, suggesting a minimal change in the folded structure of UOX. Moreover, UOX-ZBP conjugates exhibited enhanced proteolytic resistance and reduced antibody binding, compared to UOX-wild type. Overall, the present findings reveal an efficient and straightforward route for synthesizing protein-bottlebrush polymer conjugates without compromising the enzymatic activity while substantially reducing proteolytic degradation and antibody binding.

Discovery of digallic acid as XOD/URAT1 dual target inhibitor for the treatment of hyperuricemia.

The development of XOD/URAT1 dual target inhibitors has emerged as a promising therapeutic strategy for the management of hyperuricemia. Here, through virtual screening, we have identified digallic acid as a novel dual target inhibitor of XOD/URAT1 and subsequently evaluated its pharmacological properties, pharmacokinetics, and toxicities. Digallic acid inhibited URAT1 with an IC50 of 5.34 ± 0.65 µM, which is less potent than benzbromarone (2.01 ± 0.36 µM) but more potent than lesinurad (10.36 ± 1.23 µM). Docking and mutation analysis indicated that residues S35, F241 and R477 of URAT1 confer a high affinity for digallic acid. Digallic acid inhibited XOD with an IC50 of 1.04 ± 0.23 µM. Its metabolic product, gallic acid, inhibited XOD with an IC50 of 0.91 ± 0.14 µM. Enzyme kinetic studies indicated that both digallic acid and gallic acid act as mixed-type XOD inhibitors. It shares the same binding mode as digallic acid, and residues E802, R880, F914, T1010, N768 and F1009 contribute to their high affinity. The anion group (carboxyl) of digallic acid contribute significantly to its inhibition activity on both XOD and URAT1 as indicated by docking analysis. Remarkably, at a dosage of 10 mg/kg in vivo, digallic acid exhibited a stronger urate-lowering and uricosuric effect compared to the positive drug benzbromarone and lesinurad. Pharmacokinetic study indicated that digallic acid can be hydrolyzed into gallic acid in vivo and has a t1/2 of 0.77 ± 0.10 h. Further toxicity evaluation indicated that digallic acid exhibited no obvious renal toxicity, as reflected by CCK-8, biochemical analysis (CR and BUN) and HE examination. The findings of our study can provide valuable insights for the development of XOD/URAT1 dual target inhibitors, and digallic acid deserves further investigation as a potential anti-hyperuricemic drug.

Bioinspired mp20 mimicking uricase in ZIF-8: Metal ion dependent for controllable activity.

Mini protein mimicking uricase (mp20) has shown significant potential as a replacement for natural enzymes in the development of uric acid biosensors. However, the design of mp20 has resulted to an inactive form of peptide, causing of loss their catalytic activity. Herein, this paper delineates the impact of various metal cofactors on the catalytic activity of mp20. The metal ion-binding site prediction and docking (MIB) web server was employed to identify the metal ion binding sites and their affinities towards mp20 residues. Among the tested metal ions, Cu2+ displayed the highest docking score, indicating its preference for interaction with Thr16 and Asp17 residues of mp20. To assess the catalytic activity of mp20 in the presence of metal ions, uric acid assays was monitored using a colorimetric method. The presence of Cu2+ in the assays promotes the activation of mp20, resulting in a color change based on quinoid production. Furthermore, the encapsulation of the mp20 within zeolitic imidazolate framework-8 (ZIF-8) notably improved the stability of the biomolecule. In comparison to the naked mp20, the encapsulated ZIFs biocomposite (mp20@ZIF-8) demonstrates superior stability, selectivity and sensitivity. ZIF's porous shells provides excellent protection, broad detection (3-100 µM) with a low limit (4.4 µM), and optimal function across pH (3.4-11.4) and temperature (20-100°C) ranges. Cost-effective and stable mp20@ZIF-8 surpasses native uricase, marking a significant biosensor technology breakthrough. This integration of metal cofactor optimization and robust encapsulation sets new standards for biosensing applications.

An ultra-sensitive uric acid second generation biosensor based on chemical immobilization of uricase on functionalized multiwall carbon nanotube grafted palm oil fiber in the presence of a ferrocene mediator.

In this work, palm oil fiber (POF) grafted functionalized multiwall carbon nanotube (FMWCNT) decorated ferrocene (Fc) has been drop coated on a platinum electrode (Pt), in which uricase (UOx) has been chemically immobilized for sensitive and selective biosensing of uric acid (UA). Through the use of EDC/NHS, a stable bioelectrode (UOx/Fc/FMWCNT-POF/Pt) was obtained and characterized by FTIR/ATRIR, XRD, Raman, EA/EDX, TGA, SEM, TEM, CV, EIS, CA, and DPV. Results from DPV showed the rapid response of the developed bioelectrode towards UA (0.185 V) with high sensitivity (41.14 µA mM-1) and good limit of detection (19 µM) in the linear range 10-1000 µM. The low value of Michaelis-Menten constant (km = 31.364 µM) shows high affinity of the UA towards the enzyme at the electrode surface. The developed biosensor demonstrates good reproducibility, repeatability, and stability with a deviation of less than 2.5%, and was successfully applied for human blood sample analysis. The CA study revealed a fast response time (2 s) of the sensor. The work has pioneered a new addition to the class of tailorable chemical species for biosensor development and proven to be a promising new tool for point of care testing (POCT) applications.

Electrochemical detection of uric acid in human serum based on ultrasmall Ta2O5 nanoparticle anchored Pt atom with ultrahigh uricase and catalase activities.

The synthesis of ultrasmall Ta2O5 nanoparticle anchored Pt atom using aspartic acid-functionalized graphene quantum dot (Asp-GQD) is reported. The Asp-GQD was combined with tantalic acid and chloroplatinic acid to rapidly form water-soluble Ta-Asp-GQD and Pt-Asp-GQD complex. Followed by thermal annealing at 900 °C in N2 to obtain Ta2O5-Asp-GQD-Pt. The study shows that the introduction of Asp-GQD as a chelating agent and p-type semiconductor achieves to the formation of ultrasmall Ta2O5 nanoparticle, PN junction at the interface and Pt single atom anchored on the surface of Ta2O5 nanocrystals. The unique structure realizes ultrahigh uricase activity and catalase activities of Ta2O5-Asp-GQD-Pt. The Ta2O5-Asp-GQD-Pt was used as the bifunctional sensing material for the construction of an electrochemical uric acid sensor. The differential pulse voltammetric current at 0.45 V linearly increases with the increase of uric acid concentration in the range 0.001-5.00 mM with the detection limit of 0.41 µM (S/N = 3). The sensor exhibits a much better sensitivity compared with the reported methods for the detection of uric acid. The proposed analytical method has been applied to the electrochemical detection of uric acid in human serum with a spiked recovery of 95-105%. The study also offers one way to design and synthesize multifunctional sensing materials with high catalytic activity.

Rational design of mini protein mimicking uricase: Encapsulation in ZIF-8 for uric acid detection.

Five mini proteins mimicking uricase comprising 20, 40, 60, 80, and 100 amino acids were designed based on the conserved active site residues within the same dimer, using the crystal structure of tetrameric uricase from Arthrobacter globiformis (PDB ID: 2yzb) as a template. Based on molecular docking analysis, the smallest mini protein, mp20, shared similar residues to that of native uricase that formed hydrogen bonds with uric acid and was chosen for further studies. Although purified recombinant mp20 did not exhibit uricase activity, it showed specific binding towards uric acid and evinced excellent thermotolerance and structural stability at temperatures ranging from 10°C to 100°C, emulating its natural origin. To explore the potential of mp20 as a bioreceptor in uric acid sensing, mp20 was encapsulated within zeolitic imidazolate framework-8 (mp20@ZIF-8) followed by the modification on rGO-screen printed electrode (rGO/SPCE) to maintain the structural stability. An irreversible anodic peak and increased semicircular arcs of the Nyquist plot with an increase of the analyte concentrations were observed by utilizing cyclic voltammetry and electrochemical impedance spectroscopy (EIS), suggesting the detection of uric acid occurred, which is based on substrate-mp20 interaction.

A new spectrophotometric method for uric acid detection based on copper doped mimic peroxidase.

Cascade reactions catalyzed by natural uricase and mimic peroxidase (MPOD) have been applied for uric acid (UA) detection. However, the optimal catalytic activity of MPOD is mostly in acidic conditions (pH 2-5), mismatching the optimal catalytic alkaline environment of uricase. In this paper, using CuSO4 and urea as raw materials, a MPOD with high catalytic activity in alkaline environment was synthesized by hydrothermal method. Then, based on coupling reaction of uricase/UA/MPOD/guaiacol (GA) system, a novel spectrophotometric method was established to detect 5-60 µmol/L UA (limit of detection = 3.14 µmol/L (S/N = 3)) and accurately quantified serum UA (275.6 ± 39.9 µmol/L, n = 5) with 95-105% of standard addition recovery. The results were consistent with commercial UA kit (p > 0.05). The MPOD could replace natural POD to reduce the cost of UA detection due to simple preparation and cheap raw materials, and is expected to achieve the specific detection of some substances, like glucose and cholesterol, combined with glucose oxidase and cholesterol oxidase.