Construction of amperometric uric acid biosensor based on uricase immobilized on PBNPs/cMWCNT/PANI/Au composite

A chitosan-glutaraldehyde crosslinked uricase was immobilized onto Prussian blue nanoparticles (PBNPs) absorbed onto carboxylated multiwalled carbon nanotube (c-MWCNT) and polyaniline (PANI) layer, electrochemically deposited on the surface of Au electrode. The nanohybrid-uricase electrode was characterized by scanning electron microscopic (SEM), Fourier transform infrared spectroscopy (FTIR) and cyclic voltammetry. An amperometric uric acid biosensor was fabricated using uricase/c-MWCNT/PBNPs/Au electrode as working electrode, Ag/AgCl as standard and Pt wire as auxiliary electrode connected through a potentiostat. The biosensor showed optimum response within 4 s at pH 7.5 and 40 °C, when operated at 0.4 V vs. Ag/AgCl. The linear working range for uric acid was 0.005-0.8 mM, with a detection limit of 5 μM. The sensor was evaluated with 96% recovery of added uric acid in sera and 4.6 and 5.4% within and between batch of coefficient of variation respectively and a good correlation (r = 0.99) with standard enzymic colorimetric method. This sensor measured uric acid in real serum samples. The sensor lost only 37% of its initial activity after its 400 uses over a period of 7 months, when stored at 4 °C.     

Au-nanoclusters incorporated 3-amino-5-mercapto-1,2,4-triazole film modified electrode for the simultaneous determination of ascorbic acid, dopamine, uric acid and nitrite

A novel biosensor has been constructed by the electrodeposition of Au-nanoclusters (nano-Au) on poly(3-amino-5-mercapto-1,2,4-triazole) (p-TA) film modified glassy carbon electrode (GCE) and employed for the simultaneous determination of dopamine (DA), ascorbic acid (AA), uric acid (UA) and nitrite (NO₂⁻). NH₂ and SH groups exposed to the p-TA layer are helpful for the electrodeposition of nano-Au. The combination of nano-Au and p-TA endow the biosensor with large surface area, good biological compatibility, electricity and stability, high selectivity and sensitivity and flexible and controllable electrodeposition process. In the fourfold co-existence system, the linear calibration plots for AA, DA, UA and NO₂⁻ were obtained over the range of 2.1–50.1 μM, 0.6–340.0 μM, 1.6–110.0 μM and 15.9–277.0 μM with detection limits of 1.1 × 10⁻⁶ M, 5.0 × 10⁻⁸ M, 8.0 × 10⁻⁸ M and 8.9 × 10⁻⁷ M, respectively. In addition, the modified biosensor was applied to the determination of AA, DA, UA and NO₂⁻ in urine and serum samples by using standard adding method with satisfactory results.     

Chemometric-assisted kinetic-spectrophotometric method for simultaneous determination of ascorbic acid, uric acid, and dopamine

A chemometric-assisted kinetic spectrophotometric method has been developed for simultaneous determination of ascorbic acid (AA), uric acid (UA), and dopamine (DA). This method relies on the difference in the kinetic rates of the reactions of analytes with a common oxidizing agent, tris(1,10-phenanthroline) and iron(III) complex (ferritin, [Fe(phen)₃]³⁺) at pH 4.4. The changes in absorbance were monitored spectrophotometrically. The data obtained from the experiments were processed by chemometric methods of artificial neural network (ANN) and partial least squares (PLS). Acceptable techniques of prediction set, randomization t test, cross-validation, and Y randomization were applied for the selection of the best chemometric method. The results showed that feedforward artificial neural network (FFANN) is more efficient than the other chemometric methods. The parameters affecting the experimental conditions were optimized, and it was found that under optimal conditions Beer’s law is followed in the concentration ranges of 4.3–74.1, 4.3–78.3, and 2.0–33.0 μM for AA, UA, and DA, respectively. The proposed method was successfully applied to the determination of analytes in serum and urine samples.     

Transport of p-aminohippuric acid,uric acid and glucose in highly purified rabbit renal brush border membranes

A procedure for preparing highly purified brush border membranes from rabbit kidney cortex using differential and density gradient centrifugation is described. Brush border membranes prepared by this procedure were substantially free of basal-lateral membranes, mitochondria, endoplasmic reticulum and nuclear material as evidenced by an enrichment factor of less than 0.3 for (Na⁺ + K⁺)-ATPase, succinate dehydrogenase, NADPH-cytochrome c reductase and DNA. Alkaline phosphatase was enriched ten fold indicating that the membranes were enriched at least 30 fold with respect to other cellular organelles. The yield of brush border membranes was 20%.Transport of d-glucose by the membranes was identical to that previously reported except that the Arrhenius plot for temperature dependence of transport was curvilinear (E_A = 11.3–37.6 kcal/mol) rather than biphasic. Transport of p-aminohippuric acid and uric acid were increased by the presence of NaCl, either gradient or preequilibrated. However, no overshoot was obtained in the presence of a NaCl gradient, and KCl and LiCl also produced equivalent stimulation of transport suggesting a nonspecific ionic strength effect. Uptakes of p-aminohippuric acid and uric acid were not saturable, and were increased markedly by reducing the pH from 7.5 to 5.6. Probenecid (1 mM) reduced p-aminohippuric acid and uric acid (50 μM) uptake by 49% and 21%, respectively. We conclude that the uptake of uric acid and p-aminohippuric acid by renal brush border membranes of the rabbit occurs primarily by a simple solubility-diffusion mechanism.     

Electrocatalytic activity of core/shell magnetic nanocomposite

Electrically active magnetic nanocomposites (EAMNCs), Au nanoparticles/self-doped polyaniline@Fe₃O₄ (AuNPs/SPAN@Fe₃O₄) with well-defined core/shell structure, were first synthesized by a simple method. The morphology and composition of the as-synthesized AuNPs/SPAN@Fe₃O₄ nanocomposite have been characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier transform infrared (FT–IR), ultraviolet–visible (UV–Vis), X-ray powder diffraction (XRD), and thermogravimetric analysis (TGA). Horseradish peroxidase (HRP)–AuNPs/SPAN@Fe₃O₄ biocomposites were immobilized onto the surface of indium tin oxide (ITO) electrode to construct an amperometric hydrogen peroxide (H₂O₂) biosensor. The effects of HRP dosage, solution pH, and the working potential on the current response toward H₂O₂ reduction were optimized to obtain the maximal sensitivity. Under the optimal conditions, the proposed biosensor exhibited a linear calibration response in the range of 0.05 to 0.35 mM and 0.35 to 1.85 mM, with a detection limit of 0.01 mM (signal-to-noise ratio = 3). The modified electrode could virtually eliminate the interference of ascorbic acid (AA) and uric acid (UA) during the detection of H₂O₂. Furthermore, the biosensor was applied to detect H₂O₂ concentration in real samples, which showed acceptable accuracy with the traditional potassium permanganate titration.     

尿酸酶－鲁米诺化学发光传感器

研制了依赖于鲁米诺化学发光反应和固定化尿酸酶柱的测定血清尿酸的生物传感器。其测定血清样品响应时间47s。测定每份样品需时1．5min,样品体积17μl。工作曲线的线性范围1~20mg／dl。批内不精密度3．22％～4.36％,批间6.18%～7．8%。测定值回收率为93％～109％。与医院常规酶试剂盘方法比较相关系数r=0.9909。固定化尿酸酶柱室温使用,4℃冰箱保存,连续使用5个半月测定样品2000次以上,仍保持原酶柱活力的94%。     

ZnO–CuxO/polypyrrole nanocomposite modified electrode for simultaneous determination of ascorbic acid,dopamine, and uric acid

Novel zinc oxide (ZnO) nanosheets and copper oxide (Cu_xO, CuO, and Cu₂O) decorated polypyrrole (PPy) nanofibers (ZnO–Cu_xO–PPy) have been successfully fabricated for the simultaneous determination of ascorbic acid (AA), dopamine (DA), and uric acid (UA). The morphology and structure of ZnO–Cu_xO–PPy nanocomposites were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), and Raman spectroscopy. Compared with the bare glassy carbon electrode (GCE), PPy/GCE, Cu_xO–PPy/GCE, and ZnO–PPy/GCE, ZnO–Cu_xO–PPy/GCE exhibits much higher electrocatalytic activities toward the oxidation of AA, DA, and UA with increasing peak currents and decreasing oxidation overpotentials. Cyclic voltammetry (CV) results show that AA, DA, and UA could be detected selectively and sensitively at ZnO–Cu_xO–PPy/GCE with peak-to-peak separation of 150 and 154 mV for AA–DA and DA–UA, respectively. The calibration curves for AA, DA, and UA were obtained in the ranges of 0.2 to 1.0 mM, 0.1 to 130.0 μM, and 0.5 to 70.0 μM, respectively. The lowest detection limits (signal/noise = 3) were 25.0, 0.04, and 0.2 μM for AA, DA, and UA, respectively. With good selectivity and sensitivity, the current method was applied to the determination of DA in injectable medicine and UA in urine samples.     

Highly sensitive and selective uric acid biosensor based on a three-dimensional graphene foam/indium tin oxide glass electrode

A three-dimensional (3D) continuous and interconnected network graphene foam (GF) was synthesized by chemical vapor deposition using nickel foam as a template. The morphologies of the GF were observed by scanning electron microscopy. X-ray diffraction and Raman spectroscopy were used to investigate the structure of GF. The graphene with few layers and defect free was closely coated on the backbone of the 3D nickel foam. After etching nickel, the GF was transferred onto indium tin oxide (ITO) glass, which acted as an electrode to detect uric acid using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The GF/ITO electrode showed a high sensitivity for the detection of uric acid: approximately 9.44 mA mM⁻¹ in the range of 25 nM–0.1 μM and 1.85 mA mM⁻¹ in the range of 0.1–60 μM. The limit of detection of GF/ITO electrode for uric acid is 3 nM. The GF/ITO electrode also showed a high selectivity for the detection of uric acid in the presence of ascorbic acid. This electrode will have a wide range of potential application prospects in electrochemical detection.     

One-step "green" preparation of graphene nanosheets and carbon nanospheres mixture by electrolyzing graphite rob and its application for glucose biosensing

The graphene nanosheets and carbon nanospheres mixture (GNS–CNS) was prepared by electrolyzing graphite rob in KNO₃ solution under constant current, which was characterized by TEM, AFM, SEM, FT-IR, XRD, XPS, TGA and UV–vis. The nano-mixture can keep stable in water for more than one month. Based on this kind of mixture material, a novel electrochemical biosensing platform for glucose determination was developed. Cyclic voltammetry of glucose oxidase (GOD) immobilized on GNS–CNS/GCE exhibited a pair of well-defined quasi-reversible redox peaks at −0.488 V (E_pa) and −0.509 V (E_pc) by direct electron transfer between the protein and the electrode. The charge-transfer coefficient (α) was 0.51, the electron transfer rate constant was 2.64 s⁻¹ and the surface coverage of HRP was 3.18 × 10⁻¹⁰ mol cm⁻². The immobilized GOD could retain its bioactivity and catalyze the reduction of dissolved oxygen. The glucose biosensor has a linear range from 0.4 to 20 mM with detection limit of 0.1 mM. Moreover, the biosensor exhibits acceptable reproducibility and storage stability. The fabricated biosensor was further used to determine glucose in human plasma sample with the recoveries from 96.83% to 105.52%. Therefore, GOD/GNS–CNS/GCE could be promisingly applied to determine blood sugar concentration in the practical clinical analysis.     

Immobilization of rat brain acetylcholinesterase on porous gold-nanoparticle-CaCO₃ hybrid material modified Au electrode for detection of organophosphorous insecticides

An acetylcholinesterase (AChE) purified from rat brain was immobilized onto gold nanoparticles (AuNPs) assembled on the surface of porous calcium carbonate (CaCO₃) microsphere. The resulting AChE-AuNPs-CaCO₃ bioconjugate was mounted on the surface of Au electrode with the help of silica sol-gel matrix to prepare the working electrode. This electrode was connected to Ag/AgCl (3 M/saturated KCl) as standard and Pt wire as an auxiliary electrode through a potentiostat to construct an organophosphorus (OP) biosensor. The biosensor was based on inhibition of AChE by OP compounds/insecticides. The biosensor showed optimum response at pH 7.0, 30 °C, when polarized at +0.2 V. Two OP compounds, malathion and chlorpyrifos could be detected in the range of 0.1-100 nM and 0.1-70 nM, respectively at 2.0-3.0% inhibition level of AChE. The sensor was reactivated by immersing it in 0.1 mM 2-pyridine aldoxime for 10 min. The detection limit of the sensor was 0.1 nM for both malathion and chlorpyrifos. The biosensor exhibited good reusability (50 times without considerable loss) and storage stability (50% within 60 days, when stored at 4 °C).     

Purine uptake by Alcaligenes eutrophus H16

The uptake of adenine, guanine, xanthine, hypoxanthine and uric acid by whole cells was studied, using spectrophotometric techniques, ¹⁴C-labelled compounds and metabolic inhibitors. Three different non-constitutive systems were shown to maintain the uptake of adenine and that of the pairs guanine/hypoxanthine and xanthine/uric acid. —Active transport of adenine was induced by adenine only, but passive uptake was also involved. Maximum K _T values of 110–131 M were observed at the pH optimum of 8.0. —Guanine and hypoxanthine were translocated by one single mechanism as indicated by K _T and K _I values. This system was induced by both these substances but its affinity was 51/2-times higher for guanine than for hypoxanthine; it was noncompetitively stimulated by Mg²⁺. — A further system, induced by xanthine and uric acid, catalyzed the uptake of both these compounds. It exhibited two pH optima (at pH 6.6 and 7.9); inactivation by heat and stimulation or inhibition by several compounds indicated that two separate mechanisms might be involved in the uptake of xanthine and uric acid.     

A reagent less fluorescent sol-gel biosensor for uric acid detection in biological fluids

The simultaneous encapsulation of a coupled uricase-peroxidase system and amplex red in a sol-gel matrix allows one to obtain a reagent-less and ready-to-use fluorescent biosensor for the accurate detection of uric acid in highly diluted biological fluids. The detection limit of the prepared biosensor was found to be 20 nM and was linear up to 1 microM. The high sensitivity found for the biosensor permitted a reliable determination of uric acid concentrations in the presence of interfering species (e.g., ascorbic acid) just by sample dilution (up to 50000 for urine and 10000 for serum and blood). The sol-gel encapsulation preserved the hierarchy of the enzyme activity as demonstrated by the performance of the fluorescent biosensor.     

Modification of nanocrystalline TiO2 coatings with molecularly imprinted TiO2 for uric acid recognition

Combining the surface modification and molecular imprinting technique, a novel piezoelectric sensing platform with excellent molecular recognition capability was established for the detection of uric acid (UA) based on the immobilization of TiO₂ nanoparticles onto quartz crystal microbalance (QCM) electrode and modification of molecularly imprinted TiO₂ (MIT) layer on TiO₂ nanoparticles. The performance of the fabricated biosensor was evaluated, and the results indicated that the biosensor exhibited high sensitivity in UA detection, with a linear range from 0.04 to 45 μM and a limit of detection of 0.01 μM. Moreover, the biosensor presented high selectivity towards UA in comparison with other interferents. The analytical application of the UA biosensor confirmed the feasibility of UA detection in urine sample.     

3,5,2',4'-Tetrahydroxychalcone, a new non-purine xanthine oxidase inhibitor

Xanthine oxidase is a key enzyme that catalyses hypoxanthine and xanthine to uric acid and the overproduction of uric acid will lead to hyperuricemia which is an important cause of gout. In the present study, three chalcone derivatives were synthesized and evaluated for inhibitory activity against xanthine oxidase in vitro. Of the compounds, only Compound 1, 3,5,2′,4′-tetrahydroxychalcone, exhibited a significant inhibitory activity on xanthine oxidase with an IC₅₀ value of 22.5 μM. Lineweaver–Burk transformation of the inhibition kinetics data demonstrated that it was a competitive inhibitor of xanthine oxidase and Ki value was 17.4 μM. In vivo, intragastric administration of Compound 1 was able to significantly reduce serum uric acid levels and inhibited hepatic xanthine oxidase activities of hyperuricemic mice in a dose-dependent manner. Acute toxicity study in mice showed that Compound 1 was very safe at a dose of up to 5 g/kg. These results suggest that Compound 1 is a novel competitive xanthine oxidase inhibitor and is worthy of further development.     

Determination of nanomolar uric and ascorbic acids using enlarged gold nanoparticles modified electrode

Individual and simultaneous determination of 50 nM uric acid (UA) and ascorbic acid (AA) using enlarged, citrate-stabilized gold nanoparticles (AuNPs) self-assembled to 2,5-dimercapto-1,3,4-thiadiazole (DMT) monolayer modified Au (Au/DMT) electrode by an amperometric method is described for the first time. Self-assembly of AuNPs on the electrode surface was confirmed by atomic force microscopy (AFM), attenuated total reflectance FT-IR and diffuse reflectance spectral measurements. The electron transfer reaction (ETR) of [Fe(CN)₆]^3−/4− was blocked at Au/DMT electrode, whereas it was restored with a peak separation of 200 mV after the attachment of AuNPs on the Au/DMT (Au/DMT/AuNPs) electrode, which was confirmed from the ETR of the [Fe(CN)₆]^3−/4− redox couple. When the self-assembled AuNPs were enlarged by hydroxylamine seeding, the ETR of [Fe(CN)₆]^3−/4− was improved significantly with a peak separation of 100 mV. Tapping mode AFM showed that the average size of the enlarged-AuNPs (E-AuNPs) was 50-70 nm. The E-AuNPs modified electrode catalyzes the oxidation of AA and UA, separates their voltammetric signals by 200 mV, and has excellent sensitivity towards AA and UA with a detection limit of 50 nM. The practical application of the modified electrode was demonstrated by measuring the concentration of UA in blood serum and urine.     

Amperometric hydrogen peroxide biosensor based on covalently immobilizing thionine as a mediator

A novel amperometric hydrogen peroxide biosensor based on the immobilization of hemoglobin on the 2,6-pyridinedicarboxylic acid (PDC) polymer, thionine and nano-Au was successfully fabricated. In this strategy, PDC polymer acted as the matrices to covalently immobilize the thionine, and then hemoglobin was successfully adsorbed on the nano-Au which was electro-deposited on to thionine modified electrode surface. The preparation process of modified electrode was characterized with electrochemical impedance spectroscopy and atomic force microscope. The analytical performance of proposed biosensor toward H₂O₂ was investigated by cyclic voltammetry and chronoamperometry. The resulted biosensor exhibited fast amperometric response (within 6 s) to H₂O₂, and linear range was from 9.1 μM to 5.0 mM with the detection limit of 2.6 μM (S/N = 3). The apparent Michaelis–Menten constant (K _M^app) was evaluated to be 3.2 mM. Furthermore, the resulted biosensor showed good stability and reproducibility.     

Electrochemical detection of uric acid via uricase-immobilized graphene oxide

Measurement of the uric acid level in the body can be improved by biosensing with respect to the accuracy, sensitivity and time consumption. This study has reported the immobilization of uricase onto graphene oxide (GO) and its function for electrochemical detection of uric acid. Through chemical modification of GO using 1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide (NHS) as cross-linking reagents, the enzyme activity of the immobilized uricase was much comparable to the free enzyme with 88% of the activity retained. The modified GO-uricase (GOU) was then subjected to electrocatalytic detection of uric acid (UA) via cyclic voltammetry (CV). For that reason, a glassy carbon electrode (GCE) was modified by adhering the GO along with the immobilized uricase to facilitate the redox reaction between the enzyme and the substrate. The modified GOU/GCE outperformed a bare electrode through the electrocatalytic activity with an amplified electrical signal for the detection of UA. The electrocatalytic response showed a linear dependence on the UA concentration ranging from 0.02 to 0.49 mM with a detection limit of 3.45 μM at 3σ/m. The resulting biosensor also exhibited a high selectivity towards UA in the presence of other interference as well as good reproducibility.     

Selective detection of silver ions using mushroom-like polyaniline and gold nanoparticle nanocomposite-based electrochemical DNA sensor

A highly sensitive electrochemical DNA biosensor made of polyaniline (PANI) and gold nanoparticles (AuNPs) nanocomposite (AuNPs@PANI) has been used for the detection of trace concentration of Ag⁺. In the presence of Ag⁺, with the interaction of cytosine–Ag⁺–cytosine (C–Ag⁺–C), cytosine-rich DNA sequence immobilized onto the surface of AuNPs@PANI has a self-hybridization and then forms a duplex-like structure. The whole detection procedure of Ag⁺ based on the developed biosensor was evaluated by electrochemical impedance spectroscopy. On semi-logarithmic plots of the log Ag⁺ concentration versus peak current, the results show that the prepared biosensor can detect silver ions at a wide linear range of 0.01–100 nM (R = 0.9828) with a detection limit of 10 pM (signal/noise = 3). Moreover, the fabricated sensor exhibits good selectivity and repeatability. The detection of Ag⁺ was determined by Ag⁺ self-induced conformational change of DNA scaffold that involved only one oligonucleotide, showing its convenience and availability.     

血尿酸水平与急性脑梗死相关性分析

目的探讨血尿酸水平与急性脑梗死发病及预后的关系。方法检测129例急性脑梗死患者与120例健康组的血尿酸水平及相关指标进行比较,分析其相关性。结果急性脑梗死患者血尿酸水平及异常率明显增高,与对照组之间差异存在非常显著性(P0.01),在急性脑梗死患者中,血尿酸异常组高血压、高血糖、高血脂及高纤维蛋白原血症的发生率比正常组明显增高,血尿酸异常组较正常组入院第21天神经功能缺损的程度差异存在非常显著性(P0.01)。结论血尿酸水平与急性脑梗死的发病密切相关,是预后不良的预测因素。