Electrochemical Investigation on Kinetics of Xanthine Metabolism and Inhibition Effect of Febuxostat on Xanthine Oxidase Activity

Xanthine is a significant biomolecule and its concentration level in urine and blood plasma is an indicator of specified pathological states. Here, a new sensing platform was designed, which showed excellent analytical performance for xanthine. Importantly, it is the first time to investigate the kinetics of xanthine metabolic reaction by electrochemical method. The results demonstrated that the conversion of xanthine to uric acid completely conformed to the Michaelis-Menten kinetics. Furthermore, we also studied the inhibitory effect of febuxostat on xanthine oxidase activity detailed. As expected, the work may offer potential value for researchers in the treatment of hyperuricemia and gout.     

Amperometric Sensors for Determination of Hydrogen Peroxide Based on Electron Transfer Between Horseradish Peroxidase and 1，1＇－Dimethylferrocene

ＡｍｐｅｒｏｍｅｔｒｉｃＳｅｎｓｏｒｓｆｏｒＤｅｔｅｒｍｉｎａｔｉｏｎｏｆＨｙｄｒｏｇｅｎＰｅｒｏｘｉｄｅＢａｓｅｄｏｎＥｌｅｃｔｒｏｎＴｒａｎｓｆｅｒＢｅｔｗｅｅｎＨｏｒｓｅｒａｄｉｓｈＰｅｒｏｘｉｄａｓｅａｎｄ１，１＇－Ｄｉｍｅｔｈｙｌｆｅｒｒｏ...     

别嘌呤醇对黄嘌呤氧化酶的抑制作用研究

用聚苯胺黄嘌呤氧化酶电极（生物传感器）研究了别嘌呤醇对黄嘌呤对氧化酶的抑制作用，实验结果表明：别嘌呤醇对黄嘌呤氧化酶有明显的抑制作用，但这种抑制作用是可逆的，抑制剂在存在使固定酶的表观米氏常数增大但并没有影响催化反应的最大速率，所以别嘌呤是黄嘌呤氧化酶的一种可逆竞争抑制剂，抑制剂对固定酶的最适ｐＨ值没有影响，而对反应活化能略能影响。     

Direct Electrochemistry of Cholesterol Oxidase Immobilized on a Conducting Polymer: Application for a Cholesterol Biosensor

Direct electrochemistry of cholesterol oxidase (ChOx) immobilized on the conductive poly‐3′,4′‐diamine‐2,2′,5′,2″‐terthiophene (PDATT) was achieved and used to create a cholesterol biosensor. A well‐defined redox peak was observed, corresponding to the direct electron transfer of the FAD/FADH₂ of ChOx, and the rate constant (k_s) was determined to be 0.75 s^?1. Glutathione (GSH) covalently bonded with PDATT was used as a matrix for conjugating AuNPs, ChOx, and MP, simultaneously. MP co‐immobilized with ChOx on the AuNPs‐GSH/PDATT exhibited an excellent amperometric response to cholesterol. The dynamic range was from 10 to 130 μM with a detection limit of 0.3±0.04 μM.     

A Siloxane Homopolymer with Interacting Ferrocenes as a New Material for the Preparation of Sensors Based on the Detection of Hydrogen Peroxide

The electrochemical characterization of a hydrogen peroxide sensor based on a ferrocene‐containing polymer electrochemically deposited onto a platinum electrode is described. The redox polymer consists of a siloxane‐based homopolymer, with pendant electronically communicated ferrocenyl moieties. The electrodes were used as the transducer for glucose and lactate‐sensing enzyme sensors. Amperometric biosensors were prepared by immobilization of glucose oxidase (Gox) or lactate oxidase (Lox) onto these modified electrodes. The steady‐state amperometric response of the sensors is investigated as a function of the applied potential and substrate concentration. Interferences, sensitivity and stability of the sensors were also studied.     

Amperometric Study of Au‐Colloid Function on Xanthine Biosensor Based on Xanthine Oxidase Immobilized in Polypyrrole Layer

Four kinds of xanthine oxidase (XOD) based amperometric biosensors were fabricated and their analytical performances were compared. Polypyrrole (PPY)/XOD biosensor was constructed by electrochemical oxidation of pyrrole in the solution containing xanthine oxidase and pyrrole in this paper. Colloidal Au was then immobilized on the biosensor. On the other hand, electron mediator, Prussian Blue (PB), was deposited on the electrode before the immobilization of PPY/XOD to enhance electron‐transfer rate and current response. The results showed that PPY/XOD, PPY/XOD/Au‐colloid, PB/PPY/XOD and PB/PPY/XOD/Au‐colloid biosensors exhibit good response to xanthine in 1×10^?6 M and 2×10^?5 M and Michaelis‐Menten constants (K_m) of these biosensors were 242.2, 113.4, 144.5, 43.2 μmol?L^?1, respectively. The dependence of current responses with applied voltages was discussed, and different mechanisms of these biosensors were discussed. It has been found that colloidal Au can enhance the current response at the same concentration of xanthine solution and decrease the energy‐barrier of electron‐transfer reaction on the electrode.     

伞形酮对黄嘌呤氧化酶的抑制作用研究

The investigation on inhibition of xanthine oxidase by umbelliferone was carried out using a polyaniline-xanthine oxidase electrode. The experimental results indicated significant inhibition caused by umbelliferone. The apparent Michaelis-Menten constant (K'm) and the maximum rate (im) of the immobilized xanthine oxidase were both affected by umbelliferone. This indicated that umbelliferone acts as a mixed type of inhibitor of xanthine oxidase. The optimal pH of the immobilized enzyme was hardly affected by umbelliferone; The response current increases with increasing potential in the potential range 0.55~0.68V.     

Hydrogen Peroxide Biosensor Based on Immobilization of Hemoglobin on Au@Ag Nanoparticles Modified Carbon Ionic Liquid Electrode

A hydrogen peroxide (H₂O₂) biosensor based on the combination of Au@Ag core‐shell nanoparticles with a hemoglobin‐chitosan‐1‐butyl‐3‐methyl‐imidazolium tetrafluoroborate (Hb‐CHIT‐BMIM×BF₄) composite film was prepared. UV‐vis spectroscopy and transmission electron microscopy confirmed a core‐shell nanostructure of Au@Ag nanoparticle was successfully obtained. Cyclic voltammetric results showed a pair of well‐defined redox peaks appeared with the formal potential (E^O′) of ‐0.301 V (versus Ag/AgCl reference electrode) and the peak‐to‐peak separation (ΔE_p) was 84 mV in 0.1 M phosphate buffer solutions. Due to the synergetic effect of Au@Ag core‐shell nanoparticles and Hb‐CHIT‐BMIM×BF₄, the biosensor exhibited good electrocatalytic activity to the reduction of H₂O₂ in a linear range from 1.0 × 10^?6 to 1.0 × 10^?3 M with a detection limit of 4 × 10^?7 M (S/N = 3). The apparent Michaelis‐Menten constant (K_M) was estimated to be 4.4 × 10^?4 M, showing its high affinity. Thus, the study proved that the combination of Au@Ag core‐shell nanoparticles and Hb‐CHIT‐BMIM×BF₄ is able to open up new opportunities for the design of enzymatic biosensors.     

Chemiluminescent Determination of Xanthine Oxidase Activity Using A Sensitive Low-Light Detection System

Abstract

A highly sensitive and rapid chemiluminescent assay for the determination of the activity of xanthine oxidase (XOD) was developed. The chemiluminescent signal was obtained from the catalyzed oxidation of hypoxanthine, accelerated and amplified using a Fe-EDTA complex and perborate, which acts on luminol. The same luminescent mixture was previously used as detection system for immunoassays. Two different mixtures were used, which differ in their luminol and perborate content, with (CLMrho) or without (CLMb) addition of 0.1 μM rhodamine fluorophor. The response obtained from XOD standard solutions in buffer was linear from 5 to 500 U L^?1 and from 0.7 to 250 U L^?1 for CLMrho and CLMb respectively, at 25°C. 5 and 0.7 U L^?1 were the detection limits at 1 standard deviation level. The intra- and inter-assay relative standard deviations ranged from 6 to 12 % for both CLM. Measurements were made using the high performance, low-light level imaging Berthold luminograph LB-980 which allows simultaneous determination of several samples distributed on a microtiterplate. Various kinds of milk were analyzed for XOD content, which in pasteurized milk depends on the fat content and in the UHT milk disappears owing to the heat treatment.     

A Novel Hydrogen Peroxide Biosensor Based on the PDDA‐GNPs/MWNTs Nanocomposite Matrix

A novel nanocomposite designed by the assembly of the positively charged poly(diallyldimethylammonium chloride) protected gold nanoparticles (PDDA‐GNPs), and the negatively charged multi‐walled carbon nanotubes (MWCNTs) on ITO electrode via electrostatic interaction, was used as a supporting matrix for immobilizing hemoglobin (Hb) to develop a high‐performance hydrogen peroxide (H₂O₂) biosensor. The cyclic voltammetrys of immobilized Hb showed a pair of well‐defined and quasi‐reversible redox peaks with the formal potential of ‐0.205V (vs. SCE) and the peak‐to‐peak potential separation of 44 mV at a scan rate of 100 mV×s^?1 in 0.1 mol×L^?1 pH 7.0 PBS. Under the optimized experimental conditions, a linearity range for determination of H₂O₂ was from 2.0 × 10^?6 to 5.2 × 10^?4 mol×L^?1 with a correlation coefficient of 0.9994 (n = 37) and a detection limit of 8.4 × 10^?7 mol×L^?1. The biosensor displayed excellent electrochemical and electrocatalytic response to the reduction of H₂O₂, high sensitivity, long‐term stability, good bioactivity and selectivity.     

导电聚合物传感器的研究进展

导电聚合物因其具有特殊的结构和优异的物理化学性能,而成为构建传感器的一种新材料。综述了近5年来导电聚合物在生物传感器、离子传感器、气敏传感器方面的研究进展,并对导电聚合物的研究动向作了展望。     

Hydrogen Peroxide Sensor Based on Horseradish Peroxidase Combined with CaCO3 Microspheres and Gold Nanoparticles

Direct electrochemistry and electrocatalysis of horseradish peroxidase(HRP) were achieved by entrapping the enzyme between CaCO3 microspheres and gold nanoparticles through forming sandwich configuration (CaCO3-HRP-AuNPs). Polyanion, poly(styrene sulfonate)(PSS), was hybrid with CaCO3 microspheres to increase the surface negative charges for binding with HRP through electrostatic interaction. After the bioconjugate CaCO3 PSS-HRP was entrapped in chitosan based sol-gel(CS-GPTMS) film, HRP was encapsulated by in situ formation of an outer layer of AuNPs through electrochemical reduction of HAuCl4. The composite film containing AuNPs, CaCO3-PSS-HRP bioconjugates and CS-GPTMS can provide favorable microenvironment for HRP to perform direct electron transfer at glassy carbon electrode(GCE). HRP retained its bioelectrocatalytic activity and lead to sensitive and fast amperometric response for the determination of H2O2. H2O2 could be detected in a very wide linear range from 5.0×10–6 mol/L to 7.1×10–2 mol/L. The sandwich configuration of CaCO3-biomolecules-AuNPs could serve as a versatile platform for enzyme immobilization and biosensing.     

Mediatorless Hydrogen Peroxide Biosensor Based on Horseradish Peroxidase Immobilized on 4‐Carboxyphenyl Film Electrografted on Gold Electrode

A novel method to fabricate a third‐generation hydrogen peroxide biosensor was reported. The electrode was first derivatized by electrochemical reduction of in situ generated 4‐carboxyphenyl diazonium salt (4‐CPDS) in acidic aqueous solution yielded stable 4‐carboxyphenyl (4‐CP) layer. The horseradish peroxidase (HRP) enzyme was then covalently immobilized by amidation between NH₂ terminus of enzyme and COOH terminus of 4‐CP film making use of the carbodiimide chemistry. Electrodeposition conditions used to control electrode functionalization density and film electron transfer kinetics were assessed by chronoamperometry and electrochemical impedance spectroscopy. The immobilized HRP displayed excellent electrocatalytic activity towards the reduction of hydrogen peroxide (H₂O₂) without any mediators. The effect of various operational parameters was explored for optimum analytical performance. The reported biosensor exhibited fast amperometric response (within 5 s) to H₂O₂. The detection limit of the biosensor was 5 μM, and linear range was from 20 μM to 20 mM. Furthermore, the biosensor exhibited high sensitivity, good reproducibility, and long‐term stability.     

CuO Crystalline Modified Glassy Carbon Electrodes for Anodic Amperometric Determination of Hydrogen Peroxide

As an alternative selection of electrocatalytic surface modifier, the electrochemically generated copper oxides is re‐ investigated by using cyclic voltammetry (CV), scanning electron microscopy (SEM) and X‐ray photoelectron spectroscopy (XPS). Interesting phenomena have been found, which indicate that the electrodeposition from the Cu²⁺ solution under cyclic voltammetric conditions can generate a transparent Cu(OH)₂ crystalline on the surface of glassy carbon electrodes, and this crystalline can be further transferred to a novel cubic opaque CuO crystalline of about 300 nm in size by second step of cyclic voltammetry in pH 12 NaOH solution. The final electrode (denoted as nano‐CuO/GCE) can catalyze the oxidation (as well as the reduction) of H₂O₂ in basic solutions. It shows pH dependent three‐part catalytic mechanism in the range from pH 7 to pH 14. In 0.10 mol/L NaOH solution, the amperometric response at 0.15 V (vs. SCE) can give a current sensitivity as high as 139 mA/(mol·L^?1) in the rage of 5.0×10^?7?6.0×10^?4 mol/L with a lower detection limit (s/n=3) of 2.5×10^?8 mol/L, and a current sensitivity of 78.4 mA/(mol·L^?1) in the rage of 6.0×10^?4–2.0×10^?3 mol/L. This electrode also has excellent reproducibility and stability. The mechanisms for the two steps of preparation and the catalytic reactions are proposed. The nano‐CuO crystalline modified electrode may have more applications in the field of electrochemical sensing.     

基于拉伸分子动力学模拟的黄嘌呤氧化酶抑制剂解离途径的理论研究

采用拉伸分子动力学模拟的方法研究了黄嘌呤氧化酶(Xanthine oxidase, XO)抑制剂别嘌呤醇(Allopurinol)和葛根素(Puerarin)从XO离去通道解离的动态过程. 分子对接结果表明, 别嘌呤醇和葛根素均结合在XO的钼蝶呤中心(Molybdopterin, Mo-pt); 丙氨酸扫描的结果显示, Val789, Arg880, Phe911, Phe914和Val1081在XO与抑制剂的结合中起到非常重要的作用. 拉伸分子动力学模拟结果显示, 相比于葛根素, 别嘌呤醇需要更大的外力和更长的时间才能从XO中解离, 拉伸过程中Arg880, Phe1009, Thr1010, Val1011和Ala1079均对维持2种复合物的结构稳定起到重要作用, Phe649和Phe1013在抑制剂解离过程中起到门控的作用, His875起到阻碍抑制剂解离的作用.     

Carbon Electrodes Modified by Nanoscopic Iron(III) Oxides to Assemble Chemical Sensors for the Hydrogen Peroxide Amperometric Detection

In this study we show that nanoparticles of various ferric oxides (hematite, maghemite, amorphous Fe₂O₃, β‐Fe₂O₃ and ferrihydrite) incorporated into carbon paste exhibit electro‐catalytic properties towards hydrogen peroxide reduction. The modified paste electrode performances were evaluated and compared with those obtained with Prussian Blue‐modified carbon paste electrode, which represents an excellent chemical mediator towards the H₂O₂ redox reaction (as widely described in literature). The best catalytic activity was found for carbon paste modified by amorphous ferric oxide with 2–4 nm particle size, which was further tested for possible application as hydrogen peroxide sensor. At pH 7, the limit of detection was 2×10^?5 M H₂O₂ (S/N=3), the calibration curves were linear upto 8.5 mM H₂O₂ (R²=0.998), the measurement reproducibility (RSD=97%, n=4), the interelectrode reproducibility (RSD=16%, n_electrodes=5) and <3 s response time.     

Detection of Hydrogen Peroxide and Glucose Based on Immobilized C60‐Catalase Enzyme

The interaction between fullerene C60 and catalase enzyme was studied with a fullerene C60‐coated piezoelectric (PZ) quartz crystal sensor. The partially irreversible response of the C60‐coated PZ crystal sensor for catalase was observed by the desorption study, which implied that C60 could chemically react with catalase. Thus, immobilized fullerene C60‐catalase enzyme was synthesized and applied in determining hydrogen peroxide in aqueous solutions. An oxygen electrode detector with the immobilized C60‐catalase was also employed to detect oxygen, a product of the hydrolysis of hydrogen peroxide which was catalyzed by the C60‐catalase. The oxygen electrode/C60‐catalase detection system exhibited linear responses to the concentration of hydrogen peroxide and amount of immobilized C60‐catalase enzyme that was used. The effects of pH and temperature on the activity of the immobilized C60‐catalase enzyme were also investigated. Optimum pH at 7.0 and optimum temperature at 25 °C for activity of the insoluble immobilized C60‐catalase enzyme were found. The immobilized C60‐catalase enzyme could be reused with good repeatability of the activity. The lifetime of the immobilized C60‐catalase enzyme was long enough with an activity of 93% after 95 days. The immobilized C60‐catalase enzyme was also applied in determining glucose which was oxidized with glucose oxidase resulting in producing hydrogen peroxide, followed by detecting hydrogen peroxide with the oxygen electrode/C60‐catalase detection system.     

Study on Hydrogen Ion-Selective Solid Contact Electrodes Based on Decamethylcyclopentasiloxane

Hydrogen ion-selective solid contact electrode based on decamethylcyclopentasiloxane (DMCS) as ionophore was fabricated. The membrane solution was prepared by mixing DMCS, polyvinyl chloride (PVC), potassium tetrakis p-chlorophenyl borate (KTpClPB) and various plasticizers. The best performance was obtained with the sensor based on NPOE (o-nitrophenyl octyl ether) and the conducting polymer layer of poly(pyrrole), doped with NaClO₄. The electrode exhibited Nernstian-response in the range of pH 1.9–9.8 with a slope of 57.6 ± 0.2 mV per decade and fast response time within 15 s. This electrode showed good selectivity and was successfully used as an indicator electrode in the potentiometric titration.     

Enhanced Electrocatalysis for the Reduction of Hydrogen Peroxide at New Multiwall Carbon Nanotube Grafted Polydiphenylamine Modified Electrode

A new modified electrode having multiwall carbon nanotube (MWNT) grafted with polydiphenylamine (PDPA) as electrocatalytic layer is fabricated. FESEM image of the modified electrode shows a different morphology indicating the grafting of PDPA over MWNT. This morphology is in quite contrast from the conventional bilayer MWNT/conducting polymer modified electrode. The multiwall carbon nanotube grafted polydiphenylamine (MWNT‐g‐PDPA‐ME) shows excellent electrocatalytic activity towards the reduction of hydrogen peroxide. The combined presence of MWNT and PDPA as a single unit provides better sensitivity than the bilayer configuration (MWNT/PDPA‐ME). This modified electrode shows accelerated electron transfer at the interface with minimized surface fouling and surface renewability. The advantages of MWNT‐g‐PDPA‐ME over the bilayer electrode are demonstrated with chronoamperometric studies. The amperometric response to H₂O₂ obtained at ?300 mV (vs. SCE) is rapid and highly sensitive as evident from the higher (2.83×10^?3 cm³ mol^?1 s^?1) rate constant for the diffusion reduction process.     

Biosensor for Hydrogen Peroxide Based on Chitosan and Nanoparticle Complex Film–Modified Glassy-Carbon Electrodes

Abstract

A biosensor for hydrogen peroxide was fabricated by co-immobilizing cadmium telluride (CdTe) nanoparticles, chitosan, and hemoglobin (Hb) matrix. There was a pair of nearly reversible redox peaks around ?0.360 V, and the electrochemical behavior of Hb was a surface-controlled process, with an electron-transfer rate constant of 1.36 s^?1 and surface coverage of 2.62 × 10^?10 mol cm^?2. Fourier transform infrared (FT-IR) spectra and ultraviolet–visible (UV-vis) spectra indicated that Hb sustained its natural conformation. It was demonstrated that Hb in the matrix kept its bioactivity and exhibited catalytic ability toward H₂O₂, with a response ranging from 7.44 × 10^?6 to 6.95 × 10^?4 M and a detection limit of 2.23 × 10^?6 M.