Urea Biosensor Based on Electrochromic Properties of Prussian Blue

Prussian blue (PB) is an electrochromic material, which can be used as a signal transducer in the formation of optical urea biosensors. The previous researches in electrochromic properties of PB demonstrated the optical PB response to ammonium ions, which occurs when ammonium ions are interacting with PB layer at a constant 0.2 V vs Ag|AgCl|KCl_sat potential. In this work PB optical dependence on ammonium ions concentration was applied in the formation of electrochromic urea biosensor. Biosensor was formed by modifying the optically transparent indium tin oxide (ITO) coated glass electrode (glass/ITO) with Prussian blue layer and immobilizing urease (glass/ITO/PB‐urease). Calibration curve showed the linear dependency (R²=0.995) between the change of maximal absorbance (ΔA) and urea concentration in concentration range varying from 3 mM to 30 mM. The highest sensitivity (4 ΔA M^?1) of glass/ITO/PB‐urease biosensor is in the concentration range from 7 mM to 30 mM. It was determined that working principle of the glass/ITO/PB‐urease biosensor is not related to pH changes occurring during enzymatic hydrolysis of urea.     

Enzyme Urea Biosensor Based on a Modified Potentiometric PVC-Nonactin Membrane Electrode for Assay of Urea in Blood

Abstract

The developed potentiometric urea biosensor is based on a modified PVC-nonactin NH₃ -sensitive gas electrode. Membrane resistance has been optimised by incorporating lipophilic salt. The coefficient of variation for the standard urea solution is 2.45. The sensor performance has been compared with a photometric method for blood samples.     

Optical Urea Biosensor Based On Ammonium Ion Selective Membrane

ABSTRACT

An optical urea biosensor was developed by immobilizing an urease enzyme layer on a thin ammonium-selective polymer membrane. The ammonium optical membrane utilized dichlorofluorescein octadecyl ester (DCFOE) as anionic chromophore and nonactin as neutral ionophore. The urease layer was coated on the top of the ammonium layer by gelatin entrapment combined with glutaradehyde cross-linking. Hydrolysis of urea catalyzed by urease produced ammonium ion, which was extracted into the-polymer film to form complexes with nonactin. A proton was released which resulted in a color change of the optical membrane due to charge neutrality principle. The biosensor     

Amperometric Urea Biosensor Using Aminated Glassy Carbon Electrode Covered with Urease Immobilized Carbon Sheet,Based on the Electrode Oxidation of Carbamic Acid

A large oxidation current can be observed when ammonium carbamate aqueous solution is electrolyzed using a glassy carbon electrode (GCE) at a potential exceeding 1.0 V vs. Ag/AgCl and amino groups are introduced at the surface of the GCE. Aminated GCE exhibits the electrocatalytic activity of the oxidation of ammonium carbamate that is produced from urea as an intermediate product of urease reaction, and a distinct oxidation current is observed when the aminated GCE is used to oxidize the urea in the urease solution. A novel amperometric determination method to detect urea has been developed. This method is based on the electrooxidation of carbamic acid produced during urease reactions. Urease is immobilized to polymaleimidostyrene (PMS) coated on the insulated amorphous carbon sheet set on the aminated GCE surface. A good linear relationship is observed between urea concentration and the electrolytic current of the urease‐immobilized electrode in the concentration range from 0.5 mM to 21.0 mM. The proposed urea biosensor has an effective merit in that the interference resulting from ammonia and pH change caused by the urease reaction can be eliminated, differing from conventional urea biosensors.     

Use of CdSe/ZnS luminescent quantum dots incorporated within sol-gel matrix for urea detection

In this work, urea detection techniques based on the pH sensitivity of CdSe/ZnS QDs were developed using three types of sol-gel membranes: a QD-entrapped membrane, urease-immobilized membrane and double layer consisting of a QD-entrapped membrane and urease-immobilized membrane. The surface morphology of the sol-gel membranes deposited on the wells in a 24-well microtiter plate was investigated. The linear detection range of urea was in the range of 0-10 mM with the three types of sol-gel membranes. The urea detection technique based on the double layer consisting of the QD-entrapped membrane and urease-immobilized membrane resulted in the highest sensitivity to urea due to the Michaelis-Menten kinetic parameters. That is, the Michaelis-Menten constant (K_m =2.0745 mM) of the free urease in the QD-entrapped membrane was about 4-fold higher than that (K_m =0.549 mM) of the immobilized urease in the urease-immobilized membrane and about 12-fold higher than that (K_m =0.1698 mM) of the immobilized urease in the double layer. The good stability of the three sol-gel membranes for urea sensing over 2 months showed that the use of sol-gel membranes immobilized with QDs or an enzyme is suitable for biomedical and environmental applications.     

尿素酶固载于纳米二氧化钛多孔膜上的尿素生物传感器

在钛丝基体上沉积一层纳米二氧化钛(TiO2)多孔膜,然后直接将尿素酶吸附在TiO2膜上。基于TiO2膜的pH响应,发展了一种廉价的、易于微型化的pH敏尿素酶传感器。采用石英微天平、红外光谱和紫外可见光谱等手段研究尿素酶在TiO2膜上的物理固载行为。由于纳米TiO2在紫外光下光自洁特性可获得高度洁净的表面,石英微天平研究表明在光自洁后的TiO2膜上尿素酶的吸附具有很好的重复性和稳定性,吸附量为0.22mmol/g,吸附平衡常数k为3.15×105L/mol。采用电位法测定了尿素酶/TiO2复合膜电极的性能及其影响因素,在1.0 mmol/L pH 7~8 PBS,35℃,尿素的响应范围为8.5×10-5~1.5×10-1mol/L,相关系数r为0.993 7,检出限为6×10-5mol/L。     

Enhancement of Enzymatic IDE Biosensor Response Using Gold Nanoparticles. Example of the Detection of Urea

A new conductometric biosensor based on interdigitated electrodes (IDEs) has been developed for the detection of enzymatic substrates using gold nanoparticles (GNPs), synthesized bellowing the citrate process, with an average diameter of 23 nm and functionalized with urease using layer‐by‐layer technique. A detection limit of 100 µM of urea is obtained when cross‐linked urease is directly immobilized on top of the IDEs (interdigitated distance: 20 µm) whereas a detection limit of 2 µM is obtained when urease functionalized gold nanoparticles are deposited on the top of the IDEs. The use of gold nanoparticles allows the increase of the sensitivity of detection (from 10 µS/mM to 107 µS/mM) due to the decrease of the thickness of probed zone.     

基于拮抗作用检测除草剂的类囊体膜生物传感器研究

利用除草剂对植物类囊体束缚酶分解过氧化氢的拮抗作用,研制了一种快速检测痕量除草剂的电化学生物传感器.将植物类囊体用聚乙烯醇-苯乙烯吡啶(PVA-SbQ)光敏聚合剂在紫外光诱导下产生大分子网状结构进行包埋,制成生物敏感膜,并固定在铂电极表面.根据加入除草剂时类囊体膜束缚酶分解过氧化氢活性的变化,对除草剂进行测定.在含有1×10^-3mol/LNaCl,5×10^-3mol/LMgCl₂和0.01mol/LH₂O₂的Tris-HCl缓冲溶液(pH=7.4)中,基于测量0.65V处H₂O₂氧化电流的变化,可以对下列浓度的除草剂进行定量检测:百草枯3×10^-9～1.5×10^-7mol/L,敌草龙1×10^-8～3×10^-7mol/L,扑草净4×10^-8～3×10^-6mol/L,阿特拉津1×10^-7～5×10^-6mol/L,莠灭净1×10^-7～5×10^-6mol/L.利用PVA-SbQ光聚合膜固定类囊体,能够使酶的活性在低温下保持数月.     

Development of a Urea Bioprobe Based on Platinized Boron‐Doped Diamond Electrodes

Urea (CH₆ON₂) is one of the main human nitrogen‐based metabolic wastes. The concentration of urea in blood lies between 2.5–7 mM for healthy individuals, and is commonly used as an indicator for several diseases that may alter this value. Spectrophotometric methods are employed for the determination of blood urea concentration during clinical assays. Although these methods are sensitive, they make use of toxic reagents and complex reaction schemes. Therefore, in this research we present the bioelectrochemical determination of urea by the use of the protein urease (E.C.3.1.1.5) along with a nano‐platinized boron‐doped diamond electrode. This approach has been proven to be efficient and sensitive providing a platform with detection limits of 1.79 mM (S/N=3). The linear range resulted from 1 mM to 25 mM for the determination of urea, and response time of five minutes.     

醋酸纤维膜固定过氧化氢酶电极的研究

本将过氧化氢酶与经活化处理的醋酸纤维膜共价固定，并与氧电极偶合，研制成静态和流通二种过氧化氢生物电极。研究了醋酸纤维膜的活化、酶的固定化、测试介质等有关的实验条件和参数。测得静态和流通二种电极响应的线性范围分别为６．７×１０＾－５￣２．０×１０＾－３ｍｏｌ／Ｌ和１．３×１０＾－４￣１．０×１０＾－３ｍｏｌ／Ｌ。将电极用于实际试样中的回收率测定时，获得了较好的结果。     

An Electrochemical Acetylcholinesterase Biosensor Based on Fluorene(bisthiophene) Comprising Polymer for Paraoxon Detection

In this study, a novel conductive polymer comprising biosensor based on poly-2,2′-(9,9-dioctyl-9 h-fluorene-2,7-diyl)bistiophene (Poly(BT)) and acetylcholinesterase (AChE) was reported for the determination of paraoxon. This practical biosensor allowed to catalyze electrochemical oxidation of acetylthiocholine (+0.6 V vs. Ag reference). The detection range for acetylcholine chloride (AThCl) with Poly(BT)/AChE was found to be 0.025–4 mM. In pesticide analysis, wide linear ranges from 0.5 to 1 μg/L and 1 to 14 μg/L, and a low detection limit of 0.033 μg/L were estimated. Under optimum operating conditions, the developed biosensor was used for pesticide detection in milk and tap water samples, effectively.     

醋酸纤维素膜固定化脲酶的研究

酶固定膜反应器兼具有反应和分离两种功能,是酶工程领域中较活跃的研究课题.随着酶固定化技术和水平的提高,各种固定化酶生物反应器不断涌现,其中以采用固定化脲酶技术制作的人工肾最为成功.     

Modelling of the Potentiometric Response of ENFETs Based on Enzymatic Multilayer Membranes

Based on the modified site‐binding model, the response of urea‐sensitive enzymatic field effect transistors (ENFETs) is fitted. The effect of two types of surface sites (silanol and amine sites) and of additional polymeric permselective membrane on the sensitivity of the biosensor is discussed. It is found that the dependence of the slope of the sensor response versus pUrea, on the buffer concentration and on the diffusion of the urea in the enzymatic membrane can be translated by a parameter α that depends on the membrane composition. Moreover, the enhancement of the sensor sensitivity by deposition of additional permselective membranes can be illustrated by a parameter δ that depends on the association of additional permselective membranes and a buffer; the higher values of parameter δ are obtained with a Nafion membrane associated with a phosphate buffer.     

Enzymatic Biosensor Based on One-step Electrodeposition of Graphene-gold Nanohybrid Materials and its Sensing Performance for Glucose

RGO/Au/Ni electrode was manufactured by a convenient, controllable, and environmental process, which was carried out by cyclic voltammetry (CV), and in this process, graphene-gold nanohybrid materials were simultaneously deposited on the nickel foam. Then the GOx was immobilized on the RGO/Au/Ni electrode by covalent bonding, and obtained the enzymatic biosensor. Scanning electron microscope (SEM) and Raman spectroscopy were adopted to confirm the microstructure of the fabricated RGO/Au/Ni electrode. Fourier transform infrared spectroscopy (FT-IR) was used to characterize the prepared enzymatic electrode. CV, chronoamperometry, and electrochemical impedance spectroscopy (EIS) were used to characterize the electrochemical performance of the fabricated enzymatic biosensor. It is found that AuNPs were well dispersed on the wrinkled RGO sheets, and the biosensor had a high sensitivity to glucose (32.83 μA ⋅ mM⁻¹ ⋅ cm⁻²) with a wide linear range (0.15 26.15 mM), the strengths of anti-interference ability, good stability, and repeatability, etc.     

基于多壁碳纳米管的三电极血乙醇生物传感器的研究

采用丝网印刷技术在PVC基板上印制三电极,将多壁碳纳米管、麦尔多拉蓝、乙醇脱氢酶(ADH)以及氧化型辅酶Ⅰ(NAD+)依次修饰在工作电极表面,然后在该三电极表面贴一层亲水膜,形成一个5μL反应池,制成血乙醇检测新型生物传感器检测条。结果表明,此生物传感器具有良好的准确性和稳定性;检测线性范围为0.5～20mmol/L,r=0.99493;检出限为0.22mmol/L;电流达到95%稳态时间小于15s。考察了pH值、温度及干扰物对生物传感器的影响。用此生物传感器和顶空气相色谱法对10份全血标本乙醇浓度平行测试,两者相关性良好r=0.97583。利用虹吸现象吸取微量全血直接定量测定乙醇浓度是此生物传感器的特点。     

Immobilization of Urease from Pigeonpea (<Emphasis Type="Italic">Cajanus cajan</Emphasis>) on Agar Tablets and Its Application in Urea Assay

The pigeonpea urease was immobilized on agar, a common gelling substance. The tablet strips were used as moulds to cast agar tablets of uniform shape and size. The time and temperature of solidification of agar was 6 min and 44 degrees C, respectively. The 5 % agar (w/v) and 0.019 mg protein/agar tablet yielded an optimum immobilization of 51.7%. The optimum pH was shifted through 0.2 U (from 7.3 to 7.5) towards basic side upon immobilization. The optimum temperature of soluble and immobilized urease was 30 degrees C and 60 degrees C, respectively, showing the improvement in thermal stability of urease. There was an increase in K m from 3.23 to 5.07 mM after immobilization. The half-lives of soluble and immobilized urease were 21 and 53 days, respectively, at pH 7.3 and 4 degrees C. The urea was estimated in different blood samples with the help of immobilized urease and the results were consistent with those from clinical pathology laboratory through an autoanalyzer (Zydus Co., Rome, Italy).     

A Glucose Biosensor Based on Immobilization of Glucose Oxidase on Platinum Nanoparticle Doped Santa Barbara Amorphous Material-15

A novel electrochemical glucose biosensor was prepared by combining platinum nanoparticle doped Santa Barbara Amorphous Material 15 with glucose oxidase. The resulting material demonstrated high stability and reactivity for catalyzing glucose electrolytic oxidation, primarily due to the high surface area of these catalysts. This glucose biosensor was capable of interference-free determination of glucose with a linear dynamic range from 0.03 to 12.0 mmol L^?1. In addition, it has the advantages of simple preparation and good stability. The reported method is promising for the determination of glucose in human serum.     

A Micromechanical Cantilever‐Based Liposome Biosensor for Characterization of Protein‐Membrane Interaction

We have newly evaluated the interaction of lipid membrane with two different proteins of lysozyme and carbonic anhydrase from bovine (CAB) using a micro cantilever‐based liposome biosensor with a new droplet‐sealing structure. Herein 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine (DPPC) liposomes are used as model lipid membrane and are immobilized on the surface of cantilever. The interaction of DPPC liposome with the target protein causes deflection of the micro‐cantilever, which can stably be detected by measuring the resistance change of the strain gauge. The resistance change dependent on time is used to evaluate the characteristic of liposome‐protein interaction. The resistance of the cantilever‐based biosensor increases monotonously with time in both of the two protein solutions. Especially, chronological resistance change depends markedly on both the concentration and species of target proteins. Finally, these results lead us to conclude that the cantilever‐based liposome biosensor with the droplet‐sealing structure facilitates the characterization of protein‐membrane interaction. It also means that this biosensor is a promising candidate device for label‐free detection of concentration and species of different target proteins.     

Construction and Characterization of a Beet Stem Based Biosensor for Oxalate

Abstract

This report describes the construction and characterization of an oxalate-sensing electrode. The electrode is based on the incorporation of ground beet stem into the graphite paste of a graphite paste electrode. The hydrogen peroxide generated by enzymatic degradation of oxalate is monitored at a working voltage of 0.900 V vs SCE. All measurements were conducted in a succinic acid/EDTA buffer at pH 4.00. Under these conditions, the electrodes exhibit reproducible responses to oxalate. The lower limit of oxalate detection was less than 1.03 × 10^?4 M. The time to achieve a steady state response after exposure to a step change in oxalate concentration in solution is less than one minute. The magnitude of response to oxalate over the oxalate concentrations studied varies among several electrode tested as does the degree of linearity of response. An electrode studied still exhibited analytically useful responses to oxalate on the 15th day of its use. The beet stem-based electrodes display little response to glycolic acid, glucose, DL-valine, or pyruvate.     

Piezoelectric Crystal Biosensor System for Detection of Escherichia Coli

Abstract

A piezoelectric crystal biosensor system was applied to the detection of Escherichia coli. the system consists of an oscillator, a frequency counter, a flow cell and a modified piezoelectric crystal. Anti-E. coli antibody is immobilized on the surface of the crystal. It is used as an E. coli detection by measuring its resonant frequency shift due to a mass change caused by specific binding of the micro organisms to the surface. the frequency shift correlates with an E. coli concentration in the range of 10⁶?10⁸ cells·cm^?3. the resonant frequency shift is increased by further treatment to bind micro-particles modified with anti-E. coli antibody. This method allows us to improve the determination limit to 10⁵ cells · cm^?3.