Flow Injection Amperometric Detection at Enzyme‐Modified Gold Nanoelectrodes

Gold nanotubular electrode ensembles were prepared by using electroless deposition of the metal within the pores of polycarbonate particle track‐etched membranes. Glucose oxidase (Gox), used as a model enzyme, has been immobilized onto preformed self‐assembled monolayers (mercaptoethylamine or mercaptopropionic acid) on electroless gold via cross‐linking with glutaraldehyde or covalent attachment by carbodiimide coupling. Flow‐injection analysis systems in flow‐through or wall‐jet configurations using these Gox‐modified nanoelectrodes are described. The influence of different experimental parameters (i.e., applied potential, flow rate, interferents…?) on the analytical response of the sensor to glucose has been evaluated. Under optimized conditions, very reproducible results (standard deviations <4%, n=38) were obtained, linear calibration was achieved in the 2×10^?4 M to 3×10^?2 M concentration range and the detection limit was 2×10^?4 M. Moreover, no significant interferences from species like ascorbic and uric acids were observed at a potential of +0.9 V.     

Disposable Sandwich‐type Electrochemical Sensor for Selective Detection of Glucose Based on Boronate Affinity

A disposable sandwich‐type electrochemical sensor for selective detection of glucose was established. The primary receptor, 3‐aminophenylboronic acid was grafted covalently onto the surface of screen‐printed carbon electrodes through an in situ‐generated diazo‐reaction. Glucose was first captured by boronic acid group on the electrode, followed by captureing an electroactive ferroceneboronic acid (FcBA) as the secondary receptor to form bidentate glucose‐boronic complex. Electrochemical impedance spectroscopy was applied to characterize the construction of sandwich‐type disposable sensor. In the sandwich assay, current response of captured FcBA on the electrode was dependent on the concentration of glucose. The sandwich assay showed higher selectivity for glucose than that for fructose, mannose, galactose and other electroactive interferences including uric acid, ascorbic acid and dopamine, and exhibited a dynamic concentration range of glucose from 0.5 to 20.0 mmol L⁻¹. The disposable sensor demonstrated a good reproducibility with 2.2 % relative standard deviation (RSD). In addition, the disposable glucose sensor was used in detection of the trace glucose in the clinical urine samples.     

Fabrication of Photoelectrochemical Glucose Biosensor in Flow Injection Analysis System Using ZnS/CdS‐Carbon Nanotube Nanocomposite Electrode

In this work, a photoamperometric glucose biosensor based on glucose oxidase (GODx) was developed in flow injection analysis (FIA) system using ZnS‐CdS quantum dot (QD) modified multiwalled carbon nanotube/glassy carbon electrode (ZnS‐CdS/MWCNT/GCE). Cyclic voltammograms of the proposed electrode (GODx/ZnS‐CdS/MWCNT/GCE) showed a pair of well‐defined reversible redox peak attributing that direct electron transfer between the protein and electrode. The current of the reduction peak became more cathodic in the presence of O₂ due to the electrocatalytic activity of the electrode towards the reduction of dissolved O₂, but reduction current shifted to a less negative value upon addition of glucose in the solution. The obtained CV currents were affected by the irradiation of the electrode surface. Thus, the photoelectrochemical biosensing of glucose in the FIA system was studied by monitoring of the changes in the electrocatalyzed reduction peak current of dissolved O₂ at the proposed electrode dependent on glucose concentration. The proposed photoelectrochemical FIA method has a linear response to glucose ranging from of 0.01 to 1.0 mM with detection limit of 3.0 μM under optimized conditions. Photoelectrochemical biosensor was successfully fabricated in FIA system for selective, sensitive and repeatable detection of glucose and has been satisfactorily applied to determination of glucose in real sample.     

Flow Injection Analysis of Glucose in Human Serum by Chemiluminescence

Abstract

Combining the rigidly controlable conditions of Flow Injection Analysis with detection by chemiluminescence, a method for determination of glucose in serum via enzymatic degradation by glucose in serum via enzymatic degradation by glucose oxidase is described. With a Standard deviation of less than 2% the sampling frequency is 75 samples per hour. Results obtained by the proposed procedure are compared with those acquired by a routine AutoAnalyzer method used at a local hospital. It is shown that any influence on the yield of chemiluminescence due to difference in viscosity of the injected samples effectively can be eliminated.     

Minimization of Interferences in Flow Injection Amperometric Glucose Biosensor Based on Oxidation of Enzymatically‐produced H2O2

One of the major problems in amperometric biosensors based on detection of H₂O₂ produced by enzymatic reaction between oxidase enzymes and substrate is the interference of redox active compounds such as ascorbic acid (AA), dopamine (DA) and uric acid (UA). To minimize these interferences, sodium bismuthate was used for the first time as an insoluble pre‐oxidant in the flow injection (FI) amperometric glucose biosensor at a Glucose oxidase (GOx) immobilized Pt/Pd bimetallic modified pre‐anodized pencil graphite electrode (p.PGE). In this context, these interfering compounds were injected into a flow injection analysis (FIA) system using an injector which was filled with NaBiO₃. Thus, these interferents were converted into their redox inactive oxidized forms before reaching the electrode in the flow cell. While glucose was not influenced by the pre‐oxidant in the injector, the huge oxidation peak currents of the interferents decreased significantly in the biosensor. FI amperometric current time curves showed that the AA, DA and UA were minimized by 96 %, 86 %, and 98 % respectively, in the presence of an equivalent concentration of interferences in a 1.0 mM glucose solution. The proposed FI amperometric glucose biosensor exhibits a wide linear range (0.01–10 mM, R²=0.9994) with a detection limit of 2.4×10^?3 mM. Glucose levels in the artificial serum and two real samples were successfully determined using the fabricated FI amperometric biosensor.     

Flow in a Paper‐based Bioactive Channel – Study on Electrochemical Detection of Glucose and Uric Acid

Although paper‐based analysis is known for centuries, only during the last decade this simple substrate became an object of detailed microfluidic studies. In order to obtain optimum performance and separation of the analytes in a microfluidic channel, devices should be optimized, both in terms of architecture and paper properties. Enzyme immobilization methods can not only increase the storage stability but also have an impact on the flow in paper matrix, providing additional charges, and changing the porous structure of paper. Therefore it should be guaranteed that the method of choice will not obstruct the flux in the final device. Paper‐based device proposed in this study was composed of a bioactive channel, Pt working electrode, pencil drawn pseudo‐reference electrode, a buffer filled sponge providing the wicking solution and a stack of wicking pads to guarantee continuous flow. Based on our previous research we chose 4 methods of enzyme immobilization relying on different phenomena (adsorption, covalent linkage, layer‐by‐layer, capsules). Different channel architectures were also evaluated in order to achieve optimum time of the enzymatic reaction, separation of peaks and the time of measurement. Experimental results were compared with computer simulations. Final device could quantify glucose (2.0–10.0 mmol L^?1) and uric acid (0.1–1.2 mmol L^?1) in their clinical range with good repeatability.     

Glucose Biosensor Using Glucose Oxidase and Electrospun Mn2O3‐Ag Nanofibers

The highly porous Mn₂O₃‐Ag nanofibers were fabricated by a facile two‐step procedure (electrospinning and calcination). The structure and composition of the Mn₂O₃‐Ag nanofibers were characterized by SEM, TEM, XRD, EDX and SAED. The as‐prepared Mn₂O₃‐Ag nanofibers were then employed as the immobilization matrix for glucose oxidase (GOD) to construct an amperometric glucose biosensor. The biosensor shows fast response to glucose, high sensitivity (40.60 µA mM^?1 cm^?2), low detection limit (1.73 µM at S/N=3), low K_m,app value and excellent selectivity. These results indicate that the novel Mn₂O₃‐Ag nanfibers‐GOD composite has great potential application in oxygen‐reduction based glucose biosensing.     

Direct Electrochemistry of Glucose Oxidase at Reduced Graphene Oxide and β‐Cyclodextrin Composite Modified Electrode and Application for Glucose Biosensing

A simple glucose biosensor has been developed based on direct electrochemistry of glucose oxidase (GOx) immobilized on the reduced graphene oxide (RGO) and β‐cyclodextrin (CD) composite. A well‐defined redox couple of GOx appears with a formal potential of ～?0.459 V at RGO/CD composite. A heterogeneous electron transfer rate constant (K_s) has been calculated for GOx at RGO/CD as 3.8 s^?1. The fabricated biosensor displays a wide response to glucose in the linear concentrations range from 50 µM to 3.0 mM. The sensitivity and limit of detection of the biosensor is estimated as 59.74 µA mM^?1 cm^?2 and 12 µM, respectively.     

Investigation of a Biosensor Based on Phenylalanine Dehydrogenase Immobilized on a Polymer‐Blend Film for Phenylketonuria Diagnosis

We investigated a L ‐phenylalanine (L ‐phe) biosensor, functionalized through enzyme immobilization on a polymer‐blend film. The electron mediator 3,4‐dihydroxybenzaldehyde (3,4‐DHB) was employed at the electrode surface to improve direct oxidation of NADH to NAD⁺ and no additional reagents is required to be added to the sample solution. The bioactivated electrode was coated with a semi‐permeable cellulose acetate membrane in order to prevent dissolution of biofunctionalized polymer‐blend film. This constructed enzyme electrode is the first selective biosensor for phenylketonuria (PKU) detection. The sensitivity of the enzyme electrode was determined as 12.014 mA/M cm². The Michaelis–Menten and current responses as well as sensitivity of the electrode showed improved values than those of previous works. This selective biosensor presented an excellent electroanalytical response for L ‐phe, with a high steady‐state current being obtained after 20 s. The sensitivity of our biodevice is quite sufficient for the purpose of PKU detection because the reference range of clinical concern for L ‐phenylalanine concentration is C_{L ‐phe}>0.5 mM. This surface‐bioactivated enzyme electrode retained more than 80 % of its electrocatalytic activity after 16 days.     

Nickel/Copper Bilayer‐modified Screen Printed Electrode for Glucose Determination in Flow Injection Analysis

This work reports about the performance of a Ni/Cu‐modified screen printed electrodes (SPE/Ni/Cu), prepared by physical vapor deposition (PVD) in an oblique angle configuration (OAD), for non‐enzymatic glucose sensing applications. SPE/Ni/Cu electrodes showed an excellent reversibility and a catalytic behavior for detection of glucose that were controlled by the diffusion of reactants up to the active sites at the electrode surface. The study with a flow injection analysis (FIA) setup of the main experimental variables affecting the detection process has shown that the developed electrode system had an excellent glucose sensitivity of 1.04 A M⁻¹cm⁻² (R²:0.999), a linear response up to 1 mM, a limit of detection of 0.33 μM and a time of analysis of ca. 30 s per sample. The selectivity of the sensor was checked against various interferences, including ascorbic acid, uric acid, acetaminophen and other sugars, in all cases with excellent results. The feasibility of using this sensor for practical applications was successfully confirmed by determining the glucose concentration in different commercial beverages.     

Structures and Magnetic Properties of Two Copper(II) Complexes with 2,5‐Dichloro‐3,6‐Dihydroxy‐1,4‐Benzoquinone Dianionic and Tetrachloroquinone Ligand

The polynuclear copper(II) complex [Cu₂(Hdpa)₂(μ‐ClDHBQ)(ClO₄)₂]_n, 1 is bridged by ClDHBQ^?2 (2,5‐dichloro‐3,6‐dihydroxy‐1,4‐benzoquinone dianionic) and 2,2′‐dipyridylamine (Hdpa). In the axial position, Cu is connected with the oxygen atom of ClO. The perchlorate anion may be envisaged as a monodentate O‐bound ligand. Through the bond bridge of O–Cu … O–Cl, the binuclear compound [Cu₂(Hdpa)₂(μ‐ClDHBQ)(ClO₄)₂] is strung together into a long chain compound. Tetrachlorocatechol underwent partial oxidation/hydrolysis/dechlorination processes to produce ClDHBQ^?2. The other mononuclear complex [Cu(Hdpa)(TeCQ)](DMF), 2 , in which tetrachloroquinone (TeCQ) was produced by oxidation of tetrachlorocatechol (TeCC), therefore complex 2 is in the quinone form. The magnetic susceptibility measurements show antiferromagnetic coupling with J = ?11.9 cm^?1, θ = 2.6 K, and g = 2.05 for complex 1. Complex 2 exhibits the typical paramagnetic behavior of s = 1/2.     

流动注射分析血清中葡萄糖的电化学检测池

测定血清中葡萄糖含量是临床检验的重要项目之一。何立千曾对各种血糖测定方法做过综述。葡萄糖氧化酶-4-氨基安替比林-苯酚光度法(酶制剂法)虽能较好地测定血清中葡萄糖的含量,但成本较高,耗时长。因此有必要研究一种快速、廉价、稳定的分析方法。人们考虑到酶的专属性,对葡萄糖氧化酶电极做了大量工作,然而,这种电极的寿命仍不理想。     

Flow Injection Analysis of Chloramphenicol by Using a Disposable Wall‐Jet Ring Disk Carbon Electrode

A preanodized screen‐printed ring disk carbon electrode was applied to the determination of chloramphenicol (Ph? NO₂, CAP) by flow injection analysis (FIA). By setting up the first irreversible reduction reaction of Ph? NO₂ to Ph? NHOH at the disk electrode, the following reversible oxidation of hydroxylamine (Ph? NHOH) to the nitroso (Ph? NO) derivative can be monitored/collected at the ring electrode for CAP analysis. The interference from dissolved oxygen and others can thus be avoided by using this approach and precise CAP determination can be easily performed by FIA under aerobic conditions. Preanodization treatment helps to lower the overpotential of the electrochemical reaction of CAP and favors the selective detection in aqueous medium. Under the optimum conditions, ten repetitive determinations at 1 μM and 10 μM CAP resulted in relative standard deviations of less than 4%, indicating good reproducibility of the system. A linear calibration range of 0.1–20 μM with a detection limit of 0.074 μM (S/N=3) was obtained. Veterinary pharmaceutics were finally analyzed by this sensor to validate its practical applicability.     

Electron‐Transfer Mediator Microbiosensor Fabrication Based on Immobilizing HRP‐Labeled Au Colloids on Gold Electrode Surface by 11‐Mercaptoundecanoic Acid Monolayer

In this paper, a new strategy for constructing a mediator‐type amperometric hydrogen peroxide (H₂O₂) microbiosensor was described. An electropolymerized thionine film (PTH) was deposited directly onto a gold electrode surface. The resulting redox film was extremely thin, adhered well onto a substrate (electrode), and had a highly cross‐linked network structure. Consequently, horseradish peroxidase (HRP) was successfully immobilized on nanometer‐sized Au colloids, which were supported by thiol‐tailed groups of 11‐mercaptoundecanoic acid (11‐MUA) monolayer covalently bound onto PTH film. With the aid of the PTH mediator, HRP‐labeled Au colloids microbiosensor displayed excellent electrocatalytical response to the reduction of H₂O₂. This matrix showed a biocompatible microenvironment for retaining the native activity of the covalent HRP and a very low mass transport barrier to the substrate, which provided a fast amperometric response to H₂O₂. The proposed H₂O₂ microbiosensor exhibited linear range of 3.5 μM–0.7 mM with a detection limit of 0.05 μM (S/N=3). The response showed a Michaelis‐Menten behavior at larger H₂O₂ concentrations. The K_M^app value for the biosensors based on 24 nm Au colloids was found to be 47 μM, which demonstrated that HRP immobilized on Au colloids exhibited a high affinity to H₂O₂ with no loss of enzymatic activity. This microbiosensor possessed good analytical performance and storage stability.     

Selective Determination of Verapamil in Pharmaceutics and Urine Using a Boron‐doped Diamond Electrode Coupled to Flow Injection Analysis with Multiple‐pulse Amperometric Detection

This work presents a simple and low‐cost method for fast and selective determination of Verapamil (VP) in tablets and human urine samples using a boron‐doped diamond working electrode (BDD) coupled to a flow injection analysis system with multiple pulse amperometric detection (FIA‐MPA). The electrochemical behaviour of VP in 0.1 mol L⁻¹ sulfuric acid showed three merged oxidation peaks at around +1.4 V and upon reverse scan, one reduction peak at 0.0 V (vs. Ag/AgCl). The MPA detection was performed applying a sequence of three potential pulses on BDD electrode: (1) at +1.6 V for VP oxidation, (2) at +0.2 V for reduction of the oxidized product and (3) at +0.1 V for cleaning of the working electrode surface. The FIA system was optimized with injection volume of 150 μL and flow rate of 3.5 mL min⁻¹. The method showed a linear range from 0.8 to 40.0 μmol L⁻¹ (R>0.99) with a low limit of detection of 0.16 μmol L⁻¹, good repeatability (RSD<2.2 %; n=10) and sample throughput (45 h⁻¹). Selective determination of VP in urine was performed at+0.2 V due to absence of interference from ascorbic and uric acids in this potential. The addition‐recovery tests in both samples were close to 100 % and the results were similar to an official method.     

Reduced Graphene Oxide/Pt Nanoparticles/Zn‐MOF‐74 Nanomaterial for a Glucose Biosensor Construction

In this study, a new glucose biosensor was fabricated by immobilizing glucose oxidase (GOx) on platinum nanoparticles (Pt NPs) decorated reduced graphene oxide (rGO)/Zn‐MOF‐74 hybrid nanomaterial. Herein, the biosensor fused the advantages of rGO with those of porous Zn‐MOF and conductive Pt NPs. This has not only enlarged the surface area and porosity for the efficient GOx immobilization and faster mass transport, but also provided favorable electrochemical features such as high current density, remarkable electron mobility through metal nanoparticles, and improved electron transfer between the components. The GOx‐rGO/Pt NPs@Zn‐MOF‐74 coated electrode displayed a linear measurement range for glucose from 0.006 to 6 mM, with a detection limit of 1.8 μM (S/N: 3) and sensitivity of 64.51 μA mM^?1 cm^?2. The amperometric response of the enzyme biosensor demonstrated the typical behavior of Michaelis‐Menten kinetics. The obtained satisfying sensitivity and measurement range enabled fast and accurate glucose measurement in cherry juice using the fabricated biosensor. The water‐stable Zn‐MOF‐74 demonstrated higher enzyme loading capacity and can be potent supporting material for biosensor construction.     

Electrochemical Biosensor Based on Bienzyme and Carbon Nanotubes Incorporated into an Os‐complex Thin Film for Continuous Glucose Detection in Human Saliva

Saliva opens a door for noninvasive and painless glucose testing since it reflects changes in the body physiology of diabetic individuals as compared to healthy ones. In this paper, a unique, disposable saliva biosensor has been developed for accurate, low cost, and continuous glucose monitoring. The biosensor exhibits linear dependence of the catalytic current upon glucose bulk concentration over the 0.05–1.5 mM range (R=0.998). A detection limit of 0.003 mM can be calculated considering three times the standard deviation of the blank signal divided by the sensitivity of the sensor. The selectivity of the biosensor was evaluated by adding the interferent species of lactate, ascorbic acid and uric acid into in 0.5 mM glucose; the nearly negligible interference current indicates its good selectivity. The operational stability of the biosensor was measured in 1 mM glucose over a 2 h period (RSD=3.27 %). A clinical trial on real‐time noninvasive salivary glucose monitoring was carried out on 30 individuals by measuring subjects’ salivary glucose and blood glucose in parallel. The results show that there is a good correlation of glucose levels in saliva and in blood 2 h after breakfast. Thus, the disposable biosensor would be a potential alternative for continuous glucose detection in human saliva.     

Plasma‐functionalized Highly Aligned CNT‐based Biosensor for Point of Care Determination of Glucose in Human Blood Plasma

In this study, a new method for modification of vertically aligned carbon nanotube arrays (VACNTs) for selective detection of glucose was developed. VACNTs were grown by chemical vapor deposition method on a silicon substrate deposited with alumina as a buffer layer and iron as a catalyst using radio frequency (RF) sputtering and electron beam evaporation, respectively. The surface of the electrode was modified with electrodeposition of polyaniline (PANI) followed by covalent attachment of glucose oxidase (GOx). The electrode was characterized using field emission scanning electron microscopy (FESEM), micro‐Raman spectroscopy, and attenuated total reflectance Fourier transform infrared spectrometer (ATR‐FTIR) techniques. Cyclic voltammetry and differential pulse voltammetry were used to investigate the electrochemical behavior of the electrode. The fabricated electrode was successfully employed as a point‐of‐care (POC) biosensor for the detection of glucose in human blood plasma. The detection limit was 1.1 μM, and the sensitivity was 620 μA mM^?1 cm^?2 at the linear range of 2–426 μM.     

Preparation of GOD/Sol‐Gel Silica Film on Prussian Blue Modified Electrode for Glucose Biosensor Application

A novel amperometric glucose biosensor was fabricated by in situ incorporating glucose oxidase (GOD) within the sol‐gel silica film on a Prussian blue (PB) modified electrode. The method is simple and controllable, which combined the merits of in situ immobilizing biomolecules in sol‐gel silica film by electrochemical method and the synergic catalysis effects of PB and GOD molecules. Scanning electron microscopy (SEM) showed that the GOD/sol‐gel silica film was homogeneous with a large number of three‐dimensional nanopores, which not only enhanced mass transport, but also maintained the active configuration of the enzyme molecule and prevented the leakage of enzyme, therefore improved the stability and sensitivity of the biosensor. The fabricated biosensor showed fast response time (10 s), high sensitivity (26.6 mA cm^?2 M^?1), long‐term stability, good suppression of interference, and linear range of 0.01 mM–5.8 mM with a low detection limit of 0.94 μM for the detection of glucose. In addition, the biosensor was successfully applied to determine glucose in human serum samples.     

Amperometric Response of Hydrogen Peroxide at Carbon Nanotubes Paste Electrodes Modified with an Electrogenerated Poly(Fe(III)‐5‐amino‐phenantroline). Analytical Applications for Glucose Biosensing

This work reports the catalytic activity of a polymer electrogenerated from Fe(III)‐5‐amino‐1,10‐phenantroline solution at a carbon nanotubes paste electrode (CNTPE) towards the oxidation and mainly the reduction of hydrogen peroxide. The important role of carbon nanotubes on the generation of poly(Fe(III)‐5‐amino‐1,10‐phenantroline) is demonstrated through the comparison with the behavior of graphite paste electrode (CPE). The polymer electrogenerated at CNTPE largely improves the amperometric detection of hydrogen peroxide at ?0.100 V. The analytical application of the resulting electrode is demonstrated in connection with the design of a glucose biosensor based on the deposition of GOx and diluted Nafion on the top of the polymer‐modified CNTPE. The quantification of glucose in human serum samples showed a good correlation with the values obtained by the spectrophotometric technique.