3,4,9,10‐Perylenetetracarboxylic Acid/Hemin Nanocomposites Act as Redox Probes and Electrocatalysts for Constructing a Pseudobienzyme‐Channeling Amplified Electrochemical Aptasensor

A simple wet‐chemical strategy for the synthesis of 3,4,9,10‐perylenetetracarboxylic acid (PTCA)/hemin nanocomposites through π–π interactions is demonstrated. Significantly, the hemin successfully conciliates PTCA redox activity with a pair of well‐defined redox peaks and intrinsic peroxidase‐like activity, which provides potential application of the PTCA self‐derived redox activity as redox probes. Additionally, PTCA/hemin nanocomposites exhibit a good membrane‐forming property, which not only avoids the conventional fussy process for redox probe immobilization, but also reduces the participation of the membrane materials that act as a barrier of electron transfer. On the basis of these unique properties, a pseudobienzyme‐channeling amplified electrochemical aptasensor is developed that is coupled with glucose oxidase (GOx) for thrombin detection by using PTCA/hemin nanocomposites as redox probes and electrocatalysts. With the addition of glucose to the electrolytic cell, the GOx on the aptasensor surface bioelectrocatalyzed the reduction of glucose to produce H₂O₂, which in turn was electrocatalyzed by the PTCA/hemin nanocomposites. Cascade schemes, in which an enzyme is catalytically linked to another enzyme, can produce signal amplification and therefore increase the biosensor sensitivity. As a result, a linear relationship for thrombin from 0.005 to 20 nM and a detection limit of 0.001 nM were obtained.     

Properties of Dendrimer‐Encapsulated Pt Nanoparticles Doped Polypyrrole Composite Films and Their Electrocatalytic Activity for Glucose Oxidation

A type of novel electroanalytical sensing nanobiocomposite material was prepared by electropolymerization of pyrrole containing poly(amidoamine) dendrimers‐encapsulated platinum nanoparticles (Pt‐PAMAM), and glucose oxidase (GOx). The Pt nanoparticles encapsulated in PAMAM are nearly monodisperse with an average diameter of 3 nm, and they provide electrical conductivity. Polypyrrole acts as a polymer backbone to give stable and homogeneous cast thin films, and it also defines the electrical conductivity. Both Polypyrrole and PAMAM can provide a favorable microenvironment to keep the bioactivity of enzymes such as glucose oxidase. The homogeneity of GOx/Pt‐PAMAM‐PPy nanobiocomposite films was characterized by atomic force microscopy (AFM). Amperometric biosensors fabricated with these materials were characterized electrochemically using cyclic voltammetry (CV), electrochemical impedance spectra (EIS) and amperometric measurements in the presence of hydrogen peroxide or glucose. All those show the resultant biosensor sensitivity was strongly enhanced within the nanobiocomposite film. The optimized glucose biosensor displayed a sensitivity of 164 μA mM^?1 cm^?1, a linear range of 0.2 to 600 μM, a detection limit of 10 nM, and a response time of <3 s.     

Immobilization and Characterization of Glucose Oxidase on Single-Walled Carbon Nanotubes and Its Application to Sensing Glucose

The negatively charged （at pH 8.2） glucose oxidase （GOx, pI ca. 4.2） was assembled onto the surface of single-walled carbon nanotubes （SWNT）, which was covered （or wrapped） by a layer of positively charged polyelectrolyte poly（dimethyldiallylammonium chloride） （PDDA）, via the electrostatic interaction forming GOx-PDDASWNT nanocomposites. Fourier transform infrared （FTIR）, UV-Vis and electrochemical impedance spectroscopy （EIS） were used to characterize the growth processes of the nanocomposites. The results indicated that GOx retained its native secondary conformational structure after it was immobilized on the surface of PDDA-SWNT. A biosensor （Nafion-GOx-PDDA-SWNT/GC） was developed by immobilization of GOx-PDDA-SWNT nanocomposites on the surface of glassy carbon （GC） electrode using Nafion （5%） as a binder. The biosensor showed the electrocatalytic activity toward the oxidation of glucose under the presence of ferrocene monocarboxylic acid （FcM） as an electroactive mediator with a good stability, reproducibility and higher biological affinity. Under an optimal condition, the biosensor could be used to detection of glucose, presenting a typical characteristic of Michaelis-Menten kinetics with the apparent Michaelis-Menten constant of KM^app ca. 4.5 mmol/L, with a linear range of the concentration of glucose from 0.5 to 5.5 mmol/L （with correlation coefficient of 0.999） and the detection limit of ca. 83 μmol/L （at a signal-to-noise ratio of 3）. Thus the biosensor was useful in sensing the glucose concentration in serum since the normal glucose concentration in blood serum was around 4.6 mmol/L. The facile procedure of immobilizing GOx used in present work would promote the developments of electrochemical research for enzymes （proteins）, biosensors, biofuel cells and other bioelectrochemical devices.     

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.     

GOx@ZIF‐8(NiPd) Nanoflower: An Artificial Enzyme System for Tandem Catalysis

We report a facile approach to prepare an artificial enzyme system for tandem catalysis. NiPd hollow nanoparticles and glucose oxidase (GOx) were simultaneously immobilized on the zeolitic imidazolate framework 8 (ZIF‐8) via a co‐precipitation method. The as‐prepared GOx@ZIF‐8(NiPd) nanoflower not only exhibited the peroxidase‐like activity of NiPd hollow nanoparticles but also maintained the enzymatic activity of GOx. A colorimetric sensor for rapid detection of glucose was realized through the GOx@ZIF‐8(NiPd) based multi‐enzyme system. Moreover, the GOx@ZIF‐8(NiPd) modified electrode showed good bioactivity of GOx and high electrocatalytic activity for the oxygen reduction reaction (ORR), which could also be used for electrochemical detection of glucose.     

A Glucose Biosensor Based on Immobilization of Glucose Oxidase into 3D Macroporous TiO2

3D macroporous TiO₂ inverse opals have been derived from a sol‐gel procedure using polystyrene colloidal crystals as templates. EDS and SEM showed a face‐centered cubic (FCC) structure TiO₂ inverse opal was obtained. Glucose oxidase (GOx) was successfully immobilized on the surface of indium‐tin oxide (ITO) electrode modified by TiO₂ inverse opal (TiO_2(IO)). Electrochemical properties of GOx/TiO_2(IO)/ITO electrode were characterized by using the three electrodes system. The result of cyclic voltammetry showed that a couple of stable and well‐defined redox peaks for the direct electron transfer of GOx in absence of glucose, and the redox peak height enhanced in presence of 0.1 μM glucose. Compare with the ordinary structured GOx/TiO₂/ITO electrode, inverse opal structured GOx/TiO_2(IO)/ITO electrode has a better respond to the glucose concentration change. Under optimized experimental conditions of solution pH 6.8 and detection potential at 0.30 V versus saturated calomel electrode (SCE), amperometric measurements were performed. The sensitivity and the detection limit of glucose detection was 151 μA cm^?2 mM^?1 and 0.02 μM at a signal‐to‐noise ratio of 3, respectively. The good response was due to the good biocompatibility of TiO₂ and the large effective surface of the three‐dimensionally ordered macroporous structure.     

Electrochemistry and biosensing of glucose oxidase immobilized on Pt-dispersed mesoporous carbon

A method for immobilizing proteins in a carbon mesoporous material (CMM) containing platinum nanoparticles (Pt-NPs) is demonstrated. Compared to pure CMM or carbon nanotubes, CMM containing Pt-NPs enhances the electron transfer and redox properties of redox enzymes, such as glucose oxidase (GOx), due to a cooperative effect of Pt-NPs and CMM. The quasi-reversible electron transfer of GOx in this system is probed, and the apparent heterogeneous electron transfer rate constants are found to be 66% larger than in pure CMM. The GOx/Pt-CMM based glucose biosensor enables the determination of glucose at a potential of 600 mV (vs. SCE). Its detection limit is 10 times lower, and the sensitivity is 16 times higher than that of the respective biosensor without Pt-NPs.     

Avidin and Glucose Oxidase‐non‐covalently Functionalized Multi‐walled Carbon Nanotubes: A New Analytical Tool for Building a Bienzymatic Glucose Biosensor

We report an innovative supramolecular architecture for bienzymatic glucose biosensing based on the non‐covalently functionalization of multi‐walled carbon nanotubes (MWCNTs) with two proteins, glucose oxidase (GOx) (to recognize glucose) and avidin (to allow the specific anchoring of biotinylated horseradish peroxidase (b‐HRP)). The optimum functionalization was obtained by sonicating for 10 min 0.50 mg mL^?1 MWCNTs in a solution of 2.00 mg mL^?1 GOx+1.00 mg mL^?1avidin prepared in 50 : 50 v/v ethanol/water. The sensitivity to glucose for glassy carbon electrodes (GCE) modified with MWCNTs‐GOx‐avidin dispersion and b‐HRP (GCE/MWCNTs‐GOx‐avidin/b‐HRP), obtained from amperometric experiments performed at ?0.100 V in the presence of 5.0×10^?4 M hydroquinone, was (4.8±0.3) μA mM^?1 (r²=0.9986) and the detection limit was 1.2 μM. The reproducibility for 5 electrodes using the same MWCNTs/GOx‐avidin dispersion was 4.0 %, while the reproducibility for 3 different dispersions and 9 electrodes was 6.0 %. The GCE/MWCNT‐GOx‐avidin/b‐HRP was successfully used for the quantification of glucose in a pharmaceutical product and milk.     

Filling Carbon Nanotubes with Prussian Blue Nanoparticles of High Peroxidase‐Like Catalytic Activity for Colorimetric Chemo‐ and Biosensing

Facile filling of multiwalled carbon nanotubes (MWCNTs) with Prussian blue nanoparticles (PBNPs) of high peroxidase‐like catalytic activity was performed to develop novel colorimetric sensing protocols for assaying H₂O₂ and glucose. Fine control of PBNP growth was achieved by modulating the concentration ratio of K₃[Fe(CN)₆] and FeSO₄ precursors in an acidic solution containing ultrasonically dispersed MWCNTs, and thus size‐matched PBNPs could be robustly immobilized in the cavities of the MWCNTs (MWCNT‐PB_in). Unlike other reported methods involving complicated procedures and rigorous preparation/separation conditions, this mild one‐pot filling method has advantages of easy isolation of final products by centrifugation, good retention of the pristine outer surface of the MWCNT shell, and satisfactory filling yield of (24±2) %. In particular, encapsulation of PBNPs of poor dispersibility and limited functionality in dispersible and multifunctional MWCNT shells creates new and valuable opportunities for quasihomogeneous‐phase applications of PB in liquid solutions. The MWCNT‐PB_in nanocomposites were exploited as a peroxidase mimic for the colorimetric assay of H₂O₂ in solution by using 3,3′,5,5′‐tetramethylbenzidine (TMB) as reporter, and they gave a linear absorbance response from 1 μM to 1.5 mM with a limit of detection (LOD) of 100 nM . Moreover, glucose oxidase (GOx) was anchored on the outer surface of MWCNT‐PB_in to form GOx/MWCNT‐PB_in bionanocomposites. The cooperation of outer‐surface biocatalysis with peroxidase‐like catalysis of interior PB resulted in a novel cooperative colorimetric biosensing mode for glucose assay. The use of GOx/MWCNT‐PB_in for colorimetric biosensing of glucose gave a linear absorbance response from 1 μM to 1.0 mM and an LOD of 200 nM . The presented protocols may be extended to other multifunctional nanocomposite systems for broad applications in catalysis and biotechnology.     

Biological Glucose Metabolism Regulated Peptide Self‐Assembly as a Simple Visual Biosensor for Glucose Detection

A glucose oxidase (GOx)‐mediated glucose metabolism was in vitro mimicked and employed to regulate the self‐assembly of peptide‐based building blocks. In this new stimuli‐responsive self‐assembly system, two peptide‐based building blocks, respectively, having aspartic acid (gelator 1 ) and lysine (gelator 2 ) residues were designed and prepared. When adding glucose and GOx to the aqueous solution of gelator 1 or the self‐assembled fibrillar hydrogel of gelator 2 to construct glucose metabolism system, the metabolic product (gluconic acid) can trigger the protonation of the peptide molecules and induce the phase transitions of gelators 1 (sol‐gel) and 2 (gel‐sol). Because this glucose metabolism regulated peptide self‐assembly is built on the oxidation of glucose, it can be used as a simple visual biosensor for glucose detection.     

An XPS investigation on glucose oxidase and Ni/Al hydrotalcite interaction

In this work, a Ni/Al hydrotalcite (HT) was used as glucose oxidase (GOx) immobilizer. Small‐area and angle‐resolved X‐ray photoelectron spectra were recorded on HTs electrosynthesized on Pt in the absence and in the presence of GOx, and compared with those obtained for a Pt surface, modified with the electrosynthesized HT, on which a drop of GOx solution was deposited. The simultaneous electrodeposition of HT + GOx resulted in a compact deposit, thicker than the XPS sampling depth (>10 nm), that is not homogeneous in the lateral and in‐depth composition. The presence of GOx can be deduced comparing the N1s spectra of HT and HT + GOx: in the latter, the N1s component at 400 eV binding energy (BE) is predominant whilst, depending on the analyzed point, a small or no contribution from the component at 407.2 eV, due to nitrate, is revealed. Angle‐resolved XPS provides evidence on the in‐depth composition of anions, cations and GOx. The results highlight the crucial role played by nickel in GOx immobilization. On the basis of the results, it can be suggested that enzyme activity is unevenly distributed and is localized in small areas, where Ni concentration is higher. Copyright © 2010 John Wiley & Sons, Ltd.     

Direct Electrochemistry and Electrocatalysis Behaviors of Glucose Oxidase Based on Hyaluronic Acid-Carbon Nanotubes-Ionic Liquid Composite Film

Multi‐walled carbon nanotubes (MWNTs) were dispersed in the ionic liquid [BMIM][BF₄] to form a uniform black suspension. Based on it, a novel glucose oxidase (GOx)‐hyaluronic (HA)‐[BMIM][BF₄]‐MWNTs/GCE modified electrode was fabricated. UV‐vis spectroscopy confirmed that GOx immobilized in the composite film retained its native structure. The experimental results of EIS indicated MWNTs, [BMIM][BF₄] and HA were successfully immobilized on the surface of GCE and [BMIM][BF₄]‐MWNTs could obviously improve the diffusion of ferricyanide toward the electrode surface. The experimental results of CV showed that a pair of well‐defined and quasi‐reversible peaks of GOx at the modified electrode was exhibited, and the redox reaction of GOx at the modified electrode was surface‐confined and quasi‐reversible electrochemical process. The average surface coverage of GOx and the apparent Michaelis‐Menten constant were 8.5×10⁻⁹ mol/cm² and 9.8 mmol/L, respectively. The cathodic peak current of GOx and the glucose concentration showed linear relationship in the range from 0.1 to 2.0 mmol/L with a detection limit of 0.03 mmol/L (S/N=3). As a result, the method presented here could be easily extended to immobilize and obtain the direct electrochemistry of other redox enzymes or proteins.     

Ultrasensitive analysis of glucose in serum by capillary electrophoresis with LIF detection in combination with signal amplification strategies and on‐column enzymatic assay

A highly specific and sensitive method for glucose quantification in human serum samples based on on‐column enzymatic assay is described. In this method, the head of the capillary was used as a nanoliter‐microreactor, the diluted samples spiked with a novel fluorogenic reagent named 2‐[6‐(4′‐amino) phenoxy‐3H‐xanthen‐3‐on‐9‐yl] benzoic acid (APF), and the mixed enzyme solutions of glucose oxidase (GOx) and horseradish peroxidase (HRP), were individually injected into the capillary. Hydrogen peroxide (H₂O₂) generated in situ by catalytic reaction between GOx and glucose, activates APF in the presence of HRP to form a highly fluorescent product, which was electrophoretically separated from the unreacted APF and detected by the LIF detector. The proposed method allowed the determination of glucose down to 10 nM in real samples, with RSD values lower than 3.5%, which also has the potential for measurements of multicomponents in many other systems including measurement of α‐glucosidase activity and screening for its inhibitors.     

基于层-层自反应的葡萄糖氧化酶有序多层膜电极

以胱胺修饰的金电极为基础电极, 利用席夫碱反应使经高碘酸根氧化的葡萄糖氧化酶在该电极表面进行自身的层-层有序组装. 用电化学交流阻抗法对多层酶膜形成过程的跟踪结果表明, 该多层酶膜的生长是一个逐步形成的均匀过程. 用循环伏安法和I-t曲线法研究了该酶电极对葡萄糖的电催化氧化. 实验结果表明, 当采用羟基二茂铁作为人工电子转移媒介体时, 该酶电极对葡萄糖具有很好的电催化氧化功能. 该传感器制作简便, 响应迅速, 性能稳定, 催化电流与葡萄糖浓度在一定范围内成正比, 并且可以通过控制葡萄糖氧化酶的组装层数来调节该生物传感器的灵敏度与检测限.     

An Amperometric Glucose Biosensor Based on Poly (Pyrrole‐2‐Carboxylic Acid)/Glucose Oxidase Biocomposite

A newly developed amperometric glucose biosensor based on graphite rod (GR) working electrode modified with biocomposite consisting of poly (pyrrole‐2‐carboxylic acid) (PCPy) particles and enzyme glucose oxidase (GOx) was investigated. The PCPy particles were synthesized by chemical oxidative polymerization technique using H₂O₂ as initiator of polymerization reaction and modified covalently with the GOx (PCPy‐GOx) after activation of carboxyl groups located on the particles surface with a mixture of N‐(3‐dimethylaminopropyl)‐N′‐ethylcarbodiimide hydrochloride (EDC) and N‐hydroxysuccinimide (NHS). Then the PCPy‐GOx biocomposite was dispersed in a buffer solution containing a certain amount of bovine serum albumin (BSA). The resulting biocomposite suspension was adsorbed the on GR electrode surface with subsequent solvent airing and chemical cross‐linking of the proteins with glutaraldehyde vapour (GR/PCPy‐GOx). It was determined that the current response of the GR/PCPy‐GOx electrodes to glucose measured at +300 mV vs Cl reference electrode was influenced by the duration of the PCPy particles synthesis, pH of the GOx solution used for the PCPy particles modification and the amount of immobilized PCPy‐GOx biocomposite. An optimal pH of buffer solution for operation of the biosensor was found to be 8.0. Detection limit was determined as 0.039 mmol L⁻¹ according signal to noise ratio (S/N: 3). The proposed glucose biosensor was tested in human serum samples.     

Aptamer‐Based Luminescence Energy Transfer from Near‐Infrared‐to‐Near‐Infrared Upconverting Nanoparticles to Gold Nanorods and Its Application for the Detection of Thrombin

A new luminescence energy transfer (LET) system has been designed for the detection of thrombin in the near‐infrared (NIR) region by utilizing NIR‐to‐NIR upconversion lanthanide nanophosphors (UCNPs) as the donor and gold nanorods (Au NRs) as the acceptor. The use of upconverting NaYF₄:Yb³⁺,Tm³⁺ nanoparticles with sharp NIR emission peaks upon NIR excitation by an inexpensive infrared continuous wave laser diode provided large spectral overlap between the donor and the acceptor. Both the Au NRs and carboxyl‐terminated NaYF₄:Yb³⁺,Tm³⁺ UCNPs were first modified with different thrombin aptamers. When thrombin was added, a LET system was then formed because of the specific recognition between the thrombin aptamers and thrombin. The LET system was used to monitor thrombin concentrations in aqueous buffer and human blood samples. The limits of detection for thrombin are as low as 0.118 nM in buffer solution and 0.129 nM in human serum. The method was also successfully applied to thrombin detection in blood samples.     

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.     

Development of a Glucose Biosensor Using Advanced Electrode Modified by Nanohybrid Composing Chemically Modified Graphene and Ionic Liquid

Nanohybrids of chemically modified graphene (CMG) and ionic liquid (IL) were prepared by sonication to modify the electrode. The modified CMG‐IL electrodes showed a higher current and smaller peak‐to‐peak potential separation than a bare electrode due to the promoted electron transfer rate. Furthermore, the glucose oxidase (GOx) immobilized on the modified electrode displayed direct electron transfer rate and symmetrical redox potentials with a linear relationship at different scan rates. The fabricated GOx/CMG‐IL electrodes were developed selective glucose biosensor with respect to a sensitivity of 0.64 μA mM^?1, detection limit of 0.376 mM, and response time of <5 s.     

Amperometric Glucose Biosensor Based on Electrochemically Deposited Gold Nanoparticles Covered by Polypyrrole

Graphite rod (GR) modified with electrochemicaly deposited gold nanoparticles (AuNPs) and adsorbed glucose oxidase (GOx) was used in amperometric glucose biosensor design. Enzymatic formation of polypyrrole (Ppy) on the surface of GOx/AuNPs/GR electrode was applied in order to improve analytical characteristics and stability of developed biosensor. The linear glucose detection range for Ppy/GOx/AuNPs/GR electrode was dependent on the duration of Ppy‐layer formation and the linear interval was extended up to 19.9 mmol L⁻¹ after 21 h lasting synthesis of Ppy. The sensitivity of the developed biosensor was determined as 21.7 μA mM⁻¹ cm⁻², the limit of detection – 0.20 mmol L⁻¹. Ppy/GOx/AuNPs/GR electrodes demonstrated advanced good stability (the t _1/2 was 9.8 days), quick detection of glucose (within 5 s) in the wide linear interval. Additionally, formed Ppy layer decreased the influence of electroactive species on the analytical signal. Developed biosensor is suitable for the determination of glucose in human serum samples.     

Physicochemical Characteristics of Polypyrrole/(Glucose oxidase)/(Prussian Blue)‐based Biosensor Modified with Ni‐ and Co‐Hexacyanoferrates

In this work, three types of electrodes suitable for amperometric glucose biosensors were designed. One type of electrode was based on bio‐selective layer of polypyrrole/(glucose oxidase)/(Prussian Blue) (Ppy/GOx/PB) and it was used as a control electrode regarding to which electrochemical properties of two other types of electrodes were compared. During the formation of Prussian blue layers graphite electrodes were additionally modified by Ni‐hexacyanoferrate (NiHCF) and by Co‐hexacyanoferrate (CoHCF) in order to design Ppy/GOx/PB‐NiHCF and Ppy/GOx/PB‐CoHCF electrodes, respectively. Some physicochemical characteristics of all three types of electrodes were evaluated and compared. The Ppy/GOx/PB‐NiHCF electrode showed wider linear range of the calibration curve than Ppy/GOx/PB and Ppy/GOx/PB‐CoHCF electrodes. The effect of temperature on analytical performance of the Ppy/GOx/PB‐NiHCF based biosensor has been evaluated and activation energy of enzyme catalysed reaction has been calculated within the temperature range of 15 °C to 30 °C.