An Amperometric Immunosensor Based on Layer‐by‐Layer Assembly of L‐Cysteine and Nanosized Prussian Blue on Gold Electrode for Determination of Human Chorionic Gonadotrophin

A novel amperometric immunosensor based on L ‐cysteine/nanosized Prussian blue bilayer films ({NPB/L ‐cys}₂) and gold nanoparticles (nano‐Au) was fabricated for determination of human chorionic gonadotrophin (HCG). First, L ‐cys and NPB was self‐assembled by layer‐by‐layer (LBL) technology to form {NPB/L ‐cys}₂ bilayer films on the gold electrode. Subsequently, nano‐Au layer was immobilized on the {NPB/L ‐cys}₂ bilayer films by electrodepositing gold chloride tetrahydrate and then anti‐HCG was assembly on the nano‐Au layer. Finally hemoglobin (Hb) was employed to block sites against nonspecific binding. With the electrocatalytic ability of Hb and NPB for the reduction of H₂O₂, the current signal of the antigen‐antibody reaction was amplified and the enhanced sensitivity was achieved. In this study, the assembly process and performance of the immunosensor were characterized by cyclic voltammetry (CV) and the morphology was researched by scanning electron microscopy (SEM). The immunosensor performed a high sensitivity and a wide linear response to HCG in two ranges from 0.5 to 10 mIU/mL and from 10 to 200 mIU/mL with a relatively low detection limit of 0.2 mIU/mL at 3 times the background noise, as well as good stability and long‐term life.     

Sensitively Electrochemical Sensing for Sequence‐Specific Detection of Phosphinothricin Acetyltransferase Gene: Layer‐by‐Layer Films of Poly‐L‐Lysine and Au‐Carbon Nanotube Hybrid

An electrochemical DNA sensing film was constructed based on the multilayers comprising of poly‐L ‐lysine (pLys) and Au‐carbon nanotube (Au‐CNT) hybrid. A precursor film of mercaptopropionic acid (MPA) was firstly self‐assembled on the Au electrode surface. pLys and Au‐CNT hybrid layer‐by‐layer assembly films were fabricated by alternately immersing the MPA‐modified electrode into the pLys solution and Au‐CNT hybrid solution. Cyclic voltammetry was used to monitor the consecutive growth of the multilayer films by utilizing [Fe(CN)₆]^3?/4? and [Co(phen)₃]^3+/2+ as the redox indicators. The outer layer of the multilayer film was the positively charged pLys, on which the DNA probe was easily linked due to the strong electrostatic affinity. The hybridization detection of DNA was accomplished by using methylene blue (MB) as the indicator, which possesses different affinities to dsDNA and ssDNA. Differential pulse voltammetry was employed to record the signal response of MB and determine the amount of the target DNA sequence. The established biosensor has high sensitivity, a relatively wide linear range from 1.0×10^?10 mol/L to 1.0×10^?6 mol/L and the ability to discriminate the fully complementary target DNA from single or double base‐mismatched DNA. The sequence‐specific DNA related to phosphinothricin acetyltransferase gene from the transgenically modified plants was successfully detected.     

Direct Electrochemistry and Electrocatalysis of Hemoglobin/ZnO‐Chitosan/nano‐Au Modified Glassy Carbon Electrode

The highly efficient H₂O₂ biosensor was fabricated on the basis of the complex films of hemoglobin (Hb), nano ZnO, chitosan (CHIT) dispersed solution and nano Au immobilized on glassy carbon electrode (GCE). Biocompatible ZnO‐CHIT composition provided a suitable microenvironment to keep Hb bioactivity (Michaelis‐Menten constant of 0.075 mmol L^?1). The presence of nano Au in matrix could effectively enhance electron transfer between Hb and electrode. The electrochemical behaviors and effects of solution pH values were carefully examined in this paper. The (ZnO‐CHIT)‐Au‐Hb/GCE demonstrated excellently electrocatalytical ability for H₂O₂. This biosensor had a fast response to H₂O₂ less than 4 s and excellent linear relationships were obtained in the concentration range from1.94×10^?7 to 1.73×10^?3 mol L^?1 with the detection limit of 9.7×10^?8 mol L^?1 (S/N=3) under the optimum conditions. Moreover, the stability and reproducibility of this biosensor were evaluated with satisfactory results.     

Immobilization of Enzymes on the Nano‐Au Film Modified Glassy Carbon Electrode for the Determination of Hydrogen Peroxide and Glucose

A new enzyme‐based amperometric biosensor for hydrogen peroxide was developed relying on the efficient immobilization of horseradish peroxidase (HRP) to a nano‐scaled particulate gold (nano‐Au) film modified glassy carbon electrode (GC). The nano‐Au film was obtained by a chitosan film which was first formed on the surface of GC. The high affinity of chitosan for nano‐Au associated with its amino groups resulted in the formation of nano‐Au film on the surface of GC. The film formed served as an intermediator to retain high efficient and stable immobilization of the enzyme. H₂O₂ was detected using hydroquinone as an electron mediator to transfer electrons between the electrode and HRP. The HRP immobilized on nano‐Au film maintained excellent electrocatalytical activity to the reduction of H₂O₂. The experimental parameters such as the operating potential of the working electrode, mediator concentration and pH of background electrolyte were optimized for best analytical performance of amperometry. The linear range of detection for H₂O₂ is from 6.1×10^?6 to 1.8×10^?3 mol L^?1 with a detection limit of 6.1 μmol L^?1 based on signal/noise=3. The proposed HRP enzyme sensor has the features of high sensitivity (0.25 Almol^?1cm^?2), fast response time (t_90%≤10 s) and a long‐term stability (>1 month). As an extension, glucose oxidase (GOD) was chemically bound to HRP‐modified electrode. A GOD/HRP bienzyme‐modified electrode formed in this way can be applied to the determination of glucose with satisfactory performance.     

Study on the Electrochemical Behavior of Dopamine and Uric Acid at a 2‐Amino‐5‐mercapto‐[1,3,4] Triazole Self‐Assembled Monolayers Electrode

Selected from a series of structurally related heteroaromatic thiols, a newly synthesized reagent 2‐amino‐5‐mercapto‐[1,3,4] triazole (MATZ) was used to fabricate self‐assembled monolayers (SAMs) on gold electrode for the first time. The MATZ/Au SAMs was characterized by electrochemical methods and scanning electronic microscopy (SEM). In 0.04 mol/L Britton–Robinson buffer solution (pH 5), the electrochemical behavior of dopamine showed a quasireversible process at the MATZ/Au SAMs with an electrode kinetic constant 0.1049 cm/s. However, the electrochemical reaction of uric acid at the SAMs electrode showed an irreversible oxidation process, the charge‐transfer kinetics of uric acid was promoted by the SAMs. By Osteryoung square‐wave voltammetry (OSWV), the simultaneous determination of dopamine and uric acid can be accomplished with an oxidation peak separation of 0.24 V, the peak current of dopamine and uric acid were linearly to its concentration in the range of 2.5×10^?6–5.0×10^?4 mol/L for dopamine and 1×10^?6–1×10^?4 mol/L for uric acid with a detection limit of 8.0×10^?7 mol/L for dopamine and 7.0×10^?7 mol/L for uric acid. The MATZ/Au SAMs electrode was used to detect the content of uric acid in real urine and serum sample with satisfactory results.     

Electrochemical Investigation of Myoglobin in Layer‐by‐Layer Films Assembled with Sulfonated‐β‐Cyclodextrin

In this work, myoglobin (Mb) and sulfonated‐β‐cyclodextrin (S‐CD) were assembled into {S‐CD/Mb}_n layer‐by‐layer films on solid substrates. In pH 7.0 buffers, the {S‐CD/Mb}_n films assembled on electrodes showed a pair of well‐defined and nearly reversible CV peaks at about ?0.35 V vs. SCE. The stable CV response of {S‐CD/Mb}_n films could be used to electrocatalyze reduction of oxygen and hydrogen peroxide in solution. For comparison, another modified β‐cyclodextrin, carboxyethyl‐β‐cyclodextrin (C‐CD), was also assembled with Mb into {C‐CD/Mb}_n multilayer films. The driving forces of the assembly were explored and discussed.     

Preconcentration and Determination of a Phospholipid at a Surface Modified by Layer‐by‐Layer Assembly

Electrochemical oxidation of a phospholipid, phosphatidylcholine (PC), was accomplished at a 4‐aminothiophenol (ATP)‐modified gold electrode coated with a layer‐by‐layer assembly of an electrochemical catalyst (dirhodium phosphomolybdic acid), a trapping agent for PC (a cyclophane, CP, derivative, 1,4‐xylylene‐1,4‐phenylene‐diacetate), and a spacer (generation‐4 polyamidoamine dendrimer, PAMAM). The layer‐by‐layer assembly process and the trapping of PC was verified by quartz crystal microbalance measurements; Au|ATP|CP|PAMAM|CP trapped (1.5±0.4)×10^?9 mol cm^?2 of PC. The electrocatalytic oxidation of PC yielded a current that varied linearly with concentration over the range 1–50 μM; the R² value was 0.996.     

A New Enzyme Immobilization Technique Based on Thionine‐Bovine Serum Albumin Conjugate and Gold Colloidal Nanoparticles for Reagentless Amperometric Biosensor Applications

A novel enzyme immobilization technique based on thionine‐bovine serum albumin conjugate (Th‐BSA) and gold colloidal nanoparticles (nano‐Au) was developed. Thionine was covalently bound onto the BSA film with glutaraldehyde(GA) as cross‐linker to achieve Th‐BSA conjugate. The free amino groups of thionine were then used to attach nano‐Au for the immobilization of horseradish peroxidase (HRP). Such nano‐Au/Th‐BSA matrix shows a favorable microenvironment for retaining the native activity of the immobilized HRP and thionine immobilized in this way can effectively shuttle electrons between the electrode and the enzyme. The proposed biosensor displays excellent catalytic activity and rapid response for H₂O₂. The linear range for the determination of H₂O₂ is from 4.9×10^?7 to 1.6×10^?3 M with a detection limit of 2.1×10^?7 M at 3σ and a Michaelies‐Menten constant K value of 0.023 mM.     

Simultaneous Determination of Dopamine and Ascorbic Acid Using the Nano‐Gold Self‐Assembled Glassy Carbon Electrode

Electrochemical behavior of dopamine (DA) was investigated at the gold nanoparticles self‐assembled glassy carbon electrode (GNP/LC/GCE), which was fabricated by self‐assembling gold nanoparticles on the surface of L ‐cysteine (LC) modified glassy carbon electrode (GCE) via successive cyclic voltammetry (CV). A pair of well‐defined redox peaks of DA on the GNP/LC/GCE was obtained at E_pa=0.197 V and E_pc=0.146 V, respectively. And the peak separation between DA and AA is about 0.2 V, which is enough for simultaneous determination of DA and AA. The peak currents of DA and AA were proportional with their concentrations in the range of 6.0×10^?8–8.5×10^?5 mol L^?1 and 1.0×10^?6–2.5×10^?3 mol L^?1, with the detection limit of 2.0×10^?8 mol L^?1 and 3.0×10^?7 mol L^?1 (S/N=3), respectively. The modified electrode exhibits an excellent reproducibility, sensibility and stability for simultaneous determination of DA and AA in human serum with satisfactory result.     

A New Amperometric Biosensor Based on HRP/Nano-Au/L-Cysteine/Poly（o-Aminobenzoic acid）-Membrane-Modified Platinum Electrode for the Determination of Hydrogen Peroxide

The third generation amperometric biosensor for the determination of hydrogen peroxide （H2O2） has been described. For the fabrication of biosensor, o-aminobenzoic acid （oABA） was first electropolymerized on the surface of platinum （Pt） electrode as an electrostatic repulsion layer to reject interferences. Horseradish peroxidase （HRP） absorbed by nano-scaled particulate gold （nano-Au） was immobilized on the electrode modified with polymerized o-aminobenzoic acid （poABA） with L-cysteine as a linker to prepare a biosensor for the detection of H2O2. Amperometric detection of H2O2 was realized at a potential of ＋20 mV versus SCE. The resulting biosensor exhibited fast response, excellent reproducibility and sensibility, expanded linear range and low interferences. Temperature and pH dependence and stability of the sensor were investigated. The optimal sensor gave a linear response in the range of 2.99×10^-6 to 3.55×10^-3 mol·L^-1 to H2O2 with a sensibility of 0.0177 A·L^-1·mol^-1 and a detection limit （S/N = 3） of 4.3×10^-7 mol·L^-1. The biosensor demonstrated a 95% response within less than 10 s.     

Amino‐Containing Ultrafine Organosilica‐Nanoparticle‐Modified Au Electrode for the Determination of Cu(II) Ions

Using 3‐Aminopropyltriethoxysilane(APTES) as a single silica source, an amino‐rich ultrafine organosilica‐nanoparticle‐modified Au electrode was fabricated, following the formation of (3‐mercaptopropyl)‐trimethoxysilane (MPTS) monolayer on Au surface (MPTS/Au). With cetyltrimethylammonium bromide as an additive, APTES‐based gel particles on the electrode have a narrow particle size distribution of 4–7 nm and “crystal‐like” structure. AFM and electrochemical characterization confirmed the successful grafting of APTES nanoparticles on MPTS/Au. The APTES/MPTS/Au electrode is highly sensitive for the detection of copper(II) ions with a detection limit as low as 1.6×10^?12 mol L^?1 (S/N>3) by square wave voltammetry. The current is linear to copper(II) concentration between 1.6×10^?12 and 6.25×10^?10 mol L^?1.     

Multilayer Assembly of Hemoglobin and Colloidal Gold Nanoparticles on Multiwall Carbon Nanotubes/Chitosan Composite for Detecting Hydrogen Peroxide

Chitosan (CS) was chosen for dispersing multi‐wall carbon nanotubes (MWNTs) to form a stable CS‐MWNTs composite, which was first coated on the surface of a glassy carbon electrode to provide a containing amino groups interface for assembling colloidal gold nanoparticles (GNPs), followed by the adsorption of hemoglobin (Hb). Repeating the assembly step of GNPs and Hb resulted in {Hb/GNPs}_n multilayers. The assembly of GNPs onto CS‐MWNTs composites was confirmed by transmission electron microscopy. The consecutive growth of {Hb/GNPs}_n multilayers was confirmed by cyclic voltammetry and UV‐vis absorption spectroscopy. The resulting system brings a new platform for electrochemical devices by using the synergistic action of the electrocatalytic activity of GNPs and MWNTs. The resulting biosensor displays an excellent electrocatalytic activity and rapid response for hydrogen peroxide. The linear range for the determination of H₂O₂ was from 5.0×10^?7 to 2.0×10^?3 M with a detection limit of 2.1×10^?7 M at 3σ and a Michaelis–Menten constant K_M^app value of 0.19 mM.     

Electrochemical Behavior and Determination of L‐Tyrosine at Single‐walled Carbon Nanotubes Modified Glassy Carbon Electrode

Based on single‐walled carbon nanotubes (SWCNTs) modified glassy carbon electrode (GCE/SWCNTs), a novel method was presented for the determination of L ‐tyrosine. The GCE/SWCNTs exhibited remarkable catalytic and enhanced effects on the oxidation of L ‐tyrosine. In 0.10 mol/L citric acid‐sodium citrate buffer solution, the oxidation potential of L ‐tyrosine shifted negatively from +1.23 V at bare GCE to +0.76 V at GCE/SWCNTs. Under the optimized experimental conditions, the linear range of the modified electrode to the concentration of L ‐tyrosine was 5.0×10^?6–2.0×10^?5 mol/L (R₁=0.9952) and 2.7×10^?5–2.6×10^?4 mol/L (R₂=0.9998) with a detection limit of 9.3×10^?8 mol/L. The kinetic parameters such as α (charge transfer coefficient) and D (diffusion coefficient) were evaluated to be 0.66, 9.82×10^?5 cm² s^?1, respectively. And the electrochemical mechanism of L ‐tyrosine was also discussed.     

Self‐Assembled Submonolayer of β‐Cyclodextrins on Gold Electrode for the Selective Determination of 4‐Aminobiphenyl

The formation of an inclusion complex between 4‐aminobiphenyl (4‐AB) and β‐cyclodextrin molecules (β‐CD), allows the use of thiolated β‐CDs as chemi‐adsorbed material on a Au electrode as a self‐assembled submonolayer for the selective square wave voltammetric determination of 4‐AB. The submonolayer was characterized by reductive desorption and an association constant of 1.2×10⁴ L/mol was obtained. The optimization of variables yielded a linear dependence of ip/4‐AB concentration in the range of 10^?5 to 10^?4 mol/L. The selectivity of the method was evaluated in the presence of other aromatic amines obtaining better results with the modified electrode. This methodology was applied to the voltammetric determination of 4‐AB in wastewater samples.     

Electrostatic Layer‐by‐Layer Assembly of Polycation and DNA Multilayer Films by Real‐time Surface Plasmon Resonance Technique

The assembly of alternating DNA and positively charged poly‐(dimethyldiallylammonium chloride) (PDDA) multilayer films by electrostatic layer‐by‐layer adsorption has been studied. Real time surface plasmon resonance (BIAcore) technique was used to characterize and monitor the formation of multilayer films in solution in real time continuously. The results indicate that the uniform multilayer can be obtained on the poly‐(ethylenimine) (PEI) coated substrate surface. The kinetics of the adsorption of DNA on PDDA surface was also studied by real‐time BIAcore technique, and the observed rate constant was calculated using a Langmuir model (k_obs = (1.28 ± 0.08) × 10^?2s^?1).     

Amperometric Biosensor for Hydrogen Peroxide Based on Electrodeposited Sub-micrometer Gold Modified Glassy Carbon Electrode

IntroductionAmperometricbiosensorofhydrogenperoxideisofpracticalimportancebecauseofitswideapplicationsinchemical,biological,clinical,environmentalandmanyotherfields.Forimprovementofsensor抯quality,vari-ouskindsofchemicalmodificationmethodshavebeendevelopedforreducingredoxoverpotentialsofH2O2atelectrodesurfaces,increasingthedetectionsensitivity,linearrange,stabilityandlivetime.Ithasbeenshownthattheuseofsub-micrometersizedmetalparticlessuchasPt-blackcansignificantlyimprovethequalityofthebiosens…     

One‐Step Synthesis of β‐Cyclodextrin Functionalized Graphene/Ag Nanocomposite and Its Application in Sensitive Determination of 4‐Nitrophenol

β‐Cyclodextrin functionalized graphene/Ag nanocomposite (β‐CD/GN/Ag) was prepared via a one‐step microwave treatment of a mixture of graphene oxide and AgNO₃. β‐CD/GN/Ag was employed as an enhanced element for the sensitive determination of 4‐nitrophenol. A wide linear response to 4‐nitrophenol in the concentration ranges of 1.0×10^?8–1.0×10^?7 mol/L, and 1.0×10^?7–1.5×10^?3 mol/L was achieved, with a low detection limit of 8.9×10^?10 mol/L (S/N=3). The mechanism and the heterogeneous electron transfer kinetics of the 4‐nitrophenol reduction were discussed according to the rotating disk electrode experiments. Furthermore, the sensing platform has been applied to the determination of 4‐nitrophenol in real samples.     

Differential Pulse Voltammetric Determination of Selenocystine Using Selenium‐gold Film Modified Electrode

Differential pulse voltammetric determination of selenocystine (SeC) using selenium‐gold film modified glassy carbon electrode ((Se‐Au)/GC) is presented. In 0.10 mol?L^?1 KNO₃ (pH 3.20) solution, SeC yields a sensitive reduction peak at ?740 mV on (Se‐Au)/GC electrode. The peak current has a linear relationship with the concentration of SeC in the range of 5.0×10^?8–7.0×10^?4 mol?L^?1, and a 3σ detection limit of SeC is 3.0×10^?8 mol?L^?1. The relative standard deviation of the reduction current at SeC concentration of 10^?6 mol?L^?1 is 3.88% (n=8) using the same electrode, and 4.19% when using three modified electrodes prepared at different times. The content of SeC in the selenium‐enriched yeast and selenium‐enriched tea is determined. The total selenium in ordinary or selenium‐enriched tea is determined by DAN fluorescence method. The results indicate that in selenium‐enriched yeast about 20% of total selenium is present as SeC and in selenium‐enriched tea SeC is the major form of selenoamino acids. The total selenium content in selenium‐enriched tea soup is 0.09 μgSe/g accounting by 7% compared with that in selenium‐enriched tea. Hence, only a little amount of selenium is utilized by drinking tea, and most selenium still stay in tealeaf. Uncertainty are 22.4% and 16.1% for determination of SeC in selenium‐enriched yeast and selenium‐enriched tea by differential pulse voltammetry (DPV) on (Se‐Au)/GC electrode, respectively.     

Comparative Permeation Study of Different Types of Myoglobin Layer‐by‐Layer Films and the Effect of Film Permeability on Their Electrochemistry and Electrocatalysis

Three different types of myoglobin (Mb) layer‐by‐layer films were assembled respectively with TiO₂ sol‐gel by vapor‐surface deposition, TiO₂ nanoparticles, and poly(styrenesulfonate), designated as {SG‐TiO₂/Mb}_n, {NP‐TiO₂/Mb}_n, and {PSS/Mb}_n. The permeability of the films was studied and compared by rotating disk voltammetry (RDV) and electrochemical impedance spectroscopy (EIS) with different electroactive probes, showing a general permeability sequence of {SG‐TiO₂/Mb}_n>{NP‐TiO₂/Mb}_n>{PSS/Mb}_n. The electrochemical and electrocatalytic activity of Mb in these films were also investigated and compared by cyclic voltammetry (CV), RDV, and amperometry, indicating that among the three Mb films, {SG‐TiO₂/Mb}_n films demonstrated the highest maximum surface concentration of electroactive Mb and the best electrocatalytic performances toward reduction of H₂O₂. All these advantages could be attributed to the unique architecture and porous structure of {SG‐TiO₂/Mb}_n films, which could greatly facilitate the mass transport of small counterions and catalytic substrates within the films. The various influencing factors on the permeability, electrochemistry, and electrocatalysis of the Mb films were also investigated in detail.     

Layer‐by‐layer assembly of {chitosan/Pd}n multilayer film based on in‐situ photochemical reduction with excellent electrocatalytic properties

Chitosan/palladium {CTS/Pd}_n composite multilayer film was assembled based on layer‐by‐layer self‐assembly technique and in‐situ photo‐chemical reduction reaction, in which the CTS plays the role of a photo‐reduction agent and an assembly reagent. Transmission electron microscopy (TEM) shows that spherical Pd nanoparticles with diameter of 20 nm are well‐dispersed in the composite multilayer films, and the size of Pd nanoparticles increased gradually with the extension of illumination time. Besides, the {CTS/Pd}_n composite multilayer film exhibits linear, uniform and regular layer‐by‐layer growth. Furthermore, the {CTS/Pd}_n composite multilayer film presents an excellent catalytic properties for oxygen reduction, and it has potential application in energy, chemical synthesis and biological processes. Copyright © 2015 John Wiley & Sons, Ltd.