"Outer-sphere to inner-sphere" redox cycling for ultrasensitive immunosensors

This paper reports chemical-chemical (CC) and electrochemical-chemical-chemical (ECC) redox cycling, for use in ultrasensitive biosensor applications. A triple chemical amplification approach using an enzymatic reaction, CC redox cycling, and ECC redox cycling is applied toward electrochemical immunosensors of cardiac troponin I. An enzymatic reaction, in which alkaline phosphatase converts 4-aminophenyl phosphate to 4-aminophenol (AP), triggers CC redox cycling in the presence of an oxidant and a reductant, and electrochemical signals are measured with ECC redox cycling after an incubation period of time in an air-saturated solution. To obtain high, selective, and reproducible redox cycling without using redox enzymes, two redox reactions [the reaction between AP and the oxidant and the reaction between the oxidized form of AP (4-quinone imine, QI) and the reductant] should be fast, but an unwanted reaction between the oxidant and reductant should be very slow. Because species that undergo outer-sphere reactions (OSR-philic species) react slowly with species that undergo inner-sphere reactions (ISR-philic species), highly OSR-philic Ru(NH(3))(6)(3+) and highly ISR-philic tris(2-carboxyethyl)phosphine (TCEP) are chosen as the oxidant and reductant, respectively. The OSR- and ISR-philic QI/AP couple allows fast redox reactions with both the OSR-philic Ru(NH(3))(6)(3+) and the ISR-philic TCEP. Highly OSR-philic indium-tin oxide (ITO) electrodes minimize unwanted electrochemical reactions with highly ISR-philic species. Although the formal potential of the Ru(NH(3))(6)(3+)/Ru(NH(3))(6)(2+) couple is lower than that of the QI/AP couple, the endergonic reaction between Ru(NH(3))(6)(3+) and AP is driven by the highly exergonic reaction between TCEP and QI (via a coupled reaction mechanism). Overall, the "outer-sphere to inner-sphere" redox cycling in the order of highly OSR-philic ITO, highly OSR-philic Ru(NH(3))(6)(3+)/Ru(NH(3))(6)(2+) couple, OSR- and ISR-philic QI/AP couple, and highly ISR-philic TCEP allows high, selective, and reproducible signal amplification. The electrochemical data obtained by chronocoulometry permit a lower detection limits than those obtained by cyclic voltammetry. The detection limit of an immunosensor for troponin I in serum, calculated from the anodic charges in chronocoulometry, is ca. 10 fg/mL.     

Optimization of phosphatase- and redox cycling-based immunosensors and its application to ultrasensitive detection of troponin I

The authors herein report optimized conditions for ultrasensitive phosphatase-based immunosensors (using redox cycling by a reducing agent) that can be simply prepared and readily applied to microfabricated electrodes. The optimized conditions were applied to the ultrasensitive detection of cardiac troponin I in human serum. The preparation of an immunosensing layer was based on passive adsorption of avidin (in carbonate buffer (pH 9.6)) onto indium-tin oxide (ITO) electrodes. The immunosensing layer allows very low levels of nonspecific binding of proteins. The optimum conditions for the enzymatic reaction were investigated in terms of the type of buffer solution, temperature, and concentration of MgCl(2), and the optimum conditions for antigen-antibody binding were determined in terms of incubation time, temperature, and concentration of phosphatase-conjugated IgG. Very importantly, the antigen-antibody binding at 4 °C is extremely important in obtaining reproducible results. Among the four phosphatase substrates (L-ascorbic acid 2-phosphate (AAP), 4-aminophenyl phosphate, 1-naphthyl phosphate, 4-amino-1-naphthyl phosphate) and four phosphatase products (L-ascorbic acid (AA), 4-aminophenol, 1-naphthol, 4-amino-1-naphthol), AAP and AA meet the requirements most for obtaining easy dissolution and high signal-to-background ratios. More importantly, fast AA electrooxidation at the ITO electrodes does not require modification with any electrocatalyst or electron mediator. Furthermore, tris(2-carboxyethyl)phosphine (TCEP) as a reducing agent allows fast redox cycling, along with very low anodic currents at the ITO electrodes. Under these optimized conditions, the detection limit of an immunosensor for troponin I obtained without redox cycling of AA by TCEP is ca. 100 fg/mL, and with redox cycling it is ca. 10 fg/mL. A detection limit of 10 fg/mL was also obtained even when an immunosensing layer was simply formed on a micropatterned ITO electrode. From a practical point of view, it is of great importance that ultralow detection limits can be obtained with simply prepared enzyme-based immunosensors.     

An electrochemically reduced graphene oxide-based electrochemical immunosensing platform for ultrasensitive antigen detection

We present an electrochemically reduced graphene oxide (ERGO)-based electrochemical immunosensing platform for the ultrasensitive detection of an antigen by the sandwich enzyme-linked immunosorbent assay (ELISA) protocol. Graphene oxide (GO) sheets were initially deposited on the amine-terminated benzenediazonium-modified indiun tin oxide (ITO) surfaces through both electrostatic and π-π interactions between the modified surfaces and GO. This deposition was followed by the electrochemical reduction of graphene oxide (GO) for preparing ERGO-modified ITO surfaces. These surfaces were then coated with an N-acryloxysuccinimide-activated amphiphilic polymer, poly(BMA-r-PEGMA-r-NAS), through π-π stacking interactions between the benzene ring tethered to the polymer and ERGO. After covalent immobilization of a primary antibody on the polymer-modified surfaces, sandwich ELISA was carried out for the detection of an antigen by use of a horseradish peroxidase (HRP)-labeled secondary antibody. Under the optimized experimental conditions, the developed electrochemical immunosensor exhibited a linear response over a wide range of antigen concentrations with a very low limit of detection (ca. 100 fg/mL, which corresponds to ca. 700 aM). The high sensitivity of the electrochemical immunosensor may be attributed not only to the enhanced electrocatalytic activity owing to ERGO but also to the minimized background current owing to the reduced nonspecific binding of proteins.     

Hydrazine-catalyzed ultrasensitive detection of DNA and proteins

A sensitive electrochemical assay of DNA and proteins employing electrocatalytic reduction of hydrogen peroxide by labeled hydrazine on the probe immobilized surfaces was developed. The method utilizes a conducting polymer, poly-5, 2':5', 2'-terthiophene-3'-carboxylic acid (pTTCA), covalently linked to the dendrimer (DEN) and hydrazine. The detection signal was amplified by the pTTCA/DEN assembly loaded with Au nanoparticles (particle size, approximately 3.5 nm) onto which huge target DNA- or proteins-linked hydrazine labels (avidin-hydrazine) were adsorbed. The linear dynamic ranges for the electrocatalytic detection of DNA and proteins, extending from 1.0 fM to 10 microM and 10 fg/mL to 10 ng/mL, were observed, along with the detection limits of 450 aM (2700 DNA molecules in a 10-microL sample) and 4.0 fg/mL, respectively. The method eliminates the use of enzymes for DNA and protein detection and opens a way for DNA-free detection of proteins. The simplicity, good reproducibility (RSD, <4.3% for n = 10), and low detection limit of the method offer a good promise for practical DNA and protein analyses.     

Bio/Abiotic Interface Constructed from Nanoscale DNA Dendrimer and Conducting Polymer for Ultrasensitive Biomolecular Diagnosis

For sensors detecting immobilized biomarkers, the interface between the surface and the fluid medium plays an important role in determining the levels of signal and noise in the electrochemical detection process. When protein is directly immobilized on the metal electrode, denaturation of the protein by surface–protein interaction results in low activity and low signal level. A conducting polymer‐based interface can prevent the protein conformation change and alleviate this problem. A DNA dendrimer is introduced into the interfacial film on the sensor surface to further improve the sensor performance. DNA dendrimer is a nanoscale dendrite constructed of short DNA sequences, which can be easily incorporated into the abiotic conducting polymer matrix and is biocompatible with most biological species. In this work, DNA dendrimer and polypyrrole (DDPpy) form the bio/abiotic interface on electrochemical sensors. Detection of two salivary protein markers (IL‐8 and IL‐1β) and one mRNA salivary marker (IL‐8) is used to demonstrate the efficiency of the DDPpy sensor. A limit of detection (LOD) of protein of 100–200 fg mL^?1 is achieved, which is three orders of magnitude better than that without the DNA dendrimer interface. An LOD of 10 aM is established for IL‐8 mRNA. The typical sample volume used in the detection is 4 µL, thus the LOD reaches only 25 target molecules (40 yoctomole).     

Aptamer-based immunosensor on the ZnO nanorods networks

This paper presents the fabrication and characteristics of a new aptamer-based electrochemical immunosensor on the patterned zinc oxide nanorod networks (ZNNs) for detecting thrombin. Aptamers are single-stranded RNA or DNA sequence that binds to target materials with high specificity and affinity. An antibody-antigen-aptamer sandwich structure was employed to this immunosensor for detecting thrombin. First, hydrothermally grown ZNNs were patterned on the patterned 0.02 cm2 Au/Ti electrodes on a glass substrate by lift-off process. The high isoelectric point (IEP, approximately 9.5) of nanostructured ZnO makes it suitable for immobilizing proteins with low IEP. Then 5 microL of the 500 nM antibody was immobilized on the ZNNs electrode. 5 micro/L of the mixture of 1 microM aptamer labeled by ferrocene (Fc) and thrombin was dropped on the electrode for antibody-antigen binding. The peak oxidation currents of the immunosensors at various thrombin concentrations were measured by using cyclic voltammetry. The peak oxidation current was observed at 340 mV versus Ag/AgCl electrode, and the peak oxidation current increased linearly from 62.26 nA to 354.13 nA with the logarithmic concentration of thrombin in the range from 100 pM to 250 nM. Fabrication of an aptamer-based immunosensor for thrombin detection is a new attempt and the characteristics of the fabricated immunosensors showed that the fabricated aptamer-baded immunosensor worked electrochemically well and had a low detection limit (approximately 91.04 pM) and good selectivity.     

Sensitive human interleukin 5 impedimetric sensor based on polypyrrole-pyrrolepropylic acid-gold nanocomposite

A sensitive impedimetric immunosensor was constructed by using an electropolymerized nanocomposite film containing polypyrrole (PPy), polypyrrolepropylic acid (PPa), and Au nanoparticles. The nanocomposite exhibits good stability, high porosity, high hydrophilicity, and efficient probe immobilization capability. In the film, PPa enhances the hydrophilicity while providing covalent probe attachment linkers, PPy promotes the conductivity and electroactivity, and Au nanoparticles result in good conductivity, high stability, and covalent binding linkers. These combined advantages significantly improve the detection sensitivity in comparison to the conventional methods. As a model, a human interleukin 5 (IL-5) immunosensor, an important sensor for disease pathology study, clinic diagnosis, and pharmaceutical research, was fabricated with the new nanocomposite film. Various optimization works were conducted to improve the detection sensitivity. With the optimal fabrication parameters, the detection limit for IL-5 was 10 fg/mL in phosphate buffered saline (PBS) and 1 pg/mL in 1% human serum with good specificity and a dynamic range of 3 orders of magnitude. This work demonstrates a new approach to develop a sensitive and labeless impedimetric immunosensor for potential broad applications in clinical diagnosis and drug discovery.     

InP nanowire/polymer hybrid photodiode

A novel design is presented for a nanowire/polymer hybrid photodiode. n-InP nanowires are grown directly onto an indium tin oxide (ITO) electrode to increase carrier collection efficiency and to eliminate the need for an expensive substrate. Experiments show that an ohmic contact is achieved between the nanowires and the ITO electrode. The nanowires are then enveloped by a high hole mobility conjugated polymer, poly(3-hexylthiophene). Compared to the control polymer-only device, the inclusion of InP nanowires increases the forward bias current conduction by 6-7 orders of magnitude. A high rectification ratio of 155 is achieved in these photodiodes along with a low ideality factor of 1.31. The hybrid device produces a photoresponse with a fill factor of 0.44, thus showing promise as an alternative to current polymer solar cell designs.     

Electrochemical properties of ferrocene adsorbed on multi-walled carbon nanotubes electrode

The adsorbed process of ferrocene on a glassy carbon (GC) electrode modified by multi-walled carbon nanotubes (MWNTs) and electrochemical properties of the adsorbed layers are investigated. It is found that the redox process of ferrocene in solution is controlled by diffusion and surface electrochemical steps on the MWNT/GC electrode in contrast to the diffusion-controlled process of ferrocene on the GC electrode. The adsorbed ferrocene exhibits a pair of well-defined redox waves in the potential range from − 0.2 V to 0.6 V. Interestingly, two pairs of obvious redox waves for the adsorbed ferrocene are observed at the switching potential over 0.8 V and the peak current values of redox waves in more positive potential increase with the enlarging switching potential. The electrochemical reaction model of ferrocene adsorbed on the MWNT/GC electrode is proposed.     

Ultrasensitive immunoassay of protein biomarker based on electrochemiluminescent quenching of quantum dots by hemin bio-bar-coded nanoparticle tags

A hemin bio-bar-coded nanoparticle probe labeled antibody was designed by the assembly of antibody and alkylthiol-capped bar-code G-quadruplex DNA on gold nanoparticles and the interaction of hemin with the DNA to form a G-quadruplex/hemin bio-bar-code. An ultrasensitive immunoassay method was developed by combining the labeled antibody with an electrochemiluminescent (ECL) immunosensor for protein. The ECL immunosensor was constructed by a layer-by-layer modification of carbon nanotubes, CdS quantum dots (QDs), and capture antibody on a glassy carbon electrode. In air-saturated pH 8.0 PBS the immunosensor showed a carbon-nanotube-enhanced cathodic ECL emission of QDs. Upon the formation of immunocomplex, the ECL intensity decreased owing to the consumption of ECL coreactant in bio-bar-code electrocatalyzed reduction of dissolved oxygen. Using α-fetoprotein as model analyte, the quenched ECL could be used for immunoassay with a linear range of 0.01 pg mL(-1) to 1 ng mL(-1) and a detection limit of 1.0 fg mL(-1). The wide detection range and high sensitivity resulted from the enhanced ECL emission and highly efficient catalysis of the bio-bar-code. The immunosensor exhibited good stability and acceptable fabrication reproducibility and accuracy, showing great promise for clinical application.     

Conductivity mechanisms in poly(p-phenylene vinylene) light-emitting diodes at high and low bias

Polymer light-emitting diodes (LEDs) based on the structure ITO conducting glass /poly(p-phenylene vinylene)/metal (Al, In, Mg) have been investigated with particular emphasis on some anomalous and poorly understood features of the conduction mechanisms. At large forward bias (above 3 V, where electroluminescence is seen) the DC current is dominated by hole injection at the bottom ITO electrode and is not very sensitive to the top electrode metal or the fabrication conditions. It always increases exponentially with voltage at 20°C, but studies on operating voltages and apparent ideality factors as a function of thickness indicate that the conduction mechanism probably involves thermally assisted tunnelling rather than a simple Schottky diode mechanism. In contrast, the current at low forward bias (0.1–3 V) is exceptionally sensitive to the top electrode material, fabrication conditions, and operating history of the device. Anomalous behaviour is often seen, particularly with new devices and with Mg or In electrodes. With Al electrodes, particularly after a top electrode annealing step, the behaviour becomes much more stable and resistive, and logJ increases linearly with V^1/4 over 3 orders of magnitude of current. The overall behaviour at low bias is controlled by the barrier to holes at the top electrode, but the role of filamentary defects, dopants, and the insulating interfacial layer is also discussed.     

Optically transparent diamond electrode for use in ir transmission spectroelectrochemical measurements

A new analytical spectroelectrochemical methodology is reported on that utilizes an optically transparent boron-doped diamond thin film. The film was deposited on undoped Si by microwave-assisted chemical vapor deposition using a 4-h growth with a 0.5% CH4/H2 source gas mixture and 2 ppm B2H6 added for boron doping. The thin-film electrode possessed a transparency of 40-60% in the mid- and far-IR regions of the electromagnetic spectrum. The physical, electrical, optical, and electrochemical properties of the electrode were characterized by scanning electron microscopy, Raman spectroscopy, X-ray diffraction, four-point probe electrical resistance measurements, IR spectroscopy, and cyclic voltammetry. The film's electrochemical behavior was evaluated using both aqueous (Fe(CN)(6)3-/4-, methyl viologen, Ru(NH3)(6)3+/2+, and IrCl(6)2-/3-) and nonaqueous (ferrocene) redox systems. The film exhibited a low and stable background current and a nearly reversible voltammetric response for all these redox systems. The diamond/Si optically transparent electrode (OTE) and a thin-layer transmission cell were used to record the spectroelectrochemical response for 10 mM Fe(CN)(6)3-/4- in 1 M KCl. Difference IR spectra (oxidized minus reduced), recorded at various applied potentials, showed that the CN vibrational mode at 2039 cm-1 for Fe(CN)(6)4- reversibly shifted to 2116 cm-1 upon oxidation to Fe(CN)(6)3-, as expected. Difference IR spectra (oxidized minus reduced) were also recorded for 20 mM ferrocene in 0.1 M TBABF4/CH3CN. A shift of the C-H bending mode of the cyclopentadienyl ring from 823 to 857 cm-1 occurred upon oxidation of ferrocene to ferricenium. The key finding from the work is that the diamond OTE provides sensitive, reproducible, and stable spectroelectrochemical responses for aqueous and nonaqueous redox systems in the mid- and far-IR.     

Development of amperometric immunosensor using boron-doped diamond with poly(o-aminobenzoic acid)

An alternative method of a protein immunosensor has been developed at boron-doped diamond (BDD) electrode material. In order to construct the base of the immunosensor, o-aminobenzoic acid (o-ABA) was electropolymerized at an electrode by cyclic voltammetry. The poly-o-ABA-modified BDD was characterized by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). The XPS result found that carboxyl groups were formed at the electrode surface. The carboxyl groups were then used to covalently attach protein probes. The amperometric sensing of mouse IgG (MIgG) was selected as the model at the poly-o-ABA-modified BDD to compare to the poly-o-ABA-modified glassy carbon (GC) at the same condition. An antimouse IgG from goat (GaMIgG) was covalently immobilized at a poly-o-ABA-modified BDD electrode which used a sandwich-type alkaline phosphatase (ALP) catalyzing amperometric immunoassay with 2-phospho-L-ascorbic acid (AAP) as substrate. The ALP enzyme conjugated at the immunosensor can generate AAP to the electroactive species of ascorbic acid (AA), which can be determined by amperometric detection. The signal was found to be proportional with the quantity of MIgG. The limits of detection (LODs) of 0.30 (3 SD) and 3.50 ng mL(-1) (3 SD) for MIgG at BDD and GC electrodes were obtained. It also was found that the dynamic range of 3 orders of magnitude (1-1000 ng mL(-1)) was obtained at BDD, whereas at GC, the dynamic range was more narrow (10-500 ng mL(-1)). The method was applied to a real mouse serum sample that contains MIgG.     

Improvements of transparent electrode materials for GaN metal–semiconductor–metal photodetectors

Metal–semiconductor–metal (MSM) photodetectors based on GaN grown on (0 0 0 1) sapphire were fabricated and characterized. The responsivity of the Pt/GaN MSM device is low due to the blocking of incoming light by Pt electrodes. Although this problem can be partly solved by the transparent indium–tin oxide (ITO) contact, the range of operation voltage for ITO/GaN MSM devices is limited by the internal gain. Transparent multilayered electrode is proposed in this work by incorporating various intermediate layers (Ti, TiO₂, and Ti/TiO₂). The dark current of the ITO/TiO₂/GaN contact is two orders of magnitude lower than that of the ITO/Ti/GaN contact. The thin TiO₂ barrier also contributes the lower responsivity of the ITO/TiO₂/GaN structure. By introducing a thin Ti/TiO₂ interlayer at the ITO–GaN interface, a significant decrease in the dark current and an increase in responsivity can be achieved simultaneously. The photo-to-dark current contrast can reach 6×l0⁵, and the responsivity shows no discernible internal gain under a bias between 2.5 and 7.5 V.     

Poly(aniline) nanowires in sol-gel coated ITO: a pH-responsive substrate for planar supported lipid bilayers

Facilitated ion transport across an artificial lipid bilayer coupled to a solid substrate is a function common to several types of bioelectronic devices based on supported membranes, including biomimetic fuel cells and ion channel biosensors. Described here is fabrication of a pH-sensitive transducer composed of a porous sol-gel layer derivatized with poly(aniline) (PANI) nanowires grown from an underlying planar indium-tin oxide (ITO) electrode. The upper sol-gel surface is hydrophilic, smooth, and compatible with deposition of a planar supported lipid bilayer (PSLB) formed via vesicle fusion. Conducting tip AFM was used to show that the PANI wires are connected to the ITO, which convert this electrode into a potentiometric pH sensor. The response to changes in the pH of the buffer contacting the PANI nanowire/sol-gel/ITO electrode is blocked by the very low ion permeability of the overlying fluid PSLB. The feasibility of using this assembly to monitor facilitated proton transport across the PSLB was demonstrated by doping the membrane with lipophilic ionophores that respond to a transmembrane pH gradient, which produced an apparent proton permeability several orders of magnitude greater than values measured for undoped lipid bilayers.     

磷钨铁杂多酸/聚酰胺胺复合膜的制备及电催化性能

通过层层自组装方法在氧化铟锡(ITO)电极上原位制备了Dawson型磷钨铁杂多酸/聚酰胺-胺复合膜。用XPS、UV-Vis、CV、AFM分析表征复合膜的形成过程和电化学性能。结果表明: 杂多酸阴离子和聚酰胺-胺层层自组装形成多层复合膜且增长均匀; 复合膜在酸性溶液中出现了三对氧化还原峰; 用该复合膜修饰的电极稳定性好, 对亚硝酸盐和溴酸盐均具有良好的电催化活性。      

Detection of hydrazine, methylhydrazine, and isoniazid by capillary electrophoresis with a palladium-modified microdisk array electrode

A palladium particle-modified carbon fiber microdisk array electrode was designed and employed in capillary electrophoresis for the simultaneous detection of hydrazine, methylhydrazine, and isoniazid. The Pd-modified microdisk electrode had high catalytic ability for hydrazines and exhibited good reproducibility and stability. The response for hydrazine was linear over 3 orders of magnitude with a correlation coefficient of 0.993. The detection limits for hydrazine, methylhydrazine, and isoniazid were 1.2, 2.1, and 6.2 pg, respectively.     

Sensitive amperometric immunosensing using polypyrrolepropylic acid films for biomolecule immobilization

An electrochemical immunosensor was constructed using an electropolymerized pyrrolepropylic acid (PPA) film with high porosity and hydrophilicity. A high density of carboxyl groups of PPA was used to covalently attach protein probes, leading to significantly improved detection sensitivity compared with conventional entrapment methods. As a model, anti-mouse IgG was covalently immobilized or entrapped in the PPA film and used in a sandwich-type alkaline phosphatase-catalyzing amperometric immunoassay with p-aminophenyl phosphate as the substrate. With covalent binding, the detection limit for IgG in PBS buffer, pH 7.4, was 100 pg/mL with a dynamic range of 5 orders of magnitude. The covalent bonding mode in the carbonate-bicarbonate buffer, pH 9.6, further brought down the detection limit to 20 pg/mL with remarkable selectivity.     

Development of an electrochemical immunosensor for direct detection of interferon-gamma at the attomolar level

An electrochemical immunosensor for direct detection of the 15.5-kDa protein interferon-gamma (IFN-gamma) at attomolar level has been developed. Self-assembled monolayers (SAMs) of cysteine or acetylcysteine are formed on electropolished polycrystalline Au electrodes. IFN-gamma adsorbs physically to each of these SAMs. With injections of 100 mM KCl, IFN-gamma can be removed in the flow without damaging the acetylcysteine SAM. However, the cysteine SAM is affected by these KCl injections. In an on-line procedure in the flow, a specific antibody (MD-2) against IFN-gamma is covalently attached following carbodiimide/succinimide activation of the SAM. The activation of the carboxylic groups, attachment of MD-2, and deactivation of the remaining succinimide groups with ethanolamine are monitored impedimetrically at a frequency of 113 Hz, a potential of +0.2 V versus SCE, and an ac modulation amplitude of 10 mV. Plots of the real (Z') and imaginary (Z") component of the impedance versus time provide the information to control these processes. In the thermostated setup (23.0 degrees C), samples of unlabeled IFN-gamma (in phosphate buffer pH 7.4) are injected and the binding with immobilized MD-2 is monitored with ac impedance or potential-step methods. While the chronoamperometric results are rather poor, the ac impedance approach provides unsurpassed detection limits, as low as 0.02 fg mL-1 (approximately 1 aM) IFN-gamma. From a calibration curve (i.e. Z" versus the amount injected), recorded by multiple 50-microL injections of 2 pg mL(-1) of IFN-gamma, a dynamic range of 0-12 pg mL(-1) could be derived. However, when nonspecific adsorption is taken into account, which has been found to be largely reduced through injections of 100 mM KCl, a much smaller dynamic range of 0-0.14 fg mL(-1) remains. The immunosensor can be regenerated by using a sequence of potential pulses in the flow by which the SAM with attached MD-2 and bound IFN-gamma is completely removed. When the developed procedures described above are repeated, the response of the immunosensor is reproducible within 10%.     

Selective electrochemical detection using a split disk array electrode in a thin-layer radial flow system

An eight-sector array (split disk) electrode was designed for a low flow rate (<100 μL/min) amperometric detector. This electrode was fabricated photolithographically for dimensional accuracy and reproducibility. This array of a pie-shaped electrode was combined with a thin-layer radial flow cell, and a conversion efficiency of 94% was achieved at the lowest flow rate tested (0.01 mL/min). Each electrode worked free from the effects of electrochemical reactions of the other electrodes. A coulometric hydrodynamic voltammogram of reversible redox species obtained using this system exhibited a Nernstian curve. These properties enabled this electrochemical detector to be used for determining the ratio of two redox species (redox potential difference ≈ 100 mV) with small injection volume (5 μL).