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Anodization is known to be an effective way to slow down the initial corrosion rate of magnesium (Mg) and its alloys. Here, we investigated the specific use of oxy-salts to improve the corrosion resistance of anodizing coatings. Oxy-salts of silicate, phosphate, and carbonate were added separately to a sodium hydroxide alkaline electrolyte used to anodize Mg alloy AZ91D. The process was investigated in terms of anodizing behavior, the surface properties, and the corrosion behavior of AZ91D. Anodizing AZ91D using the silicate- containing electrolyte generated sparks, and produced a thicker and more corrosion-resistant layer than the other oxy-salts. In the process, MgO and SiO 2 formed Mg 2 SiO 4 at high temperatures. Coatings from the phosphate- and carbonate- containing electrolyte anodizations did not contain phosphorus or carbon. We also studied the effects of silicate concentration on the corrosion resistance and properties of the surface.

A multiwalled carbon nanotube (MWCNT)-based electrochemical biosensor is developed for monitoring microcystin-LR (MC-LR), a toxic cyanobacterial toxin, in sources of drinking water supplies. The biosensor electrodes are fabricated using vertically well-aligned, dense, millimeter-long MWCNT arrays with a narrow size distribution, grown on patterned Si substrates by water-assisted chemical vapor deposition. High temperature thermal treatment (2500 ° C) in an Ar atmosphere is used to enhance the crystallinity of the pristine materials, followed by electrochemical functionalization in alkaline solution to produce oxygen-containing functional groups on the MWCNT surface, thus providing the anchoring sites for linking molecules that allow the immobilization of MC-LR onto the MWCNT array electrodes. Addition of the monoclonal antibodies specifi c to MC-LR in the incubation solutions offers the required sensor specifi city for toxin detection. The performance of the MWCNT array biosensor is evaluated using micro-Raman spectroscopy, including polarized Raman measurements, X-ray photoelectron spectroscopy, cyclic voltammetry, optical microscopy, and Faradaic electrochemical impedance spectroscopy. A linear dependence of the electron-transfer resistance on the MC-LR concentration is observed in the range of 0.05 to 20 μ g L − 1 , which enables cyanotoxin monitoring well below the World Health Organization (WHO) provisional concentration limit of 1 μ g L − 1 for MC-LR in drinking water.

The effect of a perfluorocarbon emulsion oxygen therapeutic (PEOT) on the detection of the drugs theophylline and phenytoin was explored using a commercial enzyme multiplied immunoassay technique (EMIT®). The EMIT technique is based on the enzymatic production of NADH, which is typically detected in serum samples spectrophotometrically. Here, amperometry using the rotating disk electrode on a single drop of solution is demonstrated to detect theophylline and phenytoin in the presence of PEOT. In the study, 2,6-dichloroindophenol (DCIP) added to the immunoassay mixture is reduced by the NADH to DCIPH2. Oxidation of DCIPH2 is monitored electrochemically at +200 mV using a glassy carbon rotating disk electrode. Slopes of amperograms are proportional to the concentration of drug in the immunoassay sample. This technique yields excellent quantitative data in the therapeutic range for both drugs in 2–20 % PEOT.

Solid contact (SC) ion-selective electrodes (ISEs) have been recognized as the next generation of ISEs. In this work, the electrical conductivity and mechanical strength of a carbon nanotube (CNT) tower enable it to play the dual roles of transducer and substrate for micro SC-ISEs. The electrode had a close to Nernstian slope of 35 mV/decade aCa2+, a linear range of four orders of magnitude of calcium ion activity (10−5.6 to 10−1.8 M), and a detection limit of 1.6×10−6 M. The simplified fabrication by a one-step drop casting makes miniaturizing SC-ISEs and fabricating sensor arrays easier to achieve.

Towers of aligned multiwalled carbon nanotubes (MWCNTs) were electrochemically oxidized in aqueous 1 M NaOH. An oxidation current that decayed with time was monitored using amperometry at a fixed potential. Cyclic voltammetry showed that the background current and electrode capacitance increased after oxidation without significantly affecting the faradaic current from the reduction of ferricyanide. Oxidation in NaOH caused morphological changes and increased hydrophilicity of the MWCNT tower electrodes. XPS spectra indicated increased oxygen on the surface after oxidation, while Raman spectra indicated that a large amount of amorphous carbon was present before and after oxidation.

A label-free biosensor for Escherichia coli (E. coli) ORN 178 based on faradaic electrochemical impedance spectroscopy (EIS) was developed. α-Mannoside or β-galactoside was immobilized on a gold disk electrode using a self-assembled monolayer (SAM) via a spacer terminated in a thiol functionality. Impedance measurements (Nyquist plot) showed shifts due to the binding of E. coli ORN 178, which is specific for α-mannoside. No significant change in impedance was observed for E. coli ORN 208, which does not bind to α-mannoside. With increasing concentrations of E. coli ORN 178, electron-transfer resistance (R(et)) increases before the sensor is saturated. After the Nyquist plot of E. coli/mixed SAM/gold electrode was modeled, a linear relationship between normalized R(et) and the logarithmic value of E. coli concentrations was found in a range of bacterial concentration from 10(2) to 10(3) CFU/mL. The combination of robust carbohydrate ligands with EIS provides a label-free, sensitive, specific, user-friendly, robust, and portable biosensing system that could potentially be used in a point-of-care or continuous environmental monitoring setting.

Anodic stripping voltammetry (ASV) trace detection of the heavy metals ions Pb2+, Cd2+, Cu2+ and Zn2+ was done at highly aligned multi-wall carbon nanotube tower electrodes (CNT tower electrodes). The CNT tower electrode is one type of CNT array that consists of millions of ordered super long CNTs. Osteryoung square-wave stripping voltammetry was used for the stripping step. This electrode material shows good resolution for individual detection of these four metal ions with calculated detection limits of 12 nM, 25 nM, 44 nM and 67 nM for Pb2+, Cd2+, Cu2+ and Zn2+, respectively, for a deposition time of 120 s. The detectable limit was as low as 0.5 nM for a 10 min deposition.

Viable Escherichia coli can be detected by an immunoassay in which live bacteria captured on antibody-coated paramagnetic beads are induced to synthesize the enzyme beta-galactosidase, which catalyzes the hydrolysis of the slightly fluorescent substrate 4-methyl umbelliferyl-beta-D-galactoside to the highly fluorescent product 7-hydroxy-4-methylcoumarin for detection. The effects of bacterial strain, presence of dead bacteria, and some environmental stresses on assay performance were evaluated.

Development of biodegradable metal implants is a complex problem because it combines engineering and medical requirements for a material. This article discusses the development of sensing and corrosion control techniques that can help in the design of biodegradable metallic implants. Biodegradable metallic implants dissolve as new tissue is formed. One of the most important factors in the design of biodegradable implants is to study the active interface, which should be monitored and controlled to address the medical concern of biocompatibility. Thus miniaturized and nanotechnology-based sensors that measure the activities of the degradation process and the formation of tissue are discussed for use with in vitro and in vivo experiments. These sensors can monitor chemical components and also cell activity and can provide new knowledge about biodegradable interfaces and how to actively control the interface to provide the best bioactivity to regenerate new tissue in a short time. Development of new alloys, nano-materials, miniature sensors, corrosion control coatings, and auxiliary applications such as biodegradable drug delivery capsules is expected to open up a new era in the engineering of materials for medicine.

A rapid and convenient assay system was developed to detect viable Escherichia coli in water. The target bacteria were recovered from solution by immunomagnetic separation and incubated in tryptic soy broth with isopropyl-beta-D-thiogalactopyranoside, which induces formation of beta-galactosidase in viable bacteria. Lysozyme was used to lyse E. coli cells and release the beta-galactosidase. Beta-galactosidase converted 4-methylumbelliferyl-beta-D-galactoside to 4-methylumbelliferone (4-MU), which was measured by fluorescence spectrophotometry using excitation and emission wavelengths of 355 and 460 nm, respectively. Calibration graphs of 4-MU fluorescence intensity versus E. coli concentration showed a detection range between 8 x 10(4) and 1.6 x 10(7) cfu mL(-1), with a total analysis time of less than 3 h. The advantage of this method is that it detects viable cells because it is based on the activity of the enzyme intrinsic to live E. coli.

A new binding assay for a protein and its ligand based on a spectroelectrochemical method was demonstrated using avidin-biotin and 17beta-estradiol-antiestradiol antibody. The sensor consists of a selective film coated on an optically transparent electrode (OTE) consisting of indium tin oxide (ITO). Attenuated total reflection (ATR) was used for optical detection. The binding event of the ligand to the protein was detected using the ligand labeled with the electroactive dye Nile blue (NB). The spectroelectrochemical behaviors of NB and the labeled ligand were investigated using various ion-exchange films, such as perfluorosulfonated ionomer (Nafion), Nafion-silica, poly(acrylic acid) (PAA)-silica, poly(styrenesulfonic acid) (PSSA)-silica, and heparin-silica films, which were spin-coated on the ITO electrode. The optical signal was monitored to follow the accumulation of labeled ligand in the film and its electrochemical modulation. The signal from the labeled ligand possesses three modes of selectivity based on charge-selective partitioning, the chosen electrolysis potential, and the particular wavelength for measuring absorbance. The interaction between the labeled ligand and its protein was observed by the decrease in the changes of optical response of the labeled ligand, indicating the specific binding of labeled ligand to the protein.

In this work we fabricated 8 mm long aligned multi-walled carbon nanotube array electrodes and electrochemically deposited gold nanoparticles in a controlled manner onto the nanotube tips at the top of the array. A soldering technique was also developed to provide reliable electrical contact between the carbon nanotubes at the bottom of the array and copper on a patterned printed circuit board. The top surfaces of the nanotube tower electrodes were activated using a reactive ion etcher prior to gold deposition. Electrodeposition of Au particles on the plasma-treated tower electrodes was performed over a range of deposition times and potentials. Cyclic voltammetry and electrochemical impedance spectroscopy analyses showed that the Au-deposited carbon nanotube array electrodes were successfully fabricated. The new electrodes will be the foundation for further biosensor development using self-assembled monolayers and bio-conjugation of antibodies for novel label-free immunosensing.

A microbead based sandwich immunoassay for MS2 bacteriophage was developed using an interdigitated array (IDA) electrode with nanoscale dimensions (220 nm electrode width, 620 nm gap). The IDA was fabricated using an electron beam lithographic lift-off technique. After an antibody-assisted capture of MS2 using paramagnetic microbeads, a β-galactosidase labeled secondary antibody was used to convert p-aminophenyl-β-D-galactopyranoside (PAPG) into the redox active p-aminophenol (PAP). Amperometric detection of PAP with IDA electrodes at +300 and −200 mV vs. a Ag/AgCl reference electrode was used to measure the result, detecting MS2 concentrations as low as 10 ng/mL.

Cyclic voltammetry in a perfluorocarbon emulsion based blood substitute (PEBS) was evaluated. The intent was to determine how PEBS affects the voltammetry of four representative electroactive compounds: potassium ferricyanide, ruthenium(II) trisbipyridine (rutris), hydroquinone, and 2,6-dichloroindophenol (DCIP). Voltammograms in 0–20% PEBS-Tris buffer mixtures are affected by PEBS, but reasonably well-defined voltammograms are obtained in as much as 20% v/v. This report also shows that PEBS has only a small effect on amperometric detection of the reaction between DCIP and nicotinamide adenine dinucleotide (NADH), which has importance in clinical homogeneous immunoassays. Results support continued exploration of voltammetry/amperometry for quantitative analysis in this medium.

This paper describes the development of a label-free immunosensor based on carbon nanotube (CNT) array electrodes. Highly aligned multi-walled carbon nanotubes were grown on a Fe/Al 2 O 3 /SiO 2 /Si substrate by chemical vapor deposition (CVD). The substrate was patterned with 100 μm square blocks and 100 μm spacing between blocks. Carbon nanotube towers up to 2 mm in height grew from the blocks, and the towers were easy to peel off the silicon substrate. The harvested towers were cast in epoxy and both ends were polished; one end for electrical connection, and the other end for use as an electrode. The nanotube electrode was then electrochemically activated to open the nanotube ends and to expose COOH groups on the surface. Anti-mouse IgG was then covalently immobilized on the nanotube array. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to characterise the binding of mouse IgG to its specific antibody already immobilized on the nanotube electrode surface. A non-linear calibration plot was constructed based on the change in the electron transfer resistance at the electrode surface as a function of mouse IgG concentrations. A detection limit of 200 ng/mL and a dynamic range up to a 100 μg/mL range were obtained. Overall, it was found that the nanotube array immunosensor was easy to fabricate and has good sensitivity.

This paper describes the fabrication and evaluation of carbon nanotube (CNT) electrodes grown on stainless steel (SS) plate and wire for electrochemical sensor applications. Multi-wall carbon nanotubes with different diameters were grown on the SS plate and wire by chemical vapor deposition from an ethylene precursor. The SS provides a good electrical and mechanical connection to the CNT, and the SS is a tough substrate. The SS part of the electrode was electrically insulated from the analyte so that only the CNT were active in sensing. Cyclic voltammetry for the reduction of 6 mM K3Fe(CN)6 in a 1.0 M KNO3 supporting electrolyte was performed to examine the redox behavior of the CNT-SS electrode. The cyclic voltammograms showed sigmoidal-like shapes, indicating that mass transport around the electrodes is dominated by radial diffusion. Based on the cyclic voltammograms, the effective area of the CNT-SS electrodes and the number of individual CNTs were estimated. These results indicate that the CNT-SS plate and wire electrodes are good candidates to develop practical in vivo biosensors.

Highly aligned multi-wall carbon nanotubes (MWCNT) were synthesized in the shape of towers and used to fabricate chemical sensor electrodes. The towers were fabricated on an Fe/Al 2 O 3 /SiO 2 /Si substrate with the Fe catalyst patterned in 1 mm × 1 mm blocks with 100 μm spacing between the blocks. Thermally driven chemical vapor deposition was used for the nanotube synthesis process. Patterned MWCNT towers up to 4 mm high were grown and easily peeled off the silicon substrate. The synthesized MWCNT towers were characterized by environmental scanning electron microscopy and high resolution transmission electron microscopy. A sensor electrode was then formed by casting epoxy into the tower under pressure and polishing both ends of the tower. One end of the tower served as an electrical connection and the other as a nanoscale array electrode. Cyclic voltammetry (CV) for the reduction of 6.0 mM K 3 Fe(CN) 6 and Ru(NH 3 ) 6 Cl 3 (in a 1.0 M KNO 3 supporting electrolyte) was used to examine the surface properties of the nanotube tower electrode. The CV results showed a steady-state response up to 2 V/s attributable to radial diffusion with a high steady-state current density. Cyclic voltammetry testing of these two redox systems showed a sigmoidal shape indicating radial diffusion and high sensitivity at the electrode. Based on the CV results, the carbon nanotube array electrode is a promising candidate for future highly sensitive chemical and biosensor applications.

A fully-automated fluidic system for a bead-based immunoassay with electrochemical detection was developed. Assays for the bacteriophage MS2 and ovalbumin (OVA) were demonstrated using this system. Streptavidin-coated paramagnetic microbeads were used as a mobile solid phase. The immunoassay sandwich was made by attaching a biotinylated antibody to the streptavidin-coated beads, capturing antigen, and then exposing the antigen to an antibody conjugated with β-galactosidase. β-Galactosidase converts p -aminophenyl galactopyranoside (PAPG) to p -aminophenol (PAP), which is electrochemically oxidized to p -quinone imine (PQI). The behavior of paramagnetic microbeads in the fluidic system was investigated using beads with immobilized β-galactosidase at different concentrations of beads and flow rates for each procedure in the assay. Furthermore, the fully-automated MS2 and OVA assays were demonstrated using the fluidic system. The limits of detection for MS2 and OVA were 990 (1.6 × 10 11 particles mL −1 ) and 470 ng mL −1 , respectively.

A study comparing the electrophoretic separation performance attainable from microchips molded by masters fabricated using conventional CNC machining techniques with commercial microchips, wire imprinted microchips, and microchips from LIGA molding devices is presented. An electrophoresis-based detection system using fluorescence microscopy was used to determine the analytical utility of these microchips. The separation performance of CNC microchips was comparable to commercially available microchips as well as those fabricated from LIGA masters. The important feature of the CNC machined masters is that they have rapid design-to-device times using routinely available machining tools. This low-cost prototyping approach provides a new entry point for researchers interested in thermoplastic microchips and can accelerate the development of polymer-based lab-on-a-chip devices.

Detecting and enumerating fecal coliforms, especially Escherichia coli, as indicators of fecal contamination, are essential for the quality control of supplied and recreational waters. We have developed a sensitive, inexpensive, and small-volume amperometric detection method for E. coli beta-galactosidase by bead-based immunoassay. The technique uses biotin-labeled capture antibodies (Ab) immobilized on paramagnetic microbeads that have been functionalized with streptavidin (bead-Ab). The bead-Ab conjugate captures E. coli from solution. The captured E. coli is incubated in Luria Bertani (LB) broth medium with the added inducer isopropyl beta-D: -thiogalactopyranoside (IPTG). The induced beta-galactosidase converts p-aminophenyl beta-D: -galactopyranoside (PAPG) into p-aminophenol (PAP), which is measured by amperometry using a gold rotating disc electrode. A good linear correlation (R2 = 0.989) was obtained between log cfu mL(-1) E. coli and the time necessary to product a specific concentration of PAP. Amperometric detection enabled determination of 2x10(6) cfu mL(-1) E. coli within a 30 min incubation period, and the total analysis time was less than 1 h. It was also possible to determine as few as 20 cfu mL(-1) E. coli under optimized conditions within 6-7 h. This process may be easily adapted as an automated portable bioanalytical device for the rapid detection of live E. coli.

Immunosensing microfluidic devices are reviewed. Devices are commonly fabricated in glass, silicon, and polymers, with polymers seeing greater attention in recent years. Methods have been developed to immobilize antibodies and other molecules and resist non-specific adsorption through surface modification. The most common detection method is fluorescence, followed by electrochemistry. Various microfluidic designs have been reported for immunoassay applications. The observed trends in microfluidic immunoassay applications closely resemble the trends of general immunoassays, where large molecules are detected principally through a sandwich procedure, while competitive assays are used to detect smaller molecules. The following future trends are suggested: more sensitive detection, increased integration and miniaturization, multianalyte analysis, more robust reagents and devices, and increased functionality of surface treatments.

Nonpathogenic microorganisms are often used as simulants of biological pathogens during the initial phase of detection method development. While these simulants approximate the size, shape, and cellular organization of the microorganism of interest, they do not resemble its surface protein content, a factor particularly important in methods based on immunorecognition. Here, we develop and detect an artificial bacterial spore--B. globigii (BG) Bugbead-a particle mimicking the antigenic surface of BG spores. Two methods of spore protein extraction were compared both quantitatively (by protein concentration assay) and qualitatively (by SDS-PAGE and Western blot): extraction by mechanical disruption and extraction by chemical decoating. The former method was more efficient in producing more protein and a greater number of antigens. BG Bugbeads were made by conjugating the extracted proteins to 0.8-microm carboxyl-coated polystyrene particles via carbodiimide coupling. BG Bugbeads were successfully detected by a bead-based enzyme-labeled immunoassay with fluorescence detection with a detection limit of 6.9 x 10(3) particles/mL. Formation of the Bugbead-capture bead complex was confirmed by ESEM. The concept of a harmless artificial spore can be applied to developing improved simulants for pathogenic spore-forming microorganisms such as B. anthracis, C. botulinum, and B. cereus, which can to be used for method validation, instrument calibration, and troubleshooting.

This research is directed towards developing a more sensitive and rapid electrochemical sensor for enzyme labeled immunoassays by coupling redox cycling at interdigitated electrode arrays (IDA) with the enzyme label beta-galactosidase. Coplanar and comb IDA electrodes with a 2.4 microm gap were fabricated and their redox cycling currents were measured. ANSYS was used to model steady state currents for electrodes with different geometries. Comb IDA electrodes enhanced the signal about three times more than the coplanar IDAs, which agreed with the results of the simulation. Magnetic microbead-based enzyme assay, as a typical example of biochemical detection, was done using the comb and coplanar IDAs. The enzymes could be placed close to the sensing electrodes (approximately 10 microm for the comb IDAs) and detection took less than 1 min with a limit of detection of 70 amol of beta-galactosidase. We conclude that faster and more sensitive assays can be achieved with the comb IDA.

Alpha(1)-acid glycoprotein (AGP) consists of 183 amino acid residues and 5 carbohydrate chains and binds to basic and neutral drugs as well as steroid hormones. We investigated the structural properties and ligand-binding capacity of AGP under mild acidic conditions and its interactions with liposomes prepared from neutral or anionic lipids and the neutral drug, progesterone. Interestingly, AGP had a unique structure at pH 4.5, at which the tertiary structure changed, whereas the secondary structure remained intact. Furthermore, the binding capacity of AGP for progesterone did not significantly change under these conditions. It was also observed that AGP was strongly bound to the anionic membrane at pH 4.5, forming an alpha-helix-rich structure from the original beta-sheet-rich structure, which significantly decreased the binding capacity of AGP for progesterone. The structural transitions as well as the membrane binding were suppressed by adding NaCl. These results indicate that AGP has a unique structure on the membrane surface under mild acidic conditions. The conformational change induces binding to the membrane aided by electrostatic interaction, and AGP subsequently takes on a predominantly alpha-helical conformation.

ABTS, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate), a colorless dianion that forms a colored radical upon oxidation, was characterized with electrochemistry and spectroscopy and demonstrated to be a detectable analyte in a polymer-modified spectroelectrochemical sensor. Three positively charged polymers were incorporated into a thin film on an indium tin oxide (ITO) optically transparent electrode and used to concentrate ABTS at the electrode surface. Of the three films, poly(vinylbenzyl trimethyl ammonium chloride)-poly(vinyl alcohol) (PVTAC-PVA), poly(diallyldimethylammonium chloride)-silica (PDMDAAC-SiO2), and quaternized poly(4-vinyl-N-methylpyridinium nitrate)-silica (QPVP-SiO2), PVTAC-PVA demonstrated the best ability to absorb ABTS. Within 20 min, a change of 0.2 absorbance units at 417 nm and 13.6 μA/cm2 in anodic peak current density in cyclic voltammetry at a scan rate of 0.025 V/s were observed.

Secretion of insulin from pancreatic islets was monitored indirectly by detecting zinc. Anodic stripping voltammetric measurements of zinc were done on a bismuth-modified electrode. Comparison of the performance of bismuth-modified electrodes and mercury film electrodes showed that bismuth is an appropriate alternative for Zn detection. The bismuth-coated electrode was used to detect zinc in insulin samples and insulin secreted from pancreatic islets upon stimulation with high concentrations of K + . Detection of zinc released from pancreatic islets was done in the culture medium without any further cleanup. This detection method can be used to monitor secretion from pancreatic islets in their native environment.

The progress to electrochemical detection of a microbead-based immunoassay in small volumes has led to a reduced assay time and lower detection limits. Three electrochemical techniques are described for an immunoassay with detection in a microdrop. The techniques are amperometric detection with a rotating disk electrode (RDE), a microelectrode, and an interdigitated array (IDA) electrode. An enzyme-labeled sandwich immunoassay with mouse IgG as the model analyte is used to demonstrate the three techniques. The microbead assay is carried out in a test tube using a magnet to control bead collection. Once the immunocomplex is formed on the microbead, the beads are transferred to a microdrop where the enzyme, either alkaline phosphatase or β-galactosidase, generates 4-aminophenol (PAP). PAP is oxidized at the electrode with an applied potential of +290 mV vs. Ag/AgCl. For all three techniques, the upper limit of the dynamic range was 1000 ng/ml mouse IgG, and the detection limits were: 50 ng/ml for the RDE, 40 ng/ml for the microelectrode, and 26 ng/ml for the IDA electrode.

Insulin is stored in pancreatic islets as a zinc-insulin complex, and stimulating the islets results in the release of insulin and zinc. Simulant pancreatic islet beads have been developed using agarose beads (50-250 micro m diameter) derivatized with iminodiacetic acid that have been loaded with zinc. A qualitative comparison of the simulant beads with pancreatic islets has been made by staining with dithizone and a zinc-binding fluorescent dye, TSQ. The binding capacity of simulant beads was determined to be 34 micro mol Zn(2+)/g of dried beads using anodic stripping voltammetry. Hydrochloric acid was used to release zinc from beads to mimic the secretion of insulin from pancreatic islets and a release profile was established. The simulant beads can be used to optimize the islet isolation process and reduce the use of real islets in method development.

Alpha(1)-acid glycoprotein (AGP) is a glycoprotein that consists of 183 amino acid residues and five carbohydrate chains and binds to neutral and basic drugs. We examined the structural properties and ligand-binding capacity of AGP in interactions with reverse micelles. Also, detailed information was obtained by comparing several different states of AGP. Interaction with reverse micelles induced a unique conformational transition (beta-sheet to alpha-helices) in AGP and decreased the binding capacity for the basic drug, chlorpromazine and the steroid hormone, progesterone to AGP. These structural conformations are very similar to those observed under conditions of acidity and high ionic strength (pH 2.0, 1.5 M NaCl). This structure seems to be an intermediate between the native state and the denatured state, possibly a molten globule. The present results suggest that when AGP interacts with the biomembrane, it undergoes a structural transition to a unique structure that differs from the native and denatured states and has a reduced ligand-binding capacity.

The evolution of electrochemical immunoassay has resulted in significant improvements in assay times and detection limits. These developments are traced using a sandwich enzyme immunoassay for IgG as representative. Decreasing the volume of the immunoassay reaction chamber, improving electrochemical detection techniques, and reducing non-specific adsorption have been important factors.

Developing sensors for pathogens using real microorganisms for initial testing of the device can introduce additional variables and potential experimental hazards. The artificial microorganism “Bugbead” could be used as a simulant to evaluate the device and for methods development. Bugbead consists of a microsphere coated with proteins to represent the epitopes on a real microorganism. The advantages of the Bugbead include those of being non-pathogenic, similar in bead size to a real microorganism, and having a controllable epitope density. A well-characterized Bugbead eases the study of the device and can be prepared, stored and used safely. Bead-based sandwich immunoassay was done using, as a model system, biotinylated sheep–anti-mouse IgG as a primary antibody immobilized on streptavidin coated 2.8 μm magnetic beads. Neutravidin-dendrimer coated 0.39 μm nonmagnetic Bugbeads were labeled with biotinylated mouse and guinea pig IgGs. Alkaline phosphatase conjugated donkey–anti-guinea pig IgG was the secondary antibody. p -Aminophenyl phosphate was the phosphatase substrate and the p -aminophenol product was detected by rotating disk electrode-amperometry. The minimum detected amount was 360 Bugbeads. The capturing event between magnetic capture beads and Bugbeads was observed by optical microscopy. In simple tests of the concept, an extract containing serotype antigens of E. coli O157:H7 were coupled to Bugbeads and examined in two modified commercial test kits for this pathogen. In both cases positive results was obtained, indicating that the pathogen antigens had been successfully transferred to the microbead and were capable of simulating the assay response to the pathogen itself. These experimental results demonstrate that the Bugbead could be used in the initial steps of developing microbial sensors.

Microchip capillary electrophoresis (CE) was used with a model enzyme assay to demonstrate its potential application to combinatorial drug screening. Hydrolysis with beta-glucuronidase of the conjugated glucuronide, fluorescein mono-beta-D-glucuronide (FMG), liberated the fluorescent product, fluorescein. FMG and fluorescein were detected by fluorescence, with excitation and emission at 480 and 520 nm, respectively. Microchip CE was used to separate FMG and fluorescein. Fluorescein production was monitored to assess beta-glucuronidase activity. Michaelis-Menten enzyme kinetics analysis yielded the Km value. The results were compared with those from experiments done by conventional CE. The Km value for beta-glucuronidase with FMG is being reported for the first time as 18 microM. The inhibition of beta-glucuronidase by the competitive inhibitor D-saccharic acid-1,4-lactone (SL) was also determined using microchip CE. Reactions were done with various concentrations of inhibitor and constant beta-glucuronidase and FMG concentrations. A dose-response plot was acquired and the IC50 value for SL was determined to be 3 microM.

This paper presents the development and characterization of a generic microfluidic system for magnetic bead-based biochemical detection. Microfluidic and electrochemical detection devices such as microvalves, flow sensors, biofilters, and immunosensors have been successfully developed and individually characterized in this work. Magnetically driven microvalves, pulsed-mode microflow sensors, magnetic particle separators as biofilters, and electrochemical immunosensors have been sep-arately fabricated and tested. The fabricated microfluidic components have been surface-mounted on the microfluidic motherboard for fully integrated microfluidic biochemical detection system. A magnetic bio-bead approach has been adopted for both sampling and manipulating target biological molecules. Magnetic beads were used as both substrate of antibodies and carriers of target antigens for magnetic bead-based immunoassay, which was chosen as a proof-of-concept for the generic microfluidic bio-chemical detection system. The microfluidic and electrochemical immunosensing experiment results obtained from this work have shown that the biochemical sensing capability of the complete microfluidic subsystem is suitable for portable biochemical detection of bio-molecules. The methodology and system, which has been developed in this work, can be extended to generic bio-molecule detection and analysis systems by replacing antibody/antigen with appropriate bio receptors/reagents such as DNA fragments or oligonucleotides for application towards DNA analysis and/or high throughput protein analysis.

A bead based sandwich enzyme immunoassay coupled to electrochemical detection for ovalbumin has been developed. The enzyme label alkaline phosphatase was used to convert the substrate 4-aminophenyl phosphate to electroactive product 4-aminophenol. The detection was done in a microdrop by continuously monitoring the enzyme turnover with a rotating disk electrode. This reduces dilution of the enzyme product, a key to achieving low detection limits. The assay developed has a detection limit of 0.1 ng ml−1. Assay sensitivity in complex matrices such as food and serum was compared.

The formation of mound-like microscopic domains of biochemically active paramagnetic beads reported earlier is extended towards a miniaturized sandwich enzyme immunoassay for the model analyte mouse-IgG. After mouse-IgG is captured by the primary anti-mouse-IgG antibody (Ab) coated magnetic beads of 2.8 µm in diameter, a second anti-mouse-IgG Ab conjugated to an enzyme-label is bound to the analyte. The enzyme label alkaline phosphatase (ALP) is used to catalyze the hydrolysis of 4-aminophenyl phosphate (PAPP) to 4-aminophenol (PAP). The enzymatic activity, which depends on the concentration of captured mouse-IgG, is recorded by scanning electrochemical microscopy (SECM) where a 10 µm Pt electrode scanned over the bead domains detects PAP by oxidation. The anodic current correlates with the concentration of mouse-IgG.

A new procedure is described to deposit paramagnetic beads on surfaces to form microscopic agglomerates. By using surface-modified beads, microscopic structures with defined biochemical activity are formed. The shape and size of agglomerates were characterized by scanning electron microscopy (SEM), and the biochemical activity was mapped with scanning electrochemical microscopy (SECM). This approach is demonstrated using beads modified with anti-mouse antibodies (Ab). After allowing them to react with a conjugate of mouse IgG and alkaline phosphatase (ALP), the beads were deposited as agglomerates of well-defined size and shape. The biochemical activity was recorded in the generation-collection SECM mode by oxidizing 4-aminophenol formed in the ALP-catalyzed hydrolysis of 4-aminophenyl phosphate at the surface of the beads. The signal height correlated with both the amount of beads present in one agglomerate and the proportion of Ab binding sites saturated with the ALP mouse IgG conjugate. The feedback mode of the SECM was used to image streptavidin-coated beads after reaction with biotinylated glucose oxidase.

The application of the rotating disk electrode (RDE) directly on a micro drop of 50 μl has been developed for electrochemical detection in a bead-based immunoassay for the model analyte mouse IgG. The enzyme label alkaline phosphatase (ALP) was used for enzymatic turnover of the substrate 4-aminophenyl phosphate (PAPP) to the electroactive product 4-aminophenol (PAP). The determination of 4-aminophenol by the RDE has a linear range from 1.9 × 10 −8 to 1.9 × 10 −4 M. Direct analysis in a drop minimizes enzyme product dilution, a key requirement in achieving very low limits of detection. Furthermore, the detection scheme allows enzyme turnover to be monitored continuously, thus reducing errors associated with obtaining data at fixed times as in flow injection analysis. An unoptimized calibration curve for mouse IgG was obtained from 50 to 5000 ng/ml.

Amperometric detection with a rotating disk electrode (RDE) for a bead based enzyme immunoassay for the model antigen mouse IgG has been developed. Alkaline phosphatase was used as the enzyme label for the conversion of the substrate 4-aminophenyl phosphate to the electroactive 4-aminophenol (PAP). The detection scheme allows enzyme turnover to be monitored continuously and thus reduces errors associated with obtaining data at fixed times as in flow injection analysis. The determination of PAP by the RDE has a linear range from 5×10 −9 to 5×10 −5 M. An 800 μl electrochemical cell was used to minimize enzyme product dilution. A linear response for mouse IgG was obtained in this non-optimized system over the concentration range of 50–5000 ng ml −1 .

An amperometric immunoassay for indole-3-acetic acid (IAA) in tomato embryos was developed. The antibody against IAA was covalently bound to the chemically modified inner surface of a 22 μL silica capillary. The competitive binding of methylated IAA and an IAA alkaline phosphatase conjugate for a limited amount of antibody was accomplished in the silica capillary in 20 min. The enzymatic product p-aminophenol, produced from p-aminophenyl phosphate, was determined by flow injection analysis with amperometric detection. A detection limit for IAA of 3 pg/μL was obtained. The average concentration of IAA in mature embryos measured by this method is 0.37 ± 0.03 μg/g. Although isotope dilution GC-SIM-MS can achieve the same detection limit, the sample analysis is not feasible in such a complicated matrix and further purification and separation are required.

A competitive heterogeneous capillary enzyme immunoassay with electrochemical detection has been developed for phenobarbital in serum. The oxidized primary antibody was attached covalently to the modified interior surface of a microcapillary (22 microl). The competition between analyte phenobarbital and alkaline phosphatase labeled phenobarbital for a limited number of antibody binding sites was complete in 1.5 h. The enzymatic product (p-aminophenol) from the catalytic conversion of the substrate (p-aminophenyl phosphate) was detected by amperometric flow injection analysis. The calibration curve for phenobarbital had a detection limit of 30 microg l(-1) (2.8 pmoles or 0.65 ng) and a range of 30-3000 microg l(-1). The assay could be used to determine the phenobarbital serum concentration in a 4 microl clinical serum sample without pretreatment.

Three linear polymers of repeating amino acid units, or homopolypeptides, have been individually covalently bonded to microparticulate silica and evaluated for liquid chromatographic separations. The retention and selectivity of seven flavanones were investigated on these stationary phases and a structurally similar, commercially available reference stationary phase, Chiraspher. All three of the homopolypeptide stationary phases retain solutes in the normal-phase mode. The aromatic-containing homopolypeptide stationary phases also retain solutes in the reversed-phase mode. Selectivity values for the flavanones were higher in the normal-phase mode; chiral selectivity was observed for the amphiphilic homopolypeptide stationary phase in the reversed-phase mode. The retention mechanism of each stationary phase is suggested based on the chemical nature and conformation of the corresponding homopolypeptide ligand.

A capillary enzyme immunoassay with electrochemical detection has been developed for the determination of atrazine in water. The assay is based on competitive binding between atrazine and alkaline phosphatase-labeled atrazine for a limited number of antibody binding sites. The antibody is attached covalently to a modified capillary surface. The enzymatic product (p-aminophenol) is detected by amperometric flow injection analysis. The calibration curve for atrazine has a linear range of 0.10-10.0 μg/L and a detection limit of 0.10 μg/L. An assay including six standards can be done in less than 60 min. Intra- and inter-assay precisions at 0.1 and 5.0 μg/L are 9 and 8% and 6 and 10%, respectively. Recoveries of added atrazine from commercial bottled water, tap water, and Ohio River water at 0.50 and 5.0 μg/L range from 100 to 115%. Simple filtration is the only step needed for sample cleanup

Capillary enzyme immunoassay with flow-injection analysis for digoxin using the sequential saturation technique has been developed. Glass capillary tubes (10 cm × 0.53 mm i.d.) with immobilized digoxin antibody were used as the immunoassay reactor. The product of enzymatic reaction. 4-aminophenol, was detected amperometrically. The digoxin and the labeled digoxin binding reaction with the immobilized digoxin antibody were completed in 2 and 10 min, respectively. Digoxin was determined in a 20-μl sample with a detection limit of 10 pg ml−1 (200 fg or 260 attomoles) and a 3 orders of magnitude range.

A small-volume voltammetric detection of 4-aminophenol (PAP) has been developed using an interdigitated array (IDA) microelectrode cell in order to apply the IDA to electrochemical enzyme immunoassay. The signal of PAP at the IDA was steady state, and its magnitude was amplified compared with that of the usual single electrode due to redox cycling of PAP between the two finger sets of the IDA. A linear relationship between PAP concentration and cathodic limiting current was obtained from 1 to 1000 microM, reproducibly. The minimum sample volume in the measurement was reduced to 800 nL. High sample throughput of less than 1-min detection time per sample was achieved on 2-10-microL PAP samples. This IDA cell was applied to the electrochemical enzyme immunoassay of mouse IgG. Alkaline phosphatase was used as the enzyme label. The mouse IgG concentration was evaluated by detecting the concentration of PAP, which is the product of enzymatic reaction of the substrate, 4-aminophenyl phosphate (PAPP). Anti-mouse IgG was covalently immobilized on the glass surface of the small-volume immunowells by carbodiimide coupling. The assay range was 10-1000 ng/mL using 10-microL sample and 20-microL substrate solutions.